Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—RNA viruses
  • C07K16/102—Coronaviridae (F)
  • C07K16/104—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • 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
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/35—Valency
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/567—Framework region [FR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2469/00—Immunoassays for the detection of microorganisms
  • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

• WO 2021/224606 DOMAIN ANTIBODIES BINDING TO SARS-COV-2 £9T/£ B , 2 93 ⁇ 4 ⁇ ?3 ⁇ 4 9
• the invention provides improved single domain antibodies that target SARS-CoV-2, the use of said single domain antibodies in treating and/or preventing coronavirus, as well as the use of said single domain antibodies in the detection and diagnosis of coronavirus using various methods, assays and kits.
• Single domain antibodies or nanobodies are recombinant antigen-specific variable domains (VHHs) derived from the heavy chain only subset of camelid immunoglobulins. Their small molecular size, facile expression, high affinity and stability have combined to make them unique targeting reagents with numerous applications in the biomedical sciences. Nanobodies have emerged as alternatives to conventional antibodies for the development of diagnostic reagents, and for use in non-invasive bioimaging. The therapeutic potential of nanobodies is being explored in a number of indications, including oncology and inflammatory diseases (Revets, De Baetselier et al. 2005, Chanier and Chames 2019).
• SARS-Cov-2 SARS-nCoronavirus 2
• SARS-Cov-2 SARS-nCoronavirus 2
• NP Nucleocapsid
• the S protein plays the key role in viral attachment, fusion and entry into host cells and comprises a N-terminal (S1) subunit responsible for virus-receptor binding and C-terminal domain (S2) that mediates membrane fusion (Li 2016).
• S1 N-terminal subunit responsible for virus-receptor binding and C-terminal domain (S2) that mediates membrane fusion
• S2 C-terminal domain
• the cell surface receptor for SARS-CoV2 like the related coronavirus SARS- CoV, is angiotensin-converting enzyme 2 (ACE-2) and the interaction has been mapped to an approximately 300 amino acid receptor-binding domain (RBD) in S1 (Wan, Shang et al. 2020, Yan, Zhang et al. 2020).
• the present invention provides single domain antibodies that specifically bind to the receptor biding domain of the S-protein of SARS-CoV-2.
• a single domain antibody comprising a complementary determining region, complementary determining region 3 (CDR3), is provided.
• CDR3 complementary determining region 3
• a single domain antibody comprising a CDR2 and a CDR3 is provided.
• a single domain antibody comprising a CDR1 , a CDR2 and a CDR3 is provided.
• an anti-SARS-CoV-2 single domain antibody comprising an amino acid sequence having at least 70% identity to a sequence selected from the group consisting of: SEQ 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106 and 107 is provided.
• a polynucleotide sequence is provided encoding a single domain antibody of the invention.
• an affinity matured mutant of a single domain antibody of the invention is provided.
• composition comprising a single domain antibody of the invention.
• a single domain antibody of the invention or a pharmaceutical composition of the invention for use in medicine is provided.
• a method for the treatment of a coronavirus in a subject comprising administering to a subject a therapeutically active amount of a single domain antibody of the invention.
• a single domain antibody of the invention in the manufacture of a medicament for use in the treatment and/or prevention of a coronavirus is provided.
• methods for diagnosing a coronavirus infection in a subject are provided.
• FIG. 1 The Spike protein of SARS-CoV-2 drives infection.
• FIG. 1 Schematic representation of the Spike protein of SARS-CoV-2.
• the Spike protein is composed of the S1 and S2 subunits.
• the S1 domain contains the receptor binding domain (RBD) which is highlighted in red.
• RBD receptor binding domain
• Camelids have antibodies that are dimers of a single chain. The constant region is in black and the variable region is in grey. When the VHH domain is expressed on its own, it is termed a nanobody.
• a topology diagram shows the nanobody is composed of two b-sheets. Three loops - complementary-determining region 1 (CDR1), CDR2 and CDR3 - control antigen binding and are highlighted in darker grey.
• FIG. 1 Laboratory-matured nanobodies bind to RBD and spike proteins with high affinity. a) Maturation by mutagenesis of CDR3 region of H11 resulted in H11-D4 and H11-H4. The five changes from the parent are shown in bold.
• RBD was bound by CR3022 (immobilized as CR3022-Fc on the chip).
• H11-H4 binding occurred with similar on and off rates, indicating that H11-H4 and CR3022 recognize different epitopes on RBD.
• the response for the RBD H11-H4 mixture was larger, consistent with an H11-H4-RBD complex binding to CR3022.
• Antibody E08R was again used as a negative control.
• the spike protein shows binding to CR3022 in the presence or absence of H11-H4. Data for H11-D4 are provided in Figure 6F.
• ITC measurements show a KD of 12 ⁇ 1.5 nM and a 1:1 ratio for H11-H4 and RBD association. Replicates and data for H11-D4 are provided in Figure 7A.
• ITC measurements show a KD of 44 ⁇ 3 nM and a 1:1 ratio for association between spike protein and H11-H4. Replicates and data for H11-D4 are provided in Figure 7B.
• Biotinylated RBD was mixed with analytes at various ratios and then added to MDCKSIAT1 cells stably expressing human ACE2. The amount of biotinylated RBD bound was measured. Experiments were performed in duplicate with the mean ⁇ SD are shown.
• Biotinylated ACE2-Fc was mixed with analytes at various ratios and then added to MDCKSIAT1 cells stably expressing RBD. The amount of biotinylated ACE2-Fc bound was measured. Experiments were performed in duplicate with the mean ⁇ SD are shown.
• H11-H4-FC (6 nM, 95 % Cl 3 - 9 nM) and H11-D4-Fc (18 nM, 95 % Cl 9 - 68 nM) show potent neutralization of live wild type virus. 95 % confidence intervals are shown as dashed lines. Raw data plots are shown in Figure 8.
• H11-H4-Fc shows similar neutralization (ND504 nM) of live wild type virus in a Vero cellbased assay in Oxford.
• CR3022 is shown as a positive control for this assay system and is similar to a previous report.
• Experimental plates are shown in Figure 8.
• H11-H4 and CR3022 have different binding epitopes on RBD and show additive neutralization activities.
• H11-D4 behaved identically to H11-H4 ( Figure 2C).
• H11-D4 behaved identically to H11-H4 ( Figure 2D).
• Figure 7 ITC measurements of nanobodies binding to RBD or Spike.
• the concentration of neutralizing agent was held constant across a row and decreased on subsequent rows.
• the agent was tested against high and low virus concentrations.
• FIG. 10 Cryo-EM of the H11-H4-Spike complex.
• FIG. 12 Further analysis of the cryo-EM nanobody-Spike complexes.
• Figure 13 Further structural analysis of nanobody-RBD crystal structures.
• VHH72 black and H11-H4 (yellow) recognize different epitopes on RBD (red).
• VHH72 black and H11-H4 (yellow) recognize different epitopes on RBD (red).
• Figure 16 The key interactions that stabilize the H11-D4-RBD complex.
• Arg98 is the only change in the CDR3 loop that does not contact RBD; instead, this residue stabilizes the loop structure.
• NbRBD_H11-D4 Competition assay NbRBD_H11-D4 with ACE2-Fc IgG immobilized onto the sensor chip. NbRBD_H11-D4 is competitive with ACE-2 for binding to RBD.
• the ternary complex CR3022, RBD and NBRBD_H11-D4 shows the nanobody and antibody recognise entirely different epitopes.
• SARS-CoV or the related MERS-CoV have been shown to both block virus-receptor binding and in some cases neutralize the virus both in vitro and in vivo (Zhu, Chakraborti et al. 2007, Zhao, He et al. 2018). It has also been shown that antibodies from infected patients bind to the S protein et al. (Jan ter Meulen Edward N. van den Brink Leo L. M. Poon 2006, Prabakaran, Gan et al. 2006, Zhu, Chakraborti et al. 2007). Given the role of the S protein in the pathogenicity of the SARS-CoV2, and potential as a target for vaccine and therapeutic development (Du, He etal.
• Constant substitution refers to amino acid substitutions that do not materially affect the function of a protein (for example the ability to bind to a specific target, in particular the coronavirus spike protein of SARS-CoV-2 in the context of the invention, or the ability to elicit an immune response in a subject).
• the skilled person readily understands the properties of amino acids and can readily make a conservative substitution without materially altering the properties of the resulting polypeptide. Examples of conservative substitutions are provided in the table below.
