IT202300015579A1 - ANTIBODIES AGAINST COVID-19 AND OTHER HUMAN CORONAVIRUSES - Google Patents

Description

ANTIBODIES AGAINST COVID-19 AND OTHER HUMAN CORONAVIRUSES

DESCRIPTION

Technical field of the invention

The present invention relates to monoclonal antibodies or their antigen-binding portions having potent neutralizing activity against the Coronavirus, particularly against SARS-CoV-2 and SARS-CoV-1. The invention also relates to the use of such monoclonal antibodies or their antigen-binding portions in the therapy, prophylaxis, and diagnosis of Coronavirus-related diseases. State of the art

Human monoclonal antibodies (mAbs) represent an industrially mature technology with over 50 products already approved in the fields of cancer, inflammation, and autoimmunity. Their established safety profile and extensive development experience make mAbs ideal candidates for rapid development, especially in epidemic and pandemic settings. Until now, mAbs have been rarely used in infectious diseases, primarily because the large quantities required for therapy have made them uneconomical. However, in recent years, dramatic technological progress in isolating and screening memory B cells has enabled the identification of highly potent neutralizing mAbs and a further improvement in their potency by several orders of magnitude through established engineering procedures. This has led to a reduction in the quantity of antibodies required for therapy, thus making the non-intravenous delivery of potent neutralizing mAbs possible.

Among the many therapeutic options available, mAbs offer a number of advantages. First, they are the ones that can be developed in the shortest timeframe. Indeed, the extensive clinical experience with the safety of over 50 commercial mAbs approved for the treatment of cancer, inflammation, and autoimmunity instills high confidence in their safety, supporting the possibility of an accelerated regulatory pathway. Furthermore, the long industrial experience in the development and production of mAbs reduces the risks usually associated with the technical development of experimental products. Finally, the incredible technical progress in the field allows for shortening conventional timelines and moving from discovery to proof-of-concept studies in 5-6 months. Several candidates are currently in development in the fields of HIV, pandemic influenza, RSV, and many other infectious diseases. Perhaps the most striking demonstration of the power of mAbs for emerging infections came from the experience of Ebola. In this case, the rapidly developed potent mAbs were among the first drugs tested in the Ebola epidemic and showed remarkable efficacy in preventing mortality. Given the extraordinary effectiveness of this intervention, the potent mAb became the first and, to date, the only drug recommended by the World Health Organization (WHO) for Ebola.

The Coronaviridae family includes a large number of virus species, further classified into four genera. Seven species are of concern to humans and are highly diverse in terms of adaptation, pathogenicity, and spread. Indeed, four species are endemic and highly adapted to humans; the recently emerged and currently circulating SARS-CoV-1 and SARS-CoV-2 belong to the same genus (Betacoronavirus) and subgenus (Sarbecovirus) but are phylogenetically distinct and have strikingly different characteristics in terms of infectivity and clinical signs. These aspects demonstrate the difficulties in thoroughly sampling the entire Coronaviridae family, necessary for implementing an effective surveillance program, and therefore underscore the need for broadly active therapeutic strategies against coronaviruses.

Given the pivotal role of the transmembrane spike glycoprotein of SARS-CoV-1 and SARS-CoV-2 (S protein) in viral pathogenesis, it is considered the primary target for inducing potent neutralizing antibodies and the focus for the development of therapeutic and prophylactic tools against this virus. Indeed, the entry of SARS-CoV-1 and SARS-CoV-2 into host cells is mediated by the interaction between the S protein and human angiotensin-converting enzyme 2 (ACE2). The S protein is a trimeric class I viral fusion protein that exists in a metastable pre-fusion conformation and a stable post-fusion conformation. Each S protein monomer is composed of two distinct regions, the S1 and S2 subunits. Structural rearrangement occurs when the receptor-binding domain (RBD) present in the S1 subunit binds to the host cell membrane. This interaction destabilizes the pre-fusion state of the S protein, triggering a transition to the post-fusion conformation, which in turn leads to viral particle entry into the host cell. Single-cell RNA-seq analyses assessing ACE2 expression levels in various human organs have demonstrated that SARS-CoV-2, through binding to S proteins, can invade human cells in several important physiological systems, including the respiratory, cardiovascular, digestive, and urinary systems, increasing the potential for spread and infection. Therapeutic mAbs for the treatment of COVID-19 are being developed at an accelerated pace, unprecedented for any disease.

However, it is important to produce neutralizing mAbs or their antigen-binding portion that are effective in blocking the entry process of the virus and its variants and that can have a broader spectrum of neutralizable species/isolates. Consequently, there is an urgent need for potent, broad-spectrum antibody therapies with cross-neutralizing activity against different isolates/species of the Coronaviridae family, for use in the therapy, prophylaxis, and diagnosis of coronavirus-related diseases, particularly SARS-CoV-2 and/or SARS-CoV-1.

Summary of the invention

To identify potent mAbs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV-2 variants, and SARS-CoV-1, the inventors isolated thousands of S protein-specific memory B cells derived from people previously infected with SARS-CoV-2 and vaccinated with the coronavirus disease 2019 (COVID-19) mRNA vaccines BNT162b2 or mRNA-1273. The inventors isolated memory B cells specific for the isolated spike (S) protein using recombinant SARS-CoV-1 and SARS-CoV-2 proteins as sorting bait. Naturally produced mAbs were then screened using a microneutralization assay against SARS-CoV-1 and SARS-CoV-2. The screening strategy, described in detail in the examples, led to the identification of ten human mAbs with potent antibody activity. neutralizing antibody against Coronaviruses, in particular against SARS-CoV-2 and SARS-CoV-1. In some aspects, the invention provides a monoclonal antibody capable of neutralizing the biological activity of at least one Coronavirus, which antibody or antigen-binding portion thereof comprises a variable heavy chain (VH) domain and a variable light chain (VK) domain of a monoclonal antibody selected from the group consisting of: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

In some aspects, the invention provides a monoclonal antibody or an antigen-binding portion thereof that specifically binds to the S protein of at least one virus selected from the human severe acute respiratory syndrome (SARS) coronaviruses SARS-CoV-2, SARS-CoV-1, and their variants, wherein said antibody or said antigen-binding portion thereof has neutralizing activity. In particular, such monoclonal antibody or an antigen-binding portion thereof exhibits a 100% inhibitory concentration (IC100) of less than 100 ng/ml when tested in an in vitro neutralization assay against at least one virus selected from SARS-CoV-1, SARS-CoV-2, and their variants, e.g., against the Wuhan and Omicron variants of the SARS-CoV-2 virus (BA.5, BA.2.75, BF.7, BQ.1.1, and XBB.1.5). In some aspects, the invention provides a monoclonal antibody or an antigen-binding portion thereof that specifically comprises VK and VH domains that are at least 85%, 90%, 95%, 97%, 98%, or 99% identical in amino acid sequence to the VK and VH domains, respectively, of a monoclonal antibody selected from the group consisting of: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

In some aspects, the invention provides monoclonal antibodies or antigen-binding portions thereof that compete for binding to the S protein of at least one Coronavirus, particularly at least one virus selected from SARS-COV-1, SARS-CoV-2 and variants thereof, with any of the antibodies described herein.

In some aspects, the invention provides a monoclonal antibody or antigen-binding moiety according to any embodiment described herein, for use in prophylactic or therapeutic treatment of a viral infection or conditions or disorders resulting from such infection.

In some aspects, the invention provides a monoclonal antibody or antigen-binding moiety according to any embodiment described herein, for use in prophylactic or therapeutic treatment of an infection by at least one virus selected from SARS-CoV-2, SARS-CoV-1, and variants thereof, or conditions or disorders resulting from such infection, particularly Coronavirus Disease 2019 (COVID-19).

In some aspects, the invention provides a method for preventing or treating an infection by at least one virus selected from SARS-CoV-1, SARS-CoV-2, and variants thereof, or conditions or disorders resulting from such infection, particularly Coronavirus Disease 2019 (COVID-19), comprising administering a monoclonal antibody or antigen-binding moiety according to any embodiment described herein to a subject in need thereof.

The invention further provides a monoclonal antibody or antigen-binding moiety according to any embodiment described herein for use in the diagnosis, prophylaxis, and/or treatment of a subject who has, or is at risk of developing, a viral infection, particularly a coronavirus infection, more specifically an infection with at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants. Furthermore, the invention relates to the use of the binding molecules and/or nucleic acid molecules of the invention in the diagnosis/detection of such viral infections.

In some aspects, the invention provides a pharmaceutical composition comprising at least one or more monoclonal antibodies or antigen-binding moieties thereof according to any one of the embodiments described herein and a pharmaceutically acceptable carrier and its use in the prevention and/or treatment of infection by a Coronavirus or conditions or disorders resulting from such infection, preferably an infection by at least one virus selected from SARS-CoV-1, SARS-CoV-2 and variants thereof, in particular Coronavirus disease 2019 (COVID-19).

In some aspects, the invention provides an isolated cell line that produces the antibody or an antigen-binding portion thereof according to any of the embodiments described herein.

In some aspects, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or an antigen-binding portion thereof according to any of the embodiments described herein.

In some aspects, the invention provides a vector comprising the nucleic acid molecule encoding the antibody or an antigen-binding portion thereof of embodiments thereof according to any of the embodiments described herein, wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molecule.

In some aspects, the invention provides a non-human transgenic animal or a transgenic plant comprising the nucleic acid according to any of the preceding embodiments, wherein the non-human transgenic animal or transgenic plant expresses said nucleic acid. In some embodiments, said non-human transgenic animal is a mammal.

In some aspects, the invention contemplates the in vitro or ex vivo use of the monoclonal antibody or an antigen-binding portion thereof according to any of the embodiments described herein in the diagnosis of a Coronavirus infection, preferably an infection with at least one virus selected from SARS-CoV-1, SARS-CoV-2 and variants thereof.

In some aspects, the invention provides an in vitro method for detecting the presence of at least one Coronavirus in a sample, preferably at least one virus selected from SARS-CoV-1, SARS-CoV-2 and their variants, comprising the following steps:

i) contacting the antibody or an antigen-binding portion thereof according to any of the embodiments described herein;

ii) Detect binding of said antibody or an antigen-binding portion thereof to the S protein of said at least one coronavirus.

In some aspects, the invention provides an in vitro method for diagnosing a Coronavirus infection in a subject, preferably an infection with at least one virus selected from SARS-CoV-1, SARS-CoV-2 and their variants, comprising the following steps:

i) contacting the antibody or an antigen-binding portion thereof according to any of the embodiments described herein with a biological sample of said subject;

ii) Detect the binding of said antibody or an antigen-binding portion thereof to the S protein of said at least one Coronavirus.

In certain aspects, the invention provides a diagnostic kit comprising as a specific reagent an antibody or an antigen-binding portion thereof according to any of the embodiments described herein, said kit being intended in particular for use in a method for detecting or quantifying, in a biological sample from a patient, antibodies to coronavirus and/or coronavirus S protein, in particular anti-SARS-CoV-2 and/or anti-SARS-CoV-1 and/or SARS-CoV-2 and/or SARS-CoV-1 S protein antibodies.

In certain aspects, the invention provides the use of an antibody or an antigen-binding portion thereof according to any of the embodiments described herein for the design of a vaccine against at least one Coronavirus, in particular against at least one virus selected from SARS-CoV-1, SARS-CoV-2 and variants thereof.

In certain aspects the invention provides a mimotope specifically directed against the idiotype of an antibody or an antigen-binding portion thereof according to any of the embodiments described herein.

In certain aspects, the invention provides an anti-idiotype antibody that is specifically directed against the idiotype of an antibody or an antigen-binding portion thereof according to any of the embodiments described herein.

The invention contemplates combinations of any of the foregoing aspects and embodiments of the invention.

Brief description of the drawings

Fig. 1. Neutralizing activity of human coronavirus antibodies. The heat map shows the binding, neutralizing activity of SARS-CoV-1 and SARS-CoV-2, and the epitope region recognized by the 10 nAbs selected according to the present invention. The potency legend describes the neutralizing activity only.

Detailed description of the invention

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include the singular. Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, protein and nucleic acid genetics and chemistry, and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification, unless otherwise indicated. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989), and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), incorporated herein by reference.

The following terms, unless otherwise indicated, shall have the following meanings:

The term "polypeptide" includes native or artificial proteins, protein fragments, and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric. The term "isolated protein," "isolated polypeptide," or "isolated antibody" is a protein, polypeptide, or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins of the same species, (3) is expressed by a cell of a different species, or (4) is not present in nature. Therefore, a polypeptide synthesized or chemically synthesized in a cellular system other than the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. Examples of isolated antibodies include an anti-S protein antibody of SARS-CoV-2 and/or SARS-CoV-1 that is not associated with naturally associated components. A protein or polypeptide has been affinity purified using the SARS-CoV-2 and/or SARS-CoV-1 S protein or a portion thereof, an anti-SARS-CoV-2 and/or SARS-CoV-1 S protein antibody that was synthesized from a hybridoma or other cell line in vitro, and a human anti-SARS-CoV-2 and/or SARS-CoV-1 S protein antibody derived from a transgenic animal. A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60-75% of a sample contains a single polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80%, or 90% W/W of a protein sample, plus about 10% W/W of the protein. Proteins will typically be about 95% pure and preferably over 99% pure. Protein purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band after staining the gel with a dye well known in the art. For some purposes, higher resolution can be provided using HPLC or other means well known in the art for purification. The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but in which the remaining amino acid sequence is identical to the corresponding positions in the natural sequence. In some embodiments, fragments are at least 5, 6, 8, or 10 amino acids long. In other embodiments, the fragments are at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150, or 200 amino acids in length. The term "polypeptide analog" as used herein refers to a polypeptide that comprises a segment that has substantial identity to a portion of an amino acid sequence and that has at least one of the following properties: (1) specific binding to the S protein of at least one coronavirus under suitable binding conditions, (2) the ability to inhibit the S protein of said at least one coronavirus. Typically, polypeptide analogs comprise a conservative amino acid substitution (or insertion or deletion) from the native sequence. Analogs are typically at least 20 or 25 amino acids in length, preferably 20 or more amino acids in length. at least 50, 60, 70, 80, 90, 100, 150, or 200 amino acids in length, and may often be as long as a full-length polypeptide. Some embodiments of the invention include polypeptide fragments or polypeptide analog antibodies with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 substitutions from the germline amino acid sequence. In some embodiments, the amino acid substitutions to an anti-Coronavirus S protein antibody or antigen-binding portion thereof are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter affinity for the antigen, or (4) alter the amino acid sequence. binding to form protein complexes and (4) confer or modify other physicochemical or functional properties of such analogues, but still retain specific binding to the S protein of at least one Coronavirus. Analogues may include various muteins of a sequence different from the normally occurring peptide sequence. For example, single or multiple amino acid substitutions, preferably conservative amino acid substitutions, may be made in the normally occurring sequence, preferably in the portion of the polypeptide outside the domain or domains that form intermolecular contacts. A conservative amino acid substitution should not substantially change the structural features of the parent sequence; for example, a substituted amino acid should not alter the anti-parallel [beta] sheet that constitutes the immunoglobulin-binding domain that is present in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence. In general, glycine and proline would not be used in an anti-parallel [beta] sheet. Examples of polypeptide secondary and tertiary structures recognized in the art are described in Proteins, Structures, and Molecular Principles

; Introduction to protein structure (

; and Nature 354:105 (1991), incorporated herein by reference.

