Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—RNA viruses
  • C07K16/102—Coronaviridae (F)
  • C07K16/104—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—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
  • 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
  • C12N2770/20023—Virus like particles [VLP]
• • 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
  • C12N2770/20051—Methods of production or purification of viral material

Definitions

• the present invention relates to antibodies or antigen-binding fragments thereof against SARS- related coronavirus, pharmaceutical compositions comprising such antibodies or antigen-binding fragments thereof, kits comprising such antibodies or antigen-binding fragments thereof, and the antibodies or antigen-binding fragments thereof, the pharmaceutical compositions and the kits for use as a medicament, and in the treatment or prevention of a disease caused by SARS-related coronavirus.
• the present invention further relates to methods of treating, preventing or reducing the severity of an infection with a SARS-related coronavirus, and to nucleic acids encoding such antibodies or antigen-binding fragments thereof, expression vectors comprising such nucleic acids, host cells comprising such nucleic acids or expression vectors, and methods for the production of such antibodies or antigen-binding fragments thereof.
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• NAbs Neutralizing antibodies
• SARS-CoV-2 antibodies are directed against the spike (S) protein expressed on the virus surface, which facilitates viral entry into human cells by binding to the human ACE-2 receptor.
• S spike
• RBD receptor binding domain
• NTD N-terminal domain
• the S2 domain of the spike protein is more conserved than the S1 amongst p-coronaviruses (p -CoVs), however, mAbs targeting the S2 domain are rare and less potent (Pinto, D. et al. (2021). Science 373, 1109-1116; Sauer, M.M. et al. (2021). Nat Struct Mol Biol 28, 478-486.).
• next-generation mAbs that retain potency and effectiveness against circulating or emerging SARS-CoV-2 variants.
• SARS-related coronavirus which do not show autoreactivity and have superior neutralization potency against circulating SARS-CoV-2 variants of concern as well as emerging escape variants in comparison to antibodies currently made available to the public.
• an antibody or antigen-binding fragment thereof directed against SARS-related coronavirus, wherein the antibody or antigen-binding fragment thereof comprises the combination of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 amino acid sequence of one antibody selected from the group comprising TV1t4p2_A5 (having a CDR-H1 amino acid sequence of SEQ ID No. 27, a CDR-H2 amino acid sequence of SEQ ID No. 28, a CDR-H3 amino acid sequence of SEQ ID No. 29, a CDR-L1 amino acid sequence of SEQ ID No. 30, a CDR-L2 amino acid sequence of SEQ ID No.
• TV1t2p6_D3 having a CDR-H1 amino acid sequence of SEQ ID No. 33, a CDR-H2 amino acid sequence of SEQ ID No. 34 a CDR-H3 amino acid sequence of SEQ ID No. 35 a CDR-L1 amino acid sequence of SEQ ID No. 36 a CDR-L2 amino acid sequence of SEQ ID No. 37, a CDR-L3 amino acid sequence of SEQ ID No. 38
• TV1t4p3_E8 having a CDR-H1 amino acid sequence of SEQ ID No. 21 , a CDR-H2 amino acid sequence of SEQ ID No.
• the antibody or antigen-binding fragment thereof comprises the combination of the heavy chain variable region amino acid sequence and of the light chain variable region amino acid sequence of one antibody selected from the group comprising TV1t4p2_A5 (having the heavy chain variable region amino acid sequence of SEQ ID No. 3 and the light chain variable region amino acid sequence of SEQ ID No. 4), TV1t2p6_D3 (having the heavy chain variable region amino acid sequence of SEQ ID No. 5 and the light chain variable region amino acid sequence of SEQ ID No. 6), TV1t4p3_E8 (having the heavy chain variable region amino acid sequence of SEQ ID No. 1 and the light chain variable region amino acid sequence of SEQ ID No. 2), and TV1t3p4_E1 (having the heavy chain variable region amino acid sequence of SEQ ID No. 7 and the light chain variable region amino acid sequence of SEQ ID No. 8).
• TV1t4p2_A5 having the heavy chain variable region amino acid sequence of SEQ ID No. 3 and the light chain variable region amino acid sequence
• the amino acid sequences comprised are of one antibody selected from the group comprising TV1t4p2_A5, TV1t2p6_D3, and TV1t4p3_E8, preferably of one antibody from the group comprising TV1t4p2_A5 and TV1t2p6_D3, more preferably of antibody TV1t4p2_A5.
• the SARS-related coronavirus strain is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
• the amino acid sequences of the CDRs or of the variable regions comprised in the antibody or antigen-binding fragment thereof are from an antibody which is able to neutralize each of the SARS-CoV-2 lineages Wu01 , Alpha, Beta, Delta, BA.1 , BA.2, BA.2.12.1 , BA.4/5, BA.2.75, BA.2.75.2, BQ.1.1 , BA.4.6, XBB.1 , XBB.1 .5, and BF.7 in a pseudovirus neutralization assay as described herein with an 50% inhibitory concentration (IC50) of at most 0.07 pg/ml, preferably at most 0.06 pg/ml, more preferably at most 0.05 pg/ml, even more preferably at most 0.04 pg/ml, even more preferably at most 0.03
• IC50 50% inhibitory concentration
• a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention and at least one pharmaceutically acceptable excipient is provided, preferably wherein the pharmaceutical composition is a vaccination composition for a human subject.
• kits comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention and a container.
• an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a pharmaceutical composition according to the second aspect of the invention, or a kit according to the third aspect of the invention for use as a medicament.
• an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a pharmaceutical composition according to the second aspect of the invention, or a kit according to the third aspect of the invention for use as a vaccine.
• an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a pharmaceutical composition according to the second aspect of the invention, or a kit according to the third aspect of the invention for use in the treatment or prevention of a disease caused by SARS-related coronavirus in human subjects, preferably for use in the treatment or prevention of a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human subjects.
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a pharmaceutical composition according to the second aspect of the invention, or a kit according to the third aspect of the invention for use in prevention of infection of a human subject with SARS-related coronavirus, preferably of infection of a human subject with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• the antibody or antigen-binding fragment thereof is administered by intravenous infusion, by inhalative application, by subcutaneous injection or intramuscular injection, preferably wherein the antibody or antigen-binding fragment thereof is administered at an absolute dose of up to 4000 mg, preferably up to 2400 mg, more preferably up to 1200 mg, even more preferably up to 600 mg, even more preferably up to 300 mg, particularly preferably up to 150 mg.
• a nucleic acid is provided encoding an antibody or antigen-binding fragment thereof according to the first aspect of the invention.
• an expression vector comprising the nucleic acid of the eighth aspect of the invention in functional association with an expression control sequence.
• a host cell is provided comprising a nucleic acid according to the eighth aspect of the invention or the expression vector according to the ninth aspect of the invention.
• a method of production of an antibody or antigen-binding fragment thereof according to the first aspect of the invention comprising (a) cultivating the host cell of the tenth aspect of the invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and (b) recovering the antibody or antigenbinding fragment thereof.
