WO2024089277A2 - Anticorps - Google Patents

Anticorps Download PDF

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Publication number
WO2024089277A2
WO2024089277A2 PCT/EP2023/080151 EP2023080151W WO2024089277A2 WO 2024089277 A2 WO2024089277 A2 WO 2024089277A2 EP 2023080151 W EP2023080151 W EP 2023080151W WO 2024089277 A2 WO2024089277 A2 WO 2024089277A2
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Prior art keywords
antibody
antibodies
chain variable
variable domain
heavy chain
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PCT/EP2023/080151
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English (en)
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WO2024089277A3 (fr
Inventor
Juthanthip MONGKOLSAPAYA
Gavin Screaton
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Oxford University Innovation Limited
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Priority claimed from GBGB2215904.0A external-priority patent/GB202215904D0/en
Priority claimed from GBGB2300873.3A external-priority patent/GB202300873D0/en
Application filed by Oxford University Innovation Limited filed Critical Oxford University Innovation Limited
Publication of WO2024089277A2 publication Critical patent/WO2024089277A2/fr
Publication of WO2024089277A3 publication Critical patent/WO2024089277A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • 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

  • ANTIBODIES Field of invention The invention relates to antibodies useful for the prevention, treatment and/or diagnosis of coronavirus infections, and diseases and/or complications associated with coronavirus infections, including COVID-19.
  • Background of the invention In November 2021 Omicron BA.1, containing a raft of new spike mutations emerged and quickly spread globally. Intense selection pressure to escape the antibody response produced by vaccines or SARS-CoV-2 infection then led to a rapid succession of Omicron lineages with waves of BA.2 then BA.4/5 infection.
  • BA.1 caused the first Omicron wave, closely followed by a combination of BA.1 sub-lineage BA.1.1 and BA.2 in early 2021 (https://cov-lineages.org/lineage_list, https://nextstrain.org/nextclade/sars-cov-2) (Xia et al., 2022).
  • BA.2 became globally dominant and continues to spawn a succession of variants; first BA.2.12.1 (Huo et al., 2022), followed by BA.4 and BA.5 (Tuekprakhon et al., 2022), with BA.5 being the most widespread strain globally in June 2022 (https://cov- spectrum.org/explore/United%20Kingdom/AllSamples/Past6M).
  • biolayer interferometry (BLI) competition measurements and prior structures were used to create an interaction map of the antibody panel. It was found that potent mAbs bind to three distinct patches on the RBD, one of which is quite extended and reaches between two of the five epitopes seen in early pandemic responses. The other two correspond to diminished responses at hotspots for potent antibody binding in early pandemic responses. This corresponds to a re-focusing of the response, similar to that seen for BA.1 (Nutalai et al., 2022). The positions of mutations in the new BA.2 sub-lineages strongly overlap with the positions of antibody binding (Starr et al, 2020).
  • the invention provides an antibody capable of binding to the spike protein of coronavirus SARS-CoV-2, wherein the antibody comprises the six CDRs of antibody DP21 described herein, or of any one of the antibodies in Tables 1 to 7 as described herein.
  • the invention provides an antibody capable of binding to the spike protein of coronavirus SARS-CoV-2, wherein the antibody comprises CDRH1, CDRH2 and CDRH3, from a first antibody in Table 1 and CDRL1, CDRL2 and CDRL3 from a second antibody in Table 1, with the proviso that the first antibody and the second antibody are different.
  • the invention provides one or more polynucleotides encoding the antibody, one or more vectors comprising said polynucleotides, or a host cell comprising said vectors.
  • the invention provides a method for producing an antibody that is capable of binding to the spike protein of coronavirus SARS-CoV-2, the method comprising culturing the host cell and isolating the antibody from said culture.
  • the invention provides a pharmaceutical composition comprising: (a) the antibody, and (b) at least one pharmaceutically acceptable diluent or carrier.
  • the invention provides the antibody or the pharmaceutical composition for use in a method for treatment of a human or animal by therapy, or for use in a method of treating or preventing coronavirus infection, or a disease or complication associated with coronavirus infection.
  • the invention provides a method of treating or preventing coronavirus infection, or a disease or complication associated with coronavirus infection in a subject, comprising administering a therapeutically effective amount of the antibody or the pharmaceutical composition to said subject.
  • the invention provides a method of identifying the presence of coronavirus, or a protein fragment thereof, in a sample, comprising: (i) contacting the sample with the antibody; and (ii) detecting the presence or absence of an antibody-antigen complex, wherein the presence of the antibody-antigen complex indicates the presence of coronavirus, or a fragment thereof, in the sample.
  • the invention provides a method of treating or preventing coronavirus infection, or a disease or complication associated therewith, in a subject, the method comprising identifying the presence of coronavirus according to the method above, and treating the subject with the antibody, an anti-viral drug or an anti-inflammatory agent.
  • the invention provides the use of the antibody or the pharmaceutical composition for preventing, treating and/or diagnosing coronavirus infection, or a disease or complication associated therewith.
  • the invention provides the use of the antibody or the pharmaceutical composition for the manufacture of a medicament for treating or preventing coronavirus infection, or a disease or complication associated therewith.
  • BA.2 mAbs Generation of BA.2 mAbs.
  • A Sorting of BA.2 specific B cells.
  • B Proportion of BA.2 binding to RBD.
  • C ACE2 receptor blocking activity of mAbs.
  • D Gene usage in 25 potent BA.2 mAbs compared to potent antibodies produced following infection with early pandemic virus which have been previously reported (Dejnirattisai et al., 2021a).
  • A, B Front and back views of Mabscape antibody maps from early pandemic, Beta, BA.1 and BA.2 antibody panels.
  • Early pandemic all represents the full set of antibodies, irrespective of neutralization potency, all other panel show potent mAbs (IC50 ⁇ 100ng/ml).
  • the RBDs are shown surface rendered (grey) with the ACE2 footprint in green.
  • C, D Heatmaps of surface occupation of RBD by early pandemic, Beta, BA.1 and BA.2 antibody panels by iron heat colours (black > blue > red > orange > yellow > white hot) according to the relative level of antibody contact, calculated for each surface vertex as the number of antibodies within a 10 ⁇ radius.
  • BA.1 mutations are shown by the spikes.
  • Beta and BA.1 data are taken from (Dejnirattisai et al., 2022; Liu et al., 2021b) .
  • A Binding position and orientation of BA.2-10 viewed from front (left panel) and back (middle panel) of the RBD, and positions of the CDRs which have contact with the RBD (right panel). Only Vh (red) and Vl (blue) domains of the Fab are shown as ribbons for clarity.
  • RBD is drawn as grey surface representation with BA.4 mutation sites highlighted in magenta the additional mutation sites of all variants shown in Figure 1A are shown in cyan.
  • B -(D) Details of BA.2-10 and RBD interactions. The side chains of the RBD, Fab HC and LC are shown as grey, red and blue sticks, respectively. The yellow broken bonds represent hydrogen bonds or salt bridges.
  • E Complex of Delta-RBD/BA.2- 36 and
  • J Delta-RBD/BA.2-23.
  • O Complex of Delta-RBD/BA.2-13.
  • the drawing style and colour scheme are as in (A).
  • the drawing style and colour scheme are as in (B)-(D).
  • Figure 6. Sensitivity of Delta-RBD and LY-CoV1404 complex and sensitivity of LY-CoV1404 to Omicron subvariants containing K444T or V450P mutation indicated by the structure of its complex with RBD.
  • A) and (B) Overall structure of RBD/LY-CoV1404 (PDB ID, 7MMO) as viewed from front and back of the RBD respectively.
  • the drawing style and colour scheme are as in Figure 3A.
  • C Interactions of K444 of the RBD with CDR-H2 of LY-CoV1404.
  • D Contacts between V445 of the RBD and LY-CoV1404.
  • Serum neutralization IC50 titres (fold dilution) of lentivirus pseudotyped with the S gene of the indicated BA.2 sub-lineages.
  • Geometric mean titres are shown above each column. The single unvaccinated serum shows the lowest reactivity to BA.4/5 in Figure 12 D.
  • RBD is drawn as grey surface representation with BA.4 mutation sites highlighted in magenta and the additional mutation sites of all variants shown in Figure 1A are shown in cyan. Fabs are shown as ribbons with HC in red and LC in blue. The C1 nanobody in (B) is coloured in orange.
  • A overall structure of RBD and BA.2-23 complex.
  • B -(F) details of interactions between RBD and BA.2-23. Drawing style and colour scheme are as in Figure 5.
  • An antibody of the invention specifically binds to the spike protein of SARS-CoV- 2. In particular, it specifically binds to the receptor binding domain (RBD).
  • An antibody of the invention may comprise three, four, five or all six CDR sequences of an antibody in Table 1.
  • the antibody of the invention may comprise all six CDRs of an antibody in Table 1.
  • the antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to the heavy chain variable domain of an antibody in Table 1.
  • the antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to the light chain variable domain of an antibody in Table 1.
  • the antibody may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to the heavy chain variable domain and light chain domain, respectively, of an antibody in Table 1.
  • the antibody may comprise a heavy chain variable domain comprising the CDRH1, CDRH2 and CDRH3 of an antibody in Table 1, and a light chain variable domain comprising the CDRL1, CDRL2 and CDRL3 of the antibody in Table 1, wherein the heavy chain variable domain and the light chain variable domain comprises or consists of an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to the heavy chain variable domain and light chain variable domain, respectively of the antibody in Table 1 (i.e. the variation is in the framework regions of the antibody).
  • the antibody may be any one of the antibodies in Table 1.
  • Table 1 lists 25 individual antibodies that were identified from recovered BA.2 SARS-CoV-2 infected patients. In addition, Table 1 also lists three antibodies, DP21, DP22 and DP31, which were identified as described in Example 8. Table 1 lists the SEQ ID NOs for the heavy chain variable region and light chain variable region nucleotide and amino acid sequences, and the complementarity determining regions (CDRs) of the variable chains, of each of the antibodies.
  • the antibody in Table 1 may be selected from the group consisting of DP21, DP22, BA.2-07 and BA.2-23.
  • DP21 was found to neutralise the SARS-CoV-2 variant strains Victoria. BA.2, BA.4/BA.5, BA.4.6, BA.2.75.2 and BA.2.3.20 with an IC50 of ⁇ 0.1 ⁇ g/ml. Furthermore, DP21 was found to neutralise all the SARS-CoV-2 variant strains tested (Victoria, Delta, BA.1, BA.1.1, BA.2, BA.4/BA.5, BA.4.6, BA.2.75.2, BA.2.3.20, BJ.1, BF.7, BQ.1, BQ.1.1, BS.1, BA.2.10.4, BN.1, XBB and XBB.1) with an IC50 of ⁇ 0.25 ⁇ g/ml.
  • an antibody of the invention may comprise a CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 255, 256 and 257, respectively, and a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 258, 259 and 281, respectively.
  • the CDRL3 (QVWDSTTAV - SEQ ID NO: 281) of the antibody may comprise the sequence VWDSTTAV (SEQ ID NO: 260).