• “Deletion” as used herein refers to the removal of an amino acid in a polypeptide sequence (i.e. the replacement of one amino acid with no amino acid such that the amino acid sequence is one amino acid shorter in length). Deletion can also refer to polynucleotide sequences and the removal of one nucleic acid from a polynucleotide sequence (the replacement of one nucleic acid with no nucleic acid such that the polynucleotide sequence is one nucleic acid shorter in length).
• Identity is the degree to which two sequences are related, as determined by comparing two or more polypeptide of polynucleotide sequences. Identity can be determined using the degree of relatedness of two sequences to provide a measurement of to what extent the two sequences match. Numerous programs are well known by the skilled person for comparing polypeptide or polynucleotide sequences, for example (but not limited to the various BLAST and CLUSTAL programs. Percentage identity can be used to quantify sequence identity. To calculate percentage identity, two sequences (polypeptide or nucleotide) are optimally aligned (i.e.
• amino acid or nucleic acid residue at each position is compared with the corresponding amino acid or nucleic acid at that position.
• optimal sequence alignment can be achieved by inserting space(s) in a sequence to best fit it to a second sequence.
• the number of identical amino acid residues or nucleotides provides the percentage identity, i.e. if 9 residues of a 10 residue long sequence are identical between the two sequences being compared then the % identity is 90%. Percentage identity is generally calculated along the full length of the two sequences being compared.
• Insertion refers the addition of an amino acid in a polypeptide sequence (i.e. insertion of one amino acid means one new amino acid is added into in an existing amino acid sequence such that the amino acid sequence is one amino acid longer in length). Insertion can also refer to polynucleotide sequences and the addition of one nucleic acid to a polynucleotide sequence (i.e. insertion of one nucleic acid means one new nucleic acid is added into in an existing polynucleotide sequence such that the nucleic acid sequence is one amino acid longer in length).
• Modification refers to an alteration of an amino acid residue in a polypeptide sequence. The modification can be a substitution, deletion or insertion, as defined herein. Modification can also refer to polynucleotide sequences.
• Single domain antibody refers to a variable region of a heavy chain of an antibody, wherein the variable region is derived from a heavy chain only (i.e. devoid of a light chain) subset of camelid immunoglobulins.
• the term single domain antibody can be used interchangeably with (variable domain of camelid heavy-chain-only antibody, VHH) and Nanobody®.
• a single domain antibody is used to refer to a single heavy chain variable region that can bind the spike protein of a coronavirus, preferably SAR- CoV-2.
• the antibody can be affinity matured, humanized or modified, as described herein. This single domain antibody can be conjugated to other components.
• substitution refers the replacement of amino acid with a different amino acid. Substitution can also refer to polynucleotide sequences, i.e. the replacement of one nucleic acid with a different nucleic acid. A substitution can be a conservative substitution, as defined above.
• the single domain antibodies of the invention are based on 13 VHH sequences having positive binding to the receptor binding domain of the S protein of SARS-CoV-2, namely NbRBD_H11 , NbRBD_A7, NbRBD_F9, NbRBD_C10, NbRBD_B11, NbRDB_E11 , NbRBD_D1, NbRBD_G7, NbRBD_F5, NbRBD_G11, NbRBD_B4, NbRBD_G9 and NbRBD_C7 (amino acid sequences provided as SEQ ID NOs: 61-73, polynucleotide sequences provided as SEQ ID NOs: 74-86 respectively).
• NbRBD_H11-D4 NbRBD_H11-H4
• NbRBD_H11-H6 NbRBD_H11-A10
• NbRBD_H11-B5 NbRBD_H11-A7
• NbRBD_H11-F7 NbRBD_H11-F6, NbRBD_H11-G8
• NbRBD_H11-D1 NbRBD_H11-A9
• NbRBD_H11-C6, NbRBD_H11-E3, NbRBD_H11-F4 NbRBD_H11-C5, NbRBD_H11-C2, NbRBD_H11-B11, NbRBD_H11-A3, NbRBD_H11-D12, NbRBD_H11-D6 and NbRBD_H11-F8 (amino acid sequences provided as SEQ ID NOs:
• NbRBD_Hll-H6 GRTFSTAA 1 IRWSGGSA 2 AGSKITRSLLSDYATWPYDY 42
• NbRBD H11-F4 GRTFSTAA 1 IRWSGGSA 2 AESTITRSLLSDYATWPYDY 53 NbRBD_Hll-C5 GRTFSTAA 1 IRWSGGSA 2 AQTSYVSFLLSDYATWPYDY 54 NbRBD_Hll-C2 GRTFSTAA 1 IRWSGGSA 2 AGSRATRSLLSDYATWPYDY 55 NbRBD_Hll-Bll GRTFSTAA 1 IRWSGGSA 2 AETLNTRSLLSDYATWPYDY 56 NbRBD_H11-A3 GRTFSTAA 1 IRWSGGSA 2 ARSDNVRSLLSDYATWPYDY 57 NbRBD_Hll-D12 GRTFSTAA 1 IRWSGGSA 2 ADWGVTRSLLSDYATWPYDY 58 NbRBD H11-D6 GRTFSTAA 1 IRWSGGSA 2 ASSSVTRSLLSDYATWPYDY 59
• a single domain antibody comprising a complementary determining region, complementary determining region 3 (CDR3).
• a single domain antibody comprises a complementary determining region selected from CDR1 , complementary determining region 2 (CDR2) or complementary determining region 3 (CDR3) is provided.
• a single domain antibody comprises at least one complementary determining region selected from CDR1 , CDR2 or CDR3 is provided.
• a single domain antibody comprises at least two complementary determining regions selected from CDR1, CDR2 or CDR3.
• single domain antibody comprises three complementary determining regions: CDR1 , CDR2, and CDR3 is provided.
• a single domain antibody comprising a complementary determining region 3 (CDR3) selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 and 60 is provided, wherein the amino acid sequences of CDR3 comprise between 0 and 10 amino acid modifications.
• an single domain antibody comprising a complementary determining region 3 (CDR3) selected from the group consisting of SEQ ID NOs:3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
• the amino acid sequences of CDR3 comprise between 0 and 7 amino acid modifications.
• the CDR3 regions comprise between 0 and 6, 0 and 5, 0 and 4, 0 and 3, 0 and 2 and 0 and 1 amino acid modifications.
• the modifications can be substitutions, deletions or insertions. In one embodiment, the modifications are substitutions.
• a single domain antibody comprising a complementary determining region
• CDR3 selected from the group consisting of SEQ ID Nos: 3, 6, 9, 12, 15, 18, 21, 24, 27,
• 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 and 60 comprise between 0 and 7 amino acid modifications, wherein the CDR3 regions of amino acid sequences of SEQ ID Nos: 12, 15, 18, 27, 30, 33 and 36 comprise between 0 and 5 amino acid modifications and wherein the CDR3 regions of amino acid sequences of SEQ ID NOs: 9, 24 and 39 comprise between 0 and 3 amino acid modifications.
• the single domain antibody of the invention comprises complementary determining region 3 (CDR3) selected from the group consisting of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60 wherein the amino acid sequences of CDR3 comprise between 0 and 7 amino acid modifications.
• the complementary determining region 3 (CDR3) is SEQ ID NO: 41.
• the CDR3 regions comprise between 0 and 6, 0 and 5, 0 and 4, 0 and 3, 0 and 2 or 0 and 1 amino acid modifications.
• the modifications can be substitutions, deletions or insertions. In one embodiment, the modifications are substitutions.
• the single domain antibody of the invention may further comprise a CDR2 region.
• the CDR2 region may be defined according to a SEQ ID NO disclosed herein.
• the single domain antibody of the invention may further comprise a CDR1 region and CDR2 region.
• the CDR1 region and the CDR2 region may be defined according to a SEQ ID NO disclosed herein.
• the single domain antibody may further comprise four framework regions (FR1 , FR2, FR3 and FR4).
• an anti-SARS-CoV-2 single domain antibody wherein the single antibody domain comprises
• a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:60; wherein the amino acid sequence of CDR3 comprises between 0 and 7 amino acid modifications and wherein the amino acid sequence of CDR2 comprises between 0 and 4 amino acid modifications.
• the CDR3 regions comprise between 0 and 6, 0 and 5, 0 and 4, 0 and 3, 0 and 2 or 0 and 1 amino acid modifications.