The term "SARS-CoV-1" refers to the severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1), first identified in southern China in 2002 (World Health Organization 2020). The SARS coronavirus (SARS-CoV) is a member of the Coronaviridae family of enveloped, positive-strand RNA viruses, which, as a group, have a broad host range. It contains three major structural proteins: spike (S), membrane (M), and nucleocapsid (N). Although passive protection has been demonstrated against murine hepatitis viruses (MHV, a heavily studied coronavirus), infection has been achieved by administering mAbs specific to all major structural proteins of the virus. The spike (S) protein is the major antigenic determinant for coronaviruses.

The term SARS-CoV-2 refers to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the type of coronavirus that causes coronavirus disease 2019 (COVID-19), which was first identified in Wuhan, China in 2019 (World Health Organization 2020).

The term “variant” as used herein in relation to the SARS-CoV-1 and/or SARS-CoV-2 viruses is intended to include any known variants of those viruses, such as the Wuhan and Omicron SARS-CoV-2 variants such as BA.5, BA.2.75, BF.7, BQ.1.1, and XBB.1.5.

Where an "antibody" is referred to herein with respect to the invention, it is normally understood that an antigen-binding portion thereof may also be used. An antigen-binding portion competes with the intact antibody for specific binding. See in general, Fundamental Immunology, chapter 7.

(incorporated by reference in its entirety for all purposes). Antigen-binding moieties may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen-binding moieties include Fab, Fab?, F(ab?)2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, nanobodies, and all polypeptides that contain at least a portion of an antibody sufficient to confer specific binding of the polypeptide to the antigen.

From N-terminus to C-terminus, both the light-chain and heavy-chain variable domains comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. The assignment of amino acids to each domain in this document conforms to the IMGT convention definitions described in Lefranc et al. (2003), Developmental & Comparative Immunology 27.1 (2003): 55-77.

As used herein, an antibody referred to by number is the same as a monoclonal antibody obtained from the well of the specific selected B cell plate, for example, antibody 01J19 was obtained from well J19 of the plate with ID 01.

As used herein, an Fd fragment means an antibody fragment that consists of the VH and CH 1 domains; an Fv fragment consists of the VK or VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al, Nature 341: 544-546 (1989)) consists of a VH domain.

In some embodiments, the antibody is a single-chain antibody (scFv) in which the VL or VK and VH domains are coupled to form a monovalent molecule via a synthetic linker that allows them to be made as a single protein chain. (Science 242:423-426 (1988) and Proc. Natl Acad. ScL USA 85:5879-5883 (1988)). In some embodiments, the antibodies are diantibodies, that is, they are bivalent antibodies in which the VH and VL or VK domains are expressed on a single polypeptide chain, but using a linker that is too short to allow association between the two domains on the same chain, thus forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites. (See, for example, Holliger P. et al., Proc. Natl. Acad. ScL USA 90:6444-6448 (1993), and Structure 2:1121-1123 (1994).) In such embodiments, the CDRs may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated noncovalently. In embodiments having one or more binding sites, the binding sites may be identical to each other or may be different.

In some preferred aspects of the invention, a monoclonal antibody according to any of the embodiments described in this specification and the claims is a human antibody, i.e., a human monoclonal antibody, or an antigen-binding portion thereof.

As used herein, the term "human antibody" means any antibody in which the variable and constant domain sequences are human sequences, or any of the CDRs of the variable domain sequences are human sequences. The term includes antibodies with sequences derived from human genes but which have been modified, e.g., to reduce potential immunogenicity, increase affinity, remove cysteines that may cause undesirable folding, etc. The term includes such antibodies produced recombinantly in non-human cells, which may impart glycosylation not typical of human cells. The term "chimeric antibody" as used herein means an antibody comprising regions of two or more different antibodies.

The term "epitope" refers to any protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopes, or antigenic determinants, generally consist of chemically active surface clusters of molecules such as amino acids or carbohydrate or sugar side chains and generally exhibit specific three-dimensional structural features as well as specific charge characteristics. An epitope can be either "linear" or "conformational." In a linear epitope, all interaction points between the protein and the interacting molecule (such as an antibody) are present linearly along the protein's primary amino acid sequence. In a conformational epitope, the interaction points occur across amino acid residues on the protein that are separated from each other.

A "neutralizing antibody" (also abbreviated as nAb), an antibody with "neutralizing activity," or an antibody "capable of neutralizing biological activity" as used herein means an antibody that neutralizes a biological effect that its target (e.g., a pathogen or infectious particle) may have. A "neutralizing antibody," an antibody with "neutralizing activity," or an antibody "capable of neutralizing biological activity" as used herein is, for example, an antibody or an antigen-binding portion thereof that exhibits a 100% inhibitory concentration (IC100) of at least less than 100 ng/ml, preferably less than 50 ng/ml, or more than 100 ng/ml. preferably less than 25 ng/ml when tested in an in vitro neutralization assay against at least one Coronavirus, preferably against at least one virus selected from SARS-CoV-1, SARS-CoV-1 and their variants, performed for example as described here in the examples.

An antibody is said to bind specifically to an antigen when the dissociation constant is, for example, 1 mM, preferably <100 nM, and very preferably <10 nM. The dissociation constant can be measured by any of the methods available in the state of the art, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, surface plasmon resonance, such as BIACORE (TM). For example, the expression "binds specifically to a region of the Spike (S) protein of a Coronavirus" as here means that the antibody or an antigen-binding portion thereof causes more than 50% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay as described in the examples.

The term "polynucleotide" as used herein means a polymeric form of nucleotides at least 10 bases in length, either ribonucleotides or deoxyribonucleotides, or a modified form of both types of nucleotides. The term includes single- and double-stranded forms. The term "isolated polynucleotide" as used herein means a polynucleotide of genomic, cDNA, or synthetic origin, or a combination thereof, that by virtue of its origin (1) is not associated with all or part of a polynucleotide with which the "isolated polynucleotide" occurs in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

The term "naturally occurring nucleotides" as used herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" as used herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" as referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranylate, phosphoroamidate, and the like. See, for example, Nucl. Acids Res. 14: 9081 (1986);

J. Am. Chem. Soc.106:6077 (1984); Nucl. Acids Res.16:3209 (1988);

Anti-Cancer Drug Design 6: 539 (1991); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (Ed., Oxford University Press, Oxford England (1991)); U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90: 543 (1990), the information for which is incorporated by reference. An oligonucleotide may include a marker for detection, if desired. "Operatively linked" sequences include both expression control sequences contiguous to the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest. The term "expression control sequence" as used herein means polynucleotide sequences that are necessary to influence the expression and processing of the coding sequences to which they are linked. Expression control sequences include appropriate transcription, terminator, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., the Kozak consensus sequence); sequences that enhance protein stability; and, when desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include the promoter, ribosome binding site, and transcription termination sequence; in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. The term "carrier," as used herein, means a nucleic acid molecule capable of carrying another nucleic acid to which it has been ligated. In some embodiments, the vector is a plasmid, i.e., a circular piece of double-stranded DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. In some embodiments, vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). In other embodiments, vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and then replicate along with the host genome. Additionally, some vectors are capable of directing the expression of genes to which they are operatively ligated. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

The term "recombinant host cell" (or simply "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. It should be understood that "recombinant host cell" and "host cell" mean not only the particular host cell but also the progeny of that cell. Because some variations may arise in subsequent generations due to mutations or environmental influences, such progeny may, in fact, not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

The term "percent sequence identity" in the context of nucleotide or amino acid sequences refers to residues in two sequences that are the same when aligned for maximum match. The length of the sequence identity comparison can be a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48, or more nucleotides. Numerous algorithms are known in the art that can be used to measure nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are available programs that provide alignments and identities. of the percentage sequence of the regions of best overlap between the query and search sequences (Methods Enzymol.183:63-98 (1990); Pearson, Methods MoI. Biol.132:185-219 (2000); Methods Enzymol.266:227-258 (1996); Pearson, J. MoI. Biol 276:71-84 (1998); incorporated herein by reference). The term "substantial similarity" or "substantial sequence similarity," when referring to a nucleic acid or a fragment thereof, or amino acid means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more. preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any known sequence identity algorithm, such as FASTA, BLAST, or Gap, as discussed above. As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the GAP or BESTFIT programs using predefined gap weights provided with the programs, share at least 70%, 75%, or 80% sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 97%, 98%, or 99% sequence identity. In some embodiments, the positions of residues that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is at least 70%, 75%, or 80% of the nucleotide bases. replaced by another amino acid residue having a side-chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitution. The means for effecting this adjustment are well known to those skilled in the art. See, for example, Pearson, Methods MoI. Biol. 243: 307–31 (1994). Examples of amino acid groups that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acid substitution groups are: valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix described in Science 256:1443-45 (1992), incorporated herein by reference. A "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix. The identity Sequence homology for polypeptides is typically measured using sequence analysis software. Protein analysis software matches sequences using similarity measures assigned to various substitutions, deletions, and other changes, including conservative amino acid substitutions. For example, GCG contains programs such as "Gap" and "Bestfit" that can be used with predefined parameters specified by the programs to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and its mutein.

As used herein, the terms "label" or "labeled" refer to the incorporation of another molecule into the antibody. In one embodiment, the label is a detectable marker, for example, incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker may be therapeutic, such as a drug conjugate or toxin. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used.

Anti-Coronavirus Antibodies and Their Characterization

In one embodiment, the invention provides a monoclonal antibody or an antigen-binding portion thereof capable of neutralizing the biological activity of at least one Coronavirus, which antibody or antigen-binding portion thereof comprises a variable heavy chain (VH) domain and a variable light chain (VK) domain of a monoclonal antibody selected from the group consisting of: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

The nucleic acids encoding the variable domain portions of heavy and light chains (VH and VL) of the antibodies herein denoted as 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 and the corresponding deduced amino acid sequences can be found in the sequence list included in the description herein.

In some aspects, the invention provides a monoclonal antibody or an antigen-binding portion thereof that is capable of neutralizing the biological activity of at least one Coronavirus comprising: (a) a heavy chain variable domain amino acid sequence comprising the heavy chain variable domain amino acid sequence of an antibody selected from: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13; (b) a light chain variable domain amino acid sequence comprising the light chain variable domain amino acid sequence of an antibody selected from: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13; (c) a heavy chain variable domain of (a) and a light chain variable domain of (b); or (d) heavy chain and light chain variable domain amino acid sequences comprising heavy chain and light chain variable domain amino acid sequences, respectively, from the same antibody selected from: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

In some aspects, the invention provides a monoclonal antibody or an antigen-binding portion thereof that is capable of neutralizing the biological activity of at least one Coronavirus, comprising: (a) a heavy chain variable domain amino acid sequence comprising the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain of an antibody selected from: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13; (b) a light chain variable domain amino acid sequence comprising the light chain CDR1, CDR2, and CDR3 amino acid sequences of an antibody selected from: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13; (c) a heavy chain variable domain of (a) and a light chain variable domain of (b); or (d) the heavy chain variable domain and the light chain variable domain of (c), comprising CDR amino acid sequences of the heavy chain and light chain of the same antibody selected from: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

In some aspects, the invention provides a monoclonal antibody or an antigen-binding portion thereof that specifically binds the S protein of at least one Coronavirus, wherein the antibody comprises amino acid sequences of VH and VL from an antibody selected from: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

In some aspects, the invention provides a monoclonal antibody that is capable of neutralizing the biological activity of at least one Coronavirus, wherein said antibody comprises a heavy chain of an antibody selected from the group consisting of: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13. In some aspects, the invention provides a monoclonal antibody that is capable of neutralizing the biological activity of at least one Coronavirus, wherein said antibody comprises a light chain of an antibody selected from the group consisting of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13. In some aspects, the invention provides a monoclonal antibody that is capable of neutralizing the biological activity of at least one Coronavirus, wherein said antibody comprises a heavy chain and a light chain of the same antibody that is selected from the group consisting of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

In some aspects, the invention provides a monoclonal antibody or an antigen-binding portion thereof that is capable of neutralizing the biological activity of at least one Coronavirus comprising VL and VH domains that are at least 85%, 90%, 95%, 97%, 98%, or 99% identical in amino acid sequence to the VL and VH domains, respectively, of a monoclonal antibody selected from the group consisting of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

In some aspects, the invention provides a monoclonal antibody or an antigen-binding portion thereof that is capable of neutralizing the biological activity of at least one Coronavirus comprising light chain and heavy chain that are at least 85%, 90%, 95%, 97%, 98%, or 99% identical in amino acid sequence to the light chain and heavy chain, respectively, of a monoclonal antibody selected from the group consisting of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

Preferably, a monoclonal antibody according to any of the embodiments described in this specification and the claims is a human antibody or a human monoclonal antibody.