  • an antibody of the invention may comprise a CDRH1, CDRH2 and CDRH3 comprising or consisting of the amino acid sequences specified in SEQ ID NOs: 255, 256 and 257, respectively, and a CDRL1, CDRL2 and CDRL3 comprising or consisting of the amino acid sequences specified in SEQ ID NOs: 258, 259 and 260, respectively.
  • an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the heavy chain variable domain of antibody DP21 (i.e. SEQ ID NO: 252).
  • an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the light chain variable domain of antibody DP21 (i.e. SEQ ID NO: 254).
  • an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain, respectively, of antibody DP21 (i.e.
  • an antibody of the invention may comprise a heavy chain variable domain comprising a CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 255, 256 and 257, respectively, and a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 258, 259 and 281, respectively, wherein the heavy chain variable domain and the light chain variable domain comprises or consists of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain, respectively, of antibody DP21 (i.e.
  • an antibody of the invention may comprise a heavy chain variable domain comprising a CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 255, 256 and 257, respectively, and a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 258, 259 and 281, respectively, wherein the heavy chain variable domain and the light chain variable domain comprises or consists of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain, respectively, of antibody DP21 (i.e.
  • an antibody of the invention may comprise the heavy chain of DP21, and not the light chain of DP21.
  • the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 255, 256 and 257, respectively.
  • the antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the heavy chain variable domain of antibody DP21 (i.e. SEQ ID NO: 252).
  • the antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 252.
  • the antibody may comprise the light chain of DP21, and not the heavy chain of DP21.
  • the antibody may comprise a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 258, 259 and 281, respectively.
  • the CDRL3 (QVWDSTTAV - SEQ ID NO: 281) of the antibody may comprise the sequence VWDSTTAV (SEQ ID NO: 260).
  • the antibody may comprise a CDRL1, CDRL2 and CDRL3 comprising or consisting of the amino acid sequences specified in SEQ ID NOs: 258, 259 and 260, respectively.
  • the antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the light chain variable domain of antibody DP21 (i.e. SEQ ID NO: 254).
  • the antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 254.
  • the antibody in Table 1 may be DP22.
  • DP22 was found to neutralise all SARS-CoV-2 variant strains tested (Victoria, Delta, BA.1, BA.1.1, BA.2, BA.4/BA.5, BA.4.6, BA.2.75.2, BA.2.3.20, BJ.1, BF.7, BQ.1, BQ.1.1, BS.1, BA.2.10.4, BN.1, XBB and XBB.1) with an IC50 of ⁇ 0.5 ⁇ g/ml.
  • an antibody of the invention may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 265, 266 and 267, respectively, and a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 268, 269 and 270, respectively.
  • an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the heavy chain variable domain of antibody DP22 (i.e. SEQ ID NO: 262).
  • an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the light chain variable domain of antibody DP22 (i.e. SEQ ID NO: 264).
  • an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain, respectively, of antibody DP22 (i.e.
  • an antibody of the invention may comprise a heavy chain variable domain comprising a CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 265, 266 and 267, respectively, and a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 268, 269 and 270, respectively, wherein the heavy chain variable domain and the light chain variable domain comprises or consists of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain, respectively, of antibody DP21 (i.e.
  • an antibody of the invention may comprise the heavy chain of DP22, and not the light chain of DP22.
  • the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 265, 266 and 267, respectively.
  • the antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the heavy chain variable domain of antibody DP22 (i.e. SEQ ID NO: 262).
  • the antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 262.
  • the antibody may comprise the light chain of DP22, and not the heavy chain of DP22.
  • the antibody may comprise a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 268, 269 and 270, respectively.
  • the antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the light chain variable domain of antibody DP22 (i.e. SEQ ID NO: 264).
  • the antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 264.
  • the antibody in Table 1 may be BA.2-07.
  • BA.2-07 was found to neutralise the SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gemma, Delta, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/BA.5, BA.4.6, BA.2.75, BA.2.75.2, BA.2.3.20 and BJ.1 with an IC50 of ⁇ 0.005 ⁇ g/ml.
  • BA.2-07 was found to neutralise the SARS- CoV-2 variant strains Victoria., Alpha, Beta, Gamma, Delta, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/BA.5, BA.4.6, BA.2.75.2, BA.2.3.20, BJ.1, BF.7, BQ.1, BQ.1.1, BS.1, BA.2.10.4, BN.1, XBB and XBB.1 with an IC50 of ⁇ 0.015 ⁇ g/ml.
  • an antibody of the invention may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 55, 56 and 57, respectively, and a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 58, 59 and 60, respectively.
  • an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the heavy chain variable domain of antibody BA.2-07 (i.e. SEQ ID NO: 52).
  • an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the light chain variable domain of antibody BA.2-07 (i.e. SEQ ID NO: 54).
  • an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain, respectively, of antibody BA.2-07 (i.e.
  • an antibody of the invention may comprise a heavy chain variable domain comprising a CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 55, 56 and 57, respectively, and a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 58, 59 and 60, respectively, wherein the heavy chain variable domain and the light chain variable domain comprises or consists of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain, respectively, of antibody DP21 (i.e.
  • an antibody of the invention may comprise the heavy chain of BA.2-07, and not the light chain of BA.2-07.
  • the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 55, 56 and 57, respectively.
  • the antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the heavy chain variable domain of antibody BA.2-07 (i.e. SEQ ID NO: 52).
  • the antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 52.
  • the antibody may comprise the light chain of BA.2-07, and not the heavy chain of BA.2-07.
  • the antibody may comprise a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 58, 59 and 60, respectively.
  • the antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the light chain variable domain of antibody BA.2-07 (i.e. SEQ ID NO: 54).
  • the antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 54.
  • the antibody in Table 1 may be BA.2-23.
  • BA.2-23 was found to neutralise the SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gemma, Delta, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/BA.5, BA.4.6, BA.2.75, BA.2.75.2, BA.2.3.20 and BJ.1, with an IC50 of ⁇ 0.5 ⁇ g/ml.
  • BA.2-23 was found to neutralise the SARS- CoV-2 variant strains Victoria., Alpha, Beta, Gamma, Delta, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/BA.5, BA.4.6, BA.2.75.2, BA.2.3.20, BJ.1, BF.7, BQ.1, BQ.1.1, BS.1, BA.2.10.4, BN.1, XBB and XBB.1 with an IC50 of ⁇ 10 ⁇ g/ml.
  • an antibody of the invention may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 165, 166 and 167, respectively, and a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 168, 169 and 170, respectively.
  • an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the heavy chain variable domain of antibody BA.2-23 (i.e. SEQ ID NO: 162).
  • an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the light chain variable domain of antibody BA.2-23 (i.e. SEQ ID NO: 164).
  • an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain, respectively, of antibody BA.2-23 (i.e.
  • an antibody of the invention may comprise a heavy chain variable domain comprising a CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 165, 166 and 167, respectively, and a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 168, 169 and 170, respectively, wherein the heavy chain variable domain and the light chain variable domain comprises or consists of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain, respectively, of antibody DP21 (i.e.
  • an antibody of the invention may comprise the heavy chain of BA.2-23, and not the light chain of BA.2-23.
  • the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 165, 166 and 167, respectively.
  • the antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the heavy chain variable domain of antibody BA.2-23 (i.e. SEQ ID NO: 162).
  • the antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 162.
  • the antibody may comprise the light chain of BA.2-23, and not the heavy chain of BA.2-23.
  • the antibody may comprise a CDRL1, CDRL2 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 168, 169 and 170, respectively.
  • the antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the light chain variable domain of antibody BA.2-23 (i.e. SEQ ID NO: 164).
  • the antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 164.
  • Mixed chain antibodies of the invention An antibody of the invention may comprise a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 from a first antibody in Table 1 and a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 from a second antibody in Table 1, with the proviso that the first and second antibodies are different.
  • Such antibodies are referred to as mixed chain antibodies herein.
  • Examples of the mixed chain antibodies useful with the invention are provided in Tables 2 to 7.
  • Table 2 shows examples of mixed chain antibodies generated from antibodies in Table 1 that are derived from the same germline heavy chain IGHV 1-69.
  • Table 3 shows examples of mixed chain antibodies generated from antibodies in Table 1 that are derived from the same germline heavy chain IGHV3-9.
  • Table 4 shows examples of mixed chain antibodies generated from antibodies in Table 1 that are derived from the same germline heavy chain IGHV3-53.
  • Table 5 shows examples of mixed chain antibodies generated from antibodies in Table 1 that are derived from the same germline heavy chain IGHV1-2.
  • Table 6 shows examples of mixed chain antibodies generated from antibodies in Table 1 that are derived from the same germline heavy chain IGHV4-59.
  • an antibody of the invention comprises a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 from a first antibody in Table 1 and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 from a second antibody in Table 1, with the proviso that the first and second antibodies are different.
  • the antibody may comprise a heavy chain variable domain amino acid sequence having at least 80% sequence identity to the heavy chain variable domain from a first antibody in Table 1, and a light chain variable domain amino acid sequence having at least 80% sequence identity to the light chain variable domain from a second antibody in Table 1, with the proviso that the first and second antibodies are different.
  • the antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the heavy chain variable domain of an antibody in Table 1, and a light chain variable domain comprising or consisting of an amino acid sequence having ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the light chain variable domain of an antibody in Table 1, with the proviso that the first and second antibodies are different.
  • the antibody in Table 1 may be selected from the group consisting of: DP21, DP22, BA.2.07 and BA.2.23.
  • the first antibody and the second antibody are both selected from the group consisting of: BA.2-24, BA.2-17, BA.2-34, BA.2-05, BA.2-15, BA.2-06, BA.2-02, BA.2-11, BA.2-16 and BA.2-07.
  • the heavy chain variable domain of these antibodies are derived from IGHV 1-69.
  • the resulting mixed chain antibodies are set out in Table 2.
  • the antibody of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and/or a heavy chain variable domain and a light chain variable domain, each comprising or consisting of an amino acid sequence having at least 80% (e.g.
  • the first antibody and the second antibody are both selected from the group consisting of: BA.2-21, BA.2-28, BA.2.10 and BA.2-12.
  • the heavy chain variable domain of these antibodies are derived from IGHV 3-9.
  • the resulting mixed chain antibodies are set out in Table 3.
  • the antibody of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and/or a heavy chain variable domain and a light chain variable domain, each comprising or consisting of an amino acid sequence having at least 80% (e.g.
  • the first antibody and the second antibody are both selected from the group consisting of: BA.2-26, BA.2-23, BA.2-04, DP21, DP22 and DP31.
  • the heavy chain variable domain of these antibodies are derived from IGHV3-53. The resulting mixed chain antibodies are set out in Table 4.
  • the antibody of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and/or a heavy chain variable domain and a light chain variable domain, each comprising or consisting of an amino acid sequence having at least 80% sequence identity to the corresponding variable domain of any one of the mixed chain antibodies as set out in Table 4.