• the antibody of the first aspect of the invention for use in medicine in combination with at least one further antibody directed against SARS-related coronavirus 2 (SARS-CoV-2), wherein said further antibody has a different binding specificity.
• SARS-CoV-2 SARS-related coronavirus 2
• Figure 1 shows mean SARS-CoV-2 bNabs IC50 values of duplicates against SARS-CoV-2 variants Wu01 , Alpha, Beta, Delta, BA.1 , BA.2, BA.2.12.1 , BA.4/5, BA.2.75, BA.2.75.2, BQ.1.1 , BA.4.6, XBB.1 , XBB.1.5, and BF.7, and also against SARS-CoV-1 in pseudo virus neutralization assays; antibodies belonging to the same clonal family are marked on the left.
• Figure 2 shows mean SARS-CoV-2 bNabs IC50 values based on duplicate measurements of two independent comparative experiments between reference antibody SP1-77 from Luo S et al., 2022, and antibodies TV1t4p2_A5, and TV1t4p3_E8 according to the present invention against SARS-CoV-2 variants Wu01 , Alpha, Beta, Delta, BA.1 , BA.2, BA.2.12.1 , BA.4/5, BA.2.75, BA.2.75.2, BQ.1 .1 , BA.4.6, XBB.1 , XBB.1 .5, and BF.7.
• a or “an” entity refers to one or more of that entity; for example, "a nucleotide sequence,” is understood to represent one or more nucleotide sequences.
• the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
• “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other.
• the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
• antibody is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, humanized, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; a single variable domain (e.g., VH, VHH, VL, domain antibody), antigen binding antibody fragments, Fab, F(ab')2, Fv, disulphide linked Fv, single chain Fv, disulphide-linked scFv, diabodies, etc. and modified versions of any of the foregoing.
• immunoglobulin-like domain for example IgG, IgM, IgA, IgD or IgE
• a single variable domain e.g., VH, VHH, VL, domain antibody
• antigen binding antibody fragments Fab, F(ab')2, Fv, disulphide linked Fv,
• antibody refers to a protein, derived from a germline immunoglobulin sequence, which is capable of specifically binding to an antigen or an antigen-binding portion thereof.
• the term includes full length antibodies of any class or isotype (that is, IgA, IgD, IgE, IgG, IgM and/or IgY) and any single chain or fragment thereof.
• An antibody that specifically binds to an antigen, or antigen-binding portion thereof may bind exclusively to that antigen, or portion thereof, or it may bind to a limited number of homologous antigens, or portions thereof.
• Full-length antibodies usually comprise at least four polypeptide chains: two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds.
• IgG immunoglobulin sub-class of particular pharmaceutical interest
• the IgG class may be sub-divided into 4 sub-classes: IgG 1 , lgG2, lgG3 and lgG4, based on the sequence of their heavy chain constant regions.
• the light chains can be divided into two types, kappa and lambda, based on differences in their sequence composition.
• IgG molecules are composed of two heavy chains, interlinked by two or more disulfide bonds, and two light chains, each attached to a heavy chain by a disulfide bond.
• a heavy chain may comprise a heavy chain variable region (VH) and up to three heavy chain constant (CH) regions: CH1 , CH2 and CH3.
• a light chain may comprise a light chain variable region (VL) and a light chain constant region (CL).
• VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
• CDRs complementarity determining regions
• FR framework regions
• VH and VL regions are typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
• the hypervariable regions of the heavy and light chains form a binding domain that is capable of interacting with an antigen, while the constant region of an antibody may mediate binding of the immunoglobulin to host tissues or factors, including but not limited to various cells of the immune system (effector cells), Fc receptors and the first component (C1q) of the classical complement system.
• Antibodies of the current invention may be isolated.
• isolated antibody refers to an antibody that has been separated and/or recovered from (an)other component(s) in the environment in which it was produced and/or that has been purified from a mixture of components present in the environment in which it was produced.
• Certain antigenbinding fragments of antibodies may be suitable in the context of the current invention, as it has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
• binding portion refers to one or more fragment(s) of an antibody that retain the ability to specifically bind to an antigen, such as the spike (S) protein of SARS-CoV-2, as described herein.
• antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)S, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv; see, e.g., Bird et al., Science 242:42S-426 (1988); Huston et al., PNAS 85: 5879-5883 (1988)), dsFv, Fd (typically the VH and CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VHH, and V-NAR domains; monovalent molecules comprising a single VH and a single VL chain; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 10:949-57 (1997)); camel IgG; IgNAR; as well as
• a “human” antibody refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
• the SARS-CoV-2 antibodies described herein can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
• human antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
• human antibodies and “fully human” antibodies are used synonymously.
• a “humanized” antibody refers to a human/non-human chimeric antibody that contains one or more sequences (CDR regions or parts thereof) that are derived from a non-human immunoglobulin.
• a humanized antibody is, thus, a human immunoglobulin (recipient antibody) in which at least residues from a hyper-variable region of the recipient are replaced by residues from a hyper-variable region of an antibody from a non-human species (donor antibody) such as from a mouse, rat, rabbit or non- human primate, which have the desired specificity, affinity, sequence composition and functionality.
• donor antibody such as from a mouse, rat, rabbit or non- human primate
• FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
• An example of such a modification is the introduction of one or more so-called back-mutations, which are typically amino acid residues derived from the donor antibody.
• Humanization of an antibody may be carried out using recombinant techniques known to the person skilled in the art (see, e.g., Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. C. Lo).
• a suitable human recipient framework for both the light and heavy chain variable region may be identified by, for example, sequence or structural homology.
• fixed recipient frameworks may be used, e.g., based on knowledge of structure, biophysical and biochemical properties.
• the recipient frameworks can be germline derived or derived from a mature antibody sequence.
• CDR regions from the donor antibody can be transferred by CDR grafting.
• the CDR grafted humanized antibody can be further optimized for e.g. affinity, functionality and biophysical properties by identification of critical framework positions where re-introduction (backmutation) of the amino acid residue from the donor antibody has beneficial impact on the properties of the humanized antibody.
• the humanized antibody can be engineered by introduction of germline residues in the CDR or framework regions, elimination of immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.
• humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
• a humanized antibody will comprise at least one--typically two--variable regions, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and in which all or substantially all of the FR residues are those of a human immunoglobulin sequence.
• the humanized antibody can, optionally, also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
• Fc immunoglobulin constant region
• humanized antibody derivative refers to any modified form of the humanized antibody, such as a conjugate of the antibody and another agent or antibody.
• recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
• variable and constant regions that utilize particular human germline immunoglobulin sequences are encoded by the germline genes, but include subsequent rearrangements and mutations which occur, for example, during antibody maturation.
• the variable region contains the antigen binding domain, which is encoded by various genes that rearrange to form an antibody specific for a foreign antigen.
• the variable region can be further modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody to the foreign antigen.
