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WO2022106205A1 - Composés de liaison à la protéine de spicule du coronavirus - Google Patents

Composés de liaison à la protéine de spicule du coronavirus Download PDF

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WO2022106205A1
WO2022106205A1 PCT/EP2021/080560 EP2021080560W WO2022106205A1 WO 2022106205 A1 WO2022106205 A1 WO 2022106205A1 EP 2021080560 W EP2021080560 W EP 2021080560W WO 2022106205 A1 WO2022106205 A1 WO 2022106205A1
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Prior art keywords
vhh
protein
pbc
spike
sars
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Inventor
Nicholas Wu
Ian Wilson
Meng YUAN
Hejun LIU
Paul-Albert KÖNIG
Florian Schmidt
Sabine Normann
Jan Moritz Paul TÖDTMANN
B. Martin HÄLLBERG
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Rheinische Friedrich Wilhelms Universitaet Bonn
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Rheinische Friedrich Wilhelms Universitaet Bonn
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Definitions

  • the invention pertains to protein binding compounds, such as antibodies and antibody like compounds, specifically targeting epitopes of the Corona virus spike (S) protein which were found to stabilize a three-dimensional conformation of the protein which reduces or inhibits the viral mechanisms necessary for host cell entry.
  • the invention provides the protein binding compounds alone and in combination and multispecific constructs, as well as their use in the prevention, treatment, patient stratification and diagnosis of viral infections caused by a corona virus such as severe acute respiratory syndrome corona virus 2 (SARS C0V2).
  • corona virus such as severe acute respiratory syndrome corona virus 2 (SARS C0V2).
  • SARS-C0V-2 severe acute respiratory-syndrome coronavirus 2
  • SARS-C0V-2 severe acute respiratory-syndrome coronavirus 2
  • Studies to develop vaccines are making rapid advances, safe and effective vaccines will not be available to the broader public in the near term.
  • vaccines will likely not be suitable for immunocompromised patients, and cannot provide prophylaxis shortly before or after exposure.
  • Neutralizing antibodies or related molecules offer great potential as immediate and direct-acting antiviral agents (1). Yet, they cannot be easily and economically produced in sufficient amounts for mass application, and cannot be readily modified to include multiple specificities without major costs in yield and quality. Conventional antibodies will also have to be assessed for antibody-dependent enhancement of infection (2).
  • VHHs camelid heavy chain-only antibodies
  • Production in prokaryotic expression systems is cheap, easily upscaled, and importantly allows straight-forward protein engineering, including multivalent VHHs with enhanced functionalities (3). They have favourable biochemical properties, including high thermostability and deep tissue penetration. VHHs have been successfully developed to treat respiratory diseases by inhalator-based delivery (4).
  • the SARS-C0V-2 spike protein binds the cellular receptor ACE2 and catalyzes membrane fusion (5).
  • the spike protein is the major target of neutralizing antibodies, where binding of antibodies to the receptor binding domain (RBD) typically competes with ACE2 binding and thereby hinders infection (6).
  • RBD receptor binding domain
  • conformational flexibility of the spike protein allows each RBD to exist in two conformations: A down conformation that is thought to be less sensitive to antibody binding, and an up conformation that binds ACE2 (7, 8).
  • the inventors identified four distinct VHHs from immunized camelids that neutralize SARS-CoV- 2, characterized their binding site to the SARS-C0V-2 RBD by x-ray crystallography, defined the effects of binding to trimeric spike by cryo-electron microscopy (EM), and used the findings to design biparatopic and other multivalent VHHs with substantial neutralizing potency and protection against viral escape mutants. Surprisingly, neutralization was achieved by at least two distinct mechanisms: Competition with ACE2 binding and irreversible, pre-mature activation of the fusion machinery. The invention demonstrates the clinical potential of the described set of VHHs.
  • the invention is grounded by the surprising finding that certain protein binding compounds such as antibodies which bind to specific epitopes on the Corona virus 2 spike protein stabilize a three- dimensional conformation of the spike protein that reduces or inhibits entry of a virus into a host cell.
  • the new compounds of the invention are used alone or in combination for a treatment or prevention of an infection with a Corona virus and in particular SARS-C0V2, which is the causing virus of the Corona virus disease and pandemic of 2019 (COVID19).
  • the invention pertains to a compound or composition, optionally for use in the treatment of disease caused by an infection with a corona virus, wherein the compound or composition comprises at least a first antigen binding domain binding to a first epitope of a spike protein of the corona virus, characterized in that a binding of the at least first antigen binding domain to the first epitope of the spike protein stabilizes and/or induces an up-conformation of a receptor binding domain (RBD) of the spike protein.
  • RBD receptor binding domain
  • the invention pertains to a protein binding compound (PBC), comprising a first antigen binding domain specifically binding to an epitope of a corona virus spike (S) protein, wherein the PBC when bound to the epitope of the spike protein an up-conformation of the spike protein is induced and/or stabilized
  • PBC protein binding compound
  • S corona virus spike
  • the invention pertains to a protein binding compound (PBC), which is an isolated PBC comprising an amino acid sequence at least 80% identical to a sequence according to any one of SEQ ID NO: l to 92.
  • PBC protein binding compound
  • the invention pertains to an isolated PBC, which competes with a PBC as recited in the second or third aspect for binding to the epitope of the spike protein.
  • the invention pertains to a nucleic acid encoding for a PBC which is a protein or polypeptide according to any one of the preceding aspects.
  • the invention pertains to a recombinant host cell, comprising a PBC or an isolated nucleic acid according to any one of the preceding aspects.
  • the invention pertains to a pharmaceutical composition
  • a pharmaceutical composition comprising a PBC recited in any of the second, third or fourth aspects, an isolated nucleic acid recited in the fifth aspect, a recombinant host cell recited in the sixth aspect, together with a pharmaceutically acceptable carrier and/or excipient.
  • the invention pertains to a compound or composition of the preceding aspects for use in, or pertains to, a method of prevention or treatment of a disease.
  • the invention pertains to a compound or composition of the preceding aspects for use in, or pertaining to, a method of diagnosing of a disease.
  • the invention pertains to a method for identifying and/or characterising a compound suitable for the treatment of a disease, disorder or condition that is associated with, or caused by, a virus expressing a viral spike (S) protein, the method comprising the steps of:
  • the invention pertains to a drug delivery device, which is a nebulizer and/or inhaler, comprising a compound or composition of recited in any one of the previous aspects of the invention.
  • the invention pertains to a compound or composition, optionally for use in the treatment of disease caused by an infection with a corona virus, wherein the compound or composition comprises at least a first antigen binding domain specifically binding to a first epitope of a spike protein of the corona virus, characterized in that a binding of the at least first antigen binding domain to the first epitope of the spike protein stabilizes and/or induces an up-conformation of a receptor binding domain (RBD) of the spike protein.
  • RBD receptor binding domain
  • the invention pertains to a protein binding compound (PBC), comprising a first antigen binding domain specifically binding to an epitope of a corona virus spike (S) protein, and, optionally, wherein the PBC is able to when bound to the epitope of the spike protein an up-conformation of the spike protein is induced and/or stabilized
  • PBC protein binding compound
  • S corona virus spike
  • a PBC of the invention may be a compound of the first aspect or comprised in a composition of the first aspect.
  • corona virus is a virus of the group of RNA viruses that cause mainly respiratory diseases in mammals and birds. Respiratory tract infections of corona viruses can range from mild to lethal, the latter causing severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS), and COVID19. Corona viruses are enveloped viruses having an RNA genome. The envelope is covered with a so called “spike (S) protein”, which is a component known to be essential for host cell entry of the corona virus.
  • S spike
  • the present invention in preferred embodiments pertains to SARS-C0V2, the causing virus of the 2019 pandemic (see Hu, B., Guo, H., Zhou, P. et al. Nat Rev Microbiol (2020). https://d0i.0rg/10.1038/s41579-020-00459-7).
  • a corona virus “spike protein” or “S protein” in context of the present invention shall refer to a corona virus S protein, preferably exclusively a SARS C0V2 S protein; optionally, for the context of the present invention in preferred embodiments, the S protein shall not include SARS C0V1 S protein.
  • the spike protein exists as a homotrimer; each protomer consisting of a Si and a S2 subunit, where each resulting peplomers protrudes from the virus surface as spikes.
  • SARS C0V2 S protein has preferably an amino acid shown in SEQ ID NO: 93.
  • SARS C0V2 might in the future mutate and produce additional S protein variants, such future variants shall be included by the term insofar binding epitopes of the S protein to which the compounds and compositions of the present inventions bind according to the present disclosure remain identical or at least similar to an extend that allows for a specific binding of such inventive compounds and compositions to the S protein.
  • the amino acid sequence of the SARS C0V2 S protein may also be derived from the UniProt database in the version of October 25 2020, under the accession number: P0DTC2 (https://www.uniprot.org/uniprot/PoDTC2).
  • RBD receptor-binding domain of the S protein, which in the case of SARS-C0V2 S protein is located between amino acids 319-541 of SARS-C0V2 S protein.
  • RBD further comprises a receptor-binding motif, located between amino acids 437-508 of SARS-C0V2 S protein.
  • the term “three-dimensional conformation” or “three- dimensional structure”, “conformation” of a protein, or protein multimer, preferably a spike protein, or of a receptor binding domain (RBD) of a spike protein, as described herein, means the spatial arrangement of the amino acid sequences of this protein, or protein multimer, preferably a spike protein, or of a receptor binding domain (RBD) of a spike protein, as described herein, is composed, taken together, when they interact with one another.
  • a spike protein conformation may be preferably in the so called RBD “up” or “down” conformation, which translates in context to the spike trimer to a “one up”, “two up” or “three up” conformation of the RBD.
  • the compound or composition comprising such antigen binding domain of the invention is characterized in that a binding of the at least first antigen binding domain (e.g. of the PBC) to a trimeric spike protein stabilizes and/or induces a post fusion confirmation of the trimeric spike protein as a 2-up, more preferably a 3-up, conformation of the trimeric spike protein.
  • stabilizing (favouring or inducing) such post-fusion conformation of the spike protein using the compounds and compositions of the invention is assumed to block/inhibit the viral entry mechanism and thereby responsible for the herein disclosed therapeutic effect of the invention.
  • an RBD comprises preferably an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or most preferably 100% identical to the amino acid sequence shown in SEQ ID NO: 94 (SARS-C0V2 S protein RBD amino acid sequence).
  • antigen-binding domain shall refer to any entity of a compound or composition, such as a PBC, that has capability to direct a specific binding of the compound or composition to a target epitope.
  • antigen binding domains in context of the present invention are proteinaceous and provided in context of a protein, such as an PBC, for example such as an antibody (or “antigen-binding fragment” thereof).
  • antigen binding domains refers therefore specifically to one or more fragments of an antibody or antibody-like molecules, that retain the abilityto specifically bind to an antigen (e.g., epitope of a spike protein). It has been shown that the antigen -binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding domain” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHt domains; (iv) a Fv fragment consisting of the VL_ and VH domains of a single arm of an antibody, (v) a single domain or dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker.
  • CDR complementarity determining region
  • the two domains of the Fv fragment, Vi_ and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the Vi_ and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding domain” of an antibody.
  • antigen binding domains are provided in the form of an antigen binding domain of a VHH, or an antigen binding fragment thereof, as described herein below.
  • a PBC preferably an “antigen binding protein” (“ABP”), comprising such antigen binding domain of the invention, as used herein means a protein that specifically binds to a target antigen, such as to one or more epitope(s) displayed by or present on a target antigen.
  • the antigen of the PBCs of the invention is a corona virus spike protein, preferably of SARS-C0V2, or an orthologue (or paralogue) or other variant thereof; and the PBC can, optionally bind to one or more extracellular domains of said S protein or variant (such as the epitope(s) can be displayed by or present on one or more domains of the S protein, or a multimer thereof.
  • an antigen binding protein is an antibody (such as an VHH) (or a fragment thereof), however other forms of antigen binding protein are also envisioned by the invention.
  • the PBC may be another (non-antibody) receptor protein derived from small and robust nonimmunoglobulin “scaffolds”, such as those equipped with binding functions for example by using methods of combinatorial protein design (Gebauer & Skerra, 2009; Curr Opin Chem Biol, 13:245).
  • non-antibody PBCs include: Affibody molecules based on the Z domain of Protein A (Nygren, 2008; FEBS J 275:2668); Affilins based on gamma-B crystalline and/or ubiquitin (Ebersbach et al, 2007; J Mo Biol, 372:172); Affimers based on cystatin (Johnson et al, 2012; Anal Chem 84:6553); Affitins based on Sac7d from Sulfolobus acidcaldarius (Krehenbrink et al, 2008; J Mol Biol 383:1058); Alphabodies based on a triple helix coiled coil (Desmet et al, 2014; Nature Comms 5:5237); Anticalins based on lipocalins (Skerra, 2008; FEBS J 275:2677); Avimers based on A domains of various membrane receptors (Silverman et al,
  • epitope includes any determinant capable of being bound by an antigen binding protein, such as an antibody.
  • An epitope is a region of an antigen, such as a spike protein, that is bound by an antigen binding protein that targets that antigen, and when the antigen is a protein, includes specific amino acids that bind the antigen binding protein (such as via an antigen binding domain of said protein).
  • Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three dimensional structural characteristics, and/or specific charge characteristics.
  • antigen binding proteins specific for a particular target antigen will preferentially recognise an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm”). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.
  • the sequences being compared are typically aligned in a way that gives the largest match between the sequences.
  • One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI).
  • GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined.
  • the sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm).
  • a gap opening penalty (which is calculated as 3x the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/ 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.
  • a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 forthe PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915- 10919 for the BLOSUM 62 comparison matrix) may also be used by the algorithm.
