WO2021207433A2 - Épitopes d'anticorps neutralisant le sars-cov-2 - Google Patents

Épitopes d'anticorps neutralisant le sars-cov-2 Download PDF

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WO2021207433A2
WO2021207433A2 PCT/US2021/026286 US2021026286W WO2021207433A2 WO 2021207433 A2 WO2021207433 A2 WO 2021207433A2 US 2021026286 W US2021026286 W US 2021026286W WO 2021207433 A2 WO2021207433 A2 WO 2021207433A2
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cov
sars
spike
antibody
seq
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WO2021207433A3 (fr
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Yifan Cheng
Daniel ASARNOW
Charles CRAIK
Aashish MANGLIK
Markus Bohn
Cheng-I Wang
Yuanyu HU
Bei Wang
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Agency for Science Technology and Research Singapore
University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C07K16/102—Coronaviridae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C07K16/102—Coronaviridae (F)
    • C07K16/104—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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

  • the present invention relates to anti-SARS-CoV-2-Spike antibodies and the use of anti-SARS-CoV-2-Spike antibodies in the treatment of COVID-19.
  • BACKGROUND OF THE INVENTION [0003]
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • ACE2 host receptor angiotensin-converting enzyme 2
  • the present disclosure provides anti-SARS-CoV-2-Spike antibodies.
  • the present disclosure provides anti-SARS-CoV-2-Spike antibodies.
  • the present disclosure provides a method of preventing formation of syncytia comprising a fusion between a SARS-CoV-2 infected cell displaying a SARS-CoV- 2 Spike protein on its surface, and a second cell displaying an ACE-2 receptor on its surface, the syncytia formation mediated by the SARS-CoV-2 Spike protein binding to the ACE-2 receptor, the method comprising contacting a cell infected with SARS-CoV-2, or at risk of being infected with SARS-CoV-2 with an isolated anti-SARS-CoV-2-Spike antibody or an antigen binding fragment thereof specifically binding to the Spike protein, trapping the Spike protein in a pre-fusion state, thereby preventing the formation of the syncytia.
  • the isolated anti-SARS-CoV-2-Spike antibody or antigen binding fragment is an IgG, IgM, IgA, Fab, single domain antibody. [0008] In some embodiments, the isolated anti-SARS-CoV-2-Spike antibody or antigen binding fragment binds to a quaternary epitope of the Spike protein. [0009] In some embodiments, the isolated anti-SARS-CoV-2-Spike antibody binds to the quaternary epitope through residues in the CDR.
  • the isolated anti-SARS-CoV-2-Spike antibody or antigen binding fragment comprises a heavy chain variable domain sequence that is at least 90% identical to SEQ ID NO.7.
  • the isolated anti-SARS-CoV-2-Spike antibody or antigen binding fragment comprises a light chain variable domain sequence that is at least 90% identical to SEQ ID NO.8.
  • the isolated anti-SARS-CoV-2-Spike antibody or antigen binding fragment comprises a heavy chain variable region comprising complementarity determining regions (CDRs), wherein: a. the heavy chain CDR1 is SEQ ID NO.1; b. the heavy chain CDR2 is SEQ ID NO.2; and c.
  • CDRs complementarity determining regions
  • the heavy chain CDR3 is SEQ ID NO.3; and a light chain variable region comprising CDRs, wherein: d. the light chain CDR1 is SEQ ID NO.4; e. the light chain CDR2 is SEQ ID NO.5; and f. the light chain CDR3 is SEQ ID NO. 6.
  • the heavy chain variable region, SEQ ID NO.7 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations at E38, S57, Y58, D59, G62, S63, N64, and/or R106.
  • the mutation at position D59 is selected from D59G, D59L, D59K, D59I, D59M, D59W, D59T, D59F, or D59Y.
  • the mutation at position N64 is selected from N64R or N64I.
  • the mutation at position R106 is selected from R106E, R106D, R106N, R106Q, R106A, R106V, or R106L.
  • the light chain variable region, SEQ ID NO.8 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations at S69, L114, and/or R116.
  • the isolated anti-SARS-CoV-2-Spike antibody is a human antibody. [0019] In some embodiments, the isolated anti-SARS-CoV-2-Spike antibody is a humanized antibody. [0020] In some embodiments, the isolated anti-SARS-CoV-2-Spike antibody is a monoclonal antibody. [0021] In another aspect, the present disclosure provides method of treating a SARS-CoV-2 infection in a subject in need of such treatment, the method comprising administering to the subject an antibody described herein. [0022] In some embodiments, the antibody is in a pharmaceutical formulation comprising a pharmaceutically acceptable carrier.
  • the present disclosure provides an isolated anti-SARS-CoV-2-Spike antibody, wherein the isolated antibody or antigen binding fragment comprises a heavy chain variable domain sequence that is at least 90% identical to SEQ ID NO.7.
  • the isolated anti-SARS-CoV-2-Spike antibody or antigen binding fragment comprises a light chain variable domain sequence that is at least 90% identical to SEQ ID NO.8.
  • the isolated anti-SARS-CoV-2-Spike antibody or antigen binding fragment comprises a heavy chain variable region comprising complementarity determining regions (CDRs), wherein: a.
  • CDRs complementarity determining regions
  • the heavy chain CDR1 is SEQ ID NO.1; binds through T29, S36, Y37, E38); b. the heavy chain CDR2 is SEQ ID NO 2 (ISYDGSNK); binds through V55, I56, S57, Y58, D59, N64, Y66; and c. the heavy chain CDR3 is SEQ ID NO.3 (ARLITMVRGEDY; binds through R106, L107, T109, M110, V112, R113, G114, E115), and a light chain variable region comprising CDRs, wherein: d. the light chain CDR1 is SEQ ID NO.4 (QSISSY; binds through S36, Y38); e.
  • the light chain CDR2 is SEQ ID NO. 5 (AAS; binds through S69, G70); and f. the light chain CDR3 is SEQ ID NO.6 (QQSYNLPRT; binds through S107, Y108, N109, L114, R116).
  • the heavy chain variable region, SEQ ID NO.7 comprises at least one mutation at E38, S57, Y58, D59, G62, S63, N64, and/or R106.
  • the mutation at position D59 is selected from D59G, D59L, D59K, D59I, D59M, D59W, D59T, D59F, or D59Y.
  • the isolated anti-SARS-CoV-2-Spike antibody comprises a mutation at position N64 is selected from N64R or N64I.
  • the isolated anti-SARS-CoV-2-Spike antibody mutation at position R106 is selected from R106E, R106D, R106N, R106Q, R106A, R106V, or R106L.
  • the light chain variable region, SEQ ID NO.8, comprises a mutation at S69, L114 and/or R116.
  • the isolated anti-SARS-CoV-2-Spike antibody is a human antibody.
  • the isolated anti-SARS-CoV-2-Spike antibody is a humanized antibody. [0033] In some embodiments, the isolated anti-SARS-CoV-2-Spike antibody is a monoclonal antibody. [0034] In some embodiments, isolated polynucleotide composition comprising a first nucleic acid encoding the VH region of any of the amino acid sequences described herein, and a second nucleic acid encoding the VL region of any of the amino acid sequences described herein. [0035] In some embodiments, the present disclosure provides a plasmid comprising at least one polynucleotide sequence of any of the polynucleotide compositions described herein.
  • the present disclosure provides a host cell comprising a polynucleotide sequence described herein. [0037] In some embodiments, the present disclosure provides a method of producing antibodies comprising culturing the host cell under conditions that promote the production of the antibodies described herein. [0038] In some embodiments, the present disclosure provides pharmaceutical compositions comprising as an active ingredient, at least one isolated antibody or antigen binding fragment thereof described herein and a pharmaceutically acceptable carrier.
  • the present disclosure provides pharmaceutical composition described herein for use in preventing or retarding formation of SARs-CoV-2 mediated syncytia formation by binding to RBD and stabilizing a SARs-CoV-2 Spike protein conformation retarding or preventing S1 shedding and trapping a pre-fusion state of the SARs-CoV-2.
  • BRIEF DESCRIPTION OF THE DRAWINGS [0040] The invention may be better understood from the following detailed description when read in conjunction with the accompanying drawings.
  • Figure 1 shows isolation of SARS-CoV-2 receptor-blocking antibodies from a na ⁇ ve human library
  • A Blocking of ACE2/RBD (SARS-CoV-2) interactions by 27 Fab clones, tested by competition ELISA.
  • the samples used in the assays were unpurified Fab from bacterial supernatant, hence the percentages of blocking were not indicative of their true potency.
  • Red arrows indicate the 6 clones in subsequent studies.
  • B KD of Fab based on 1:1 Langmuir fitting and apparent KD of IgG based on 1:2 bivalent analyte fitting of BLI sensorgrams for immobilized Fc-RBD.
  • Vero E6 cells were transfected with furin recognition mutation of SARS-CoV-2 S-protein (R682RAR to A682AAR)-GFP. After 48 hours, the cell culture medium was changed to DMEM (no serum) and treated WT/WO antibodies and incubated for 1 hour at 37°C. The cells were then treated WT/WO trypsin 15 ⁇ g/ml for 2 hours at 37°C. Cells were fixed with 4%PFA and stained with DAPI.
  • FIG. 4 shows Structures of Spike-Fab complexes.
  • A Schematic representation of three Fabs, 5A6 (goldenrod), 2H4 (purple), and 3D11 (sky blue), bound to the SARS-CoV-2 Spike protein. All Fabs are shown in relation to the complex formed by an open RBD (red) and the extracellular domain of ACE2 (rose brown).
  • FIG. 5 shows binding mode and epitope of 5A6.
  • A A tour through the primary interface between Spike RBD and 5A6, using three immediately adjacent cross-sections along the viewing axis. Spike residues and labels are colored in coral or by heteroatom, and 5A6 residues are colored goldenrod, with labels for VH residues in black and VL residues in gray. Fab residue labels use the IMGT numbering system (Lefranc et al., 2003). Predicted hydrogen bonds are shown as dashed gray lines.
  • the interface features extensive hydrogen bonding, numerous hydrophobic contacts, and multiple salt bridges.
  • a aromatic cluster formed by 5A6 VL Y38 and Y108, and RBD F486, a salt bridge between 5A6 VH R106 and RBD E484, and a cation-pi interaction between Fab VH R112 and RBD Y449 are particularly notable.
  • the secondary interface between 5A6 and the neighboring open RBD comprises mostly hydrogen bonds, many of which involve main-chain atoms. Atom and label colors are as in (A).
  • An interesting feature is stabilization of an alternate conformation of RBD R408 by 5A6 VL T20 and T88.
  • FIG. 6 shows spike functional modulation by receptor-blocking antibodies
  • PDB 1hzh
  • FIG. 6 shows spike functional modulation by receptor-blocking antibodies
  • the Spike trimer When serially bound by ACE2 or an orthosteric mimetic antibody like 2H4, the Spike trimer passes through a series of conformations that eventually permit S1 shedding and the S2 post-fusion transition that mediates membrane fusion.
  • allosteric antibodies such as 3D11 can advance the trimer directly to the end of the opening process, potentiating formation of syncytia through fusion of neighboring cells. Allosteric opening most likely contributes to lower potency in a high-affinity receptor- blocking antibody, and might even suggest the possibility antibody-dependent enhancement of infection.
  • the Spike might instead be recognized by 5A6, which inhibits membrane fusion and syncytia formation by preventing S1 shedding and trapping the pre-fusion trimer.
  • Figure 7 shows blocking of ACE2/SARS-CoV-2 RBD interaction by 1F4, 2H4, 3D11, 3F11, 5A6 and 6F8 IgGs tested by competition ELISA, Related to Figure 1. Data are presented as means ⁇ SD in triplicates and are representative of two independent experiments.
  • Figure 8 shows binding avidity of 1F4, 2H4, 3D11, 3F11, 5A6 and 6F8 IgG antibodies to SARS-CoV-2 Spike RBD proteins tested by ELISA, Related to Figure 1.
  • Figure 9 shows binding affinity of five Fab clones to SARS-CoV-2 Spike RBD protein measured by biolayer interferometry, Related to Figure 1 Fab binding to immobilized Fc-RBD was tested using a range of Fab concentrations from 100 nM to 3.125 nM (in 2-fold dilution). A representative set of measurements is shown with sensorgrams in black and curve fittings in red.
  • Figure 10 shows binding avidity of six IgGs to the RBD by biolayer interferometry, Related to Figure 1 IgG Binding to immobilized Fc-RBD was tested using a range of IgG concentrations from 12.5 nM to 0.39 nM (in 2-fold dilutions). The anti-Fc sensor chip was quenched with excess irrelevant, same-isotype IgG to prevent confounding from antibody binding directly to the chip. Representative sensorgrams are shown in black, with curve fittings in red. [0051] Figure 11 shows the potency of 2H4, 3D11 and 5A6 IgG antibodies in neutralizing live SARS-CoV-2 virus assays determined by measuring the viral genome copy number (GCN), Related to Figure 2.