• the constant region will change in further response to an antigen (i.e., isotype switch).
• the rearranged and somatically mutated nucleic acid molecules that encode the light chain and heavy chain immunoglobulin polypeptides in response to an antigen cannot have sequence identity with the original nucleic acid molecules, but instead will be substantially identical or similar (i.e., have at least 80% identity).
• a “chimeric antibody” refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.
• Alternative antibody formats include alternative scaffolds in which the one or more CDRs of the antigen-binding portion can be arranged onto a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer or an EGF domain.
• a suitable non-immunoglobulin protein scaffold or skeleton such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer or an EGF domain.
• domain refers to a folded protein structure which retains its tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
• single variable domain refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It, therefore, includes complete antibody variable domains such as VH, VHH and VL and modified antibody variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.
• a single variable (V) domain is capable of binding an antigen or epitope independently of a different variable region or domain.
• a “domain antibody” or”dAbTM” may be considered the same as a “single variable domain”.
• a single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent nurse shark and Camelid VHH dAbsTM.
• Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains.
• Such VHH domains may be humanized according to standard techniques available in the art, and such domains are considered to be "single variable domains".
• VH includes camelid VHH domains.
• An antigen-binding fragment may be provided by means of arrangement of one or more CDRs on non-antibody protein scaffolds.
• Protein Scaffold as used herein includes but is not limited to an immunoglobulin (Ig) scaffold, for example an IgG scaffold, which may be a four chain or two chain antibody, or which may comprise only the Fc region of an antibody, or which may comprise one or more constant regions from an antibody, which constant regions may be of human or primate origin, or which may be an artificial chimera of human and primate constant regions.
• Ig immunoglobulin
• isotype refers to the antibody class (e.g., lgG1 , lgG2, lgG3, lgG4, IgM, lgA1 , lgA2, IgD, and IgE antibody) that is encoded by the heavy chain constant region genes.
• antibody class e.g., lgG1 , lgG2, lgG3, lgG4, IgM, lgA1 , lgA2, IgD, and IgE antibody
• Constant variations also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Such conservative substitutions are within the definition of the classes of the peptides of the invention.
• the biological activity of the peptides can be determined by standard methods known to those of skill in the art and described herein.
• the term "substantial homology" indicates that two polypeptides, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate amino acid insertions or deletions, in at least about 80% of the amino acids, at least about 90% to 95%, or at least about 98% to 99.5% of the amino acids.
• the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
• vectors e.g., non-episomal mammalian vectors
• vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
• certain vectors are capable of directing the expression of genes to which they are operatively linked.
• Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors”).
• expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
• plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
• viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
• recombinant host cell (or simply “host cell”), as used herein, is intended to refer to a cell that comprises a nucleic acid that is not naturally present in the cell, and can be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny cannot, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.
• effector function refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event that results therefrom.
• exemplary “effector functions” include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcyR-mediated effector functions such as ADCC and antibody dependent cell-mediated phagocytosis (ADCP), and downregulation of a cell surface receptor (e.g., the B cell receptor; BCR).
• CDC complement dependent cytotoxicity
• Fc receptor binding FcyR-mediated effector functions
• ADCP antibody dependent cell-mediated phagocytosis
• BCR B cell surface receptor
• Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).
• Fc receptor or “FcR” is a receptor that binds to the Fc region of an immunoglobulin.
• FcRs that bind to an IgG antibody comprise receptors of the FcyR family, including allelic variants and alternatively spliced forms of these receptors.
• the FcyR family consists of three activating (FcyRI, FcyRIII, and Fc.RIV in mice; FcyRIA, FcyRIIA, and FcyRIIIA in humans) and one inhibitory (FcyRIIB) receptor.
• FcyRIIB inhibitory receptor
• NK cells selectively express one activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans) but not the inhibitory FcyRIIB in mice and humans.
• Human lgG1 binds to most human Fc receptors and is considered equivalent to murine lgG2a with respect to the types of activating Fc receptors that it binds to.
• the constant region may be modified to stabilize the antibody, e.g., to reduce the risk of a bivalent antibody separating into two monovalent VH-VL fragments.
• residue S228 residue numbering according to the EU index
• P proline
• Antibodies or fragments thereof can also be defined in terms of their complementarity-determining regions (CDRs).
• complementarity-determining region refers to the regions of an antibody in which amino acid residues involved in antigen binding are situated.
• the region of hypervariability or CDRs can be identified as the regions with the highest variability in amino acid alignments of antibody variable domains.
• Databases can be used for CDR identification such as the Kabat database, the CDRs e.g., being defined as comprising amino acid residues 24-34 (CDR1), 50-59 (CDR2) and 89-97 (CDR3) of the light-chain variable region, and 31-35 (CDR1), SO-
• CDRs can be defined as those residues from a "hypervariable loop" (residues 26-33 (L1), 50-52 (L2) and 91-96 (L3) in the light-chain variable region and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable region (Chothia and Lesk, J. Mol. Biol 196: 901-917 (1987)).
• the CDR regions of the antibody sequences described herein are preferably defined according to the numbering scheme of IMGT which is an adaptation of the numbering scheme of Chothia (ImMunoGeneTics information system®; Lefranc, M.-P. et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 27, 209-212 (1999).; http://imgt.org).
• the terms “specific binding,” “selective binding,” “selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen.
• the antibody binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
• a non-specific antigen e.g., BSA, casein
• the binding affinity between two molecules, e.g. an antibody, or fragment thereof, and an antigen, through a monovalent interaction may be quantified by determination of the equilibrium dissociation constant (KD).
• KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the SPR method.
• the rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and dissociation rate constant kd (or koff), respectively.
• the specificity of an interaction and the value of an equilibrium binding constant can be determined directly by well-known methods.
• Standard assays to evaluate the ability of ligands (such as antibodies) to bind their targets are known in the art and include, for example, ELISAs, Western blots, RIAs, and flow cytometry analysis.
• the binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as SPR.
• SPR standard assays known in the art, such as SPR.
• naturally-occurring as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
• a “polypeptide” refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain.
• One or more amino acid residues in the protein can contain a modification such as, but not limited to, glycosylation, phosphorylation or disulfide bond formation.
• a “protein” can comprise one or more polypeptides.
• nucleic acid molecule is intended to include DNA molecules and RNA molecules.
• a nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.
• ug and uM are used interchangeably with “pg” and “pM,” respectively.
• administering refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
• Different routes of administration for the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
• parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
• an antibody described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
• Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
• the present inventors have dedicated themselves to solving the problem of the present invention and were successful to find novel human monoclonal antibodies against SARS-related coronavirus having superior neutralization potency against all currently circulating SARS-CoV-2 variants of concern, most importantly including XBB.1.5, as well as emerging variants in comparison to publicly available neutralizing antibodies.
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• SARS-CoV-2 humoral immunity matures continuously in the months following infection, e.g. due to memory B cells acquiring somatic mutations within 6 months after infection, leading to more potently neutralizing antibodies.