According to a preferred aspect, a monoclonal antibody or an antigen-binding portion thereof according to any of the embodiments described in this specification and the claims is capable of neutralizing biological activity or exhibits neutralizing activity against at least one Coronavirus selected from SARS-CoV-1, SARS-CoV-2, OC43, HKU1, 229E, and NL63, and variants thereof, more preferably at least one Coronavirus selected from SARS-CoV-1, SARS-CoV-2, and variants thereof.

In another preferred aspect, a monoclonal antibody or an antigen-binding portion thereof according to any of the embodiments described in this specification and the claims is capable of neutralizing biological activity or exhibits neutralizing activity against at least two, at least three, or at least four species belonging to the Coronaviridae family, in particular at least SARS-CoV-2 and SARS-CoV-1.

In certain aspects, a monoclonal antibody or an antigen-binding portion thereof according to any of the embodiments described in this disclosure and the claims specifically binds to a region of the Spike (S) protein of said at least one Coronavirus, in particular the S protein of SARS-CoV-2 and/or SARS-CoV-1 and at least partially inhibits the binding of the S protein to a receptor.

In one embodiment said region is (i) in the S1 domain of said S protein; or (ii) in the S2 domain of said S protein; or (iii) in the S protein trimer of said at least one Coronavirus in its pre-fusion conformation or in its post-fusion conformation or in a combination of (i) with (ii) or in a combination of (i) with (iii) or in a combination of (ii) with (iii) or a combination of (i) with (iv) or a combination of (ii) with (iv).

Preferably, said region is the S protein trimer of SARS-CoV-2 and/or SARS-CoV-1 and/or any variants thereof. In another aspect, said region is the NTD domain of the S protein of SARS-CoV-2 and/or SARS-CoV-1 and/or any of their variants.

In one embodiment, the invention provides a monoclonal antibody or antigen-binding moiety that specifically binds to at least one Coronavirus, wherein said antibody or antigen-binding moiety thereof elicits an amount equal to or greater than 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein of said at least one Coronavirus as measured by a neutralization binding (NOB) assay.

In certain aspects, a monoclonal antibody or an antigen-binding portion thereof according to any of the embodiments described herein that specifically binds to the S protein of at least one coronavirus, wherein said antibody or antigen-binding portion thereof has neutralizing activity. In particular, such monoclonal antibody or antigen-binding portion thereof exhibits a 100% inhibitory concentration (IC100) of less than 100 ng/ml, preferably less than 50, 25, 20, 10, 8, 6, 5, 4, 3, 2, or 1 ng/ml, when tested in an in vitro neutralization assay against said at least one coronavirus, for example, against at least one virus selected from SARS-CoV-1, SARS-CoV-2, and their variants, at a 100 TCID50 viral dose.

One type of amino acid substitution that can be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, but not limited to, alanine or serine. In one embodiment, there is a substitution of a non-canonical cysteine. The substitution can be made in a CDR region or framework region of a variable domain or in the constant domain of an antibody. In some embodiments, the cysteine is canonical. Another type of amino acid substitution that can be made is to change any potential proteolytic site in the antibody. Such sites can occur in a CDR region or framework region of a variable domain or in the constant domain of an antibody. Substituting cysteine residues and removing proteolytic sites can reduce the risk of heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution is to change any potential proteolytic site in the antibody. Such sites can occur in a CDR region or framework region of a variable domain or in the constant domain of an antibody. Substituting cysteine residues and removing proteolytic sites can reduce the risk of heterogeneity in the antibody product and thus increase its homogeneity. Elimination of asparagine-glycine pairs, which form potential deamidation sites, by altering one or both residues. In some embodiments, the C-terminal lysine of the heavy chain of the anti-Coronavirus S protein antibody of the invention is cleaved. In various embodiments of the invention, the heavy and light chains of the anti-Coronavirus S protein antibodies may optionally include a signal sequence.

The class and subclass of antibodies according to the present invention can be determined by any method known in the art. In general, the class and subclass of an antibody can be determined using antibodies specific for a particular class and subclass of antibodies. Such antibodies are commercially available. The class and subclass can be determined by ELISA, Western blot (immunoblot), and other techniques. Alternatively, the class and subclass can be determined by sequencing all or part of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences with known amino acid sequences of various immunoglobulin classes and subclasses, and determining the class and subclass of the antibodies.

In some embodiments, the antibody according to any of the embodiments described herein is an IgG, IgM, IgE, IgA, or IgD molecule. In one embodiment, the antibody is an IgG and is a subclass of IgG1, IgG2, IgG3, IgG4. In yet another embodiment, the human antibody subclass is IgG1.

In some embodiments of the invention, the antibodies according to any of the embodiments described herein bind to the S protein of said at least one coronavirus, and in particular at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants, with high affinity. In some embodiments, the antibodies according to any of the embodiments described herein bind with high affinity to the protein trimer of the S protein of SARS-CoV-2 and/or SARS-CoV-1. In some embodiments, the antibodies bind to the N-terminal domain or receptor-binding domain (RBD) of the S protein of SARS-CoV-2 and/or SARS-CoV-1. In another embodiment, the antibody binds to the S1 or S2 domains of the S protein of SARS-CoV-2 and/or SARS-CoV-1. The affinity The binding affinity and dissociation rate of an anti-SARS-CoV-2 S protein antibody with SARS-CoV-2 S protein can be determined by methods known in the art. The binding affinity can be measured by ELISA, RIA, flow cytometry, surface plasmon resonance, such as BIACORE (TM). The dissociation rate can be measured by surface plasmon resonance. Preferably, the binding affinity and dissociation rate are measured by surface plasmon resonance. More preferably, the binding affinity and dissociation rate are measured using BIACORE (TM). Whether an antibody has substantially the same KD as an anti-Coronavirus S protein antibody can be determined using methods known in the art.

The invention further provides a monoclonal antibody that binds to the S protein of at least one Coronavirus, in particular at least one virus selected from SARS-CoV-1, SARS-CoV-2 and their variants, and competes or cross-competes with and/or binds the same epitope of an antibody selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13. If two antibodies compete with each other to bind to said S protein of said at least one Coronavirus, they are said to cross-compete.

Whether an antibody binds to the same epitope or cross-competes for binding with an anti-protein S antibody of the present invention can be determined using methods known in the art. In one embodiment, the antibody of the invention is allowed to bind to the S protein of said at least one coronavirus under saturating conditions and then the ability of the test antibody to bind to the S protein is measured. If the test antibody is able to bind to the S protein simultaneously with the antibody of the present invention, the test antibody binds to a different epitope than the antibody of the present invention. However, if the test antibody is unable to bind simultaneously with the S protein of said at least one coronavirus, the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the antibody of the present invention, or the binding of the antibody of the present invention may be impaired. induce a conformational change in the S protein of said at least one coronavirus that prevents or reduces binding of the test antibody. This experiment can be performed using ELISA, RIA, BIACORE (TM), flow cytometry, or other methods known in the art. To verify whether an antibody according to the present invention competes with another antibody capable of binding the S protein of said at least one coronavirus, the competition method described above can be used in two directions, i.e., determining whether the reference antibody blocks the test antibody and vice versa. In one embodiment, the experiment is performed using ELISA. Methods for determining the KD are discussed further below.

In another embodiment, the invention provides an antibody that inhibits, blocks, or reduces the binding of the S protein of said coronavirus to a receptor, specifically angiotensin-converting enzyme 2 (ACE2). In another embodiment, the invention provides an antibody that inhibits, blocks, or decreases viral entry mediated by the S protein of the coronavirus into cells. In another embodiment, the invention provides an antibody against the S protein of SARS-CoV-2 and/or against SARS-CoV-1 or their variants that inhibits, blocks, or reduces the fusion of viral and cellular membranes. In another embodiment, the invention provides an antibody that reduces viral load. In another embodiment, the invention provides an antibody that inhibits, blocks, or decreases the severity, for any period of time, of symptoms or conditions resulting from infection with SARS-CoV-2 and/or SARS-CoV-1. In some embodiments, the invention provides an antibody that inhibits, blocks, or decreases the severity of symptoms or conditions resulting from SARS-CoV-2 and/or SARS-CoV-1 infection by 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% for one day, one week, one month, 6 months, one year, or for the rest of the subjects' lives. In some embodiments, the invention provides an antibody that can perform any combination of the preceding embodiments.

In some embodiments, the mAb constant region of the antibodies is modified to extend their half-life and reduce the risk of antibody-dependent enhancement (ADE) of the disease. For example, two different and alternative sets of mutations in their constant domains can be applied to improve the therapeutic activity of mAbs (M252Y/S254T/T256E according to Dall'Acqua et al., 2006; M428L/N434S as reported by Zalevsky et al., 2010).

In some embodiments, in order to reduce the risk of antibody-dependent enhancement (ADE) of disease, mutations that eliminate binding to Fc receptors will be introduced in the Fc portion of the IgG1 molecule as previously described (L234A/L235A as in

, 2010; P329G LALA as in ). All of these modifications can be performed by site-directed mutagenesis, for example using the Agilent Quick-Change II Site-Directed Mutagenesis Kit, according to the manufacturer's recommendations. In some embodiments, the antibody comprises the variable regions of an antibody selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 and a mutant IgG1 constant region backbone, which contains one or more of the following sets of mutations: L234A/L235A (as in , 2001; , 2010), P329G (as in 2016); M428L/N434S (as in 2010). Preferably the antibody comprises the variable regions of an antibody selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 and a constant region backbone of mutant IgG1, which contains all three sets of such mutations.

Nucleic acids, vectors, host cells, and recombinant methods for antibody production Nucleic acids

The present invention also includes nucleic acid molecules encoding antibodies or antigen-binding portions thereof according to any of the embodiments described herein. In some embodiments, different nucleic acid molecules encode a heavy chain and a light chain of an antibody according to the present invention. In other embodiments, the same nucleic acid molecule encodes a heavy chain and a light chain of an antibody according to the present invention. In one embodiment, the nucleic acid encodes an antibody, or an antigen-binding portion thereof, of the invention. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a VK amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions and/or 1, 2, or 3 non-conservative substitutions with respect to the germline. The substitutions may be located in the CDR regions, framework regions, or the constant domain. In some embodiments, the nucleic acid molecule encodes a VK amino acid sequence comprising one or more variants from the germline sequence that are identical to variations found in the VK of one of the antibodies selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

In some embodiments, the nucleic acid molecule encodes at least three amino acid substitutions from the germline sequence found in the VK of one of the antibodies selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the VK amino acid sequence of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 or a variant or portion thereof. In some embodiments, the nucleic acid encodes an amino acid sequence comprising the light chain CDRs of one of said antibodies listed above. In some embodiments, said portion is a contiguous portion comprising CDR1-CDR3. In some embodiments, the nucleic acid encodes the amino acid sequence of the light chain CDRs of said antibody. In some embodiments, said portion encodes a contiguous region from CDR1-CDR3 of the light chain of an antibody according to the present invention.

In some embodiments, the nucleic acid molecule encodes a VK amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to a VK amino acid sequence of a VK region of any of the antibodies 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13. The nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions, such as those described above, to a nucleotide sequence encoding the amino acid sequence of a VK region. In another embodiment, the nucleic acid encodes a full-length light chain of a selected antibody 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 or a light chain comprising a mutation, such as that described herein.

In yet another embodiment, the nucleic acid molecule encodes the variable domain of the heavy chain (VH) comprising a human gene sequence of the VH1, VH3, or VH4 family or a sequence derived therefrom. In some embodiments, the nucleic acid molecule encodes one or more amino acid mutations from the germline sequence that are identical to amino acid mutations present in the VH of one of the monoclonal antibodies 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding at least a portion of the VH amino acid sequence of a monoclonal antibody selected from monoclonal antibodies 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13, all three CDR regions, a contiguous portion including CDR1-CDR3, or the entire VH region, with or without a signal sequence. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence of one of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 or said sequence without the signal sequence. In some preferred embodiments, the nucleic acid molecule comprises at least a portion of the nucleotide sequence of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 or said sequence without the signal sequence. In some embodiments, said portion encodes the VH region (with or without a signal sequence), a CDR3 region, all three CDR regions, or a contiguous region between CDR1-CDR3.

In some embodiments, the nucleic acid molecule encodes a VH amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the VH amino acid sequences of any of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

The nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions, such as those described above, to a nucleotide sequence encoding the amino acid sequence of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 or encoding a VH region thereof.

In another embodiment, the nucleic acid encodes a full-length heavy chain of an antibody selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 or a heavy chain having the amino acid sequence of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 with or without a signal sequence or a heavy chain comprising a mutation, such as one of the variants discussed herein. Furthermore, the nucleic acid may comprising the nucleotide sequence of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 with or without a signal sequence, or a nucleic acid molecule encoding a heavy chain comprising a mutation, such as one of the variants discussed herein.