  • GCN viral genome copy number
  • Figure 12 shows Cryo-EM densities and resolution estimation, related to Figure 4. Density maps colored by local resolution, Fourier shell correlation curves, and particle orientation distributions for the structures reported in this work. All maps use the same local resolution scale, shown at the top right of the figure.
  • A The apo Spike, with all RBDs closed.
  • B The apo Spike, with one RBD open.
  • C The Spike:3D11 complex.
  • D Refinement of Spike:3D11, focused on the Fab variable domains and RBD 16 epitope.
  • E Spike:2H4 complexes with one, two, or three Fabs bound.
  • FIG. 13 shows additional structural details, related to Figure 4.
  • A Eight subclasses of the Spike:3D11 complex, determined using symmetry relaxation in Relion 3.1. At left, top views show that these classes vary in the occupancy of Fab at each RBD. Only Fabs are colored, with missing or weak Fab densities are indicated by black ellipses. At right, two extremum classes and one intermediate show relative motion of the RBDs and NTDs, relaxations which likely contribute to the S2 unsheathing that eventually permits Spike- mediated membrane fusion.
  • Fabs, RBDs, and NTDs are colored, and a dashed line delineates Fab and RBD.
  • the general direction of S2-opening movements, as observed in the classes, are indicated by black arrows.
  • the quaternary epitope bound by 5A6 is cryptic because the Fab stabilizes unique conformations of RBDs, observed only the the complex, that contribute to the epitope.
  • RBDs from Spike:5A6 complex I goldenrod
  • the hinge regions connecting the Fc domain of an IgG antibody to each of its two Fabs are 23 residues long, approximately 10 of which are flexible due to disulfide bonds. Assuming a standard polypeptide length of about 3.5 ⁇ per residue, each hinge might extend as far as 35 ⁇ , allowing for some 70 ⁇ separation between the two Fabs.
  • the shortest gaps between Fabs in the Spike:2H4 and Spike:5A6 complexes are ⁇ 90 ⁇ , however variation of the elbow angles between Fab V and C domains could reduce the effective separation.
  • Different Fab clones have elbow angles across just over 90° (Stanfield et al., 2006), and changes as great as 37° have been observed between multiple structures of the same clone (Wilson and Stanfield, 1994). For example, a 15° elbow bend might reduce separation by 10 ⁇ at each Fab (20 ⁇ total), to about the maximum length of the hinge.
  • Bivalent, IgG-bound states thus likely differ in Fab elbow angle and feature some relaxation of the RBDs, in order to support the avid binding of IgG antibodies to Spike trimer observed in our experiments.
  • the Spike:2H4 IgG sample contains numerous, relatively small particles, and some of its 2D class averages resemble monomeric Spike (classes 4 and 6) or two Spike monomers crosslinked by antibody (class 3).
  • the Spike:3D11 IgG sample much of the protein is contained within stereotypical aggregates approximately 150 nm in size. Notable 2D averages resemble a Fab bound to Spike RBD (classes 3-5), rare Spike trimers in the closed conformation (class 2), and a potential Spike dimer or pair of monomers crosslinked by antibody (class 6).
  • Spike:5A6 IgG is a well-behaved sample (despite the crowded micrograph).
  • FIG 14 shows Cryo-EM processing, Related to Figure 4 (A) A micrograph drawn from the Spike:5A6 complex dataset, representative of those 11 obtained for the Fab complexes. (B) Selected 2D class averages of Spike:5A6 particles, evincing clear secondary structure and multiple Fabs bound to the RBDs. (C) Cryo-EM image processing workflow for Spike alone, leading to structures of the trimer with all RBDs closed, and with one RBD open or in an intermediate state.
  • IC50 was calculated by variable slope four parameter non-linear regression model using Graphpad PRISM 7 Software or ⁇ Quest GraphTM IC50 23 Calculator from AAT Bioquest, Inc (https://www.aatbio.com/tools/ic50-calculator) with 24 top and bottom constrains set at 100% and 0% respectively.
  • Figure 17 shows variable domain identity to the germline sequences.
  • Figure 18 shows binding kinetics of antibodies and Fabs measured against Fc-RBD by BLI or Spike trimer by SPR.
  • Figure 19 shows Cryo-EM data collection, refinement and validation statistics.
  • Figure 20 shows an overview of the 5A6 binding surface.
  • Figure 21 shows an overview of the 5A6 binding surface, the main contributor from this loop is Tyr32 according to the sequence numbering in Figure 46 and/or Figure 47 (or the corresponding position according to IMGT numbering).
  • L1 should be seen in context with L3.
  • Figure 22 shows an overview of the 5A6 binding surface. L2 is not forming a proper interface at this point. Diversifying upstream of Gly57 according to the sequence numbering in Figure 46 and/or Figure 47 (or the corresponding position according to IMGT numbering)might give a chance for a second interaction.
  • Ser56 according to the sequence numbering in Figure 46 and/or Figure 47 might be a good option for a point mutation to introduce pi-stacking (Tyr) or a hydrogen bond.
  • Figure 23 shows an overview of the 5A6 binding surface. Tyr92 according to the sequence numbering in Figure 46 and/or Figure 47 (or the corresponding position according to IMGT numbering)provides the main interaction. Pro95 according to the sequence numbering in Figure 46 and/or Figure 47 (or the corresponding position according to IMGT numbering) might be involved in constraining the loop in the desired geometry.
  • Arg96 according to the sequence numbering in Figure 46 and/or Figure 47 (or the corresponding position according to IMGT numbering) is too distant to interact with the RBD Glu residue to productively interact and could become (for example) A) hydrophobic (Ala > Ile > Leu) or B) part of an engineered hydrogen bonding network (as an e.g. Asp) with HC residue Val50 according to the sequence numbering in Figure 46 (or the corresponding position according to IMGT numbering).
  • FIG. 24 shows an overview of the 5A6 binding surface.
  • Glu33 or E38 according to IMGT numbering
  • IMGT numbering is a suboptimal amino acid at this position. Neutralizing the charge (Asn > Gln) is an obvious choice.
  • a hydrophobic can be placed here (A > I > L) and even a Gly compares very favorably with Glu (there is actually not a single amino acid that could be worse at this position, anything is better than Glu).
  • H1 forms an extended interface with H2.
  • FIG. 25 shows an overview of the 5A6 binding surface.
  • H2 green
  • H1 forms a contiguous interface with H1 and contains Val50 (or V55 according to IMGT numbering) which engages V483 (red circle).
  • Val50 or V55 according to IMGT numbering
  • V483A potential hydrogen bonding network if both become Tyr.
  • Figure 26 shows an overview of the 5A6 binding surface, in particular H3 (blue).
  • Figure 27 shows an overview of the 5A6 binding surface.
  • Figure 28 shows an overview of the 5A6 binding surface.
  • Figure 29 shows ddg predictions with rosetta with limited backbone flexibility (cartoon of the structure is magenta and the ddg model is green). The ddg prediction accommodated V483A (in spike) and attempted to reconfigure a network around E38 (VH, CDR1) interacting with R116 (also known as raw sequence R96) (VL, CDR3).
  • E38 when mutated to Q (red) could bond with E484 and allow R116 (also known as raw sequence R96) (orange in the structure, blue in the ddg model) to interact with N40 (VH, FR2, yellow) to maintain the scaffold.
  • Figure 30 shows V483 in green sticks with hydrogens and L114 (VL, CDR3, magenta) in the ddg model (green) to show favorable interactions in the WT.
  • Figure 31 shows single point mutants at heavy chain position S52 according to the sequence numbering in Figure 46 (or S57 according to IMGT numbering). Mutations that improve binding (at least 5x slower Koff compared to 5A6 WT) are highlighted in Green.
  • Figure 32 shows single point mutants at heavy chain position Y53 according to the sequence numbering in Figure 46 (or Y58 according to IMGT numbering). Mutations that improve binding (at least 5x slower Koff compared to 5A6 WT) are highlighted in Green. Mutations that weaken binding (at least 5x faster Koff compared to 5A6 WT) are highlighted as Red.
  • Figure 33 shows single point mutants at heavy chain position D54 according to the sequence numbering in Figure 46 (or D59 according to IMGT numbering). Mutations that improve binding (at least 5x slower Koff compared to 5A6 WT) are highlighted in Green.
  • Figure 34 shows single point mutants at heavy chain position G55 according to the sequence numbering in Figure 46 (or G62 according to IMGT numbering). Mutations that improve binding (at least 5x slower Koff compared to 5A6 WT) are highlighted in Green. Mutations that weaken binding (at least 5x faster Koff compared to 5A6 WT) are highlighted as Red.
  • Figure 35 shows single point mutants at light chain position S56 according to the sequence numbering in Figure 46 (or S69 according to IMGT numbering). Mutations that improve binding (at least 5x slower Koff compared to 5A6 WT) are highlighted in Green.
  • Figure 36 shows single point mutants at heavy chain position N57 according to the sequence numbering in Figure 46 (or N64 according to IMGT numbering). Mutations that improve binding (at least 5x slower Koff compared to 5A6 WT) are highlighted in Green. Mutations that weaken binding (at least 5x faster K off compared to 5A6 WT) are highlighted as Red.
  • Figure 37 shows single point mutants at light chain position L94 according to the sequence numbering in Figure 47 (or L114 according to IMGT numbering). Mutations that improve binding (at least 5x slower Koff compared to 5A6 WT) are highlighted in Green.
  • FIG. 38 shows single or double point mutants at light chain R96 and S56 according to the sequence numbering in Figure 46 and Figure 47 (or light chain R116 and S69 according to IMGT numbering). Mutations that improve binding (at least 5x slower Koff compared to 5A6 WT) are highlighted in Green. Mutations that weaken binding (at least 5x faster Koff compared to 5A6 WT) are highlighted as Red.
  • Figure 39 shows single point mutants at heavy chain E33 according to the sequence numbering in Figure 47 (or Y58 according to IMGT numbering) with single or double mutants at light chain R96 and S56 according to the sequence numbering in Figure 46 and Figure 47 (or R116 and S69 according to IMGT numbering).
  • Mutations that improve binding (at least 5x slower Koff compared to 5A6 WT) are highlighted in Green.
  • Mutations that weaken binding at least 5x faster K off compared to 5A6 WT) are highlighted as Red.
  • Figure 40 shows binding ELISA of supernatants of 27 Fab clones to the antigen protein of biotinylated RBD-mFc.
  • FIG. 41 shows blocking of spike RBD protein binding to ACE2-His by supernatants of 27 Fab clones.
  • the 27 Fab clones were tested in an ELISA assay to assess their potency in blocking spike protein binding to the recombinant ACE2 protein (ACE2 with His tag), with an irrelevant Fab clone used as a negative control.
  • Figure 42 shows a binding ELISA of 19 IgGs to the antigen protein of biotinylated RBD-mFc.
  • FIG. 43 shows blocking of spike RBD protein binding to ACE2-Fc by 19 IgGs.
  • the 19 antibody clones were tested in an ELISA assay to assess their potency in blocking spike protein binding to the recombinant ACE2 protein (ACE2 with human Fc tag), with an irrelevant IgG1 used as a negative control antibody.
  • Figure 44 shows pseudotyped virus neutralization assay by 19 IgGs.
  • Figure 45 shows 5A6 IMGT numbering.
  • Figure 46 shows the variable heavy chain of 5A6 with corresponding raw sequence numbers, IMGT numbering, and Kabat numbering. The figure also shows prospective mutation essential and prospective mutation desired positions. Red shows primary interface residues, purple shows secondary interface residues. T29, S36, Y37, E38 interface with T470, I472, G482, V483, E484, F490 to form a buried surface.
  • E38 receives electrophilic polarization by the side-chain of E484 and forms a hydrogen bond with the backbone amide nitrogen of E484.
  • Y37 forms a parallel-displaced ⁇ - ⁇ interaction with F490.
  • V55, I56, S57, Y58, D59, N64, Y66 form a buried interface with T470, E471, I472, N481, G482, V483.
  • the positively charged ring edge of Y58 forms an anion- ⁇ interaction with E471.
  • Y66 forms a hydrogen bond with N481.
  • R106, L107, T109, M110, V112, R113, G114, E115 form a buried interface with E484, G485, Y489, F490, L492, Q493, S494.
  • R106 forms a salt bridge with E484.
  • R113 forms a cation- ⁇ interaction with Y449.
  • Figure 47 the variable light chain of 5A6 with corresponding raw sequence numbers, IMGT numbering, and Kabat numbering. Red shows primary interface residues, purple shows secondary interface residues. The figure also shows prospective mutation essential and prospective mutation desired positions.
  • R18, T20, T22, S28, S36, S65, S77, S79, G80, S83, G84, T85, D86, T88, T90 form a buried interface with Y369, A372, F374, S375, T376, F377, K378, G404, D405, R408, Q414, K417, V503, G504, Y508.
  • the backbone carbonyl oxygen of S83 forms a hydrogen bond with the backbone amide nitrogens of F377, K378.
  • the backbone amide nitrogen of T85 forms a hydrogen bond with the backbone carbonyl oxygen of S375.
  • D86, R24 form a hydrogen-bonding-network with Y508.