• the CDR2 regions comprise between 0 and 3, 0 and 2, 0 and 4, 0 and 1 amino acid modifications.
• an anti-SARS-CoV-2 single domain antibody wherein the single antibody domain comprises
• a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:60; wherein the amino acid sequence of CDR3 comprises between 0 and 7 amino acid modifications and wherein the amino acid sequence of CDR2 comprises between 0 and 4 amino acid modifications.
• the CDR3 regions comprise between 0 and 6, 0 and 5, 0 and 4, 0 and 3, 0 and 2 or 0 and 1 amino acid modifications.
• the CDR2 regions comprise between 0 and 3, 0 and 2, 0 and 4, 0 and 1 amino acid modifications.
• an anti-SARS-CoV-2 single domain antibody wherein the single antibody domain comprises
• a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:44; wherein the amino acid sequence of CDR3 comprises between 0 and 7 amino acid modifications and wherein the amino acid sequence of CDR2 comprises between 0 and 4 amino acid modifications.
• the CDR3 regions comprise between 0 and 6, 0 and 5, 0 and 4, 0 and 3, 0 and 2 or 0 and 1 amino acid modifications.
• the CDR2 regions comprise between 0 and 3, 0 and 2, 0 and 4, 0 and 1 amino acid modifications.
• an anti-SARS-CoV-2 single domain antibody wherein the single antibody domain comprises
• the single domain antibody of the invention may further comprise a CDR1 region.
• the CDR1 region may be defined according to a SEQ ID NO disclosed herein.
• the single domain antibody may further comprise four framework regions (FR1, FR2, FR3 and FR4).
• an anti-SARS-CoV-2 single domain antibody wherein the single antibody domain comprises
• CDR1 comprising SEQ ID NO:1 , a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:41;
• CDR1 comprising SEQ ID NO: 1 , a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:42;
• CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2 and CDR3 comprising SEQ ID NO:56;
• CDR1 comprising SEQ ID NO:1
• CDR2 comprising SEQ ID NO:2
• CDR3 comprising SEQ ID NO:57
• CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:60; wherein the amino acid sequence of CDR3 comprises between 0 and 7 amino acid modifications, wherein the amino acid sequence of CDR2 comprises between 0 and 4 amino acid modifications and wherein the amino acid sequence of CDR1 comprises between 0 and 4 amino acid modifications.
• the CDR3 regions comprise between 0 and 6, 0 and 5, 0 and 4, 0 and 3, 0 and 2 or 0 and 1 amino acid modifications.
• the CDR2 regions comprise between 0 and 3, 0 and 2, 0 and 4, 0 and 1 amino acid modifications.
• the CDR1 regions comprise between 0 and 3, 0 and 2, 0 and 4, 0 and 1 amino acid modifications.
• an anti-SARS-CoV-2 single domain antibody wherein the single antibody domain comprises
• CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:43;
• CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:44;
• CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:48;
• CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:49;
• CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:60; wherein the amino acid sequence of CDR3 comprises between 0 and 7 amino acid modifications, wherein the amino acid sequence of CDR2 comprises between 0 and 4 amino acid modifications and wherein the amino acid sequence of CDR1 comprises between 0 and 4 amino acid modifications.
• an anti-SARS-CoV-2 single domain antibody wherein the single antibody domain comprises
• CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:43;
• CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NO:44; wherein the amino acid sequence of CDR3 comprises between 0 and 7 amino acid modifications, wherein the amino acid sequence of CDR2 comprises between 0 and 4 amino acid modifications and wherein the amino acid sequence of CDR1 comprises between 0 and 4 amino acid modifications.
• the CDR3 is SEQ ID NO: 41.
• an anti-SARS-CoV-2 single domain antibody comprising: a CDR1 comprising SEQ ID NO: 1 , a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 and 60.
• an anti-SARS-CoV-2 single domain antibody comprising: a CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 40, 41 , 42, 43 and 44.
• an anti-SARS-CoV-2 single domain antibody comprising: a CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2 and a CDR3 comprising SEQ ID NOs: 41.
• the single chain antibody comprises four framework regions.
• the framework regions separate the CDR sequences.
• the four framework regions are framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3) and framework region 4 (FR4) and are interspersed between the CDR1, CDR2 and CDR3 (i.e FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4).
• the single domain antibody of the invention comprises or essentially consists of four framework regions (FR1 , FR2, FR3 and FR4) and three CDRs (CDR1, CDR2 and CDR3).
• the single domain antibody of the invention consists of four framework regions (FR1 , FR2, FR3 and FR4) and three CDRs (CDR1, CDR2 and CDR3).
• the one or more amino acid modifications are in the CDR region or regions. In other embodiments, the one or more amino acid modifications are in the framework regions, i.e. not in the CDR region or regions. In some embodiments, the one or more polynucleotide modifications are in the CDR region or regions. In other embodiments, the one or more polynucleotide modifications are in the framework regions, i.e. not in the CDR region or regions.
• the CDR3 regions comprise between 0 and 7, 0 and 6, 0 and 5, 0 and 4, 0 and 3, 0 and 2 or 0 and 1 amino acid modifications.
• the CDR2 regions comprise 0 and 4, 0 and 3, 0 and 2 or 0 and 1 amino acid modifications.
• the CDR1 regions comprise 0 and 4, 0 and 3, 0 and 2 or 0 and 1 amino acid modifications.
• the modifications can be substitutions, deletions or insertions. In one embodiment, the modifications are substitutions.
• a single domain antibody of the invention comprising one or more modifications has a binding affinity for the receptor binding domain of the SARS-CoV-2 S- protein that is substantially equal to, or better than (for example, a lower Kd value) than the specified sequence without any modifications.
• an anti-SARS-CoV-2 single domain antibody comprising an amino acid sequence having at least 70% identity to a sequence selected from the group consisting of: SEQ 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101 , 102, 103, 104, 105, 106 and 107 is provided.
• Each of these sequences comprises three CDR regions (CDR1 , CDR2 and CDR3) and four framework regions (FR1 , FR2, FR3 and FR4).
• the amino acid sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity, optionally 75-100%, 80-100%, 85-100%, 90-100%, 91-100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100% identity to a sequence selected from the group consisting of: SEQ 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106 and 107.
• an anti-SARS-CoV-2 single domain antibody comprising SEQ 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106 and 107 is provided.
• an anti-SARS-CoV-2 single domain antibody consisting or essentially consisting of SEQ 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106 and 107 is provided.
• At least herein and throughout means, in some embodiments, the recited percentage up to 100%.
• at least 75% can mean, in some embodiments, 75% to 100%.
• an anti-SARS-CoV-2 single domain antibody comprising an amino acid sequence having at least 70% identity to a sequence selected from the group consisting of: SEQ ID NO: 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106 and 107 is provided.
• Each of these sequences comprises three CDR regions (CDR1 , CDR2 and CDR3) and four framework regions (FR1, FR2, FR3 and FR4), wherein the CDR3 region has been affinity matured.
• the amino acid sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity, optionally 75-100%, 80-100%, 85-100%, 90-100%, 91-100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100% identity to a sequence selected from the group consisting of: SEQ 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106 and 107.
• an anti-SARS-CoV-2 single domain antibody comprising SEQ 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106 and 107 is provided.
• an anti-SARS-CoV-2 single domain antibody consisting or essentially consisting of SEQ 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106 and 107 is provided.
• an anti-SARS-CoV-2 single domain antibody comprising an amino acid sequence having at least 70% identity to a sequence selected from the group consisting of: SEQ ID NOs: 87, 88, 89, 90 and 91.
• the amino acid sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity, optionally 75-100%, 80-100%, 85-100%, 90-100%, 91-100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100% identity to a sequence selected from the group consisting of: of SEQ ID NOs: 87, 88, 89, 90 and 91.
• the amino acid sequence is selected from the group consisting of SEQ ID NOs: 87, 88, 89, 90 and 91.
• an anti-SARS-CoV-2 single domain antibody comprising an amino acid sequence having at least 70% identity to SEQ ID NOs: 88.
• the amino acid sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity, optionally 75-100%, 80-100%, 85-100%, 90-100%, 91-100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100% identity SEQ ID NO: 88.
• the amino acid sequence is SEQ ID NO: 88.
• a polynucleotide sequence is provided encoding a single domain antibody of the invention.
• the polynucleotide is DNA or RNA.
