WO2022052733A1 - Anticorps neutralisant sans effet ade pour sars-cov-2 - Google Patents

Anticorps neutralisant sans effet ade pour sars-cov-2 Download PDF

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WO2022052733A1
WO2022052733A1 PCT/CN2021/112201 CN2021112201W WO2022052733A1 WO 2022052733 A1 WO2022052733 A1 WO 2022052733A1 CN 2021112201 W CN2021112201 W CN 2021112201W WO 2022052733 A1 WO2022052733 A1 WO 2022052733A1
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seq
cov
sars
antigen
antibody
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王乔
陆路
周韵娇
刘泽众
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复旦大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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

Definitions

  • the present invention relates to neutralizing antibodies and antigen-binding fragments thereof without ADE effect against SARS-CoV-2 novel coronavirus, and methods for preparing and using the neutralizing antibodies and antigen-binding fragments thereof.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • 2019-nCoV or HCoV-19 coronaviridae Study Group of the International Committee on Taxonomy of, 2020; Jiang et al, 2020a; Jiang et al, 2020c.
  • SARS-CoV-2 along with SARS-CoV-1 identified in 2003 (sometimes called SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) identified in 2012, belongs to the genus ⁇ -CoV (Zhou et al. People, 2020; Zhu et al., 2020).
  • the viral genome is packaged by the nucleocapsid (N) protein and surrounded by an envelope containing structural proteins.
  • N nucleocapsid
  • S-protein trimerizes and mediates viral entry into host cells (Li, 2016) and is a major target of neutralizing antibodies in humans (Jiang et al., 2020b; Premkumar et al., 2020; Wu, F. et al., 2020). It contains 1273 amino acids, including a large extracellular domain (S-ECD), a transmembrane helix and a small intracellular C-terminus.
  • S-ECD small extracellular domain
  • the two main domains within S-ECD have been identified as the S1 head region and the S2 stalk region, and the critical receptor-binding domain (RBD) is located in the S1 part.
  • SARS-CoV-2 RBD human receptor angiotensin-converting enzyme-2 (ACE2)
  • ACE2 human receptor angiotensin-converting enzyme-2
  • the virus invades the host cell through Fc receptor-mediated internalization of the virus-antibody immune complex. This phenomenon has been demonstrated for dengue, coronavirus and other viruses (Eroshenko et al., 2020; Iwasaki and Yang, 2020; Katzelnick et al., 2017; Miner and Diamond, 2017; Salje et al., 2018).
  • SARS-CoV-1 infection S-protein-binding antibodies promote ACE2-independent viral internalization into macrophages, monocytes, and B cells (Jaume et al., 2011; Wang et al., 2014; Yip et al., 2014).
  • the uptake of viral particles via the ADE pathway may lead to elevated production of proinflammatory cytokines.
  • proinflammatory cytokines During SARS-CoV-2 infection, significantly elevated IgG antibody responses (Zhang et al., 2020; Zhao et al., 2020) and significantly increased levels of proinflammatory cytokines in serum ( Huang et al., 2020; Wang, J. et al., 2020).
  • IgG antibody responses Zinct al., 2020; Zhao et al., 2020
  • proinflammatory cytokines in serum Huang et al., 2020; Wang, J. et al., 2020.
  • SARS-CoV-2 mutants were recently discovered in India (Lopez et al., 2021; Harvey et al., 2021). The current study suggests that mutations in these SARS-CoV-2 mutant strains may increase the affinity of RBD for its cellular receptor ACE2 and confer resistance to vaccine sera and many monoclonal antibodies.
  • Convalescent plasma or serum from individuals vaccinated by mRNA vaccine or inactivated virus vaccine showed a significant reduction in neutralizing activity against these emerging circulating mutants (Chen et al, 2021b; Wang et al, 2021a; Wang et al, 2021b ; Wang et al, 2021c; Weisblum et al, 2020; Wibmer et al, 2021).
  • Such immune evasion has also been reported for several monoclonal antibodies in clinical stage, such as LY-CoV555, CB6 and REGN10933 (Wang et al., 2021b; Wang et al., 2021c).
  • the inventors selected convalescent individuals with high levels of serum IgG neutralizing activity against SARS-CoV-2, and isolated individuals expressing the same Ig variable gene segments and highly similar CDR3 sequences Many extended clones of memory B cells of closely related antibodies. Nearly half of the isolated antibodies targeted five different antibody-binding epitopes on the RBD domain of the S-protein. Characterization of neutralizing and enhancing activity of these antibodies identified a series of antibodies that not only effectively neutralized SARS-CoV-2 but also did not induce ADE effects. Of particular interest is that certain antibodies of the invention retain sensitivity to a range of SARS-CoV-2 strains with a 1 amino acid mutation. In addition, the inventors identified RBD domain-binding antibodies that effectively broadly neutralized both SARS-CoV-1 and SARS-CoV-2 viruses without ADE effects.
  • beta-CoV-B beta-coronavirus lineage B
  • SARS-CoV-2 SARS-CoV-2
  • SARS-CoV-1 SARS-CoV-1
  • bat SARSr-CoV WIV1 bat SARSr-CoV WIV1.
  • members of the antibody family that competed for binding with XG014 showed reduced levels of cross-reactivity and induced antibody-dependent cell fusion, suggesting a unique recognition mode for XG014.
  • the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to the S protein of SARS-CoV-2, characterized in that the isolated antibody or antigen-binding fragment thereof binds
  • the fragment comprises a heavy chain variable region VH comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3 and a light chain variable region VL comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 are selected from the following amino acid sequences:
  • the heavy chain variable region VH and light chain variable region VL are selected from the following amino acid sequences:
  • the isolated antibody or antigen-binding fragment thereof specifically binds the RBD domain of the S protein.
  • the isolated antibody or antigen-binding fragment thereof specifically binds the S1 domain of the S protein but not the RBD domain.
  • the isolated antibody or antigen-binding fragment thereof specifically binds the S2 domain of the S protein.
  • the present invention also provides an isolated antibody or an antigen-binding fragment thereof that specifically binds to the RBD domain of the SARS-CoV-2 S protein, characterized in that the isolated antibody or an antigen-binding fragment thereof comprising a heavy chain variable region VH comprising a complementarity determining region CDRH1, CDRH2 and CDRH3 and a light chain variable region VL comprising a complementarity determining region CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 are selected from the following amino acid sequences:
  • the heavy chain variable region VH and light chain variable region VL are selected from the following amino acid sequences:
  • the single amino acid mutant of SARS-CoV-2 comprises V341I, F342L, V367F, R408I, A435S, G476S and V483A mutations in the RBD domain.
  • the isolated antibodies or antigen-binding fragments thereof described herein neutralize SARS-CoV-2 and single amino acid mutants thereof in vitro with a 50% inhibitory concentration IC50 value of less than 10 ⁇ g/ml, such as by cell-based The luciferase activity was determined.
  • the isolated antibodies or antigen-binding fragments thereof described herein neutralize SARS-CoV-2 and single amino acid mutants thereof in vitro with a 50% inhibitory concentration IC50 value of less than 25 ng/ml.
  • the present invention also provides an isolated antibody or an antigen-binding fragment thereof that specifically binds to the RBD domain of the SARS-CoV-2 S protein, characterized in that the isolated antibody or an antigen-binding fragment thereof comprising a heavy chain variable region VH comprising a complementarity determining region CDRH1, CDRH2 and CDRH3 and a light chain variable region VL comprising a complementarity determining region CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 are selected from the following amino acid sequences:
  • the heavy chain variable region VH and light chain variable region VL are selected from the following amino acid sequences:
  • the isolated antibody or antigen-binding fragment thereof neutralizes SARS-CoV-2 in vitro with a 50% inhibitory concentration IC50 value of less than 25 ng/ml and in vitro with an IC50 value of less than 10 ng/ml and SARS-CoV-1, as determined by cell-based luciferase activity.
  • the present invention also provides an isolated antibody or an antigen-binding fragment thereof that specifically binds to the RBD domain of the SARS-CoV-2 S protein, characterized in that the isolated antibody or an antigen-binding fragment thereof comprising a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH comprising CDRH1 of SEQ ID NO:27, CDRH2 of SEQ ID NO:55, SEQ ID NO:83 CDRH3 of SEQ ID NO: 139, CDRL2 of SEQ ID NO: 167 and CDRL3 of SEQ ID NO: 195,
  • said isolated antibody or antigen-binding fragment thereof cross-neutralizes SARS-CoV-2, SARS-CoV-2 mutant strains, SARS-CoV-1 and bat SARSr-CoV WIV1.
  • the heavy chain variable region VH and light chain variable region VL comprise the amino acid sequences of SEQ ID NO: 111 and SEQ ID NO: 223, respectively.
  • the SARS-CoV-2 mutant is selected from the group consisting of B.1.1.7, B.1.351, P.1, B.1.617.1 and B.1.617.2.
  • the Fc receptor-binding site of an isolated antibody or antigen-binding fragment thereof described herein includes the GRLR mutations G236R and L328R.
  • the isolated antibody or antigen-binding fragment thereof described herein is a monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody, chimeric antibody, multispecific antibody or antibody fragment thereof .
  • the antigen-binding fragment is a Fab fragment, Fab' fragment, F(ab') 2 fragment, Fv fragment, diabody or single chain antibody molecule.
  • the isolated antibody or antigen-binding fragment thereof described herein is of the IgGl type, IgG2 type, IgG3 type, or IgG4 type.
  • the present invention also provides an isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof described herein.
  • the present invention also provides an expression vector comprising the polynucleotide described herein.
  • the present invention also provides a host cell comprising the expression vector described herein.
  • the present invention also provides a method of producing an isolated antibody or antigen-binding fragment thereof as described herein, characterized in that the method comprises culturing a host as described herein under conditions that permit expression of the antibody or antigen-binding fragment thereof cells, and recovering antibodies or antigen-binding fragments thereof produced by the host cells.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.
  • the present invention also provides the use of the isolated antibody or antigen-binding fragment thereof in the preparation of a medicament for treating or preventing SARS-CoV-2 infection in a subject in need thereof.
  • the present invention also provides the use of the isolated antibody or antigen-binding fragment thereof in the preparation of a medicament for treating or preventing infection by SARS-CoV-2 and a single amino acid mutant thereof in a subject in need.
  • the present invention also provides the use of the isolated antibody or antigen-binding fragment thereof in the preparation of a medicament for treating or preventing SARS-CoV-2 and/or SARS-CoV-1 infection in a subject in need.
  • the present invention also provides that the isolated antibody or its antigen-binding fragment is used in the preparation of SARS-CoV-2, SARS-CoV-2 mutant strain, SARS-CoV-1 and/or SARS-CoV-2 for treating or preventing a subject in need thereof. or use in drugs for bat SARSr-CoV WIV1 infection.
  • the present invention provides methods for treating or preventing SARS-CoV-2 infection in a subject in need thereof, the methods comprising administering to the subject an effective amount of at least one isolated antibody disclosed herein or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof does not produce an antibody-dependent enhancement (ADE) effect.
  • ADE antibody-dependent enhancement
  • the invention provides methods for effectively neutralizing SARS-CoV-2 virus without antibody-dependent enhancement (ADE) effects in a subject in need thereof, the methods comprising administering to the subject An effective amount of at least one antibody or antigen-binding fragment thereof disclosed herein.
  • AD antibody-dependent enhancement
  • the present invention provides a method for treating or preventing SARS-CoV-2 and/or SARS-CoV-1 infection in a subject in need thereof, the method comprising administering to the subject an effective amount of at least An antibody or antigen-binding fragment thereof disclosed herein, wherein the antibody or antigen-binding fragment thereof does not produce an antibody-dependent enhancement (ADE) effect.
  • ADE antibody-dependent enhancement
  • the present invention provides a method for treating or preventing SARS-CoV-2, SARS-CoV-2 mutant, SARS-CoV-1 and/or bat SARSr-CoV WIV1 infection in a subject in need thereof
  • a method comprising administering to a subject an effective amount of at least one antibody or antigen-binding fragment thereof disclosed herein, wherein the isolated antibody or antigen-binding fragment thereof cross-neutralizes SARS-CoV-2, SARS-CoV-2 2 mutants, SARS-CoV-1 and bat SARSr-CoV WIV1.
  • the invention provides an isolated antibody or antigen-binding fragment thereof disclosed herein for use in the treatment or prevention of SARS-CoV-2 infection in a subject in need thereof, wherein the antibody or antigen-binding fragment thereof does not Produces antibody-dependent enhancement (ADE) effects.
  • AD antibody-dependent enhancement
  • the invention provides the antibodies or antigen-binding fragments thereof disclosed herein for use in effectively neutralizing SARS-CoV-2 virus without antibody-dependent enhancement (ADE) effects in a subject in need thereof.
  • AD antibody-dependent enhancement
  • the invention provides an antibody or antigen-binding fragment thereof disclosed herein for use in the treatment or prevention of SARS-CoV-2 and/or SARS-CoV-1 infection in a subject in need thereof, wherein the antibody or antigen-binding fragments thereof do not produce antibody-dependent enhancement (ADE) effects.
  • AD antibody-dependent enhancement
  • the invention provides an antibody or antigen-binding fragment thereof disclosed herein for use in the treatment or prophylaxis of SARS-CoV-2, SARS-CoV-2 mutants, SARS-CoV-1 in a subject in need thereof and/or bat SARSr-CoV WIV1 infection, wherein said isolated antibody or antigen-binding fragment thereof cross-neutralizes SARS-CoV-2, SARS-CoV-2 mutants, SARS-CoV-1 and bat SARSr-CoV WIV1.
  • the invention provides neutralizing antibodies or antigen-binding fragments thereof directed against SARS-CoV-2 that bind an epitope in the S-protein (SEQ ID NO: 225) .
  • the epitope is located at RBD (aa 319-541), S1 (aa 14-685), S2 (aa 686-1213) or S-ECD (aa 14-1213) of SEQ ID NO: 225 in the domain.
  • the antibody or antigen-binding fragment thereof binds an epitope in an amino acid sequence that is at least 90% identical to SEQ ID NO: 225 or a subsequence thereof above.
  • Figure 1 shows a schematic representation of the different domains of the SARS-CoV-2 S-protein.
  • Figure 2 shows age and sex histograms for 24 donors.
  • Figure 3 shows antibody responses in individuals recovering from SARS-CoV-2 infection.
  • AE ELISA of serum samples. From 24 volunteers, including 16 SARS-CoV-2 convalescent individuals (donors #1 to #16) and 8 SARS-CoV-2-unexposed uninfected individuals (donors #17 to #24)
  • Various serum dilutions of SARS-CoV-2 RBD (A), S1 (B), S2 (C), S-ECD (D) and N (E) proteins were evaluated by ELISA (top panel).
  • ELISA area under the curve (ELISAAUC) values were calculated by PRISM software and presented as a histogram (lower panel).
  • Figure 4 shows the gating strategy for cell sorting. Double positive (bait protein-PE + and bait protein-APC + ) cells were single-cell sorted for antibody cloning.
  • Figure 5 shows the frequency of B lymphocytes recognizing SARS-CoV-2 protein in uninfected control donors (upper panel) and selected donor #16 (lower panel).
  • Representative flow cytometry plots showing binding of biotinylated Avi-Tag-labeled RBD (A), chemically biotinylated S-ECD (B), and biotinylated Avi-Tag-labeled S-ECD (C) Percentage of CD19 + CD20 + B cells. All bait proteins were labeled with either allophycocyanin (APC) or phycoerythrin (PE) for a dual fluorescent-dye sorting strategy. The experiment was repeated at least 2 times.
  • APC allophycocyanin
  • PE phycoerythrin
  • Figure 6 shows an antibody pie chart for Donor #16.
  • a total of 292 sequenced antibodies were cloned with naturally paired Ig heavy and light chains.
  • Antibodies with the same combination of IGH and IGL variable gene sequences and closely related CDR3s are grouped together and represented as different slices. A total of 25 slices were identified. Individually cloned antibodies are shown as a light gray slice.
  • the VH, VL gene and CDR3 amino acid sequences for paired IGH and IGL are shown for the expanded cloned antibodies and 48 selected antibodies (XG001-XG048).
  • Figure 7 shows the V(D)J amino acid sequence alignment of representative antibodies from the same extended clone.