  • S36 interfaces with F486 to form a buried surface.
  • Y38 forms a hydrogen bonding network with N487, Y489 and a T- shaped ⁇ - ⁇ interaction with F486.
  • S69 and G70 interface with Y449 to form a buried surface.
  • S107, Y108, N109, L114, R116 interface with V483, E484, G485, F186 to form a buried surface.
  • SARS-CoV-2 forms a face-to-face ⁇ - ⁇ interaction with F486.
  • R116 forms a hydrogen bond with the backbone carbonyl oxygen of E484.
  • ACE2 angiotensin-converting enzyme 2
  • the virus enters cells by fusion of the viral envelop with cellular plasma membranes and alternatively by endocytosis and subsequent fusion of the viral envelope with endosomal membranes.
  • the SARS-CoV-2 Spike protein comprises two subunits, S1 and S2, and is responsible for target recognition and mediating viral entry.
  • the Spike protein Upon binding to the host cell receptor through the receptor binding domain (RBD) at the tip of the S1 subunit, the Spike protein undergoes dramatic conformational changes and proteolytic processing. Further shedding of the S1 subunit exposes the S2 subunit fusion peptide, which inserts into the host cell membrane and induces viral fusion.
  • RBD receptor binding domain
  • Both SARS-CoV-2 and SARS-CoV use ACE2 as the entry receptor to infect host cells.
  • the RBD binds to ACE2 via the receptor binding motif (RBM), a small patch made up of about 20 amino acids.
  • syncytia multinucleated giant cells
  • Synctia are associated with lung tissue damage in SARS-CoV and MERS-CoV infections, and have been widely observed in autopsies of patients afflicted with severe COVID-19. Synctia are also implicated in chronic cardiovascular injury due to COVID-19. Blocking the RBD/ACE2 interaction is useful in the treatment of coronaviruses, for example COVID-19.
  • Receptor engagement to a Spike RBD locks it in the open conformation and triggers a cooperative process in which the Spike conformational ensemble is driven towards further opening by successive rounds of receptor binding. This process culminates in unsheathing of the S1-ACE2 subcomplex. S1 shedding in turn facilitates the post-fusion state transition, leading to membrane fusion and viral entry.
  • Spike protein on the surface of infected cells can also mediate ACE2-dependent fusion of neighboring cells to form multinucleated giant cells, presumably through the same cycle of proteolysis and conformational transitions. Genetic variation in Spike, inside and outside the RBM, can influence function by altering the conformational equilibrium of the trimer.
  • Infection by SARS-CoV-2 is initiated by binding of viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. While antibodies that block this interaction are in emergency use as early COVID-19 therapies, precise determinants of neutralization potency remain unknown. A series of antibodies was discovered that all potently block ACE2 binding, yet exhibit divergent neutralization efficacy against live virus. Strikingly, these neutralizing antibodies can either inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in COVID-19 patients.
  • ACE2 host receptor angiotensin-converting enzyme 2
  • Cryo-EM structures are used to reveal the conformational landscapes of Spike-Fab complexes and provide 3D mapping of antibody-Spike epitopes, which are used to draw mechanistic insights that explain distinct bioactivities in the known intermediates in Spike opening (see Figure 6).
  • Multiple cryogenic electron microscopy structures of Spike-antibody complexes reveal distinct binding modes that not only block ACE2 binding, but also alter the Spike protein conformational cycle triggered by ACE2 binding.
  • the data in the present disclosure shows that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion, with profound implications in COVID-19 pathology and immunity.
  • the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • antigen binding domain or “ABD” herein is meant a set of six Complementary Determining Regions (CDRs) that, when present as part of a polypeptide sequence, specifically binds a target antigen as discussed herein.
  • CDRs Complementary Determining Regions
  • these CDRs are generally present as a first set of variable heavy CDRs (vhCDRs or VHCDRs or CDR-HC or CDRH) and a second set of variable light CDRs (vlCDRs or VLCDRs or CDR-LC or CDRL), each comprising three CDRs: vhCDR1, vhCDR2, vhCDR3 (also referred to herein as CDRH1, CDRH2, CDRH3) for the heavy chain and vlCDR1, vlCDR2 and vlCDR3 (also referred to herein as CDRL1, CDRL2, CDRL3) for the light chain.
  • vhCDRs or VHCDRs or CDR-HC or CDRH variable heavy CDRs
  • vlCDRs or VLCDRs or CDR-LC or CDRL variable light CDRs
  • the CDRs are present in the variable heavy and variable light domains, respectively, and together form an Fv region.
  • the six CDRs of the antigen binding domain are contributed by a variable heavy and variable light chain.
  • the set of 6 CDRs are contributed by two different polypeptide sequences, the variable heavy domain (vh or VH; containing the vhCDR1, vhCDR2 and vhCDR3) and the variable light domain (vl or VL; containing the vlCDR1, vlCDR2 and vlCDR3), with the C-terminus of the vh domain being attached to the N- terminus of the CH1 domain of the heavy chain and the C-terminus of the vl domain being attached to the N-terminus of the constant light domain (and thus forming the light chain).
  • VH and VL domains are covalently attached, generally through the use of a linker as outlined herein, into a single polypeptide sequence, which can be either (starting from the N-terminus) vh-linker-vl or vl-linker-vh, with the former being generally preferred (including optional domain linkers on each side, depending on the format used.
  • the CDRs are separated by framework regions in each of the variable heavy and variable light domains: for the light variable region, these are FR1-vlCDR1-FR2- vlCDR2-FR3-vlCDR3-FR4, and for the heavy variable region, these are FR1-vhCDR1-FR2- vhCDR2-FR3-vhCDR3-FR4, with the framework regions showing high identity to human germline sequences.
  • Antigen binding domains of the invention include, Fab, Fv and scFv.
  • linker herein is meant a linker used in scFv and/or other antibody structures.
  • linker peptide may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr.
  • the linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity.
  • the linker is from about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length.
  • linkers of 1 to 20 amino acids in length may be used, with from about 5 to about 10 amino acids finding use in some embodiments.
  • Useful linkers include glycine-serine polymers, including for example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least one (and generally from 3 to 4), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • glycine-serine polymers including for example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least one (and generally from 3 to 4), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • non-proteinaceous polymers including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers, that is may find use as linkers.
  • linker sequences may include any sequence of any length of CL/CH1 domain but not all residues of CL/CH1 domain; for example the first 5-12 amino acid residues of the CL/CH1 domains.
  • Linkers can be derived from immunoglobulin light chain, for example C ⁇ or C ⁇ .
  • Linkers can be derived from immunoglobulin heavy chains of any isotype, including for example C ⁇ 1, C ⁇ 2, C ⁇ 3, C ⁇ 4, C ⁇ 1, C ⁇ 2, C ⁇ , C ⁇ , and C ⁇ .
  • Linker sequences may also be derived from other proteins such as Ig-like proteins (e.g., TCR, FcR, KIR), hinge region-derived sequences, and other natural sequences from other proteins.
  • the linker is a “domain linker”, used to link any two domains as outlined herein together. While any suitable linker can be used, many embodiments utilize a glycine-serine polymer, including for example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least one (and generally from 3 to 4 to 5) as well as any peptide sequence that allows for recombinant attachment of the two domains with sufficient length and flexibility to allow each domain to retain its biological function.
  • the term “antibody” is used in the broadest sense and includes, for example, an intact immunoglobulin or an antigen binding portion.
  • Antigen binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • antibody includes traditional tetrameric antibodies of two heavy chains and two light chains, as well as antigen binding fragments such as Fv, Fab and scFvs.
  • the invention provides bispecific antibodies that include at least one antigen binding domain as outlined herein.
  • modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a protein.
  • a modification may be an altered carbohydrate or PEG structure attached to a protein.
  • amino acid modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence.
  • amino acid modification is always to an amino acid coded for by DNA, e.g., the 20 amino acids that have codons in DNA and RNA.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid.
  • the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism.
  • the substitution M252Y refers to a variant polypeptide, in this case an Fc variant, in which the methionine at position 252 is replaced with tyrosine.
  • a protein which has been engineered to change the nucleic acid coding sequence but not change the starting amino acid is not an “amino acid substitution”; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution.
  • variant protein or “protein variant”, or “variant” as used herein is meant a protein that differs from that of a parent protein by virtue of at least one amino acid modification.
  • Protein variant may refer to the protein itself, a composition comprising the protein, or the amino sequence that encodes it.
  • the protein variant has at least one amino acid modification compared to the parent protein, e.g., from about one to about seventy amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent.
  • the parent polypeptide for example an Fc parent polypeptide, is a human wild type sequence, such as the Fc region from IgG1, IgG2, IgG3 or IgG4.
  • variant sequence herein will preferably possess at least about 80% identity with a parent protein sequence, and most preferably at least about 90% identity, more preferably at least about 95%-98%-99% identity.
  • variant protein can refer to the variant protein itself, compositions comprising the protein variant, or the DNA sequence that encodes it.
  • antibody variant or “variant antibody” as used herein is meant an antibody that differs from a parent antibody by virtue of at least one amino acid modification
  • IgG variant or “variant IgG” as used herein is meant an antibody that differs from a parent IgG (again, in many cases, from a human IgG sequence) by virtue of at least one amino acid modification
  • immunoglobulin variant or “variant immunoglobulin” as used herein is meant an immunoglobulin sequence that differs from that of a parent immunoglobulin sequence by virtue of at least one amino acid modification.
  • Fc variant or “variant Fc” as used herein is meant a protein comprising an amino acid modification in an Fc domain.
  • the Fc variants of the present invention are defined according to the amino acid modifications that compose them.
  • M252Y or 252Y is an Fc variant with the substitution tyrosine at position 252 relative to the parent Fc polypeptide.
  • M252Y/S254T/T256E defines an Fc variant with the substitutions M252Y, S254T and T256E relative to the parent Fc polypeptide.
  • the identity of the wild type amino acid may be unspecified, in which case the aforementioned variant is referred to as 252Y/254T/256E.
  • amino acid position numbering is according to IMGT numbering. All IMGT numbering was done using ANARCI. (See Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) PMID: 12477501; hereby entirely incorporated by reference).
  • the EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporated by reference.)
  • the modification can be an addition, deletion, or substitution. Substitutions can include naturally occurring amino acids and, in some cases, synthetic amino acids. See Figure 46 and Figure 47 for examples of the numbering system used herein.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the peptidyl group may comprise naturally occurring amino acids and peptide bonds.
  • Fab or "Fab region” as used herein is meant the polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full-length antibody, antibody fragment or Fab fusion protein.
  • Fv or “Fv fragment” or “Fv region” as used herein is meant a polypeptide that comprises the VL and VH domains of a single antigen binding domain (ABD).
  • amino acid and amino acid identity as used herein is meant one of the 20 naturally occurring amino acids that are coded for by DNA and RNA.
  • parent polypeptide as used herein is meant a starting polypeptide that is subsequently modified to generate a variant.
  • the parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide.
  • Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it.
  • parent immunoglobulin as used herein is meant an unmodified immunoglobulin polypeptide that is modified to generate a variant
  • parent antibody as used herein is meant an unmodified antibody that is modified to generate a variant antibody.
  • parent antibody includes known commercial, recombinantly produced antibodies as outlined below.
  • hetero constant region herein is meant the CH1-hinge-CH2-CH3 portion of an antibody, generally from human IgG1, IgG2 or IgG4.
  • target antigen as used herein is meant the molecule that is bound specifically by the variable region of a given antibody.
  • target cell as used herein is meant a cell that expresses a target antigen.
  • variant region as used herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the V.kappa., V.lamda., and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively.
  • wild type or WT herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations.
  • a WT protein has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.
  • position as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the EU index for antibody numbering.
  • residue as used herein is meant a position in a protein and its associated amino acid identity. For example, Asparagine 297 (also referred to as Asn297 or N297) is a residue at position 297 in the human antibody IgG1.
  • the antibodies of the present invention are generally recombinant. “Recombinant” means the antibodies are generated using recombinant nucleic acid techniques in exogenous host cells.
  • Percent (%) amino acid sequence identity with respect to a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific (parental) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects “sequence identity.”
  • Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • invention sequence The degree of identity between an amino acid sequence of the present invention
  • parental amino acid sequence is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "invention sequence,” or the length of the parental sequence, whichever is the shortest. The result is expressed in percent identity.
  • two or more amino acid sequences are at least 50%, 60%, 70%, 80%, or 90% identical. In some embodiments, two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.
  • Specific binding or “specifically binds to” or is “specific for” a particular antigen or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.
  • Kassoc or “Ka”, as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction
  • Kdis or “Kd,” as used herein, is intended to refer to the dissociation rate of a particular antibody- antigen interaction
  • K D is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M).
  • K D values for antibodies can be determined using methods well established in the art.
  • the method for determining the K D of an antibody is by using surface plasmon resonance, for example, by using a biosensor system such as a BIACORE® system.
  • the K D of an antibody is determined by Bio-Layer Interferometry.
  • the K D value is measured with the immobilized.