• Such nucleic acid sequences may be in the form of a genetic construct.
• an anti-SARS-CoV-2 single domain antibody comprising a polynucleotide sequence having at least 70% identity to a sequence selected from the group consisting of: SEQ 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 and 128 is provided.
• the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to a sequence selected from the group consisting of: SEQ 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127 and 128.
• an anti-SARS-CoV-2 single domain antibody comprising SEQ 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120,
• an anti-SARS- CoV-2 single domain antibody consisting or essentially consisting of 74, 75, 76, 77, 78, 79, 80,
• an anti-SARS-CoV-2 single domain antibody comprising a polynucleotide sequence having at least 70% identity to a sequence selected from the group consisting of: SEQ ID NO: 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127 and 128 is provided.
• the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to a sequence selected from the group consisting of: SEQ ID NO: 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 and 128.
• an anti-SARS-CoV-2 single domain antibody comprising SEQ 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127 and 128 is provided.
• an anti-SARS-CoV-2 single domain antibody consisting or essentially consisting of SEQ 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 and 128 is provided.
• an anti-SARS-CoV-2 single domain antibody comprising a polynucleotide sequence having at least 70% identity to a sequence selected from the group consisting of SEQ ID NOs: 108, 109, 110, 111 and 112.
• the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 108, 109, 110, 111 and 112.
• the polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 108, 109, 110, 111 and 112.
• the polynucleotide sequence is SEQ ID NO: 109.
• the single domain antibodies of the invention bind to the receptor binding domain of the SARS- CoV-2 S-protein.
• the single domain antibodies of the invention block or modulate the binding between the receptor binding domain of a coronavirus, in particular the SARS-CoV-2 spike (S) protein, and the angiotensin converting enzyme 2 receptor (ACE2 receptor).
• S SARS-CoV-2 spike
• ACE2 receptor angiotensin converting enzyme 2 receptor
• the single domain antibodies of the invention inhibit binding of the receptor binding domain of the SARS-CoV-2 spike (S) protein to the ACE2 receptor, wherein binding of the receptor binding domain of the SARS-CoV-2 spike (S) protein to the ACE2 receptor is inhibited by at least 10%, optionally at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%.
• Percentage inhibition of binding to the ACE2 receptor can be measured in numerous ways, as well understood by the skilled person, including but not limited to surface plasmon resonance.
• the single domain antibodies of the invention can neutralize coronavirus infection.
• the single domain antibodies of the invention can neutralize SARS-CoV-2 infection.
• the single domain antibodies have an ND50 value of less than 100nM less than 10nM, less than 5nM, less than 1nM, less than 0.5nM or less than 0.1nM.
• the single domain antibodies have an ND50 value of less than 0.1 nM less than 10pM, less than 5pM, less than 1pM, less than 0.5pM or less than 0.1pM.
• the ND50 value can be determined using any standard neutralization assay, including that disclosed herein.
• the single domain antibodies of the invention can prevent non-neutralized SARS- CoV-2 infection from spreading.
• the single domain antibodies of the invention have a Kd value for SARS- CoV-2 spike protein, in particular the receptor binding domain of the spike protein, of less than 100nM, less than 50nM, less than 20nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, less than 1nM, less than 0.5nM or less than 0.1nM.
• a Kd value for SARS- CoV-2 spike protein in particular the receptor binding domain of the spike protein, of less than 100nM, less than 50nM, less than 20nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, less than 1nM, less than 0.5nM or less than 0.1nM.
• the single domain antibodies of the invention have a Kd value for SARS-CoV-2 spike protein of less than 100pM less than 10pM, less than 5pM, less than 1pM, less than 0.5pM or less than 0.1pM. Binding affinity of an antibody can be measured according to several standard well-known techniques, including for example surface plasma resonance.
• a single domain antibody of the invention having one or more modifications as specified herein has a binding affinity value that is within 20% (i.e. within the range of 20% below or 20% above the binding affinity value of the corresponding single domain antibody without one or more modifications) of the binding affinity value of the corresponding single domain antibody without one or more modifications.
• the binding affinity value of single domain antibody of the invention having one or more modifications as specified herein is within 10%, optionally 5%, 4%, 3%, 2% or 1% of the binding affinity value of the corresponding single domain antibody without one or more modifications.
• the single domain antibodies of the invention can modulate, reduce or prevent coronavirus infectivity.
• the single domain antibodies or pharmaceutical compositions of the invention can modulate, block or inhibit the fusion of a coronavirus to a target host cell.
• the single domain antibodies or pharmaceutical compositions of the invention can modulate, block or inhibit entry of coronavirus into a target host cell.
• an affinity matured mutant of a single domain antibody of the invention is provided.
• the CDR1 of the single domain antibody of the invention is affinity matured.
• the CDR2 of the single domain antibody of the invention is affinity matured.
• the CDR3 of the single domain antibody of the invention is affinity matured.
• CDR3 is affinity matured and either CDR1 or CDR2 are also affinity matured.
• CDR3 is affinity matured and CDR2 is also affinity matured.
• CDR3 is affinity matured and CDR1 is also affinity matured.
• each of CDR1 , CDR2 and CDR3 are affinity matured.
• At least one, at least two, at least three or all four of the framework regions are affinity matured.
• each of CDR1 , CDR2, CDR3, FR1, FR2, FR3 and FR4 are affinity matured.
• the affinity of the affinity matured mutant of a single domain antibody of the invention has a higher affinity for SARS- CoV-2 receptor binding domain (RBD) than the parental antibody from which it was derived.
• the CDR3 sequences of SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60 are affinity matured variants of the CDR3 sequence SEQ ID NO: 3 (NbRBD_H11). It has been surprisingly discovered that variation in an seven amino acid long region of the CDR3 region of a single domain antibody of the invention results in particularly improved affinity for SARS-CoV-1 spike protein, wherein the seven amino acid sequence starts at the second amino acid in the CDR3 sequence and ends at the eight amino acid in the sequence (i.e. position 1 refers to the starting alanine (A) in the CDR3 sequence).
• the CDR3 region comprises modifications in a seven amino acid long region of the CDR3, wherein the seven amino acid long region starts at position 2 of the CDR3 and ends at position 8 of the CDR3.
• a humanized single domain antibody of the invention is provided. Humanization requires the modification of the amino acid sequence of the antibody. Methods to reduce the immunogenicity of the single domain antibodies of the invention include CDR grafting on to a suitable antibody framework scaffold or remodelling variable surface residues, e.g. by site- directed mutagenesis. Methods of humanization of Nanobodies® are known to the skilled person, see for example Vincke et al., 2009.
• the CDR1 of the single domain antibody of the invention is humanized.
• the CDR2 of the single domain antibody of the invention is humanized.
• the CDR3 of the single domain antibody of the invention is humanized.
• At least one or at least two of the CDR1 , CDR2 and CDR3 are humanized. In one embodiment, each of CDR1 , CDR2 and CDR3 are humanized. In one embodiment, at least one, at least two, at least three or all four of the framework regions (FR1, FR2, FR3 and FR4) are humanized. In one embodiment, each of CDR1 , CDR2, CDR3, FR1 , FR2, FR3 and FR4 are humanized. In some embodiments, the single domain antibodies are conservatively humanised, for example to retain better antigen binding.
• the single domain antibody of the invention is fused to or conjugated to an Fc region. In one embodiment, the single domain antibody of the invention is fused to or conjugated to a scFv region. In one embodiment, the single domain antibody of the invention is a bivalent or polyvalent antibody. In one embodiment, the single domain antibody of the invention is bispecific or multispecific. In one embodiment, the single domain antibody is a chimeric antibody. In one embodiment, the single domain antibody comprises two VHH chains, wherein the VHH chains can be any of the single domain antibodies disclosed herein.
• immunologically active molecules comprising the sequences of the invention, for example immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain, such as Fab, F(ab’)2, Fv, scFv, dAb, Fd; and diabodies.
• immunoglobulin isotypes e.g., IgG, IgE, IgM, IgD and IgA
• fragments which comprise an antigen binding domain such as Fab, F(ab’)2, Fv, scFv, dAb, Fd
• proteins or fusion expression products comprising the single domain antibodies of the invention are provided.
• a vector suitable for expressing a single domain antibody sequence of the invention is provided.
• the vector may be a plasmid, viral vector, cosmid, phage or artificial chromosome.