  • XG001/XG002, XG003/XG004 and XG020/XG044, respectively, were derived from closely expanding B cell clones. Consensus amino acid residues are shown in grey boxes.
  • Figure 8 shows antibody epitopes in the SARS-CoV-2 S protein.
  • A-D Binding of human monoclonal antibodies to different domains of the SARS-CoV-2 S-protein. Representative area under the curve (AUC) of ELISA using S-ECD domain (A), RBD domain (B), S1 domain (C) and S2 domain (D) from at least 2 independent experiments is shown value. PBS was used as a negative control.
  • E Based on antigen-binding assay, 48 antibodies were classified into 5 types, RBD-binding, S1 but not RBD-binding, S2-binding, S-ECD-binding, and no binding.
  • F Competitive ELISA defines 4 groups of RBD-binding antibodies.
  • the primary antibody (x-axis) was non-biotinylated and used to block epitopes, while the secondary antibody (y-axis) was biotinylated for detection by streptavidin-HRP.
  • the results of the competitive ELISA are shown as percent binding by the second biotinylated antibody compared to a PBS-blocked reference, and are color-coded: black, 0%–25%; dark grey, 25%–50%; Light grey, 50%–75%; white, >75%. All antibodies tested blocked the binding of their own biotinylated forms.
  • XG008 and XG038 bind 2 mutually exclusive sub-epitopes of RBD group III.
  • Anti-HBs antibody H004 Wang, Q. et al., 2020
  • Weak RBD binders (XG015, XG042, XG045, XG047) were excluded from this assay. Representative 2 experiments.
  • Figure 9 shows a competitive ELISA and 4 major RBD antibody panels.
  • A-B Competitive ELISA to validate 5 non-competing epitopes on RBD.
  • a representative antibody is selected from each antibody group, wherein XG011 is selected from RBD Group I, XG025 is selected from RBD Group II, XG008 and XG038 are selected from RBD Group III, and XG014 is selected from RBD Group IV.
  • XG008 and XG038 bind 2 mutually exclusive sub-epitopes in RBD group III.
  • the primary antibody was an unlabeled RBD-binding antibody and was used to block epitopes on coated RBD (A) or coated S-ECD protein (B).
  • the secondary antibody was a biotinylated RBD antibody for streptavidin-HRP dependent detection. PBS-blocking wells are the normalized reference.
  • Figure 10 shows antibodies from 4 RBD panels, with IGHV and IGKV/IGLV labeled.
  • Figure 11 shows in vitro neutralizing activity.
  • A Neutralizing potency of representative human monoclonal antibodies using a luciferase-based SARS-CoV-2 pseudovirus. Luciferase signal (a surrogate for infection) was determined in the presence of various concentrations of the indicated monoclonal antibodies and normalized to a no antibody control (dashed line). Test antibodies with no neutralizing ability in the assay are shown as grey lines.
  • B In vitro neutralization assay against SARS-CoV-2 pseudovirus. IC50 values are shown for each antibody. All experiments were repeated a minimum of 2 times. The abbreviation "nn" means "no neutralization in the assay”.
  • E and F Quantitative reverse transcription PCR results (E) and corresponding IC50 values (F) of the in vitro neutralization assay against SARS-CoV-2 euvirus.
  • the SARS-CoV-2 N-protein RNA copy number was calculated based on a standard curve of N-protein DNA samples drawn using 7 concentrations of N-protein DNA samples containing 10-fold serial dilutions. At least 2 independent experiments were performed.
  • G Cluster analysis identified antibody epitopes associated with neutralizing activity. Unsupervised hierarchical clustering using SARS-CoV-2 pseudovirus neutralization data in the presence of 45 monoclonal antibodies at 9 dilutions. Clusters A, B, C and D were identified. Antibodies (XG021, XG034, XG039) with no ELISA binding against the tested antigens were excluded. Antibodies with different epitopes on the S-protein or RBD are color-coded or shape-coded, respectively.
  • Figure 12 shows an immunofluorescence-based in vitro neutralization assay against SARS-CoV-2 euvirus.
  • AG Different mAbs XG005(A), XG008(B), XG013(C), XG014(D), XG016(E), XG017(F) and XG020 after SARS-CoV-2 euvirus infection
  • G Immunofluorescence staining of SARS-CoV-2 N-protein in the presence of. Immunofluorescence using an anti-N-protein polyclonal antibody (Gu et al., 2020) was used to visualize infected cells, with DAPI staining showing total cell density.
  • Figure 13 shows the cross-neutralizing activity of monoclonal antibodies.
  • AB In vitro neutralization assays of XG014 (A) and XG038 (B) against SARS-CoV-2 pseudoviruses with different RBD mutations. Luciferase activity was measured, normalized and considered a surrogate for infection. The reference used for normalization had no added antibody (dashed line).
  • CD In vitro neutralization experiments against SARS-CoV-1 pseudovirus (C) and corresponding IC50 values (D) for each antibody. The abbreviation "nn” means "no neutralization in the assay”. Test antibodies with no neutralizing ability in the assay are shown as light grey lines in (C). At least 2 experiments were performed with the indicated representative antibodies.
  • Figure 14 shows the in vitro neutralization assay against SARS-CoV-1 pseudovirus by XG014 and XG041 antibodies.
  • A-B In vitro neutralization assay of XG014 (A) and XG038 (B) against SARS-CoV-1 pseudovirus. Luciferase activity was determined and normalized using the luciferase signal of a reference sample to which no antibody was added (dashed line).
  • Figure 15 shows in vitro antibody-dependent viral entry by monoclonal antibodies.
  • A-B The ADE effect of antibodies was determined using in vitro Raji cells expressing a luciferase-expressing SARS-CoV-2 pseudovirus in the presence of different dilutions of the antibody. Antibodies that induce high levels of luciferase signal are shown in (A), including 9 RBD-binding antibodies and 2 S1-binding but not RBD-binding antibodies (B). The presence of all other antibodies tested showed background or low levels of luciferase signal. The luciferase signal induced by antibody XG003 at 2 ⁇ g/ml was used as a normalization reference, and others were expressed as fold change in luciferase activity.
  • FIG. 16 shows the antibody-dependent enhancement (ADE) effect by monoclonal antibodies in Raji cells.
  • A In vitro Raji cell-dependent assay using luciferase-expressing SARS-CoV-2, representative monoclonal antibodies induce ADE effects.
  • the luciferase signal induced by antibody XG003 at 2 ⁇ g/ml was used as a normalization reference, and others were expressed as fold change in luciferase activity.
  • XG005 and XG016 induced high levels of luciferase signal, whereas antibodies XG014 and XG038 did not induce ADE.
  • XG006 induced viral entry only at high concentrations, whereas the opposite was true for XG029.
  • Figure 17 shows cluster analysis identifying RBD epitopes associated with antibody neutralizing or enhancing effects.
  • A Unsupervised hierarchical clustering using SARS-CoV-2 pseudovirus neutralization (NEU) and Raji cell antibody-dependent enhancement (ADE) effect data. Clusters X, Y and Z were identified. Antibodies (XG021, XG034, XG039) with no ELISA binding against the tested antigens were excluded. This cluster analysis was repeated using other group neutralization and ADE values.
  • BC Violin plots of ADEAUC (B) or IC50 values (C) of antibodies in the three clusters. Statistical analysis was performed using Dunn's Kruskal-Wallis multiple comparison test.
  • Figure 18 shows cluster analysis identifying RBD group IV epitopes associated with ADE effects.
  • ADE antibody-dependent enhancement
  • FIG. 19 Cryo-EM data collection and processing of XG014 bound to SARS-CoV-2 S trimer.
  • A Representative electron micrographs and 2D classification results of XG014-bound SARS-CoV-2 S.
  • B Locally resolved map of fully reconstructed and locally refined RBD-XG014.
  • C Gold standard Fourier shell correlation curves of XG014-bound SARS-CoV-2 S trimer ( ⁇ ) and locally refined RBD/XG014 interface region ( ⁇ ). The horizontal dashed line indicates the 0.143 cutoff.
  • D Flow chart of data processing for XG014-conjugated SARS-CoV-2 S trimers.
  • Figure 20 Cryo-EM data collection and processing of XG005 bound to SARS-CoV-2 S trimer.
  • A Representative electron micrographs and 2D classification results of XG005-bound SARS-CoV-2 S.
  • B Locally resolved map of fully reconstructed and locally refined RBD-XG005.
  • C Gold standard Fourier shell correlation curves of XG005-bound SARS-CoV-2 S trimer ( ⁇ ) and locally refined RBD/XG005 interface region ( ⁇ ). The horizontal dashed line indicates the 0.143 cutoff.
  • D Flow chart of data processing for XG005-bound SARS-CoV-2 S trimers.
  • FIG. 21 Four monoclonal antibodies with non-overlapping epitopes.
  • A Competitive ELISA of four monoclonal antibodies against recombinant SARS-CoV-2 RBD protein. The primary antibody was non-biotinylated and was added to each well in a combination of three antibodies to block the coated RBD, while the corresponding secondary biotinylated antibodies were each used to detect binding. The results of the competitive ELISA are expressed as percent binding of the second biotinylated antibody and are shown as follows: black 0%-25%; dark grey 26%-50%; light grey 51%-75%; white >76%. Representative of two replicate experiments.
  • FIG. 22 In vitro neutralization of monoclonal antibodies.
  • A Antibody binding to SARS-CoV-2, its mutants, and recombinant S-ECD or RBD protein of SARS-CoV-1. The area under the curve (AUC) was calculated by PRISM software. Experiments were performed at least twice.
  • B-C In vitro neutralization assay using SARS-CoV-2 pseudovirus in Huh-7 cells. The percent inhibition of infection in the presence of the indicated antibodies XG011, XG014, XG017 or XG025 is shown.
  • the insertion in (C) is an 11 amino acid insertion KTRNKSTSRRE between Y248 and L249.
  • FIG. 23 In vitro neutralization assay.
  • A Quantification of the S protein of SARS-CoV-2 mutant pseudoviruses. To accurately evaluate the neutralizing activity of monoclonal antibodies, Western blot analysis was used to quantify the SARS-CoV-2 S protein to ensure that similar amounts of pseudoviruses were used in the experiments.
  • B-C Immunofluorescent staining of SARS-CoV-2 N protein against the in vitro neutralization assay of SARS-CoV-2 true virus. Immunofluorescence using an anti-N protein polyclonal antibody (Zhou et al., 2021b) was used to evaluate the neutralizing effect of XG014. Scale bar 400 ⁇ m.
  • (D-E) In vitro neutralization activity of XG014 against SARS-CoV-2 mutant pseudovirus in Caco-2 (D) and Calu-3 cells (E). Experiments were performed at least twice and data are shown as mean ⁇ SEM.
  • (F-G) In vitro neutralization assay using SARS-CoV (F) or SARSr-CoV WIV1 (G) pseudovirus in A549 cells overexpressing ACE2. The infection rate of samples without added antibody was used as a reference to normalize the percent inhibition of viral infection. All in vitro neutralization experiments were performed at least twice and data are shown as mean ⁇ SEM. N/D, not detected.
  • Figure 24 In vitro neutralization assay. In vitro neutralizing activity of XG014 against SARS-CoV-2 mutant strains B.1.617.1 and B.1.617.2 pseudoviruses in Huh-7 cells. Experiments were performed at least twice and data are shown as mean ⁇ SEM.
  • FIG. 25 Cryo-EM structure of SARS-CoV-2 S trimer complexed with XG014Fab.
  • A Molecular surface schematic of the structure of the SARS-CoV-2 S-XG014 complex, with the three RBDs 'downward' when viewed along two orthogonal directions. Each SARS-CoV-2 protomer is shown in a different color. Exemplary downward conformational RBDs are indicated by arrows. The XG014 light and heavy chains are represented in different colors, respectively.
  • B Cartoon illustration of the XG014-RBD region.
  • C Key interactions between XG014 heavy chain and SARS-CoV-2 RBD. Hydrogen bonds are indicated by dashed lines.
  • FIG. 26 Amino acid sequence alignment of the RBD region.
  • the amino acid sequences of the RBD regions from SARS-CoV-2, SARS-CoV-1 (i.e. SARS-CoV) and SARSr-CoV WIV1 were aligned.
  • Identical amino acid residues are shown with a dark background, arrows indicate residues involved in the major interaction between RBD and XG014.
  • FIG. 27 Comparison of XG014 epitope with other antibodies that bind all "down" SARS-CoV-2 S trimers.
  • A Schematic representation of the surface of two adjacent RBDs (labeled RBD and RBD') in all closed S trimers. ACE2 binding residues and popular mutation sites 417, 484 and 501 are shown in different colors.
  • BH Schematic representation of the surfaces of two adjacent RBDs in all closed S trimers, including XG014 (this application), S2M11 (PDB ID: 7K43), C144 (PDB ID: 7K90), COVOX-316 (PDB ID: 7ND7), 2-4 (PDB ID: 6XEY), 1-57 (PDB ID: 7LS9), and S309 (PDB ID: 6WPS).
  • FIG. 28 XG005 and XG016, but not XG014, induce S protein-mediated membrane fusion.
  • A Competitive ELISA for three neutralizing antibodies (XG014, XG005 and XG016) targeting the same set of epitopes. The results of the competitive ELISA are expressed as percent binding of biotinylated antibody and are shown as follows: black 0%-25%; dark grey 26%-50%; light grey 51%-75%; and white >76%. Representative of two replicate experiments.
  • C-D In vitro neutralization assay using SARS-CoV-2 pseudovirus in Huh-7 cells. Experimental results show percent infection inhibition by XG005 (C) or XG016 (D) normalized to infection without the addition of antibody. Data are shown as mean ⁇ SEM. All experiments were repeated at least twice.
  • E-F In vitro neutralization assay using SARS-CoV-1 (E) or SARSr-CoV WIV1 (F) pseudovirus in Huh-7 cells. The infection rate of samples without added antibody was used as a reference to normalize the percent inhibition of viral infection. All in vitro neutralization experiments were performed at least twice and data are shown as mean ⁇ SEM. N/D, not detected.
  • FIG. 1 Schematic illustration of antibody-dependent S protein-mediated membrane fusion between Raji cells expressing Fc ⁇ receptor II (FcyRII) and HEK-293T cells overexpressing SARS-CoV-2 S protein.
  • H XG005 and XG016, but not XG014 and PBS, promote syncytia formation between Raji cells and HEK-293T cells overexpressing the SARS-CoV-2 S protein. Scale bar of fluorescence images 200 ⁇ m.
  • I The number of fusion cells induced by different concentrations of the indicated antibodies.
  • J Fluorescence images of SARS-CoV-2 S protein-mediated membrane fusion in the presence of XG005 or XG005-GRLR. Scale bar 200 ⁇ m.
  • K The number of fusion cells induced by XG005 and XG005-GRLR. All in vitro neutralization and cell fusion experiments were performed at least twice.
  • FIG. 29 XG014, but not XG005 or XG016, inhibits S protein-mediated membrane fusion.
  • A Schematic representation of S protein-mediated membrane fusion. Monoclonal antibody blocking the S protein-ACE2 interaction inhibits S protein-mediated membrane fusion. Such S protein-mediated membrane fusion occurs between HEK-293T cells overexpressing SARS-CoV-2 or SARS-CoV-1 S protein and ACE2-positive Huh-7 or Caco-2 or Calu-3 cells.
  • B-C XG014 inhibits syncytia formation between Huh-7 cells and HEK-293T cells overexpressing the S protein of SARS-CoV-2 or SARS-CoV-1. Representative fluorescence image (B), scale bar 400 ⁇ m.
  • XG014 inhibits SARS-CoV-2 S protein-mediated syncytia formation in Caco-2 or Calu-3 cells. Scale bar of fluorescence image (D) 400 ⁇ m. The number of fusion cells induced by different concentrations of XG014 (E). White arrows in (B) and (D) indicate examples of syncytia.
  • F-G The induction of syncytia formation with Raji cells by XG005 and XG016 (FIG. 28G) was potently inhibited by EK1. Representative fluorescence images (F), scale bar 200 ⁇ m. Number of fused cells in the presence of antibody and EK1 (G).
  • FIG. 30 Cryo-EM structure of SARS-CoV-2 S trimer complexed with XG005.