  • a preferred method of determining similarity between a protein or nucleic acid is that provided by the Blast searches supported at Uniprot supra (e.g., http://www.uniprot.org/uniprot/Q5DX21); in particular for amino acid identity, those using the following parameters: Program: blastp; Matrix: blosum62; Threshold: 10; Filtered: false; Gapped: true; Maximum number of hits reported: 250.
  • GAP program can be adjusted if so desired to result in an alignment that spans at least about 10, 15, 20, 25, 30, 35, 40, 45, 50 or other number of contiguous amino acids of the target polypeptide or region thereof.
  • the herein disclosed compounds or compositions comprise a second antigen binding domain binding to a second epitope of the spike protein, wherein the first and second epitope are (i) identical, (ii) overlapping, or (iii) distinct.
  • the second antigen binding domain may, in the case of (i), be identical, in the cases of (ii) and (iii) are not identical.
  • the first antigen binding domain and the second antigen binding domain may be comprised within one protein complex or are provided in separate protein complexes. In context of the present invention such combinations of antigen binding domains either in one complex or in separate complexes but in one composition (e.g.
  • a binding of the second antigen binding domain to the second epitope of the spike protein in accordance with the invention may (further) stabilize and/or induce a post fusion conformation of the trimeric spike protein and/or an up-conformation of a receptor binding domain (RBD) of the spike protein, and preferably wherein a binding of the second antigen binding domain to a trimeric spike protein stabilizes and/or induces a 2-up, more preferably a 3-up, conformation of the trimeric spike protein.
  • RBD receptor binding domain
  • connection is realized by using a linker moiety that covalently or non- covalently connects the first and the second antigen binding domains.
  • linker moiety may be any molecule suitable to stably link the first and the second antigen binding domain to each other.
  • the linker of the invention may be a synthetic amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having the function of a hinge region.
  • linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci.
  • Exemplary linkers include simple peptide linkers, or complex protein domain linkers, such as antibody constant domains (such as human CH1 and/or CH3).
  • the peptide linkers utilized in certain embodiments of the invention are used to link the antigen binding domains of two or more antibodies, antigen-binding sites, and/or antibody fragments comprising the different antigenbinding sites (e.g. scFv, full length antibody or VHH) together to form a multispecific antibody according to the invention.
  • the peptide linkers are glycine-rich peptides with at least 5 amino acids, preferably of at least 10 amino acids, more preferably between 10 and 50 amino acids.
  • the term “specifically binds” refers to a non-random binding reaction between two molecules, such as a reaction between an antigen binding domain, or the PBC in which such domain is comprised, and the antigen to which it is directed.
  • a PBC that specifically binds to an antigen means that the antibody binds to the antigen with an affinity (KD) of less than about 10-5 M, for example less than about to -6 M, io ⁇ 7 M, to -8 M, io ⁇ 9 M or to -10 M or lesser.
  • KD refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • an antibody e.g., a VHH of the present invention
  • an antigen e.g., S protein
  • KD dissociation equilibrium constant
  • KD is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i. e., Kd/Ka) and is expressed as a molar concentration (M).
  • KD values for antibodies can be determined using methods well established in the art such as surface plasmon resonance (BIAcore®), ELISA and KINEXA).
  • a preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a BIAcore® system or by ELISA.
  • Ka (or “K-assoc”), as used herein, refers broadly to the association rate of a particular antibody-antigen interaction
  • Kd or “K-diss”
  • a PBC of the invention in certain embodiments is a compound selected from a polypeptide, peptide, glycoprotein, a peptidomimetic, ubiquitin-like protein, an antibody or antibody-like molecule; a nucleic acid such as a DNA or RNA, including variants or derivatives thereof such as a peptide nucleic acid (PNA); a carbohydrate such as a polysaccharide or oligosaccharide and the like, including variants or derivatives thereof; a lipid such as a fatty acid and the like, including variants or derivatives thereof; or a small organic molecules including but not limited to small molecule ligands, small cell-permeable molecules, and peptidomimetic compounds.
  • PNA peptide nucleic acid
  • the first antigen binding domain specifically binds to an epitope of the S protein which involves at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or most preferably all of the amino acid residues of the S protein selected from E484, T470, 1468, F490, Y351, L492, L455, Y453, Q493, S494, L452, R403, Y449, G447, G446 and K444 (as compared to SEQ ID NO: 93).
  • the invention involves a second antigen binding domain such second antigen binding domain may not bind to such afore selected epitope.
  • PBC protein binding compounds
  • such PBC can preferentially comprise at least one complementarity determining region (CDR), such as one from an antibody (in particular from a VHH), and in particular embodiments the PBC can comprise a CDR having an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to (preferably, at least 90% sequence identity to), or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a CDR sequence set forth in Table 1 herein.
  • CDR complementarity determining region
  • complementarity determining region refers broadly to one or more of the hyper- variable or complementarity determining regions (CD Rs) found in the variable regions of light or heavy chains of an antibody. See, for example: “IMGT”, Lefranc et al, 20003, Dev Comp Immunol 27:55; Honegger & Pliickthun, 2001, J Mol Biol 309:657, Abhinandan & Martin, 2008, Mol Immunol 45:3832, Rabat, et al. (1987): Sequences of Proteins of Immunological Interest National Institutes of Health, Bethesda, Md.
  • a PBC can comprise at least one complementarity determining region (CDR).
  • CDR3 complementarity determining region 3
  • a PBC of the invention comprises at least one complementarity determining region 3 (CDR3), such as one having an amino acid sequence with at least 8o%, 85%, 90% or 95% (preferably at least 90%) sequence identity to, or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from those heavy and light chain CDR3 sequences shown in Table 1, e.g.
  • amino acid sequence selected from SEQ ID Nos 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, and 91, or having an amino acid sequence having no more than three, preferably no more than two or one, amino acid substitution(s), deletion(s), insertion(s) or addition(s) compared to these sequences.
  • a PBC of the invention may, alternatively or as well as a CDR3 sequence, comprise at least one CDRt, and/or at least one CDR2 (such as one from an antibody, in particular from a VHH).
  • a PBC of the invention comprises at least one such CDR3, as well as at least one such CDRt and at least one such CDR2, more preferably where each of such CDRs having an amino acid sequence with at least 80%, 85%, 90% or 95% (preferably at least 90%) sequence identity to, or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from the corresponding (heavy and light chain) CDRt, CDR2 and CDR3 sequences shown in Table 1.
  • a PBC of the invention can be an antibody (such as an VHH) or an antigen binding fragment thereof.
  • the term “antibody” may be understood in the broadest sense as any immunoglobulin (Ig) that enables binding to its epitope.
  • An antibody as such is a species of a PBC.
  • Full length “antibodies” or “immunoglobulins” are generally heterotetrameric glycoproteins of about 150 kDa, composed of two identical light and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulphide bond, while the number of disulphide linkages varies between the heavy chain of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulphide bridges. Each heavy chain has an amino terminal variable domain (VH) followed by three carboxy terminal constant domains (CH).
  • VH amino terminal variable domain
  • CH carboxy terminal constant domains
  • Each light chain has a variable N-terminal domain (VL) and a single C-terminal constant domain (CL).
  • 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
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDRt, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to cells or factors, including various cells of the immune system (e.g., effector cells) and the first component (Ctq) of the classical complement system.
  • a preferred form of an antibody of the invention includes a heavy-chain antibody or VHH, being those which consist only of heavy chains and lack the two light chains usually found in antibodies.
  • Heavychain antibodies include the hcIgG (IgG-like) antibodies of camelids such as dromedaries, camels, llamas and alpacas, and the IgNAR antibodies of cartilaginous fishes (for example sharks).
  • Yet other forms of antibodies include single-domain antibodies (sdAb, called Nanobody by Ablynx, the developer) being an antibody fragment consisting of a single monomeric variable antibody domain. Single-domain antibodies are typically produced from heavy-chain antibodies, but may also be derived from conventional antibodies.
  • the antibody of the invention is a VHH comprising an antibody heavy chain variable region, or an antigen binding fragment thereof, and not comprising an antibody light chain variable region, nor an antigen binding fragment thereof.
  • Antibodies can include, for instance, chimeric, humanized, (fully) human, or hybrid antibodies with dual or multiple antigen or epitope specificities, antibody fragments and antibody sub-fragments, e.g., Fab, Fab' or F(ab')2 fragments, single chain antibodies (scFv) and the like (described below), including hybrid fragments of any immunoglobulin or any natural, synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • a PBC of the invention can comprise an antibody heavy chain, or an antigen binding fragment thereof, and/or an antibody light chain, or an antigen binding fragment thereof.
  • a PBC of the invention can comprise an antibody heavy chain variable region, or an antigen binding fragment thereof, and/or an antibody light chain variable region, or an antigen binding fragment thereof, and in yet further embodiments, a PBC of the invention can comprise an antibody heavy chain variable region CDR1, CDR2, and CDR3, and/or an antibody light chain variable region CDR1, CDR2, and CDR3.
  • the PBC comprises an antibody heavy chain sequence, or the fragment thereof, comprising a CDRt having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85 and 89, or having no more than three, preferably no more than two or one, amino acid substitution(s), deletion(s), insertion(s) or addition(s) compared to these sequences; and/or a CDR2 having at 80% sequence identity to an amino acid sequence selected from SEQ ID Nos.
  • the PBC may be an antibody, preferably a VHH, or an antigen binding fragment thereof, composed of the first antigen binding domain comprising an antibody heavy chain sequence comprising CDRt to CDR3 sequences in the following combination: in each case independently, optionally with not more than three or two, preferably one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • Preferred embodiments and aspects of the present invention pertain to the antibodies, or the herein described variants thereof, designated as VHH-E, VHH-N, VHH-U, VHH-V, and VHH-W.
  • the such first antigen binding domain is preferably derived from VHH-E, or at least binds specifically to the same or overlapping epitope, and wherein the optional second antigen binding domain binds specifically to the same, or overlapping, epitope of any of VHH-U, VHH-V, and VHH- W.
  • an PBC of the invention can comprise an antibody heavy chain sequence having at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity to, or having no more than ten, nine, eight, seven, six, five, four, three, two or one, preferably no more than three, two or one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from SEQ ID NOs. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, or 92 (eg, a full length sequence disclosed in table 1 - preferred are sequences 20, 56, 84, 88, or 92).
  • one preferred embodiment of the invention pertains to multimeric PBCs, comprising more than one (or more) antigen binding domains, preferably wherein such antigen binding domains may bind different antigenic epitopes, such as different epitopes on the S protein.
  • Such multimeric PBCs of the invention are preferably bispecific molecules, for examples having a first and a second antigen binding site which are derived from any combination of binding sites (and their herein disclosed variants) of the VHH disclosed in table 1.
  • Preferred combinations for such multi- or bispecific PBCs are derived from a combination of one or more antigen binding domains of VHH-E, VHH-N, VHH-U, VHH-V, and VHH-W.
  • Such preferred PBCs comprise a first antigen binding domain derived from VHH-E, or any of its fragments or variants, and a second antigen binding domain derived from any of the other VHH of table 1, preferably of an antigen binding domain selected from any one of VHH-N, VHH-U, VHH-V, and VHH-W.
  • the present invention therefore, in some particular preferred aspects and embodiments pertains to a PBC, comprising a first and a second antigen binding domain, wherein the first antigen binding domain comprises optionally a CDR1 having an amino acid sequence shown in SEQ ID NO: 17, optionally a CDR2 having an amino acid sequence shown in SEQ ID NO: 18, and a CDR3 having an amino acid sequence shown in SEQ ID NO: 19; in each case independently, optionally with not more than three or two, preferably one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences; and preferably wherein the first antigen binding domain comprises said CDR1, CDR2 and CDR3; and wherein said second antigen binding domain comprises:
  • the antigen binding domains of such PBC may be connected via a linker as described herein elsewhere. Further the first and/or second antigen binding domain is monovalent or divalent or multivalent.
  • the PBC of the invention may have an additional further antigen binding domain, specifically binding to for example albumin.
  • Such further antigen binding domain maybe provided as an antibody specifically binding albumin.
  • a PBC of the invention which is a variant of VHH-E may have preferably an amino acid substitution, addition or deletion in any of the following amino acid positions of S105; G106, S53, S54, and/or D55 SEQ ID NO: 20. According to the structural data obtained in context of the invention, the selected amino acid positions are not relevant for VHH antigen binding.
  • a PBC of the invention which is a variant of VHH-U/W may have preferably an amino acid substitution, addition or deletion in any of the following amino acid positions G107; S108, G55, G56, and/or S57 of SEQ ID NO: 84 or 92 (respectively). According to the structural data obtained in context of the invention, the selected amino acid positions are not relevant for VHH antigen binding.
  • a PBC of the invention which is a variant of VHH-V may have preferably an amino acid substitution, addition or deletion in any of the following amino acid positions A96; T97, S53, D54 and/or G55 of SEQ ID NO: 88 (respectively). According to the structural data obtained in context of the invention, the selected amino acid positions are not relevant for VHH antigen binding.
  • Preferred for the such variants are amino acid substitutions, preferably of amino acid residues with similar properties such as exchanges between S - T, G - A, and D - E.
  • overlapping epitope in the context of the present specification refers to an epitope that is partially identical to a certain epitope.
  • the invention pertains to a protein binding compound (PBC), which is an isolated PBC comprising an amino acid sequence at least 80% identical to a sequence according to any one of SEQ ID NO: l to 92.
  • PBC protein binding compound
  • the invention pertains to an isolated PBC, which competes with a PBC as recited in the second or third aspect for binding to the epitope of the spike protein.