• longitudinal analyses of serum antibodies showed that renewed antigen contact expands the response against SARS-CoV-2 in potency and breadth.
• breakthrough infections with newly emerging variants of concern (VOC) can also increase and improve the plasma antibody response.
• the present inventors addressed the need for broadly neutralizing antibodies effective against a wide range of emerging variants by longitudinally investigating the humoral immune response on a molecular level following repeated vaccination and Omicron breakthrough infection.
• the inventors isolated and characterized 656 monoclonal antibodies (mAbs) from SARS- CoV-2-specific memory B cells to identify the antibodies of the present invention which are able to neutralize an extensive range of SARS-CoV-2 variants with high potency and superiority in breadth compared to antibodies that are already used in the clinic. These new bNAbs may hold great therapeutic or prophylactic potential with a chance to also react against future variants.
• mAbs monoclonal antibodies
• antibodies TV1t4p2_A5 and TV1t4p3_E8 of the present invention have been tested in comparative experiments against a reference antibody SP1-77 which was previously reported as supposedly being capable of neutralizing a high diversity of variants of SARS-CoV-2 (cf. (Luo, S. et al. An antibody from single human V H -rearranging mouse neutralizes all SARS-CoV-2 variants through BA.5 by inhibiting membrane fusion. Sci. Immunol. 7, eadd5446 (2022)).
• the present invention provides antibodies or antigen-binding fragments thereof directed against SARS-related coronavirus, wherein the antibody or antigen-binding fragment thereof comprises the combination of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 amino acid sequence of one antibody selected from the group comprising TV1t4p2_A5 (having a CDR-H1 amino acid sequence of SEQ ID No. 27, a CDR-H2 amino acid sequence of SEQ ID No. 28, a CDR-H3 amino acid sequence of SEQ ID No. 29, a CDR-L1 amino acid sequence of SEQ ID No. 30, a CDR-L2 amino acid sequence of SEQ ID No. 31 , a CDR-L3 amino acid sequence of SEQ ID No.
• the antibody or antigen-binding fragment thereof comprises the combination of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 amino acid sequence of one antibody selected from the group comprising TV1t4p2_A5 (having a CDR
• TV1t2p6_D3 (having a CDR-H1 amino acid sequence of SEQ ID No. 33, a CDR-H2 amino acid sequence of SEQ ID No. 34 a CDR-H3 amino acid sequence of SEQ ID No. 35 a CDR-L1 amino acid sequence of SEQ ID No. 36 a CDR-L2 amino acid sequence of SEQ ID No. 37, a CDR-L3 amino acid sequence of SEQ ID No. 38), TV1t4p3_E8 (having a CDR-H1 amino acid sequence of SEQ ID No. 21 , a CDR-H2 amino acid sequence of SEQ ID No. 22, a CDR-H3 amino acid sequence of SEQ ID No.
• a CDR-L1 amino acid sequence of SEQ ID No. 24 a CDR-L2 amino acid sequence of SEQ ID No. 25, a CDR-L3 amino acid sequence of SEQ ID No. 26
• TV1t3p4_E1 having a CDR- H1 amino acid sequence of SEQ ID No. 39, a CDR-H2 amino acid sequence of SEQ ID No. 40, a CDR-H3 amino acid sequence of SEQ ID No. 41 , a CDR-L1 amino acid sequence of SEQ ID No. 42, a CDR-L2 amino acid sequence of SEQ ID No. 43, a CDR-L3 amino acid sequence of SEQ ID No. 44).
• the antibodies which have been generated and described herein, may be used and claimed as the complete monoclonal human antibody or as any functional or antigen-binding fragment thereof.
• the antibody or any kind of functional or antigenbinding fragment thereof should at least comprise the complementarity determining regions (CDR) 1 to 3 of the heavy chain and CDR 1 to 3 of the light chain of the antibody.
• the CDR regions of the antibody sequences described herein are preferably defined according to the numbering scheme of IMGT which is an adaptation of the numbering scheme of Chothia (ImMunoGeneTics information system®; Lefranc, M.-P. et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 27, 209-212 (1999); http://imgt.org).
• IMGT is an adaptation of the numbering scheme of Chothia (ImMunoGeneTics information system®; Lefranc, M.-P. et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 27, 209-212 (1999); http://imgt.org).
• the CDRs can be easily and unambiguously determined by a skilled person.
• the light and heavy chain variable region sequences of the preferred antibodies and antigen-binding fragments thereof described herein with the internal designations TV1t4p3_E8, TV1t4p2_A5, TV1t2p6_D3, and TV1t3p4_E1 are as follows (CDRs marked in bold):
• the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence of antibody TV1t4p2_A5
• the antibody or antigen-binding fragment thereof comprises a light chain variable region amino acid sequence of antibody TV1t4p2_A5
• the antibody comprises a heavy chain variable region amino acid sequence of SEQ ID No. 3 and a light chain variable region amino acid sequence of SEQ ID No. 4, or the antibody comprises a heavy chain variable region amino acid sequence of SEQ ID No. 5 and a light chain variable region amino acid sequence of SEQ ID No. 6, or the antibody comprises a heavy chain variable region amino acid sequence of SEQ ID No. 1 and a light chain variable region amino acid sequence of SEQ ID No. 2, or the antibody comprises a heavy chain variable region amino acid sequence of SEQ ID No. 7 and a light chain variable region amino acid sequence of SEQ ID No. 8.
• the antibody consists of two heavy chains of sequence SEQ ID NO. 3 and two light chains of sequence SEQ ID NO. 4, or the antibody consists of two heavy chains of sequence SEQ ID NO. 5 and two light chains of sequence SEQ ID NO. 6, or the antibody consists of two heavy chains of sequence SEQ ID NO. 1 and two light chains of sequence SEQ ID NO. 2, or the antibody consists of two heavy chains of sequence SEQ ID NO. 7 and two light chains of sequence SEQ ID NO. 8.
• the CDR sequences of the light and heavy chain variable region sequences of the antibodies and antigen-binding fragments thereof described herein are as follows:
• the antibody used as a source for sequences comprised in the antibody or antigen-binding fragment thereof according to the present invention is selected from the group comprising TV1T4p2_A5, TV1t2p6_D3, and TV1t4p3_E8, preferably from the group comprising TV1t4p2_A5 and TV1t2p6_D3, more preferably of antibody TV1t4p2_A5.
• the antibody used as a source for sequences comprised in the antibody of the invention is TV1t4p2_A5. In another embodiment of the present invention, the antibody used as a source for sequences comprised in the antibody of the invention is TV1t2p6_D3. In one embodiment of the present invention, the antibody used as a source for sequences comprised in the antibody of the invention is TV1t4p3_E8. In another embodiment of the present invention, the antibody used as a source for sequences comprised in the antibody of the invention is TV1t3p4_E1 .