  • A Molecular surface schematic of the structure of the SARS-CoV-2 S-XG005 complex, with the three RBDs 'downward' when viewed along two orthogonal directions. Each SARS-CoV-2 protomer is shown in a different color. XG005 light and heavy chains are represented in different colors, respectively.
  • B Cartoon illustration of the XG005-RBD region.
  • C Key interactions between XG005 and SARS-CoV-2 RBD. Hydrogen bonds are indicated by dashed lines.
  • D Localization of ACE2 relative to XG005 bound to the SARS-CoV-2 RBD.
  • E Comparison of XG014-RBD and XG005-RBD structures.
  • F Schematic representation of the molecular surface of the SARS-CoV-2 RBD showing the differences and similarities between the XG005 and XG014 epitopes. Common residues involved in both XG005 and XG014 interactions as well as specific residues involved in XG005 or XG014 binding are indicated.
  • FIG. XG014 has prophylactic and therapeutic effects against SARS-CoV-2 in vivo.
  • A Illustration of prophylaxis and treatment regimens.
  • B Changes in body weight in prevention, treatment and control groups.
  • C-D SARS-CoV-2 viral mRNA titers in lung (C) and intestine (D) 4 days post-challenge. RT-qPCR was used to quantify SARS-CoV-2 viral mRNA load.
  • E-F Dot plots showing the correlation between body weight change (x-axis) and SARS-CoV-2 viral mRNA titers (y-axis) in lung (E) and intestine (F). Spearman rank correlation coefficient (r) and significance (p).
  • antibodies or antigen-binding fragments thereof that bind the SARS-CoV-2 S protein.
  • the antibody or antigen-binding fragment thereof binds an epitope in the RBD, S1, S2, or S-ECD domain of the S protein.
  • the antibody or antigen-binding fragment thereof prevents SARS-CoV-2 virus entry into host cells, thereby treating or preventing SARS-CoV-2 infection in a subject.
  • the antibody is a human monoclonal antibody.
  • the S protein domain-binding antibodies or antigen-binding fragments thereof of the invention can neutralize SARS-CoV-2 in a subject without producing antibody-dependent enhancement (ADE) effect. Accordingly, the present invention can be used in various methods and compositions for treating or preventing SARS-CoV-2 infection in a subject.
  • XG014 is super efficient and broadly neutralizing without enhancing S protein-mediated membrane fusion.
  • Cryo-electron microscopy revealed that XG014 recognizes a conserved epitope outside the receptor binding motif (RBM) in the RBD and that it locks all three RBDs of the S trimer to "down" or “off” "Conformation that sterically hinders receptor binding.
  • RBM receptor binding motif
  • XG014 is effective in preventing and treating SARS-CoV-2 in human ACE2-transgenic (hACE2-Tg) mice, suggesting that XG014 is developed as a broad-spectrum and highly effective SARS-CoV-2 inhibitory efficiency and has the potential for clinical development.
  • S-protein also known as the spike protein, is one of the viral structural proteins that mediate viral entry into host cells.
  • the SARS-CoV-2 S-protein contains 1273 amino acids (SEQ ID NO: 225), including a large extracellular domain (S-ECD), a transmembrane helix and a small intracellular C-terminus.
  • S-ECD small extracellular domain
  • the two main domains within S-ECD have been identified as the S1 head region and the S2 stalk region, and the critical receptor-binding domain (RBD) is located in the S1 part. See Figure 1.
  • amino acid sequence of the S protein of SARS-CoV-2 virus is as follows:
  • amino acid sequence of the S protein of SARS-CoV-1 virus is as follows:
  • amino acid sequence of the S protein of SARSr-CoV WIV1 virus is as follows:
  • polynucleotide or “nucleic acid” includes both single- and double-stranded nucleotide polymers.
  • Nucleotides comprising polynucleotides can be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotides. Such modifications include: base modifications, such as bromouridine and inosine derivatives; ribose modifications, such as 2',3'-dideoxyribose; and internucleotide linkage modifications, such as phosphorothioate, dithio Phosphate, selenophosphate, diselenophosphate and phosphoramidate, etc.
  • oligonucleotide means a polynucleotide comprising 200 nucleotides or less. In certain embodiments, the oligonucleotides are 10-60 bases in length. In other embodiments, the oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 nucleotides in length. Oligonucleotides can be single-stranded or double-stranded, such as those used to construct mutant genes. Oligonucleotides can be sense or antisense oligonucleotides. Oligonucleotides can include labels for detection assays, including radioactive, fluorescent, hapten, or antigenic labels. Oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.
  • vector means any molecule or entity (eg, nucleic acid, plasmid, phage or virus) used to transfer protein-encoding information to a host cell.
  • expression vector refers to a vector suitable for transformation of a host cell and containing nucleic acid sequences that direct and/or regulate the expression of one or more heterologous coding regions to which it is operably linked.
  • Expression constructs can include, but are not limited to, sequences that affect or regulate transcription, translation; and, if present, sequences that affect RNA splicing of the coding region to which it is operably linked.
  • operably linked means that the components to which the term is used are in a relationship that allows the components to perform their inherent function under suitable conditions.
  • a regulatory sequence in a vector "operably linked" to a protein-coding sequence is ligated with the protein-coding sequence so that expression of the protein-coding sequence is accomplished under conditions compatible with the transcriptional activity of the regulatory sequence.
  • the term "host cell” means a cell that has been or can be transformed with a nucleic acid sequence and thereby express a gene of interest. Regardless of whether the progeny is morphologically or genetically identical to the original parent cell, as long as the gene of interest is present in the progeny, the term includes the progeny of said parent cell.
  • transfection means that a cell takes up foreign or exogenous DNA, which is "transfected” when the exogenous DNA is introduced into the cell membrane.
  • Various transfection techniques are well known in the art and disclosed herein. See, e.g., Graham et al, 1973, Virology 52:456; Sambrook et al, 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al, 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197.
  • the techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.
  • transformation refers to a change in the genetic properties of a cell that is transformed when it is modified to contain new DNA or RNA.
  • a cell is transformed when the cell's native state is genetically modified by the introduction of new genetic material through transfection, transduction, or other techniques.
  • the transformed DNA can recombine with cellular DNA by physically integrating into the cellular chromosome, or can remain transiently unreplicated as an extrachromosomal element, or can replicate independently as a plasmid.
  • a cell is considered to have been "stably transformed” when the transformed DNA replicates as the cell divides.
  • amino acid includes its ordinary meaning in the art. Conventional one-letter amino acid codes and three-letter amino acid codes, as shown in Table 1, are used herein.
  • Stereoisomers of the 20 conventional amino acids eg D-amino acids
  • unnatural amino acids such as ⁇ -, ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids may also be present in the antibodies of the invention.
  • suitable components eg D-amino acids, unnatural amino acids such as ⁇ -, ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids may also be present in the antibodies of the invention.
  • suitable components eg D-amino acids, unnatural amino acids such as ⁇ -, ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids may also be present in the antibodies of the invention.
  • suitable components eg D-amino acids, unnatural amino acids such as ⁇ -, ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamic acid, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ⁇ -N-methylarginine and other similar amino acids and imino acids (eg 4 -Hydroxyproline).
  • the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, consistent with standard usage and convention.
  • a “mutant" of a protein comprises an amino acid sequence in which one or more amino acid residues are inserted, deleted, and/or substituted in the amino acid sequence relative to another protein sequence. Mutants include fusion proteins.
  • identity refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecule sequences as determined by aligning and comparing the sequences. "Percent identity” means the percentage of identical residues between amino acids or nucleotides within the molecules being compared, calculated based on the smallest sized molecule being compared. For these calculations, gaps (if any) in the alignment are preferably resolved by a specific mathematical model or computer program (ie, an "algorithm").
  • Methods that can be used to calculate aligned nucleic acid or polypeptide identities include those described in: Computational Molecular Biology, (Lesk, AM ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith , edited by DW), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (edited by Griffin, AM and Griffin, HG), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (edited by Gribskov, M. and Devereux, J.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.
  • the sequences to be compared are generally aligned in such a way that the greatest match between the sequences is obtained.
  • An example of a computer program that can be used to determine percent identity is the GCG package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI).
  • GAP is used to align two polypeptides or polynucleotides for which percent sequence identity is to be determined. Sequences are aligned according to their respective amino acid or nucleotide best match (the "match span" is determined by an algorithm).
  • Gap opening penalty (calculated as 3x mean diagonal, where the "average diagonal” is the average of the diagonals of the comparison matrix used; the “diagonal” is the A matrix assigns each perfect amino acid match a score or value) and a gap extension penalty (which is usually 1/10 times the gap opening penalty) and a comparison matrix such as PAM 250 or BLOSUM 62.
  • the algorithm also uses standard comparison matrices (for PAM 250 comparison matrices, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5:345-352; for BLOSUM 62 comparison matrices, see Henikoff et al. , 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919).
  • Certain alignment designs used to align two amino acid sequences can result in only a short region of the two sequences matching, and such a small aligned region can have extremely high levels even if there is no significant relationship between the two full-length sequences. sequence identity.
  • the chosen alignment method (GAP program) can be adjusted if it is desired to generate an alignment spanning at least 50 or other numbers of contiguous amino acids of the target polypeptide.
  • derivative refers to a molecule that includes chemical modifications other than insertions, deletions or substitutions of amino acids (or nucleic acids).
  • derivatives include covalent modifications including, but not limited to, chemical bonding to polymers, lipids, or other organic or inorganic moieties.
  • the chemically modified antigen binding protein may have a longer circulating half-life than the chemically unmodified antigen binding protein.
  • chemically modified antigen binding proteins can improve the ability to target desired cells, tissues and/or organs.
  • the derivatized antigen-binding protein is covalently modified to comprise one or more water-soluble polymeric linkers including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol .
  • the derivatized antigen binding protein comprises one or more polymers including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymers poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols such as ethylene glycol, and polyvinyl alcohol and mixtures of said polymers.
  • the derivatives are covalently modified with polyethylene glycol (PEG) subunits.
  • PEG polyethylene glycol
  • one or more water-soluble polymeric bodies are bonded at one or more specific positions (e.g., at the amino terminus of the derivative).
  • one or more water-soluble polymers are randomly attached to one or more side chains of the derivative.
  • PEG is used to improve the therapeutic capacity of an antibody or antigen-binding fragment thereof.
  • PEG is used to improve the therapeutic capacity of humanized antibodies. Certain such methods are discussed, for example, in US Pat. No. 6,133,426, which is incorporated herein by reference for any purpose.
  • antibody refers to an intact immunoglobulin of any isotype or a fragment thereof that can compete with an intact antibody for specific binding to a target antigen, and includes, for example, chimeric antibodies, humanized antibodies, whole human antibodies, and bispecific antibodies .
  • An "antibody” is a class of antigen binding proteins.
  • An intact antibody will generally contain at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, eg, antibodies naturally occurring in camels may contain only heavy chains.
  • Antibodies may be derived from only a single source, or may be "chimeric," ie, different portions of the antibody may be derived from two different antibodies.
  • Antigen binding proteins, antibodies or binding fragments can be produced in hybridomas; by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, mutants and fragments thereof.
  • antibodies include monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, respectively , humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates”) and fragments thereof.
  • Naturally occurring antibody building blocks typically comprise tetramers.
  • Each tetramer typically consists of two identical pairs of polypeptide chains, each pair having a full-length "light chain” (in some embodiments, about 25 kDa) and a full-length "heavy chain” (in some In embodiments, about 50-70 kDa).
  • the amino-terminal portion of each chain typically contains a variable region of about 100-110 or more amino acids, which is typically responsible for antigen recognition.
  • the carboxy-terminal portion of each chain is generally defined as the constant region responsible for effector function.
  • Human light chains are generally divided into kappa and lambda light chains.
  • Heavy chains are generally classified as mu, delta, gamma, alpha, or epsilon chains, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgM has subclasses including, but not limited to, IgM1 and IgM2.
  • IgA is likewise subdivided into subclasses including, but not limited to, IgA1 and IgA2.
  • variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D” region of about 10 or more amino acids.
  • J Fundamental Immunology
  • the heavy chain also includes a "D” region of about 10 or more amino acids.
  • different antibody variable regions vary widely in amino acid sequence, even among antibodies of the same species.
  • Antibody variable regions generally determine the specificity of a particular antibody for its target.
  • Variable regions typically exhibit the same general structure of relatively conserved framework regions (FRs) linked by three hypervariable regions, also known as complementarity determining regions or CDRs.
  • the CDRs from each pair of two chains are typically positioned through the framework regions, which CDRs allow binding of specific epitopes.
  • Both light and heavy chain variable regions generally comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminal to C-terminal. Amino acid assignments to each domain are generally consistent with the following definitions: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk, J. Mol.
  • the CDR regions in heavy chains are commonly referred to as H1, H2, and H3, and are numbered sequentially in the direction from the amino-terminus to the carboxy-terminus.
  • the CDR regions in the light chain are commonly referred to as L1, L2, and L3, and are numbered sequentially in the direction from the amino-terminus to the carboxy-terminus.
  • light chain includes full-length light chains and fragments thereof having variable region sequences sufficient to confer binding specificity.
  • a full-length light chain comprises a variable region domain VL and a constant region domain CL .
  • the variable region domain of the light chain is located at the amino terminus of the polypeptide.
  • Light chains include kappa and lambda chains.
  • heavy chain includes full-length heavy chains and fragments thereof having variable region sequences sufficient to confer binding specificity.
  • the full-length heavy chain contains the variable region domain VH and three constant region domains CH1 , CH2 and CH3 .
  • the VH domain is at the amino terminus of the polypeptide, while the CH domain is at the carboxy terminus, and CH3 is closest to the carboxy terminus of the polypeptide.
  • Heavy chains can be of any isotype, including IgG (including IgGl, IgG2, IgG3, and IgG4 subtypes), IgA (including IgAl and IgA2 subtypes), IgM, and IgE.
  • antigen-binding fragment includes, but is not limited to, Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, diabodies, and single chain antibody molecules.
  • the "Fc" region comprises two heavy chain fragments containing antibody CH1 and CH2 domains.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains.
  • Fab fragments comprise the CH1 and variable regions of one light chain and one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a “Fab'fragment” comprises a light chain and part of a heavy chain containing the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, so there is a gap between the two Fab' fragments.
  • An interchain disulfide bond can form between the two heavy chains to form an F(ab') 2 molecule.
  • F(ab') 2 fragment contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains, thereby forming an interchain between the two heavy chains disulfide bonds.
  • the F(ab') 2 fragment therefore consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
  • Fv fragments comprise variable regions from both heavy and light chains, but lack constant regions.
  • Single chain antibodies are Fv molecules in which the heavy and light chain variable regions are joined together by elastic linkers to form a single polypeptide chain, thereby forming the antigen binding region.
  • Single chain antibodies are described in detail in International Patent Application Publication No. WO 88/01649 and US Patent Nos. 4,946,778 and 5,260,203, the contents of which are incorporated herein by reference.
  • Monoclonal antibody refers to a homogeneous population of antibodies having a single molecular composition. Monoclonal antibodies can be nonspecific or multispecific.
  • Domain antibodies are immunologically functional fragments of immunoglobulins that contain only heavy or light chain variable regions.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • a “multispecific antibody” is an antibody that targets more than one antigen or epitope.
  • a “bispecific”, “dual specific” or “bifunctional” antibody, respectively, is a hybrid antibody having two distinct antigen binding sites. The two binding sites of the bispecific antibody will bind two different epitopes, which may be located on the same or different protein targets.
  • An antibody is "selective" when it binds one target more tightly than it binds a second target.
  • a “recombinant antibody” is an antibody prepared by recombinant techniques. Methods and techniques for making recombinant antibodies are well known in the art.
  • Bispecific or diabodies are usually artificial hybrid antibodies with two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be prepared by a variety of methods including, but not limited to, fusing hybridomas or linking Fab' fragments. See, eg, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).
  • Each individual immunoglobulin chain is usually composed of several "immunoglobulin domains", each of which consists of approximately 90-110 amino acids and has a characteristic folding pattern. These domains are the basic units that make up antibody polypeptides.
  • the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains.
  • the heavy chain C region typically contains one or more domains that may be responsible for effector functions. The number of heavy chain constant region domains will depend on the isotype. For example, an IgG heavy chain contains three C -region domains called CH1, CH2 , and CH3 . Antibodies can be provided of any of these isotypes and subtypes.