  • the K D value is measured with the antibody (e.g., parent mouse antibody, chimeric antibody, or humanized antibody variants) immobilized.
  • the K D value is measured in a bivalent binding mode.
  • the K D value is measured in a monovalent binding mode.
  • a “disease” includes a state of health of an animal, including a human, wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a “disorder” in an animal, including a human includes a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • the terms “treatment”, “treating”, “treat”, and the like, refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof or reducing the likelihood of a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and/or relieving one or more disease symptoms.
  • Treatment is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition.
  • treatment encompasses delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g., in the case of a vaccine.
  • mammal refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. In some embodiments, the mammals are from the order Carnivora, including felines (cats) and canines (dogs).
  • the mammals are from the order Artiodactyla, including bovines (cows) and swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • the mammal is a human. In some embodiments, the mammal is cynomolgus monkey.
  • An “effective amount” or “therapeutically effective amount” of a composition includes that amount of the composition which is sufficient to provide a beneficial effect to the subject to which the composition is administered.
  • an “effective amount” of a delivery vehicle includes that amount sufficient to effectively bind or deliver a composition.
  • subject or “subject” or “patient” is meant any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cynomolgus monkey, cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.
  • a first therapy is administered during the entire course of administration of a second therapy; where the first therapy is administered for a period of time that is overlapping with the administration of the second therapy, e.g., where administration of the first therapy begins before the administration of the second therapy and the administration of the first therapy ends before the administration of the second therapy ends; where the administration of the second therapy begins before the administration of the first therapy and the administration of the second therapy ends before the administration of the first therapy ends; where the administration of the first therapy begins before administration of the second therapy begins and the administration of the second therapy ends before the administration of the first therapy ends; where the administration of the second therapy begins before administration of the first therapy begins and the administration of the first therapy ends before the administration of the second therapy ends.
  • “in combination” can also refer to regimen involving administration of two or more therapies. “In combination with” as used herein also refers to administration of two or more therapies which may be administered in the same or different formulations, by the same or different routes, and in the same or different dosage form type.
  • “Encoding” includes the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if, for example, transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • the term “nucleic acid” includes RNA or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide or polynucleotide.
  • nucleotide sequence includes the ordering of nucleotides in an oligonucleotide or polynucleotide in a single-stranded form of nucleic acid.
  • nucleic acid construct it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes including non-native nucleic acid sequences, and the like.
  • operably linked includes a polynucleotide in functional relationship with a second polynucleotide, e.g., a single-stranded or double- stranded nucleic acid moiety comprising the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized, upon the other.
  • a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region. The order specified when indicating operably linkage is not important.
  • the phrases: “the promoter is operably linked to the nucleotide sequence” and “the nucleotide sequence is operably linked to the promoter” are used interchangeably herein and are considered equivalent.
  • the nucleic acid encoding the desired protein further comprises a promoter/regulatory sequence
  • the promoter/regulatory sequence is positioned at the 5' end of the desired protein coding sequence such that it drives expression of the desired protein in a cell.
  • oligonucleotide refers to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • the backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups.
  • the term “recombinant,” as applied to a polynucleotide means the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures resulting in a construct distinct and/or different from a polynucleotide found in nature. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
  • promoter includes a DNA sequence operably linked to a nucleic acid sequence to be transcribed such as a nucleic acid sequence encoding a desired molecule.
  • a promoter is generally positioned upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors.
  • a “vector” is capable of transferring gene sequences to target-cells.
  • vector construct means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target-cells, which can be accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element.
  • the term includes cloning, and expression vehicles, as well as integrating vectors.
  • regulatory element as used herein includes a nucleotide sequence which controls some aspect of the expression of nucleic acid sequences.
  • regulatory elements illustratively include an enhancer, an internal ribosome entry site (IRES), an intron, an origin of replication, a polyadenylation signal (pA), a promoter, an enhancer, a transcription termination sequence, and an upstream regulatory domain, which contribute to the replication, transcription, and/or post-transcriptional processing of a nucleic acid sequence.
  • regulatory elements can also include cis-regulatory DNA elements as well as transposable elements (TEs).
  • TEs transposable elements
  • control element or “control sequence” is a nucleotide sequence involved in an interaction of molecules contributing to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature.
  • Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3’ direction) from the promoter.
  • an amino acid residue is “phosphorylated” used herein means that a phosphate group is ester-linked to the side chain of the amino acid residue.
  • Typical amino acid residues that may be phosphorylated include serine (Ser), threonine (Thr), and tyrosine (Tyr).
  • the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
  • the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975].
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
  • compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.
  • Antibodies [00144] The present disclosure provides novel anti-SARS-CoV-2-Spike antibodies. Such antibodies bind human SARS-CoV-2. [00145] Table 1 lists exemplary peptide sequences of heavy chain variable regions and light chain variable regions that, in combination as designated in Table 1, are exemplary antibodies to SARS-CoV-2. In some embodiments, the heavy chain variable region and the light chain variable region are arranged in a Fab format.
  • the anti-SARS-CoV-2-Spike antibodies in the present disclosure include a heavy chain variable region having an amino acid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:7 and a light chain variable region having an amino acid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8.
  • the anti-SARS-CoV-2-Spike antibodies include a vhCDR1 comprising SEQ ID NO:1, a vhCDR2 comprising SEQ ID NO:2, a vhCDR3 comprising SEQ ID NO:3, a vlCDR1 comprising SEQ ID NO:4, a vlCDR2 comprising SEQ ID NO:5, and a vlCDR3 comprising SEQ ID NO:6.
  • one or more of such 6 CDRs have from 1, 2, 3, 4 or 5 amino acid modifications.
  • a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-SARS-CoV-2- Spike antibodies retain binding to human SARS-CoV-2.
  • the anti-SARS-CoV-2-Spike antibodies include a heavy chain variable region having an amino acid sequence identical to any of the clones listed in Table 1.
  • the anti-SARS-CoV-2-Spike antibodies include a light chain variable region having an amino acid sequence identical to any of the clones listed in Table 1.
  • the anti-SARS-CoV-2-Spike antibodies include a heavy chain variable region and a light chain variable region having an amino acid sequence identical to any of the clones listed in Table 1.
  • variants in the framework regions retain at least about 80, 85, 90 or 95% identity to a germline sequence.
  • variations are made in the framework regions that retain at least 80, 85, 90 or 95% identity to the germline gene sequences, while keeping 6 CDRs unchanged.
  • variations are made in both the framework regions that retain at least 80, 85, 90 or 95% identity to the germline gene sequences, and the 6 CDRs.
  • the CDRs can have amino acid modifications (e.g., from 1, 2, 3, 4 or 5 amino acid modifications in the set of CDRs (that is, the CDRs can be modified as long as the total number of changes in the set of 6 CDRs is less than 6 amino acid modifications, with any combination of CDRs being changed; e.g., there may be one change in vlCDR1, two in vhCDR2, none in vhCDR3, etc.).
  • amino acid modifications e.g., from 1, 2, 3, 4 or 5 amino acid modifications in the set of CDRs (that is, the CDRs can be modified as long as the total number of changes in the set of 6 CDRs is less than 6 amino acid modifications, with any combination of CDRs being changed; e.g., there may be one change in vlCDR1, two in vhCDR2, none in vhCDR3, etc.).
  • amino acid sequences of CDRs and/or variable regions of a heavy chain and a light chain from those described herein and combining them with amino acid sequences of framework regions and/or constant regions of a heavy chain and a light chain of an antibody as appropriate, a person skilled in the art will be able to design an anti-SARS- CoV-2 antibody according to the present invention.
  • the antibody framework regions and/or constant region (Fc domain) described in the current invention can derive from an antibody of any species, such as from human, rabbit, dog, cat, mouse, horse or monkey.
  • the constant region is derived from human, and includes a heavy chain constant region derived from those of IgG, IgA, IgM, IgE, and IgD subtypes or variants thereof, and a light chain constant region derived from kappa or lambda subtypes or variants thereof.
  • the heavy chain constant region is derived from a human IgG, including IgG1, IgG2, IgG3, and IgG4.
  • the amino acid sequence of the heavy chain constant region is at least 80%, 85%, 90%, or 95% identical to a human IgG1, IgG2, IgG3, or IgG4 constant region.
  • the amino acid sequence of the constant region is at least 80%, 85%, 90%, or 95% identical to an antibody constant region from another mammal, such as rabbit, dog, cat, mouse, horse or monkey.
  • the antibody constant region includes a hinge, a CH2 domain, a CH3 domain and optionally a CH1 domain.
  • the antibodies described herein can be derived from a mixture from different species, e.g., forming a chimeric antibody and/or a humanized antibody. In general, both “chimeric antibodies” and “humanized antibodies” refer to antibodies that combine regions from more than one species.
  • chimeric antibodies traditionally comprise variable region(s) from a mouse (or rat, in some cases) and the constant region(s) from a human.
  • Humanized antibodies generally refer to non-human antibodies that have had the variable-domain framework regions swapped for sequences found in human antibodies.
  • the entire antibody, except the CDRs is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs.
  • the CDRs some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region.
  • Humanized antibodies can also be generated using mice with a genetically engineered immune system, as described for example in Roque et al., 2004, Biotechnol. Prog.20:639-654, entirely incorporated by reference.
  • a variety of techniques and methods for humanizing and reshaping non-human antibodies are well known in the art (See Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited therein, all entirely incorporated by reference).
  • Humanization methods include but are not limited to methods described in Jones et al., 1986, Nature 321:522-525; Riechmann et al.,1988; Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol.160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci, USA 89:4285-9, Presta et al., 1997, Cancer Res.57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad.
  • Humanization or other methods of reducing the immunogenicity of nonhuman antibody variable regions may include resurfacing methods, as described for example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, entirely incorporated by reference.
  • Other humanization methods may involve the grafting of only parts of the CDRs, including but not limited to methods described in Tan et al., 2002, J. Immunol.169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirely incorporated by reference.
  • the antibodies of the current invention comprise a heavy chain variable region derived from a particular human germline heavy chain immunoglobulin gene and/or a light chain variable region derived from a particular human germline light chain immunoglobulin gene.
  • Such antibodies may contain amino acid differences as compared to the human germline sequences, due to, for example, naturally- occuring somatic mutations or intentional introduction of site-directed mutation.
  • a humanized antibody typically is at least 80% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the antibody as being derived from human sequences when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences).
  • a humanized antibody may be at least 95, 96, 97, 98 or 99%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene.
  • a humanized antibody derived from a particular human germline sequence will display no more than 10-20 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene.
  • the humanized antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.
  • the antibodies of the current disclosure are humanized and affinity matured, as is known in the art.
  • Structure-based methods may be employed for humanization and affinity maturation, for example as described in US Patent No 7,657,380. Selection based methods may be employed to humanize and/or affinity mature antibody variable regions, including but not limited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem.272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem.271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.
  • the present disclosure provides novel SARS-CoV-2 antibodies.
  • the anti- SARS-CoV-2-Spike antibodies described herein block the interaction between viral Spike protein and host receptor angiotensin-converting enzyme 2 (ACE2).
  • ACE2 host receptor angiotensin-converting enzyme 2
  • the anti-SARS-CoV-2-Spike antibodies bind human SARS-CoV-2 with high affinities.
  • the antibodies have neutralizing activity against SARS-CoV-2.
  • the SARS-CoV-2 antibodies block Spike mediated cell-cell fusion.
  • the SARS-CoV-2 antibodies block Spike mediated cell-cell fusion as an IgG but not as an antibody fragment, Fab. In some embodiments, the SARS-CoV-2 antibodies inhibit syncytia formation. The cell-cell fusion process induces formation of syncytia, a component of tissue damage in patients with severe COVID-19,
  • nucleic acids encoding these antibodies, as well as host cells that include such nucleic acids are described in the present disclosure. Also provided in the present disclosure are methods of using such antibodies to treat COVID-19 and pharmaceutical compositions and administration of pharmaceutical compositions comprising anti-SARS-CoV-2-Spike antibodies.
  • the anti-SARS-CoV-2-Spike antibodies described herein block the interaction between Spike protein to ACE2. In some embodiments, the anti- SARS-CoV-2-Spike antibodies described herein block the interaction between Spike protein to ACE2 and also alter the Spike protein conformational cycle triggered by ACE2 binding. In some embodiments, the anti-SARS-CoV-2-Spike antibodies described herein block the interaction between Spike protein to ACE2 and also alter the Spike protein conformational cycle triggered by ACE2 binding.
  • the anti-SARS-CoV-2-Spike antibodies described herein block the interaction between SARS-CoV-2 Spike protein to ACE2 and also alter the SARS-CoV-2 Spike protein conformational cycle triggered by ACE2 binding. Stabilization of different Spike conformations leads to modulation of Spike- mediated membrane fusion.
  • the anti-SARS-CoV-2-Spike antibodies block Spike mediated cell-cell fusion. Modulation of Spike-mediated membrane fusion affects COVID-19 pathology and immunity. Inhibition of Spike-mediated cell-cell fusion can inhibit the formation of syncytia, a component of tissue damage in patients with severe COVID-19.