A nucleic acid molecule encoding the heavy or light chain of an antibody or portions thereof according to the invention can be isolated from any source that produces such an antibody. In various embodiments, the nucleic acid molecules are isolated from a B cell isolated from an animal immunized with the S protein of at least one coronavirus, such as SARS-CoV-2 and/or SARS-CoV-1 or their variants, or from an immortalized cell derived from such a B cell that expresses or encodes an antibody to the S protein of SARS-CoV-2 and/or SARS-CoV-1. Methods for isolating mRNA encoding an antibody are well known in the art. See, for example, Sambrook et al. The mRNA can be used to produce cDNA for use in polymerase chain reaction (PCR) or in cDNA cloning of antibody genes. In one embodiment, the nucleic acid molecule is isolated from a B cell isolated from an animal immunized with the S protein of at least one coronavirus, such as SARS-CoV-2 and/or SARS-CoV-1 or their variants. The nucleic acid is isolated from a hybridoma having as its fusion partner a human immunoglobulin-producing cell derived from a non-human transgenic animal. In a more preferred embodiment, the human immunoglobulin-producing cell is isolated from a XENOMOUSE animal. In another embodiment, the human immunoglobulin-producing cell comes from a non-human, non-murine transgenic animal, as described above. In another embodiment, the nucleic acid is isolated from a non-human, non-transgenic animal. Nucleic acid molecules isolated from a non-human, non-transgenic animal may be used, for example, for humanized antibodies. In some embodiments, a nucleic acid encoding a heavy chain of an antibody of the invention may comprise a nucleotide sequence encoding a VH domain of the invention joined in-frame to a nucleotide sequence encoding a heavy chain constant domain from any source. Similarly, a nucleic acid molecule encoding a light chain of an antibody of the invention may comprise a nucleotide sequence encoding a VK domain of the invention joined in-frame to a nucleotide sequence encoding a constant domain light chain from any source. In a further aspect of the invention, nucleic acid molecules encoding the variable domain of the heavy (VH) and/or light (VL or VK) chains are "converted" into full-length antibody genes. In one embodiment, nucleic acid molecules encoding the VH or VL or VK domains are converted into full-length antibody genes by insertion into an expression vector that already encodes the heavy (CH) or light (CL) constant domains, such that the VH segment is operably linked to the CH segment(s) within the vector and/or the VL or VK segment is operably linked to the CL segment within the vector. In another embodiment, nucleic acid molecules encoding the VH and/or VL or VK domains are converted into full-length antibody genes by, for example, linking a nucleic acid molecule encoding a VH and/or VL or VK domain to a nucleic acid molecule encoding a CH and/or CL domain using standard molecular biological techniques. Nucleotide sequences of human immunoglobulin heavy and light chain constant domain genes are known in the art. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publ. 91-3242, 1991. Nucleic acid molecules encoding the full-length heavy and/or light chains can then be expressed from a cell into which they have been introduced and the antibody isolated.

The nucleic acid molecules can be used to recombinantly express large quantities of the antibodies of the invention. The nucleic acid molecules can also be used to produce chimeric antibodies, bispecific antibodies, single-chain antibodies, immunoadhesins, diabodies, mutated antibodies, and antibody derivatives, as described below. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules can be used to humanize the antibody, also as described below.

In another embodiment, a nucleic acid molecule of the invention is used as a probe or PCR primer for a specific antibody sequence. For example, the nucleic acid can be used as a probe in diagnostic methods or as a PCR primer to amplify DNA regions that could be used, among other things, to isolate additional nucleic acid molecules encoding variable domains of antibodies. In some embodiments, the nucleic acid molecules are oligonucleotides. In some embodiments, the oligonucleotides are derived from highly variable domains of the heavy and light chains of the antibody of interest. In some embodiments, the oligonucleotides encode, in whole or in part, one or more of the following: of the CDRs of antibodies 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 or their variants as described herein.

Vectors

The invention provides vectors comprising nucleic acid molecules encoding the heavy chain of an antibody of the invention or an antigen-binding portion thereof. The invention also provides vectors comprising nucleic acid molecules encoding the light chain of such antibodies or an antigen-binding portion thereof. The invention further provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof. In some embodiments, the antibodies or antigen-binding portions of the invention are expressed by inserting DNA encoding partial or full-length light and heavy chains, obtained as described above, into expression vectors such that the genes are operably linked to the necessary expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The antibody gene is ligated into a vector such that the transcriptional and translational control sequences within the vector serve the intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the host cell being used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In one embodiment, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector using standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). A convenient vector is one that encodes a functionally complete human immunoglobulin CH or CL sequence, with appropriate restriction sites designed such that any VH, VL, or VK sequence can be easily inserted and expressed, as described above. In such vectors, splicing typically occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C domain, as well as in splice regions occurring within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein). In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. Those skilled in the art will understand that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of host cell to be transformed, the desired protein expression level, etc. Preferred regulatory sequences for expression in mammalian host cells include viral elements that drive high levels of protein expression in mammalian cells, such as retroviral LTR-derived promoters and/or enhancers, cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma, and potent mammalian promoters such as native immunoglobulin and actin promoters. For a further description of viral regulatory elements and their sequences, see, for example, U.S. Patent No. 5,111,511.

No. 5,168,062, U.S. Patent No. 4,510,245, and U.S. Patent No. 4,968,615. Methods for expressing antibodies in plants are known in the art, including a description of promoters and vectors, as well as plant transformation. See, for example, U.S. Patent No. 6,517,529, incorporated herein by reference. Methods for expressing polypeptides in bacterial or fungal cells, e.g., yeast cells, are also well known in the art. In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may feature additional sequences, such as sequences that regulate vector replication in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells in which it is expressed. The vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017, incorporated herein by reference). For example, the selectable marker gene typically confers resistance to drugs, such as G418, hygromycin, or methotrexate, in a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification), the neo gene (for G418 selection), and the glutamate synthase gene.

Non-hybridoma host cells and methods of recombinant protein production

The nucleic acid molecules encoding antibodies and vectors according to the present invention comprising these nucleic acid molecules can be used for transfection or transformation of a suitable mammalian, plant, bacterial, or yeast host cell. Transfection or transformation can be accomplished by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nuclei. Additionally, nucleic acid molecules can be introduced into mammalian cells using viral vectors. Methods for transforming cells are well known in the art (see, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporated herein by reference). Methods for transforming plant cells are well known in the art, including, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art. Mammalian cell lines available as expression hosts are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, but are not limited to, Chinese hamster ovary (CHO) cells, N50 cells, SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and various other cell lines. Preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potatoes, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris. Furthermore, expression of antibodies of the invention by production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthase gene expression system (the GS system) is a common approach to enhance expression under certain conditions. The GS system is discussed in whole or in part in relation to European patents Nos. 0 216846, 0256 055, 0323997, and 0338 841. Antibodies expressed by different cell lines or in transgenic animals are likely to have different glycosylation patterns. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies.

Transgenic animals and plants

The monoclonal antibodies of the invention can also be produced transgenically by generating a mammal or plant transgenic for the immunoglobulin heavy and light chain sequences of interest and producing the antibody in a form recoverable from them. In connection with transgenic production in mammals, the antibodies of the invention can be produced and recovered in the milk of goats, cows, or other mammals. See, for example, U.S. Patent Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957, incorporated herein by reference. In some embodiments, transgenic non-human animals comprising human immunoglobulin loci are immunized with the S protein of at least one coronavirus, such as at least one coronavirus according to any of the variants described herein, or an immunogenic portion thereof, as described above. Methods for producing antibodies in plants are described, for example, in U.S. Patents 6,046,037 and 5,959,177, which are incorporated herein by reference.

In some embodiments, transgenic non-human animals or plants are produced by introducing one or more nucleic acid molecules encoding an antibody of the invention into the animal or plant by standard transgenic techniques. See Hogan and U.S. Pat. No. 6,417,429, supra. The transgenic cells used to produce the transgenic animal may be embryonic stem cells or somatic cells or a fertilized egg. The transgenic non-human organisms may be heterozygous chimeric, non-chimeric, and homozygous nonchimeric. See, for example, Hofian et al. Manipulation of the Mouse Embryo: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999), all incorporated herein by reference. In some embodiments, the transgenic non-human animals feature a targeted disruption and replacement with a target construct encoding a heavy chain and/or light chain of interest. In one embodiment, the transgenic animals comprise and express nucleic acid molecules encoding heavy and light chains that bind specifically to the S protein of said at least one coronavirus, and preferably to (i) the S1 domain of the S protein of SARS-CoV-2 and/or SARS-CoV-1; (ii) the S2 domain of the S protein of SARS-CoV-2 and/or SARS-CoV-1; or (iii) both (i) and (ii). In one embodiment, the transgenic animals comprise and express nucleic acid molecules encoding heavy and light chains that bind specifically to the human S protein of SARS-CoV-2 and/or SARS-CoV-1 or variants thereof. In some embodiments, the transgenic animals comprise nucleic acid molecules encoding a modified antibody, such as a single-chain antibody, a chimeric antibody, or a humanized antibody. The antibodies of the invention can be produced in any transgenic animal. In one embodiment, the nonhuman animals are mice, rats, sheep, pigs, goats, cattle, or horses. The nonhuman transgenic animal expresses said encoded polypeptides in blood, milk, urine, saliva, tears, mucus, and other body fluids. Class Change

Another aspect of the invention provides a method for converting the class or subclass of an antibody of the invention into another class or subclass. In some embodiments, a nucleic acid molecule encoding a VL or VK or VH that does not include sequences encoding CL or CH is isolated using methods well known in the art. The nucleic acid molecule is then operably linked to a nucleotide sequence encoding a CL or CH from a desired immunoglobulin class or subclass. This can be achieved using a vector or a nucleic acid molecule comprising a CL or CH chain, as described above. For example, the class of an anti-SARS-CoV-2 S protein antibody, originally IgM, can be converted to IgG. Furthermore, class conversion can be used to convert one IgG subclass to another, for example, from IgG1 to IgG2. Another method for producing an antibody of the invention comprising a desired isotype includes the steps of isolating a nucleic acid encoding a heavy chain of an antibody of the invention and a nucleic acid encoding a light chain of an antibody of the invention, isolating the sequence encoding the VH region, ligating the VH sequence to a sequence encoding a constant domain of the heavy chain of the desired isotype, expressing the light chain gene and the heavy chain construct in a cell, and harvesting the antibody with the desired isotype.

Modified antibodies

In another embodiment, a fusion antibody or immunoadhesive may be made that comprises all or a portion of an antibody of the invention linked to another polypeptide. In one embodiment, only the variable domains of the antibody according to any of the embodiments described herein are bound to the polypeptide. In yet another embodiment, the VH domain of an antibody of the invention is linked to a first polypeptide, while the VK domain of an antibody of the invention is linked to a second polypeptide that associates with the first polypeptide such that the VH and VK domains can interact with each other to form an antigen-binding site. In yet another embodiment, the VH domain is separated from the VK domain by a linker such that the VH and VL domains can interact with each other (see below under single-chain antibodies). The VH-linker-VK antibody is then bound to the polypeptide of interest. The fusion antibody is This method is useful for targeting a polypeptide to a cell or tissue expressing the coronavirus S protein, such as a cell or tissue expressing the SARS-CoV-2 and/or SARS-CoV-1 S protein. The polypeptide can be a therapeutic agent, such as a toxin, chemokine, or other regulatory protein, or it can be a diagnostic agent, such as an easily visualized enzyme, such as horseradish peroxidase. Fusion antibodies can also be created in which two (or more) single-chain antibodies are linked together. This is useful if you want to create a bivalent or polyvalent antibody on a single polypeptide chain or if you want to create a bispecific antibody or nanobody. To create a single-chain antibody (scFv), the DNA fragments encoding VH and VK are operably linked to another fragment encoding a flexible linker, for example encoding the amino acid sequence (GIy4 -Ser)3, so that the VH and VK sequences can be expressed as a contiguous single-chain protein, with the VK and VH domains joined by the flexible linker. See, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. ScL USA 85:5879-5883 (1988); McCafferty et al., Nature 348:552-554 (1990). The single-chain antibody can Antibodies can be monovalent, if only a single VH and VK are used, bivalent, if two VH and VK are used, or polyvalent, if more than two VH and VK are used. Bispecific or polyvalent antibodies can be generated that bind specifically to the S protein of SARS-CoV-2 and/or SARS-CoV-1 or their variants and to another molecule. Bispecific antibodies or antigen-binding fragments can be produced by a variety of methods including hybridoma fusion or Fab fragment linkage. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al, J. Immunol. 148: 1547-1553 (1992). Additionally, bispecific antibodies can be formed as "diabodies" or "Janusin". In some embodiments, the bispecific antibody binds to two different epitopes of the S protein of SARS-CoV-2 and/or SARS-CoV-1. In some embodiments, the bispecific antibody has a first heavy chain and a first light chain from monoclonal antibodies 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13, and a heavy chain and light chain from additional antibodies. In some embodiments, the additional light chain and heavy chain are also from one of the monoclonal antibodies identified above, but are different from the first heavy and light chains. In some embodiments, the modified antibodies described above are prepared using one or more variable domains or CDR regions from a monoclonal antibody provided herein.

Derivatized and labeled antibodies

An antibody or an antigen-binding portion thereof of the invention may be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, antibodies or portions thereof are derivatized such that the neutralizing activity against said at least one coronavirus is not adversely affected by the derivatization or labeling. Accordingly, the antibody portions and antibodies of the invention are intended to comprise both intact and modified forms of the antibodies described herein. For example, an antibody or an antibody portion of the invention may be functionally linked (by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detection agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can be used to target the antibody. mediate the association of the antibody or antibody moiety with another molecule (such as a streptavidin core region or a polyhistidine tag). One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or different types, e.g., to create bispecific antibodies). Suitable crosslinking agents include those that are heterobifunctional, with two distinctly reactive groups separated by an appropriate spacer (e.g., mmaleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from , Rockford, II. [0179]. Another type of derivatized antibody is a labeled antibody. Useful detection agents with which an antibody or antigen-binding moiety of the invention can be derivatized include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, phycoerythrin, 5-dimethylaminenapthalenesulfonyl chloride, lanthanide phosphors, and the like. An antibody may also be labeled with enzymes that are useful for detection, such as horseradish peroxidase, [beta]-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When an antibody is labeled with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product, which is detectable. An antibody may also be labeled with enzymes that are useful for detection, such as horseradish peroxidase, [beta]-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. An antibody can also be labeled with biotin and detected by indirect measurement of avidin or streptavidin binding. An antibody can also be tagged with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags). In some embodiments, the markers are attached by spacers of various lengths to reduce potential steric hindrance. An antibody can also be labeled with a radiolabeled amino acid. The radiolabel can be used for diagnostic and therapeutic purposes. For example, the radiolabel can be used to detect cells or tumors expressing SARS-CoV-2 and/or SARS-CoV-1 S proteins using radiography or other diagnostic techniques. Additionally, the radiolabel can be used therapeutically as a toxin for cancer cells or tumors. In some embodiments, the antibody can be used to detect SARS-CoV-2 and/or SARS-CoV-1 S proteins using radiography or other diagnostic techniques. be labeled with a paramagnetic, radioactive, or florigenic ion that is detectable during imaging. In some embodiments, the paramagnetic ion is chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), or erbium(III). In other embodiments, the radioactive ion is iodine-123, technetium-99, indium-111, rhenium-188, rhenium-186, copper-67, iodine-131, yttrium-90, iodine-125, astatine-211, and gallium-67. In other embodiments, the antibody of the invention is labeled with an X-ray imaging agent such as lanthanum(III), gold(III) lead(II), and bismuth(III).