• a host cell comprising an expression vector or plasmid, wherein the expression vector or plasmid comprises a polynucleotide of the invention is provided.
• the host cell comprises a polynucleotide of the invention integrated within the genome of the host cell.
• the host cell is a prokaryotic cell, for example a bacterial cell, or a eukaryotic cell, for example a yeast cell or mammalian cell.
• the host cell is Escherichia coli or CHO cells.
• a method for producing a single domain antibody of the invention comprising the steps of (a) culturing a host cell as provided herein under conditions suitable for producing a single domain antibody to obtain a culture containing single domain antibodies and (b) isolating said single domain antibodies from the culture.
• compositions may comprise, consist essentially of or consist of the single domain antibodies of the invention.
• a pharmaceutical composition comprising single domain antibodies of the invention.
• the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
• the pharmaceutical composition may be formulated according to route of administration.
• the pharmaceutical composition is formulated for oral, nasal, ocular, buccal, vaginal, rectal, transdermal, intravenous, intramuscular or subcutaneous administration.
• the pharmaceutical composition is formulated for administration by inhalation, optionally nasal and or oral inhalation.
• Pharmaceutical compositions in this form may include aerosols, fine particles or dust.
• the composition or pharmaceutical composition optionally comprises one or more pharmaceutically acceptable excipients.
• the composition or pharmaceutical composition optionally comprises one or more pharmaceutically acceptable adjuvants.
• composition or pharmaceutical composition is optionally admixed with one or more pharmaceutically acceptable diluents, excipients or carriers.
• suitable excipients for the different forms of pharmaceutical compositions described herein may be found in the "Handbook of Pharmaceutical Excipients, 2 nd Edition, (1994), Edited by A Wade and PJ Weller.
• composition or pharmaceutical composition may comprise one or more additional components.
• the composition or pharmaceutical composition additionally comprises a pharmaceutically acceptable carrier.
• the carrier is suitable for pulmonary delivery.
• the composition or pharmaceutical composition additionally comprises a therapeutically active agent.
• the composition or pharmaceutical composition may be joined or conjugated to a protein or biologically active molecule.
• the composition or pharmaceutical composition is part of a fusion protein and fused to one or more proteins or biologically active molecules.
• the protein or biologically active molecule may be a fluorescent protein, a bioluminescent protein, a split fluorescent protein (i.e split into two or more parts that will join together in the presence of drug), a split bioluminescent protein, a biosensor, a fluorescent biosensor or a split or hinged biosensor.
• a vaccine comprising single domain antibodies of the invention.
• the vaccine comprises a polynucleotide encoding a single domain antibody of the invention is provided.
• compositions, pharmaceutical compositions and vaccines of the invention can elicit an immune response in a subject, preferably an immune response to SARS-CoV-2.
• the immune response is a protective immune response.
• the immune response that reduces the symptoms or severity of SARS-CoV-2 in a subject.
• administration of the antibodies of the present invention prevents or substantially reduces non-neutralised virus from replicating and/or spreading. This is supported by plaque reduction neutralisation test data: in the presence of antibodies of the present invention, including but not limited to H11-H4-Fc, plaques formed are smaller than would normally be expected ( Figure 9).
• antibodies of the present invention including but not limited to H11-H4-Fc, are capable of forming plaques that are 5% smaller than in the presence of a positive control, for example CR3022.
• the plaques are 10% smaller than in the presence of a positive control (for example CR3022); in some embodiments, plaques are 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% smaller than in the presence of a positive control (for example CR3022).
• a pharmaceutical device for example an inhaler, suitable to administer the pharmaceutical compositions of the invention is also provided.
• the pharmaceutical device for example an inhaler, comprises a single domain antibody of the invention.
• kits may include instructions for use and/or additional pharmaceutically active components.
• the single domain antibodies and the additional pharmaceutically active components may be formulated together, or alternatively in some embodiments, the single domain antibodies and the additional pharmaceutically active components may be present separately in the kit.
• a single domain antibody of the invention or a pharmaceutical composition of the invention for use in medicine.
• the single domain antibodies or pharmaceutical compositions of the invention can be used to treat a coronavirus, optionally Middle Eastern respiratory syndrome (MERS-CoV) or severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), preferably COVID-19.
• the single domain antibodies or pharmaceutical compositions of the invention can be used to block or modify the interaction of the spike protein of a coronavirus, in particular SARS-CoV-2, with its target, angiotensin converting enzyme 2 receptor.
• the single domain antibodies or pharmaceutical compositions of the invention block, reduce or inhibit binding of the spike protein of a coronavirus, in particular SARS-CoV-2, with its target, angiotensin converting enzyme 2 (ACE2) receptor.
• ACE2 angiotensin converting enzyme 2
• the single domain antibodies or pharmaceutical compositions of the invention can neutralize coronavirus and/or can modulate, reduce or prevent coronavirus infectivity.
• the single domain antibodies or pharmaceutical compositions of the invention can modulate, block or inhibit the fusion of coronavirus to a target host cell.
• the single domain antibodies or pharmaceutical compositions of the invention can modulate, block or inhibit entry of coronavirus into a target host cell.
• the single domain antibodies of the invention or pharmaceutical compositions of the invention can be used for the treatment or prophylaxis of coronavirus infection, in particular COVID-19.
• a single domain antibody of the invention or a pharmaceutical composition of the invention for use in the treatment or prophylaxis of a coronavirus infection optionally Middle Eastern respiratory syndrome (MERS-CoV) or severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), preferably COVID-19.
• MERS-CoV Middle Eastern respiratory syndrome
• SARS-CoV-1 severe acute respiratory syndrome coronavirus 1
• a method for the treatment of a coronavirus in a subject comprising administering to a subject a therapeutically active amount of a single domain antibody of the invention.
• the subject is a mammal, preferably a human.
• the use of a single domain antibody of the invention in the manufacture of a medicament for use in the treatment and/or prevention of a coronavirus is provided. In one embodiment, the use of a single domain antibody of the invention in the manufacture of a medicament for use in the treatment of a coronavirus is provided.
• the coronavirus is selected from the group consisting of MERS-CoV, SARS-CoV-1 and COVID-19. In one embodiment, the coronavirus is COVID-19.
• the invention may relate to treating a subject displaying severe symptoms of COVID-19 or alternatively to treating a subject showing milder symptoms of COVID-19.
• the single domain antibodies of the invention are useful for treating a cytokine storm associated with a coronavirus infection.
• methods for the detection of a coronavirus protein such as MERS-CoV, SARS-CoV-1 and SARS-CoV-2 are provided.
• a method for the detection of a SARS-CoV-2 protein is provided.
• a method for detecting the presence of a coronavirus S-protein is provided.
• a method for a method for detecting the presence of a SARS-CoV-2 S-protein is provided.
• a method for detecting a coronavirus protein in a sample comprises the steps of (a) contacting a sample with the single domain antibodies of the invention and (b) detecting the antibody-antigen complex, wherein the presence of the complex indicates the presence of coronavirus protein.
• step (a) of the method the sample is contacted with the single domain antibodies under suitable conditions for an antibody-antigen complex to form.
• the antigen is the coronavirus protein.
• a method for detecting the presence of a coronavirus S-protein is provided.
• a method for a method for detecting the presence of a SARS-CoV-2 S- protein, optionally the receptor binding domain of the S-protein is provided.
• the sample can be a biological sample, optionally a bodily fluid such as blood, serum, nasal secretions, sputum, plasma, urine or spinal fluid.
• the biological sample is bodily fluid obtained using a throat or nasal swab.
• the biological sample is a tissue sample.
• the sample can be obtained from or isolated from a mammal, preferably a human.
• the sample is obtained from or isolated from a subject who is suspected to have coronavirus.
• Detecting the presence of coronavirus protein in a sample from a subject provides a positive indication that the subject is infected with coronavirus.
• the results of the method of detection are used to diagnose a subject in relation to coronavirus.
• the presence of coronavirus protein in the method of detection would provide a positive diagnosis for coronavirus.
• the method of detection may also be used to provide a prediction of outcome in relation to infection of coronavirus infection.
• a method for detecting coronavirus protein in a subject comprises the steps of (a) administering to a subject a single domain antibody of the invention and (b) detecting the presence of an antibody-antigen complex, wherein the presence of the complex indicates the presence of coronavirus protein in the subject.