  • the anti-SARS-CoV-2-Spike antibodies described herein block the interaction between Spike protein to ACE2 and also inhibit syncytia. In some embodiments, the anti-SARS-CoV-2-Spike antibodies described herein block the interaction between Spike protein to ACE2 and also inhibit cell-cell fusion and syncytia formation in addition to blocking receptor binding. In some embodiments, the anti-SARS- CoV-2-Spike antibodies described herein block the interaction between Spike protein to ACE2 and also inhibit syncytia by trapping the pre-fusion state. [00160] In some embodiments, the anti-SARS-CoV-2-Spike antibodies function as orthosteric receptor mimetics.
  • the anti-SARS-CoV-2-Spike antibodies function as orthosteric receptor mimetics conducive to the same cooperative processes as receptor binding.
  • An example of an antibody that functions as an orthosteric receptor mimetic is 2H4.
  • anti-SARS-CoV-2-Spike antibodies that function in the manner of 2 H4 neither inhibit or promote syncytia formation.
  • the anti-SARS-CoV-2-Spike antibodies function as allosteric effectors.
  • the anti-SARS-CoV-2-Spike antibodies function as allosteric effectors that advance Spike directly to the final stages of S2 unsheathing.
  • an antibody that functions as an allosteric effector is 3D11.
  • anti-SARS-CoV-2-Spike antibodies that function in the manner of 3D11 promote syncytia formation.
  • the anti-SARS-CoV-2-Spike antibodies function by stabilizing a Spike conformation that prohibits S1 shedding and traps the pre-fusion state.
  • stabilizing a Spike conformation that prohibits S1 shedding and traps the pre-fusion state prevents both targeted viral fusion and Spike-mediated cell-cell fusion.
  • synergy between receptor blockade and pre-fusion trapping allows for prevention of targeted viral fusion and Spike-mediated cell-cell fusion.
  • an antibody that acts by stabilizing a Spike conformation that prohibits S1 shedding and traps the pre-fusion state is 5A6.
  • anti-SARS-CoV-2-Spike antibodies that function by stabilizing a Spike conformation that prohibits S1 shedding and traps the pre-fusion state inhibit syncytia formation.
  • anti-SARS-CoV-2-Spike antibodies that function in the manner of 5A6 inhibit syncytia formation.
  • the D614G mutant is known to occupy states with multiple open RBDs, and has been found to shed the S1 subunit less readily than the original SARS-CoV-2 Spike protein.
  • the effects of 3D11 and 5A6 are mediated by open RBD conformations that represent immediately available binding sites for 3D11 and present the full quaternary epitope of 5A6.
  • the reduced shedding of the more stable D614G Spike may also assist the pre-fusion trapping activity of 5A6, while conveying resistance against trimer denaturation by 3D11.
  • the quaternary epitope recognized by 5A6 conveys cooperative binding as well as avidity, and both aspects may hinder viral escape via mutations in Spike protein.
  • binding of the anti-SARS-CoV-2-Spike antibodies to human is measured by ELISA. In some embodiments, binding of the anti-SARS-CoV-2- Spike antibodies to human SARS-CoV-2 is measured by FACS. In such embodiments, antibodies described herein display an EC50 that can range from 0.05nM to 2.5nM as measured by either ELISA or FACS. In such embodiments, antibodies described herein display an EC50 that can range from 0.1nM to 2.2nM as measured by either ELISA or FACS. [00166] In some embodiments, the anti-SARS-CoV-2-Spike antibodies described herein bind human SARS-CoV-2 with high affinities.
  • the K D value can be measured with the antigen immobilized or with the antibody immobilized.
  • the K D value can also be measured in a monovalent or a bivalent binding mode.
  • the K D values between the antibodies and human SARS-CoV-2 can be about 5 ⁇ 10 -2 M or less, 2.5 ⁇ 10 -2 M or less, 1 ⁇ 10 -2 M or less, 5 ⁇ 10 -3 M or less, 2.5 ⁇ 10 -3 M or less, 1 ⁇ 10 -3 M or less, 5 ⁇ 10 -4 M or less, 2.5 ⁇ 10 -4 M or less, 1 ⁇ 10 -4 M or less, 5 ⁇ 10 -5 M or less, 2.5 ⁇ 10 -5 M or less, 5 ⁇ 10 -6 M or less, 2.5 ⁇ 10 -6 M or less, 1 ⁇ 10 -6 M or less, 5 ⁇ 10 -7 M or less, 2.5 ⁇ 10 -7 M or less, 1 ⁇ 10 -7 M or less, 5 ⁇ 10 -8 M or less, 1 ⁇ 10 -8 M or less, 1 ⁇ 10 -9 M or less
  • the K D value can be 1 ⁇ 10 -6 M or less, 5 ⁇ 10 -7 M or less, 2.5 ⁇ 10 -7 M or less, 1 ⁇ 10 -7 M or less, 5 ⁇ 10 -8 M or less, 2.5 ⁇ 10 -8 M or less, 1 ⁇ 10 -8 M or less, 5 ⁇ 10 -9 M or less, 1 ⁇ 10- 9 M or less, 5 ⁇ 10 -10 M or less, 1 ⁇ 10 -10 M or less, 5 ⁇ 10 -11 M or less, 1 ⁇ 10 -11 M or less, 5 ⁇ 10 -12 M or less, or 1 ⁇ 10 -12 M or less.
  • the K D values range from about 0.1 nM to about 1 ⁇ M, about 0.25 nM to about 500 nM, 0.5 nM to about 250 nM, 1 nM to about 100 nM M, or about 2 nM to about 50 nM.
  • the binding affinities of the anti-SARS-CoV-2-Spike antibodies described herein are compared with other anti-SARS-CoV-2-Spike antibodies.
  • the anti-SARS-CoV-2-Spike antibodies described herein have higher binding affinity than other antibodies.
  • One advantage of having a higher binding affinity than 4C7 is that the antibodies described herein can be more efficacious in modulating immune response to SARS-CoV-2.
  • the anti-SARS-CoV-2 antibodies display low immunogenicity when administered into human subjects. These antibodies can contain an Fc domain derived from human IgG1, human IgG2 or human IgG3. In some embodiments, these antibodies are humanized using the framework regions derived from human immunoglobulins. [00169] Effects of the anti-SARS-CoV-2 antibodies on cell function can be assayed using a variety of methods known in the art and described herein, including for example, by the method described in Example 1. Accordingly, the anti-SARS-CoV-2 antibodies can serve as SARS-CoV-2 antagonists. [00170] The anti-SARS-CoV-2-Spike antibodies described herein bind human SARS- CoV-2.
  • the anti- SARS-CoV-2 antibodies bind human SARS-CoV-2 with high affinities. In some embodiments, the antibodies have neutralizing activity against SARS-CoV-2. In some embodiments, the SARS-CoV-2 antibodies block Spike mediated cell-cell fusion as an IgG but not as an antibody fragment Fab. In some embodiments, the SARS-CoV-2 antibodies inhibit syncytia formation. The cell-cell fusion process induces formation of syncytia, a component of tissue damage in patients with severe COVID-19. [00171] In some embodiments, anti- SARS-CoV-2 antibodies described act as SARS- CoV-2 antagonists, and block interaction of SARS-COV-2 with Spike protein.
  • anti- SARS-CoV-2 antibodies prevent syncytia formation.
  • anti- SARS-CoV-2 antibodies include antibodies that contain a heavy chain variable region comprising an amino acid sequence at least about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:7, and a light chain variable region comprising amino acid sequence at least about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8; and/or a vhCDR1 comprising SEQ ID NO:1, a vhCDR
  • the anti-SARS-CoV-2-Spike antibodies described herein provide a method of preventing formation of syncytia comprising a fusion between a SARS-CoV-2 infected cell displaying a SARS-CoV-2 Spike protein on its surface, and a second cell displaying an ACE-2 receptor on its surface, the syncytia formation mediated by the SARS-CoV-2 Spike protein binding to the ACE-2 receptor, the method comprising contacting a cell infected with SARS-CoV-2, or at risk of being infected with SARS-CoV-2 with an isolated anti-SARS-CoV-2-Spike antibody or an antigen binding fragment thereof specifically binding to the Spike protein, trapping the Spike protein in a pre-fusion state, thereby preventing the formation of the syncytia.
  • the isolated anti- SARS-CoV-2-Spike antibody or antigen binding fragment is an IgG, IgM, IgA, Fab, single domain antibody.
  • the isolated anti-SARS-CoV-2-Spike antibody or antigen binding fragment binds to a quaternary epitope of the Spike protein. Examples of binding to a quaternary epitope of the Spike protein can be found in Figures 21-30.
  • the quaternary epitope of the binder is composed of both the ACE2 interacting amino acid residues in one RBD as well as amino acids in an adjacent RBD.
  • the isolated anti-SARS-CoV-2-Spike antibody binds to the quaternary epitope through residues in the CDR. Examples of binding to a quaternary epitope of the Spike protein can be found in Figures 21-30.
  • the quaternary epitope of the binder is composed of both the ACE2 interacting amino acid residues in one RBD as well as amino acids in an adjacent RBD addition to framework amino acid residues binding to an adjacent RBD where the secondary binding is achieved through framework residues of the antibody. In some embodiments, the binding is within 4 angstroms.
  • the isolated antibody or antigen binding fragment comprises a light chain variable domain sequence that is at least 90% identical to SEQ ID NO.7.
  • the isolated antibody or antigen binding fragment comprises a heavy chain variable domain sequence that is at least 90% identical to SEQ ID NO.8.
  • the isolated anti-SARS-CoV-2-Spike antibody or antigen binding fragment comprises a heavy chain variable region comprising complementarity determining regions (CDRs), wherein: a. the heavy chain CDR1 is SEQ ID NO.1 (GFTFSSYE) b. the heavy chain CDR2 is SEQ ID NO 2 (ISYDGSNK); and c. the heavy chain CDR3 is SEQ ID NO.3 (QQSYNLPRT), and a light chain variable region comprising CDRs, wherein: d.
  • CDRs complementarity determining regions
  • the light chain CDR1 is SEQ ID NO.4 (QSISSY)
  • the light chain CDR2 is SEQ ID NO.5 (AAS);
  • the light chain CDR3 is SEQ ID NO.6 (QQSYNLPRT).
  • the heavy chain variable region, SEQ ID NO.7 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations.
  • Exemplary positions for the mutations include, without limitation, any of the positions E33, S52, Y53, D54, G55, S56, N57, L94, and/or R96 according to the sequence numbering in Figure 46, alone or in combination (or the corresponding position according to IMGT numbering).
  • Exemplary positions for the mutations include, without limitation, any of the positions E38, S57, Y58, D59, G62, S63, N64, R106 according to IMGT numbering, alone or in combination.
  • the heavy chain variable region, SEQ ID NO.7 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations at E38, S57, Y58, D59, G62, S63, N64, and/or R106.
  • the mutation at position D59 is selected from D59G, D59L, D59K, D59I, D59M, D59W, D59T, D59F, or D59Y.
  • the mutation at position N64 is selected from N64R or N64I.
  • the mutation at position R106 is selected from R106E, R106D, R106N, R106Q, R106A, R106V, or R106L.
  • the heavy chain variable region, SEQ ID NO.7 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations. Exemplary positions for the mutations include, without limitation any of the positions listed in Figures 31 to 39 which are numbered according to the sequence numbering in Figure 46, alone or in combination (or the corresponding position according to IMGT numbering).
  • the isolated antibody or antigen binding fragment comprises a heavy chain variable domain sequence that is at least 90% identical to SEQ ID NO.7.
  • the light chain variable region, SEQ ID NO.8 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations.
  • An exemplary position for the mutations include, without limitation position S69, L114 and/or R116 according to IMGT numbering.
  • the light chain variable region, SEQ ID NO.8 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations at any of the positions listed in Figures 31 to 39, alone or in combination.
  • the isolated antibody or antigen binding fragment comprises a light chain variable domain sequence that is at least 90% identical to SEQ ID NO.8.
  • the isolated antibody is a human antibody. In some embodiments, the isolated antibody is a humanized antibody. [00182] In some embodiments, the isolated antibody is a monoclonal antibody. [00183] In some embodiments, the isolated antibody is effective in treating a SARS- CoV-2 infection.
  • the isolated antibody is useful in preventing or retarding formation of SARs-CoV-2 mediated syncytia formation by binding to RBD and stabilizing a SARs-CoV-2 Spike protein conformation retarding or preventing S1 shedding and trapping a pre-fusion state of the SARs-CoV-2.
  • the isolated anti-SARS-CoV-2-S1 antibody or antigen binding fragment comprises a heavy chain variable region comprising complementarity determining regions (CDRs), wherein: a.
  • the heavy chain CDR1 is SEQ ID NO.1 (GFTFSSYE; binds through T28, S31, Y32, E33 according to the sequence numbering in Figure 46 (or the corresponding position according to IMGT numbering))
  • the heavy chain CDR2 is SEQ ID NO 2 (ISYDGSNK; binds through V50, I51, S52, Y53, D54, N57, Y59 according to the sequence numbering in Figure 46 (or the corresponding position according to IMGT numbering))
  • the heavy chain CDR3 is SEQ ID NO.