  • neutralize refers to binding a ligand and preventing or reducing the biological effect of the ligand. This can be achieved, for example, by directly blocking the binding site on the ligand or by binding the ligand and altering the binding capacity of the ligand by indirect means (eg structural or energetic changes of the ligand).
  • the antibody or fragment can substantially inhibit binding of a ligand to its binding partner.
  • the neutralizing antibody reduces the binding of RBD to its receptor ACE2.
  • the neutralizing ability is characterized and/or described via a competition assay. In certain embodiments, neutralizing ability is described as a 50% inhibitory concentration ( IC50 ) value.
  • antibody-dependent enhancement (ADE) effect means that after certain virus-specific antibodies bind to the virus, the virus-bound antibody can bind to cells expressing FcR on the surface through its Fc region to mediate the entry of the virus into these cells, resulting in enhanced the infectious process of the virus.
  • ADE antibody-dependent enhancement
  • the immune system can produce a wide variety of specific antibodies against the virus particles and their components.
  • these antibodies only those directed against the surface S protein of the virus can bind to virus particles; while those directed against other components and components of the virus cannot bind to the virus particles, because these components are encapsulated inside the virus, and the antibodies Unable to get in touch with it.
  • These antibodies that bind to the S protein on the surface of the virus are various, and each has a different binding site on the S protein. Some of these antibodies have their binding sites coincident with the binding sites of cellular receptors on the S protein on the surface of the virus. Such antibodies not only bind to viral particles, but also compete with cell surface receptors for binding to viral particles.
  • antibodies can prevent the binding of viral surface proteins to cellular receptors, thereby inhibiting viral infection.
  • Such antibodies are called neutralizing antibodies.
  • most of the antibodies that can bind to virus particles but do not have in vitro neutralizing effect can also play a very important role in the human body through the Fc region of their constant region.
  • the Fc region of an antibody can bind to Fc receptors on effector cells, thereby recruiting various effector cells: recruit natural killer cells, which induce antibody-dependent cytotoxicity (ADCC); recruit complement molecules that bind to serum, which induce complement-dependent cells. Toxicity (CDC); recruitment of macrophages, triggering antibody-dependent cell-mediated phagocytosis (ADCP).
  • ADE effects complicate vaccine development.
  • There are four serotypes of dengue virus and after the human body is infected with a certain serotype of dengue virus, the body produces antibodies against this serotype with life-long protection (Guzman MG, 2007).
  • the life-threatening dengue hemorrhagic fever symptoms caused by dengue virus do not occur during the first infection (Screaton G, 2015), but rather after a second infection with a different serotype (30% difference in amino acid sequence). ⁇ 35%). This is because the concentration or affinity of antibodies against a certain serotype of dengue virus induced after the first infection is not sufficient to neutralize the virus of another serotype in the second infection, and instead promotes the enhancement of viral infection. This phenomenon is called the ADE effect.
  • the antibody that binds to the virus particle causes the virus particle to be phagocytosed by cells possessing Fc receptors or complement receptors through its Fc region, and instead promotes its infection of such cells.
  • viral particles enter the host cell through endocytosis, and the fusion process of the viral membrane and the host membrane is completed in the lysosome.
  • Antibody-dependent endocytosis instead promotes the phagocytosis of viral particles by host cells, aggravating viral infection. Therefore, whether an antibody inhibits or promotes viral infection is the result of the balance between its neutralizing potency and ADE effect; and the effect of serum on virus is the comprehensive performance of all antibody molecules (neutralizing potency and ADE effect) contained in it.
  • target refers to a molecule or portion of a molecule capable of being bound by an antibody or antigen-binding fragment thereof.
  • a target may have one or more epitopes.
  • the target is an antigen.
  • the use of "antigen” in the phrase “antigen-binding fragment” refers only to a protein sequence comprising an antigen that can be bound by an antibody. In this case, the protein is not necessarily exogenous or capable of inducing an immune response.
  • Compet when used in the context of competing for neutralizing antibodies of the same epitope, it means competition between antibodies, which is determined by an assay in which the antibody to be detected or its antigen
  • the binding fragment prevents or inhibits (eg reduces) specific binding of the reference antibody to a common antigen (eg, the S protein or a domain thereof).
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • sandwich competition Assays see, eg, Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, eg, Kirkland et al., 1986, J. Immunol.
  • solid-phase Direct labeling assay solid phase direct labeling sandwich assay (see eg Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling of RIA with I-125 label ( See, eg, Morel et al., 1988, Molec. Immunol. 25 :7-15); solid-phase direct biotin-avidin EIA (see, eg, Cheung, et al., 1990, Virology 176 :546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32 :77-82).
  • the assay involves the use of purified antigen bound to a solid surface or cell bearing either an unlabeled detection antibody and a labeled reference antibody.
  • Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the antibody being tested.
  • the antibody being tested is present in excess.
  • a competing antibody is present in excess, it will inhibit (eg decrease) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% % or 75% or more specific binding of the reference antibody to a common antigen.
  • binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • an antigen refers to a molecule or molecular portion capable of being bound by a selective binding agent such as an antibody or immunologically functional fragment thereof.
  • a selective binding agent such as an antibody or immunologically functional fragment thereof.
  • an antigen can be administered to an animal to generate antibodies capable of binding the antigen.
  • An antigen may possess one or more epitopes capable of interacting with different antibodies.
  • epitopes includes any determinant capable of being bound by an antibody.
  • Epitopes are regions of an antigen that are bound by antibodies targeting that antigen. When the antigen is a protein, it contains the specific amino acids that are in direct contact with the antibody. In most cases, epitopes are located on proteins, but in some cases, they can be located on other kinds of molecules, such as nucleic acids.
  • Epitopic determinants can include chemically active surface clusters of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and/or specific charge characteristics.
  • expanded clone also known as B cell expanded clone or expanded clone antibody
  • B cell expanded clone or expanded clone antibody refers to the germinal center of the lymph node after the initial B cell binds to the antigen. In the germinal center, B cells continue to expand with the help of T cells. The variable regions are further mutated, eventually resulting in very strong binding antibodies.
  • Antibodies produced by the same extended clone are generally considered to have great similarity in function and properties, such as similar CDR sequences, binding to the same epitope, and achieving the same biological effect.
  • the term "individually cloned” is also referred to as a single cloned antibody, and no antibody with very similar CDR sequences has been found in experiments. It is very likely that the limited experimental sample did not find the expanded clone antibody, so this antibody is referred to as a single clone.
  • therapeutically effective amount refers to the determined amount of an antibody or antigen-binding fragment thereof of the invention that produces a therapeutic response in a mammal.
  • the therapeutically effective amount is readily determined by one of ordinary skill in the art.
  • patient and “subject” are used interchangeably and include human and non-human animal subjects as well as subjects with a formally diagnosed disease, a subject with an unspecified disease, a subject receiving medical treatment, Subjects at risk of developing disease, etc.
  • treatment includes therapeutic treatment, prophylactic treatment, and use in reducing a subject's risk of developing a disease or other risk factor. Treatment does not require a complete cure of the disease, but includes embodiments in which symptoms are alleviated or underlying risk factors are alleviated.
  • prevention does not require 100% elimination of the likelihood of an event. More precisely, it means that the probability of an event occurring in the presence of the antibody or method is reduced.
  • Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection).
  • Enzymatic reactions and purification techniques can be performed according to manufacturer's instructions, or can be accomplished according to methods commonly used in the art or as described herein.
  • the foregoing techniques and methods can generally be practiced according to conventional methods well known in the art, as well as set forth in the various general and more specific references that are cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is hereby incorporated by reference for any purpose.
  • the present disclosure provides potent antibodies or antigen-binding fragments thereof (hereinafter collectively referred to as "antibodies”) capable of binding SARS-CoV-2 and neutralizing its activity, which antibodies do not produce ADE effects.
  • the present invention is based, at least in part, on the understanding that the binding of the SARS-CoV-2 RBD to its human receptor angiotensin-converting enzyme-2 (ACE2) is a critical initial step for viral entry into target cells, and that blocking this interaction with antibodies may be a Promising approaches for the treatment and prevention of SARS-CoV-2 infection.
  • the present inventors also identified a relationship between enhancing and neutralizing activity and antibody epitopes of the SARS-CoV-2 S protein domain.
  • the inventors unexpectedly discovered a series of antibodies capable of neutralizing SARS-CoV-2 without ADE effect, and in particular found a series of antibodies capable of cross-neutralizing SARS-CoV-2 and SARS-CoV-1 without ADE effect antibody.
  • the present inventors have also found that a series of SARS-CoV-2 mutant strains show retention of sensitivity to certain antibodies of the present invention.
  • the inventors have also found that ⁇ -coronavirus lineage B ( ⁇ -CoV-B), including SARS-CoV-2, SARS-CoV-2 mutants, SARS-CoV-1, and bat SARSr-CoV WIV1, can effectively neutralize antibody.
  • the antibodies described herein neutralize SARS-CoV-2 in vitro with a 50% inhibitory concentration ( IC50 ) value of less than 10 ⁇ g/ml. In some embodiments, the antibodies described herein neutralize SARS-CoV-2 in vitro with a 50% inhibitory concentration ( IC50 ) value of less than 0.1 ⁇ g/ml. In some embodiments, the antibodies described herein neutralize SARS-CoV-2 in vitro with a 50% inhibitory concentration ( IC50 ) value of less than 50 ng/ml. In some embodiments, the antibodies described herein neutralize SARS-CoV-2 in vitro with a 50% inhibitory concentration ( IC50 ) value of less than 25 ng/ml.
  • the antibodies described herein neutralize SARS-CoV-2 in vitro with a 50% inhibitory concentration (IC50 ) value of about 1-20 ng/ml.
  • IC50 50% inhibitory concentration
  • the IC50 values of the antibodies described herein were measured by luciferase activity as described in the cell-based in vitro neutralization assays in the Examples.
  • the antibodies described herein neutralize SARS-CoV-1 in vitro with a 50% inhibitory concentration ( IC50 ) value of less than 10 ⁇ g/ml. In some embodiments, the antibodies described herein neutralize SARS-CoV-1 in vitro with a 50% inhibitory concentration ( IC50 ) value of less than 0.1 ⁇ g/ml. In some embodiments, the antibodies described herein neutralize SARS-CoV-1 in vitro with a 50% inhibitory concentration ( IC50 ) value of less than 50 ng/ml. In some embodiments, the antibodies described herein neutralize SARS-CoV-1 in vitro with a 50% inhibitory concentration ( IC50 ) value of less than 25 ng/ml.
  • the antibodies described herein neutralize SARS-CoV-1 in vitro with a 50% inhibitory concentration ( IC50 ) value of less than 10 ng/ml. In some embodiments, the antibodies described herein neutralize SARS-CoV-1 in vitro with a 50% inhibitory concentration ( IC50 ) value of less than 6 ng/ml. In some embodiments, the antibodies described herein neutralize SARS-CoV-1 in vitro with a 50% inhibitory concentration ( IC50 ) value of about 1-6 ng/ml.
  • the IC50 values of the antibodies described herein were measured by luciferase activity as described in the cell-based in vitro neutralization assays in the Examples.
  • the antibodies described herein do not produce an ADE effect, as measured by the area under the ADE curve (AUC) and potentiation efficacy as described in the cell-based in vitro potentiation assays in the Examples.
  • AUC area under the ADE curve
  • the present invention relates to isolated antibodies or antigen-binding fragments thereof against SARS-CoV-2.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to the SARS-CoV-2 S protein.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds S-ECD.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds the S1 domain.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds the S2 domain.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to RBD.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2, the isolated antibody or antigen-binding fragment thereof comprising:
  • CDRHs heavy chain complementarity determining regions
  • CDRH1 selected from the following amino acid sequences: SEQ ID NOs: 1-28;
  • CDRH2 selected from the following amino acid sequences: SEQ ID NOs: 29-56;
  • CDRH3 selected from the following amino acid sequences: SEQ ID NOs: 57-84;
  • CDRLs one or more light chain complementarity determining regions
  • CDRL1 selected from the following amino acid sequences: SEQ ID NOs: 113-140;
  • CDRL2 selected from the following amino acid sequences: SEQ ID NOs: 141-168;
  • CDRL3 having a sequence of amino acids selected from the group consisting of: SEQ ID NOs: 169-196;
  • the isolated antibody or antigen-binding fragment thereof can effectively neutralize SARS-CoV-2 without ADE effect.
  • the isolated antibody or antigen-binding fragment thereof comprises at least one CDRH of A) and at least one CDRL of B). In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises at least two CDRHs of A) and at least two CDRLs of B). In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2, the isolated antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • the isolated antibody or antigen-binding fragment thereof can effectively neutralize SARS-CoV-2 without ADE effect.
  • the amino acid sequence of the heavy chain variable region (VH) has at least 85%, 90%, 95%, 96%, 97%, 98% of the amino acid sequence of any one of SEQ ID NOs: 85-112 %, 99% or 100% sequence identity.
  • the amino acid sequence of the light chain variable region (VL) has 85%, 90%, 95%, 96%, 97%, 98% of the amino acid sequence of any one of SEQ ID NOs: 197-224 , 99% or 100% sequence identity.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2, the isolated antibody or antigen-binding fragment thereof comprising:
  • One or more CDRHs selected from the following:
  • CDRH1 selected from the amino acid sequences of SEQ ID Nos: 7, 8, 11-21, 24-28;
  • CDRH2 selected from the amino acid sequences of SEQ ID NOs: 35, 36, 39-49, 52-56;
  • CDRLs selected from the following:
  • CDRL1 selected from the amino acid sequences of SEQ ID NOs: 119, 120, 123-133, 136-140;
  • CDRL2 selected from the amino acid sequences of SEQ ID NOs: 147, 148, 151-161, 164-168;
  • a CDRL3 selected from the amino acid sequences of SEQ ID NOs: 175, 176, 179-189, 192-196;
  • the antibody or antigen-binding fragment thereof specifically binds to the RBD domain of protein S, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without ADE effect.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2, the isolated antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • the antibody or antigen-binding fragment thereof specifically binds to the RBD domain of protein S, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without ADE effect.
  • the amino acid sequence of the heavy chain variable region (VH) is at least 85%, 90%, 95% identical to any one of SEQ ID NOs: 91, 92, 95-105, 108-112 , 96%, 97%, 98%, 99% or 100% sequence identity.
  • the amino acid sequence of the light chain variable region (VL) has 85%, 90%, 95%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2, the isolated antibody or antigen-binding fragment thereof comprising:
  • One or more CDRHs selected from the following:
  • CDRH1 selected from the amino acid sequences of SEQ ID NOs: 1-6, 22, 23;
  • CDRH2 selected from the amino acid sequences of SEQ ID NOs: 29-34, 50, 51;
  • CDRH3 selected from the amino acid sequences of SEQ ID NOs: 57-62, 78, 79;
  • CDRLs selected from the following:
  • CDRL1 selected from the amino acid sequences of SEQ ID NOs: 113-118, 134, 135;
  • CDRL2 selected from the amino acid sequences of SEQ ID NOs: 141-146, 162, 163;
  • CDRL3 selected from the amino acid sequences of SEQ ID NOs: 169-174, 190, 191;
  • the antibody or antigen-binding fragment thereof specifically binds to the S1 but not the RBD domain of the S protein, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without ADE effects.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2, the isolated antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • the antibody or antigen-binding fragment thereof specifically binds to the S1 but not the RBD domain of the S protein, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without ADE effects.
  • the amino acid sequence of the heavy chain variable region (VH) is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the amino acid sequence of the light chain variable region (VL) is 85%, 90%, 95%, 96%, 97% identical to any one of SEQ ID NOs: 197-202, 218, 219 %, 98%, 99% or 100% sequence identity.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2, the isolated antibody or antigen-binding fragment thereof comprising:
  • One or more CDRHs selected from the following:
  • CDRLs selected from the following:
  • CDRL1 selected from the amino acid sequences of SEQ ID NOs: 121, 122;
  • CDRL2 selected from the amino acid sequences of SEQ ID NOs: 149, 150;
  • CDRL3 selected from the amino acid sequences of SEQ ID NOs: 177, 178;
  • the antibody or antigen-binding fragment thereof specifically binds to the S2 domain of the S protein, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without an ADE effect.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2, the isolated antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • the antibody or antigen-binding fragment thereof specifically binds to the S2 domain of the S protein, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without an ADE effect.