Compositions and kits

The invention relates to compositions comprising any of the antibodies or their antigen-binding portions of the invention and one or more pharmaceutically acceptable excipients and/or carriers. In some embodiments, the composition may comprise antibodies or a binding portion thereof according to any of the preceding embodiments. In some embodiments, the subject of treatment is a human. In other embodiments, the subject is a veterinary subject. In some embodiments, an anti-SARS-CoV-2 and/or SARS-CoV-1 antagonist antibody that binds to the S1 domain and one that binds to the S2 domain or antigen-binding portions of one or both are both administered to a subject, together or separately. In some embodiments, the antibodies are in a composition comprising a pharmaceutically acceptable carrier. In another embodiment, one or more of the antagonist antibodies of the invention are administered in combination with one or more excipients and/or carriers. Additional antagonistic antibodies that bind different epitopes on the S protein, that bind the S protein of at least one coronavirus from different isolates of SARS-CoV-2 and/or SARS-CoV-1, and/or that bind different stages of SARS-CoV-2 and/or SARS-CoV-1 or their variants (i.e., early, mid, or late-stage viruses). As used herein, a "pharmaceutically acceptable carrier" means any solvent, dispersion medium, coating, antibacterial and antifungal agents, absorption retarders, and isotonic and similar agents that are physiologically compatible. Examples of pharmaceutically acceptable carriers include water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and similar agents, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride. Further examples of pharmaceutically acceptable substances are wetting agents or minor amounts of excipients such as wetting or emulsifying agents, preservatives, or buffers, which increase the shelf life or efficacy of the antibody. The compositions of the present invention may be in a variety of forms, such as liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended route of administration and the therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization in humans. The preferred route of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In one embodiment, the antibody is administered by infusion or intravenous injection. In yet another embodiment, the antibody is administered by intramuscular or subcutaneous injection. The therapeutic compositions are typically sterile and stable under manufacturing and storage conditions. The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for a high drug concentration. Sterile injectable solutions may be prepared by incorporating the antibody according to any of the embodiments of the invention in the required amount in an appropriate solvent with one or a combination of the ingredients listed above, as required, followed by filter sterilization. Dispersions are generally prepared by incorporating the active compound into a sterile carrier containing a base dispersion medium and the other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, which produce a powder of the active ingredient plus any other desired ingredients from a previously filter-sterilized solution. The appropriate fluidity of a solution may vary. The absorption of injectable compositions can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size when dispersed, and by the use of surfactants. Prolonged absorption of injectable compositions can be achieved by including an absorption-retarding agent in the composition, such as monostearate salts and gelatin. The antibodies of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous, intramuscular, or intravenous infusion. As understood by one skilled in the art, the route and/or mode of administration will vary based on the desired results. Other modes of administration include intraperitoneal, intrabronchial, transmucosal, intraspinal, intrasnovial, intraaortic, intranasal, ocular, otic, topical, and vestibular. In some embodiments, the active compound of the antibody compositions may be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous, intramuscular, or intravenous infusion. A controlled-release formulation can be prepared with a carrier that will protect the antibody from rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyesters, and polylactic acid, can be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, sustained-release and controlled-release drug delivery systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). The invention also provides compositions suitable for inhalation administration, comprising one or more of the antibodies described herein. Any of the antibodies of the invention may be conveniently delivered to a subject in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges, e.g., gelatin for use in an inhaler or insufflator, may be formulated as containing a powdered mixture of the compound and a suitable powdered base such as lactose or starch. Dellamary et al. (2004) J Control Release.; 95(3):489-500 discloses formulations for the pulmonary delivery of antibodies. The invention also provides compositions, suitable for administration through the oral mucosa, that comprise one or more of any of the antibodies described herein. Oral transmucosal delivery refers to the transport of a carrier vehicle across a mucosal membrane into the oral cavity, pharyngeal cavity, or esophagus and may be contrasted, for example, with traditional oral delivery, in which drug absorption occurs in the intestine. Accordingly, routes of administration in which antibodies are absorbed through the buccal, sublingual, gingival, pharyngeal, and/or esophageal mucosa are all included in "oral transmucosal delivery," as that term is used therein. For transmucosal mucosal administration, any antibody of the invention may be formulated, for example, as chewing gum (see U.S. Patent No. 5,711,961) or buccal patches (see, e.g., U.S. Patent No. 5,298,256). The invention also provides compositions suitable for administration through the vaginal mucosa, which comprise one or more antibody-containing compounds. of any of the antibodies described herein. The antibodies of the invention may be formulated as a vaginal suppository, foam, cream, tablet, capsule, ointment, or gel. In some embodiments, the compositions comprising the antibodies are formulated with permeants appropriate for the transmucosal barrier to be permeated. Such permeants are generally known in the art and include, for example, bile salts for transmucosal administration and fusidic acid derivatives. In some embodiments, an antibody of the invention may be administered orally, for example, with an inert diluent or an available edible vehicle. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the antibodies may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention other than parenterally, it may be necessary to coat the compound or co-administer it with a material to prevent its inactivation. Additional active compounds may also be incorporated into the compositions. In some embodiments, an inhibitory antibody of the invention is co-formulated with and/or co-administered with one or more additional therapeutic agents, particularly antiviral agents. These therapeutic agents include, but are not limited to, antibodies that bind to other targets, photosensitizers, androgens, estrogens, nonsteroidal anti-inflammatory agents, antihypertensive agents, analgesics, antidepressants, antibiotics, anticancer agents, anesthetics, antiemetics, anti-infectives, contraceptives, antidiabetic agents, steroids, anti-allergic agents, chemotherapeutic agents, anti-migraine agents, smoking cessation agents, antiviral agents, immunosuppressants, thrombolytic agents, lipid-lowering agents, and anti-obesity agents. Therapeutic agents also include peptide analogues that inhibit the activity of these agents. of the S protein of at least one coronavirus such as SARS-CoV-1 and/or SARS-CoV-2 or their variants, antibodies or other molecules that prevent the entry of said coronavirus into a cell, including but not limited to preventing the binding of the S proteins to a receptor such as the ACE2 receptor, and agents that inhibit the expression of the S protein of said coronavirus. In one embodiment, additional agents that inhibit the expression of the S protein of said coronavirus include an antisense nucleic acid capable of hybridizing to an mRNA of the S protein of said coronavirus, such as a hairpin RNA or siRNA, locked nucleic acids (LNAs), or ribozymes. Sequence-specific nucleic acids capable of inhibiting gene function through RNA interference are well known in the art. Such combination therapies may require lower doses of the neutralizing antibody and the co-administered agents, thus avoiding possible toxicities or complications associated with the various monotherapies. In certain specific embodiments, the therapeutic agent(s) co-formulated with and/or co-administered with an anti-SARS-CoV-2 S protein inhibitory antibody of the invention is an antimicrobial agent. Antimicrobial agents include antibiotics (e.g., antibacterials), antiviral agents, antifungal agents, and antiprotozoal agents. Non-limiting examples of antimicrobial agents are sulfonamides, trimethoprim-sulfamethoxazole, quinolones, penicillins, and cephalosporins. Compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding moiety of the invention. A "therapeutically effective amount" refers to an effective amount, at the dosages, and for the periods of time necessary, to achieve the desired therapeutic outcome. A "therapeutically effective amount" refers to an effective amount, at the dosages, and for the periods of time necessary, to achieve the desired therapeutic outcome. A "therapeutically effective amount" refers to an effective amount, at the dosages, and for the periods of time necessary, to achieve the desired therapeutic outcome. The therapeutically effective amount of the antibody or antibody portion may vary depending on factors such as disease status, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody or its antibody portion are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an effective amount, at the dosages and for the periods of time necessary, to achieve the desired prophylactic outcome. Typically, because a prophylactic dose is used in individuals before or at an early stage of the disease, the prophylactic effective amount may be lower than the therapeutically effective amount.

Dosing regimens may be adjusted to provide the desired optimal response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be reduced or increased proportionally as indicated by the needs of the therapeutic situation.

It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosing. The unit dosage form used herein refers to physically discrete units suitable as unit dosages for the mammalian subjects to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specifications for the unit dosage forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the antibody or a portion thereof and the particular therapeutic or prophylactic effect to be achieved, and (b) the inherent limitations of the art of compounding such an antibody for the treatment of sensitivity in individuals.

An exemplary and non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody moiety of the invention is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg, more preferably 0.1 to 25, 0.1 to 10, or 0.1 to 3 mg/kg. In some embodiments, a formulation contains 5 mg/mL of antibody in a buffer of 20 mM sodium citrate, pH 5.5, 140 mM NaCl, and 80 mg/ml polysorbate 80. It should be noted that dosage values may vary with the type and severity of the condition being alleviated. It is further understood that for any specific subject, specific dosing regimens should be adjusted over time based on the individual needs and professional judgment of the person administering or supervising the administration of the compositions, and that the dosing ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Another aspect of the present invention provides kits comprising an antibody, or antigen-binding portion, of the invention or a composition comprising such an antibody or antigen-binding fragment. A kit may include, in addition to the antibody or composition, diagnostic or therapeutic agents. A kit may also include instructions for use in a diagnostic or therapeutic method, as well as packaging materials such as, but not limited to, ice, dry ice, Styrofoam, foam, plastic, cellophane, shrink wrap, bubble wrap, cardboard, and peanut starch. In one embodiment, the kit includes the antibody or a composition comprising it and a diagnostic agent that can be used in a method described below. In yet another embodiment, the kit includes the antibody or a composition comprising it and one or more therapeutic agents that may be used in a method described below.

In one embodiment, the antibodies or their binding portion or a composition comprising such antibodies according to any of the embodiments described herein are for use in the prevention or treatment of patients infected with Coronavirus, particularly infected with SARS-CoV-2 and/or SARS-CoV-1 or their variants. The use of such antibodies and compositions of said Abs includes, but is not limited to, passive immunization in persons at risk of contracting the infection (e.g., occupationally exposed personnel, persons living in endemic areas) and treatment of acute cases, whether hospitalized or not. The invention also relates to compositions for inhibiting viral infection, and in particular Coronavirus infection, more particularly infection with SARS-CoV-2 and/or SARS-CoV-1 or their variants in a mammal comprising a quantity of an antibody of the invention in combination with a quantity of an antiviral agent, wherein the quantity The antibody and antiviral agent are together effective in inhibiting viral replication, viral infection of new cells, or viral loads.

Diagnostic methods of use

The antibodies according to the invention can also serve as diagnostic tools for the rapid detection of an infection by at least one coronavirus. In another aspect, the invention provides diagnostic methods. The antibodies according to any of the embodiments described herein can be used to detect the S protein of said at least one coronavirus, particularly a virus selected from SARS-CoV-2, SARS-CoV-1, and their variants, in a biological sample in vitro, ex vivo, or in vivo. In one embodiment, the invention provides a method for diagnosing the presence or location of said one or more coronaviruses in a subject who needs it. The antibodies of the invention can be used in a conventional immunoassay, including, but not limited to, an ELISA, an RIA, flow cytometry, tissue immunohistochemistry, Western blot (immunoblot), or immunoprecipitation. The antibodies of the invention can be used to detect the S protein of coronaviruses from humans. The invention provides a method for detecting the Coronavirus S protein in a biological sample, particularly S proteins from at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants, comprising contacting the biological sample with an antibody of the invention and detecting the bound antibody. In one embodiment, the antibody of the invention is directly labeled with a detectable marker. In another embodiment, said antibody (the first antibody) is unlabeled and a second antibody or other molecule capable of binding the anti-S protein antibody is labeled. As is known to one skilled in the art, a second antibody capable of specifically binding the particular species and class of the first antibody is chosen. For example, if the selected antibody is a human IgG, the secondary antibody could be an anti-human IgG. Other molecules that can bind antibodies include, but are not limited to, protein A and protein G, both of which are commercially available, e.g., from Pierce Chemical Co. Examples of biological samples for use in the diagnostic methods described herein include urine, feces, blood, saliva, biopsies, cerebrospinal fluid, nasopharyngeal and oropharyngeal lavage, sputum, endotracheal aspirate, bronchoalveolar lavage, or other biological samples obtainable from a human subject. Suitable markers for the antibody or secondary antibody have been described above and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, [beta]-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol. In other embodiments, protein S can be analyzed in a biological sample using a competitive immunoassay using protein S standards labeled with a detectable substance and an unlabeled antibody of the invention. In this assay, the biological sample, the labeled protein S standards, and the antibody are combined, and the amount of labeled protein S standard bound to the unlabeled antibody is determined. The amount of coronavirus protein S in the biological sample is inversely proportional to the amount of labeled protein S standard bound to the antibody. The immunoassays described above can be used for a variety of purposes. For example, the antibodies can be used to detect the S protein in cultured cells or as a diagnostic test in samples from a subject, particularly the S protein from at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants. Diagnostic methods according to any embodiment described herein can be followed by a further step of administering at least one antibody of the invention to the positive subject, for example, according to any of the therapeutic methods described herein.