  • Antibodies derived from IGHV3-53 may be used to produce mixed-chain antibodies with antibodies from IGHV3-66 (although none were present in the high affinity group of antibodies discussed herein). (see, e.g. Dejnirattisai, Wanwisa, et al.
  • the first antibody is selected from the group consisting of BA.2-26, BA.2-23, BA.2-04, DP21, DP22 and DP31
  • the second antibody is an antibody from IGHV3-66.
  • the second antibody is selected from the group consisting of BA.2-26, BA.2- 23, BA.2-04, DP21, DP22 and DP31
  • the first antibody is an antibody from IGHV3- 66.
  • the heavy chain variable domain of these antibodies are derived from IGHV3-53 and IGVH3-66.
  • the first antibody and the second antibody are both selected from the group consisting of: BA.2-09 and BA.2-03.
  • the heavy chain variable domain of these antibodies are derived from IGHV 1-2.
  • the resulting mixed chain antibodies are set out in Table 5.
  • the antibody of the invention may comprise all six CDRs (CDRH1- 3 and CDRL1-3), and/or a heavy chain variable domain and a light chain variable domain, each comprising or consisting of an amino acid sequence having at least 80% (e.g. ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%)sequence identity to the corresponding variable domain of any one of the mixed chain antibodies as set out in Table 5.
  • the first antibody and the second antibody are both selected from the group consisting of: BA.2-30 and BA.2-25.
  • the heavy chain variable domain of these antibodies are derived from IGHV 4-59.
  • the resulting mixed chain antibodies are set out in Table 6.
  • the antibody of the invention may comprise all six CDRs (CDRH1- 3 and CDRL1-3), and/or a heavy chain variable domain and a light chain variable domain, each comprising or consisting of an amino acid sequence having at least 80% (e.g. ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to the corresponding variable domain of any one of the mixed chain antibodies as set out in Table 6.
  • the first antibody and the second antibody are both selected from the group consisting of: BA.2-36 and BA.2-33.
  • the heavy chain variable domain of these antibodies are derived from IGHV 4-61.
  • the resulting mixed chain antibodies are set out in Table 7.
  • the antibody of the invention may comprise all six CDRs (CDRH1- 3 and CDRL1-3), and/or a heavy chain variable domain and a light chain variable domain, each comprising or consisting of an amino acid sequence having at least 80% (e.g. ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to the corresponding variable domain of any one of the mixed chain antibodies as set out in Table 7.
  • the constant region domains of an antibody molecule of the invention if present, may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required.
  • the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains.
  • the constant regions are of human origin.
  • human IgG i.e. IgG1, IgG2, IgG3 or IgG4 constant region domains may be used.
  • the constant region is a human IgG1 constant region.
  • An antibody of the invention may be or may comprise a modification from the amino acid sequence of an antibody in Tables 1 to 7, whilst maintaining the activity and/or function of the antibody. The modification may a substitution, deletion and/or addition.
  • the modification may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and/or deletions from the amino acid sequence of an antibody in Tables 1 to 7.
  • the modification may comprise an amino acid substituted with an alternative amino acid having similar properties.
  • Some properties of the 20 main amino acids, which can be used to select suitable substituents, are as follows:
  • the modification may comprise a derivatised amino acid, e.g. a labelled or non- natural amino acid, providing the function of the antibody is not significantly adversely affected.
  • Modification of antibodies of the invention as described above may be prepared during synthesis of the antibody or by post-production modification, or when the antibody is in recombinant form using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.
  • Antibodies of the invention may be modified (e.g. as described above) to improve the potency of said antibodies or to adapt said antibodies to new SARS-CoV-2 variants.
  • the modifications may be amino acid substitutions to adapt the antibody to substitutions in a virus variant.
  • the known mode of binding of an antibody to the spike protein e.g. by crystal structure determination, or modelling
  • This information can then be used to identify possible substitutions of the antibody that will compensate for the change in the epitope characteristics. For example, a substitution of a hydrophobic amino acid in the spike protein to a negatively changes amino acid may be compensated by substituting the amino acid from the antibody that interacts with said amino acid in the spike protein to a positively charged amino acid.
  • Methods for identifying residues of an antibody that may be substituted are encompassed by the present disclosure, for example, by determining the structure of antibody-antigen complexes as described herein.
  • the antibodies of the invention may contain one or more modifications to increase their cross-lineage neutralisation property.
  • E484 of the spike protein which is a key residue that mediates the interaction with ACE2, is mutated in almost all currently circulating SARS-CoV-2 strains (see e.g. Figure 1A, demonstrating that all strains shown comprise an E484A or E484R mutation) resulting in differing neutralisation effects of the antibodies.
  • antibodies that bind to E484 can be modified to compensate for the changes in E484 of the spike protein.
  • E484 is mutated from a positively charge to negatively charged amino acid in the BA.2.3.20 SAR-CoV-2 strain, when compared to the original Wuhan strain.
  • the amino acid residues of antibodies that bind to or near E484 may be mutated to compensate for the change in charge.
  • Antibodies of the invention may be isolated antibodies.
  • An isolated antibody is an antibody which is substantially free of other antibodies having different antigenic specificities.
  • the term 'antibody' as used herein may relate to whole antibodies (i.e. comprising the elements of two heavy chains and two light chains inter-connected by disulphide bonds) as well as antigen-binding fragments thereof.
  • Antibodies typically comprise immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin immunoglobulin
  • HCVR heavy chain variable region
  • VL light chain variable region
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab’ and F(ab’)2 fragments, scFvs, and Fab expression libraries
  • An antibody of the invention may be a monoclonal antibody.
  • Monoclonal antibodies (mAbs) of the invention may be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example those disclosed in “Monoclonal Antibodies: a manual of techniques”(Zola H, 1987, CRC Press) and in “Monoclonal Hybridoma Antibodies: techniques and applications” (Hurrell JGR, 1982 CRC Press).
  • An antibody of the invention may be multispecific, such as bispecific.
  • a bispecific antibody of the invention binds two different epitopes.
  • the epitopes may be in the same protein (e.g. two epitopes in spike protein of SARS-CoV-2) or different proteins (e.g.
  • a bispecific antibody of the invention may bind to two separate epitopes on the spike protein of SARS-CoV-2.
  • the bispecific antibody may bind to the NTD of the spike protein and to the RBD of the spike protein.
  • the bispecific antibody may bind to two different epitopes in the RBD of the spike protein.
  • One or more (e.g. two) antibodies of the invention can be coupled to form a multispecific (e.g. bispecific) antibody. Methods to prepare multispecific, e.g. bispecific, antibodies are well known in the art.
  • An antibody may be selected from the group consisting of single chain antibodies, single chain variable fragments (scFvs), variable fragments (Fvs), fragment antigen- binding regions (Fabs), recombinant antibodies, monoclonal antibodies, fusion proteins comprising the antigen-binding domain of a native antibody or an aptamer, single-domain antibodies (sdAbs), also known as VHH antibodies, nanobodies (Camelid-derived single- domain antibodies), shark IgNAR-derived single-domain antibody fragments called VNAR, diabodies, triabodies, Anticalins, aptamers (DNA or RNA) and active components or fragments thereof.
  • scFvs single chain variable fragments
  • Fvs variable fragments
  • Fabs fragment antigen- binding regions
  • the constant region domains of an antibody molecule of the invention may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required.
  • the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains.
  • the constant regions are of human origin.
  • human IgG i.e. IgG1, IgG2, IgG3 or IgG4 constant region domains may be used.
  • the constant region is a human IgG1 constant region.
  • the light chain constant region may be either lambda or kappa.
  • Antibodies of the invention may be mono-specific or multi-specific (e.g. bi- specific).
  • a multi-specific antibody comprises at least two different variable domains, wherein each variable domain is capable of binding to a separate antigen or to a different epitope on the same antigen.
  • An antibody of the invention may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human or humanised antibody. Typically, the antibody is a human antibody. Fully human antibodies are those antibodies in which the variable regions and the constant regions (where present) of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, but not necessarily from the same antibody.
  • the antibody of the invention may be a full-length antibody.
  • the antibody of the invention may be an antigen-binding fragment.
  • An antigen- binding fragment of the invention binds to the same epitope of the parent antibody, i.e. the antibody from which the antigen-binding fragment is derived.
  • An antigen-binding fragment of the invention typically retains the parts of the parent antibody that interact with the epitope.
  • the antigen-binding fragment typically comprise the complementarity- determining regions (CDRs) that interact with the antigen, such as one, two, three, four, five or six CDRs.
  • the antigen-binding fragment further comprises the structural scaffold surrounding the CDRs of the parent antibody, such as the variable region domains of the heavy and/or light chains.
  • the antigen-binding fragment retains the same or similar binding affinity to the antigen as the parent antibody.
  • an antigen-binding fragment does not necessarily have an identical sequence to the parent antibody.
  • the antigen-binding fragment may have ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, 100% sequence identity with the respective CDRs of the parent antibody.
  • the antigen-binding fragment may have ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, 100% sequence identity with the respective variable region domains of the parent antibody.
  • the non-identical amino acids of a variable region are not in the CDRs.
  • Antigen-binding fragments of antibodies of the invention retain the ability to selectively bind to an antigen.
  • Antigen-binding fragments of antibodies include single chain antibodies (i.e. a full-length heavy chain and light chain); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH), scFv.
  • An antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma R et al., 1998, J. Immunol. Methods, 216, 165-181).
  • an antibody of the invention may be able to neutralise at least one biological activity of SAR-CoV-2 (a neutralising antibody), particularly to neutralise virus infectivity. Neutralisation may also be determined using IC50 or IC90 values.
  • the antibody may have an IC50 value of ⁇ 0.1 ⁇ g/ml, ⁇ 0.05 ⁇ g/ml, ⁇ 0.01 ⁇ g/ml ⁇ 0.005 ⁇ g/ml or ⁇ 0.002 ⁇ g/ml.
  • an antibody of the invention may have an IC 50 value of between 0.0001 ⁇ g/ml and 0.1 ⁇ g/ml, sometimes between 0.0001 ⁇ g/ml and 0.05 ⁇ g/ml or even between 0.0001 ⁇ g/ml and 0.001 ⁇ g/ml.
  • the IC 50 values of some of the antibodies of Table 1 are provided in Tables 8 and 11.
  • the ability of an antibody to neutralise virus infectivity may be measured using an appropriate assay, particularly using a cell-based neutralisation assay, as is known in the art.
  • the neutralisation ability may be measured in a focus reduction neutralisation assay (FRNT) where the reduction in the number of cells (e.g. human cells) infected with the virus (e.g. for 2 hours at 37 oC) in the presence of the antibody is compared to a negative control in which no antibodies were added.
  • An antibody of the invention may block the interaction between the spike protein of SAR-CoV-2 with the cell surface receptor, angiotensin-converting enzyme 2 (ACE2), of the target cell, e.g. by direct blocking or by disrupting the pre-fusion conformation of the spike protein.
  • ACE2 angiotensin-converting enzyme 2
  • Blocking of the interaction between spike and ACE2 can be total or partial.