  • Compet when used in the context of PBCs (e.g., antigen binding domain) that compete for binding for the same antigen (or epitope displayed by such antigen) means competition between PBCs as may be determined by an assay in which the PBC (e.g., antibody or binding fragment thereof) being tested prevents or inhibits (e.g., reduces) binding of a reference PBC (e.g., a ligand, or a reference antibody) to a common antigen (e.g., SARS- C0V2 Spike protein).
  • a reference PBC e.g., a ligand, or a reference antibody
  • the PBC is isolated and/or substantially pure.
  • isolated refers to a protein that is purified from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.
  • An isolated PBC according to the invention maybe a recombinant, synthetic or modified (non-natural) PBC.
  • isolated refers to a nucleic acid or cells that is/are purified from DNA, RNA, proteins or polypeptides or other contaminants (such as other cells) that would interfere with its therapeutic, diagnostic, prophylactic, research or other use, or it refers to a recombinant, synthetic or modified (non-natural) nucleic acid.
  • an isolated PBC or nucleic acid or cells is/are substantially pure.
  • a “recombinant” protein or nucleic acid is one made using recombinant techniques. Methods and techniques for the production of recombinant nucleic acids and proteins are well known in the art.
  • an PBC of the invention may bind to (e.g., via one or more epitope(s) displayed by one or more EC domain(s) of) a corona virus S protein or a paralogue, orthologue or other variant thereof with a KD that is less than 1000 nM, such as less than about too nM, or in particular, less than about 30 nM).
  • the PBC of the invention will bind (e.g. said epitope(s) of) said S protein or variant with a KD that is less than 10 nM, such as an KD of about 2 nM.
  • the PBC is an antibody or an antigen binding fragment thereof, and the antibody is a monoclonal antibody, or wherein the antigen binding fragment is a fragment of a monoclonal antibody.
  • Such antibodies may also non VHH antibodies.
  • mAb refers to an antibody obtained from a population of substantially identical antibodies based on their amino acid sequence. Monoclonal antibodies are typically highly specific. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (e.g. epitopes) of an antigen, each mAb is typically directed against a single determinant on the antigen. In addition to their specificity, mAbs are advantageous in that they can be synthesized by cell culture (hybridomas, recombinant cells or the like) uncontaminated by other immunoglobulins. The mAbs herein include for example chimeric, humanized or human antibodies or antibody fragments.
  • Monoclonal antibodies in accordance with the present invention may be prepared by methods well known to those skilled in the art. For example, mice, rats or rabbits maybe immunized with an antigen of interest together with adjuvant. Splenocytes are harvested as a pool from the animals that are administered several immunisations at certain intervals with test bleeds performed to assess for serum antibody titers. Splenocytes are prepared that are either used immediately in fusion experiments or stored in liquid nitrogen for use in future fusions. Fusion experiments are then performed according to the procedure of Stewart & Fuller, J. Immunol. Methods 1989, 123:45-53. Supernatants from wells with growing hybrids are screened by e.g.
  • ELISA enzyme-linked immunosorbent assay
  • ELISA-positive cultures are cloned either by limiting dilutions or fluorescence-activated cell sorting, typically resulting in hybridomas established from single colonies.
  • the ability of an antibody, including an antibody fragment or sub-fragment, to bind to a specific antigen can be determined by binding assays known in the art, for example, using the antigen of interest as the binding partner.
  • a PBC of the invention is an antibody or an antigen binding fragment thereof, wherein the antibody is a human antibody a humanised antibody or a chimeric-human antibody, or wherein the antigen binding fragment is a fragment of a human antibody a humanised antibody or a chimeric-human antibody.
  • Human antibodies can also be derived by in vitro methods. Suitable examples include but are not limited to phage display (CAT, Morphosys, Dyax, Biosite/Medarex, Xoma, Yumab, Symphogen, Alexion, Affimed) and the like.
  • phage display a polynucleotide encoding a single Fab or Fv antibody fragment is expressed on the surface of a phage particle (see e.g., Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et al., J Mol Biol 222: 581 (1991); U.S. Patent No. 5,885,793).
  • Phage are “screened” to identify those antibody fragments having affinity for target.
  • certain such processes mimic immune selection through the display of antibody fragment repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to target.
  • high affinity functional neutralizing antibody fragments are isolated.
  • a complete repertoire of human antibody genes may thus be created by cloning naturally rearranged human V genes from peripheral blood lymphocytes (see, e.g., Mullinax et al., Proc Natl Acad Sci (USA), 87: 8095-8099 (1990)) or by generating fully synthetic or semisynthetic phage display libraries with human antibody sequences (see Knappik et al 2000; J Mol Biol 296:57; de Kruif et al, 1995; J Mol Biol 2481:97).
  • mice are capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies.
  • a preferred embodiment of transgenic production of mice and antibodies is disclosed in U.S. Patent Application Serial No. 08/759,620, filed December 3, 1996 and International Patent Application Nos. WO 98/24893, published June 11, 1998 and WO 00/76310, published December 21, 2000. See also Mendez et al., Nature Genetics, 15:146-156 (1997). Through the use of such technology, fully human monoclonal antibodies to a variety of antigens have been produced.
  • XenoMouse® lines of mice are immunized with an antigen of interest, e.g.an epitope of the S protein as described herein), lymphatic cells (such as B-cells) are recovered from the hyper-immunized mice, and the recovered lymphocytes are fused with a myeloid-type cell line to prepare immortal hybridoma cell lines.
  • lymphatic cells such as B-cells
  • myeloid-type cell line to prepare immortal hybridoma cell lines.
  • mice are also commercially available: eg, Medarex - HuMab mouse, Kymab - Kymouse, Regeneron - Velocimmune mouse, Kirin - TC mouse, Trianni - Trianni mouse, OmniAb - OmniMouse, Harbour Antibodies - H2L2 mouse, Merus - MeMo mouse. Also are available are “humanised” other species: rats: OmniAb - OmniRat, 0MT - UniRat. Chicken: OmniAb - OmniChicken.
  • humanised antibody refers to immunoglobulin chains or fragments thereof (such as Fab, Fab', F(ab')2, Fv, or other antigen-binding sub-sequences of antibodies), which contain minimal sequence (but typically, still at least a portion) derived from nonhuman immunoglobulin.
  • humanised antibodies are human immunoglobulins (the recipient antibody) in which CDR residues of the recipient antibody are replaced by CDR residues from a non-human species immunoglobulin (the donor antibody) such as a mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • the framework sequence of said antibody or fragment thereof may be a human consensus framework sequence.
  • humanised antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximise antibody performance.
  • the humanised antibody will comprise substantially all of at least one, and typically at least two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanised antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, which (eg human) immunoglobulin constant region may be modified (eg by mutations or glycoengineering) to optimise one or more properties of such region and/or to improve the function of the (eg therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life.
  • an immunoglobulin constant region typically that of a human immunoglobulin, which (eg human) immunoglobulin constant region may be modified (eg by mutations or glycoengineering) to optimise one or more properties of such region and/or to improve the function of the (eg therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life.
  • Fc modification for example, Fc engineering or Fc enhancement
  • chimeric antibody refers to an antibody whose light and/or heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant regions which are identical to, or homologous to, corresponding sequences of different species, such as mouse and human.
  • variable region genes derive from a particular antibody class or subclass while the remainder of the chain derives from another antibody class or subclass of the same or a different species. It covers also fragments of such antibodies.
  • a typical therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species may be used.
  • a PBC of the invention comprises an antigen binding domain of an antibody wherein the antigen binding domain is of a human antibody.
  • PBC comprises an antigen binding domain of an antibody or an antigen binding fragment thereof, which is a human antigen binding domain; (ii) the antibody is a monoclonal antibody, or wherein the antigen binding fragment is a fragment of a monoclonal antibody; and (iii) the antibody is a human antibody or a humanised antibody, or wherein the antigen binding fragment is a fragment of a human antibody, a humanised antibody or a chimeric-human antibody.
  • Light chains of human antibodies generally are classified as kappa and lambda light chains, and each of these contains one variable region and one constant domain. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon chains, and these define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • Human IgG has several subtypes, including, but not limited to, IgGt, lgG2, lgG3, and IgGq.
  • Human IgM subtypes include IgM, and lgM2.
  • Human IgA subtypes include IgAt and lgA2.
  • the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains ten or twelve heavy chains and ten or twelve light chains.
  • Antibodies according to the invention may be IgG, IgE, IgD, IgA, or IgM immunoglobulins, in the case of camelid antibodies or their fragments, such antibodies are IgG 2b/2c (otherwise known as IgG 2/3).
  • the PBC of the invention is an IgG antibody or fragment thereof. In some embodiments, the PBC of the invention is an IgE antibody or fragment thereof. In some embodiments, the PBC of the invention is an IgD antibody or fragment thereof. In some embodiments, the PBC of the invention is an IgA antibody or fragment thereof. In some embodiments, the PBC of the invention is an IgM antibody or fragment thereof.
  • the PBC of the invention is, comprises or is derived from an IgG immunoglobulin or fragment thereof; such as a human, human-derived IgG immunoglobulin, or a rabbit- or rat-derived IgG, and/or an IgG2 immunoglobulin, or fragment thereof.
  • the PBC of the invention comprises or is derived from a rat-derived IgG
  • the PBC is, comprises or is derived from, a rat IgG2a or IgG2b immunoglobulin.
  • the PBC of the invention comprises or is derived from a human-derived IgG
  • the PBC of the invention comprises or is derived from a human IgGt, IgG2 or IgG4
  • the PBC of the invention is, comprises or is derived from a human IgGt or IgG2
  • a PBC is an antibody wherein the antibody is an IgG, IgE, IgD, IgA, or IgM immunoglobulin; preferably an IgG immunoglobulin.
  • An PBC of the invention where comprising at least a portion of an immunoglobulin constant region (typically that of a human immunoglobulin) may have such (eg human) immunoglobulin constant region modified - for example eg by glycoengineering or mutations - to optimise one or more properties of such region and/or to improve the function of the (eg therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life.
  • an immunoglobulin constant region typically that of a human immunoglobulin
  • modified - for example eg by glycoengineering or mutations - to optimise one or more properties of such region and/or to improve the function of the (eg therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life.
  • Antibody fragments include “Fab fragments”, which are composed of one constant and one variable domain of each of the heavy and the light chains, held together by the adjacent constant region of the light chain and the first constant domain (CH1) of the heavy chain. These may be formed by protease digestion, e.g. with papain, from conventional antibodies, but similar Fab fragments may also be produced by genetic engineering. Fab fragments include Fab’, Fab and “Fab-SH” (which are Fab fragments containing at least one free sulfhydryl group).
  • Fab’ fragments differ from Fab fragments in that they contain additional residues at the carboxy terminus of the first constant domain of the heavy chain including one or more cysteines from the antibody hinge region.
  • Fab’ fragments include “Fab’-SH” (which are Fab’ fragments containing at least one free sulfhydryl group).
  • antibody fragments include F(ab‘)2 fragments, which contain two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains (“hinge region”), such that an interchain disulphide bond is formed between the two heavy chains.
  • a F(ab’)2 fragment thus is composed of two Fab’ fragments that are held together by a disulphide bond between the two heavy chains.
  • F(ab’)2 fragments may be prepared from conventional antibodies by proteolytic cleavage with an enzyme that cleaves below the hinge region, e.g. with pepsin, or by genetic engineering.
  • an “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
  • Single-chain antibodies or “scFv” are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region.
  • An “Fc region” comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulphide bonds and by hydrophobic interactions of the CH3 domains.
  • the PBC of the invention is an antibody fragment selected from the list consisting of: Fab’, Fab, Fab’-SH, Fab-SH, Fv, scFv and F(ab’)2.
  • a PBC of the herein disclosed invention may be provided as a fusion protein of the PBC (for example a VHH, or multi-specific VHH as described ) fused to a heterologous polypeptide chain, such as Fc immunoglobulin domains, albumin binding proteins, or tags or labels, and/or the PBC conjugated to another chemical moiety such as an effector group or a labelling group.
  • a heterologous polypeptide chain such as Fc immunoglobulin domains, albumin binding proteins, or tags or labels
  • another chemical moiety such as an effector group or a labelling group.
  • Fusion proteins that have an increased half life compared to the VHH are fusions to albumin, albumin antibodies, albumin binding peptides or a fusion to albumin binding domains.
  • the invention pertains to a nucleic acid encoding for a PBC which is a protein or polypeptide according to any one of the preceding aspects, hence preferably such PBC is an antigen binding protein (ABP).
  • the component encoded by a nucleic acid of the invention may be all or part of one chain of an antibody of the invention; or the component may be a binding domain of a VHH of said ABP.
  • the component encoded by such a nucleic acid may be all or part of one or other of the chains of an antibody of the invention; for example, the component encoded by such a nucleic acid may be an ABD of the invention.
  • the nucleic acids of the invention may also encode a fragment, derivative, mutant, or variant of an ABP of the invention, and/or represent components that are polynucleotides suitable and/or sufficient for use as hybridisation probes, polymerase chain reaction (PCR) primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense or inhibitory nucleic acids (such as RNAi/siRNA/shRNA or gRNA molecules) for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing.
  • PCR polymerase chain reaction
  • a nucleic acid of the invention comprises a nucleic acid having a sequence encoding a heavy or light chain CDR, a combination of heavy and/or light chain CDR1, CDR2 and CDR3 or a heavy or light chain variable domain, in each case as displayed in Table 1, or a functional fragment thereof.
  • the nucleic acid according to the invention may be a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof, optionally linked to a polynucleotide to which it is not linked in nature.
  • such nucleic acid may comprise one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 20, in particular between 1 and about 5, or preferably all instances of a particular nucleotide in the sequence) unnatural (e.g. synthetic) nucleotides; and/or such nucleic acid may comprise (e.g. is conjugated to) another chemical moiety, such as a labelling group or an effector group; for example, a labelling group or an effector group as described elsewhere herein.