  • the amino acid sequence of the heavy chain variable region (VH) has at least 85%, 90%, 95%, 96%, 97%, 98% of the amino acid sequence of any one of SEQ ID NOs: 93, 94 %, 99% or 100% sequence identity.
  • the amino acid sequence of the light chain variable region (VL) is 85%, 90%, 95%, 96%, 97%, 98% of any one of SEQ ID NOs: 205, 206 , 99% or 100% sequence identity.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof against SARS-CoV-2 selected from the antibodies shown in Table 2 below:
  • the isolated antibody or antigen-binding fragment thereof can effectively neutralize SARS-CoV-2 without ADE effect.
  • the isolated antibodies or antigen-binding fragments thereof of the invention also include the corresponding CDR sequences shown in Table 2 containing substitutions, deletions or insertions of no more than 2 amino acids at one or more places.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof against SARS-CoV-2 selected from the antibodies shown in Table 3 below:
  • the isolated antibody or antigen-binding fragment thereof can effectively neutralize SARS-CoV-2 without ADE effect.
  • the isolated antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) having at least 80% sequence identity to the amino acid sequence shown in Table 3; and/or having a The amino acid sequences shown in Table 3 are light chain variable regions (VL) with at least 80% sequence identity.
  • the isolated antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) having at least 90% sequence identity to the amino acid sequence shown in Table 3; and/or having a The amino acid sequences shown in Table 3 are light chain variable regions (VL) with at least 90% sequence identity.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2 selected from the antibodies shown in Table 4 below:
  • the antibody or antigen-binding fragment thereof specifically binds to the RBD domain of protein S, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without ADE effect.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof against SARS-CoV-2 selected from the antibodies shown in Table 5 below:
  • Antibody name VH SEQ ID NO VL SEQ ID NO 91 203 XG011 92 204 XG017 95 207 96 208 97 209 XG036 98 210 XG025 99 211 100 212 XG041 101 213 XG010 102 214 103 215 XG008 104 216 105 217 108 220 XG022 109 221 XG038 110 222 XG014 111 223 112 224
  • the antibody or antigen-binding fragment thereof specifically binds to the RBD domain of protein S, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without ADE effect.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof against SARS-CoV-2 selected from the antibodies shown in Table 6 below:
  • the antibody or antigen-binding fragment thereof specifically binds to the S1 but not the RBD domain of the S protein, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without ADE effects.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2 selected from the antibodies shown in Table 7 below:
  • the antibody or antigen-binding fragment thereof specifically binds to the S1 but not the RBD domain of the S protein, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without ADE effects.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof against SARS-CoV-2 selected from the antibodies shown in Table 8 below:
  • the antibody or antigen-binding fragment thereof specifically binds to the S2 domain of the S protein, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without an ADE effect.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2 selected from the antibodies shown in Table 9 below:
  • the antibody or antigen-binding fragment thereof specifically binds to the S2 domain of the S protein, wherein the isolated antibody or antigen-binding fragment thereof is capable of effectively neutralizing SARS-CoV-2 without an ADE effect.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2 selected from the antibodies shown in Table 10 below:
  • the isolated antibody or antigen-binding fragment thereof can effectively neutralize SARS-CoV-2 and its single amino acid mutant without ADE effect.
  • the single amino acid mutant strain of SARS-CoV-2 comprises V341I, F342L, V367F, R408I, A435S, G476S and V483A mutations in the RBD.
  • the isolated antibody or antigen-binding fragment thereof comprises CDRH1 of SEQ ID NO:27, CDRH2 of SEQ ID NO:55, CDRH3 of SEQ ID NO:83, CDRH3 of SEQ ID NO:139 Antibodies to CDRL1, CDRL2 of SEQ ID NO: 167, CDRL3 of SEQ ID NO: 195, or antigen-binding fragments thereof.
  • the isolated antibody or antigen-binding fragment thereof comprises CDRH1 of SEQ ID NO:28, CDRH2 of SEQ ID NO:56, CDRH3 of SEQ ID NO:84, CDRH3 of SEQ ID NO:140 CDRL1 of SEQ ID NO: 168, CDRL2 of SEQ ID NO: 168, antibodies to CDRL3 of SEQ ID NO: 196, or antigen-binding fragments thereof.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2 selected from the antibodies shown in Table 11 below:
  • the isolated antibody or antigen-binding fragment thereof can effectively neutralize SARS-CoV-2 and its single amino acid mutant without ADE effect.
  • the single amino acid mutant strain of SARS-CoV-2 comprises V341I, F342L, V367F, R408I, A435S, G476S and V483A mutations in the RBD.
  • the isolated antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof comprising the VH of SEQ ID NO: 111 and the VL of SEQ ID NO: 223.
  • the isolated antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof comprising the VH of SEQ ID NO: 112 and the VL of SEQ ID NO: 224.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof against SARS-CoV-2 selected from the group consisting of: CDRH1 comprising SEQ ID NO:27, SEQ ID NO:55 CDRH2, CDRH3 of SEQ ID NO:83, CDRL1 of SEQ ID NO:139, CDRL2 of SEQ ID NO:167, CDRL3 of SEQ ID NO:195 and an antibody or antigen-binding fragment thereof, and an antibody comprising SEQ ID NO:28
  • the isolated antibody or antigen-binding fragment thereof is capable of effectively cross-neutralizing SARS-CoV-2 and SARS-CoV-1 without ADE effect.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2 selected from the group consisting of: VH comprising SEQ ID NO: 111 and SEQ ID NO: 223 An antibody or antigen-binding fragment thereof to the VL of SEQ ID NO: 112, and an antibody or antigen-binding fragment thereof comprising the VH of SEQ ID NO: 112 and the VL of SEQ ID NO: 224, wherein the isolated antibody or antigen-binding fragment thereof can effectively cross and SARS-CoV-2 and SARS-CoV-1 without ADE effect.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof against SARS-CoV-2, the isolated antibody or antigen-binding fragment thereof comprising CDRH1 of SEQ ID NO:27, CDRH2 of SEQ ID NO:55, CDRH3 of SEQ ID NO:83, CDRL1 of SEQ ID NO:139, CDRL2 of SEQ ID NO:167, CDRL3 of SEQ ID NO:195, wherein the isolated antibody or antigen-binding fragment thereof is capable of cross-neutralizing SARS-CoV -2, SARS-CoV-2 mutant strains, SARS-CoV-1 and bat SARSr-CoV WIV1.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof directed against SARS-CoV-2, the isolated antibody or antigen-binding fragment thereof comprising the VH of SEQ ID NO: 111 and the VL of SEQ ID NO: 223, wherein the isolated antibody or antigen-binding fragment thereof is capable of cross-neutralizing SARS-CoV-2, SARS-CoV-2 mutant strains, SARS-CoV-1 and bat SARSr-CoV WIV1.
  • the SARS-CoV-2 mutant is selected from the group consisting of B.1.1.7, B.1.351, P.1, B.1.617.1 and B.1.617.2.
  • B.1.1.7 is the code for the British mutant (named Alpha mutant by the World Health Organization)
  • B.1.351 is the code for the South African mutant (named Beta mutant by the World Health Organization)
  • P.1 refers to Brazil
  • the code for the mutant strain named Gamma mutant by the World Health Organization
  • B.1.617.1 and B.1.617.2 are the codes for the Indian mutant (B.1.617.2 was named Delta mutant by the World Health Organization).
  • the bat SARSr-CoV WIV1 is highly similar to SARS-CoV-1, both derived from the Chinese horseshoe bat and can bind to the same human receptors. It has also been found that this new virus has been able to replicate rapidly in cultured human respiratory tissue, indicating that the virus can directly infect humans, and this transmissibility has exceeded expectations (Ge et al., 2013; Menachery et al., 2016 ).
  • the epitopes recognized by the antibodies of the present invention are as follows:
  • mutants may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions in VH and/or VL that do not adversely affect antibody properties.
  • sequence identity to a VH or VL amino acid sequence of the invention may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • Exemplary modifications are conservative amino acid substitutions, eg, in the antigen binding site or in framework, which do not adversely alter the properties of the antibody. Conservative substitutions can also be made to improve antibody properties, such as stability or affinity.
  • amino acids encoded by genes can be divided into four categories: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine); (3) non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polarities (glycine, aspartate amide, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes collectively classified as aromatic amino acids.
  • the amino acid repertoire can be grouped into: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine), (3) lipid family (glycine, alanine, valine, leucine, isoleucine, serine, threonine), wherein serine and threonine are optionally grouped separately as hydroxyl-containing aliphatic; (4) Aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur (cysteine and methionine) (Stryer (editor), Biochemistry, 2nd edition, WH Freeman and Co., 1981).
  • any native residue in the polypeptide can also be replaced with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al (1998) Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al (1998) Adv. Biophys. 35:1-24). Desired amino acid substitutions can be determined by those skilled in the art when such substitutions are desired. The resulting antibody mutants can be tested for characteristics using the assays described herein.
  • Amino acid substitutions can be performed, for example, by PCR mutagenesis (US Pat. No. 4,683,195).
  • libraries of mutants can be generated using known methods, such as using random (NNK) codons or non-random codons (eg DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp)) and then screen the library for mutants with the desired properties.
  • variable regions one from a heavy chain and one from a light chain
  • alternative embodiments may include a single heavy chain variable region or a single light chain variable region .
  • a single variable region can be used to screen for variable domains capable of forming two-domain-specific antigen-binding fragments. The screening can be accomplished by phage display screening methods using, for example, the hierarchical dual combinatorial method disclosed in International Patent Publication No. WO92/01047.
  • Antibodies of the invention can be prepared using a variety of techniques for producing antibodies.
  • monoclonal antibodies can be produced using the hybridoma method proposed by Kohler and Milstein in Nature 256:495, 1975.
  • a mouse or other host animal such as a hamster, rat or monkey
  • S protein or fragment thereof is immunized with the S protein or fragment thereof, after which splenocytes from the immunized animal are fused with myeloma cells using standard methods to form hybridomas Cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
  • Colonies produced by a single immortalized hybridoma cell are screened for production of antibodies with desired properties such as binding specificity, cross-reactivity or lack of binding specificity, lack of cross-reactivity, and affinity for antigen.
  • Various host animals can be used to prepare the antibodies of the present invention.
  • Balb/c mice can be used to make antibodies.
  • Various techniques can be used to humanize antibodies made in Balb/c mice and other non-human animals to generate more human-like sequences.
  • Exemplary humanization techniques involving selection of human acceptor frameworks are known to those skilled in the art and include CDR grafting (US Pat. No. 5,225,539), SDR grafting (US Pat. No. 6,818,749), resurfacing (Padlan, Mol. Immunol 28:489-499, 1991), specificity determining residue resurfacing (US Patent Publication No. 20100261620), human remodeling (or human framework remodeling) (U.S. Patent Publication No. US 2009/0118127), superhumanization (US Patent No. 7,709,226) and directed selection (Osbourn et al (2005) Methods 36:61-68, 2005; US Patent No. 5,565,332).
  • Humanization can be further optimized by introducing modified framework support residues to preserve binding affinity (backmutation) using disclosed techniques such as those described in International Patent Publication No. WO90/007861 and International Patent Publication No. WO92/22653 antibodies to improve their selectivity or affinity for the desired antigen.
  • Transgenic mice carrying human immunoglobulin (Ig) loci in the genome can be used to generate human antibodies against the protein of interest, as described, for example, in International Patent Publication No. WO90/04036, US Patent No. 6150584, International Patent Publication No. WO99/ 45962, International Patent Publication No. WO02/066630, International Patent Publication No. WO02/43478; Lonberg et al (1994) Nature 368:856-9; Green et al (1994) Nature Genet.
  • Ig immunoglobulin
  • the endogenous immunoglobulin locus in such mice can be disrupted or deleted, and at least one complete or partial human immunoglobulin can be combined by homologous or non-homologous recombination using transchromosomes or minigenes.
  • the protein locus was inserted into the mouse genome.
  • Human antibodies can be selected from phage display libraries in which phage are engineered to express human immunoglobulins or portions thereof, such as Fabs, single chain antibodies (scFvs), or unpaired or paired antibody variable regions (Knappike et al (2000) J. Mol. Biol. 296:57-86; Krebs et al (2001) J. Immunol. Meth. 254:67-84; Vaughanet al (1996) Nature Biotechnology 14:309-314; Sheets et al (1998) PITAS (USA) 95 : 6157-6162; Hoogenboom and Winter, (1991) J. Mol. Biol. 227:381; Marks et al (1991) J. Mol. Biol. 222:581).
  • Fabs single chain antibodies
  • scFvs single chain antibodies
  • Antibodies of the invention can be isolated, for example, from phage display libraries that express the heavy and light chain variable regions of antibodies as fusion proteins with the phage pIX coat protein, as described by Shi et al (2010) J . Mol. Biol. 397:385-96 and International Patent Publication No. WO09/085462.
  • the library can be screened for phage binding to the S protein, and the resulting positive clones can be further characterized, and Fabs isolated from clone lysates and expressed as full-length IgG.
  • phage display methods for isolating human antibodies are described, for example, in US Pat. Nos.
  • immunogenic antigens and production of monoclonal antibodies can be performed by any suitable technique, such as recombinant protein production.
  • the immunogenic antigen can be administered to the animal as a purified protein or protein mixture (including whole cells, cell extracts, or tissue extracts), or the antigen can be formed de novo in the animal from nucleic acid encoding the antigen or a portion thereof.
  • Another embodiment of the invention is an isolated polynucleotide encoding any of the antibody heavy chain variable regions and/or antibody light chain variable regions of the invention. Also within the scope of the invention are multiple polynucleotides encoding the same antibody of the invention in view of the degeneracy or codon preference of the genetic code in a given expression system.
  • the polynucleotide sequences encoding the VH or VL of the antibodies of the invention, or fragments thereof, may be operably linked to one or more regulatory elements, such as promoters or enhancers, that allow for expression of the nucleotide sequence in the intended host cell .
  • a polynucleotide can be a cDNA.
  • Another embodiment of the present invention is a vector comprising the polynucleotide of the present invention.
  • Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors or any other suitable for introducing the synthetic polynucleotides of the invention into a given organism or genetic background by any means a.
  • a polynucleotide optionally linked to a constant region and encoding the light chain variable region and/or heavy chain variable region of an antibody of the invention is inserted into an expression vector.
  • the light and/or heavy chains can be cloned into the same or different expression vectors.
  • DNA fragments encoding immunoglobulin chains can be operably linked to control sequences in one or more expression vectors that ensure expression of the immunoglobulin polypeptides.
  • control sequences include signal sequences, promoters (eg, naturally associated or heterologous promoters), enhancer elements, and transcription terminator sequences, which are selected to be consistent with those selected for expression.
  • the antibody is compatible with host cells. Once the vector is incorporated into an appropriate host, the vector maintains the host under conditions suitable for high-level expression of the protein encoded by the incorporated polynucleotide.
  • Suitable expression vectors are typically replicable in the host organism as episomal or as part of the host chromosomal DNA.
  • expression vectors typically contain a selectable marker, such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance, to allow detection of those cells transformed with the desired DNA sequence .
  • Suitable promoter and enhancer elements are known in the art.
  • exemplary promoters include lacl, lacZ, T3, T7, gpt, ⁇ P, and trc.
  • exemplary promoters include light and/or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus very early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in retroviral long terminal repeats; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art.
  • exemplary promoters are constitutive promoters, such as ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, etc.; or regulated promoters, such as GAL1 promoter, GAL10 promoter , ADH2 promoter, PH05 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter and AOX1 (eg, for Pichia). Selection of suitable vectors and promoters is within the ability of one of ordinary skill in the art.
  • Bacterial vectors pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, Uppsala, Sweden).
  • Eukaryotic vectors pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia).
  • host cell refers to a cell into which a vector has been introduced. It should be understood that the term “host cell” is intended to refer not only to a particular test cell, but also the progeny of such cells, but also to stable cell lines produced from a particular test cell. Certain modifications may occur in the progeny, due to mutation or due to environmental influences, and such progeny may therefore differ from the parental cell but are still encompassed within the scope of the term "host cell” as used herein.
  • host cells can be eukaryotic cells, prokaryotic cells, plant cells or archaeal cells.