  • an antibody of the invention may reduce spike-ACE2 formation by ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 99% or 100%.
  • Blocking of spike-ACE2 formation can be measured by any suitable means known in the art, for example, by ELISA. Most antibodies showing neutralisation also showed blocking of the interaction between the spike protein and ACE2. Furthermore, a number of non-neutralising antibodies are good ACE2 blockers.
  • an antibody of the invention may have an affinity constant (K D ) value for the spike protein of SARS-CoV-2 of ⁇ 5nM, ⁇ 4nM, ⁇ 3nM, ⁇ 2nM, ⁇ 1nM, ⁇ 0.5nM, ⁇ 0.4nM, ⁇ 0.3nM, ⁇ 0.2nM or ⁇ 0.1nM.
  • K D affinity constant
  • the KD value can be measured by any suitable means known in the art, for example, by ELISA or Surface Plasmon Resonance (Biacore) at 25 °C.
  • Binding affinity (K D ) may be quantified by determining the dissociation constant (Kd) and association constant (Ka) for an antibody and its target.
  • the antibody may have an association constant (Ka) of ⁇ 10000 M -1 s -1 , ⁇ 50000 M -1 s -1 , ⁇ 100000 M -1 s -1 , ⁇ 200000 M -1 s -1 or ⁇ 500000 M -1 s -1 , and/or a dissociation constant (Kd) of ⁇ 0.001 s -1 , ⁇ 0.0005 s -1 , ⁇ 0.004 s -1 , ⁇ 0.003 s -1 , ⁇ 0.002 s -1 or ⁇ 0.0001 s -1 .
  • An antibody of the invention is preferably able to provide in vivo protection in coronavirus (e.g.
  • SARS-CoV-2) infected animals For example, administration of an antibody of the invention to coronavirus (e.g. SARS-CoV-2) infected animals may result in a survival rate of ⁇ 30%, ⁇ 40%, ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 95% or 100%. Survival rates may be determined using routine methods.
  • Antibodies of the invention may have any combination of one or more of the above properties.
  • Antibodies of the invention may bind to the same epitope as, or compete for binding to SARS-CoV-2 spike protein with, any one of the antibodies described herein (i.e. in particular with antibodies with the heavy and light chain variable regions described above).
  • Fc regions An antibody of the invention may or may not comprise an Fc domain.
  • the antibodies of the invention may be modified in the Fc region in order to improve their stability. Such modifications are known in the art. Modifications may improve the stability of the antibody during storage of the antibody.
  • the in vivo half-life of the antibody may be improved by modifications of the Fc-region. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulphide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.
  • an antibody of the invention may be modified to promote the interaction of the Fc domain with FcRn.
  • the Fc domain may be modified to improve the stability of the antibody by affecting Fc and FcRn interaction at low pH, such as in the endosome.
  • the M252Y/S254T/T256E (YTE) mutation may be used to improve the half- life of an IgG1 antibody.
  • the antibody may be modified to affect the interaction of the antibody with other receptors, such as Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RIIB, Fc ⁇ RIII, and Fc ⁇ R. Such modifications may be used to affect the effector functions of the antibody.
  • an antibody of the invention comprises an altered Fc domain as described herein below.
  • an antibody of the invention comprises an Fc domain, but the sequence of the Fc domain has been altered to modify one or more Fc effector functions.
  • an antibody of the invention comprises a “silenced” Fc region.
  • an antibody of the invention does not display the effector function or functions associated with a normal Fc region.
  • An Fc region of an antibody of the invention does not bind to one or more Fc receptors.
  • an antibody of the invention does not comprise a CH2 domain.
  • an antibody of the invention does not comprise a CH3 domain.
  • an antibody of the invention comprises additional CH 2 and/or CH 3 domains.
  • an antibody of the invention does not bind Fc receptors.
  • an antibody of the invention does not bind complement.
  • an antibody of the invention does not bind Fc ⁇ R, but does bind complement.
  • an antibody of the invention in general may comprise modifications that alter serum half-life of the antibody.
  • an antibody of the invention has Fc region modification(s) that alter the half-life of the antibody. Such modifications may be present as well as those that alter Fc functions.
  • an antibody of the invention has modification(s) that alter the serum half-life of the antibody.
  • an antibody of the invention may comprise a human constant region, for instance IgA, IgD, IgE, IgG or IgM domains.
  • human IgG constant region domains may be used, especially of the IgG1 and IgG3 isotypes when the antibody molecule is intended for therapeutic uses where antibody effector functions are required.
  • the antibody heavy chain comprises a CH1 domain and the antibody light chain comprises a CL domain, either kappa or lambda.
  • the antibody heavy chain comprises a CH1 domain, a CH2 domain and a CH3 domain and the antibody light chain comprises a CL domain, either kappa or lambda.
  • the four human IgG isotypes bind the activating Fc ⁇ receptors (Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa), the inhibitory Fc ⁇ RIIb receptor, and the first component of complement (C1q) with different affinities, yielding very different effector functions (Bruhns P. et al., 2009. Specificity and affinity of human Fc ⁇ receptors and their polymorphic variants for human IgG subclasses. Blood.113(16):3716-25), see also Jeffrey B. Stavenhagen, et al. Cancer Research 2007 Sep 15; 67(18):8882-90.
  • an antibody of the invention does not bind to Fc receptors.
  • the antibody does bind to one or more type of Fc receptors.
  • the Fc region employed is mutated, in particular a mutation described herein.
  • the Fc mutation is selected from the group comprising a mutation to remove or enhance binding of the Fc region to an Fc receptor, a mutation to increase or remove an effector function, a mutation to increase or decrease half-life of the antibody and a combination of the same.
  • reference is made to the impact of a modification it may be demonstrated by comparison to the equivalent antibody but lacking the modification.
  • modifications may be present at M252/S254/T256 + H44/N434 that alter serum half-life and in particular M252Y/S254T/T256E + H433K/N434F may be present.
  • it is desired to increase half-life.
  • it may be actually desired to decrease serum half-life of the antibody and so modifications may be present that decrease serum half-life.
  • Numerous mutations have been made in the CH 2 domain of human IgG1 and their effect on ADCC and CDC tested in vitro (Idusogie EE. et al., 2001. Engineered antibodies with increased activity to recruit complement. J Immunol.166(4):2571-5).
  • alanine substitution at position 333 was reported to increase both ADCC and CDC.
  • a modification at position 333 may be present, and in particular one that alters ability to recruit complement.
  • Lazar et al. described a triple mutant (S239D/I332E/A330L) with a higher affinity for Fc ⁇ RIIIa and a lower affinity for Fc ⁇ RIIb resulting in enhanced ADCC (Lazar GA. et al., 2006).
  • modifications at S239/I332/A330 may be present, particularly those that alter affinity for Fc receptors and in particular S239D/I332E/A330L .
  • an antibody of the invention may have a modified hinge region and/or CH1 region.
  • the isotype employed may be chosen as it has a particular hinge regions.
  • Public V-regions also described as public V-genes herein, are the V regions of the germline heavy chain and light chain regions that are found in a large proportion of the antibody responses to SARS-CoV-2 found within the population. In this application, the V regions are specific responses to the original BA.2 SARS-CoV-2 variant.
  • an antibody “derived” from a specific v-region refers to antibodies that were generated by V(D)J recombination using that germline v-region sequence.
  • the germline IGHV1-69 v-region sequence may undergo somatic recombination and somatic mutation to arrive at an antibody that specifically binds to the spike protein of SARS-CoV-2.
  • the nucleotide sequence encoding the antibody does not comprise a sequence identical to the IGHV1-69 germline sequence, nevertheless, the antibody is still derived from this v-region.
  • An antibody of the invention typically comprises no more than 20 non-silent mutations in the v-region, when compared to the germline sequence, such as no more than 17 non-silent mutations.
  • An antibody of the invention typically comprises between 5-20 non-silent mutations in the v-region, when compared to the germline sequence, such as between 7-17, 8-15 and 8-13 non-silent mutations.
  • Germline v-region sequences are well known in the art, and methods of identifying whether a certain region of an antibody is derived from a particular germline v-region sequence are also well known in the art.
  • an antibody of the invention derives from a v-region selected from IGHV1-69, IGHV3-9, IGHV3-53, IGHV1-2, IGHV4-59, IGHV4-61, IGHV3-15 or IGHV3-48.
  • an antibody of the invention is encoded by a v-region selected from IGHV1-69, IGHV3-9, IGHV3-53, IGHV1-2, IGHV4-59, IGHV4-61, IGHV3-15 or IGHV3-48 and having 5-20 non-silent nucleotide mutations, or 7-17 non-silent mutations, such as 8-15 or 8-13 non-silent mutations when compared to the naturally occurring germline sequence.
  • a silent mutation are defined herein is a change in the nucleotide sequence without a change in the amino acid sequence for which the nucleotide sequence encodes.
  • a non-silent mutation is therefore a mutation that leads to a change in the amino acid sequence encoded by the nucleotide sequence.
  • the inventors have surprisingly found that the light chain variable region of two antibodies having the same heavy chain v-region may be exchanged to produce a mixed- chain antibody comprising the heavy chain variable region of a first antibody and the light chain variable region of a second antibody.
  • the two antibodies may both comprise a heavy chain variable region derived from IGHV3-53.
  • both antibodies also comprise a light chain variable region derived from the same light chain v- region, although this is not essential because, for example, the light chain of antibody BA.2-23 may be matched with any heavy chain variable region derived from IGHV3-53 and lead to a potent neutralising antibody.
  • an antibody of the invention comprises the CDRs of an heavy chain variable domain of an antibody derived from a major public v-region selected from IGHV1-69, IGHV3-9, IGHV3-53/3-66, IGHV1-2, IGHV4-59, IGHV4-61, such as antibodies BA.2-24, BA.2-17, BA.2-34, BA.2-05, BA.2-15, BA.2-06, BA.2-02, BA.2-11, BA.2-16 and BA.2-07 for IGHV1-69, antibodies BA.2-21, BA.2-28, BA.2-10 and BA.2-12 for IGHV3-9, BA.2-26, BA.2-23, BA.2-04, DP21, DP22 and DP31 for IGHV3-53/3-66, antibodies BA.2-09 and BA.2-03 for IGHV1-2, antibodies BA.2-30 and BA.2-25 for IGHV4-59, or antibodies BA.2-36 and BA.
  • an antibody of the invention comprises the heavy chain variable domain of an antibody derived from a major public v-region selected from IGHV1-69, IGHV3-9, IGHV3-53/3-66, IGHV1-2, IGHV4-59, IGHV4-61, such as antibodies BA.2-24, BA.2-17, BA.2-34, BA.2-05, BA.2-15, BA.2-06, BA.2-02, BA.2-11, BA.2-16 and BA.2-07 for IGHV1-69, antibodies BA.2-21, BA.2-28, BA.2-10 and BA.2-12 for IGHV3-9, BA.2-26, BA.2-23, BA.2-04, DP21, DP22 and DP31 for IGHV3-53/3-66, antibodies BA.2-09 and BA.2-03 for IGHV1-2, antibodies BA.2-30 and BA.2-25 for IGHV4-59, or antibodies BA.2-36 and BA.2-33 for IGHV4-61.