• the SARS-related coronavirus strain is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
• the amino acid sequences of the CDRs or of the variable regions comprised in the antibody or antigen-binding fragment thereof according to the present invention are from an antibody which is able to neutralize each of the SARS-CoV-2 lineages Wu01 , Alpha, Beta, Delta, BA.1 , BA.2, BA.2.12.1 , BA.4/5, BA.2.75, BA.2.75.2, BQ.1.1 , BA.4.6, XBB.1 , XBB.1 .5, and BF.7 in a pseudovirus neutralization assay as described in the description with an IC50 of at most 0.07 pg/ml, preferably at most 0.06 pg/ml, more preferably at most 0.05 pg/ml, even more preferably at most 0.04 pg/ml, even more preferably at most 0.03 pg/ml, particularly preferably at most 0.025 pg/ml.
• the pseudovirus neutralization assay for determining IC50 values is to be carried out as described in the section “Pseudovirus assay to determine IgG and plasma SARS-CoV-2 neutralizing activity ” below.
• additional antibodies or antigen-binding fragments thereof are provided which are directed against SARS-related coronavirus, wherein the amino acid sequences of the CDRs or of the variable regions comprised therein are from an antibody which is also able to neutralize each of the SARS-CoV-2 lineages Wu01 , Alpha, Beta, Delta, BA.1 , BA.2, BA.2.12.1 , BA.4/5, BA.2.75, BA.2.75.2, BQ.1.1 , BA.4.6, XBB.1 , XBB.1.5, and BF.7 in a pseudovirus neutralization assay as described in the description.
• These antibodies carry the internal designations TV1t4p2_H8, TV1t2p7_F11 , TV1t4p1_F7, and TV1t2p4_D11.
• the light and heavy chain variable region sequences of these additional antibodies and antigen-binding fragments thereof described herein are as follows (CDRs marked in bold):
• the antibody or antigen-binding fragment thereof comprises the combination of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 amino acid sequence of one antibody selected from the group comprising TV1t4p2_H8 (having a CDR-H1 amino acid sequence of SEQ ID No. 45, a CDR-H2 amino acid sequence of SEQ ID No. 46, a CDR-H3 amino acid sequence of SEQ ID No. 47, a CDR-L1 amino acid sequence of SEQ ID No. 48, a CDR-L2 amino acid sequence of SEQ ID No. 49, a CDR-L3 amino acid sequence of SEQ ID No.
• TV1t4p2_H8 having a CDR-H1 amino acid sequence of SEQ ID No. 45, a CDR-H2 amino acid sequence of SEQ ID No. 46, a CDR-H3 amino acid sequence of SEQ ID No. 47, a CDR-L1 amino acid sequence of SEQ ID No. 48, a CDR-
• TV1t2p7_F11 having a CDR- H1 amino acid sequence of SEQ ID No. 51 , a CDR-H2 amino acid sequence of SEQ ID No. 52, a CDR-H3 amino acid sequence of SEQ ID No. 53, a CDR-L1 amino acid sequence of SEQ ID No. 54, a CDR-L2 amino acid sequence of SEQ ID No. 55, a CDR-L3 amino acid sequence of SEQ ID No. 56
• TV1t4p1_F7 having a CDR-H1 amino acid sequence of SEQ ID No. 57, a CDR-H2 amino acid sequence of SEQ ID No. 58 a CDR-H3 amino acid sequence of SEQ ID No.
• the CDR sequences of the light and heavy chain variable region sequences of the additional antibodies and antigen-binding fragments thereof described herein with the internal designations TV1t4p2_H8, TV1t2p7_F11 , TV1t4p1_F7, and TV1t2p4_D11 are as follows:
• the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence of antibody TV1t4p2_H8 (SEQ ID No. 9), or a heavy chain variable region amino acid sequence of antibody TV1t2p7_F11 (SEQ ID No. 11), or a heavy chain variable region amino acid sequence of antibody TV1t4p1_F7 (SEQ ID No. 13), or a heavy chain variable region amino acid sequence of antibody TV1t2p4_D11 (SEQ ID No. 15).
• the antibody or antigen-binding fragment thereof comprises a light chain variable region amino acid sequence of antibody TV1t4p2_H8 (SEQ ID No. 10), or a light chain variable region amino acid sequence of antibody TV1t2p7_F11 (SEQ ID No. 12), or a light chain variable region amino acid sequence of antibody TV1t4p1_F7 (SEQ ID No. 14), or a light chain variable region amino acid sequence of antibody TV1t2p4_D11 (SEQ ID No. 16).
• the antibody comprises a heavy chain variable region amino acid sequence of SEQ ID No. 9 and a light chain variable region amino acid sequence of SEQ ID No. 10, or the antibody comprises a heavy chain variable region amino acid sequence of SEQ ID No. 11 and a light chain variable region amino acid sequence of SEQ ID No. 12, or the antibody comprises a heavy chain variable region amino acid sequence of SEQ ID No. 13 and a light chain variable region amino acid sequence of SEQ ID No. 14, or the antibody comprises a heavy chain variable region amino acid sequence of SEQ ID No. 15 and a light chain variable region amino acid sequence of SEQ ID No. 16.
• the antibody consists of two heavy chains of sequence SEQ ID NO. 9 and two light chains of sequence SEQ ID NO.
• the antibody consists of two heavy chains of sequence SEQ ID NO. 11 and two light chains of sequence SEQ ID NO. 12 or the antibody consists of two heavy chains of sequence SEQ ID NO. 13 and two light chains of sequence SEQ ID NO. 14, or the antibody consists of two heavy chains of sequence SEQ ID NO. 15 and two light chains of sequence SEQ ID NO. 16.
• the antibody used as a source for sequences comprised in the antibody of the invention is TV1t4p1_G7.
• This antibody is able to efficiently cross-neutralize the SARS-related coronavirus strain SARS-CoV-1 in addition to efficient neutralization of different variants of SARS-CoV-2.
• the antibody used as a source for sequences comprised in the antibody of the invention is TV1t4p3_H5.
• This antibody is able to efficiently cross- neutralize the SARS-related coronavirus strain SARS-CoV-1 in addition to efficient neutralization different variants of SARS-CoV-2.
• the light and heavy chain variable region sequences of the antibodies TV1t4p1_G7 and TV1t4p3_H5 described herein are as follows (CDRs marked in bold):
• the antibody has a heavy chain variable region amino acid sequence of antibody TV1t4p1_G7 (SEQ ID No. 17), or a heavy chain variable region amino acid sequence of antibody TV1t4p3_H5 (SEQ ID No. 19).
• the antibody has a light chain variable region amino acid sequence of antibody TV1t4p1_G7 (SEQ ID No. 18), or a light chain variable region amino acid sequence of antibody TV1t4p3_H5 (SEQ ID No. 20).
• the antibody comprises a heavy chain variable region amino acid sequence of SEQ ID No. 17 and a light chain variable region amino acid sequence of SEQ ID No. 18, or the antibody comprises a heavy chain variable region amino acid sequence of SEQ ID No. 19 and a light chain variable region amino acid sequence of SEQ ID No. 20.