  • Escherichia coli, bacilli (such as Bacillus subtilis) and other enterobacteriaceae (such as Salmonella, Serratia) and various pseudomonas Pseudomonas species are examples of prokaryotic host cells.
  • Other microorganisms such as yeast can also be used for expression. Saccharomyces (eg, S. cerevisiae) and Pichia are examples of suitable yeast host cells.
  • Exemplary eukaryotic cells can be derived from mammalian, insect, avian or other animal sources.
  • Mammalian eukaryotic cells include immortalized cell lines, such as hybridoma or myeloma cell lines, such as SP2/0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NSO (European Cell Culture Collection (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines.
  • An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196).
  • Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61) or DG44.
  • Another embodiment of the present invention is a method of producing an antibody of the present invention, the method comprising culturing a host cell of the present invention under conditions that allow expression of the antibody, and then recovering the antibody produced by the host cell.
  • Methods for preparing and purifying antibodies are well known in the art. Once all antibodies, dimers thereof, individual light and/or heavy chains, or other antibody fragments (such as VH and/or VL) have been synthesized (chemically or recombinantly), they can be purified according to standard procedures, Such standard procedures include ammonium sulfate precipitation, affinity chromatography columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, etc.
  • HPLC high performance liquid chromatography
  • Antibody can be substantially pure, for example, at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99% pure, or More pure, eg free of contaminants (such as cellular debris, macromolecules other than the test antibody, etc.).
  • Another embodiment of the present invention is a method for preparing an antibody of the present invention, the method comprising:
  • Antibodies are recovered from host cells or culture medium.
  • Polynucleotides encoding specific VH or VI sequences of the invention are incorporated into vectors using standard molecular biology methods. Host cell transformation, culturing, antibody expression and purification are accomplished using well-known methods.
  • the present invention provides pharmaceutical compositions comprising the antibodies described herein, together with a pharmaceutically acceptable carrier.
  • the antibodies of the present invention can be formulated into pharmaceutical compositions comprising an effective amount of the antibody as the active ingredient in a pharmaceutically acceptable carrier.
  • carrier refers to a diluent, adjuvant, excipient or vehicle with which the active compound is administered.
  • vehicles can be liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulates.
  • compositions can be sterilized by well-known conventional sterilization techniques (eg, filtration).
  • the compositions may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, stabilizers, thickening agents, lubricants and coloring agents, and the like, as required to approximate physiological conditions.
  • concentration of the antibodies of the invention in such pharmaceutical formulations can vary widely, i.e., from less than about 0.5%, usually to at least about 1% to as much as 15%, 20%, 25%, 30%, by weight. % by weight, 35% by weight, 40% by weight, 45% by weight, or 50% by weight, and will be selected according to the particular mode of application chosen, primarily based on the desired dosage, fluid volume, viscosity, and the like.
  • Suitable vehicles and formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Ed., Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092, see especially pp. 958-989.
  • the mode of administration of the antibodies of the invention in the methods of the invention described herein can be by any suitable route, such as parenteral administration, eg, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, mucosal (oral, intranasal, intravaginal, rectal) or other means known to the skilled artisan, which are well known in the art.
  • parenteral administration eg, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous
  • pulmonary mucosal (oral, intranasal, intravaginal, rectal) or other means known to the skilled artisan, which are well known in the art.
  • the antibodies in the methods of the invention described herein can be administered to a patient by any suitable route, eg, parenterally, intramuscularly, subcutaneously, or intraperitoneally by intravenous (i.v.) infusion or bolus injection.
  • Intravenous infusions may be administered, eg, over 15, 30, 60, 90, 120, 180, or 240 minutes, or over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours .
  • a therapeutically effective amount of an antibody of the invention is administered to a subject in the methods of the invention described herein.
  • a "therapeutically effective amount" of an antibody can be determined by standard research techniques. For example, in vitro assays can be employed to help identify optimal dosage ranges.
  • a therapeutically effective amount of an antibody of the invention can be determined by administering the antibody to relevant animal models well known in the art. Selection of a specific effective dose can be determined by one of skill in the art based on consideration of several factors (eg, via clinical trials). Such factors include the disease to be treated or prevented, the symptoms involved, the patient's weight, the patient's immune status, and other factors known to the skilled artisan.
  • Effective doses can be derived from dose response curves derived from in vitro or animal model test systems.
  • Antibodies of the invention can be tested for efficacy and effective doses using any of the models described herein.
  • Antibodies in the methods of the invention can be used in one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, Administration is repeated after three months, four months, five months, six months or more.
  • the course of treatment can also be repeated, as with chronic administration. Repeated administrations can be the same dose or different doses.
  • Antibodies in the methods of the invention may also be administered prophylactically to reduce the risk of infection in a subject with SARS-CoV-2/SARS-CoV-1, delaying the onset of SARS-CoV-2/SARS-CoV-1 infection , and/or reduce the risk of relapse of SARS-CoV-2/SARS-CoV-1 infection in remission.
  • Antibodies in the methods of the invention described herein can be stored lyophilized and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective for conventional protein formulations, and well-known lyophilization and reconstitution techniques can be employed.
  • Human embryonic kidney 293T (HEK293T) cells, human hepatoma Huh-7 cells, Calu-3 cells, Caco-2 cells and African green monkey kidney Vero-E6 cells (Xia et al., 2020a) were obtained from the American Type Culture Collection Center (ATCC), A549 cells stably expressing ACE2 were obtained from Dr. Yang Xuanming, School of Life Science and Biotechnology, Shanghai Jiaotong University. The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS heat-inactivated fetal bovine serum
  • Raji cells human Burkitt lymphoma B lymphoblasts obtained from ATCC (Jaume et al., 2011) were maintained in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. All cell lines were cultured at 37 °C in 5% CO .
  • Human embryonic kidney HEK293F suspension cells ATCC were cultured using HEK293 serum-free OPM-293-CD05 medium (OPM Biosciences) at 37°C in 5% CO 2 while shaking at 100 rpm.
  • the SARS-CoV-2 true virus nCoV-SH01 (GenBank: MT121215.1) used in this study was isolated from infected patients in the Biosafety Level 3 (BSL-3) laboratory of Shanghai Medical College, Fudan University (Wu, Y . et al., 2020a).
  • the SARS-CoV-2 virus propagates in Vero-E6 cells.
  • the concentrated viral stocks were aliquoted and stored in liquid nitrogen.
  • the pseudovirus mutant B.1.1.7 used in this study contains deletions of 69H, 70V and 144Y, and also contains N501Y, A570D, P681H, T716I, S982A and D1118H mutations.
  • the pseudovirus mutant B.1.351 contains L18F, D80A, D215G, R246I, K417N, E484K, N501Y, D614G and A701V point mutations and amino acid deletions at positions 242-244.
  • the pseudovirus mutant P.1 contains L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I and V1176F amino acid point mutations.
  • B.1.617.1 contains D111D, E154K, L452R, E484Q, D614G, P681R, Q1071H and H1101D point mutations.
  • B.1.617.2 contains T19R, G142D, EFR156-158G, L452R, T478K, D614G, P681R and D950N point mutations.
  • SPF Specific pathogen-free mice
  • E. coli Trans5 ⁇ (TransGen Biotech) was grown at 37°C with shaking at 230 rpm.
  • EK1 peptide (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 253) (Xia et al., 2020), synthesized by Kangbei Bio Co., Ltd. (Ningbo, China), dissolved in water and stored at -20°C until use.
  • Peripheral blood samples were collected from SARS-CoV-2 patients at Zhoushan Hospital in Zhejiangzhou. Serum samples were heat-inactivated at 56°C for 60 minutes, separated from clotted whole blood by centrifugation, and stored in aliquots at -80°C. After drawing 400 ml of blood from donor #16, human peripheral blood mononuclear cells (PBMCs) were isolated using a cell separation tube with a frit barrier. Isolated PBMCs were resuspended in 90% heat-inactivated FBS supplemented with 10% dimethyl sulfoxide (DMSO) and cryopreserved in liquid nitrogen.
  • DMSO dimethyl sulfoxide
  • BLI was performed on the Octet RED96 system (ForteBio) to analyze the binding kinetics of monoclonal antibodies against SARS-CoV-2 RBD (Kactus Biosystems, China). Measurements were performed using a streptavidin (SA) biosensor. Antigens and monoclonal antibodies were diluted with PBST (PBS, 0.02% TritonX-100) in black 96-well plates (Greiner Bio-One). To immobilize the antigen on the SA biosensor surface, Avi-labeled SARS-CoV-2 RBD was biotinylated using the BirA biotinylation kit (Avidity). Before each assay, the SA biosensor was pre-wetted in distilled water for at least 10 min.
  • PBST PBS, 0.02% TritonX-100
  • Serum or primary antibody was serially diluted 1:3 in PBS (maximum concentration, 1:10 for serum samples, 10 ⁇ g/ml for monoclonal antibody samples) for a total of 8 dilutions and added to incubate at room temperature 1 hour. Visualization was performed with HRP-conjugated goat anti-human IgG (Thermo Fisher Scientific) or HRP-conjugated mouse anti-human IgG Fab (GenScript). The area under the curve (AUC) was calculated for each antibody by using PRISM software analysis to evaluate antigen-binding capacity.
  • Pseudoviruses were generated as previously reported (Xia et al., 2020a). Briefly, plasmid pNL4-3.luc.RE (an HIV-1 backbone expressing a luciferase reporter) and pcDNA3.1-SARS-CoV-1-S were transfected using the transfection reagent VigoFect (Vigorous Biotechnology, Beijing). /pcDNA3.1-SARS-CoV-2-S (encoding the S-protein of SARS-CoV-1 or SARS-CoV-2) was co-transfected into HEK293T cells. The supernatant containing released pseudovirions was harvested 72 hours after transfection.
  • SARS-CoV-2 pseudovirus mutants were generated as described above, except that a plasmid of pcDNA3.1-SARS-CoV-2 with corresponding mutations in the S-protein was used. These plasmids were constructed by a site-directed mutagenesis kit (Yeasen Biotech, Shanghai) using the plasmid of pcDNA3.1-SARS-CoV-2-S as a template.
  • diluted antibody or serum samples were incubated with SARS-CoV-1 or SARS-CoV-2 pseudoviruses at 37 °C for 30 min before adding Huh-7 cells for infection.
  • different antigens RBD, S1, S2 or S-ECD proteins
  • RBD, S1, S2 or S-ECD proteins were incubated at different concentrations with 5 ⁇ g/ml of purified IgG from donor #16 for 1 hour at 37°C, then Incubation with SARS-CoV-2 pseudovirus. After half an hour of incubation, the mixture was finally added to Huh-7 cells for infection. After 12 h of incubation, the supernatant was replaced with fresh DMEM medium supplemented with 2% FBS.
  • Absolute luciferase values were measured and relative values were calculated by normalizing to virus-only control wells in the same lane. For example, the absolute luciferase value in pseudovirus-only control wells (considered the reference) was 5x104 , while adding a neutralizing serum sample reduced this to 1x104 . Therefore, the normalized luciferase value was calculated to be 100% in the pseudovirus only control and 20% for this neutralizing serum. Because many aspects, such as pseudovirus concentration, cultured cell concentration, cell state, immunofluorescence readout, etc., differ significantly between plates and assays, normalization is required to combine data obtained from different assays for comparison. For serum neutralization assays ( Figures 3F, 3G), the inverse of the serum dilution that resulted in 50% inhibition compared to pseudovirus alone was reported as the 50 % neutralization titer (NT50).
  • cells were fixed in 4% paraformaldehyde/PBS for 20 minutes, washed with PBS and permeabilized with 0.1% Triton X-100/PBS at room temperature. After blocking with 3% BSA, cells were incubated with anti-N polyclonal antibody (Gu et al., 2020) at a 1:1000 dilution overnight at 4°C and visualized with donkey anti-mouse IgG AlexaFluor488 (Thermo Fisher Scientific) . Nuclei were stained with DAPI. Cells were visualized using an Eclipse Ti-S inverted fluorescence microscope (Nikon).
  • the primers and probes used are as follows: SARS-CoV-2-NF (5'-GGG GAA CTT CTC CTG CTA GAT, SEQ ID NO: 226), SARS-CoV-2-NR (5'-CAGACA TTT TGC TCT CAA GCT G, SEQ ID NO: 227) and SARS-CoV-2-N-probe (5'-FAM-TTG CTG CTG CTT GAC AGA TT-TAMRA-3', SEQ ID NO: 228).
  • SARS-CoV-2-NF 5'-GGG GAA CTT CTC CTG CTA GAT, SEQ ID NO: 226)
  • SARS-CoV-2-NR 5'-CAGACA TTT TGC TCT CAA GCT G, SEQ ID NO: 227)
  • SARS-CoV-2-N-probe 5'-FAM-TTG CTG CTG CTT GAC AGA TT-TAMRA-3', SEQ ID NO: 228).
  • the procedure for quantitative reverse transcription PCR was performed using the Mastercycler ep realplex Real-time PCR System (Eppendorf) as follows: 95°C for 5 minutes; 40 cycles of 95°C for 10 seconds, 50°C for 30 seconds, and 72°C for 30 seconds.
  • Codon-optimized wild-type cDNA of the SARS-CoV-2 receptor-binding domain (RBD) (amino acids 333–530) together with the Avi-Tag tag (GLNDIFEAQKIEWHE, SEQ ID NO: 229) was synthesized (GENEWIZ) and cloned into the pACgp67 vector with a C-terminal 8 ⁇ His tag for purification.
  • SARS-CoV-2 RBD was expressed using the Bac-to-Bac baculovirus system.
  • the extracted bacmid DNA was then transfected into Sf9 cells using Cellfectin II Reagent (Invitrogen). Low titer virus is harvested and then amplified to generate high titer virus stocks.
  • the supernatant containing secreted unglycosylated RBD was harvested 72 h post infection and RBD protein was captured and purified by Ni-NTA resin (GE Healthcare). SDS-PAGE analysis revealed purified recombinant protein in excess of 95% purity.
  • RBD protein (GenScript) expressed and purified from recombinant baculovirus-infected insect Sf9 cells was chemically biotinylated using the EZ-Link TM Sulfo-NHS-LC-Biotin kit (Thermo Fisher Scientific) according to the manufacturer's instructions.
  • bait protein-PE and bait protein-APC were prepared by incubating 3 ⁇ g of biotinylated RBD or 25 ⁇ g of biotinylated S-ECD protein with streptavidin-PE (eBioscience) or streptavidin- Prepared by APC (BD Biosciences).
  • B cells Purification of B cells, dual fluorescent dye labeling of bait protein-conjugated B cells, and single-cell sorting experiments were performed as previously described (Escolano et al., 2019; Robbiani et al., 2017; Wang, Q. et al., 2020). Briefly, thawed and washed PBMCs with RPMI medium were incubated with CD19 microbeads (Miltenyi Biotec) for positive selection of B lymphocytes.
  • CD19 microbeads Miltenyi Biotec
  • Reverse primer for the first round of nested PCR R1-HC (5'-GGAAGG TGT GCA CGC CGC TGG TC, SEQ ID NO: 234).
  • Reverse primer for the second round of nested PCR R2-HC (5'-GTT CGG GGA AGT AGT CCT TGA C, SEQ ID NO: 235).
  • Primers for immunoglobulin kappa light chain Forward primer mix for 1st/2nd round of nested PCR: F1-Kappa(5'-ATG AGG STC CCY GCT CAG CTG CTG G, SEQ ID NO: 236)+ F2-Kappa(5'-CTC TTC CTC CTG CTA CTC TGG CTC CCA G, SEQ ID NO: 237)+F3-Kappa(5'-ATT TCT CTG TTG CTC TGGATC TCT G, SEQ ID NO: 238)+F4- Kappa (5'-ATG ACC CAG WCT CCA BYC WCC CTG, SEQ ID NO: 239).
  • Reverse primer for kappa light chain (first PCR): R1-Kappa (5'-GTT TCT CGT AGT CTG CTT TGC TCA, SEQ ID NO: 240).
  • Reverse primer for second PCR R2-Kappa (5'-GTG CTG TCC TTG CTG TCC TGC T, SEQ ID NO: 241).