• a method for detecting coronavirus protein in a subject comprises the steps of (a) administering to a subject a single domain antibody of the invention, (b) obtaining a sample from a subject and contacting the sample with a single domain antibody of the invention and (c) detecting the antibody-antigen complex, wherein the presence of the complex indicates the presence of coronavirus protein in the subject.
• the antigen is the coronavirus protein.
• a method for detecting coronavirus protein in a subject comprises the steps of (a) obtaining a sample from a subject, (b) contacting a sample from the subject with a single domain antibody of the invention and (c) detecting the antibody-antigen complex, wherein the presence of the complex indicates the presence of coronavirus protein in the subject.
• the sample may be an isolated sample (i.e. previously obtained from a subject).
• a method for diagnosing coronavirus infection in a subject comprises the steps of (a) administering to a subject a single domain antibody of the invention and (b) detecting the presence of an antibody-antigen complex, wherein the presence of the complex provides a positive diagnosis of coronavirus in the subject.
• a method for diagnosing coronavirus infection in a subject comprises the steps of (a) administering to a subject a single domain antibody of the invention, (b) obtaining a sample from a subject and contacting the sample with a single domain antibody of the invention and (c) detecting the antibody-antigen complex, wherein the presence of the complex provides a positive diagnosis of coronavirus in the subject.
• a method for diagnosing coronavirus infection in a subject comprises the steps of (a) obtaining a sample from a subject, (b) contacting a sample from the subject with a single domain antibody of the invention and (c) detecting the antibody-antigen complex, wherein the presence of the complex provides a positive diagnosis of coronavirus in the subject.
• a method for diagnosing coronavirus infection in a subject comprising (a) contacting a sample with a single domain antibody of the invention, (b) detecting the number of antibody-polypeptide complexes and (c) detecting the presence of coronavirus in the sample, wherein the presence of the complex provides a positive diagnosis of coronavirus in the subject.
• the sample may be an isolated sample (i.e. previously obtained from a subject).
• the method comprises the step of comparing the sample with reference sample values for levels of the antibody-antigen complex.
• An antigen-antibody complex value above that of the reference sample value can provide a positive indication of coronavirus infection.
• the sample may be an isolated sample (i.e. previously obtained from a subject).
• the single domain antibody of the invention may further comprise a marker such as a radiolabelled marker, imaging marker, MRI-marker, fluorescent marker or other detectable marker.
• a marker such as a radiolabelled marker, imaging marker, MRI-marker, fluorescent marker or other detectable marker.
• Such antibodies can be used in each of the detection or diagnosis methods described herein to enable the detection of the antibody in the subject in real time.
• Such antibodies can also be used in each of the detection or diagnosis methods described herein to enable the detection of the antibody in a sample, such as a tissue or blood sample, isolated or obtained from a subject.
• an assay to detect a coronavirus comprises (a) contacting a sample obtained from a patient with a single domain antibody of the invention, wherein the single domain antibody comprises a detectable label or reporter molecule to selectively isolate the coronavirus in the patient sample.
• an assay to detect coronavirus comprises (a) contacting a sample obtained from a patient with a fusion protein comprising a single domain antibody of the invention and a biosensor, optionally a fluorescent or hinged biosensor,
• the assay may for example be an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay, a radioimmunoassay (RIA) or a fluorescence-activated cell sorting (FACS).
• the detectable label or reporter molecule can be a fluorescent or chemical molecule (e.g. fluorescein isothiocyanate, or rhodamine), a biosensor, a radioisotope or enzyme (e.g.
• kits comprising (a) a detectable marker (b) a single domain antibody of the invention.
• the detectable label or reporter molecule can be a fluorescent or chemical molecule (e.g. fluorescein isothiocyanate, or rhodamine), a biosensor, optionally a fluorescent or hinged biosensor or a radioisotope or enzyme (e.g. alkaline phosphatase, b-galactosidase, horseradish peroxidase or luciferase).
• the methods described herein can be in vitro or ex vivo.
• the methods described herein can also be performed in vivo.
• Recombinant Spike ectodomain was expressed by transient transfection in HEK293S GnTI cells (ATCC CRL-3022) for 9 days at 30 °C.
• Conditioned media was dialysed against 2xPBS buffer.
• the Spike ectodomain was purified by immobilised metal affinity chromatography using Talon resin (Takara Bio) charged with cobalt followed by size exclusion chromatography using HiLoad 16/60 Superdex 200 column in 150 mM NaCI, 10 mM HEPES (pH 8.0), 0.02% NaN 3 at 4 °C.
• the final fractions containing the Spike ectodomain were identified by reducing SDS- PAGE, pooled, concentrated using a 100 kDa MWCO concentrator (Amicon Ultra, Merck), and stored at 16 °C.
• Codon optimised Genblocks for the receptor binding domain (RBD amino acids 330-532) of SARS-CoV2 (Genbank MN908947), and human Angiotensin Converting Enzyme 2 (ACE-2 amino acids 19-615) were inserted into the vector pOPINTTGneo (Nettleship, Watson et al. 2015) incorporating a C-terminal BirA-His6 tag and pOPINTTGneo- 3C-Fc to make C-terminal fusions to Human IgG Fc.
• Recombinant RBDs and CR3022 Fab fragments were transiently expressed in Expi293TM (ThermoFisher Scientific, UK) and proteins were purified from culture supernatants by immobilised metal affinity using an automated protocol implemented on an AKTAxpress (GE Healthcare, UK) (Nettleship, Rahman-Huq et al. 2009) followed by a Superdex 200 10/300GL column, using phosphate-buffered saline (PBS) pH 7.4 buffer. Purified protein were biotinylated in vitro by incubation with biotin protein ligase (Avidity LLC, Co, USA).
• Mutations in the CDR3 of NbNH11 were introduced by PCR using seven pairs of forward and reverse primers forward primers (H11_AM_CDR3_F1-7 in combination with H11_AM_CDR3_R1-7).
• the mutated fragments were amplified with the primers H11_Phd_F and H11_Phd_R (Table 3a), digested with Sfil restriction enzyme and cloned into pADL-23c phagemid (Antibody Design Laboratories, San Diego CA, USA).
• the ligated vector was transformed into TG1 cells by electroporation to give a phage library consisting approximately 2 x 10 9 independent clones. Two rounds of biopanning of the library were carried out on 5 nM and 1 nM of RBD, respectively, as described above and positive phage identified by ELISA and sequenced.
• the phagemids amplified from the selected clones were transformed into the WK6 E. coli strain and grown in TB medium (supplemented with 100 pg/mL ampicillin and 1mM MgCI2), shaking at 225 rpm and 37 oC, with induction of protein expression by 1mM IPTG at OD ⁇ 1.2, and then grown overnight, shaking at 225 rpm and 20 0C.
• the bacterial cells were pelleted and re-suspended in TES buffer (0.2 M Tris pH8, 0.5 mM EDTA, 0.5 M sucrose) overnight, followed by 2 hours in TES/4 buffer (TES diluted 4x in water).
• the supernatant was harvested through centrifugation at 16800 rpm and 40C, and subsequently diluted 10x in GF buffer (20 mM Tris pH7.5 and 150 mM NaCI).
• the proteins were purified through an immobilised metal affinity using an automated protocol implemented on an AKTAxpress (GE Healthcare, UK) (Nettleship, Watson et al. 2015) followed by a Hiload 16/60 superdex 75 or a Superdex 75
• VHH phage display library (Abcore Inc. Ramona, CA, USA) constructed in the vector pADL- 20c and comprising approximately 1 x 10 10 independent clones was inoculated into 2xTYA (2xTY supplemented with 100 pg/mL ampicillin) and infected with M13 helper phage to obtain a library of VHH-presenting phages.
• Phages displaying VHHs specific for the SARS-CoV-2 RBD were enriched after two rounds of bio-panning on 50 nM and 5 nM of RBD, respectively, through capturing with DynabeadsTM M-280 (Thermo Fisher Scientific).
• the Dynabeads and phages were firstly blocked with StartingBlockTM (PBS) Blocking Buffer (Thermo Fisher Scientific) for 30 minutes; the phages were incubated with the RBD for 1 hour, and then 5 minutes with the Dynabeads (Thermo Fisher Scientific); and subsequently washed 6 times with PBS supplemented with 0.05 % Tween 20 and 1 time with PBS.