  • the nucleic acid of the invention may be isolated or substantially pure.
  • the nucleic acid of the invention may be recombinant, synthetic and/or modified, or in any other way non-natural.
  • a nucleic acid of the invention may contain at least one nucleic acid substitution (or deletion) modification (such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 such modifications, in particular between 1 and about 5 such modifications, preferably 2 or 3 such modifications) relative to a product of nature, such as a human nucleic acid.
  • Nucleic acids encoding antibody polypeptides may be isolated from B-cells of mice, rats, all camelid species, such as llamas and alpacas, sharks, chicken or rabbits that have been immunized with an S protein antigen or fragment thereof, such as one or more epitopes of the S protein (such as of an RBD thereof).
  • the nucleic acid may be isolated by conventional procedures such as PCR.
  • Changes can be introduced by mutation into the sequence of a nucleic acid of the invention. Such changes, depending on their nature and location in a codon, can lead to changes in the amino acid sequence of a polypeptide (e.g., an antigen binding protein) that it encodes. Mutations can be introduced using any technique known in the art.
  • one or more particular amino acid residues may be changed using, for example, a site-directed mutagenesis protocol.
  • one or more randomly selected residues maybe changed using, for example, a random mutagenesis protocol.
  • a mutant polypeptide can be expressed and screened for a desired property. Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues.
  • nucleic acid of the invention may not alter the amino acid sequence of the encoded polypeptide, but may lead to changes to its stability and/or effectiveness of expression of the encoded polypeptide.
  • codon optimisation the expression of a given polypeptide sequence may be improved by utilising the more common codons for a given amino acid that are found for the species in which the nucleotide is to be expressed.
  • codon optimisation and alternative methods (such as optimisation of CpG and G/C content), are described in, for example, Hass et al, 1996 (Current Biology 6:315); WO1996/O9378; W02006/015789 and WO 2002/098443).
  • the invention relates to a nucleic acid construct (NAC) comprising at least one nucleic acid of the invention (such as described above).
  • NAC nucleic acid construct
  • Such an NAC can comprise one or more additional features permitting the expression of the encoded ABP or component of said ABP (eg the ABD) in a cell (such as in a host cell).
  • NACs of the invention include, but are not limited to, plasmid vectors, viral vectors, mRNA, non-episomal mammalian vectors and expression vectors, for example, recombinant expression vectors.
  • the nucleic acid constructs of the invention can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a cell, such as a host cell, (see below).
  • the nucleic acid constructs of the invention will be, typically, recombinant nucleic acids, and/or may be isolated and/or substantially pure. Recombinant nucleic acids will, typically, be non-natural; particularly if they comprise portions that are derived from different species and/or synthetic, in-vitro or mutagenic methods.
  • nucleic acid or NAC of the invention may, as further described herein elsewhere, be used in therapy to deliver such nucleic acid or NAC by administration to a subject to cells involved with the disorder to be treated.
  • Delivery systems for targeting a nucleic acid or NAC to a specific cell or tissue are well known in the art and include viral delivery systems where such nucleic acid or NAC of the invention is introduced into or combined with a viral vector.
  • the invention relates to a cell (such as a host cell and/or a recombinant host cell) comprising one or more nucleic acid or NAC of the invention.
  • a cell such as a host cell and/or a recombinant host cell
  • such cell is capable of expressing the ABP (or component thereof) encoded by said NAC(s).
  • an ABP of the invention comprises two separate polypeptide chains (e.g. a heavy and light chain of an IgG)
  • the cell of the invention may comprise a first NAC that encodes (and can express) the heavy chain of such ABP as well as a second NAC that encodes (and can express) the light chain of such ABP; alternatively, the cell may comprise a single NAC that encodes both chains of such ABP.
  • a (host) cell of invention may be one of the mammalian, prokaryotic or eukaryotic host cells as described elsewhere herein, in particularly where the cell is a E. coli cell, a Komagataella phaffii cell or a Chinese hamster ovary (CHO) cell.
  • the (host) cell is a human cell; in particular it may be a human cell that has been sampled from a specific individual (eg an autologous human cell).
  • a specific individual eg an autologous human cell
  • such human cell can be propagated and/or manipulated in-vitro so as to introduce a NAC of the present invention.
  • the utility of a manipulated human cell from a specific individual can be to produce an ABP of the invention, including to reintroduce a population of such manipulated human cells into a human subject, such as for use in therapy.
  • the manipulated human cell may be introduced into the same human individual from which it was first sampled; for example, as an autologous human cell.
  • the human cell that is subject to such manipulation can be of any germ cell or somatic cell type in the body.
  • the donor cell can be a germ cell or a somatic cell selected from the group consisting of fibroblasts, B cells, T cells, dendritic cells, keratinocytes, adipose cells, epithelial cells, epidermal cells, chondrocytes, cumulus cells, neural cells, glial cells, astrocytes, cardiac cells, oesophageal cells, muscle cells, melanocytes, hematopoietic cells, macrophages, monocytes, and mononuclear cells.
  • the donor cell can be obtained from any organ or tissue in the body; for example, it can be a cell from an organ selected from the group consisting of liver, stomach, intestines, lung, pancreas, cornea, skin, gallbladder, ovary, testes, kidneys, heart, bladder, and urethra.
  • the PBCs, ABPs, nucleic acids or NACs (or the cells, such as host cells) of the invention may be formulated into a pharmaceutical composition appropriate to facilitate administration to animals or humans.
  • pharmaceutical composition means a mixture of substances including a therapeutically active substance (such as a PBC of the invention) for pharmaceutical use.
  • the invention pertains to a pharmaceutical composition
  • a pharmaceutical composition comprising a PBC recited in any of the second, third or fourth aspects, an isolated nucleic acid recited in the fifth aspect, a recombinant host cell recited in the sixth aspect, together with a pharmaceutically acceptable carrier and/ or excipient.
  • the pharmaceutical composition of the invention may comprise between 0.1% and 100% (w/w) active ingredient (for example, a PBC of the invention), such as about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 8% 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably between about 1% and about 20%, between about 10% and 50% or between about 40% and 90%.
  • active ingredient for example, a PBC of the invention
  • compositions are intended to include any and all solvents, solubilisers, fillers, stabilisers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • solvents solubilisers, fillers, stabilisers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • diluents emulsifying agents
  • humectants humectants
  • dispersion media coatings, antibacterial or antifungal agents, isotonic and absorption
  • the pharmaceutical composition of (or for use with) the invention is, typically, formulated to be compatible with its intended route of administration.
  • routes of administration include oral, parenteral, e.g., intrathecal, intra-arterial, intravenous, intradermal, intramuscular, subcutaneous, oral, transdermal (topical) and transmucosal administration.
  • the compounds of (or for use with) the invention are typically delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebuliser.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebuliser.
  • the present invention provides the possibility delivering an effective amount of protein binding constructs, such as preferably the VHH of the invention, for the treatment of a pulmonary disease in mammals via the pulmonary route, which, according to one specific embodiment, is provided by inhaling a pharmaceutical dosage formulation with an inhaler device.
  • protein binding constructs such as preferably the VHH of the invention
  • the invention pertains to a drug delivery device, which is a nebulizer and/or inhaler, comprising a compound or composition of recited in any one of the previous aspects of the invention.
  • the device should generate from the formulation an aerosol cloud of the desired particle size of the fine solid particles or liquid droplets (distribution) at the appropriate moment of the mammal's inhalation cycle, containing the right dose of the PBC of the invention.
  • formulation requirements formulation, particle size, time and dose
  • VHH-VE construct of the invention is preferably used in an inhaler device, because surprisingly it could be shown that nebulizing does not induce fragmentation of the VHH.
  • nebulizers such as e.g. vibrating mesh nebulizers, metered dose inhalers, metered dose liquid inhalers, and dry powder inhalers.
  • Devices taking into account optimized and individualized breathing pattern for controlled inhalation manoeuvres may also be used (see e.g. AKITA® technology on page 157 of “Pulmonary Drug Delivery”, Bechtold- Peters and Luessen, eds., supra).
  • nebulizers have been classified into two main types: air-jet (pneumatic) and ultrasonic devices. Recently, a third type, vibrating-mesh nebulizers has been commercialized (Newman, S., Gee-Turner, A., 2005. The Omron MicroAir Vibrating mesh technology nebuliser, a 21st century approach to inhalation therapy. J. Appl. Ther. Res. 5, 29-33).
  • Air-jet nebulizers convert liquid into aerosols by means of a high velocity gas passing through a narrow “venturi” nozzle. The fluid in the nebulizer reservoir is drawn up a feed tube and emerges as fine filaments that collapse into aerosol droplets due to surface tension.
  • a high frequency vibrating piezoelectric crystal is employed to generate the aerosol.
  • a fountain of fluid is produced at the air-fluid interface. Small droplets are generated from the lower regions of the fountain whilst large droplets are generated from the apex.
  • baffles in the nebulizer trap and recycle the large (primary) aerosol droplets, whilst small (secondary) droplets are released for inhalation.
  • the aerosol output comprises aerosolized droplets and solvent vapour which saturates the outgoing air. This induces cooling of the nebulizer fluid and increases solute concentration in the residual volume (Cockcroft, D.
  • Ultrasonic nebulizers are generally unsuitable for delivery of suspensions (Taylor, K. M. G., McCallion, 0. N. M., 2002. Ultrasonic nebulizers. In: Swarbrick, J., Boylan, J. C. (Eds.), Encyclopedia of Pharmaceutical Technology, 2nd ed. Marcel Dekker, Inc., New York, pp. 2840-2847) and liposomes (Elhissi. A. M. A., Taylor, K. M.
  • Vibrating-mesh nebulizers may overcome the drawbacks of air-jet and ultrasonic nebulizers. Vibrating-mesh devices employ perforated plates which vibrate in order to generate the aerosol. These nebulizers do not heat the fluid during atomization and have been shown to be suitable for delivery of suspensions (Fink, J. B., Simmons, B. S., 2004. Nebulization of steroid suspension: an in vitro evaluation of the Aeroneb Go and Pari LC Plus nebulizers. Chest 126, 816S), and delicate structures such as liposomes (Wagner, A., Vorauer-Uhl, K., Katinger, H., 2006.
  • Vibrating-mesh nebulizers are divided into passively and actively vibrating-mesh devices (Newman, S., Gee-Turner, A., 2005. The Omron MicroAir Vibrating mesh technology nebuliser, a 21st century approach to inhalation therapy. J. Appl. Ther. Res. 5, 29-33). Passively vibrating- mesh devices (e.g.
  • Omron MicroAir NE-U22 nebulizer employ a perforated plate having up to 6000 tapered holes, approximately 3_min diameter.
  • a vibrating Piezo-electric crystal attached to a transducer horn induces “passive” vibrations in the perforated plate positioned in front of it, resulting in extrusion of fluid through the holes and generation of the aerosol.
  • Actively vibrating-mesh devices e.g. Aeroneb Pro nebulizer
  • the invention pertains to a compound or composition of the preceding aspects for use in, or pertains to, a method of treatment of a disease
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic (also referred to as “prevention” or “preventing”) in terms of completely or partially preventing a disease or symptom thereof and/or maybe therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; (c) relieving the disease symptom, i.e., causing regression of the disease or symptom; (d) limiting spread of a virus from one cell to another within an individual, e.g., limiting spread of a virus from an infected epithelial cell to other, uninfected, epithelial cells within an individual (which is preferable in accordance with the invention, which without being bound to theory, provides means and methods for reducing viral entry into a host cell); (e) limiting replication of a virus within an individual; (f) limiting entry of a virus into a cell in an individual; and (g) reducing the number of viruses in an individual or in
  • subject is used interchangeably herein to refer to a bird or mammal, including, but not limited to, chicken, murines (rats, mice), felines, pholidota, chiroptera, non-human primates (e.g., simians), humans, canines, ungulates, etc.
  • a “subject” is a human, and can also be referred to as a “patient.”
  • a disease to be treated in the various aspects and embodiments of the invention are selected from diseases caused by a virus, preferably a virus that causes a respiratory disease.
  • the virus is preferably a virus that is characterized by a host cell entry mechanism that uses a spike protein, or variants thereof.
  • diseases include Corona virus caused primary or secondary diseases.
  • the treatment is preventive or prophylactic (i.e., it protects the patient against infection), whereas if the administration is performed after infection or initiation of the disease, the treatment is therapeutic (i.e., it combats the existing infection).
  • the compounds or composition of the invention are used in a therapeutic aspect.
  • the compounds or compositions of the invention may in addition be used to prevent a virally caused disease in a subject that is suspected of developing the disease, or who to a subject that is a member risk group.
  • risk group is characterized by having an increased risk for a fatal event or outcome in the case of an infection with the virus.
  • Elderly subjects such as subjects of 50 years or older (preferably 60 years or older, most preferably 70 years or older) are risk groups, or subjects having another immune compromising condition, or being generally of poor health.
  • a therapeutic or preventive use in context of the present invention is preferably a use in the treatment or prevention of COVID19, or any later (subsequent to 2019) variant of the disease.
  • the invention relates to a method for the treatment of a disease, disorder or condition in a mammalian subject by administering a product to the subject wherein the product is an PBC of the invention, or of a variant thereof.
  • the invention relates to a product for use in medicine, wherein the product is a compound that is PBC of the invention, or of a variant thereof.
  • the modulating compound such as an PBC stabilizes (favors or induces) a S protein up-conformation (such as of SARS-C0V2- S protein; and/ or wherein the product is selected from the list consisting of a mono or multi specific PBC, nucleic acid, NAC or recombinant host cell of the invention, in particular a PBC of the invention.