  • the light chain CDR1 is SEQ ID NO.4 (QSISSY; binds through S30, Y31 according to the sequence numbering in Figure 47 (or the corresponding position according to IMGT numbering))
  • the light chain CDR2 is SEQ ID NO.
  • the isolated anti-SARS-CoV-2-S1 antibody or antigen binding fragment comprises a heavy chain variable region comprising complementarity determining regions (CDRs), wherein: a.
  • the heavy chain CDR1 is SEQ ID NO.1; binds through T29, S36, Y37, E38)
  • the heavy chain CDR2 is SEQ ID NO 2 (ISYDGSNK); binds through V55, I56, S57, Y58, D59, N64, Y66
  • the heavy chain CDR3 is SEQ ID NO.3 (ARLITMVRGEDY; binds through R106, L107, T109, M110, V112, R113, G114, E115), and a light chain variable region comprising CDRs, wherein: d. the light chain CDR1 is SEQ ID NO.4 (QSISSY; binds through S36, Y38) e.
  • the light chain CDR2 is SEQ ID NO.5 (AAS; binds through S69, G70); and f. the light chain CDR3 is SEQ ID NO.6 (QQSYNLPRT; binds through S107, Y108, N109, L114, R116), wherein the positions are according to IMGT numbering.
  • the heavy chain variable region, SEQ ID NO.7 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations at any of the positions E33, S52, Y53, D54, G55, S56, N57, L94, and/or R96 (or the corresponding position according to IMGT numbering).
  • the heavy chain variable region, SEQ ID NO.7 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations at any of the positions E38, S57, Y58, D59, G62, S63, N64, R106 according to IMGT numbering, alone or in combination.
  • the light chain variable region, SEQ ID NO.7 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations at any of the positions listed in Figures 31 to 39.
  • the isolated antibody or antigen binding fragment comprises a heavy chain variable domain sequence that is at least 90% identical to SEQ ID NO.7.
  • the light chain variable region, SEQ ID NO.8, comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations at position S56 and R96 according to the sequence numbering in Figure 47. In some embodiments, the light chain variable region, SEQ ID NO.8, comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations at position S69, L114, and/or R116 according to IMGT numbering. In some embodiments, the light chain variable region, SEQ ID NO.8, comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations at position L114 and/or R116 according to IMGT numbering.
  • the light chain variable region, SEQ ID NO.8, comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mutations at any of the positions listed in Figures 31 to 39.
  • the isolated antibody or antigen binding fragment comprises a light chain variable domain sequence that is at least 90% identical to SEQ ID NO.8.
  • the isolated antibody is a human antibody.
  • the isolated antibody is a humanized antibody.
  • the isolated antibody is a monoclonal antibody.
  • the isolated antibody is effective in treating a SARS-CoV-2 infection.
  • the isolated antibody is useful in preventing or retarding formation of SARs-CoV-2 mediated syncytia formation by binding to RBD and stabilizing a SARs-CoV-2 Spike protein conformation retarding or preventing S1 shedding and trapping a pre-fusion state of the SARs-CoV-2.
  • mutations to the heavy chain variable region of 5A6 may be made according to the positions suggested in Figure 46.
  • mutations to the light chain variable region of 5A6 may be made according to Figure 47.
  • mutations to the heavy chain variable region of SEQ ID NO: 7 may be made according to the positions suggested in Figure 46.
  • mutations to the light chain variable region of SEQ ID NO: 8 may be made according to Figure 47. In some embodiments, mutations to the CDRs of 5A6 may be made according to the positions suggested in Figure 46 and/or Figure 47.
  • a SARS-Cov-2-Spike antibody is effective in preventing syncytia formation if it has a k off ⁇ 7.14E-3/sec. In some embodiments, a SARS- Cov-2-Spike antibody may be effective in preventing syncytia formation if the k off is in the range of 5X ⁇ k off ⁇ 10X as compared to 5A6 WT.
  • a SARS-Cov-2-Spike antibody is effective in preventing syncytia formation if it has a k off > 10X as compared to 5A6 WT.
  • a SARS-Cov-2-Spike antibody is effective in preventing syncytia formation if it has a k off ⁇ 5X as compared to 5A6 WT.
  • a SARS-Cov-2-Spike antibody may be effective in preventing syncytia formation if the koff is in the range of 5X ⁇ k off ⁇ 10X as compared to 5A6 WT.
  • a SARS-Cov-2-Spike antibody it is uncertain if a SARS-Cov-2-Spike antibody is effective in preventing syncytia formation if it has a k off > 10X as compared to 5A6 WT.
  • a SARS-Cov-2-Spike antibody has a k off ⁇ 7.14E- 3/sec.
  • a SARS-Cov-2-Spike antibody has a k off in the range of 5X ⁇ k off ⁇ 10X as compared to 5A6 WT.
  • a SARS-Cov-2-Spike antibody has a k off > 10X as compared to 5A6 WT.
  • a SARS-Cov-2-Spike antibody has a k off ⁇ 7.14E- 3/sec. In some embodiments, a SARS-Cov-2-Spike antibody does not have a k off in the range of 5X ⁇ k off ⁇ 10X as compared to 5A6 WT. In some embodiments, a SARS-Cov-2-Spike antibody does not have a k off > 10X as compared to 5A6 WT. [00190] In some embodiments, a SARS-Cov-2-Spike antibody has a k off ⁇ 5X as compared to 5A6 WT.
  • a SARS-Cov-2-Spike antibody does not have a k off in the range of 5X ⁇ k off ⁇ 10X as compared to 5A6 WT. In some embodiments, a SARS- Cov-2-Spike antibody does not have a k off > 10X as compared to 5A6 WT.
  • Exemplary mutations that may be made in order to improve k off are disclosed in Figures 31-39 and 46-47. In Figures 31-39, mutations that improve binding (at least 5x slower K off compared to 5A6 WT) are highlighted in Green. Mutations that weaken binding (at least 5x faster Koff compared to 5A6 WT) are highlighted as Red.
  • mutations with improved K off are selected from D59G, D59L, D59K, D59I, D59M, D59W, D59T, D59F, or D59Y. In some embodiments, mutations with improved Koff are selected from N64R or N64I.
  • a preferred mutation to the variable heavy region of 5A6 is to R98 according to the raw sequence numbering in Figure 47 (R106 according to IMGT numbering). The mutations VH R98 according to the raw sequence numbering in Figure 46 (R106 according to IMGT numbering) interacts directly with Spike E484 via a charge-charge interaction.
  • Spike residue 484 is mutated from a negatively charged E to positive charged K.
  • This E484K mutation strongly affects the affinity of 5A6 due to abrogating the salt bridge with 5A6 R98 according to the raw sequence numbering in Figure 46 (R106 according to IMGT numbering).
  • Compensatory charge swaps in 5A6 including R98E, R98D according to the raw sequence numbering in Figure 46 (R106E, R106D according to IMGT numbering) will likely restore affinity.
  • Exemplary antibodies of the invention form, one or more primary interactions with the epitope through one or more CDR of one or more of the light and heavy chains. In some embodiments, the antibodies form one or more secondary interactions with the epitope.
  • the invention provides an antibody, and methods of using the antibody as described herein.
  • An exemplary antibody of the invention includes particular amino acid residues interfacing with amino acids on the epitope.
  • an amino acid residue at position 36 interacts with F486 of the epitope.
  • An exemplary interaction is formation of a buried surface.
  • the amino acid residue is S36.
  • an amino acid residue at position 38 interacts with one or both of N487, Y489, and F486.
  • An exemplary interaction is a hydrogen bonding network between the amino acid at position 38 and N487 and Y489, and a T-shaped ⁇ - ⁇ interaction with F486.
  • An exemplary amino acid residue is Y38.
  • amino acid residues at positions 69 and 70 interface with Y449 of the epitope.
  • An exemplary interaction is the formation of a buried surface.
  • Exemplary residues at positions 69 and 70 include S69 and G70.
  • one, two, three, four and/or five of the amino acids at positions 107, 108, 109, 114 and 116 interact with V483, E484, G485 and F186.
  • amino acid residues at positions 107, 108, 109, 114 and 116 are S107, Y108, N109, L114, and R116.
  • amino acid residues at positions 29, 36, 37, and 38 interact with amino acid residues in the epitope, T470, I472, G482, V483, E484, F490.
  • An exemplary interaction is formation of a buried surface.
  • the amino acid residue at 38 receives electrophilic polarization by the side-chain of E484 and forms a hydrogen bond with the backbone amide nitrogen of E484.
  • the residue at 37 forms a parallel-displaced ⁇ - ⁇ interaction with F490.
  • the amino acid residues at 29, 36, 37, and 38 are T29, S36, Y37, and E38.
  • one, two, three, four, five, six or seven amino acids at 55, 56, 57, 58, 59, 64 and/or 66 interact with T470, E471, I472, N481, G482, V483.
  • An exemplary interaction is formation of a buried interface.
  • the amino acid residues are V55, I56, S57, Y58, D59, N64, and Y66.
  • the amino acid residue at position 58 forms an anion- ⁇ interaction with E471.
  • the amino acid residue at position 66 forms a hydrogen bone with N481.
  • the antibody interacts with the epitope via amino acid residues at positions 106, 107, 109, 110, 112, 113, 114, and/or 115.
  • the amino acids at these positions interact with amino acid residues E484, G485, Y489, F490, L492, Q493, S494 of the epitope.
  • An exemplary interaction is formation of a buried interface.
  • the amino acid residue at 106 forms a salt bridge with E484.
  • the amino acid residue at 113 forms a cation- ⁇ interaction with Y449.
  • the amino acid residues of heavy chain CDR3 are R106, L107, T109, M110, V112, R113, G114, and E115.
  • light chain residues at one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 of the amino acid residues at 18, 20, 22, 28, 36, 65, 77, 79, 80, 83, 84, 85, 86, 88, and/or 90 interacts with Y369, A372, F374, S375, T376, F377, K378, G404, D405, R408, Q414, K417, V503, G504, and/or Y508 of the epitope.
  • An exemplary interaction is formation of a buried interface.
  • the backbone carbonyl oxygen of the residue at 83 forms a hydrogen bond with the backbone amide nitrogens of F377, and/or K378.
  • the residue at one or more of 86 and/or 24 form a hydrogen-bonding-network with Y508.
  • the amino acid residues of the antibody are R18, T20, T22, S28, S36, S65, S77, S79, G80, S83, G84, T85, D86, T88, and T90.
  • Nucleic acids of the invention are also provided, as well as expression vectors containing such nucleic acids and host cells transformed with such nucleic acids and/or expression vectors.
  • the protein sequences depicted herein can be encoded by any number of possible nucleic acid sequences due to the degeneracy of the genetic code.
  • Table 2 gives exemplary nucleic acids encoding the heavy chain variable region and light chain variable region of the antibodies described herein.
  • nucleic acid compositions encoding the anti-SARS-CoV-2-Spike antibodies and/or SARS-CoV-2-binding domains are also provided.
  • the nucleic acid compositions generally include a first nucleic acid encoding the heavy chain variable region and a second nucleic acid encoding the light chain variable region.
  • a single nucleic acid encoding the heavy chain variable region and light chain variable region, separated by a linker described herein, can be made.
  • the nucleic acid compositions generally include a first nucleic acid encoding the heavy chain and a second nucleic acid encoding the light chain, which will, upon expression in a cell, spontaneously assemble into the “traditional” tetrameric format of two heavy chains and two light chains.
  • the nucleic acids encoding the components of the invention can be incorporated into expression vectors, and depending on the host cells, used to produce the antibodies of the invention. These two nucleic acids can be incorporated into a single expression vector or into two different expression vectors.
  • the nucleic acids can be operably linked to any number of regulatory elements (promoters, origin of replication, selectable markers, ribosomal binding sites, inducers, etc.) in an expression vector.
  • the expression vectors can be extra-chromosomal or integrating vectors.
  • the nucleic acids and/or expression vectors of the current invention can be introduced into any type of host cells, which are well known in the art, including mammalian, bacterial, yeast, insect and fungal cells. After transfection, single cell clones can be isolated for cell bank generation using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix.
  • Clones can be cultured under conditions suitable for bio- reactor scale-up and maintained expression of the antibodies.
  • the antibodies can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.
  • D. Therapeutic Applications [0001] The current disclosure provides a method of treating a subject with SARS-CoV-2, and the method includes administering to the subject an effective amount of an anti-SARS- CoV-2 antibody described herein, or a pharmaceutical composition containing an anti- SARS-CoV-2 antibody.
  • the methods of treating a subject with SARS-CoV-2 by the present disclosure comprises administering to the subject an effective amount of an anti- SARS-CoV-2 antibody that acts as a SARS-CoV-2 antagonist, or by administering a pharmaceutical composition containing an antagonistic anti-SARS-CoV-2 antibody.