  • the invention provides a method of generating an antibody that binds specifically to the spike protein of SARS-CoV-2 (e.g. a SARS-CoV-2 strain of the Victoria, Alpha, Beta, Gamma, Delta, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4, BA.5, BA.4.6, BA.2.75, BA.2.75.2, BA.2.3.20, BJ.1, BF.7, BQ.1, BQ.1.1, BS.1, BA.2.10.4, BN.1, XBB and/or XBB.1), the method comprising identifying two or more antibodies derived from the same light chain and/or heavy chain v-regions, replacing the light chain of a first antibody with the light chain of a second antibody, to thereby generate a mixed- chain antibody comprising the heavy chain of the first antibody and the light chain of the second antibody.
  • SARS-CoV-2 e.g. a SARS-CoV-2 strain of the Victoria, Alpha, Beta, Gamma, Delta, BA.1, BA
  • the method further comprises determining the affinity for and/or neutralisation of SARS-CoV-2 of the mixed-chain antibody.
  • the method may further comprise comparing the affinity of the mixed-chain antibody with that of the first and/or second antibodies.
  • the method may further comprise selecting a mixed chain antibody that has the same or greater affinity than the first and/or second antibodies.
  • the heavy chain v-region is IGHV 3-53 and/or the light chain v- region is IGLV Kappa 1-33.
  • the heavy chain v-region is IGHV 3-53 and/or the light chain v-region is IGLV Kappa 3-9.
  • the invention provides an antibody that specifically binds to the BA.2 variant of SARS-CoV-2, wherein the antibody has a v-region derived from IGHV1-69. It has been surprisingly discovered that antibody responses to infection with the BA.2 variant of SARS-CoV-2 is biased towards antibodies with a heavy chain variable region derived from IGHV1-69.
  • the antibody of the invention comprises the CDRH1, CDRH2 and CDRH3 from BA.2-24, BA.2-17, BA.2-34, BA.2-05, BA.2-15, BA.2-06, BA.2-02, BA.2- 11, BA.2-16 and BA.2-07.
  • the invention provides an antibody that specifically binds to the BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4, BA.5, BA.4.6, BA.2.75, BA.2.75.2, BA.2.3.20, BJ.1, BF.7, BQ.1, BQ.1.1, BS.1, BA.2.10.4, BN.1, XBB and/or XBB.1 variants of SARS-CoV-2, wherein the antibody has a v-region derived from IGHV3-53.
  • the antibody of the invention comprises the CDRH1, CDRH2 and CDRH3 from BA.2-26, BA.2-23, BA.2- 04, DP21, DP22 and DP31.
  • Antibody conjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein-coupling agents known in the art.
  • An antibody, of the invention may be conjugated to a molecule that modulates or alters serum half-life.
  • An antibody, of the invention may bind to albumin, for example in order to modulate the serum half-life.
  • an antibody of the invention will also include a binding region specific for albumin.
  • an antibody of the invention may include a peptide linker which is an albumin binding peptide. Examples of albumin binding peptides are included in WO2015/197772 and WO2007/106120 the entirety of which are incorporated by reference.
  • Polynucleotides, vectors and host cells The invention also provides one or more isolated polynucleotides (e.g. DNA) encoding the antibody of the invention.
  • the polynucleotide sequence is collectively present on more than one polynucleotide, but collectively together they are able to encode an antibody of the invention.
  • the polynucleotides may encode the heavy and/or light chain variable regions(s) of an antibody of the invention.
  • the polynucleotides may encode the full heavy and/or light chain of an antibody of the invention.
  • one polynucleotide would encode each of the heavy and light chains.
  • Polynucleotides which encode an antibody of the invention can be obtained by methods well known to those skilled in the art. For example, DNA sequences coding for part or all of the antibody heavy and light chains may be synthesised as desired from the corresponding amino acid sequences.
  • a polynucleotide of the invention may be provided in the form of an expression cassette, which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the antibody of the invention in vivo.
  • the invention also provides one or more expression cassettes encoding the one or more polynucleotides that encoding an antibody of the invention. These expression cassettes, in turn, are typically provided within vectors (e.g.
  • the invention provides a vector encoding an antibody of the invention.
  • the invention provides vectors which collectively encode an antibody of the invention.
  • the vectors may be cloning vectors or expression vectors.
  • a suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide of the invention.
  • the polynucleotides, expression cassettes or vectors of the invention are introduced into a host cell, e.g. by transfection.
  • the invention also provides a host cell comprising the one or more polynucleotides, expression cassettes or vectors of the invention.
  • the polynucleotides, expression cassettes or vectors of the invention may be introduced transiently or permanently into the host cell, allowing expression of an antibody from the one or more polynucleotides, expression cassettes or vectors.
  • host cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast, or prokaryotic cells, such as bacteria cells.
  • Particular examples of cells include mammalian HEK293, such as HEK293F, HEK293T, HEK293S or HEK Expi293F, CHO, HeLa, NS0 and COS cells, or any other cell line used herein, such as the ones used in the Examples.
  • the cell line selected will be one which is not only stable, but also allows for mature glycosylation.
  • the invention also provides a process for the production of an antibody of the invention, comprising culturing a host cell containing one or more vectors of the invention under conditions suitable for the expression of the antibody from the one or more polynucleotides of the invention, and isolating the antibody from said culture.
  • Pharmaceutical composition provides a pharmaceutical composition comprising an antibody of the invention.
  • the composition may comprise a combination (such as two, three or four) of the antibodies of the invention.
  • the pharmaceutical composition may also comprise a pharmaceutically acceptable carrier.
  • the composition of the invention may include one or more pharmaceutically acceptable salts.
  • a "pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.
  • Suitable pharmaceutically acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers include water, buffered water and saline. Other suitable pharmaceutically acceptable carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • compositions of the invention may comprise additional therapeutic agents, for example an anti-viral agent.
  • the anti-viral agent may bind to coronavirus and inhibit viral activity. Alternatively, the anti-viral agent may not bind directly to coronavirus but still affect viral activity/infectivity.
  • the anti-viral agent could be a further anti-coronavirus antibody, which binds somewhere on SARS-CoV-2 other than the spike protein.
  • examples of an anti-viral agent useful with the invention include Remdesivir, Lopinavir, ritonavir, APN01, and Favilavir.
  • the additional therapeutic agent may be an anti-inflammatory agent, such as a corticosteroid (e.g. Dexamethasone) or a non-steroidal anti-inflammatory drug (e.g. Tocilizumab).
  • the additional therapeutic agent may be an anti-coronavirus vaccine.
  • the pharmaceutical composition may be administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally or orally.
  • kits comprising antibodies or other compositions of the invention and instructions for use.
  • the kit may further contain one or more additional reagents, such as an additional therapeutic or prophylactic agent as discussed herein.
  • additional reagents such as an additional therapeutic or prophylactic agent as discussed herein.
  • the invention further relates to the use of the antibodies and the pharmaceutical compositions, described herein, e.g. in a method for treatment of the human or animal body by therapy, or in a diagnostic method.
  • the method of treatment may be therapeutic or prophylactic.
  • the invention relates to methods of treating coronavirus (e.g. SARS- CoV-2) infections, a disease or complication associated therewith, e.g. COVID-19.
  • the method may comprise administering a therapeutically effective amount of an antibody, a combination of antibodies, or a pharmaceutical composition of the invention.
  • the method may further comprise identifying the presence of coronavirus, or fragments thereof, in a sample, e.g. SARS-CoV-2, from the subject.
  • the invention also relates to an antibody, a combination of antibodies, or a pharmaceutical composition according to the invention for use in a method of treating coronavirus (e.g. SARS-CoV-2) infections, a disease or complication associated therewith, e.g. COVID-19.
  • the invention also relates to a method of formulating a composition for treating coronavirus (e.g.
  • said method comprises mixing an antibody, a combination of antibodies, or a pharmaceutical composition according to the invention with an acceptable carrier to prepare said composition.
  • the invention also relates to the use of an antibody, a combination of antibodies, or a pharmaceutical composition according to the invention for treating coronavirus (e.g. SARS-CoV-2) infections or a disease or complication associated therewith, e.g. COVID- 19.
  • the invention also relates to the use of an antibody, a combination of antibodies, or a pharmaceutical composition according to the invention for the manufacture of a medicament for treating or preventing coronavirus (e.g.
  • the invention also relates to preventing, treating or diagnosing coronavirus infection caused by any SARS-CoV-2 strain.
  • the coronavirus infection may be caused by any SARS-CoV-2 strain.
  • the SARS-CoV-2 strain may be the earliest identified Wuhan strain (hCoV- 19/Wuhan/WIV04/2019 (WIV04); GISAID accession no. EPI_ISL_402124), and variants thereof.
  • the SARS-CoV-2 strain may be a member of lineage A, A.1, A.2, A.3, A.5, B, B.1, B.1.1, B.2, B.3, B.4, B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), delta, kappa, and/or lambda.
  • the SARS-CoV-2 strain may be a member of lineage A.23.1, B.1.1.7 (alpha), B.1.351 (beta), B.1.258, B.1.526.2, B.1.616, B.1.617.1 (kappa), B.1.617.2 (delta), C36.3, C.37 (lambda), P.1 (gamma), B.1.1.529 (omicron), BA.1, BA.1.1, BA.2 BA.4, BA.5, BA.4.6, BA.2.75, BA.2.75.2, BA.2.3.20, BJ.1, BF.7, BQ.1, BQ.1.1, BS.1, BA.2.10.4, BN.1, XBB or XBB.1.
  • the strain may be an as-yet-unidentified strain of SARS-CoV-2 comprising mutations in the RBD already identified in the existing strains, as shown in Figure 1A. It has been surprisingly shown that certain antibodies retain neutralising activity against synthetic SARS-CoV-2 strains comprising combinations of mutations that render the strain more resistant to neutralisation from antibodies generated against early strains SARS-CoV-2.
  • the SARS-CoV-2 strain may comprise one or more mutations, e.g. in the spike protein, relative to the hCoV-19/Wuhan/WIV04/2019 (WIV04) (GISAID accession no. EPI_ISL_402124).
  • the SARS-CoV-2 strain may be a modified hCoV- 19/Wuhan/WIV04/2019 (WIV04) strain comprising one or more modifications, e.g. in the spike protein.
  • the mutation may be the mutations (e.g. substitutions) observed in the BA.2 strain of SARS-CoV-2.
  • the mutation may be the mutations (e.g. substitutions) observed in the BA.1, BA.1.1, BA.2 BA.4, BA.5, BA4.6, BA.2.75, BA.2.75.2, BA.2.3.20, BJ.1, BF.7, BQ.1, BQ.1.1, BS.1, BA.2.10.4, BN.1, XBB or XBB.1 strains of SARS-CoV-2.
  • the methods and uses of the invention may comprise inhibiting the disease state (such as COVID-19), e.g. arresting its development; and/or relieving the disease state (such as COVID-19), e.g. causing regression of the disease state until a desired endpoint is reached.