  • the invention also relates to method of treating or preventing a disease, disorder or condition in a mammalian subject in need thereof, comprising administering to said subject at least once an effective amount of modulating compound as desired above, or, and in particular administering to said subject at least once an effective amount of the PBC, the NAC, the (host) cells, or the pharmaceutical composition as described above.
  • the invention also relates to the use of a product of the invention as describe above, or a modulating compound as described above (in particular an PBC of the invention) for the manufacture of a medicament, in particular for the treatment of a disease, disorder or condition in a mammalian subject, in particular where the disease, disorder or condition is one as set out herein.
  • the modulating compound is one described above, and/or is an PBC, NAC, a (host) cell, or a pharmaceutical composition of the present invention; in particular is an PBC of the invention.
  • the invention pertains to a compound or composition of the preceding aspects for use in, or pertains to, a method of treatment of a disease.
  • a compound or composition of the invention in particular one or more of the herein disclosed PBC of the invention, or a variant thereof, can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders and/or conditions associated with the undesired presence of a viral S protein.
  • Viral S- protein comprising viral particles, positive cells for such viral particles or proteins, or cells positive for a virus or a viral s protein, or a variant thereof, and/or associated with a viral infection can be so detected.
  • the disease, disorder and/or conditions so detected, diagnosed, or monitored can be one of those described elsewhere herein.
  • the diseases, disorders or conditions is a disorder caused by a viral infection, such as corona virus infection.
  • the invention relates to a method for determining the presence or an amount of an S protein or of a virus or virus particle comprising the s protein in a biological sample from a subject or in or on any environmental sample (such as a sample taken from any surface), the method comprising the steps of:
  • a biological sample will (preferably) comprise solid material, liquid, cells or tissue of the subject, or an extract of such cells or tissue.
  • the method will also comprise a step of:
  • detection and/or determination methods can be practiced as a method of diagnosis, such as a method of diagnosis whether a mammalian subject (such as a human subject or patient) has a disease or disorder associated with a corona virus infection.
  • the biological sample is one obtained from a mammalian subject like a human patient.
  • the term “biological sample” is used in its broadest sense and can refer to a bodily sample obtained from the subject (eg, a human patient).
  • the biological sample can include a clinical sample, i.e., a sample derived from a subject.
  • samples can include, but are not limited to: peripheral bodily fluids, which may or may not contain cells, e.g., blood, urine, plasma, mucous, bile pancreatic juice, supernatant fluid, and serum; tissue or fine needle biopsy samples; lung biopsy samples or sections (or cells thereof), and archival samples with known diagnosis, treatment and/or outcome history.
  • Bio samples may also include sections of tissues, such as frozen sections taken for histological purposes.
  • the term “biological sample” can also encompass any material derived by processing the sample. Derived materials can include, but are not limited to, cells (or their progeny) isolated from the biological sample, nucleic acids and/or proteins extracted from the sample. Processing of the biological sample may involve one or more of, filtration, distillation, extraction, amplification, concentration, fixation, inactivation of interfering components, addition of reagents, and the like.
  • the biological sample is a plasma (or serum) sample (previously taken) from the subject.
  • Such detection, determination and/or diagnosis methods can be conducted as an in-vitro method, and can be, for example, practiced using the kit of the present invention (or components thereof).
  • the biological sample is a tissue sample from the subject, such as a sample of a respiratory system of the subject. Such sample can be taken from the lung, or the upper respiratory tract, such as the mouth, nose, or the trachea.
  • determination and/or diagnosis method of the invention can comprise, such as in a further step, comparing the detected amount (or activity of) of (eg protein) the applicable biomarker (ie viral S protein or a variant thereof) with a standard or cut-off value; wherein a detected amount greater than the standard or cut-off value indicates a phenotype (or a risk of developing a phenotype) that is associated with the presence or infection of the virus.
  • Such a standard or cut-off value may be determined from the use of a control assay or may be pre-determined from one or more values obtained from a study or a plurality of samples having known phenotypes.
  • a cut-off value for a diagnostic test may be determined by the analysis of samples taken from patients in the context of a controlled clinical study, and determination of a cut-off depending on the desired (or obtained) sensitivity and/or specificity of the test.
  • Examples of methods useful in the detection of include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA), which employ PBC (eg of the present invention) such as an antibody or an antigen-binding fragment thereof, preferably a VHH, that specifically binds to such virus or viral s protein.
  • immunoassays such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA), which employ PBC (eg of the present invention) such as an antibody or an antigen-binding fragment thereof, preferably a VHH, that specifically binds to such virus or viral s protein.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • the PBC typically, will be labelled with a detectable labelling group.
  • labelling groups fall into a variety of classes, depending on the assay in which they are to be detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active moieties; d) optical dyes; enzymatic groups (e.g.
  • labelling groups include, but are not limited to, the following: radioisotopes or radionuclides, fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognised by a secondary reporter (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.).
  • suitable labelling groups include, but are not limited to, the following: radioisotopes or radionuclides, fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, lucifer
  • the labelling group is coupled to the PBC via spacer arms of various lengths to reduce potential steric hindrance.
  • Various methods for labelling proteins are known in the art and may be used.
  • the PBC may be labelled with a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.).
  • any one of the herein disclosed compounds or compositions, preferably such PBC of the invention are provided as kits for use in the diagnostic methods of the invention.
  • the invention pertains to a method for identifying and/or characterising a compound suitable for the treatment of a disease, disorder or condition that is associated with, or caused by, a virus expressing a viral spike (S) protein, the method comprising the steps of: bringing into contact a candidate compound and a spike protein (or a variant or fragment thereof); and
  • the invention pertains to a method for identifying and/or characterising a compound suitable for the treatment of a disease, disorder or condition that is associated with, or caused by, a virus expressing a viral spike (S) protein, the method comprising the steps of:
  • ACE2 angiotensin converting enzyme 2
  • the PBC used in the screening method is a VHH-E, VHH-N, VHH-U, VHH-V, or VHH-W, or a functional variant thereof.
  • the screening method of the invention is predicated on the observation that the inventive PBC stabilize an up-conformation of the RBD.
  • the at least two cells express a viral spike protein.
  • a truncation of the SARS-C0V2 spike protein is preferably a C terminal truncation of 10 to 30 amino acids, preferably of about 20 amino acids, more preferably of 18 amino acids.
  • the at least two cells are composed of a first cell comprising a first detectable label and a second cell comprising a second detectable label, wherein the first and the second detectable label are different.
  • detectable labels allows a detection and/or quantification of cell fusion of the first and the second cell, for example by microscopy.
  • Such detectable labels are exemplary shown herein elsewhere.
  • Preferred examples of detectable labels for such uses comprise the expression of fluorescent proteins within the at least two cells, such as GFP (and other fluorescent, for example EGFP, RFP, RFP-t, DsRed, tagged versions of proteins etc).
  • the term “comprising” is to be construed as encompassing both “including” and “consisting of’, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention.
  • “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other.
  • a and/or B is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
  • the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question.
  • the term typically indicates deviation from the indicated numerical value by ⁇ 20%, ⁇ 15%, ⁇ 10%, and for example ⁇ 5%.
  • the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect.
  • a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.
  • the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect.
  • a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.
  • Figure 1 shows that camelid VHHs against two epitopes on SARS-C0V-2 Spike RBD neutralize infection.
  • A Strategy to identify VHHs against SARS-C0V-2 spike RBD.
  • B Binding of too nM HA-tagged VHHs to immobilized SARS-C0V-2 spike RBD or control protein MBP was quantified by ELISA with HRP- coupled anti-HA. Unrelated VHH SN was used as a negative control.
  • C SARS-C0V-2 spike-pseudotyped VSV AG eGFP was incubated with the indicated concentrations of HA-tagged VHHs at 37 0 C for th, followed by infection of Vero E6 cells for 8 h.
  • eGFP-positive cells were measured by flow cytometry to quantify infection. Normalized values from three independent experiments ⁇ SEM and IC50 values are plotted.
  • D SARS-C0V-2 was incubated with the indicated concentrations of HA-tagged VHHs as in (C), followed by plaque assay on Vero E6 cells. Plaques were enumerated 3 days post infection and normalized values of three independent experiments ⁇ SEM and IC50 values are plotted.
  • E, F Biotinylated SARS-C0V-2 spike RBD was immobilized on surface plasmon resonance spectroscopy chips.
  • E Indicated HA-tagged VHHs were injected for 90 s, followed by dissociation for 120 s.
  • KD Dissociation constants
  • F Epitope binning was performed by first injecting a single VHH for 120 s, followed by injection of 1:1 mixtures of the first VHH in combination with VHH E, U, V, or W for 80 s.
  • Figure 2 shows that camelid VHHs against two epitopes on SARS-C0V-2 Spike RBD neutralize infection.
  • A Colloidal Coomassie stained SDS-PAGE gel of 2 pg HA-tagged VHHs used in Fig. 1B-F, Fig. 7A, Fig. 2B-C, and Fig. 8A.
  • B Binding of indicated concentration of HA-tagged VHHs to immobilized SARS-C0V-2 spike RBD or control protein MBP was quantified by ELISA with HRP-coupled anti-HA.
  • C, D SARS-C0V-2 spike-pseudotyped VSV AG eGFP was incubated with the indicated concentrations of HA- tagged or LPETG-tagged (VHH 72 and Tyi) VHHs at 37°C for lh, followed by infection of Vero E6 cells for 8h. eGFP-positive cells were measured by flow cytometry to quantify infection. Normalized values from three independent experiments ⁇ SEM are plotted.
  • E SARS-C0V-1 spike-pseudotyped VSV AG eGFP was incubated with the indicated concentrations of LPETG-tagged VHH 72 and infection of Vero E6 cells quantified as in C. Normalized values from three independent experiments ⁇ SEM are plotted.
  • FIG. 3 shows that X-ray crystallography defines binding sites of neutralizing VHHs on the SARS-C0V-2 RBD.
  • A-C Crystal structure of SARS-C0V-2 spike RBD in complex with VHH E and VHH U at 1.87 A (A) and detailed interaction interface of RBD (in white) with VHH E (B) and RBD with VHH U (C), respectively.
  • D-E Crystal structure of SARS-C0V-2 spike RBD in complex with VHH V at 2.55 A (D) and detailed interaction interface of RBD with VHH V (E). Escape mutants (see Fig. 9) in the RBD are labeled with asterisks.
  • F Overview of binding sites of all four neutralizing VHHs on the RBD and their overlap with ACE2 based on pdb 6M0J (38). Steric clashes are indicated with dashed circles.
  • FIG. 4 shows that X-ray crystallography defines binding sites of neutralizing VHHs on the SARS-C0V-2 RBD.
  • A-B Crystal structure of SARS-C0V-2 spike RBD in complex with VHH W at 2.73 A
  • A and detailed interaction interface of RBD with VHH W with view focusing on epitope residues involved in escape mutations
  • B and detailed interaction interface of RBD with VHH W with view focusing on epitope residues involved in escape mutations
  • FIG. D Comparison of interaction interface of RBD (in white) with ACE2 (left) or VHH E (right), respectively.
  • E HEK 293T cells transiently expressing ACE2-tagRFP-t were incubated with DyLight 488-labeled SARS- C0V-2 spike RBD in the presence of the indicated concentrations of VHHs. RBD bound to ACE2-positive cells was quantified by flow cytometry. Normalized data from three independent experiments ⁇ SEM is plotted. [164] Figure 5: shows that cryo-electron microscopy structures reveal that VHHs stabilize SARS-C0V-2 spike trimers with RBDs in the up conformation.
  • VHH E binds to SARS-C0V-2 in a 3-up conformation; the resolution is 2.95 A (0.143 FSC).
  • C, D VHH V binds to SARS- C0V-2 in a 2-up conformation with all VHH binding sites occupied at a resolution of 3.14 A, and VHH V binding to the RBD in the up- or the down-conformation.
  • FIG. 6 shows that cell surface-displayed VHHs reduce infection.
  • Vero cells doxycycline- inducibly expressing the indicated VHHs as a cell surface-displayed fusion of VHH-mIgG2a Fc-PDGFR0 TM were cultivated in the presence of 1 pg/ mb doxycycline for 24b, followed by infection with VSV AG eGFP pseudotyped with SARS-C0V-2 spike (A) or SARS-C0V-1 spike (B). 8h post infection, eGFP-positive cells were measured by flow cytometry to quantify infection. Average values from three independent experiments ⁇ SEM are plotted.
  • FIG. 7 shows that multivalent VHH fusions potentiate neutralization of SARS-C0V-2.
  • SARS-C0V-2 spike-pseudotyped VSV AG eGFP was incubated with 2-fold serial dilutions of 0.25 pM VHH E, 1 pM VHH U, 1 pM VHH V or the indicated combinations containing 50% of each VHH at 37 0 C for th.
  • Vero E6 cells were subsequently incubated with the mixtures and infection quantified as in Fig. 1B. Normalized values from three independent experiments ⁇ SEM are plotted.
  • F, G Cryo-EM reconstruction (F) and atomic models (G) of VHH VE in complex with trimeric SARS-C0V-2 spike.
  • the biparatopic VHH binds to SARS-C0V-2 in the 3-up conformation; the resolution is 2-53 A (0.143 FSC).
  • J Flp-In 293 T-REx cell lines expressing SARS-C0V-2 S A18 as well as either eGFP or mCherry were treated with 1 LIM of the indicated VHHs. Representative images were recorded after 6h or t2h as in Fig. 5E, and were from the same data set as those in Fig. 5E. Scale bars represent too pm.
  • FIG. 8 shows that multivalent VHH fusions potentiate neutralization of SARS-C0V-2.
  • SARS-C0V-2 spike-pseudotyped VSV AG eGFP was incubated with 2-fold serial dilutions of 0.25 pM VHH E, 1 pM VHH U, 1 pM VHH W or the indicated combinations containing 50% of each VHH at 37 0 C for th.