Therapeutic methods of use

In another embodiment, the invention provides a method for neutralizing at least one coronavirus, particularly at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants, by administering an antibody according to any of the embodiments described herein to a patient in need. Any type of antibody described herein can be used therapeutically. In various embodiments, said antibody is a human antibody. In some embodiments, the antibody, or its antigen-binding portion, binds to the S1 domain of said coronavirus S protein. In some embodiments, the patient is a human patient. Alternatively, the patient may be a mammal infected with said at least one coronavirus, for example, infected with at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants. In one embodiment, the invention provides methods for treating, aiding in the treatment, preventing, or aiding in the prevention of an infection by at least one coronavirus, preferably at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants, and conditions or disorders resulting from such infection, in a subject by administering to the subject a therapeutically effective or prophylactically effective amount of an antibody of the invention. Antibodies and antigen-binding fragments thereof that are antagonistic to the S protein of said coronavirus can be used as therapeutics for this infection. The antibody can be administered locally or systemically. The therapeutic compositions comprising one or more antibodies according to any of the embodiments described herein can be administered to the subject, for example, orally, nasally, vaginally, buccally, rectally, through the eye, or through the lungs, in a variety of acceptable pharmaceutical dosage forms, which will be familiar to those skilled in the art. For example, antibodies may be administered nasally using a nasal insufflator device. Antibodies may also be administered to the eye in a gel formulation. For example, prior to administration, a formulation containing one or more antibodies according to any of the embodiments described herein may be conveniently contained in a two-compartment unit dose container, one compartment containing a lyophilized antibody preparation and the other compartment containing normal saline solution. The antibody dosage will generally be between 0.1 and 100 mg/kg, more preferably between 0.5 and 50 mg/kg, more preferably between 1 and 20 mg/kg, and most preferably between 1 and 10 mg/kg. The serum antibody concentration may be measured by any method known in the art.

In another embodiment, the antibodies of the present invention are administered to the subject in combination with other therapeutic agents. In one embodiment, the additional therapeutic agents may alone treat the symptoms of coronavirus infection and may optionally synergize with the effects of the antibodies. The additional agent administered may be selected by a person skilled in the art to treat the infection. Co-administration of the antibody with an additional therapeutic agent (combination therapy) includes the administration of a composition comprising the antibody and the additional therapeutic agent, as well as the administration of two or more separate compositions, one comprising the antibody and the others comprising the additional therapeutic agents. Furthermore, while co-administration or combination therapy generally means that the antibody and additional therapeutic agents are administered simultaneously, it also includes instances where the antibody and other therapeutic agents are administered at different times. For example, the antibody may be administered simultaneously. The antibody can be administered once every three days, while the additional therapeutic agent is administered once daily. Alternatively, the antibody can be administered before or after treatment with the additional therapeutic agent, such as after a patient has failed therapy with the additional agent. Similarly, administration of any of the antibodies of the invention can be administered before or after another therapy.

This antibody and one or more additional therapeutic agents (combination therapy) may be administered once, twice, or at least for a period of time until the condition is treated, palliated, or cured. Preferably, combination therapy is administered multiple times. Combination therapy can be administered from three times daily to once every six months. Administration may be based on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once monthly, once every two months, once every three months, and once every six months, or it may be administered continuously via a minipump. Combination therapy may be administered orally, mucosally, buccally, intranasally, inhalably, intravenously, subcutaneously, intramuscularly, or parenterally. In some aspects, the invention provides a method for treating, preventing, or alleviating the symptoms of at least one Coronavirus-mediated disease, particularly a disease mediated by a virus selected from SARS-CoV-1 and/or SARS-CoV-1 or variants thereof, in a subject in need thereof, comprising the step of administering to said subject an antibody or antigen-binding moiety according to any of the preceding embodiments, further comprising at least one additional therapeutic agent selected from the group consisting of: (a) one or more antibodies from the group consisting of: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13;

And

(b) one or more antibodies that specifically bind said S protein of a plurality of Coronavirus strains; and/or

(c) one or more neutralizing antibodies that do not bind the S protein of said Coronavirus; and/or

(d) one or more agents that bind the Coronavirus S protein receptor; and/or

(e) one or more antiviral agents.

In some aspects, the invention provides a kit for the treatment, prevention or alleviation of symptoms of a disease mediated by at least one Coronavirus in a subject in need thereof, in particular a disease mediated by a virus selected from SARS-CoV-1 and/or SARS-CoV-1 or their variants, comprising a) one or more antibodies of the group consisting of: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13; and

(b) one or more antibodies that specifically bind the S protein of a plurality of Coronavirus strains; and/or

(c) one or more neutralizing antibodies that do not bind said S protein; and/or

(d) one or more agents that bind the Coronavirus S protein receptor; and/or

(e) one or more antiviral agents.

The human monoclonal antibody or an antigen-binding portion thereof described herein may also be advantageously used as a diagnostic reagent in an in vitro method for detecting coronavirus antibodies, particularly SARS-CoV-2 and/or SARS-CoV-1 antibodies, in a biological sample previously obtained from a patient (such as serum, plasma, blood, or any other suitable biological material obtained from the patient, preferably a human). These antibodies may be found in the biological sample obtained from the patient, for example, following previous exposure to the virus or because a monoclonal antibody of the invention had previously been administered to the patient for therapeutic, prophylactic, or research purposes. Therefore, a diagnostic kit comprising the human monoclonal antibody or an antigen-binding portion thereof described herein, as a specific reagent, also falls within the scope of the invention, said kit being specifically designed for the detection and/or quantification of coronavirus antibodies in a biological sample previously obtained from a patient.

The human monoclonal antibody or an antigen-binding portion thereof described herein may also be advantageously used for the design of a coronavirus vaccine. As disclosed in Rappuoli, Rino et al. "Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design." The Journal of experimental medicine vol. 213, 4 (2016): 469-81. doi:10.1084/jem.20151960," human mAb can be used to identify protective antigens/epitopes. Structural characterization of the Ab-antigen complex can be used to instruct antigen design. Therefore, the scope of the invention also includes a method or use of the human monoclonal antibody or its antigen-binding portion described herein for the design of a vaccine against a coronavirus, in particular against the SARS-CoV-2 virus, the SARS-CoV-1 virus, and/or variants thereof.

The human monoclonal antibody or an antigen-binding portion thereof described herein may be used to prepare mimotopes, such as antiidiotypic antibodies, peptides, truncated S proteins, or artificial forms, or others, capable of evoking the antibodies described herein. Among these, antiidiotypic antibodies are preferred. Antiidiotypic antibodies are antibodies specifically directed against the idiotype of the neutralizing antibodies used for their manufacture, and are therefore capable of mimicking the key epitopes they recognize. The production of antiidiotypic antibodies is conducted using methods known per se that need not be further explained in detail herein. Therefore, mimotopes, preferably antiidiotypic antibodies, directed against an antibody of the invention also fall within the scope of the invention. The human monoclonal antibody or an antigen-binding portion thereof described herein may be used to manufacture antiidiotypic antibodies according to methods known per se. Anti-idiotype antibodies are antibodies specifically directed against the idiotype of the broad-spectrum neutralizing antibodies used to prepare them and, as such, are capable of mimicking the key epitopes they recognize. Therefore, anti-idiotype antibodies directed against a monoclonal antibody of the invention are also included within the scope of the invention.

The following experimental section is provided for illustrative and non-limiting purposes only and is not intended to limit the scope of the invention as defined in the appended claims. The claims are an integral part of the specification.

EXAMPLES

1. Materials and methods

Donor Enrollment and Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) In the current study, six donors immunized with at least three doses of mRNA vaccine and infected at least twice with SARS-CoV-2 were enrolled. This cohort was termed superhybrid immunity (SHI). Human peripheral blood mononuclear cells (PBMCs) were collected from these donors and used to perform single-cell sorting of spike (S) protein-specific class-switched memory B cells (MBCs). To identify broadly reactive human monoclonal antibodies (mAbs), both SARS-CoV-1 and SARS-CoV-2 S proteins were used as sorting bait. A total of 4,505 S-protein-specific MBCs were isolated from all donors and tested in a cytopathic effect-based neutralization assay (CPE-MN) to identify SARS-CoV-2 neutralizing antibodies (nAbs) and through our pseudotyping platform to identify SARS-CoV-1 nAbs. A total of 365 nAbs were identified, displaying varying levels of neutralization potency and breadth of coverage against the Wuhan SARS-CoV-2 virus and its variants, and SARS-CoV-1. From all identified nAbs, ten antibodies were selected, here named 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13. To assess the binding breadth of the selected nAbs, the antibodies were characterized for their ability to bind to the virus. to bind to the SARS-CoV-2 S protein trimer, the receptor-binding domain (RBD), the N-terminal domain (NTD), and the S2 domain, and to the S protein of other human coronaviruses, including SARS-CoV-1, OC43, HKU1, 229E, and NL63. Furthermore, nAbs targeting the SARS-CoV-2 S RBD protein were further characterized through a competition assay to identify the region of the epitope targeted by these antibodies. The specific characteristics of these newly discovered antibodies are reported here.

Functional characterization of human coronavirus neutralizing nAbs

The ten nAbs that showed broad functional activity were initially characterized for their binding activity to the SARS-CoV-2 S protein trimer, the receptor-binding domain (RBD), the N-terminal domain (NTD), and the S2 domain, and to the S protein of other human coronaviruses, including SARS-CoV-1, OC43, HKU1, 229E, and NL63. All antibodies recognized the SARS-CoV-2 S protein trimer. One antibody, 04N13, also recognized the NTD domain of the SARS-CoV-2 S protein, while the remaining nAbs bound to the RBD (Fig. 1, left panel). Among the 10 antibodies, two, 02G11 and 03O07, were shown to also recognize the SARS-CoV-1 S protein (Fig. 1, middle panel). The S proteins of OC43, HKU1, 229E, and NL63 were not recognized by any of our antibodies. The neutralizing potency of the nAbs against the Wuhan and Omicron SARS-CoV-1 and SARS-CoV-2 variants was subsequently evaluated. Of the two antibodies capable of binding the SARS-CoV-1 S protein, 03O07 showed extreme neutralizing potency against SARS-CoV-1 with a 50% inhibitory concentration (IC50) of 56.0 ng ml-1 (Fig. 1, right panel). In contrast, all nAbs neutralized the Wuhan and Omicron SARS-CoV-2 virus (BA.5, BA.2.75, BF.7, BQ.1.1, and XBB.1.5) showing different ranges of neutralizing potency (Fig. 1, right panel). The 100% inhibitory concentration (IC100) ranged from 9.4 ? 37.7 ng ml-1 for 01J19, 15.5 ? 110.8 ng ml-1 for 02K18, 9.5 ? 167.4 ng ml-1 for 05F22, 15.2 ? 107.6 ng ml-1 for 01J18, 3.0 ? 53.5 ng ml-1 for 02K05, 4.7 ? 3767.5 ng ml-1 for 01B20, 126.8 ? 405.8 ng ml-1 for 02G11, 67.9 ? 479.9 ng ml-1 for 03O07, 21.0 ? 627.5 ng ml-1 for 05N18, and 11.1 ? 97.8 ng ml-1 for 02N13, (Fig.1, right panel).

Finally, the nine RBD-targeting nAbs were classified based on their ability to compete with Class 1/2 J08<1>, Class 3 S309<2>, and Class 4 CR3022<3> antibodies. Most antibodies (5/9; 55.7%), 01J19, 02K18, 05F22, 01J18, and 02K05, competed with J08 and thus targeted the Class 1/2 region (Fig. 1, right panel). Subsequently, two antibodies (2/9; 22.2%), 02G11 and 03O07 and one antibody (1/9; 11.1%), 01B20, targeted the Class 4 and Class 3 epitope region, respectively. Finally, one antibody (1/9; 11.1%), 05N18, did not compete with any of the three antibodies and therefore we were unable to identify the target epitope region (Fig. 1, right panel).

Genetic characterization of human coronavirus neutralizing nAbs

The genes encoding the heavy and light chains of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 have been characterized. Table 1 lists the complete nucleotide and amino acid sequences of the heavy and light chains of these latter antibodies. Table 2 shows the rearrangements of the immunoglobulin heavy chain V, D, and J (IGHV; IGHD; IGHJ) and light chain V and J (IGLV; IGLJ) genes, the length of the complementary determining region 3 (CDR3) of the heavy chains (H-CDR3) and light chains (L-CDR3), and the heavy and light chain V gene mutation frequencies (%). Overall, a higher level of mutations was observed. A higher rate of V gene mutations was observed in heavy chains (mean 8.35%) compared to light chains (mean 4.08%). Similarly, longer H-CDR3s (mean 16.4 amino acids) were observed compared to L-CDR3s (mean 9.70 amino acids) (Table 2). Several heavy and light chain rearrangements were used in the present nAb panel. The majority of nAbs (4/10; 40.0%) were encoded by IGHV3-66 germlines paired with several heavy chain and light chain D and J genes (Table 2). 02K18 and 05F22 utilized identical rearrangements of the V, D, and J heavy chain genes (IGHV3-66;IGHD1-26;IGHJ4-1) and the V and J light chain genes (IGKV3-15;IGKJ2-1) with major differences in the CDR3 lengths of both chains (Table 2). Similarly, 02G11 and 03O07 shared the same heavy (IGHV1-46;IGHD5-5;IGHJ4-1) and light (IGKV3-D15;IGKJ1-1) chain rearrangements with nearly identical CDR3 lengths and V gene mutation levels. Four antibodies (01J19, 02K05, 01B20, and 02N13) in our panel utilized different heavy V genes than the rest of the nAbs. These included IGHV4-31 (01J19), IGHV3-53 (02K05), IGHV1-3 (01B20), and IGHV3-48 (02N13) (Table 2).

Table 1. Sequences of neutralizing nAbs against human coronavirus

Table 2. Genetic characteristics of human coronavirus-neutralizing nABs

Declaration pursuant to Article 170bis

Pursuant to Article 170bis of the Italian Industrial Property Code, the applicant of this patent application declares that: 5 - For the biological material containing microorganisms or genetically modified organisms, the subject of or used in the aforementioned patent application, the obligations deriving from national or EU regulations apply, and in particular, the provisions of paragraph 6 of Legislative Decrees no. 206 of 12 April 2001 and no. 224 of 8 July 2003, 10 concerning such modifications, have been complied with; - The donors of the human blood samples used in this patent application have given their written informed consent. The study has been approved by the local ethics committees.