  • the methods encompassed by the present disclosure comprise methods of treating a subject with SARS-CoV-2, for example, by administering anti-SARS-CoV-2 antibodies that includes a heavy chain variable region comprising an amino acid sequence at least about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:7, and a light chain variable region comprising amino acid sequence at least about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8; and/or a vhCDR1 comprising SEQ ID NO
  • the current disclosure also provides a method of modulating an immune response in a subject, and the method includes administering to the subject an effective amount of an anti-SARS-CoV-2 antibody described herein, or a pharmaceutical composition containing an anti- SARS-CoV-2 antibody.
  • the methods of modulating an immune response encompassed by the present disclosure comprises stimulating an immune response in a subject, and in further embodiments, such methods comprise administering to the subject an effective amount of an anti-SARS-CoV-2 antibody that acts as a SARS-CoV-2 antagonist, or by administering a pharmaceutical composition containing an antagonistic anti-SARS-CoV-2 antibody.
  • the methods encompassed by the present disclosure comprise methods of modulating an immune response in a subject, for example, by administering anti- SARS-CoV-2 antibodies that includes a heavy chain variable region comprising an amino acid sequence at least about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:7, and a light chain variable region comprising amino acid sequence at least about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8; and/or a vhCDR1 comprising SEQ ID NO:1,
  • the Anti-SARS-CoV-2-Spike antibodies described herein can be used in combination with additional therapeutic agents to treat Covid-19.
  • the present disclosure also features pharmaceutical compositions/formulations that contain a therapeutically effective amount of an anti-SARS-CoV-2 antibody described herein.
  • the composition can be formulated for use in a variety of drug delivery systems.
  • One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985.
  • the antibodies of the present disclosure can exist in a lyophilized formulation or liquid aqueous pharmaceutical formulation.
  • the aqueous carrier of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation.
  • Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
  • the antibodies of the present disclosure could exist in a lyophilized formulation including the proteins and a lyoprotectant.
  • the lyoprotectant may be sugar, e.g., disaccharides. In certain embodiments, the lyoprotectant is sucrose or maltose.
  • the lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and/or a preservative.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • a patient’s dose can be tailored to the approximate body weight or surface area of the patient.
  • Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those skilled in the art, especially in light of the dosage information and assays disclosed herein.
  • the dosage can also be determined through the use of known assays for determining dosages used in conjunction with appropriate dose-response data.
  • An individual patient's dosage can be adjusted as the progress of the disease is monitored. Blood levels of the targetable construct or complex in a patient can be measured to see if the dosage needs to be adjusted to reach or maintain an effective concentration.
  • Pharmacogenomics may be used to determine which targetable constructs and/or complexes, and dosages thereof, are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308: 43-53, 2001; Steimer et al., Clinica Chimica Acta 308: 33-41, 2001). [0012] Doses may be given once or more times daily, weekly, monthly or yearly, or even once every 2 to 20 years.
  • Administration of the present invention could be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter or by direct intralesional injection. This may be administered once or more times daily, once or more times weekly, once or more times monthly, and once or more times annually.
  • Antibodies were discovered that all exhibit effective receptor blockade but have different neutralization potencies against SARS-CoV-2. Taking these antibodies as mechanistic probes, it was shown that bivalent binding and receptor blockade are not the sole determinants of potent neutralization, In addition to blocking ACE2, these antibodies either inhibit or enhance syncytia formation in Vero E6 cells, suggesting that potentiation of cell- cell fusion by antibodies may compromise the effectiveness of viral neutralization in treatment of severe COVID-19.
  • One potently neutralizing and potentially therapeutic antibody, designated 5A6 uniquely inhibits cell-cell fusion and syncytia formation and blocks receptor binding.
  • cryo-EM cryogenic electron microscopy
  • Stepwise binding BLI assays show that Fab fragments 5A6 and 3D11 have non-overlapping footprints on the RBD, while 5A6 shares at least partially overlapping epitopes with the other four antibodies (Figure 1D).
  • C SARS-CoV-2 neutralization by receptor blocking antibodies
  • Six receptor-blocking antibodies were evaluated for neutralizing activity against SARS-CoV-2 pseudovirus in CHO-ACE2 cells with a luciferase reporter (Figure 2A), and against live SARS-CoV-2 (Young et al., 2020) in Vero E6 cells by cell viability (Figure 2B).
  • the antibodies neutralize pseudovirus with IC50 values ranging from 75.5 to 428.3 ng/mL, but while neutralization of live virus is 11- to 20-fold less potent for other antibodies, 5A6 retains similar potency with IC50 of 140.7 ng/ml ( ⁇ 2- fold weaker). This difference in neutralization potency in live virus and pseudovirus assays is most likely due to the non-replicating nature of pseudoviruses, which are thereby more sensitive to blockade of viral entry.
  • a live virus neutralization of a subset of antibodies was validated (2H4, 3D11 and 5A6) using a RT-qPCR to quantify viral replication (Figure 11), and observed similar trends as obtained from cell viability assays ( Figure 2B).
  • 3D11 IgG exhibits a similarly high signal at saturation, but with lower affinity for the pseudoviral particles, despite having higher affinity than 5A6 for immobilized RBD or Spike trimer (Figure 2E, Figure 15).
  • 2H4 and 1F4 IgGs saturate immobilized pseudovirus at 1/3 the density of 5A6 or 3D11, while neutralizing live virus slightly more effectively than 3D11.
  • 2H4 is an orthosteric receptor-mimetic antibody [00213] Multiple structures of 2H4 Fab were determined to be bound to Spike with resolution sufficient for unambiguously docking a model of 2H4, but precluding precise modelling of the epitope and complementarity determining regions (CDRs) ( Figure 12E). Three major conformational states were identified by 3D classification, revealing either one, two, or three 2H4 Fabs bound to the Spike trimer. The receptor blocking activity of 2H4 is straightforward, as it recognizes an epitope that overlaps much of the ACE2 interface ( Figure 4A).
  • Binding of the 2H4 Fab is compatible with both major RBD conformations, and the structures are drawn from an ensemble of quaternary states reminiscent of those that follow ACE2 binding, and lead to S1 shedding and spike- mediated membrane fusion (Benton et al.2020) ( Figure 4B).
  • the first of three predominant states features 2H4 bound to one open RBD, and the other two RBDs closed.
  • the second state adds a second copy of 2H4, on a closed RBD counter-clockwise from the first.
  • Density inspection and 3D variability analysis (3DVA) reveal that the third RBD is primarily open, and a trajectory of opening states correlate with binding the second Fab.
  • the final state features three Fabs bound, and a strictly open third RBD.
  • 3D11 allosterically blocks ACE2 binding and triggers Spike opening
  • the Spike:3D11 complex is relatively homogeneous, with only one major state (Figure 4D), and we determined its structure to ⁇ 3.0 ⁇ resolution ( Figure 12C). All three RBDs are bound to 3D11 Fab in the open conformation, with the Fab making a right angle to the long axis of the RBD, via an epitope exposed only in the open state and outside the RBM ( Figure 4A).
  • the epitope partly overlaps those of some other antibodies that bind outside the RBM (Liu et al., 2020; Yuan et al., 2020), but is distinct from those (Barnes et al., 2020; Pinto et al., 2020) that bind freely to a closed RBD (Figure 13D). Clashes between 3D11 and Spike NTDs also prevent 3D11 binding to closed RBDs, indicating 3D11 binds only to open RBDs.
  • 3D11 Although its epitope does not significantly overlap with the ACE2-RBD interface, 3D11 nevertheless effectively blocks ACE2 binding and stabilizes a quaternary state of the Spike, with three open RBDs and NTDs, that closely resembles the penultimate stage of ACE2-induced Spike opening (Benton et al., 2020). We therefore term 3D11 an allosteric receptor-mimetic antibody, which does not directly target the ACE2 interface, yet prevents ACE2 binding and enhances Spike-mediated fusion by rapidly advancing the Spike conformational cycle to its final stages. H.
  • 5A6 traps a pre-fusion conformation to inhibit spike-mediated fusion
  • Multiple states of the Spike:5A6 complex were resolved to better than 3.0 ⁇ , with local resolution sufficient for accurate modelling of the Fab-RBD interface (Figure S6G).
  • 5A6 recognizes surface loops near the tip of the RBD, which are solvent exposed even when all RBDs are closed.
  • the binding geometry is permissive of any trimer configuration and any stoichiometry, without steric constraints from Spike or Fab.
  • all 5A6 complexes feature at least two 5A6 Fabs bound to an open RBD counter-clockwise adjacent in turn from a closed RBD ( Figure 4F).
  • biopanning was performed using SARS-CoV-2 RBD (YP_009724390.1) (Arg319-Phe541) with a mouse Fc tag (Sino Biological, 40592-V05H) biotinylated using the EZ-Link NHS-PEG4-Biotin labelling kit (Thermo Fisher Scientific, #A39259).
  • biotinylated SARS-CoV-2 RBD-mFc protein was immobilized on M280 streptavidin- coated magnetic beads (Life Technologies, #11205D); 3.5 x 1012 cfu phage in 1mL 1% casein-PBS blocking buffer was used in the first round, and 1.64 x 1011 cfu phage were used in the second round.
  • binders to mouse Fc were removed by pre-incubation of phage with 2 ⁇ M mouse IgG before mixing with the RBD-mFc antigen. After two rounds of biopanning, the Fabs of selected clones were expressed in E.
  • Fabs were reformatted into human IgG in the pTT5 vector (National Research Council of Canada) and the IgG antibodies were expressed using ExpiCHO expression system (Thermo Fisher Scientific) by transient co-transfection of plasmids expressing the heavy and light chain of each antibody clone. Eight days after transfection, ExpiCHO-S cell suspension was centrifuged for 10 min at 2000 rpm and filtered with 0.22 ⁇ m filter to remove the cells and debris.
  • Antibodies were then purified from the culture supernatant using Protein G resin (Merck Millipore) following the manufacturer’s instructions. After elution, the purified antibodies were dialyzed at 4°C. for 4-20 hours against 1x PBS, for 3 times and concentrated to 1-2 mg/ml using 10MWCO Vivaspin 20 (Sartorius).
  • Fab production and purification [00223] The tag-less Fab fragments were produced using the ExpiCHO transient expression system. Eight days after transfection, ExpiCHO-S cell suspension was centrifuged and filtered; and Fab was purified from the filtered culture supernatant using cation exchange chromatography (CIEX) on AKTA FPLC System (GE Healthcare).
  • CIEX cation exchange chromatography
  • the supernatant was concentrated to 2 ml using 10MWCO Vivaspin 20 (Sartorius), diluted 1:20 in Buffer A (20 mM Sodium Acetate, pH 5.2), filtered through 0.22 ⁇ m filter, and loaded onto Mono-S 5/50 GL column at a flow rate of 1ml/min.
  • Fab fragments were eluted in Buffer B (20 mM Sodium Acetate, pH 5.2 with 1 M Sodium Chloride) with a sequential linear gradient of 0% to 5% in 5 min, 5% to 15% in 30 min, and 15% to 100% in 20 min of Buffer B injection at a flow rate of 1 ml/min.
  • the resulting purified Fab fragments were dialyzed at 4°C for 4-20 hours against 4 liters of 20 mM Histidine, 150 mM NaCl, pH 6.6, for 3 times and concentrated to 1-2 mg/ml using 10MWCO Vivaspin 6 (Sartorius).
  • NeutrAvidin protein (Thermo Fisher Scientific, #31000) was coated at 5 ⁇ g/ml onto 96-well ELISA plates in coating buffer (8.4 g/L NaHCO3, 3.56 g/L Na2CO3, pH 9.5) overnight at 4°C. After blocking with 1% Casein (Thermo Fisher Scientific, #A37528) for two hours, biotinylated antigen at 0.2 ⁇ g/ml was added to the plates and captured by NeutrAvidin during one-hour incubation at room temperature. After washing with 0.05% PBST for 5 times, the IgG antibodies were added at different concentrations with 3-fold dilutions in triplicate and incubated for one hour.
  • a stable cell line expressing human ACE2, CHO-ACE2 (a kind gift from Professor Yee-Joo Tan, IMCB, A*Star) (Ng et al., 2014) was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated FBS, 1% MEM Non-Essential Amino Acids Solution (Gibco, 11140-050) and 0.5 mg/ml of GeneticinTM Selective Antibiotic (Gibco, 10131-027). Every 2-3 days, cells were passaged by dissociating the cells with StemProTM AccutaseTM Cell Dissociation Reagent (Gibco, A1110501).
  • Grids were blotted using Whatman #1 filter paper for 8 or 10 seconds at a blot force of 0 at 4°C and 100% humidity using a Mark IV Vitrobot (Thermo Fisher) and plunge frozen into liquid ethane.
  • Samples were loaded onto a Titan Krios transmission electron microscope (Thermo Fisher) equipped with a Gatan K3 direct electron detector (Gatan) and a Quantum GIF energy filter (Gatan) operated with a 20 eV slit width during image acquisition.
  • the K3 camera was operated in CDS mode using super resolution. A nominal magnification of 105,000x was used, for a pixel size of 0.835 ⁇ (0.4175 ⁇ super resolution pixel size) at the sample.