  • Vero E6 cells were subsequently infected and infection quantified as in Fig. 1B. Normalized values from three independent experiments ⁇ SEM are plotted.
  • FIG. 9 shows that simultaneous targeting of two independent epitopes with neutralizing VHHs prevents viral escape.
  • A Genome structure of VSV SARS-C0V-2 S A18 eGFP.
  • B-E Evolution experiment.
  • B Replication-competent VSV SARS-C0V-2 S A18 eGFP at an MOI of 0.5 was incubated with different concentrations of the indicated VHHs and allowed to replicate on Vero E6 cells in 12-wells for 4 days. The fraction of infected (eGFP positive) cells and CPE was estimated microscopically and is plotted according to the indicated grey tone code.
  • VHH E Wildtype VSV SARS-C0V-2 spike eGFP or plaque-purified escape mutants of VHH E (St-tf, Spike S494P), VHH U (Sl-2h, Spike S371P), VHH V (Sl-3b, Spike K378Q), VHH W (St-4a, Spike S371P), and VHH LaM-4 (St-toa, wt spike) at an MOI of 0.5 were incubated with the indicated VHH concentrations, and used for Vero E6 infection experiments as described in Fig. 1B. Infection was quantified by flow cytometry and normalized data from three independent experiments ⁇ SEM is plotted.
  • FIG. 10 shows that VHH VE neutralizes pseudotyped viruses with SARS-C0V-2 spike of emerging SARS-C0V-2 variants.
  • VSV AG eGFP pseudotyped with spike of the indicated SARS-C0V-2 variants was incubated with the indicated concentrations of untagged VHH VE (produced in Pichia pastoris) at 37°C for 1 h, followed by infection of Vero E6 cells for 16 h.
  • eGFP-positive cells were measured by flow cytometry to quantify infection. Normalized values from three independent experiments ⁇ SEM are plotted.
  • FIG. 11 shows that VHH VE neutralizes authentic SARS-C0V-2 variants.
  • the indicated SARS- C0V-2 variants were incubated with the indicated concentrations of untagged VHH VE (produced in Pichia pastoris) at 37°C for 1 h, followed by plaque assay on Vero E6 cells. Plaques were enumerated 3 days post infection and normalized values of three independent experiments ⁇ SEM and are plotted.
  • FIG. 12 shows that VHH VE does not aggregate or form relevant amounts of adducts when nebulized.
  • Three samples of VHH VE (P1-P3) were independently nebulized with an AerogenSolo device at a concentration of 60 mg/mL, and subsequently analyzed by gel filtration with a Superdex 75 Increase 10/300 GL and compared with the original material (Po). The area under the curve of the monomeric fraction was determined and is displayed on the left.
  • FIG. 13 shows that VHH VE does not lose neutralizing activity during nebulization.
  • Three samples of VHH VE (P1-P3) were independently nebulized with an AerogenSolo device at a concentration of 60 mg/mL, and subsequently used to test neutralization of SARS-C0V-2 spike pseudo typed VSV AG eGFP as described in Figure 10.
  • FIG. 14 shows that VHH VE protects mice from SARS-C0V-2 infection.
  • K18-ACE2 transgenic C57BL/6 mice B6.Cg-Tg(Kt8-ACE2)2Prlmn/J) were intranasally infected with the indicated authentic
  • Figure 15 shows an alignment of multiple sequence variants of the VHH-VE of the invention. 4 variants a-d of the VHH VE is shown. SEQ ID Nos: 100-105 show the sequences of the VHH-VE original construct and the respective variants.
  • SEQ ID NO: 93 shows the amino acid sequence of SARS-C0V2 spike protein: EXAMPLES
  • Example 1 Provision of VHHs against epitopes on SARS-C0V-2 spike RBD
  • Camelid VHHs that bind two different epitopes on the SARS-C0V-2 spike RBD neutralize infection One alpaca and one llama were immunized with the receptor binding domain (RBD) of SARS-C0V-2 spike as well as formalin-inactivated SARS-C0V-2 to elicit neutralizing heavy chain-only antibodies (Fig. 1A). 23 candidate VHHs (Fig. 2A) were identified by phage display, and confirmed binding of 10 hits by ELISA (Fig. 1B and Fig. 2B).
  • VSV replication-deficient vesicular stomatitis virus
  • SARS-C0V-2 spike A18 Fig. 1C and Fig. 2C
  • VHHs, VHHs E, U, V, and W potently neutralized infection in a dose-dependent manner, with an IC50 value of 60 nM for the most potent VHH, VHH E.
  • Three published VHHs from a synthetic library did not neutralize (9), while the published VHH Tyt (10) neutralized infection at comparable levels, although no complete neutralization was observed.
  • SARS-CoV-t-specific VHH VHH 72 (11) did not impair infection with SARS-CoV-2-pseudotyped virus as a monomer (Fig. 2D), but potently inhibited SARS-CoV-1-pseudotyped virus (Fig. 2E). None of the four VHH hits neutralized SARS-CoV-1-pseudotyped virus (Fig. 2F). Plaque reduction neutralization tests (PRNTs) with SARS-C0V-2 on Vero E6 cells confirmed the neutralizing activity, yielding IC50 values ranging from 69 to 305 nM (Fig. 1D).
  • Virus neutralization was further microscopically validated by quantifying the replication of an independent mNeonGreen expressing clone of SARS-C0V-2 on human Caco2 cells in the presence of VHHs over time (fig. 2G).
  • the binding affinities of the VHHs to the RBD were measured by surface plasmon resonance (SPR) (Fig. 1E, Fig 2H), and the dissociation constants of 2 nM (VHH E), 21 nM (VHH U), 9 nM (VHH V) and 22 nM (VHH W) in the kinetic mode were obtained.
  • VHHs bind to two distinct regions: U, V, and W competed for the same binding interface on RBD (interface UVW), while VHH E bound to an independent RBD interface (interface E), and could bind to the RBD at the same time as U, V, or W (Fig. 1F).
  • Binding epitopes of neutralizing VHHs on the spike RBD The x-ray crystal structures of complexes of VHH E and VHH U with the SARS-C0V-2 RBD at 1.87 A (Fig. 3, A to C), VHH V with the RBD and Fab CC12.3 (6) at 2.55 A (Fig 3, D to E) and VHH W with the RBD and Fab CC12.3 at 2.73 A (fig. 4, A to B) were determined.
  • VHH E and U bind to distinct epitope sites on the RBD (Fig. 3A).
  • VHH E employs its complementarity determining region (CDR) 1 and its unusually long CDR3 (22 amino acids) to bind to the ACE2 interface of the RBD (Fig.
  • VHH E binds at an orientation that markedly differs from that of other neutralizing antibodies (Fig. 3F and Fig. 4C). Its CDR3 adopts an extended beta-hairpin conformation and binds to the receptor binding site (RBS) using polar and hydrophobic interactions (Fig. 3B). Interestingly, 16 of 27 epitope residues that bind VHH E (Fig. 3B) are also involved in ACE2 binding (15) (Fig. 4D).
  • VHH U binds to a distinct epitope on RBD, it should also compete with ACE2 for binding to RBD due to steric hindrance (Fig. 3F). VHH U uses its three CDRs to bind the RBD. The epitope of VHH U overlaps with the binding site of SARS-C0V-1 neutralizing antibody CR3022 (12) (Fig. 4C) and SARS-C0V-1 neutralizing VHH VHH 72 also covers the equivalent interface in the SARS-C0V-1 RBD (11).
  • VHH V binds to a similar epitope to VHH U and W
  • VHH V approached the RBD with a markedly distinct angle.
  • CDR3 was mainly involved in binding.
  • no major changes in the overall structure of the RBD were observed on VHH U binding.
  • binding of VHH V should also compete with ACE2 binding due to steric hindrance (Fig. 3F).
  • RBDs exist in an equilibrium of up- and down- conformations in the context of the trimeric spike on virions. Most unperturbed spike trimers exist in the configuration with none or one RBD up, while the 3-up conformation is barely populated at all (7, 8, 16). Only the up conformation of the RBD is compatible with ACE2 (17) binding, which likely induces further conformational changes that favor secondary proteolytic cleavage, dissociation of the St subdomain, and eventually conversion to the post-fusion conformation. However, it is unknown how many RBDs must be in the up conformation to permit these transitions.
  • cryo-electron microscopy structures of the soluble, trimeric spike complex bound to individual VHHs using cryo-EM (7, 18) were determined.
  • the initial analysis of the inventors focused on one representative VHH binding each of the two identified epitopes.
  • Ab-initio reconstruction of VHH E bound to the trimeric spike revealed that the predominant complex contained all RBDs in the up conformation (3-up) with all three VHH E binding sites populated (Fig. 5A to B). This suggests that binding of VHH E stabilizes (or induces) the 3-up conformation, perhaps by mimicking the engagement of multiple peptidase domains of ACE2.
  • the cryo-EM structures of trimeric spike in complex with VHH V also showed full coverage of all VHH binding sites (Fig. 5 C to D).
  • the major population of VHH V:spike complexes contained two RBDs in the up-conformation and one in the down-conformation. Apart from the RBD state, the overall structure of the trimeric spike and the RBD was not substantially changed by any of the VHHs. Importantly, the VHHs altered the abundance of the spike trimer populations with a 2- or 3-up configuration and confirmed the binding mode to be similar to the binding modes identified by macromolecular crystallography described above.
  • ACE2 can only bind to RBDs in the up-conformation and is expected to trigger conformational changes required for secondary proteolytic processing and fusion. Premature activation of these steps will inactivate the fusion machinery, as the energetically favored post-fusion conformation is irreversible.
  • a HEK 293-based cell line was generated that inducibly expresses the SARS-C0V-2 spike protein on the cell surface. Lacking the expression of the cognate receptor ACE2, the SARS-C0V-2 spike is not expected to mediate fusion of these cells.
  • Activation of the fusion machinery has two potential outcomes: Either it facilitates infection by inducing fusion of viral membranes with cellular membranes (requiring proximity of both membranes), or it prematurely induces conformational changes of the spike in the absence of suitable membranes, thus irreversibly inactivating the fusion capacity.
  • SARS-C0V-2 susceptible Vero cells were generated exposing neutralizing VHHs at their plasma membrane as fusions to an Fc fragment and a transmembrane domain (Fig. 6 A to B).
  • VHHs E, U, V, and W on their surface were partially protected from infection by SARS-C0V-2, but not SARS-CoV-1-pseudotyped virus. In contrast VHH 72 inhibited both viruses. Importantly, no enhancement of infection was observed.
  • VHH E As the most potent neutralizing VHH VHH E did not induce fusion more efficiently than the other VHHs, it is possible that the observed fusogenic activity is more pronounced in case of VHH U and W, while the effect of VHH E is mainly mediated by inhibition of virus attachment by competing ACE2 binding, or by interfering with the loss of St.
  • Multivalent VHH fusions potentiate neutralization of SARS-C0V-2: In the course of natural adaptive immune responses, viruses face a polyclonal antibody response, which may enhance neutralizing activity by additive or synergistic effects (22). To test whether the four neutralizing VHHs act synergistically, the neutralizing activity of combinations of individual VHHs was compared. For direct comparison, starting concentrations of the individual VHHs were established that led to similar doseresponse curves in a two-fold dilution series (Fig. 7A and Fig. 8A). Then neutralizing activities of the individual VHHs were compared with mixtures of VHHs containing 50% of the concentration of each VHH combined.
  • VHHs U and V, U and W, or V and W Combinations of VHHs competing for the same epitope (VHHs U and V, U and W, or V and W) exhibited additive inhibition, i.e. mixtures containing half the concentrations of two VHHs neutralized to the same extent as the individual VHHs alone at full concentration (Fig. 7A, Fig. 8A).
  • VHHs E and U, E and V, or E and W mixtures containing 50% of the two VHHs neutralized more efficiently than 100% of each of the individual VHHs.
  • Combinations of VHHs binding independent epitopes were also more potent in preventing SARS-C0V-2 mNeonGreen replication in human cells (Fig 7B, Fig. 8B).
  • VHH EV The reverse construct (VHH EV) with a (GGGGS)3 linker was thus expected to either bind to two different RBDs within a trimeric complex, to only employ one of its binding sites, or to induce some strain or distortion on the targeted RBD.
  • the biparatopic VHHs VHH VE and EV were produced in bacteria, purified and subsequently subjected to SPR analysis (Fig. 7C). With apparent dissociation constants of 84 pM (VE) and 200 pM (EV), the biparatopic VHHs bound to the RBD 22- and 9-fold stronger than the best individual VHH (VHH E). While binding to trimeric spike (Fig. 8C) was confirmed as well, the more complex binding behavior precluded the application of defined models required to calculate affinities.
  • VHH EE and VHH EEE connected by GS-rich linkers of 15 aa length were produced and similarly their neutralizing activity was tested.
  • VHH EE and VHH EEE neutralized VSV AG eGFP (SARS-C0V-2 S) with IC50 values in the pM range (930 and 520 pM, respectively), improving neutralizing potential 73- or 133-fold, respectively, as compared to VHH E (Fig. 7H).
  • IC50 values in PRNT with wildtype virus were even lower, reaching 180 and 170 pM for VHH EE and EEE (Fig. 7I).
  • VHH W bivalent fusions
  • VHHs EE, EEE, and W did not induce obvious cell fusion after 6 or I2h.
  • VHH VE is able to bind to a single spike monomer, to stabilize the RBD in the up conformation, and to substantially enhance VHH-triggered cell fusion
  • E and V and perhaps E and W
  • VHH E binding alone already stabilizes the RBD 3-up conformation this may involve additional conformational changes, possibly facilitating proteolytic processing of S2 to S2’, or the bona fide dissociation of St.