  • Primers for immunoglobulin lambda light chain Forward primer mix for 1st/2nd round of nested PCR: Forward primer mix for lambda light chain: F1-Lambda(5'-GGT CCT GGG CCC AGT CTG TGC TG, SEQ ID NO: 242)+F2-Lambda(5'-GGT CCT GGG CCC AGT CTG CCC TG, SEQ ID NO: 243)+F3-Lambda(5'-GCT CTG TGA CCT CCT ATG AGC TG, SEQ ID NO:244)+F4-Lambda(5'-GGT CTC TCT CSC AGC YTG TGC TG, SEQ ID NO: 245)+F5-Lambda(5'-GTT CTT GGG CCA ATT TTA TGC TG, SEQ ID NO: 246) +F6-Lambda(5'-GGT CCA ATT CYC AGG CTG TGG TG,
  • Reverse primer for lambda light chain (first PCR): R1-Lambda (5'-CAC CAG TGT GGC CTT GTT GGC TTG, SEQ ID NO: 249).
  • Reverse primer for the second PCR R2-Lambda (5'-CTC CTC ACT CGA GGG YGG GAA CAG AGT G, SEQ ID NO: 250). PCR products amplified from each single cell were loaded onto a 2% agarose gel for electrophoresis and purified for Sanger sequencing.
  • Heatmaps and cluster dendrograms were generated using the Pretty Heatmaps (pheatmap and hclust) R package.
  • SARS-CoV-2 or SARS-CoV-1 S protein-mediated cell fusion assays were performed as previously described (Liu et al., 2020b; Xia et al., 2020). Briefly, the pAAV-IRES-EGFP-SARS-CoV-2-S or pAAV-IRES-EGFP-SARS-CoV-1-S plasmids were transfected into HEK-293T cells using the transfection reagent VigoFect.
  • HEK-293T cells overexpressing the S protein of SARS-CoV-2 or SARS-CoV-1 were incubated with different concentrations (1:4 serial dilutions) of XG014 (maximum concentration 30 ⁇ g/ml) for 30 min, followed by addition to inoculated Huh -7 or Caco-2 or Calu-3 cells.
  • SARS-CoV-1S-mediated cell fusion occurred only in the presence of trypsin (80 ng/mL), which was not required for SARS-CoV-2S-mediated cell fusion.
  • Cells were treated with 4% paraformaldehyde after 5 hours of incubation, and the number of confluent cells was counted using a fluorescence microscope (Nikon Eclipse Ti-S) in 5 randomly selected fields of view.
  • Raji cells were used to assay for enhanced cell membrane fusion by antibodies (XG005, XG014, XG016 or XG005-GRLR). Raji cells were seeded into 96-well plates treated with 0.01% poly-L-lysine. Serial dilutions (1:4) of antibody (maximum concentration 10 ⁇ g/ml) were mixed with HEK-293T cells overexpressing SARS-CoV-2 S protein and incubated at 37°C for 30 minutes. The mixture was applied to Raji cells and incubated for an additional 24 hours. Cells were fixed and confluent cell counts were performed under a fluorescence microscope in 5 randomly selected fields of view.
  • Antibody-mediated EK1 inhibition of cell fusion was performed as previously described (Wu et al., 2020a). Briefly, 5 ⁇ g of antibody and various concentrations of EK1 were incubated with S-overexpressing HEK-293T cells for 30 min and then added to Raji cells.
  • Vero-E6 cells were seeded into 96-well plates. After 24 hours, XG011 (80 ⁇ g/ml), XG014 (0.16 ⁇ g/ml), XG017 (3.2 ⁇ g/ml) or XG025 (16 ⁇ g/ml) antibodies were serially diluted 1:4 in DMEM medium for a total of 6 dilution and incubated with SARS-CoV-2 true virus for 30 minutes. The mixture was then applied to Vero-E6 cells and incubated for a further 2 hours. Subsequently, 1% carboxymethylcellulose (Sigma, USA) was added, followed by incubation for another 72 hours. Finally, PBS containing 4% paraformaldehyde and 1% crystal violet was added for fixation and staining. After rinsing with water, plaque numbers were counted and percent plaque reduction normalized using PBS-treated samples.
  • SARS-CoV-2 S ectodomain a gene encoding the SARS-CoV-2 S ectodomain (residues 1-1208, GenBank: MN908947), which has a furin cleavage site (residues 1-1208), was synthesized "GSAS" substitutions at 682-285) and mutation of the S2 domain ("PP" substitutions at residues 986-987) and addition of a T4 fibritin trimerization motif and a twin-strep-II tag at the C-terminus, and insertion into The mammalian expression vector pCAGGS was used for expression, as previously described (Wrapp et al., 2020).
  • Plasmids were transiently transfected into HEK293F cells using polyethyleneimine. The supernatant was harvested after 72 hours and purified by affinity chromatography. The S protein was further purified by using a Superose 6 Increase 10/300 column (GE Healthcare) in 20 mM Tris pH 8.0 and 200 mM NaCl.
  • cryo-grid preparation For cryo-grid preparation, purified S protein and XG014 Fab were mixed in a 1:1.5 molar ratio and incubated on ice for 10 s to obtain cryo-EM samples at a total concentration of 0.7 mg/ml. Then, a 3.0 ⁇ l aliquot of the sample was placed on a fresh glow-discharged 300 mesh perforated carbon-coated gold grid (C-flat, 1.2/1.3, Protochips Inc.), which was wiped through filter paper for 7 seconds, and inserted into liquid ethane using a Vitrobot (FEI). Afterwards, the grids were transferred to liquid nitrogen for storage.
  • C-flat, 1.2/1.3, Protochips Inc. a fresh glow-discharged 300 mesh perforated carbon-coated gold grid
  • the S trimer was mixed with XG005Fab at a molar ratio of 1:1.7 (S monomer:Fab), incubated at 4°C for 1 h, and further passed through Superose6Increase 10/300 Column (GE Healthcare) purification. Peak tubes were concentrated to 0.4 mg/mL in 20 mM Tris pH 8.0 and 200 mM NaCl.
  • Cryo-EM data were collected on a Titan Krios microscope (Thermo Fisher) operating at 300 kV, equipped with a K2 peak direct detector (Gatan) and a GIF quantum energy filter (Gatan) set to a slit width of 20 eV.
  • Particles are initially automatically picked using the Laplacian-of-Gaussian method, followed by two-dimensional (2D) classification. The top average was used as a 2D reference for template picking, yielding 286,984 particles. Extract 2 ⁇ 2 binned particles ( pixels) and perform the conventional 2D classification, 3D initial model, 3D classification and 3D self-refinement process, followed by subsequent re-concentration and re-extraction binning 1 ⁇ 1 ( pixels). Finally, 109,304 particles were classified and subjected to self-refinement, CTF refinement and Bayesian refinement, resulting in Density map for overall resolution.
  • Post-processing was also performed with masking of only the RBD domain and antibody VH-VL variable domains to obtain a binding interface.
  • Overall resolution density map Overall, 32281 NRAb2 particles were selected and then subtracted for focus refinement, yielding Overall resolution density map. 23,948 NRAb3 particles were selected and then subtracted for focus refinement, yielding Overall resolution density map.
  • the focused refinement map of NRAb was fitted to and merged with the XG005 trimer map using the "vop maximum" recommended in UCSF Chimera (Pettersen et al., 2004).
  • the SARS-CoV-2 S trimer (PDB ID: 6VSB), Fab 38-3-11A heavy chain (PDB ID: 6Z3P) and The structure of the Fab NA-45 light chain (PDB ID: 6PZE) was manually fitted to the final map using Chimera (Pettersen et al., 2004) and further manually corrected by real-space refinement in COOT (Emsley et al., 2010) .
  • the atomic model was further refined by position and B-factor refinement in real space using Phenix (Afonine et al., 2018).
  • the SARS-CoV-2 S protein with 2 upward RBDs was extracted from the atomic model (PDB ID: 7A29).
  • the initial model of the XG005Fab was generated from the homologous structures of VH and VL (PDB ID: 5K8A and 6KTR) using the SWISS-MODEL website (Waterhouse et al., 2018).
  • Initial models of S and antibodies were fitted to the synthetic map of the SARS-CoV-2 S trimer-XG005 complex using Chimera (Pettersen et al., 2004) and then manually adjusted with COOT (Emsley et al., 2010).
  • Several rounds of repeated real-space refinement are further performed in PHENIX (Afonine et al., 2018).
  • mice Eighteen SPF human ACE2 transgenic (hACE2-Tg) mice were randomly assigned to three groups of 6 mice each, which were defined as PBS control, prevention and treatment groups, respectively.
  • a single intraperitoneal injection of 20 mg/kg (approximately 0.5 mg) of XG014 antibody was performed 4 hours before and 2 hours after SARS-CoV-2 challenge, respectively.
  • an equal volume of PBS was administered for the control group. All mice were infected intranasally with 0.5 x 105 PFU SARS-CoV-2 true virus. Mice body weights were monitored for 5 consecutive days. Lungs and intestines were collected 4 days after infection.
  • RT-qPCR was used to quantify the mRNA levels of the SARS-CoV-2 virus.
  • the isolated RNA was reverse transcribed using the First Strand cDNA Synthesis Kit (Takara, Japan) and amplified by real-time PCR using the SYBR Green PCR Kit (Takara, Japan).
  • the gene for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control to measure cytokine levels. Collected lungs were fixed in 4% paraformaldehyde and stained with hematoxylin and eosin (H&E).
  • donor sera from post-infection recovery showed significantly higher ELISA binding potency to each of the SARS-CoV-2 S-protein domains (RBD, S1, S2, and S-ECD) (FIGS. 3A-3D; FIG. 1).
  • the N-protein in the virion also elicited robust antibody responses after infection (Figure 3E).
  • luciferase signal (a surrogate for infection) was then compared at different dilutions of serum or purified IgG antibodies ( Figures 3F-3H). Although 5 individuals (donors #5, #6, #7, #8, and #16) achieved half-maximal neutralizing titers (NT50) above 2000 for serum samples (Fig. 3F), only 1 The individual (donor #16) showed more pronounced neutralization of the purified IgG fraction ( NT50 : 1.1 ⁇ g/ml; Figure 3H). This difference may be attributable to the neutralizing activity of IgM or IgA antibodies in those convalescent individuals.
  • Serum neutralizing activity against the SARS-CoV-1 pseudovirus was significantly lower in convalescent individuals, however slightly higher than in uninfected donors (Fig. 3G).
  • Fig. 3G Serum neutralizing activity against the SARS-CoV-1 pseudovirus was significantly lower in convalescent individuals, however slightly higher than in uninfected donors (Fig. 3G).
  • SARS-CoV-2 RBD- or S-ECD-binding B cells were identified using a dual fluorescent dye labeling strategy (Fig. 4) (Wang, Q. et al., 2020). Unexposed uninfected controls showed background levels of bait protein-specific B cells, while donor #16 with high serum neutralizing activity showed a different population of bait protein-binding B cells.
  • Gated double positive cells (bait protein-PE + and bait protein-APC + ) were single-cell sorted and immunoglobulin heavy chain (IGH; IgG isotype) and light chain (IGL or IGK) genes were nested PCR reactions were amplified from sorted single cells (Robbiani et al., 2017; Scheid et al., 2009b; Wang, Q. et al., 2020). Overall, 292 paired heavy and light chain variable regions were obtained from RBD-binding and S-ECD-binding IgG + memory B cells ( Figure 6).
  • RBD-binding antibodies bind overlapping or non-overlapping epitopes
  • a competitive ELISA was performed. Antibodies with weak levels of ELISA binding (XG015, XG042, XG045, XG047) were excluded.
  • the coated RBD protein was first pre-incubated with a non-biotinylated primary antibody, followed by a biotinylated secondary antibody. As expected, all tested antibodies competed with themselves, while the control antibody anti-HBs H004 (Wang, Q. et al., 2020) failed to block any secondary antibody recognizing RBD.
  • a neutralization assay was performed using a luciferase-expressing SARS-CoV-2 pseudovirus to infect Huh-7 cells (Xia et al., 2020a; Xia et al. , 2020b) and calculated their 50% inhibitory concentrations ( IC50 ) ( Figures 11A and 11B).
  • the neutralizing activity of these antibodies varied significantly, ranging from potent neutralizing antibodies to almost no neutralizing activity.
  • the most potent antibodies showed IC50 values of 6-15 ng/ml, of which 4 were RBD-bound, XG005 ( IC50 : 6.1 ng/ml), XG014 ( IC50 : 14.4 ng/ml), XG016 ( IC50 : 9.1 ng/ml) and XG038 ( IC50 : 12.7ng/ml), and 1 of which is S1-bound, but not RBD-bound, XG027 ( IC50 : 15.7ng/ml) ( Figure 11A and 11B) .
  • Example 5 Against naturally occurring SARS-CoV-2 RBD mutants and SARS-CoV-1 cross-neutralizing activity
  • SARS-CoV-2 viruses mutate slowly, and several SARS-CoV-2 strains with only 1 amino acid mutation in the RBD region have been reported, such as V341I, F342L, V367F, R408I, A435S, G476S, and V483A (Ou et al., 2020).
  • SARS-CoV-2 strains with only 1 amino acid mutation in the RBD region have been reported, such as V341I, F342L, V367F, R408I, A435S, G476S, and V483A (Ou et al., 2020).
  • the 2 most potent monoclonal antibodies, XG014 and XG038, were singled out for use against several SARS-CoV-2s harboring these reported RBD mutations Pseudoviruses were subjected to neutralization assays ( Figures 13A and 13B).
  • Antibody XG014 remained sensitive to all SARS-CoV-2 mutants tested ( Figure 13A), while antibody XG038 showed reduced neutralization against 2 of the 7 virus mutants (G476S and V483A; Figure 13A) 13B). Therefore, a single amino acid mutation in the RBD domain can confer SARS-CoV-2 resistance to human monoclonal antibodies.
  • XG006 enhanced viral infection of Raji cells only at high concentrations, but lost the enhancement effect when diluted, while XG005 induced viral infection at all dilutions tested (Figure 15A; Figure 16A).
  • the area under the ADE curve (AUC) and enhancement efficacy were calculated as previously reported (Bardina et al., 2017; Robbiani et al., 2019) ( Figure 15C; Figures 16B and 16C), and a positive correlation was observed between these 2 values correlation (FIG. 16D).
  • FIG. 16E and 16F there was no significant correlation between the calculated IC50 values for neutralizing antibodies and the corresponding ADE AUC and potentiation efficacy values
  • Cluster X antibodies are characterized by virus neutralizing activity and ADE effect.
  • Cluster Y antibodies showed potent neutralizing activity, but low levels of ADE effect, whereas antibodies from Cluster Z were non-neutralizing and did not induce viral entry, ie, no ADE effect ( Figure 17A).
  • Further comparison using ADE AUC and neutralizing IC50 confirmed that cluster X antibody induced more antibody-dependent viral entry in Raji cells (Fig. 17B), and that antibodies from clusters X and Y were more potent neutralizing antibodies (Fig. 17C) ).
  • RBD epitopes were only associated with neutralizing activity, not ADE effects.
  • Antibodies in cluster Y had neutralizing activity but did not induce or induced low levels of viral entry in Raji cells ( Figures 17A and 17D). These antibodies are directed against many different epitopes, including RBD group II and RBD group III antibody binding epitopes. These types of antibodies were not found in other clusters, i.e. those recognizing only group II epitopes or only group III epitopes; rather, they were found only in cluster Y ( Figures 17A and 17D), indicating antibodies directed against these epitopes Is closely related to neutralizing activity and does not induce potentiating effects.
  • a number of human monoclonal antibodies have been identified from convalescent individuals with SARS-CoV-2 or SARS-CoV-1 and evaluated for their potential passive antibody administration for prevention and treatment (Andreano et al., 2020; Brouwer et al., 2020 ; Cao et al, 2020; Chen et al, 2020; Chi et al, 2020; Ju et al, 2020; Kreer et al, 2020; Liu, L. et al, 2020; Pinto et al, 2020; 2020; Rogers et al, 2020; Seydoux et al, 2020; Wan et al, 2020; Wec et al, 2020; Wu, Y.
  • IGHV usage of anti-SARS-CoV-2 antibodies in this study and others was very different, and a recent study reported that IGHV3-53 was the IGHV gene most frequently used to target RBD (Yuan et al., 2020). However, in donor #16 of the present study, multiple lineages from IGHV1 to IGHV7 were identified, but only 1 IGHV3-53 antibody, suggesting that IGHV usage varies significantly among individuals.