• the antibody consists of two heavy chains of sequence SEQ ID NO. 17 and two light chains of sequence SEQ ID NO. 18, or the antibody consists of two heavy chains of sequence SEQ ID NO. 19 and two light chains of sequence SEQ ID NO. 20.
• the CDR sequences of the light and heavy chain variable region sequences of the antibodies TV1t4p1_G7 and TV1t4p3_H5 described herein are as follows:
• the antibody or antigen-binding fragment thereof comprises the combination of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 amino acid sequence of one antibody selected from the group comprising TV1t4p1_G7 (having a CDR-H1 amino acid sequence of SEQ ID No. 69, a CDR-H2 amino acid sequence of SEQ ID No. 70, a CDR-H3 amino acid sequence of SEQ ID No. 71 , a CDR-L1 amino acid sequence of SEQ ID No. 72, a CDR-L2 amino acid sequence of SEQ ID No. 73, a CDR-L3 amino acid sequence of SEQ ID No.
• TV1t4p1_G7 having a CDR-H1 amino acid sequence of SEQ ID No. 69, a CDR-H2 amino acid sequence of SEQ ID No. 70, a CDR-H3 amino acid sequence of SEQ ID No. 71 , a CDR-L1 amino acid sequence of SEQ ID No.
• TV1t4p3_H5 having a CDR-H1 amino acid sequence of SEQ ID No. 75, a CDR-H2 amino acid sequence of SEQ ID No. 76, a CDR-H3 amino acid sequence of SEQ ID No. 77, a CDR-L1 amino acid sequence of SEQ ID No. 78, a CDR-L2 amino acid sequence of SEQ ID No. 79, a CDR-L3 amino acid sequence of SEQ ID No. 80).
• the antibodies or antigen-binding fragments thereof as described herein further encompass antibody amino acid sequences being at least 80% identical to the sequences as defined above as long as they are still directed against the spike (S) protein of SARS-CoV-2 as in SEQ ID NO. 81 , preferably as long as they are still directed against the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 as in SEQ ID NO. 82.
• the specific sequence of the spike (S) protein of SARS-CoV-2 or the receptor-binding domain thereof against which the antibodies or antigen-binding fragments thereof should be directed may be taken from one of the commonly known SARS-CoV-2 variants Wu01 , Alpha, Beta, Delta, BA.1 , BA.2, BA.2.12.1 , BA.4/5, BA.2.75, BA.2.75.2, BQ.1.1 , BA.4.6, XBB.1 , XBB.1 .5, and BF.7.
• the antibody or antigen-binding fragment thereof according to the present invention does not display autoreactivity defined as detectable binding when tested against permeabilized HEp-2 cells using an antinuclear antibody (ANA) testing kit (NOVA-Lite HEp-2 ANA kit; Inova Diagnostics) at concentrations of 100 pg/ml of the antibody or antigen-binding fragment thereof.
• ANA antinuclear antibody
• sequence variations encompassed herein are meant to include sequences having trivial mutations, i.e., conservative mutations, of the antibody amino acid sequence which do not interfere with structural folds and the affinity of the antibody to the spike (S) protein.
• the deviations in the amino acid sequence leading to an at least 80%, 85%, 90% or 95% overall identity to the individualized sequences explicitly disclosed herein are present exclusively outside the CDR regions of the antibodies according to the invention.
• the present invention encompasses antibody amino acid sequences having 1 , 2, 3, 4, 5, or 6 mutations within the constant regions of the antibody.
• the antibodies according to the present invention are preferably of human origin.
• at least the sequences outside the CDRs, such as framework and constant regions of the antibody are preferably of human origin or can be attributed to human origin.
• the antibodies of the present invention are preferably monoclonal.
• the antibody is a monoclonal antibody or a fragment thereof that retains binding specificity and ability to neutralize infectious pathogen.
• the antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody.
• the antibody may be an antibody comprising an Fc domain of any human IgG isotype (e.g. IgG 1 , lgG2, lgG3, or lgG4).
• the antigen-binding compound consists of or comprises a Fab, Fab', Fab'-SH, F(ab)2, Fv, a diabody, single-chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments.
• an antibody or antigen-binding fragment directed against the spike (S) protein of SARS-CoV-2 means an antibody binding to the spike (S) protein of SARS-CoV-2 with an at least 10-fold, more preferably at least 50-fold, particularly preferably at least 100-fold increased affinity compared to unrelated epitopes, proteins or protein regions.
• Gapped BLAST is utilized as described by Altschul et al. (Nucleic Acids Res. (1997) 25: 3389-3402).
• Altschul et al. Nucleic Acids Res. (1997) 25: 3389-3402).
• the default parameters of the respective programs are used.
• antibody amino acid sequences form part of the invention which consist of or comprise a nucleic acid sequence being at least 85% identical to the sequences defined above and disclosed herein, more preferably at least 90% identical, even more preferred at least 95% identical.
• the SARS-related coronavirus strain is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which may alternatively be referred to as SARS-related coronavirus 2 in the art.
• the SARS-related coronavirus strains are severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1) or the Bat SARS-like coronavirus WIV-1 , preferably the severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1).
• the antibody or antigen-binding fragment thereof is directed against the ectodomain of the spike (S) protein of SARS-CoV-2.
• the antibody or antigen-binding fragment thereof is directed against the Wu01 spike (S) homotrimer of SARS-CoV-2 as described in Hoffmann M. et al. (2020). Cell 181 , 271-280 (EPI_ISL_406716. This virus isolate has been studied intensively and is best understood at the time of filing. However, preferably, the antibody or antigen-binding fragment thereof should also be directed against equivalent sequences of other virus variants. According to one specific embodiment, the antibody or antigen-binding fragment thereof is directed against the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 (SEQ ID NO. 82).
• RBD receptor-binding domain
• the antibody or antigen-binding fragment thereof does not display autoreactivity against human cells defined as a detectable binding pattern when tested against permeabilized HEp-2 cells using an antinuclear antibody (ANA) testing kit (NOVA-Lite HEp-2 ANA kit; Inova Diagnostics) at concentrations of 100 pg/ml of the antibody or antigen-binding fragment thereof.
• ANA antinuclear antibody
• other assays known in the art may be used to determine or exclude autoreactivity of antibodies or antigen-binding fragments thereof.
• antibody designations may be used. It is pointed out that the antibodies consist of heavy and light chains which also form part of the present description. If reference is made to an antibody by its designation or to a SEQ ID NO., it should be understood that these ways of reference are interchangeable.
• the present invention further relates to a pharmaceutical composition
• a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the invention as defined and further described herein and at least one pharmaceutically acceptable excipient.
• the pharmaceutical composition is a vaccination composition for a human and/or animal subject.
• the pharmaceutical composition is capable of conferring passive immunity against SARS-CoV-2 to a subject, preferably a human subject.
• the present invention also encompasses a kit comprising an antibody or antigen-binding fragment thereof according to the invention as defined and further described herein and a container.