  • the methods and uses of the invention may comprise the amelioration or the reduction of the severity, duration or frequency of a symptom of the disease state (such as COVID-19) (e.g.
  • the symptoms or complications may be fever, headache, fatigue, loss of appetite, myalgia, diarrhoea, vomiting, abdominal pain, dehydration, respiratory tract infections, cytokine storm, acute respiratory distress syndrome (ARDS) sepsis, and/or organ failure (e.g. heart, kidneys, liver, GI, lungs).
  • coronavirus e.g. SARS-CoV-2
  • e.g. SARS-CoV-2 coronavirus
  • the methods and uses of the invention may comprise preventing the coronavirus infection from occurring in a subject (e.g. humans), in particular, when such subject is predisposed to complications associated with coronavirus infection.
  • the invention also relates to identifying subjects that have a coronavirus infection, such as by SARS-CoV-2.
  • the methods and uses of the invention may involve identifying the presence of coronavirus (e.g.
  • SARS-CoV-2 SARS-CoV-2
  • the detection may be carried out in vitro or in vivo.
  • the invention relates to population screening.
  • the invention relates to identifying any SARS-CoV-2 strain, as described herein.
  • the invention may also relate to a method of identifying escape mutants of SARS- CoV-2, comprising contacting a sample with a combination of antibodies of the invention and identifying if each antibody binds to the virus.
  • escape mutants refers to variants of SARS-CoV-2 comprising non-silent mutations that may affect the efficacy of existing treatments of SARS-CoV-2 infection.
  • the non-silent mutations is on an epitope recognised by a prior art antibody and/or antibodies described herein that specifically binds to an epitope of SARS-CoV-2, e.g. on the spike protein of SARS-CoV-2. If the antibody does not bind to the target, it may indicate that the target comprises a mutation that may alter the efficacy of existing SARS-CoV-2 treatments.
  • the methods and uses of the invention may include contacting a sample with an antibody or a combination of the antibodies of the invention, and detecting the presence or absence of an antibody-antigen complex, wherein the presence of the antibody-antigen complex indicates that the subject is infected with SARS-CoV-2.
  • in vitro detection techniques include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
  • In vivo techniques include introducing into a subject a labelled anti-analyte protein antibody.
  • the antibody can be labelled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the detection techniques may provide a qualitative or a quantitative readout depending on the assay employed.
  • the invention relates to methods and uses for a human subject in need thereof.
  • non-human animals such as rats, rabbits, sheep, pigs, cows, cats, or dogs is also contemplated.
  • the subject may be at risk of exposure to coronavirus infection, such as a healthcare worker or a person who has come into contact with an infected individual.
  • a subject may have visited or be planning to visit a country known or suspected of having a coronavirus outbreak.
  • a subject may also be at greater risk, such as an immunocompromised individual, for example an individual receiving immunosuppressive therapy or an individual suffering from human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS).
  • HIV human immunodeficiency syndrome
  • AIDS acquired immune deficiency syndrome
  • the subject may be asymptomatic or pre-symptomatic.
  • the subject may be early, middle or late phase of the disease.
  • the subject may be in hospital or in the community at first presentation, and/or later times in hospital.
  • the subject may be male or female. In certain embodiments, the subject is typically male.
  • the subject may not have been infected with coronavirus, such as SARS-CoV-2.
  • the subject may have a predisposition to the more severe symptoms or complications associated with coronavirus infections.
  • the method or use of the invention may comprise a step of identifying whether or not a patient is at risk of developing the more severe symptoms or complications associated with coronavirus.
  • the subject may or may not have been diagnosed to be infected with coronavirus, such as SARS-CoV- 2.
  • the invention relates to analysing samples from subjects.
  • the sample may be tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • the sample may be blood and a fraction or component of blood including blood serum, blood plasma, or lymph.
  • the sample is from a throat swab, nasal swab, or saliva.
  • the antibody-antigen complex detection assays may be performed in situ, in which case the sample is a tissue section (fixed and/or frozen) of the tissue obtained from biopsies or resections from a subject.
  • the antibodies pharmaceutical compositions and combinations may be administered subcutaneously, intravenously, intradermally, orally, intranasally, intramuscularly or intracranially.
  • the antibodies pharmaceutical compositions and combinations are administered intravenously or subcutaneously.
  • the dose of an antibody may vary depending on the age and size of a subject, as well as on the disease, conditions and route of administration.
  • Antibodies may be administered at a dose of about 0.1 mg/kg body weight to a dose of about 100 mg/kg body weight, such as at a dose of about 5 mg/kg to about 10 mg/kg.
  • Antibodies may also be administered at a dose of about 50 mg/kg, 10 mg/kg or about 5 mg/kg body weight.
  • a combination of the invention may for example be administered at a dose of about 5 mg/kg to about 10 mg/kg for each antibody, or at a dose of about 10 mg/kg or about 5 mg/kg for each antibody.
  • a combination may be administered at a dose of about 5 mg/kg total (e.g.
  • the antibody or combination of antibodies of the invention may be administered in a multiple dosage regimen. For example, the initial dose may be followed by administration of a second or plurality of subsequent doses. The second and subsequent doses may be separated by an appropriate time.
  • the antibodies of the invention are typically used in a single pharmaceutical composition/combination (co-formulated).
  • the invention also generally includes the combined use of antibodies of the invention in separate preparations/compositions.
  • the invention also includes combined use of the antibodies with additional therapeutic agents, as described above. Combined administration of the two or more agents and/or antibodies may be achieved in a number of different ways. In one embodiment, all the components may be administered together in a single composition.
  • each component may be administered separately as part of a combined therapy.
  • the antibody of the invention may be administered before, after or concurrently with another antibody, or binding fragment thereof, of the invention.
  • the antibody of the invention may be administered before, after or concurrently with an anti-viral agent or an anti-inflammatory agent.
  • the antibody contains a detectable label. Methods of attaching a label to an antibody are known in the art, e.g.
  • the antibody may be indirect labelled, e.g. by reactivity with another reagent that is directly labelled.
  • indirect labelling include detection of a primary antibody using a fluorescently-labelled secondary antibody and end-labelling of a DNA probe with biotin such that it can be detected with fluorescently-labelled streptavidin.
  • the detection may further comprise: (i) an agent known to be useful for detecting the presence of coronavirus, , e.g. SARS-CoV-2, or a protein or a fragment thereof, e.g.
  • kits for detecting the presence of coronavirus e.g. SARS-CoV-2, in a sample.
  • the kit may comprise: a labelled antibody or a combination of labelled antibodies of the invention; means for determining the amount of coronavirus, e.g.
  • the labelled antibody or the combination of labelled antibodies can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect coronavirus, e.g. SARS-CoV-2, in a sample.
  • the kit may further comprise other agents known to be useful for detecting the presence of coronavirus, as discussed above.
  • the antibodies or combinations of antibodies of the invention are used in a lateral flow test.
  • the lateral flow test kit is a hand-held device with an absorbent pad, which based on a series of capillary beds, such as pieces of porous paper, microstructured polymer, or sintered polymer.
  • the test runs the liquid sample along the surface of the pad with reactive molecules that show a visual positive or negative result.
  • the test may further comprise using other agents known to be useful for detecting the presence of coronavirus, e.g. SARS-CoV-2, or a fragment thereof, as discussed above, such as anti- an anti-nucleocapsid antibody.
  • coronavirus e.g. SARS-CoV-2
  • a fragment thereof such as anti- an anti-nucleocapsid antibody.
  • different applications of the disclosed antibodies combinations, or pharmaceutical compositions of the invention may be tailored to the specific needs in the art.
  • gaps can be introduced in a first sequence for optimal alignment with a second sequence).
  • the nucleotide or amino acid residues at each position are then compared.
  • a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the nucleotides or amino acids are identical at that position.
  • sequence comparison is carried out over the length of the reference sequence.
  • test sequence For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: 3, SEQ ID NO: 3 would be the reference sequence.
  • the skilled person would carry out an alignment over the length of SEQ ID NO: 3, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 3. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 3. If the sequence is shorter than SEQ ID NO: 3, the gaps or missing positions should be considered to be non-identical positions.
  • the skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences.
  • a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • CDRH heavy chain
  • CDRL light chain variable domain
  • Example 1 Emerging BA.2, BA.4 and BA.5 sublineages At present a number of lineages are growing rapidly from within both the BA.2 and BA.5 branches. Most striking is the large degree of convergent evolution, particularly at antigenic RBD positions such as 346, 444, 452, 460, 486, 490, 493 and 494.
  • BA.4/5 branches which contain L452R, F486V and the reversion R493Q
  • BA.4.6 and BF.7 R346T
  • BA.4.7 R346S
  • BQ.1 K444T, N460K
  • BQ.1.1 R346T, K444T, N460K
  • BA.2.75 branch which contains G339H, G446S, N460K and the reversion R493Q
  • BA.2.75.2 R346T and F486S and BA.2.75 mutations
  • BN.1 aka BA.2.75.5.1 with R346T, K356T, F490S and BA.2.75 mutations
  • BM.1.1.1 aka BA.2.75.3.1.1.1 with R346T, F486S, F490S and BA.2.75 mutations).
  • BJ.1 (aka BA.2.10.1.1; G339H, R346T, L368I, V445P, G446S, V483A and F490V), BA.2.10.4 (G446S, F486P, S494P and the R493Q reversion), BS.1 (aka BA.2.3.2.1; R346T, L452R, N460K, G476S and the Q493R reversion), BA.2.3.20 (K444R, N450D, L452M, N460K, E484R and the Q493R reversion), and finally a BJ.1 x BM.1.1.1 (aka BA.2.75.3.1.1.1) recombinant, XBB (which relative to BA.2 contains R346T, L368I, V445P, G446S, N460K, F486S, F490S and the Q493R reversion).
  • BJ.1 (aka BA.2.10.1.1; G
  • BA.2 variant lineages contain deletions or mutations in the NTD, often similar to those seen in the VOCs, for example ⁇ 144 in BJ.1, BS.1, and BA.2.10.4 (previously seen in Alpha and BA.1) and NSP12 G671S in BJ.1, XBB and BA.2.10.4 (previously seen in Delta).
  • FRNT focus reduction neutralization assays
  • FIG. 4A B shows antibody binding sites on the RBD surface for four sets of responses following the pandemic
  • Figure 4C D.
  • the BA.2 mAbs segregate into 3 adjacent clusters, two of which (left shoulder and neck in our anatomical definition of RBD topology (Dejnirattisai et al., 2021a)) are hotspots for the binding of potent mAbs against early pandemic virus (Dejnirattisai et al., 2021a), whilst the third cluster, which is termed herein the right chest, spans between the neck and right flank epitopes in early pandemic responses (note that in the early responses right flank binders were not found to be potent (IC50 ⁇ 100ng/ml)).