  • Image processing [00239] Dose-weighted, motion-corrected sums down-sampled to the physical pixel size were obtained from the super-resolution DED movies using UCSF Motioncor2 (Zheng et al., 2017).
  • CTF estimation was performed in cryoSPARC (Punjani et al., 2017) followed by blob-based particle picking, 2D classification, ab initio modelling, 3D classification, and 3D refinement.
  • particles were instead picked using templates generated from the apo trimer structure, and the apo trimer was likewise used as an initial model in 3D classification.
  • the resolution of the interface between the Spike RBD and the 5A6 Fab was further improved using na ⁇ ve focused refinements. Processing details are given in Figure 17 and Figure 14.
  • Pseudotyped viral particles expressing SARS-CoV-2 Spike protein were produced by transfecting of 30 million 293T cells with 12 ⁇ g pMDLg/pRRE (a gift from Didier Trono, Addgene #12251), 6 ⁇ g pRSV-Rev (a gift from Didier Trono, Addgene #12253), 24 ⁇ g pHIV-Luc-ZsGreen (a gift from Bryan Welm, Addgene #39196) and 12 ⁇ g pTT5LnX-CoV-SP (expressing SARS-CoV-2 Spike protein, Genbank: YP_009724390.1, a kind gift from DSO National Laboratories) using Lipofectamine 2000 transfection reagent (Invitrogen, 11668-019).
  • the transfected cells were cultured at 37°C incubator for 3 days. Viral supernatant was harvested, centrifuged at 700 g for 10min to remove cell debris and filtered through a 0.45 ⁇ m filter unit (Sartorius, #16555). Lenti-X p24 rapid titer kit (Takara Bio, #632200) was used to quantify the viral titres following the manufacturer instructions.
  • pTT5LnX-CoV-SP plasmid with D614G mutation was generated using QuickChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent, #210513) and was used to generate mutant pseudovirus expressing SARS-CoV-2 Spike protein carrying D614G mutation.
  • pseudovirus particles [00244] Purification of pseudovirus particles [00245] To concentrate and purify the pseudovirus particles expressing the SARS-CoV-2 Spike glycoproteins, pre-cleared 40 mL viral supernatant was concentrated by 20% sucrose gradient centrifugation at 10,000 g for 4 hours at 4°C in an SW41 Ti rotor with no brake. Upon removal of supernatant, 1mL of PBS was added to the virus pellet and left at 4°C overnight. Concentrated virus was further purified by an OptiPrep (60% [wt/vol] iodixanol; #07820; STEMCELL Technologies Inc) velocity gradient. Iodixanol gradients were prepared in PBS in 1.2% increments ranging from 6 to 18%.
  • Pseudoviruses were layered onto the top of the gradient and centrifuged for 1.5 hours at 200,000 g in an SW41 Ti rotor. Gradient fraction that contained pseudovirus pellet was collected. [00246] Pseudovirus neutralization assay [00247] CHO-ACE2 cells were seeded at a density of 3.2 x 104 cells in 100 ⁇ L of complete medium without Geneticin in 96-well Flat Clear Bottom Black Polystyrene TC- treated Microplates (Corning, #3904).
  • IgG or Fab antibodies were incubated in a 96-well flat-bottom cell culture plate (Costar, #3596) with an equal volume of pseudovirus (12 ng of p24) at the final volume of 50 ⁇ L at 37°C for one hour, and the mixture was added to the monolayer of pre-seeded CHO-ACE2 cells in triplicate. After one hour of pseudovirus infection at 37°C, 150 ⁇ l of culture medium was added to each well and the cells were further incubated for another 48 hours. Upon removal of culture medium, cells were washed twice with sterile PBS, and then lysed in 20 ⁇ L of 1x Passive lysis buffer (Promega, E1941) with gentle shaking at 37°C for 30 minutes.
  • 1x Passive lysis buffer Promega, E1941
  • Luciferase activity was then assessed using a Luciferase Assay System (Promega, E1510) on a Promega GloMax Luminometer. The relative luciferase units (RLU) were converted to percent neutralization and plotted with a non-linear regression curve fit using PRISM.
  • RLU relative luciferase units
  • the potency of 2H4, 3D11 and 5A6 IgG antibodies in neutralizing live SARS-CoV-2 virus assays was also determined by measuring the viral genome copy number (GCN).25 ⁇ l of 100 TCID50 of SARS-CoV-2 live virus (hCoV-19/Singapore/3/2020) was mixed with an equal volume of serially diluted 2H4, 3D11 or 5A6 IgG antibodies and incubated at 37°C for one hour before the mixture was added to 50 ⁇ l of 4x105 Vero E6 C1008 cells in suspension.
  • GCN viral genome copy number
  • the infected cells were incubated at 37°C incubator for 48 hrs after which supernatant was harvested and viral GCN was determined by subsequent RT- qPCR targeting the E gene using the RESOLUTE 2.0 kit as per manufacturer’s instructions. Briefly, 2.5 ⁇ l of supernatant was diluted with 2.5 ⁇ l of Milli-Q water and added to 20 ⁇ l of RT-PCR master mix. PCR was carried out as follows: reverse transcription at 55°C for 15min, inactivation at 95°C for 4min, followed by 45 cycles of amplification consisting of denaturation at 95°C for 3s and annealing/extension at 62°C for 30s and GCN values determined by comparing Ct values against a logGCN standard curve.
  • MucilAirTM HAE human airway epithelia reconstituted from human primary cells obtained from nasal or bronchial biopsies were provided by Epithelix SARL (Geneva, Switzerland) and maintained in air-liquid interphase with specific culture medium in Costar Transwell inserts (Corning, NY, USA) according to the manufacturer’s instructions.
  • the potency of 5A6 IgG was tested in neutralizing a live virus strain (BetaCoV/France/IDF0571/2020) isolated from one of the first COVID-19 cases confirmed in France: a 47-year old female patient hospitalized in January 2020 in the Department of Infectious and Tropical Diseases, Bichat Claude Bernard Hospital, Paris (Lescure et al., 2020).
  • the complete viral genome sequence was obtained using Illumina MiSeq sequencing technology, was then deposited after assembly on the GISAID EpiCoV platform (Accession ID EPI_ISL_411218) under the name BetaCoV/France/IDF0571/2020.
  • Opti-MEM medium Gibco, Thermo Fisher Scientific
  • Opti-MEM medium Gibco, Thermo Fisher Scientific
  • Viral suspensions were pre-incubated 60 min with antibody 5A6 IgG (75 ng/ml or 150 ng/ml) or an anti-Ebola glycoprotein control antibody (150 ng/ml) before infection.
  • a control infection was performed in absence of antibody.
  • Viral replication was quantified as the measured copy number of the viral genomes inside, and at the apical poles of, nasal and bronchial HAE.
  • Samples collected from apical washes at 48 hours post-infection were separated into 2 tubes: one for TCID50 viral titration (stored at - 80°C) and one for RT- qPCR.
  • HAE cells were harvested in RLT buffer (Qiagen) and total RNA was extracted using the RNeasy Mini Kit (Qiagen) for subsequent RT-qPCR.
  • ⁇ TEER trans epithelial electrical resistance
  • EOM2 Epithelial Volt/Ohm Meter for TEER
  • Ohm/cm2 Ohm/cm2
  • Vero E6 cells were transfected with S protein bearing furin recognition mutation (R682RAR to A682AAR) with C-terminal GFP tag by Lipofectamin 2000 (Invitrogen) and were cultured on ⁇ -Slide 8 well chamber slides (Ibidi). The transfection efficiency was monitored by percentage of GFP positive cells and optimized within 15-30% to achieve the best signal-to-noise ratio in the following cell-cell fusion assay.
  • Anti-human IgG Fc (AHC) sensors were first loaded with 1 ⁇ g/ml supplemented with 0.1% Tween-20 and 0.1% BSA) for 5 min to establish a stable baseline. The sensors were then dipped into different concentrations of each Fab from 100 nM to 3.125 nM in two- fold dilutions for 6 min, and then in kinetics buffer again for 10 min to measure association and dissociation. Assays were run at 25°C and data was analysed on the Octet System Data Acquisition Software version 9.0.0.4. using the 1:1 Langmuir binding model.
  • the AR2G sensor tips (ForteBio) were activated in freshly prepared 20mM EDC (1-ethyl-3-[3- dimethylaminopropyl]-carbodiimide hydrochloride), 10mM NHS (N-hydroxysuccinimide) solution and the 5A6 antibody was immobilized to the sensor tips using a concentration of 7.5 ⁇ g/ml of 5A6 in 10 mM sodium acetate pH 6 buffer. After quenching in 1M ethanolamine, the 5A6-immobilized sensor tips were dipped in 5 ⁇ g/ml of tagless RBD for 600s, then in 10 ⁇ g/ml of the second antibody for 300s. The assay was run at 25°C.
  • Sensor tips were regenerated in 10 mM glycine at pH 2.7 and neutralized in PBS with 0.1% Tween-20 before another cycle of sandwich assay was performed. Each sensor tip was used in a total of 3 cycles. Data analysis was done in the Octet System Data Acquisition Software version 9.0.0.4.
  • IgG and Fab affinity for Spike trimer by SPR [00261] StreptagII-tagged prefusion S ectodomain, diluted to 10 ⁇ g/mL in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% PS-20, pH 7.4, was captured on a StreptactinXT-immobilized (Iba Life Sciences) CM5 Series S sensor chip at an average level of 224 or 347 RU (response units) for IgG and Fab kinetics measurements, respectively using a Biacore T200 (Cytiva Life Sciences).2-fold serial dilutions of purified IgG from 12.5 nM to 0.39 nM or Fab from 100 nM to 3.125 nM were flowed over the captured prefusion S ectodomain at 30 ⁇ L/minute for 90 seconds followed by 420 seconds of dissociation flow.
  • EC50 values were calculated by non-linear regression analysis on the binding curves using GraphPad Prism and IC50 values were calculated either using the [Inhibitor] vs response variable slope four parameter non-linear regression model of GraphPad Prism, or the four parameter logistic regression model in the Quest GraphTM IC50 Calculator from AAT Bioquest, Inc (https://www.aatbio.com/tools/ic50- calculator).
  • ANOVA One-way analysis of variance

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Abstract

L'invention concerne de nouveaux anticorps anti-Spike du SARS-CoV-2, des compositions pharmaceutiques comprenant de tels anticorps, et des méthodes thérapeutiques d'utilisation de tels anticorps et compositions pharmaceutiques.
PCT/US2021/026286 2020-04-07 2021-04-07 Épitopes d'anticorps neutralisant le sars-cov-2 Ceased WO2021207433A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
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CN113999301A (zh) * 2021-12-07 2022-02-01 江苏中新医药有限公司 抗SARS-CoV-2(COVID-19)S蛋白RBD单克隆抗体
WO2022139680A1 (fr) * 2020-12-21 2022-06-30 Chugai Seiyaku Kabushiki Kaisha Molécules de liaison à sars-cov-2 et leurs utilisations
WO2024068777A1 (fr) 2022-09-28 2024-04-04 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Protéines ace2 modifiées présentant une activité améliorée contre le sars-cov-2
WO2024249362A1 (fr) * 2023-06-01 2024-12-05 The Regents Of The University Of California Isolement de phages d'anticorps rares et criblage biologique par discrimination (rapide)

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CN114213531B (zh) * 2021-12-07 2023-04-25 中国人民解放军军事科学院军事医学研究院 抗新型冠状病毒中和抗体、其抗原结合片段及其应用
AR128082A1 (es) * 2021-12-23 2024-03-20 Novavax Inc ANTICUERPOS CONTRA LA ESPÍCULA (S) DEL SARS-CoV-2 Y SU USO EN EL TRATAMIENTO DE COVID-19

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WO2007044695A2 (fr) * 2005-10-07 2007-04-19 Dana-Farber Cancer Institute Anticorps diriges contre le sras-cov et procedes d'utilisation de ceux-ci
JP2012502649A (ja) * 2008-09-19 2012-02-02 メディミューン，エルエルシー Ｃｄ１０５を対象とする標的結合剤およびその使用

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022139680A1 (fr) * 2020-12-21 2022-06-30 Chugai Seiyaku Kabushiki Kaisha Molécules de liaison à sars-cov-2 et leurs utilisations
CN113999301A (zh) * 2021-12-07 2022-02-01 江苏中新医药有限公司 抗SARS-CoV-2(COVID-19)S蛋白RBD单克隆抗体
CN113999301B (zh) * 2021-12-07 2023-07-28 江苏中新医药有限公司 抗SARS-CoV-2(COVID-19)S蛋白RBD单克隆抗体
WO2024068777A1 (fr) 2022-09-28 2024-04-04 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Protéines ace2 modifiées présentant une activité améliorée contre le sars-cov-2
WO2024249362A1 (fr) * 2023-06-01 2024-12-05 The Regents Of The University Of California Isolement de phages d'anticorps rares et criblage biologique par discrimination (rapide)

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