List of sequences in the description

>SEQ ID NO:1 Amino acid sequence of the heavy chain variable domain of the antibody here defined as 01J19 (01J19_VH) QVQLQQWGPGLVKPSQTLSLACTVSGGSISSGSSYWTWIRQQPGKGLEWIGFIFYSESTHH KFFSGNTYYNPSLKSRVIISVDTSKNQFSLRLNSVTAADTAVYYCAREVGGDFWSDYNMP GDYHQIDVWGKGTTVIVSA

>SEQ ID NO:2 Amino acid sequence of the heavy chain variable domain of the antibody defined here as 02K18 (02K18_VH) QVQLVQSGGGLVQPGGSLRLSCVASEIIVSSNYMSWVRQAPGKRLEWVSALFSGGSTFYA DSIKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDLGPVGGSDYWGQGTHVIVSS

>SEQ ID NO:3 Amino acid sequence of the heavy chain variable domain of the antibody here defined as 05F22 (05F22_VH) EVQLVESGGGVVQPGGSLRVSCEASEIIVSSNYMSWVRQAPGKGLEWVSTVFSGGSTFYA ESVKGRFTISTDNSKNTLYLHMNRLRVEDTAVYYCTRDLGVVGGTDYWGQGALVTVSS

>SEQ ID NO:4 Amino acid sequence of the heavy chain variable domain of the antibody here defined as 01J18 (01J18_VH) QVQLVESGGGLVQPGGSLRLSCAASTLTVSRNYMNWVRQAPGTGLEWVSVIYPGGTTYY ADSVKGRFTVSRDDSKNILFLQMDSLRVEDTAMYYCSRPIMGATSGMDVWGQGTMVTVS SASTKGPSVFPL

>SEQ ID NO:5 Amino acid sequence of the heavy chain variable domain of the antibody defined here as 02K05 (02K05_VH) QVQLVESGGGLIQPGGSLRLSCEASEIIVNWNYMSWVRQAPGKGLEWVSIIYSGGSTFYAD SVKGRFTISTDKSNNTLYLQMHTLRAEDTAVYYCARTLPQFEGFDIWGQGTMVTVSS

>SEQ ID NO:6 Amino acid sequence of the heavy chain variable domain of the antibody here defined as 01B20 (01B20_VH) QVQLVQSGDEVKKPGASVKISCKTSGFSLTSHALHWVRQAPGQRLEWMGWIGISADEGNT KYSHKFEGRLTISRDTSASTAYMDLSSLSSDDTAMYYCATHSGTYQVDYFDHWGQGTLVT VAS

>SEQ ID NO:7 Amino acid sequence of the heavy chain variable domain of the antibody here defined as 02G11 (02G11_VH) QVQLVQSGAEVKKPGASVKVSCKAFGYTFSDHYMHWLRQAPGQGPEWMGVVIPINGGTS YAQEFQDRLTVTRDKSTSTVYMELSSLTYEDTAVYYCARDPVGPDNFGYSLLDAKYFFDL WGQGTLVTVSS

>SEQ ID NO:8 Amino acid sequence of the heavy chain variable domain of the antibody here defined as 03O07 (03O07_VH) QVQLVQSAAEVKKPGASVKVSCRAFGYTFSDHYMHWLRQAPGQGPEWMGVVIPINGGTT YAQNFQDRLTVTRDKSTSSVYMELSSLTYEDTAVYYCARDPVGPDNFGYSLLDAKYYFDY WGQGTLVTVSS

>SEQ ID NO:9 Amino acid sequence of the heavy chain variable domain of the antibody here defined as 05N18 (05N18_VH) QVQLVESGGELVQPGGSLTLSCAASEIVVSLNYMRWVRQAPGKGLEWVSLIYAGGSTFYA DSVKGRFTISRDNSKNTLYLQMRSLRVEDSAVYYCARDLVTDGMDVWGQGTTVTVSS

>SEQ ID NO:10 Amino acid sequence of the heavy chain variable domain of the antibody here defined as 02N13 (02N13_VH) QVQLVESGGGLVQPGGSLRLSCAASGFHFGTYSFNWVRQAPGKGLEWVSYISSSSSTIFYA DSVEGRFTISRDDARGSLFLQMNSLRAEDTAIYYCARDPRYYRDSPYYYPLDSWGQGTLV TVSS

>SEQ ID NO:11 Nucleotide sequence of the heavy chain variable domain of the antibody herein defined as 01J19 (01J19_VH) CAGGTGCAGCTGCAGCAGTGGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCC TCGCCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTTCTTCCTACTGGACCTGGATC CGCCAGCAGCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCT TTTACAGCGAGAGCACCCACCACAAGTTTTTCAGTGGGAACACCTACTACAACCCGTC CCTCAAGAGTCGAGTGATCATTTCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAGG CTGAACTCCGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGAGGTAGGCG GCGATTTTTGGAGTGATTATAACATGCCCGGGGACTACCACCAGATTGACGTCTGGGG CAAAGGGACCCGGTCATCGTCTCCGCA

>SEQ ID NO:12 Nucleotide sequence of the heavy chain variable domain of the antibody defined here as 02K18 (02K18_VH) CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGTAGCCTCTGAAATCATCGTCAGTAGCAACTACATGAGTTGGGTCCGCCAG GCTCCAGGGAAGAGGCTGGAGTGGGTCTCAGCTCTTTTTAGTG GAGGCAGCACTTTCTACGCAGACTCCATAAAGGGCAGATTCACCATCTCCAGAGACGA TTCCAAGAACACACTGTATCTTCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTG TATTACTGTGCGAGAGAGATCTCGGTCCGGTGGGAGGTTCTGACTACTGGGGCCAGGGAA CCCACGTCATCGTCTCCTCA

>SEQ ID NO:13 Nucleotide sequence of the heavy chain variable domain of the antibody herein defined as 05F22 (05F22_VH) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTAGTCCAGCCTGGGGGGTCCCTGAGA GTCTCCTGTGAAGCCTCCGAAATCATCGTCAGCAGCAACTACATGAGTTGGGTCCGCC AGGCTCCAGGAAAGGGGCTGGAGTGGGTCTCAACTGTTTTTAGTGGTGGCAGCACATT TTATGCTGAGTCCGTGAAGGGCAGATTCACCATCTCCACAGACAATTCCAAGAACACG CTGTATCTTCACATGAACAGGCTGAGAGTCGAAGACACGGCTGTCTACTATTGTACGA GAGATCTCGGTGTGGTGGGAGGAACTGATTACTGGGGCCAGGGAGCCCTGGTCACCGT CTCCTCA

>SEQ ID NO:14 Nucleotide sequence of the heavy chain variable domain of the antibody herein defined as 01J18 (01J18_VH) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGCTCCCTGAGAC TCTCCTGTGCAGCCTCTACACTCACCGTCAGTAGAAACTACATGAACTGGGTCCGCCAG GCTCCAGGGACGGGGCTGGAGTGGGTCTCGGTGATCTATCCCGGTGGTACGACATATT ATGCAGACTCCGTGAAGGGCAGATTCACCGTCTCCAGAGACGATTCCAAGAACATACT GTTTCTTCAAATGGACAGTCTGAGGGTCGAGGACACGGCTATGTATTACTGTTCGAGAC CAATAATGGGAGCTACTTCGGGTATGGACGTGTGGGGCCAAGGGACCATGGTCACCGT CTCCTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGC

>SEQ ID NO:15 Nucleotide sequence of the heavy chain variable domain of the antibody defined here as 02K05 (02K05_VH) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGAAGCCTCTGAAATCATCGTCAATTGGAACTACATGAGCTGGGTCCGCCAG GCTCCAGGGAAGGGGCTGGAGTGGGTCTCAATTATTTATAGTGGAGGAAGCACATTCT ACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCACCGACAAATCCAACAACACGCT GTATCTTCAAATGCACACCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGG ACCCTCCCGCAGTTTGAAGGTTTTGATATATGGGGCCAAGGGACAATGGTCACCGTCTC TTCA

>SEQ ID NO:16 Nucleotide sequence of the heavy chain variable domain of the antibody defined here as 01B20 (01B20_VH) CAGGTGCAGCTGGTGCAGTCTGGGGATGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG ATTTCCTGCAAGACTTCTGGATTCAGCCTCACTAGCCATGCTCTGCATTGGGTGCGCCA GGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCGGGATCAGCGCTGATGAGGG TAACACAAAATATTCACATAAATTCGAGGGCAGACTCACCATTTCCAGGGACACATCC GCGAGCACAGCCTACATGGACCTGAGCAGCCTGAGCTCTGACGACACGGCTATGTATT ACTGTGCAACACATAGTGGGACCTACCAAGTTGACTACTTTGACCACTGGGGCCAGGG AACCCTGGTCACCGTCGCCTCA

>SEQ ID NO:17 Nucleotide sequence of the heavy chain variable domain of the antibody here defined as 02G11 (02G11_VH) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTAAAGAAGCCTGGGGCCTCAGTGAAG GTTTCCTGCAAGGCATTTGGATACACCTTCAGCGACCACTATATGCACTGGCTGCGACA GGCCCCTGGACAGGGGCCTGAGTGGATGGGAGTAGTCATCCCTATTAATGGTGGCACA AGTTACGCACAGGAGTTCCAGGACAGACTCACCGTGACCAGGGACAAGTCGACCAGC ACAGTCTACATGGAGCTGAGCAGCCTGACATATGAAGACACGGCCGTCTATTACTGTG CGAGAGATCCAGTGGGGCCGGACAACTTTGGTTACTCTCTGTTGGACGCCAAATACTTC TTTGACCTCTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA

>SEQ ID NO:18 Nucleotide sequence of the heavy chain variable domain of the antibody defined here as 03O07 (03O07_VH) CAGGTGCAGCTGGTGCAGTCTGCGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGG TTTCCTGCAGGGCATTTGGATACACCTTCAGCGACCACTATATGCACTGGCTGCGACAG GCCCCTGGACAGGGGCCTGAGTGGATGGGAGTAGTCATCCCTATTAATGGTGGCACAA CTTACGCACAGAACTTCCAGGACAGACTCACCGTGACCAGGGACAAGTCGACCAGCTC AGTCTACATGGAGCTGAGCAGCCTGACATATGAAGACACGGCCGTCTATTACTGTGCG AGAGATCCGGTGGGGCCGGACAACTTTGGTTACTCTCTGTTGGACGCCAAATACTACTT TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

>SEQ ID NO:19 Nucleotide sequence of the heavy chain variable domain of the antibody herein defined as 05N18 (05N18_VH) CAGGTGCAGCTGGTGGAGTCTGGGGGAGAGTTGGTCCAGCCGGGGGGGTCCCTGACCC TCTCCTGTGCAGCCTCTGAAATCGTCGTCAGTCTCAATTACATGAGGTGGGTCCGCCAG GCTCCAGGGAAGGGGCTGGAGTGGGTCTCACTTATTTATGCCG GTGGCAGTACATTCTACGCGGACTCCGTGAAGGGAAGATTCACCATCTCCAGAGACAA TTCCAAGAACACACTGTATCTTCAAATGAGAAGCCTGAGAGTTGAGGACTCGGCTGTA TATTACTGTGCGAGAGATCTGGTTACCGACGGTATGGACGTCTG GGGCCAAGGGACCACGGTCACCGTCTCCTCA

>SEQ ID NO:20 Nucleotide sequence of the heavy chain variable domain of the antibody here defined as 02N13 (02N13_VH) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCCACTTCGGCACCTACAGTTTCAACTGGGTCCGCCAA GCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTA

GCAGTAGTACCATTTTCTACGCAGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGAC GATGCCAGGGGCTCTCTGTTTCTGCAAATGAACAGCCTGAGAGCGGAGGACACGGCTA TCTATTACTGTGCGAGAGACCCCCGATATTATCGTGATAGTCC TTATTATTACCCCCTTGACTCTTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

>SEQ ID NO:21 Amino acid sequence of the variable light chain domain of the antibody here defined as 01J19 (01J19_Vk) DIVMTQSPLSLPVTPGEPASISCRSSQNLLQSNGHNYLDWYLQKPGQSPQLLIYLSSNRASG VPDRFSGSGSGTHFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIK

>SEQ ID NO:22 Amino acid sequence of the variable light chain domain of the antibody defined here as 02K18 (02K18_Vk) EIVMTQSPVTLSVSPGARVTLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGVPAR FSGSGSETEFSLTISSLQSEDFAIYFCQQYDNWPPDYTFGQGTKLEIK

>SEQ ID NO:23 Amino acid sequence of the variable light chain domain of the antibody here defined as 05F22 (05F22_Vk) EIVMTQSPVTLSVSPGERATLSCRASQSVYSNLAWYQQKPGQAPRLLIYEASTRATDIPARF SGGSGTDFTLTISSLQSEDFAIYFCQQYTNWPPGYTFGQGTKLEIK

>SEQ ID NO:24 Amino acid sequence of the light chain variable domain of the antibody here defined as 01J18 (01J18_Vk) AIQMTQSPSSLSASVGDRVTIHCQASQDINNYLNWYQQKPGKAPQLLIYDASNLETGVPSR FSGSGSGTYFTFTISSLQPEDIATYYCQQHDNIPPTFGQGTKVEIK

>SEQ ID NO:25 Amino acid sequence of the light chain variable domain of the antibody here defined as 02K05 (02K05_Vk) AIQMTQSPSSLSASVGDRVTITCRASQGISTFLNWYQQKPGKAPKLLIYSASSLHSGVPSRFS GSGSGTDFTLTISNLQPEDFATYYCQQSDSTPLYTFGQGTMLEIK

>SEQ ID NO:26 Amino acid sequence of the variable light chain domain of the antibody here defined as 01B20 (01B20_Vk) AIQMTQSPSTLSASVGDRVTITCRASQSINNWLAWYQQKPGKAPKLLIFDVSSLDSGVPSRF SGGSGTEFTLTISSLQPDDFATYYCQQYNGYPWTFGQGTKVEIK

>SEQ ID NO:27 Amino acid sequence of the variable light chain domain of the antibody here defined as 02G11 (02G11_Vk) EIVMTQSPATLSVSPGERATLSCRASHSVGSNLAWYQHKPGQAPRLLIYGASTRATGIPARF SGGSGTEFTLTISSLQSEDFAIYYCQNYNEWTRTFGQGTKVEVK