• PBS StartingBlockTM
• Thermo Fisher Scientific Blocking Buffer
• the retained phages were eluted through incubation with TBSC buffer (10 mM Tris pH 7.4, 137 mM NaCI, 1 mM CaCh) and 1 mg/ml_ trypsin (Sigma-Aldrich) for 30 min.
• the collected phages were amplified in exponentially growing TG1 E. coli cells and plated on 2xTY agar plates supplemented with 100 pg/mL ampicillin. Enrichment after each round of panning was determined by plating the cell culture with 10-fold serial dilutions. After the second round of panning, 93 individual clones were picked to inoculate 2xTYA and were grown overnight, shaking at 250 rpm and 37 °C. The next day, the overnight culture was used to inoculate 2xTYA and infected with M13 helper phage to obtain clonal VHH-presenting phages. Enzyme-linked immunosorbent assays
• the wells of microtiter plates (Greiner high and medium binding) were coated with 5 pg/mL neutravidin in PBS pH 7.4 overnight at 4 °C. The next day, the wells were coated with 50 nM biotinylated RBD, and then blocked with 3 % milk powder in PBS pH 7.4. Supernatant of clonal phage was added into each well, binding was detected by incubating the wells with HRP-
• Nanobody H11 was immobilized onto the sample flow cell of the sensor chip.
• the reference flow cell was left blank.
• Nanobody H 11 was injected over the two flow cells at a range of 8 concentrations prepared by serial two-fold dilutions from 2.5 mM, at a flow rate of 30 pL/min, with an association time of 60 s and a dissociation time of 60 s.
• the data were fitted to a 1:1 binding model and to calculate KD using GraphPad Prism 8.
• Nanobody H11-H4 / H11-D4 was injected over the two flow cells at a range of five concentrations prepared by serial two-fold dilutions from 50 nM, at a flow rate of 30 pL/min using a single-cycle kinetics program with an association time of 60 s and a dissociation time of 60 s.
• Running buffer was also injected using the same program for background subtraction. All data were fitted to a 1:1 binding model using the Biacore T200
• H4 / H11-D4 and 0.1 mM Spike (5) a mixture of 1 mM E08R Fab and 0.1 mM Spike; (6) 0.1 mM
• ITC measurements were carried out using an iTC200 MicroCalorimeter (GE Healthcare) at 25 °C.
• Spike, RBD and nanobody were prepared and dialyzed in the same buffer, i.e. , PBS.
• Nanobody was titrated into Spike or RBD solution corresponding to approximately 72 mM nanobody and 6 mM Spike or 250 mM nanobody and 25 mM RBD.
• Each experiment consisted of an initial injection of 0.4 mI_ followed by 16 injections of 2.4 mI_ nanobody solution into the cell containing either Spike or RBD, while stirring at 750 rpm.
• Data acquisition and analysis were performed using the Origin scientific graphing and analysis software package (OriginLab).
• nM number of sites
• DH calories/mole
• AS calories/mole/degree
• K binding constant in molar -1
• MDCK-SIAT1 cells were stably transfected with codon-optimized human ACE2 cDNA (NM_021804.1) using a second-generation lentiviral vector system and FACS sorted for highly expressing population.
• Cells (3 x 10 4 per well) were seeded the day before the assay on a flat- bottomed 96-well plate.
• RBD-6H amino acid 340-538; NITN.GPKK
• EZ-link Sulfo-NHS-Biotin A39256; Life Technologies).
• a serial half-log dilution (ranging 1 mM to 0.1 nM) of analytes and controls were performed in a U-bottomed 96 well plate in 30 m ⁇ - volume. An equal volume of 25 nM of biotinylated RBD was added and 50 m ⁇ - of each of the resulting mixtures were added to the MDCK-ACE2 cells for 1 hour.
• MDCK-SIAT1 cells were stably transfected with RBD (amino acids 340-538 NITN.GPKK) fused to the transmembrane and cytoplasmic domain of haemagglutinin H7
• SARS-CoV-2 (Australia/VIC01/2020) 43 which was diluted to a concentration of 933 pfu/mL (70 pfu/50 pL) and mixed 50:50 in minimal essential medium (MEM) (Life Technologies, California, USA) containing 1 % fetal bovine serum (FBS) (Life Technologies) and 25 mM HEPES buffer (Sigma,
• Virus was allowed to adsorb onto cells at 37 °C for a further hour in a humidified box, then the cells were overlaid with MEM containing 1.5 % carboxymethylcellulose (Sigma), 4 % (v/v) FBS and 25mM HEPES buffer.
• Plaque reduction neutralization tests in Oxford were performed using passage 4 of SARS- CoV-2 Victoria/01 /2020 43 using established methodology 45 .
• virus stock (9.75x10 4 pfu/mL) was diluted by 10 and by 100 in Dulbecco’s Modification of Eagle’s Medium containing 1 % FBS (D1; 100 m ⁇ ) was mixed with nanobody-Fc (100 m ⁇ ) diluted in D1 so as give a final concentrations of H11-H4 at 100, 32, 10, 3.2 nM for measurement.
• solutions with CR3022 333, 167, 84 and 42 at nM were prepared. Each experiment was performed in triplicate in 24 well tissue culture plate.
• the plate was incubated at room temperature for 30 minutes and 0.5 mL of a single cell suspension of Vero E6 cells in D1 at 5 x 10 5 /mL was added. The plates were incubated for a further 2 h at 37 °C before being overlain with 0.5 mL of D1 supplemented with carboxymethyl cellulose (1.5 %). The resulting cultures were incubated for a further 4 days at 37 °C before plaques were revealed by staining the cell monolayers with amido black in acetic acid/methanol ( Figure 9a, b, c, d). To probe whether
• CR3022 and H11-H4 were additive, solutions of H11-H4 at 100, 32, 10, 3.2 nM were each incubated for 30 mins with CR3022 at a final concentration of 84 nM. The resulting mixtures were analyzed as described above in triplicate experiments and the wells are shown in Figure 9d.
• Oxford neutralisation used Vero Ccl-81 (from a stock that was originally from ATCC).
• PHE neutralisation used VeroE6 Cells purchased from ECACC.
• Cell based competition assays used MDCK-SIAT1 cells derived from a commercial source (Sigma-Aldrich). All mammalian protein expressions were performed with purchased 293Expi cells (ThermoFisher Scientific) and E. coli cells.
• Nanobody complex with Spike, preparation and cryo-EM data collection Purified spike protein in 10 mM Hepes, pH 8, 150 mM NaCI, was incubated with H11-H4 purified in 50 mM Tris, pH 7, 150 mM NaCI, at a molar ratio of 1:3.6 (Spike trimer: nanobody) at 16 °C overnight. Spike protein was used at a final concentration of 1 mg/ml_. The mixture was centrifuged at 21000g, 16 °C prior to grid preparation. For H11-D4 - Spike a mixture in the molar ratio of 1:6 (Spike trimer: nanobody) was incubated at 20 °C for ten minutes.
• Frozen grids were first screened on a Glacios microscope operating at 200 kV (Thermo Fisher Scientific) before imaging on a Titan Krios G2 (Thermo Fisher Scientific) at 300 kV. Movies (40 frames each) were collected in compressed tiff format on a K3 detector (Gatan) in super resolution counting mode using a custom EPU version 2.5 (Thermo Fisher Scientific) (Table
• Pixel size (A/pix) (Super 0.415 0.53 resolution)
• Motion correction and alignment of 2x binned super-resolution movies was performed using Relion (v3.1) 46 with a 5 x 5 patch-based alignment.
• CTF-estimation of full-frame non-weighted micrographs was performed using GCTF (v1.06) and non-template-driven particle picking was then performed within cryoSPARC (v2.14.1 -live) 47 followed by multiple rounds of 2D classification.
• the resulting 2D class averages consistent with Spike trimer were used for template-driven particle picking before further rounds of 2D and 3D classification with C1 symmetry.
• An initial model for Spike was generated using PDB 6VXX 26 and rigid body fitted into the map using Chimera 49 followed by Coot 50 .
• the H11-D4-RBD crystal structure was superimposed onto the naked Spike model in Coot and checked for fit in the density.
• S1/S2 domains split into subdomains for each subunit (residues 27-307; 308-321 and 591-700; 322-333 and 529- 590; 701-1147) were then independently rigid body fitted in Coot 50 before a final real space refinement with PHENIX 51 with hydrogen atoms added using ReadySet 51 resulting in a final correlation coefficient of 0.8.