• the present invention is also directed to the antibody or antigen-binding fragment thereof according to the invention as defined and further described herein, the pharmaceutical composition as described herein and the kit for use as a medicament, preferably for use as a vaccine.
• the antibody or antigen-binding fragment thereof according to the invention as defined and further described herein, the pharmaceutical composition as described herein and the kit as described herein are provided for use in conferring passive immunity to a subject and/or provided for use in the form of a composition capable of conferring passive immunity to a subject.
• the present invention is also directed to the antibody or antigen-binding fragment thereof according to the invention as defined and further described herein, the pharmaceutical composition as described herein and the kit for use in the treatment or prevention of a disease caused by SARS-related coronavirus in human or animal subjects, preferably for use in the treatment or prevention of a disease caused by SARS-related coronavirus 2 (SARS-CoV-2) in human or animal subjects.
• SARS-CoV-2 SARS-related coronavirus 2
• the present invention is directed to the antibody or antigen-binding fragment thereof according to the invention as defined and further described herein, the pharmaceutical composition as described herein and the kit for use in prevention of infection of a human and/or animal subject with SARS-related coronavirus, preferably of infection of a human and/or animal subject with SARS- related coronavirus 2 (SARS-CoV-2).
• SARS-CoV-2 SARS-related coronavirus 2
• the present invention is directed to a method of treating or preventing a SARS- related coronavirus infection or reducing the severity of disease in a human and/or animal subject comprising administering a therapeutically effective amount of at least one antibody and/or antigenbinding fragment thereof as described herein to said subject, preferably wherein the SARS-related coronavirus is SARS-CoV-2.
• an antibody and/or antigen-binding fragment thereof according to the invention is administered to a patient in need thereof by intravenous injection or infusion, subcutaneous injection, intramuscular injection, or inhalative application, preferably by intravenous injection.
• the antibody or antigen-binding fragment thereof is administered by intravenous infusion at an absolute dose of up to 4000 mg, preferably up to 2000 mg, more preferably up to 1200 mg, even more preferably up to 600 mg, even more preferably up to 300 mg, particularly preferably up to 150 mg.
• the dosage of an antibody or antigen-binding fragment thereof of the invention to be administered to a subject can further vary depending on such things as the severity of the symptoms exhibited as well as the age, sex, and health of the subject.
• an antibody according to the invention is administered to a patient in need thereof by inhalative application.
• the antibody is administered by inhalative application, wherein it is provided in a liquid pharmaceutical composition which is nebulized by a mesh nebulizer or a jet nebulizer prior to administration.
• a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
• routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation and inhaled through the mouth), transdermal (e.g., topical), transmucosal, and rectal administration.
• the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to human beings.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
• the methods of the invention may comprise pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent.
• pulmonary administration e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent.
• the methods of the invention may also comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion).
• a composition formulated for parenteral administration by injection e.g., by bolus injection or continuous infusion.
• the pharmaceutical formulation of the present invention may be provided in liquid form or may be provided in lyophilized form.
• the present invention relates to a nucleic acid encoding an antibody or antigen-binding fragment thereof as described herein.
• the present invention relates to an expression vector comprising the nucleic acid as described herein in functional association with an expression control sequence.
• the present invention relates to a host cell comprising a nucleic acid as described herein. In one aspect, the present invention relates to a host cell comprising an expression vector as described herein.
• the present invention relates to a method of production of an antibody or antigenbinding fragment as described herein, comprising (a) cultivating a host cell as described herein under conditions allowing expression of the antibody or antigen-binding fragment thereof, and (b) recovering the antibody or antigen-binding fragment thereof.
• the present invention relates to an antibody or antigen-binding fragment thereof against SARS-related coronavirus 2 (SARS-CoV-2) as described herein for use in medicine in combination with at least one further antibody directed against SARS-related coronavirus 2 (SARS-CoV-2), wherein said further antibody has a different binding specificity.
• SARS-CoV-2 SARS-related coronavirus 2
• the present invention is also directed to the use of the antibody or antigen-binding fragment thereof according to the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treatment of a disease caused by SARS-related coronavirus in human or animal subjects, preferably for treatment or prevention of COVID-19 in human or animal subjects.
• Plasma and PBMCs were isolated by density gradient centrifugation (Histopaque, Sigma Aldrich) as advised in the manufacturer’s instructions. Plasma aliquots were stored at -80°C and PBMCs at -150°C in 10% DMSO (Sigma-Aldrich) and 90% (v/v) FBS (Sigma-Aldrich).
• HEK-293T-ACE2 cells were maintained in T75 tissue culture flasks (Sarstedt) with DMEM (Gibco) containing 10% FBS (Sigma Aldrich), 1 mM L-Glutamine (Gibco), 1 mM Sodium pyruvate (Gibco) and 1 % Penicillin-Streptomycin (Gibco) at 37°C and 5% CO2.
• B cells were isolated from PBMCs through use of magnetic CD19 microbeads (Miltenyi Biotec) and subsequently stained with anti-CD20-Alexa700, anti-IgG-PE, DAPI, and labeled SARS-CoV-2 spike protein in FACS buffer (PBS with 2% FCS and 0.1 % 0.5 M EDTA) for 20 min at 4°C in the dark. Spike binding was determined by a double-staining with Wu01 spike protein marked with DyLight 488 and DyLight 650 (DyLight Antibody Labeling Kit, Thermo Fisher) for time points 1 and 2.
• Heavy and light chain genes of sorted B cells were amplified using a single-cell PCR protocol previously described. Following cell lysis, Superscript IV reverse transcriptase (Thermo Fisher), random hexamer primers (Invitrogen), RNasin (Promega), and RNaseOUT (Thermo Fisher) were used to create cDNA from RNA. The cDNA formed the template for following heavy and light chain amplification through semi-nested PCR with PlatinumTaq HotStart DNA polymerase (Thermo Fisher), V gene-specific forward primer mixes, reverse primers, and KB extender. Quality check of PCR products was ensured with gel electrophoresis.
• Sequences of the single-cell PCR products were used to order double-stranded DNA fragments (eBlocks, IDT DNA) for antibody production.
• Expression vector overhangs were added to the DNA fragments to enable sequence- and ligation-independent cloning (SLIC).
• DNA fragments were inserted into human antibody expression vectors (lgG1 heavy-, kappa-, or lambda chain) as previously published.
• 25 ng eBlock DNA fragment was added to 80 ng cut vector for heavy, kappa, or lambda chain and 0.2 pl T4 DNA polymerase (3,000 units/ml) in NEBuffer 2.1. This mix was incubated for 2:30 min at 24 °C and then for at least 10 min on ice.
• the product of the SLIC reaction was used to transform DH5a competent bacteria.
• Monoclonal antibodies were produced in a 24 well format and transfection supernatant used for further assays without purification.