  • Example 4 Structural analysis To obtain more detailed insight into selected BA.2 mAbs we determined the cryo- EM structure of the complex of Delta-RBD with BA.2-23 and BA.2-36, along with the early pandemic mAb-45 (Dejnirattisai et al., 2021a) and EY6A (Zhou et al., 2020) and also the X-ray structures of Delta-RBD with BA.2-10 and EY6A, Delta-RBD with BA.2-13 and C1 nanobody, and Delta-RBD with BA.2-36 ( Figures 5, 6, 9, 14 and 15). BA.2-10 (IGHV3-9) binds at the right chest region of the RBD as expected from the competition mapping.
  • IGHV3-9 binds at the right chest region of the RBD as expected from the competition mapping.
  • BA.2-13 (IGHV3-15) also binds to the right chest region but higher and more towards the midline of the RBD than BA.2-10 and in a very different orientation, so that its LC contact area on the RBD largely overlaps with the footprint of BA.2-10 HC.
  • BA.2-10 CDR-H3 overlaps the CDR-H3 contacts of BA.2-13.
  • BA.2-13 interacts with RBD residues 346, 444-445, 450 and 452 ( Figure 5O-5S). Variants containing the R346T mutation either seriously reduce or completely knockout neutralization of BA.2-13.
  • BA.2.3.20 which contains K444R, N450D and L452M mutations, also completely knocks out the activity of BA.2-13.
  • BA.2-13 contacts RBD residues 452 and 484, it is not sensitive to mutations L452R or E484K as it fully neutralizes Beta, Gamma and Delta variants.
  • BA.2-36 (IGHV4-61) binds to the right chest region in a similar position and orientation to BA.2-13 ( Figure 5E-6I). It also contacts RBD residues 346, 444, 450 and 452, explaining its similar variant neutralization profile to BA.2-13, with neutralization seriously reduced or completely knocked out for variants containing the R346T mutation and byBA.2.3.20.
  • BA.2-23 is an IGHV3-53 family antibody and binds the left shoulder in the expected position.
  • BA.2.3.20 which has a number of mutations across the top of the shoulders, neck and at the back of the RBD ( Figure 1A, B) also caused large disruption; 13/25 mAbs were knocked out with only 6/25 retaining IC50 activity at ⁇ 100 ng/ml.
  • BJ.1 which has a different set of mutations across the top of the RBD
  • activity of 13/25 mAbs was completely knocked out with only 9/25 retaining IC50 activity at ⁇ 100 ng/ml.
  • Activity of most mAb was reduced or completely knocked out against BQ.1, BQ.1.1, XBB and XBB.1, whilst activity of some mAb was preserved on BN.1, and BA.2.10.4.
  • Omi-42 Only a single mAb, Omi-42 was unaffected by all variants. Omi-42 is unusual as it binds at the back of the left shoulder of the RBD ( Figure 4, right panel) (Nutalai et al., 2022) in a region that has not yet been targeted for mutation by the set of newly emerging BA.2 variants, perhaps because of the relative rarity of antibodies binding in this region ( Figures 1B,4).
  • Example 6 Escape from therapeutic mAbs The activity of a panel of mAbs that have been developed for clinical use (Dong et al., 2021; Sun and Ho, 2020; Weinreich et al., 2021; Yuan et al., 2022) was tested.
  • Example 7 Severe knock down of serum neutralization titres Neutralization on serum collected 28 days following a third dose of Pfizer BNT162b2 vaccine (Polack et al., 2020) was tested. In cases infected with BA.1, BA.2 and BA.4/5. The characteristics of these subjects are described in the methods.
  • BA.2.75.2 BA.2.75 showed only a modest reduction compared to BA.2.
  • BA.2 antibodies cluster tightly in two of the three regions characteristic of potent early pandemic responses, although mutations have knocked out many of the so-called ‘public’ responses, including gene families IGHV3-53/66, diminishing the potent left shoulder responses.
  • the panel likely contains a mix of both, and the strong representation of weaker neutralizing right flank antibodies in the early responses suggests that the chest binders which overlap these may have been matured to higher affinity and selected because this portion of the RBD has been subjected to less mutational change in BA.1 and BA.2.
  • the change away from antibodies binding more directly on the ACE2 footprint is highlighted by the substantial role in early responses played by public gene families such as IGHV3-53/66 (He et al., 2022; Yuan et al., 2020), IGHV1-69, IGHV3-9 and IGHV1-58 (Nutalai et al., 2022).
  • IGHV1-58 binding was knocked out by the G496S mutation in BA.1 (Dejnirattisai et al., 2022) whilst, as described herein, the BA.2 IGHV1-69 mAbs are almost all knocked out by BA.4.
  • the few members of the most frequently discovered gene family of all, IGHV3-53/66, that are active against BA.2 are severely affected by the F486S mutation in BA.75.2 as are the larger number of IGHV3-53/66 mAb isolated following BA.1 infection.
  • Bacterial Strains and Cell Culture Vero (ATCC CCL-81) and VeroE6/TMPRSS2 cells were cultured at 37 °C in Dulbecco’s Modified Eagle medium (DMEM) high glucose (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 2 mM GlutaMAX (Gibco, 35050061) and 100 U/ml of penicillin–streptomycin.
  • DMEM Modified Eagle medium
  • FBS fetal bovine serum
  • GlutaMAX Gibco, 35050061
  • Human mAbs were expressed in HEK293T cells cultured in FreeStyleTM 293 Expression Medium (Cat# 12338018, GibcoTM) at 37 °C with 5% CO2.
  • HEK293T ATCC CRL-112678 cells were cultured in DMEM high glucose (Sigma- Aldrich) supplemented with 10% FBS, 1% 100X Mem Neaa (Gibco) and 1% 100X L- Glutamine (Gibco) at 37 °C with 5% CO 2 .
  • DMEM high glucose Sigma- Aldrich
  • FBS 1% 100X Mem Neaa
  • HEK293T cells were cultured in DMEM high glucose (Sigma) supplemented with 2% FBS, 1% 100X Mem Neaa and 1% 100X L-Glutamine at 37 °C for transfection.
  • BA.2 RBD were expressed in HEK293T (ATCC CRL-11268) cells cultured in FreeStyleTM 293 Expression Medium (Cat# 12338018, GibcoTM) at 37 °C with 5% CO2.
  • E.coli DH5 ⁇ bacteria were used for transformation and large-scale preparation of plasmids. A single colony was picked and cultured in LB broth at 37 °C at 200 rpm in a shaker overnight..
  • Diagnosis was confirmed through reporting of symptoms consistent with COVID-19, hospital presentation, and a test positive for SARS-CoV-2 using reverse transcriptase polymerase chain reaction (RT-PCR) from an upper respiratory tract (nose/throat) swab tested in accredited laboratories and lineage sequence confirmed through national reference laboratories in the United Kingdom.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • a blood sample was taken following consent at least 14 days after PCR test confirmation.
  • Clinical information including severity of disease (mild, severe or critical infection according to recommendations from the World Health Organisation) and times between symptom onset and sampling and age of participant was captured for all individuals at the time of sampling.
  • Diagnosis was confirmed through reporting of symptoms consistent with COVID-19 or a positive contact of a known Omicron case, and a test positive for SARS-CoV-2 using reverse transcriptase polymerase chain reaction (RT-PCR) from an upper respiratory tract (nose/throat) swab tested in accredited laboratories and lineage sequence confirmed through national reference laboratories.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • a blood sample was taken following consent at least 10 days after PCR test confirmation.
  • Clinical information including severity of disease (mild, severe or critical infection according to recommendations from the World Health Organisation) and times between symptom onset and sampling and age of participant was captured for all individuals at the time of sampling.
  • Diagnosis was confirmed through reporting of symptoms consistent with COVID-19, hospital presentation, and a test positive for SARS-CoV-2 using reverse transcriptase polymerase chain reaction (RT-PCR) from an upper respiratory tract (nose/throat) swab tested in accredited laboratories and lineage sequence confirmed through national reference laboratories in the United Kingdom.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • a blood sample was taken following consent at least 14 days after PCR test confirmation.
  • Clinical information including severity of disease (mild, severe or critical infection according to recommendations from the World Health Organisation) and times between symptom onset and sampling and age of participant was captured for all individuals at the time of sampling.
  • Sera from Pfizer vaccinees Pfizer vaccine serum was obtained from volunteers who had received three doses of the BNT162b2 vaccine.
  • Vaccinees were Health Care Workers, based at Oxford University Hospitals NHS Foundation Trust, not known to have prior infection with SARS-CoV-2 and were enrolled in the OPTIC Study as part of the Oxford Translational Gastrointestinal Unit GI Biobank Study 16/YH/0247 [research ethics committee (REC) at Sale & The Humber – Sheffield] which has been amended for this purpose on 8 June 2020.
  • the study was conducted according to the principles of the Declaration of Helsinki (2008) and the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines. Written informed consent was obtained for all participants enrolled in the study. Participants were sampled approximately 28 days (range 25-56) after receiving a third “booster dose of BNT162B2 vaccine.
  • BA.2 S-specific single B cells sorting was performed as previously described (Dejnirattisai et al., 2021a). Briefly, PBMC were stained with LIVE/DEAD Fixable Aqua dye (Invitrogen) followed by recombinant trimeric S-twin-Strep of BA.2.
  • IgG+ memory B cells were gated as CD19+, IgG+, CD3-, CD14-, CD56-, CD16-, IgM-, IgA- and IgD-, and S+ was further selected and single cells were sorted into 96-well PCR plates with 10 ⁇ l of catching buffer (Tris, Nuclease free- H2O and RNase inhibitor). Plates were briefly centrifuged at 2000xg for 1 min and left on dry ice before being stored at -80 °C. Cloning and expression of BA.2 S-specific human mAbs BA.2 S-specific human mAbs were cloned and expressed as described previously (Dejnirattisai et al., 2021a).
  • genes for Ig IGHV, Ig V ⁇ and Ig V ⁇ were recovered from positive wells by RT-PCR. Genes encoding Ig IGHV, Ig V ⁇ and Ig V ⁇ were then amplified using Nested-PCR by a cocktail of primers specific to human IgG. PCR products of HC and LCs were ligated into the expression vectors of human IgG1 or immunoglobulin ⁇ -chain or ⁇ -chain by Gibson assembly (Gibson, 2011).
  • plasmids encoding HCs and LCs were co-transfected by PEI-transfection into a HEK293T cell line, and supernatants containing mAbs were collected and filtered 4-5 days after transfection, and the supernatants were further characterized or purified.
  • ACE2 binding inhibition assay by ELISA MAXISORP immunoplates were coated with 5 ⁇ g/ml of purified ACE2-His protein overnight at 4 °C and then blocked by 2% BSA in PBS. Meanwhile, mAbs were serially diluted and mixed with 2.5 ⁇ g/ml of recombinant BA.1 trimeric S-twin-Strep.