• the H11-D4-RBD crystal structure was used as reference structure restraints during refinement of the Spike owing to the density. Rounds of manual inspection in Coot 50 and real space refinement with PHENIX 51 resulted in the final model. Data processing and refinement statistics are shown in Table 4.
• 3D classification was done using emd_21374 low pass filtered to 60 A. Initially the data was processed as C3 but was relaxed to C1 as the RBD and nanobody density were poor. The best C1 3D class was selected for further refinement, CTF refinement, and particle polishing within Relion. The coordinates from the Spike-H11-D4 structure were rigid- body docked into the Spike-
• H 11 -D4-RBD-CR3022 and H11-H4-RBD-CR3022 crystallography Purified RBD, Fab CR3022 and H11-D4 were mixed together at a molar ratio of 1:1:1 to a final concentration of approximately 7 mg/ml_ and incubated at room temperature for one hour. Initial screening was performed in 96-well plates using the nanoliter sitting-drop vapor diffusion method. The best crystals were grown in condition containing 0.1 M sodium citrate tribasic dihydrate, pH 5.0, 10 % (w/v) polyethylene glycol 6000.
• Model rebuilding was done with COOT 50 , initially refined with PHENIX 51 then with REFMAC5 58 aided by PDB-REDO 59 , MOLPROBITY 60 and the TLSMD server 61 .
• Each nanobody was mixed with 8.7 mg of RBD at 2.9 mg/mL at a molar ratio nanobody: RBD 1.1:1 and the complex was incubated for 3 h in a cold room under agitation at 2 rpm.
• RBD in the complex was deglycosylated by the addition of 0.4 mg of EndoH glycosidase and incubated overnight at room temperature, under agitation at 2 rpm.
• the mixture was then concentrated to 1 ml_ with a 5 kDa MWCO concentrator and injected on gel filtration using a Superdex 200 10/300 (GE) in 50 mM Tris pH 7, 150 mM NaCI.
• the surface on RBD that contacts H11-D4 is formed by Lys444-Phe456 and Gly482-Ser494 ( Figure 15D). These two stretches of RBD sequence comprise over 90 % of buried surface area and make all the hydrogen bonds with H11-D4 ( Figure 15C).
• H11-H4 binds to RBD with a KD of 12 ⁇ 1.5 nM
• H11-D4 with a KD of 39 ⁇ 2 nM and both showed a 1:1 stoichiometry ( Figure 2e, Figure 7a).
• a single binding event was observed with a 1:1 nanobody:monomer (3:1 nanobody:Spike) stoichiometry and a K D of 44 ⁇ 3 nM for H11-H4 and K D of 79 ⁇ 2 nM for H11-
• a bivalent Fc- nanobody fusion competes with ACE2 for RBD binding
• nanobodies were fused to the Fc domain of human lgG1 to produce a homodimeric chimeric protein capable of bivalent binding (Figure 3a).
• the ability of these constructs to block ACE2 binding to RBD was tested in two assays.
• MDCK-SIAT1 cells stably expressing human ACE2 (MDCK-ACE2) were seeded on plates and the ability of various analytes (H11-H4-Fc, H11-D4-Fc, ACE2-Fc, CR3022 18 and VHH72-Fc) to block binding of RBD was measured ( Figure 3b).
• VHH72 25 is a nanobody isolated from a llama immunized with Spike from SARS-CoV-1 which is cross reactive against Spike from SARS-CoV-2.
• the MDCK- ACE2 cell binding assay yielded an
• H11-H4-Fc and H11-D4-Fc neutralize virus
• the chimeric fusions were tested in a plaque reduction neutralization test at the Public Health
• the cryo-EM single particle structure of this variant of Spike has been shown to be trimeric with a predominantly ‘up-down-down’ arrangement of the three RBDs 13 .
• the maps clearly identified additional density at all three RBDs in the H11-D4 and H11-H4 complexes ( Figure 10, 11).
• Nanobodies rely on three variable loops denoted CDR1 , CDR2 and CDR3 to form the antigen binding site (Figure 1c).
• CDR1 , CDR2 and CDR3 three variable loops denoted CDR1 , CDR2 and CDR3 to form the antigen binding site.
• Figure 1c crystal structures of the H11-H4-SARS-CoV-2 RBD complex and the H11-D4-SARS-CoV-2 RBD complex were determined to 1.85 and 1.80 A resolution respectively (Table 5). Both crystal structures have a single copy of the complex in the asymmetric unit. Superposition of the two complexes has confirmed that both nanobodies recognize the same epitope (Figure 4b). Comparison of the structures shows that the entire complex superimposes with an r.m.s.d. 1.0 A over 322 Ca atoms, but the individual RBD’s superimpose with an r.m.s.d. 0.5 A over 195
• RBD ( Figure 4d,e).
• the surface on RBD which contacts H11-H4 is formed by Lys444 to Phe456 and Gly482 to Ser494 ( Figure 4f). These two stretches of RBD sequence comprise 90
• Arg52 from CDR2 of H11-H4 was found at the heart of a network of interactions, including RBD residues Glu484 with which it made a bivalent salt link and Phe490 with which it made a p-cation interaction 28 (Figure 4h). Arg52 also forms hydrogen bonds to the backbone carbonyl of Ser103 and side chain of Tyr109 ( Figure 4h) that may stabilize the conformation of the CDR3 loop.
• the seven-residue stretch of H11-H4 CDR3 region which varied during maturation, contributes over 60 % of the surface area buried by the complex and makes five hydrogen bonds to RBD (Figure 4g).
• the Spike trimer exists in an equilibrium between the all ‘down’ configuration and mixed ‘up down’ states 13 .
• the Spike protein can only bind to ACE2 with the RBD in the ‘up’ state 11 and this results in dissociation of the trimer.
• SARS-CoV- 2 Spike binds to ACE2 with a 10 to 20-fold higher affinity (KD of ⁇ 15 nM) than SARS-CoV-1 Spike, a fact that has been proposed to drive its higher transmissibility 1331 .
• Neutralizing antibodies that have been identified to date for SARS-CoV-1 bind to the RBD of the Spike protein and many do so by blocking ACE2 binding 32 but CR3022 operates by a different mechanism 18 .
• H11-H4 and H11-D4 which differ in sequence at five residues within the CDR3 loop (Figure 2a) and have shown some subtle differences in properties ( Figure 2, 3). Since the H11-H4 nanobody has the higher affinity for RBD ( Figure 2e,f), the discussion focuses on this variant but unless explicitly stated is equally valid for H11-D4.
• H11-H4 binds with high affinity to RBD ( Figure 2b,e,f), blocks ACE2 binding ( Figures 2c, 3b, c) and neutralize the virus (Figure 3d,e).
• Our analysis has suggested that H11-H4 would bind to both the ‘all down’ as well as ‘two down one up’ conformations of RBD within the Spike ( Figure 4a and Figure 2c).
• the epitope on SARS-CoV-2 RBD that is recognized by H11-H4 overlaps only to a limited degree with the ACE2 binding region ( Figure
• This region of SARS-CoV-2 RBD has several sequence changes when compared to
• SARS-CoV-1 RBD Figure 1b.
• the Pro469 - Pro470 turn in the SARS-CoV-1 RBD structure 33 is very different to the structure at Val483 - Glu484 in SARS-CoV-2. Additional sequence and structural changes between SARS-CoV-1 and SARS-CoV-2 (Tyr442->Leu455, Trp476-
• SARS-CoV-1 and SARS-CoV-2 raises the possibility SARS-CoV-2 variants may emerge that retain ACE2 receptor binding but are no longer recognized by H11-H4 or its relatives. At least some of the plausible escape mutations would perturb the position of Phe486 which inserts into a cleft in ACE2, an interaction important to SARS-CoV-2’s increased affinity 30 .
• the rapid pipeline from naive library screen to maturation and thorough characterization does offer the possibility that new nanobodies could be generated against SARS-CoV-2 viruses that have escaped H11-H4.
• Vero cells have been described 35 . Some, but not all, of these nanobodies blocked ACE2 binding and no molecular insights into their mode of action were reported 35 . A preprint has reported a llama antibody, Ty1, that neutralizes pseudovirus and blocks ACE2 binding 36 , but the coordinates of the EM structure were not available. The use of convalescent serum has shown clinical promise in patients severely ill with SARS-
• VHHs high-affinity llama single domain antibodies
• VHH-Fc VHH-Fc antibody

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