• 1.25 pg of each heavy and light chain plasmid were mixed with branched polyethylenimine (PEI, Sigma Aldrich) to transfect 2.5 ml HEK-293-6E cells (cell density 0,8x10/ml) in a 24 well plate (Sarstedt).
• HEK-293-6E cells were cultured in Freestyle 293 Expression Medium (Gibco) containing 0.2% Penicillin + Streptomycin (Gibco). After 7 days in New Brunswick S41 i incubator shaking at 250 rpm (37°C and 6% CO2), plates were centrifuged for 5 min at 400 g (4°C) and transfection supernatant was harvested.
• 96 well ELISA plates (Greiner) were coated with 2.5 pg/ml anti-IgG (Jackson Immunoresearch, AffiniPure Goat Anti-Human IgG) in PBS at 4°C over night. All following steps were performed at RT.
• Goat Anti-Human IgG-HRP (Southern Biotech) was added as a secondary antibody in a 1 :2500 dilution in PBS 5% milk. One hour later, plates were washed again and ABTS Solution (Life Technologies) was used as a substrate for readout at 415 nm with reference at 695 nm with a TECAN Microplate Reader (Software XFIour4). IgG concentrations in transfection supernatants were calculated relating the measured OD values of sample and standard.
• Greiner flat bottom 96 well plates were coated with 2 pg/ml SARS-CoV-2 trimeric spike protein (Wu01 and BA.1 HexaPro protein as described below). After incubation at 4°C over night, plates were washed with PBS (Gibco) containing 0.05% Tween (Carl Roth) before blocking with PBS 5% milk (Panreac AppliChem, nonfat dried milk powder) for one hour at RT. All following steps were performed at RT. Plates were washed and transfection supernatants adjusted to a starting concentration of 10 pg/ml were added in a 1 :5 dilution series. An antibody with known spike reactivity was used as a positive control.
• a negative control an HIV-reactive antibody was used. After 1.5 h incubation, plates were washed and 1 :2500 Goat Anti-Human IgG-HRP (Southern Biotech) in 5% milk was added for one hour. After washing, ABTS Single Solution (Life Technologies) was added as a substrate and the signal was measured at 415 nm with reference at 695 nm using a TECAN Microplate Reader (Software XFIour4). Detection of spike RBD-binding IgG by chemiluminescent microparticle immunoassay (CMIA)
• IgG targeting the receptor binding domain (RBD) of the spike protein was quantified by the chemiluminescent microparticle immunoassay (CMIA) SARS-CoV-2 IgG II Quant (Abbott) on the automated system Alinity i (Abbott).
• CMIA chemiluminescent microparticle immunoassay
• Abbott SARS-CoV-2 IgG II Quant
• Abbott automated system Alinity i
• Anti-RBD IgG titers were reported in binding antibody unit per milliliter (BAU/mL), according to the World Health Organization (WHO) international standard.
• a 50 ml transfection format was applied for antibodies presenting good Omicron neutralization activity in screening with 24 well transfection supernatant.
• 25 pg heavy chain and 25 pg light chain plasmids were combined with 2.25 ml DPBS (Gibco).
• 170 pl polyethylenimine (PEI, Sigma Aldrich) were added. Vortexing the mix was followed by 10 min incubation at RT.
• the transfection solution was added dropwise to 50 ml HEK-293-6E cells (density 0.8x10/ml) in culture flasks. After 7 days shaking at 37°C and 6% CO2, plates were centrifuged at 4000 rpm at 4°C for 25 min.
• Pseudovirus particles were produced by cell transfection with plasmids coding for HIV-1 Tat, HIV-1 Gag/Pol, HIV-1 Rev, luciferase followed by an IRES and ZsGreen, and the SARS-CoV-2 spike protein as previously described. After transfection of HEK-293T cells with the pseudovirus encoding plasmids using FUGENE 6 Transfection Reagent (Promega) and incubation for 48 h and 72 h at 37°C and 5% CO2, supernatant was harvested and stored at -80°C. The virus batches were titrated and used for infection of HEK-293T-ACE2 cells.
• Luciferase activity was measured after incubation for 48 h at 37°C and 5% CO2 by addition of luciferin/lysis buffer (10 mM MgCI2, 0.3 mM ATP, 0.5 mM Coenzyme A, 17 mM IGEPAL (all Sigma-Aldrich), and 1 mM D-Luciferin (GoldBio) in Tris-HCL) and read-out in a microplate reader (Berthold Technologies, Tristar 2). Batches showing around 1000-fold higher RLUs in contrast to non-infected cells were accepted for further use in assays.
• Pseudovirus neutralization assays were done as previously published. Transfection supernatants containing monoclonal antibodies were directly used in this assay without prior purification. Plasma was heat-inactivated for 45 min at 56°C. For one hour (37°C and 5% CO2), a dilution series of each sample was incubated with the pseudovirus supernatants from the HEK-293T transfection in DMEM high glucose medium (Gibco, with 10% FBS, 1 mM L-Glutamine, 1 mM Sodium pyruvate, 1 % Penicillin-Streptomycin) in 96 well plates.
• DMEM high glucose medium Gibco, with 10% FBS, 1 mM L-Glutamine, 1 mM Sodium pyruvate, 1 % Penicillin-Streptomycin
• Luciferase activity was measured after addition of luciferin-containing lysis buffer (10 mM MgCI2, 0.3 mM ATP, 0.5 mM Coenzyme A, 17 mM IGEPAL (all Sigma-Aldrich), and 1 mM D-Luciferin (GoldBio) in Tris-HCL) in relative luminescence units (RLUs) using a Tristar 2 reader (Berthold Technologies). Untreated virus control wells (without antibodies) were used on every plate to show the range of RLUs, same as negative controls (virus only and cells only).
• SARS-CoV-2 HexaPro spike trimer MN908947, A. A. 16-1208, RRAR to GSAS, F817P, A892P, A899P, A942P, K986P, V987P, N- terminal BM40 signal peptide, C-terminal T4 foldon followed by a Twin strep tag, 139 kDa; SARS- CoV-2 HexaPro BA.1 spike trimer: MN908947, A. A.
• furin site RRAR to GSAS, T19I, L24S, 25PPAins, G142D, V213G, G339D, S371 F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681 H, N764K, D796Y, Q954H, N969K, including the stabilizing mutations: F817P, A892P, A899P, A942P, K986P, V987P, N-terminal BM40 signal peptide, C-terminal T4 foldon followed by a Twin strep tag, 139 kDa.
• HEK293 EBNA cell lines were generated employing the sleeping beauty transposon system.
• the expression constructs (2 pg) were co-transfected with the transposase plasmid (10:1) into HEK293 EBNA cells, and after stringent puromycin selection (3 mg/mL; Sigma), cells were transferred into triple flasks and protein production was induced by the addition of doxycycline (0.5 mg/mL, Sigma).
• Supernatants of confluent cultures were harvested every 3 days, filtered and the different spike proteins purified by applying it to Strep-TactinXT (IBA Lifescience, Goettingen, Germany) resin.

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