  • Antibody-S protein mixtures were incubated at 37°C for 1 hr. After incubation, the mixtures were transferred into the ACE2-coated plates and incubated for 1 hr at 37 °C. After wash, StrepMAB-Classic (2-1507-001, iba) was diluted at 0.2 ⁇ g/ml by 2% BSA and used as primary antibody followed by Goat anti-mouse IgG-AP (#A16093, Invitrogen) at 1:2000 dilution. The reaction was developed by adding PNPP substrate and stopped with NaOH. The absorbance was measured at 405nm. The ACE2/S binding inhibition was calculated by comparing to the antibody-free control well. IC50 was determined using the Probit program from the SPSS package.
  • Pseudovirus plasmid construction and lentiviral particles production Pseudotyped lentivirus expressing SARS-CoV-2 S proteins from ancestral strain (Victoria, S247R), BA.2 and BA.4/5 were constructed as described previously (Nie et al., 2020; Liu et al., 2021; Nutalai et al., 2022; Tuekprakhon et al., 2022).
  • BA.2.3.20 To create BA.2.3.20, we introduced M153T, N164K, H245N, G257D, K444R, N450D, L452M, and N460K, as well we changed L452R in BA.2 into L452M, and reversed 493R in BA.2 to 493Q as in the ancestral strain.
  • BA.2.10.4 was generated by introducing W64R, G446S, F486P, R493Q, S494P and P1143L into BA.2 backbone, as well as deleting aa142-144 and 243-244.
  • BS.1 To create BS.1, we added aa144 deletion, G257V, R346T, L452R, G476S, N460K, F486S, F490S, and Q493H reversion into BA.2 backbone.
  • BA.2.75 vector was used as backbone with addition of R346T, K357T and F490S.
  • V83A To construct XBB, we added V83A, aa144 deletion, H146Q, Q183E, V213E, R346T, L368I, V445P, G446S, N460K, F486S, and F490S into BA.2 backbone, also change 339D in BA.2 to 339H, reverse 493Q to H.
  • XBB.1 was constructed by adding G252V into XBB. Same method was used to construct BF.7, BA4.6, BQ.1 and BQ.1.1 with new RBD mutations from BA.2 lineages.
  • R346T was added into BA.4 backbone.
  • R346T and N658S were introduced into BA.4 backbone.
  • BQ.1 was constructed by introducing K444T and N460K into BA.4 backbone, while BQ.1.1 was to add R346T into BQ.1 construct.
  • the resulting pcDNA3.1 plasmid carrying S gene was used for generating pseudoviral particles together with the lentiviral packaging vector and transfer vector encoding luciferase reporter.
  • Stable HEK293T/17 cells expressing human ACE2 were then added to the mixture at 1.5 ⁇ 10 4 cells/well.48 hr post infection, culture supernatants were removed and 50 ⁇ L of 1:2 Bright-Glo TM Luciferase assay system (Promega, USA) in 1 ⁇ PBS was added to each well. The reaction was incubated at room temperature for 5 mins and firefly luciferase activity was measured using CLARIOstar® (BMG Labtech, Ortenberg, Germany). The percentage neutralization was calculated relative to the control. Probit analysis was used to estimate the dilution that inhibited half maximum pseudotyped lentivirus infection (PVNT50).
  • Fabs were digested from purified IgGs with papain using a Pierce Fab Preparation Kit (Thermo Fisher), following the manufacturer’s protocol.
  • Competition assays of anti-Omicron BA.2 RBD mAbs Competition assays of anti-Omicron BA.2 RBD mAbs were performed on an Octet Red 96e machine (Sartorius) using Octet Anti-HIS (HIS2) Biosensors (Sartorius). His- tagged Omicron BA.2 RBD dissolved in the running buffer (10 mM HEPES, pH 7.4 and 150 mM NaCl) was used as the ligand and was first immobilized onto the biosensors.
  • the biosensors were then washed with the running buffer to remove unbound RBD.
  • Each biosensor was dipped into different saturating mAbs (Ab1) to saturate the bound RBD, except one biosensor was dipped into running buffer in this step, acting as the reference.
  • All biosensors were washed with the running buffer again and dipped into wells containing the same competing antibody (Ab2).
  • the y axis values of signals of different saturating antibodies in this step were divided by the value of the reference channel to get ratio results of different Ab1-Ab2 pairs. Ratio results close to 0 indicated total competition while 1 indicated no competition.
  • Antibody mapping to RBD surface All BA.2 antibodies and several antibodies with previously solved structures (mAb- 45, -58, -278, EY6A AZD8895, AZD1061) (Dejnirattisai et al., 2021a) were used in a competition assay prepared for antibody mapping to the RBD surface. Antibody mapping was carried out using mabscape (Dejnirattisai et al., 2021a) and cluster4x (Ginn, 2020).
  • Mid-point positions of mAb-45, -58, -278, EY6A AZD8895, AZD1061 were calculated from crystal structures and used to seed the analysis in 1000 Monte Carlo runs, whereas several known structural positions were not included in the analysis and used as a cross-check. A total of 178 Monte Carlo runs formed a single cluster with the lowest score and these were used to calculate average positions for BA.2 antibodies.
  • Diffraction data for Delta RBD/EY6A/BA2-10 and Delta RBD/BA2-36 were collected at 100 K at beamline I03 of Diamond Light Source, UK, using the automated queue system that allows unattended automated data collection (https://www.diamond.ac.uk/Instruments/Mx/I03/I03- Manual/Unattended-Data-Collections.html).3600 diffraction images of 0.1o each were collected from a single crystal of the Delta-RBD/BA.2-10/EY6A complex.7200 diffraction images were collected from two crystals for Delta-RBD/BA.2-36 complex.360o of diffraction data for Delta RBD/BA.2-13/C1 were collected at beamline I04.
  • the one clear class average containing 167492 particles, commensurate with an RBD decorated with four fabs, was then refined using non-uniform refinement before unbinning and further refinement to a final reported resolution of 2.9 ⁇ resolution (-92.9 reported global b-factor).
  • Phylogenetic tree The phylogenetic tree was generated by pruning the Nextclade reference tree (https://nextstrain.org/nextclade/sars-cov-2/21L) which contains one sequence per Pango lineage. The tree was generated with a Snakemake workflow using the Augur toolchain.
  • the workflow is available at: https://github.com/neherlab/nextclade_data_workflows/tree/27bf7e0b4f62cbbbc9a8ac96db 1587cd76b3ae10
  • the topology of the tree was constrained using the Usher tree that incorporates nearly all sequences available through GISAID (Turakhia et. al, 2021).
  • the tree was pruned using a BioPython script and visualized using Figtree. Quantification and statistical analysis Statistical analyses are reported in the results and figure legends. Neutralization was measured on pseudovirus. The percentage reduction was calculated and IC 50 determined using the probit program from the SPSS package.
  • Example 8 Potently neutralising antibodies isolated following Delta infection DP21 and DP22 antibodies were generated from two volunteers who had recovered from sequence confirmed Delta infection having previously been vaccinated with 2 doses of the Pfizer-BioNtech vaccine. Samples were taken 31-33 days after symptom onset. Memory B cells were isolated by staining PBMC with trimeric double Strep-tagged Delta Spike (S), and IgG + B cells binding Delta S were single cell sorted.
  • S trimeric double Strep-tagged Delta Spike
  • IgVH and IgVL sequences were recovered by Reverse-transcript PCR (RT-PCR) following by Nested- PCR, and full-length heavy chain (HC) and light chain (LC) expressing plasmids were created using Gibson assembly. Assembly products were transfected into HEK-293T cells in 24-well plates, and supernatant harvested and tested in ELISA and neutralisation assay against Delta variant. Potent neutralising antibodies were expressed by transfecting 293T cells and purified by ProteinA/G beads.
  • RT-PCR Reverse-transcript PCR
  • HC heavy chain
  • LC light chain
  • Example 9 Cross reactivity of DP21, DP22, DP31 and SARS1-34
  • live virus neutralisation assay of DP21 and DP22 were performed using the following pseudo-viruses: Victoria, Delta, BA.1, BA.2, BA.3, BA.1.1, BA.2.12.1, BA.4 and BA.5
  • Figure 10A pseudoviral neutralization assays on Victoria, Delta, BA.1, BA.2, BA.3, BA.4, BA.2.75, BA.4.6, BA.2.75.2, BA.2.3.20 and BA.4 with multiple RBD mutations
  • Figure 10B pseudoviral neutralization assays on Victoria, BA.2, BA.4/5, BA.2.75, BA.4.6, BA.2.75.2, BA.2.3.20, BF.7, BQ.1, BJ.1, BS.1, BA.2.10.4, BN.1, XBB and XBB.1
  • Figure 11A pseudoviral neutralization assays on Victoria, BA.2, BA.4/5, BA.2.75, BA.4.6, BA.2.75.2, BA.
  • a neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science 369, 650-655. Dejnirattisai, W., Huo, J., Zhou, D., Zahradnik, J., Supasa, P., Liu, C., Duyvesteyn, H.M.E., Ginn, H.M., Mentzer, A.J., Tuekprakhon, A., et al. (2022). SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses. Cell 185, 467-484 e415.
  • a human coronavirus evolves antigenically to escape antibody immunity.
  • Pre-clustering data sets using cluster4x improves the signal-to-noise ratio of high-throughput crystallography drug-screening analysis.
  • Macromolecular structure determination using X-rays, neutrons and electrons recent developments in Phenix. Acta Crystallogr D Struct Biol 75, 861-877. Liu, C., Ginn, H.M., Dejnirattisai, W., Supasa, P., Wang, B., Tuekprakhon, A., Nutalai, R., Zhou, D., Mentzer, A.J., Zhao, Y., et al. (2021a). Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum. Cell 184, 4220-4236 e4213.
  • Beta mAb response underscores the antigenic distance to other SARS-CoV-2 variants. Cell, Host and Microbe 30, 53-68.
  • Starr TN Greaney AJ, Hilton SK, Ellis D, Crawford KHD, 3,ns AS, Navarro MJ, Bowen JE, Tortorici MA, Walls AC et al. (2020). Deep Mutational Scanning of SARS- CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding. Cell 182,1295-1310.
  • Starr TN Greaney AJ, Hannon WW, Loes AN, Hauser, K, Dillen, J.R., Ferri, E., Farrell, AG, Dadonaite, B., McCallum, M. et al. (2022).
  • SARS-CoV-2 variants of concern alpha, beta, gamma and delta have extended ACE2 receptor host ranges.JGenVirol103.doi:10.1099/jgv.0.001735. Tuekprakhon, A., Nutalai, R., Dijokaite-Guraliuc, A., Zhou, D., Ginn, H.M., Selvaraj, M., Liu, C., Mentzer, A.J., Supasa, P., Duyvesteyn, H.M.E., et al. (2022). Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum.
  • LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants. Cell Rep 39, 110812. Winter, G. (2010). xia2: an expert system for macromolecular crystallography data reduction. Journal of applied crystallography 43, 186-190.

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Abstract

La présente invention concerne des anticorps capables de se lier à la protéine de spicule du coronavirus SARS-CoV-2, ainsi que des procédés et des utilisations de ceux-ci dans la prévention, le traitement et/ou le diagnostic d'infections à coronavirus et de maladies et/ou de complications associées à des infections à coronavirus, y compris la COVID-19.
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