  • the homodimeric and -trimeric VHHs EE and EEE did not induce fusion despite a substantial boost in neutralizing activity.
  • VHH EEE binding of VHH EEE is likely more potent in competing with ACE2 binding of virions due to higher avidity. Yet, the marked difference from VHH E alone, which did induce activation of the spike, suggests that cross-linking the individual spike subunits by EEE may in addition prevent crucial conformational changes necessary for fusion by restraining the available conformational landscape. This may include rearrangements required to enhance proteolytic processing or the conformational changes that go along with loss of St. Of note, unlike described for more distantly related coronaviruses, St subunits dissociating from SARS-C0V-1 spike after receptor engagement are monomeric (23). This implies that individual St subunits dissociate in the course of fusion activity, a step likely perturbed by homodimeric and -trimeric VHH E (and V).
  • VSV eGFP SARS-CoV-2 S replicated well in ACE2-containing Vero cells but did not infect BHK cells lacking ACE2, as expected.
  • VSV eGFP SARS-CoV-2 S was pre-incubated at an MOI of 0.5 with different concentrations of individual or combinations of VHHs before adding the mixture to Vero E6 cells (Fig. 9B).
  • Virus treated with control VHH (LaM-4 (27)) infected and killed cells within 1 or 2 days.
  • Virus incubated with combinations of VHHs E and U, E and V, or E and W were only able to substantially replicate in the presence of 0.1 LIM total VHH in the first round, i.e. non-neutralizing concentrations as determined in single round infection experiments (Fig 7, A to B, and fig. 8, A to B). Together, this experiment suggested that combining two neutralizing VHHs targeting independent epitopes prevented rapid emergence of fully resistant mutants.
  • virions treated with cocktails of E and U, or E and V were inhibited by similar VHH concentrations as in the first round, suggesting that no mutants escaping both VHHs had emerged.
  • virus replication in the presence of VHH EV and VE was tested using identical conditions. To validate the comparability of the experiments, selection with individual VHHs was repeated with identical results (data not shown). Virus replication in the presence of EV and VE only occurred at 4 nM VHH in the first round of selection and no substantial improvement was detected in a second selection round, suggesting that no escape mutants targeting both interfaces had emerged.
  • the RBD sequences encoded in viral RNAs were determined, which were purified from infected cells after one or two rounds of evolution. Sequence variants using a customized analysis pipeline were found to be of high quality and that no mutations accumulated in virions replicating in the presence of control VHHs. However, for virus replicating in the presence of individual neutralizing VHHs, around 75% of all reads contained a single point mutation in the structurally defined E or UVW interfaces of the RBD after one round of selection, while almost 100% of all reads exhibited the same mutations after two rounds of evolution. Escape mutants selected with VHH E contained an S494P mutation located in the key site in the VHH E:RBD interface.
  • Virus replicating in the presence of the VHH combinations E and U, E and V, or E and W accumulated individual point mutations in the interface with VHH E in the first round of evolution, but no virus that was mutated in both interfaces emerged.
  • the second round of evolution still no virus variants containing mutations in both interfaces developed in the presence of VHH E and U, or E and V, while some virus with mutations in the E and W interface emerged in the presence of VHH E and W.
  • Virus in the presence of permissive concentrations of EV or VE did not accumulate any escape mutations in the first round of evolution (Fig. 9D).
  • VHH E escape mutant ‘St-tf (spike S494P) was no longer sensitive to VHH E, but was still neutralized by VHH U, V, and W.
  • VHHs EV and VE neutralized the S494P escape mutant better than V alone, suggesting that the VHH E component of the bivalent VHHs still helped neutralize, perhaps because the escape mutant allowed residual binding of VHH E if stabilized by the additional VHH V interface.
  • VHH U and W escape mutants ‘St-2h’ and ‘St-4a’ both carrying spike S371P were still sensitive to VHH E, but were completely resistant to VHHs U, V, and W, demonstrating that a single point mutation in this interface allows escape from different VHHs.
  • VHHs targeting more than one epitope potently suppresses the emergence of escape mutants.
  • the combination of monomeric VHHs illustrates the additional effects particularly well, as protection was dramatically improved as compared to individual VHHs, where replication was observed at 25 to 125-fold higher concentrations. Fusions of VHHs targeting distinct epitopes (biparatopic VHHs) were even more potent, and, importantly, also performed better on escape mutants.
  • Example 4 The VHH combination VE provides an increased neutralization efficacy across various SARS-CoV virus variants
  • VHH VE can efficiently neutralize all clinically relevant SARS-C0V-2 variants (wt, beta, gamma, delta), as shown by neutralization assays with SARS-C0V-2 spike pseudotyped VSV (Fig.10 and Table 2) as well as authentic clinical virus isolates (Fig. 11 and Tab. 3). VHH VE also neutralizes additional variants of interest, including A.23.1 (Uganda), B.1.429 (California), B.1.526 (New York), and B.1.617.1 (India). Importantly, the bivalent approach can overcome partial or full resistance of individual variants to single nanobodies. None of the clinically observed variants escapes individua VHHs V and E as well as the escape variants S371P, 0.3780 (both VHH V), and S494P (VHH E).
  • VHH VE neutralizes pseudotyped viruses with SARS-C0V-2 spike of emerging SARS- C0V-2 variants.
  • VSV AG eGFP pseudotyped with spike of the indicated SARS-C0V-2 variants was incubated with the various concentrations of HA-His-tagged VHHs (produced in E. coli WK6) or untagged VHH VE (produced in Pichia pastoris) with and without the albumin-binding domain (ABD) of Streptococcus strain G148-GA3 at 37°C for 1 h. Incubation was followed by infection of Vero E6 cells for 16 h. eGFP-positive cells were measured by flow cytometry to quantify infection.
  • Inhibitory concentration 50 (IC50) values were calculated as the half-maximal inhibitory dose using 4-parameter nonlinear regression (GraphPad Prism).
  • mNb6 Schott al., PMID: 33154106
  • Nb2i-34 Xiang et al., PMID: 33154108
  • VHH VE neutralizes authentic SARS-C0V-2 variants.
  • the indicated SARS-C0V-2 variants were incubated with various concentrations of untagged VHH VE (produced in Pichia pastoris) at 37°C for 1 h, followed by plaque assay on Vero E6 cells. Plaques were enumerated 3 days post infection and normalized values of three independent experiments were used to calculate inhibitory concentration 50 (IC50) values as the half-maximal inhibitory dose using 4-parameter nonlinear regression (GraphPad Prism).
  • IC50 inhibitory concentration 50
  • VHH VE was nebulized and subsequently analyzed for macroscopic aggregation (dynamic light scattering), soluble aggregates (gel filtration, Fig. 12), covalent adducts (SDS-PAGE), and neutralizing activity (Fig. 13).
  • VHH VE with a C-terminal fusion to the albumin-binding domain (ABD) of Streptococcus strain G148-GA3 extended the serum half-life to 42.6 h (i.v.), 45.7 h (s.c.), and 50.7 h (i.m.), respectively. This suggests that systemic application of half-life extended VHH VE is a viable treatment option.
  • C-terminal fusion of the albumin binding domain (ABD) did not affect biological activity (see Table 2).
  • Therapeutic efficacy of VHH VE-ABD (applied intraperitoneally) against SARS-C0V-2 wt, beta, and delta was demonstrated in transgenic mice expressing human ACE2 (Fig. 14).
  • the inventors produced a variety of derivatives of VHH VE based on the structure in an attempt to stabilize interactions with the target by introducing favourable charges or increasing the rigidity of loops and linkers. All variants were expressed well in bacteria, purified, and tested in neutralization experiments. All variants exhibited a comparable potency of neutralization. A sequence alignment is shown in Figure 15 indicating that various amino acid positions can be changed.
  • Flp-In 293 T-REx cells inducibly expressing SARS-C0V-2 Spike A18 with a C-terminal Strep2-HA tag were generated using the Flp-In system (Thermo Fisher Scientific) according to the manufacturer’s recommendations, and cultivated in DMEM supplemented with 10% FBS, Glutamax, 4 pg/mL blasticidine S, and 50 pg/mL hygromycin B.
  • Vero E6 cells inducibly expressing cell surface-displayed VHHs were generated using lentiviral transduction with plnducer2o-based vectors and cultivated in DMEM supplemented with 10% FBS, Glutamax, and 500 pg/mL G418.
  • VHHs were expressed as fusions of IgK signal peptide-HA-VHH-m!gG2a Fc-myc-PDGFRP(TM).
  • SARS-C0V-2 clinical isolate SARS-CoV-2/human/Germany/Heinsberg-ot/2O2O was isolated from a throat swap of an infected patient at the University of Bonn, Germany, and was used for plaque reduction assays (39).
  • Vero E6 cells were infected with SARS-C0V-2 at a multiplicity of infection (MOI) of o.oi.
  • MOI multiplicity of infection
  • Supernatants were harvested 2 days post infection (p.i.), cleared by centrifugation, and quantified by plaque assay.
  • virus was produced in cells covered with Opti-MEM: Clarified supernatants containing virus were mixed with 37% formaldehyde to obtain a final concentration of 4% formaldehyde and incubated at 4°C for 4 h.
  • the virus suspension was overlaid on 7 ml of a 30% sucrose cushion in 20 mM Hepes pH 7.4, 150 mM NaCl and virions were sedimented by ultracentrifugation in a SW 32 TI rotor at 4 0 C, 30.000 rpm for 2.5 h.
  • Inactivated virus pellets from 28 mb of virus-containing supernatants were resuspended in too pl of 20 mM Hepes pH 7.4, 150 mM NaCl and the virus suspensions from five ultracentrifuge tubes were pooled. Inactivation of the virus was verified by the lack of replication in a Vero E6 infection experiment. Isolate SARS-CoV-2/human/USA/WA-CDC-WAt/2O2O for in vivo experiments was obtained from the CDC (Atlanta, GA, USA).
  • Recombinant SARS-C0V-2 clone icSARS-CoV-2-mNG expressing mNeonGreen 40
  • SARS-CoV-2/human/USA/WA-CDC-WAt/2O2O 40
  • WRCEVA World Reference Center for Emerging Viruses and Arboviruses
  • UTMB Universality of Texas Medical Branch
  • Viral titers were determined based on mNeonGreen expression after serial dilutions.
  • VSV AG eGFP pseudotyped with VSV G VSV AG eGFP (VSV G)
  • VSV G VSV AG eGFP
  • VACV T7-expressing vaccinia virus
  • pVSV eGFP dG a kind gift from Connie Cepko, Harvard Medical School, Addgene plasmid # 31842
  • T7-based expression vectors for VSV polymerase (pL), phosphoprotein (pP), nucleoprotein (pN), and glycoprotein (pG) all kind gifts from Sean Whelan, Harvard Medical School
  • VSV AG eGFP VSV G was amplified in BSR-T7/5 cells transiently transfected with VSV G expression vector pMD2.G (a kind gift from Didier Trono, EPFL, Addgene plasmid # 12259) at 34° C.
  • VSV AG eGFP SARS-C0V-1 SA18
  • VSV AG eGFP SARS-C0V-2 SA18
  • BSR-T7/ 5 cells transiently transfected with pCAGGS SARS-C0V-1 SA18 or pCAGGS SARS-C0V-2 SA18, respectively, followed by infection with VSV AG eGFP (VSV G) and cultivation at 34 0 C.
  • pCAGGS SARS-C0V-1 SA18 encodes aa 1-1237 of SARS-C0V-1 strain Frankfurt 1 (cloned from a template kindly provided by Stephan Poehlmann, German Primate Center), while pCAGGS SARS-C0V-2 SA18 encodes aa 1-1255 of SARS-CoV-2/human/China/Wuhan-Hu-i/2Oi9 (cloned from a codon-optimized template kindly provided by Stephan Poehlmann). C-terminal truncations were introduced to avoid retention of spike proteins in the ER/Golgi and maximize exposure at the plasma membrane. Viruscontaining supernatants were clarified by centrifugation and stored at -8o° C.
  • Viral titers were determined by infection of Vero E6 with dilution series of the virus for 8 hours, followed by quantification of green fluorescent cells by flow cytometry. Supernatants were shown to be free of VSV G-pseudotyped virus, as no infection of BSR-T7/5 cells was observed.
  • Virus was amplified in Vero E6 cells at 34 0 C and virus- containing supernatants were clarified and stored at -8o° C. Viral titers were determined by infection of Vero E6 with dilution series of the virus for 8 hours, followed by quantification of green fluorescent cells by flow cytometry. Chimeric virus strain VSV eGFP SARS-CoV-2 SA18 was confirmed to infect primate ACE2 expressing VeroE6 cells, but not BSR-T7/5 cells.
  • RBD receptor-binding domain
  • SARS-CoV-2/human/China/Wuhan-Hu-i/2Oi9 GenBank: QHD43416.1
  • coding sequence was cloned into a customized pFastBac vector and fused with an N-terminal gp6y signal peptide and C-terminal His6 tag.
  • a recombinant bacmid DNA was generated using the Bac-to- Bac system (Thermo Fisher Scientific).
  • Baculovirus was generated by transfecting Sf9 cells with purified bacmid DNA using FuGENE HD (Promega), and subsequently used to infect suspension cultures of High Five cells (Thermo Fisher Scientific) at an MOI of 5 to 10. Infected High Five cells were cultivated at 28 °C for 72h, shaking at 110 r.p.m. for protein expression. The supernatant was then concentrated using a 10 kDa MW cutoff Centramate cassette (Pall Corporation). The RBD protein was purified by Ni-NTA, followed by size exclusion chromatography, and buffer exchanged into 20 mM Tris-HCl pH 7.4 and 150 mM NaCl.