>SEQ ID NO:28 Amino acid sequence of the variable light chain domain of the antibody here defined as 03O07 (03O07_Vk)

EIVMTQSPATLSVSPGERATLSCRASHSVGSNLAWYQHKPGQAAPRLLIYDASTRVTGIPARF SSGSGSGTDFTLTISSLQSEDFAIYYCQHYNEWTRTFGQGTKVEIK

>SEQ ID NO:29 Amino acid sequence of the light chain variable domain of the antibody here defined as 05N18 (05N18_Vk) AIQMTQSPSFLSASVGDRVTIACRASQGIASSLAWYQQKPGKAPKLLIFAASTLQSGVPSRF SGSGSGTEFALTISGLQPEDFATYYCQQLDASTITFGPGTKVEIK

>SEQ ID NO:30 Amino acid sequence of the variable light chain domain of the antibody here defined as 02N13 (02N13_Vk) AIQMTQSPFSLSASVGDRVTITCQASQDITNSLNWFQQKPGKAPNLLIYGASNLETGVSTRF SGGSGTHFTFTISSLQPEDVATYYCQQYGDFPPYSFGQGTKLEIK

>SEQ ID NO:31 Nucleotide sequence of the variable light chain domain of the antibody herein defined as 01J19 (01J19_Vk) GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTC CATCTCCTGCAGGTCTAGTCAGAACCTCCTTCAAAGTAATGGACACAACTATTTGGATT GGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCT ATTTGAGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCCGGC ACACATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACT GCATGCAAGCTCTACAAACTCCTCGAACGTTCGGCCAAGGGACCAAGGTGGAAATCAA

A

>SEQ ID NO:32 Nucleotide sequence of the light chain variable domain of the antibody defined here as 02K18 (02K18_Vk) GAAATTGTGATGACGCAGTCTCCAGTCACCCTGTCTGTGTCTCCAGGGGCAAGAGTCA CCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAA ACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTGTCC CAGCCAGGTTCAGTGGCAGTGGGTCTGAGACAGAGTTCAGTCTCACCATCAGCAGCCT GCAGTCTGAAGATTTTGCTATTTATTTTTGTCAGCAGTATGATAACTGGCCTCCGGACT ACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

>SEQ ID NO:33 Nucleotide sequence of the light chain variable domain of the antibody herein defined as 05F22 (05F22_Vk) GAAATTGTGATGACGCAGTCTCCAGTCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCA CCCTCTCCTGCAGGGCCAGTCAGAGTGTTTACAGCAACTTAGCCTGGTATCAGCAGAA ACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAAGCATCCACCCGGGCCACTGATATCC CTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG CAGTCTGAAGACTTTGCAATTTATTTCTGTCAGCAGTATACTAACTGGCCTCCGGGGTA CACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

>SEQ ID NO:34 Nucleotide sequence of the light chain variable domain of the antibody here defined as 01J18 (01J18_Vk) GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC CATTCATTGCCAGGCGAGTCAGGACATTAACAACTATTTAAATTGGTATCAACAGAAA CCAGGGAAAGCCCCTCAGCTCCTGATCTACGATGCATCCAATT TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACATATTTTACTTT CACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGCATGAT AATATCCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAA

A

>SEQ ID NO:35 Nucleotide sequence of the light chain variable domain of the antibody defined here as 02K05 (02K05_Vk) GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTGAC CATCACTTGCCGGGCAAGTCAGGGCATTAGCACCTTTTTAAATTGGTATCAGCAGAAA CCAGGGAAAGCCCCTAAACTCCTGATCTATTCTGCATCCAGTTTGCACAGTGGGGTCCC CTCAAGGTTCAGTGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAATCTGC AACCTGAAGATTTCGCAACTTACTACTGTCAACAGAGTGACAGTACCCCTCTGTACACT TTTGGCCAGGGGACCATGCTGGAGAT CAAA

>SEQ ID NO:36 Nucleotide sequence of the light chain variable domain of the antibody defined here as 01B20 (01B20_Vk) GCCATCCAGATGACCCAGTCTCCATCCACCCTGTCTGCATCTGTAGGAGACAGAGTCAC CATCACTTGCCGGGCCAGTCAGAGTATTAATAACTGGTTGGCCTGGTATCAGCAGAAA CCAGGGAAAGCCCCTAAGCTCCTGATCTTTGATGTCTCCAGTTTGGACAGTGGGGTCCC ATCAAGGTTTAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTG CAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATGGTTATCCGTGGACGTT CGGCCAAGGGACCAAGGTGGAAATCAAA

>SEQ ID NO: Nucleotide sequence of the variable light chain domain of the antibody here defined as 02G11 (02G11_Vk) GAAATTGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCA CCCTCTCCTGCAGGGCCAGTCACAGTGTTGGCAGCAACCTAGCCTGGTACCAGCACAA ACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGCATCCACCAGGGCCACTGGCATCC CAGCCAGATTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCT GCAGTCTGAAGATTTTGCAATTTATTACTGTCAGAACTATAATGAGTGGACACGGACGT TCGGCCAAGGGACCAAGGTGGAAGTCAAA

>SEQ ID NO:38 Nucleotide sequence of the light chain variable domain of the antibody herein defined as 03O07 (03O07_Vk) GAAATTGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCA CCCTCTCCTGCAGGGCCAGTCACAGTGTTGGCAGCAACTTAGCCTGGTACCAGCACAA ACCTGGCCAGGCTCCCAGACTCCTCATCTATGATGCATCCACCAGGGTCACTGGCATCC CAGCCAGATTCAGCGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCT GCAGTCTGAAGATTTTGCAATTTATTACTGTCAGCACTATAATGAGTGGACACGGACGT TCGGCCAAGGGACCAAGGTGGAAATCAAA

>SEQ ID NO:39 Nucleotide sequence of the variable light chain domain of the antibody here defined as 05N18 (05N18_Vk) GCCATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGAGTCAC CATCGCTTGCCGGGCCAGTCAGGGCATTGCCAGTTCTTTAGCCTGGTATCAACAAAAAC CAGGGAAAGCCCCTAAGCTCCTGATCTTTGCTGCGTCCACTT TGCAAAGTGGAGTCCCATCAAGGTTCAGCGGCAGCGGATCTGGGACAGAATTCGCTCT CACAATCAGCGGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAACTTGATG CTTCCACGATCACTTTCGGCCCTGGGACCAAAGTGGAAATCAA

A

>SEQ ID NO:40 Nucleotide sequence of the variable light chain domain of the antibody defined here as 02N13 (02N13_Vk) GCCATCCAGATGACCCAGTCTCCATTCTCCCTGTCTGCATCTGTTGGAGACAGAGTCAC CATCACTTGCCAGGCGAGTCAGGACATTACCAACTCTTTGAATTGGTTTCAGCAGAAAC CAGGGAAAGCCCCTAACCTCCTGATCTACGGTGCGTCCAATT TGGAAACAGGGGTCTCAACACGGTTCAGTGGAAGTGGATCTGGGACACATTTTACTTT CACCATCTCCAGCCTGCAGCCTGAAGATGTTGCAACATATTATTGTCAACAATATGGTG ATTTCCCTCCGTACTCTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

S protein sequences

Sequences of the surface glycoprotein of severe acute respiratory syndrome coronavirus 2 are known in the art. The sequences of the S1 ("Spike_rec_bind") and S2 ("Coronavirus S2 glycoprotein") domains are in GenBank under the ID QHD43416.1.

Claims (16)

1. Monoclonal antibody or an antigen-binding portion thereof capable of neutralizing the biological activity of at least one Coronavirus, preferably at least one virus selected from SARS-CoV-2, SARS-CoV-1 and their variants, which antibody or antigen-binding portion thereof comprises a variable heavy chain domain (VH) and a variable light chain domain (VK), wherein: said VH has the amino acid sequence set out in SEQ ID NO:1 and said VK has the amino acid sequence SEQ ID NO:21; or said VH has the amino acid sequence set forth in SEQ ID NO:2 and said VK has the amino acid sequence SEQ ID NO:22; or said VH has the amino acid sequence set forth in SEQ ID NO:3 and said VK has the amino acid sequence SEQ ID NO:23; or said VH has the amino acid sequence set forth in SEQ ID NO:4 and said VK has the amino acid sequence SEQ ID NO:24; or said VH has the amino acid sequence set forth in SEQ ID NO:5 and said VK has the amino acid sequence SEQ ID NO:25; or said VH has the amino acid sequence set forth in SEQ ID NO:6 and said VK has the amino acid sequence SEQ ID NO:26; or said VH has the amino acid sequence set forth in SEQ ID NO:7 and said VK has the amino acid sequence SEQ ID NO:27; or said VH has the amino acid sequence set forth in SEQ ID NO:8 and said VK has the amino acid sequence SEQ ID NO:28; or said VH has the amino acid sequence set forth in SEQ ID NO:9 and said VK has the amino acid sequence SEQ ID NO:29; or said VH has the amino acid sequence set forth in SEQ ID NO:10 and said VK has the amino acid sequence SEQ ID NO:30. 2. A monoclonal antibody or an antigen-binding portion thereof according to claim 1, wherein said VL and said VK are at least 85% identical in amino acid sequence, preferably at least 95% identical in amino acid sequence, more preferably at least 98% identical or at least 99% identical in amino acid sequence of: VH with SEQ ID NO:1 and VK with SEQ ID NO:21; or VH with SEQ ID NO:2 and VK with SEQ ID NO:22; or VH with SEQ ID NO:3 and VK with SEQ ID NO:23; or VH with SEQ ID NO:4 and VK with SEQ ID NO:24; or VH with SEQ ID NO:5 and VK with SEQ ID NO:25; or VH with SEQ ID NO:6 and VK with SEQ ID NO:26; or VH with SEQ ID NO:7 and VK with SEQ ID NO:27; or VH with SEQ ID NO:8 and VK with SEQ ID NO:28; or VH with SEQ ID NO:9 and VK with SEQ ID NO:29; or VH with SEQ ID NO:10 and VK with SEQ ID NO:30. 3. A monoclonal antibody or an antigen-binding portion thereof according to claims 1 or 2, wherein said antibody or an antigen-binding portion thereof is a human monoclonal antibody. 4. A monoclonal antibody or an antigen-binding portion thereof according to any of claims 1 to 3, wherein said antibody or antigen-binding portion thereof exhibits a 100% inhibitory concentration (IC100) of less than 100 ng/ml when tested in an in vitro neutralization assay against at least one virus selected from SARS-CoV-2, SARS-CoV-1 and their variants. 5. Human monoclonal antibody or an antigen-binding portion thereof that competes for binding to the S protein of at least one Coronavirus with any of the antibody or antigen-binding portion thereof according to any of claims 1 to 4, preferably at least one Coronavirus selected from SARS-CoV-1, SARS-CoV-2 and their variants. 6. Monoclonal antibody or an antigen-binding portion thereof according to any of claims 1 to 5 for use in the prophylactic or therapeutic treatment of a virus infection or conditions or disorders resulting from such infection, in particular for use in the prevention and/or treatment of an infection by at least one Coronavirus. 7. Monoclonal antibody or an antigen-binding moiety according to any of claims 1 to 6, for use in a prophylactic or therapeutic treatment of an infection by at least one virus selected from SARS-CoV-2, SARS-CoV-1 and their variants, or conditions or disorders resulting from such infection, in particular Coronavirus Disease 2019 (COVID-19). 8. A pharmaceutical composition comprising one or more monoclonal antibodies or an antigen-binding portion thereof according to any of claims 1 to 7 and a pharmaceutically acceptable carrier. 9. Composition according to claim 8, for use in the prophylactic or therapeutic treatment of a virus infection or conditions or disorders resulting from such an infection, in particular for use in the prevention and/or treatment of a Coronavirus infection, more particularly for use in the prophylactic or therapeutic treatment of an infection by at least one virus selected from SARS-CoV-2, SARS-CoV-1 and their variants, or conditions or disorders resulting from such an infection, in particular for use in the prevention and/or treatment of a SARS-CoV-2 and/or SARS-CoV-1 infection. 10. In vitro or ex vivo use of a monoclonal antibody or an antigen-binding portion thereof according to any of claims 1 to 7 in the diagnosis of an infection by at least one Coronavirus, preferably by at least one virus selected from SARS-CoV-2, SARS-CoV-1 and their variants. 11. In vitro method for detecting the presence of at least one Coronavirus in a sample comprising the following steps: (i) contacting the antibody or an antigen-binding portion thereof according to any of claims 1 to 7 with said sample; ii) Detecting the binding of said antibody or an antigen-binding portion thereof to the S protein of said at least one Coronavirus, preferably wherein said Coronavirus is at least one virus selected from SARS-CoV-2, SARS-CoV-1 and their variants. 12. In vitro method for the diagnosis of an infection by at least one Coronavirus in a subject which includes the following steps: i) contacting the antibody or an antigen-binding portion thereof according to any of claims 1 to 7 with a biological sample of said subject; ii) Detecting the binding of said antibody or an antigen-binding portion thereof to the S protein of said at least one Coronavirus, preferably wherein said Coronavirus is at least one virus selected from SARS-CoV-2, SARS-CoV-1 and their variants. 13. A diagnostic kit comprising as a specific reagent an antibody or an antigen-binding portion thereof according to any of claims 1 to 7, said kit being intended in particular for use in a method for detecting or quantifying, in a sample from a patient, antibodies against coronavirus and/or the coronavirus S protein, in particular antibodies against Sars-Cov-2 and/or SARS-CoV-1 and/or SARS-CoV-2 and/or SARS-CoV-1 S protein. 14. Use of an antibody or an antigen-binding portion thereof according to any of claims 1 to 7 for the design of a vaccine against at least one Coronavirus, in particular against at least one virus selected from SARS-CoV-2, SARS-CoV-1 and their variants. 15. A mimotope specifically directed against the idiotype of an antibody or an antigen-binding portion thereof according to any of claims 1 to 7. 16. An anti-idiotype antibody that is specifically directed against the idiotype of an antibody or an antigen-binding portion thereof according to any of claims 1 to 7.