  • SARS-CoV-1 belongs to the same subfamily as SARS-CoV-2 and shares the same human receptor, ACE2, it is likely that the immune system accidentally produces antibodies with partial cross-reactivity.
  • ACE2 human receptor 2
  • H014 a phage antibody isolated from RBD-immunized mice, showed IC50s of 150 ng/ml and 450 ng/ml for SARS-CoV-1 and -2, respectively (Lv et al., 2020).
  • antibodies isolated from SARS-CoV-1 infected individuals have been shown to cross-neutralize against SARS-CoV-2; for example, antibody ADI-55688 (4 ng/ml and >100 ng for SARS-CoV-1 and -2, respectively) IC50 /ml), ADI-56046 ( IC50 of 20ng/ml and 50ng/ml for SARS-CoV-1 and -2, respectively) and S309 (120ng/ml for SARS-CoV-1 and -2, respectively) and IC50 of 79ng/ml).
  • SARS-CoV-2 mutates slowly, so monitoring individual amino acid changes and understanding their potential phenotypic relevance is critical (Korber et al., 2020). Point mutations in MERS-CoV and SARS-CoV-1 have been shown to confer resistance to naturally occurring neutralizing antibodies (Sui et al., 2008; Tang et al., 2014; terMeulen et al., 2006). In the present study, 2 reported virus mutants, V483A and G476S, showed potent neutralizing potency against one monoclonal antibody, XG038 , with IC50s 20 or 80 times higher, respectively, than wild-type strains ( Figure 13B) .
  • SARS-CoV-2 patients with severe disease often have robust IgG antibody responses. This association between higher IgG titers and worse outcomes suggests an ADE effect of SARS-CoV-2 (Cao, 2020).
  • the ADE effect of SARS-CoV-2 antibodies was successfully identified and evaluated by utilizing a Raji cell-based model for studying antibody-dependent viral entry.
  • Example 8 Efficient and broad-spectrum neutralizing activity of XG014
  • the inventors have identified monoclonal antibodies that recognize four sets of non-overlapping RBD epitopes. Due to their significant neutralizing activity against SARS-CoV-2, four representative groups of neutralizing antibodies (XG011, XG014, XG017, XG025) from each epitope group ( Figure 21A) were selected for further study.
  • the antibody XG017 has normal binding ability, but the antibody XG017 cannot bind the S-ECD of B.1.351 and P.1 and the RBD of B.1.351, which may be attributed to Mutations contained in the RBD region (FIG. 22A).
  • XG014 showed broad and potent medium against all four mutants (B.1.1.7 British Alpha mutant, B.1.351 South African Beta mutant, P.1 Brazilian Gamma mutant and B.1.617.2 Indian Delta mutant) and activity (Fig. 22B, Fig. 24), while XG017 was unable to neutralize pseudoviruses of SARS-CoV-2 mutant strains B.1.351 and P.1 (Fig. 22B), consistent with the ELISA binding data (Fig. 22B). Neutralizing activity was further tested against pseudoviruses carrying single or double mutations (see Methods).
  • XG014 The lungs and intestines are the major organs for SARS-CoV-2 infection (Lamers et al., 2020).
  • in vitro neutralization assays were performed on the human lung cell line Calu-3 and the human intestinal cell line Caco-2 using true and pseudoviruses, respectively. Consistent with the eukaryotic experiments in Vero-E6 cells (Fig. 21C), XG014 was able to effectively inhibit SARS-CoV-2 eukaryotic infection in Calu-3 and Caco-2 cells with IC50 determined in both cell lines The values were all 0.007 ⁇ g/ml (FIG. 22D).
  • XG014 exhibits particularly potent and broad in vitro neutralizing activity against ⁇ -CoV-B, including SARS-CoV-2, its various mutants (including Alpha, Beta, Gamma, and Delta mutants), SARS-CoV-1 and bat SARSr-CoV WIV1.
  • cryo-EM cryo-electron microscopy
  • the interaction between the heavy chain and the RBD is mainly attributed to a large number of hydrophobic interactions.
  • the 16-residue-long hydrophobic complementarity determining region CDR-H3 (HQYGYNYGYFYYYIDV) was inserted into a hydrophobic cavity formed by RBD residues F338, F342, V367, L368, F374, W436, L441 and glycans linked to N343 residues (Fig. 25C).
  • hydrophilic interactions further enhanced XG014 binding by forming hydrogen bonds between CDR-H2 and both T345 and R346, and between CDR-H3 and residues 439-441.
  • residues N32 on CDR-L1 of the light chain and residues T94 on CRD-L3 also participate in the interaction by forming two hydrogen bonds with N440 (Fig. 25D), the latter in SARS-CoV-2, SARS- Strictly conserved among CoV-1 and SARSr-CoV WIV1 ( Figure 26).
  • a striking feature of XG014 is that it also contacts adjacent RBDs through its light chain and buries additional surface area (Figure 25B). This contact is mainly through van der Waals interactions, without specific hydrogen bonds or hydrophobic interactions. Furthermore, structural modeling suggested that the major epitope of XG014 did not overlap with the ACE2 binding site ( Figure 25E).
  • the structural data are also fully consistent with the cross-neutralizing activity of XG014 against ⁇ -CoV-B, including SARS-CoV-2 mutant strains, SARS-CoV-1 and bat SARSr-CoV WIV1, as most key contact residues are conserved (Fig. 25F; Fig. 26).
  • Residues 417, 484 and 501 identified as critical mutation sites in recently circulating SARS-CoV-2 mutant strains (Grubaugh et al., 2021; Supasa et al., 2021; Xie et al., 2021), did not participate in the Interaction of XG014. Therefore, XG014 retains its neutralizing activity against these recent SARS-CoV-2 mutants.
  • XG014 has a unique antigen-binding epitope
  • the S2M11 Nanobody and C144Fab span two adjacent RBDs and thus rely on quaternary interactions ( Figures 27C and 27D).
  • both S2M11 and C144 inserted their CRD-H3 into the hydrophobic gap of the adjacent RBD.
  • the gap is formed by residues 338-342, 367-368 and 436, which overlap with the XG014 epitope.
  • N343 is also specifically involved in S2M11 and C144 binding, which may also partly explain their closed S trimer interaction.
  • Monoclonal antibody S309 is a well-researched new coronavirus S protein antibody. It can bind to the S trimer protein with three RBDs in the "down" closed state and the S trimer with only one RBD "up”. protein (Pinto et al., 2020).
  • XG005, XG016 and XG014 also recognize the S-ECD and RBD proteins of SARS-CoV-2 mutant strains B.1.1.7, B.1.351 and P.1, only XG014 binds the S-ECD of SARS-CoV-1 ( Figure 28B).
  • XG005 and XG016 although highly effective at neutralizing SARS-CoV-2 pseudovirus and its mutant strains ( Figures 28C and 28D), did not neutralize SARS-CoV-1 or SARSr-CoV WIV1 pseudoviruses ( Figures 28E and 28E and 28F). This indicated that the levels of broad-spectrum neutralizing activity of XG005 and XG016 were significantly reduced compared to XG014.
  • FIG. 28G To evaluate the possible impact of antibody-dependent S protein-mediated membrane fusion, ACE2-negative but SARS-CoV-2 S protein-overexpressing HEK-293T cells and Fc ⁇ receptor II (Fc ⁇ RII) + /ACE2 - Raji cells ( a human B lymphoblastoid line), a cell fusion assay was further established (FIG. 28G). Both XG005 and XG016 induced membrane fusion in a dose-dependent manner between these two cell lines ( Figures 28H and 28I). In contrast, XG014 had no effect on membrane fusion, similar to the PBS-treated control group ( Figures 28H and 28I).
  • EK1 a peptide targeting the HR1 domain of the S protein, was then used to block six-helix bundle formation, thus inhibiting SARS-CoV-2 S protein-mediated membrane fusion (Xia et al., 2020).
  • the presence of EK1 completely blocked S protein-mediated membrane fusion in a dose-dependent manner, highlighting a critical role during S protein-mediated membrane fusion ( Figures 29F and 29G).
  • a competitive ELISA using recombinant ACE2 protein showed that XG005 and XG016, but not XG014, blocked the interaction of the SARS-CoV-2 RBD with the cellular receptor ACE2, suggesting overlapping but not identical among the three antibodies Antigen binding mode (FIG. 28A).
  • cryo-EM structure of the SARS-CoV-2 S trimer complexed with XG005Fab was determined, and the overall Resolution (Figure 30A). To improve the resolution of these regions, each RBD/XG005Fab interface region was locally refined to the resolution separately (Fig. 20). Similar to XG014, XG005 binds all three RBDs. Unlike the XG014 structure, however, most XG005-bound S particles displayed two "up" RBD and one "down” RBD conformations (Figure 30A).
  • the RBD epitope of XG005 shares common residues with the RBD epitope of XG014, mainly located in the monocyclic region (L437-450) and overlapping with the ACE2 binding site ( Figure 30D and 30E). Both XG005 and XG014 interact with this region through hydrophilic interactions, including hydrogen bonds and salt bridges. Additionally, XG014 and XG005 each have its own unique epitope, with XG014 targeting a particularly conserved hydrophobic gap away from the RBM, while XG005 directly targeting the less conserved ACE2 binding residues ( Figures 30E and 30F). These indicate the different neutralization mechanisms and characteristics of XG014 and XG005.
  • XG014 could be developed as a preventive or therapeutic antibody against SARS-CoV-2 infection
  • human ACE2 transgenic (hACE2-Tg) mice were used and injected intraperitoneally before or after SARS-CoV-2 true virus challenge Antibody (FIG. 31A). Mild or no significant weight loss was observed for both prevention and treatment groups ( Figure 31B). In contrast, mice in the control group suffered severe weight loss (FIG. 31B).
  • SARS-CoV-2 is a positive-strand RNA virus that is inherently prone to mutation (Zhou et al., 2021a).
  • SARS-CoV-2 mutants B.1.1.7 British Alpha mutant, B.1.351 South African Beta mutant, P.1 Brazilian Gamma mutant, B.1.617.2 Indian Delta mutant
  • Wang et al., 2021b; Zhou et al., 2021a have been distributed globally spread and has attracted considerable attention in terms of the efficacy of vaccines and monoclonal antibodies.
  • the RBD region is responsible for binding to the host receptor ACE2 and thus contains multiple conformationally neutralizing epitopes for blocking ACE2 interactions (Du et al., 2009; Liu et al., 2020b; Zhou et al., 2021b ).
  • mutations in RBDs, especially in the interaction surface between ACE2 and RBD (RBM motif) not only increase receptor affinity, leading to higher infectivity, but also escape antibody immune responses.
  • the RBM region has been reported to be highly variable in these mutant strains (Thomson et al., 2021), so antibodies targeting epitopes outside the RBM domain have a broad spectrum of recognition capabilities and can improve neutralizing activity against a large number of escape mutations.
  • Applicants have identified and functionally and structurally defined a highly conserved antibody epitope on the S protein, which is located outside the RBM.
  • Antibody XG014 targeting this epitope was highly resistant to escape mutations located in the RBM, such as E484K and N501Y, thereby neutralizing B.1.1.7, B.1.351 and P.1 mutant pseudoviruses.
  • XG014 neutralized SARS-CoV-1 and bat SARSr-CoV WIV1, suggesting that XG014 targets neutralizing epitopes conserved across multiple ⁇ -CoVs.
  • RBD residues 338-346 FGEVFNATR
  • N343 glycan which are important for the XG014 interaction, are in close contact with several antibodies (316, S2M11 and Fab309), resulting in 338-346 being an epitope hotspot.
  • a nanobody has recently been reported to mimic ACE2-RBD binding to trigger conformational changes and activate the SARS-CoV-2 fusion mechanism (Koenig et al., 2021). Therefore, it is speculated that some antibodies, such as XG005 and XG016, may drag SARS-CoV-2 virions to host cells by interacting with Fc receptors and change the S protein conformation to induce virion-cell fusion.
  • the inventors' data further support this conclusion that most XG005-bound SARS-CoV-2 S trimer particles have two "up” RBDs, even for the most common of the S constructs used in the study. The conformation has only one "up” RBD and two “down” RBDs (Wrapp et al., 2020).
  • XG005 induces a rearrangement of the RBD into an "up” state, which is necessary for receptor binding as well as further conformational changes required for membrane fusion and viral entry.
  • XG014 induced all RBDs to the "down” state, thereby limiting the conformational changes of the RBDs.
  • Cell fusion is mediated by the viral S protein on S protein-expressing cells (or SARS-CoV-2-infected cells) together with ACE2 receptors on ACE2-expressing cells, but can also be independent of ACE2 receptors and via Fc ⁇ R- mediated pathway.
  • the conformational plasticity of immunogens plays an important role in vaccine design. For example, the introduction of two consecutive proline residues stabilizes the S protein of ⁇ -CoV in the prefusion conformation, thereby significantly increasing their immunogenicity (Pallesen et al., 2017). This strategy has been widely used in the development of COVID-19 vaccines (Dagotto et al., 2020). Based on the antigenic surface targeted by XG014, it was speculated that vaccination with a stable S protein with all RBDs in a "down" state might induce more XG014-like antibodies, thereby increasing the range and potency of neutralizing antibodies induced.
  • S309 another cross-neutralizing antibody against SARS-CoV-2 and SARS-CoV-1, also locks the RBD in the "down” conformation, further illustrating that the presence is associated with locking the RBD in the "down” state of multiple neutralizing epitopes. Since it has been reported that double-cysteine mutants (S383C and D985C) form disulfide bonds to lock all RBDs in the "down” state (Henderson et al., 2020), such RBD-locked S proteins were used as vaccines to boost immune responses may be a promising strategy for broadening cross-neutralizing antibody responses.
  • the conserved epitope on the outside of the RBM targeted by XG014 suggests that this antigenic surface can be exploited as a promising target for designing pan-vaccine against ⁇ -CoV-B.
  • the super-potent broadly neutralizing monoclonal antibody XG014 represents a very clinically promising candidate for the prevention or treatment of SARS-CoV-2, SARS-CoV-2 mutants, SARS-CoV-1 and possibly new SARSr-CoV infections Use of promising drug candidates.
  • IMGT/V-QUEST the highly customized and integrated system for IG and TR standardized VJ and VDJ sequence analysis. Nucleic Acids Res.36(Web Server issue ), W503-508.DOI:10.1093/nar/gkn316.
  • a neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2.Science.DOI:10.1126/science.abc6952.
  • Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH-and cysteine protease-independent FcgammaR pathway.J Virol.85(20),10582-10597.DOI:10.1128/JVI.00671- 11.
  • a human neutralizing antibody targets the receptor-binding site of SARS-CoV-2. Nature 584,120-124.
  • Tortorici MA, Beltramello, M., Lempp, FA, Pinto, D., Dang, HV, Rosen, LE, McCallum, M., Bowen, J., Minola, A., Jaconi, S., et al. ( 2020).
  • Ultrapotent human antibodies protect again SARS-CoV-2 challenge via multiple mechanisms. Science 370, 950-957.
  • SARS-CoV-2501Y.V2 escapes neutralization by South African COVID-19 donor plasma.Nat Med 27,622-625.
  • IgBLAST an immunoglobulin variable domain sequence analysis tool. Nucleic Acids Res. 41 (Web Server issue), W34-40. DOI: 10.1093 /nar/gkt382.

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Abstract

La présente invention concerne un anticorps monoclonal neutralisant sans effet de renforcement dépendant des anticorps (ADE) pour le nouveau coronavirus SARS-CoV-2 et un fragment de liaison à l'antigène de celui-ci, et un procédé pour préparer et utiliser l'anticorps neutralisant et le fragment de liaison à l'antigène de celui-ci. La présente invention vérifie la relation entre l'effet ADE et l'activité neutralisante de l'anticorps et un épitope de liaison de l'anticorps sur un domaine de la protéine S de SARS-CoV-2, trouve une série d'anticorps capables de neutraliser SARS-CoV-2 mais n'ayant aucun effet ADE, et en particulier trouve une série d'anticorps capables de neutraliser de manière croisée SARS-CoV-2 et SARS-CoV-1 et n'ayant aucun effet ADE. La présente invention constate en outre que certains anticorps restent encore sensibles à de nombreuses souches mutantes du SARS-CoV-2.
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