  • Fluorescent RBD was produced by reaction with DyLight 488 NHS ester (Thermo Fisher Scientific) in 100 mM sodium pohosphate pH 8.0, 150 mM NaCl, pH 8.0, followed by desalting with 7K MWCO Zeba spin desalting columns (Thermo Fisher Scientific).
  • VHH coding sequences were cloned into pHEN6-based bacterial, periplasmic expression vectors with C-terminal HA-His6 or LPETG-His6 tags using Gibson cloning (New England Biolabs). Multivalent VHHs were either directly cloned into pHEN6, or first assembled in pBluescript II (KS) + cloning vectors lacking promoters by Gibson cloning, followed by subcloning into pHEN6 using conventional ligation with T4 ligase. VHHs were produced in Escherichia coli WK6 transformed with the respective expression vectors.
  • KS pBluescript II
  • CC12.3 Fab was produced as described before (12). In brief, the coding sequences of heavy and light chain of CC12.3 Fab were cloned into phCMVs. ExpiCHO cells were transiently co-transfected at a ratio of 2:1 (HC:LC) using ExpiFectamineTM CH0 Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The supernatant was collected at 10 days post-transfection. The Fabs were purified with a CaptureSelectTM CH1-XL Affinity Matrix (Thermo Fisher Scientific) followed by size exclusion chromatography.
  • Soluble, trimeric SARS-C0V-2 spike was expressed as a fusion of aa 1-1208 of SARS-CoV- 2/human/China/Wuhan-Hu-i/20i9 containing the mutations R682G, R683S, R685S, K986P, V987P (S2- P), a C-terminal T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrepTag and an His8 tag from a mammalian expression vector based on paH as described before (7).
  • SARS-C0V-2 spike HexaPro mutant with additional proline mutations was used (42).
  • protein was produced in FreeStyle 293F cells transfected with FreeStyle MAX reagent (Thermo Fisher Scientific) or Expi293F cells transfected with ExpiFectamine 293 (Thermo Fisher Scientific).
  • the ectodomain was purified from filtered supernatant on Streptactin XT resin (IBA Lifesciences), followed by gel filtration on a HiLoad® 16/600 Superdex 200 column in 50 mM Tris pH 8, 200 mM NaCl. Soluble spike protein was biotinylated using UV-traceable ChromaLink Biotin (SoluLink).
  • VHH plasmid libraries in the M13 phagemid vector pD were generated as described before (43).
  • RNA from peripheral blood lymphocytes was extracted and used as a template to generate cDNA using three sets of primers (random hexamers, oligo(dT), and primers specific for the constant region of the alpaca heavy chain gene).
  • VHH coding sequences were amplified by PCR using VHH- specific primers, cut with Asci and Notl, and ligated into pJSC linearized with the same restriction enzymes.
  • E. coli TGt cells (Agilent) were electroporated with the ligation reactions and the obtained ampicillin- resistant colonies were harvested, pooled, and stored as glycerol stocks.
  • SARS-C0V-2 spike RBD-specific VHHs were obtained by phage display and panning with a protocol modified from Schmidt et al. (43).
  • E. coli TGt cells containing the VHH library were infected with helper phage VCSM13 to produce phages displaying the encoded VHHs as pill fusion proteins. Phages in the supernatant were purified and concentrated by precipitation. Phages presenting RBD-specific VHHs were enriched using enzymatically or chemically biotinylated RBDs immobilized to MyOne Streptavidin Tt Dynabeads (Thermo Fisher Scientific). The retained phages were used to infect E.
  • VHHs leaked into the supernatant were tested for specificity using ELISA plates coated with control protein MBP or SARS-C0V-2 RBD. Bound VHHs were detected with HRP-coupled rabbit anti-E-Tag antibodies (Bethyl), HRP-coupled MonoRabTM Rabbit Anti-Camelid VHH Antibody (GenScript), and the chromogenic substrate TMB. Reactions were stopped with t M HC1 and absorption at 450 nm was recorded. Positive candidates were sequenced, and representative VHHs cloned into bacterial expression vectors for further analysis.
  • SARS-C0V-2 spike RBD or the control protein mannose binding protein (MBP) in PBS were immobilized on ELISA plates at a concentration of lpg/mL over night.
  • Serial 10-fold dilutions of HA-tagged VHHs in 10% FBS/PBS were incubated with the immobilized antigen, followed by incubation with HRP-coupled anti-HA (clone 6E2, 1:5000, Cell Signaling), and the chromogenic substrate TMB. Reactions were stopped with 1 M HC1 and absorption measured at 450 nm.
  • VHH E/VHH U/RBD, VHH V/CC12.3/RBD, and VHH W/CC12.3/RBD complexes were formed by mixing each of the protein components at an equimolar ratio and incubated overnight at 4°C.
  • the final concentration for the complexes used for crystallization screening ranged from 13.5 to 17.8 mg/ml.
  • Crystallization screening was set-up by the vapor diffusion method in sitting drops containing 0.1 pl of protein and 0.1 pl of reservoir solution with the 384 conditions of the JCSG Core Suite (Qiagen) using our custom-designed robotic CrystalMation system (Rigaku) at Scripps Research. Diffraction-quality crystals were obtained in the following conditions:
  • VHH E/VHH U/RBD complex (13.5 mg/mL): 20% PEG-3000, 0.1 M citrate pH 5.5 at 2O°C,
  • VHH V/CC12.3/RBD complex (17.8 mg/mL): 20% PEG 3350, 0.2 M Na2HP04, pH 9.1 at 2O°C,
  • VHH W/CC12.3/RBD complex (17.6 mg/mL): 1.0 M Li-chloride, 10% PEG-6000, 0.1 M Bicine pH 9.0 at 2O°C.
  • a 3-pl aliquot of the sample solution was applied to glow-discharged UltrAuFoil Gold 200 mesh (R 2/2 geometiy) grids (Quantifoil Micro Tools GmbH) or CryoMatrix® holey grids with amorphous alloy film (Zhenjiang Lehua Technology Co., Ltd) in a Vitrobot Mk IV (Thermo Fisher Scientific) at 4 °C and 100% humidity (blot 10 s, blot force 3).
  • Cryo-EM data collection was performed with EPU Thermo Fisher Scientific) using a Krios Gsi transmission-electron microscope (Thermo Fisher Scientific) operated at 300 keV in the Karolinska Institutet 3D-EM facility.
  • Images were acquired in nanoprobe EFTEM mode with a slit width of 20 eV using a GIF 967 energy filter (Ametek) and a K3 detector (Ametek) during 1.5 seconds with a dose rate of 66.7 e-/px/s resulting in a total dose of 0.81 e-/A2 fractionated into 60 movie frames.
  • Motion correction, CTF-estimation, fourier binning (to 1.02 A/px), picking and extraction in 600 pixel boxes were performed on the fly using Warp (50).
  • VHH E 6,468
  • VHH V 9,861
  • VHH VE 12,606
  • VHH VE 6,468
  • VHH V 9,861
  • VHH VE 12,606
  • the structure of the spike protein trimer PDB:6ZXN (53) was used as a starting model for model building.
  • the VHH structures bound to RBD were determined using models built with the crystallography data from this study. Structure refinement and manual model building were performed using COOT and PHENIX (48) in interspersed cycles with secondary structure and geometry restrained. All structure figures and all EM density-map figures were generated with UCSF ChimeraX (54).
  • HEK 293T cells were transfected with expression vectors for ACE2-tagRFP-t and detached in PBS with 1 mM EDTA. 0.04 LIM DyLight 488-labeled SARS-C0V-2 RBD was incubated with different concentrations of VHHs at room temperature (RT) for 20 min. ACE2-expressing cells were subsequently incubated with RBD-VHH mixtures on ice for 20 min. ACE2-tagRFP-t and DyLight 488 positive cells were subsequently quantified using a MACS Quant VYB flow cytometer.
  • Flp-In 293 T-REx cells inducibly expressing SARS-C0V-2 spike Ai8-Strep2-HA were reversely transfected with pCAGGS eGFP or pCAGGS mCherry using Lipofectamine 2000 (Thermo Fisher Scientific). 4 h post transfection, cells were detached by scraping, washed, and seeded in tissue culture- treated CellCarrier-96 Ultra Microplates (Perkin Elmer) at a density of 100,000 cells per well in phenol red-free full medium without antibiotics. 2 h later, SARS-C0V-2 spike A18 expression was induced by the additions of doxycycline at a final concentration of 1 pg/mL. Cells were treated with the indicated concentrations of VHHs 20 h post transfection. Representative images were recorded using the lox objective of a Leica SP8 confocal microscope after 6 h. Cells were fixed 12 h post treatment and representative images recorded accordingly.
  • Virus mixtures were incubated at 37 0 C for lh, and subsequently used to infect Vero E6 cells. The inoculum was removed after lh and cells were covered with 0.5 mL full DMEM (FBS). After 7I1 at 37 0 C (8h p.i.), cells were trypsinized, fixed, and eGFP-positive cells quantified using a MACS Quant VYB flow cytometer. Inhibitory concentration 50 (IC50) values were calculated using the ‘log(inhibitor) vs. normalized response’ equation in GraphPad Prism.
  • Plaque reduction assays with SARS-CoV-2/human/Germany/Heinsberg-oi/2O2O were performed in Vero E6 cells. 24-well plates were seeded with 1.5-105 Vero E6 cells per well. The next day, VHHs at a concentration of 1 pM were subjected to a two-fold dilution series. 120 pl of each VHH dilution was mixed with 120 pl of OptiPROTM SFM cell culture media (Thermo Fisher Scientific) containing 80 pfu of SARS- C0V-2. After 1 h at 37°C, 200 pl of each mixture was added to 24-well plates.
  • virus was replicated in the presence of serial dilutions of VHHs on Vero E6 cells (26). 1.4-105 cells/well were seeded into 12-well plates. The next day, 0.5 mL virus dilution (equivalent to an MOI of 0.5) in DMEM (3% FBS, PS) was incubated with 0.5 mL VHH dilution in DMEM (3% FBS, PS) at room temperature for 30 min. The mixture was subsequently transferred to 12-wells with Vero E6 cells and cultivated at 37 0 C for four days.
  • eGFP-positive cells were evaluated and selected wells harvested for further cultivation: Cells were lysed in RLT PLUS buffer containing 1% P-mercaptoethanol, followed by purification of RNA with the RNeasy Mini Kit (Qiagen). Supernatants were cleared and stored at 4 0 C. 1:5 dilutions in DMEM (3% FBS, PS) were used to infect cells for a second round of selection under otherwise identical conditions. eGFP-positive cells and CPE were evaluated 5 days post infection and selected supernatants and cell lysates harvested.
  • FASTQ files were examined using FastQC (Version 0.11.9) and multiqc (Version 1.9).
  • SARS-C0V-2 RBD reference was indexed using bowtie2-build (Version 2.4.1) and samtools (Version 1.10).
  • the reads where aligned using bowtie2 (Version 2.4.1).
  • Conversion into BAM file, sorting and indexing was performed using samtools.
  • BAM files were examined with QualiMap (Version 2.2.2-dev) and multiqc. Variant calling was performed using default GATK HaplotypeCaller (Version 4.1.8.1), and variants were inspected in bam.files using IGV.
  • Vero E6 cells were amplified from individual virus plaques grown on Vero E6 cells. 1.4-105 Vero E6 cells per well were seeded into 12-wells. The next day, 10-fold serial dilutions of supernatants from virus replicated in the presence of VHH E, U, V, W, E and U, E and V, E and W, or La-Mq (round 1) were prepared and incubated in the presence or absence of 1 pM of VHHs at RT for 30 min. Vero E6 cells were infected for 30 min at 37 0 C, followed by overlay of cells with 0.75% agarose, MEM, 2% FBS, PS, 25 mM Hepes with or without the respective VHHs.
  • VHH-resistant fluorescent plaques emerged from supernatants of the combinations VHH E and U, E and V, or E and W.
  • a plug of the agarose on top of marked plaques was removed with a Pasteur pipette and incubated in 500 uL DMEM for 4I1 at 4 0 C.
  • 48-wells were infected with 250 iiL of this virus dilution and cultivated in DMEM (2% FBS, PS, 25 mM Hepes) at 37 0 C until all cells were eGFP positive.
  • RNA was reversely transcribed into cDNA using a SARS-C0V-2 spike specific primer.
  • the RBD coding sequence flanked by additional 70 bp was amplified by PCR and sequenced by Sanger sequencing. 6/6 plaques resistant to VHH E exhibited the point mutation S494P, 5/5 plaques resistant to VHH U and 5/5 plaques resistant to VHH W encoded the point mutant S371P, and 8/8 plaques resistant to VHH V contained the point mutation K378Q. 4/4 plaques from virus cultivated in the presence of control VHH La-Mq did not contain any mutations. Selected clones were amplified in 15 cm dishes of Vero E6 cells and cleared supernatants stored at -8o° C.

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Abstract

L'invention concerne des composés de liaison à des protéines, tels que des anticorps et des composés de type anticorps, ciblant spécifiquement des épitopes de la protéine de spicule (S) du Coronavirus qui ont été trouvés pour stabiliser une conformation tridimensionnelle de la protéine qui réduit ou inhibe les mécanismes viraux nécessaires à l'entrée de la cellule hôte. L'invention concerne les composés de liaison à des protéines seuls et en combinaison et des constructions multispécifiques, ainsi que leur utilisation dans la prévention, le traitement, la stratification des patients et le diagnostic d'infections virales provoquées par un coronavirus tel que le syndrome respiratoire aigu sévère du Coronavirus 2 (SARS CoV2).
PCT/EP2021/080560 2020-11-18 2021-11-03 Composés de liaison à la protéine de spicule du coronavirus Ceased WO2022106205A1 (fr)

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