Antibodies against covid-19 and other human coronaviruses

Abstract

This invention relates to a monoclonal antibody or its antigen-binding portion thereof, which has potent neutralizing activity against coronaviruses, particularly against at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants. The invention also relates to the use of such monoclonal antibodies or their antigen-binding portions in the treatment, prevention, and diagnosis of coronaviruses, particularly SARS-CoV-2 and / or SARS-CoV-1-related diseases.

Description

[0001] Technical Field of the Invention

[0002] This invention relates to a monoclonal antibody or its antigen-binding moiety thereof, which has potent neutralizing activity against coronaviruses, particularly SARS-CoV-2 and SARS-CoV-1. The invention also relates to the use of such monoclonal antibodies or their antigen-binding moiety in the treatment, prevention, and diagnosis of coronavirus-related diseases. Background Technology

[0003] Human monoclonal antibodies (mAbs) are an industrially mature technology with over 50 approved products in the fields of cancer, inflammation, and autoimmune diseases. Their comprehensive safety assessments and extensive development experience make mAbs ideal drug candidates for rapid development, especially in the context of infectious diseases and pandemics. To date, the application of mAbs in infectious diseases has been limited, primarily due to the large quantities required for treatment, making them uneconomical. However, recent remarkable technological advancements in the isolation and screening of memory B cells have enabled the identification of highly potent neutralizing mAbs and further enhancement of their potency by several orders of magnitude through established engineered procedures. This possibility reduces the number of antibodies required for treatment, thus enabling non-intravenous delivery of potent neutralizing mAbs.

[0004] Among the many available treatment options, mAbs offer a number of advantages. First, they are drugs that can be developed in the shortest possible timeframe. In fact, the extensive clinical experience with the safety of over 50 commercially available monoclonal antibodies approved for the treatment of cancer, inflammation, and autoimmune diseases provides a high degree of confidence in their safety profile, supporting the possibility of accelerated regulatory pathways. Furthermore, long-standing industrial experience in developing and manufacturing monoclonal antibody mAbs reduces the risks often associated with the development of investigational product technologies. Finally, this remarkable technological advancement in the field has shortened the traditional timeline, from discovery to proof-of-concept trials to just 5-6 months. Currently, several drug candidates are under development in the areas of HIV, influenza, RSV, and many other infectious diseases. Perhaps the most striking example of the efficacy of mAbs in emerging infectious diseases comes from the Ebola outbreak. In this outbreak, a rapidly developed potent mAb was among the first drugs tested during the Ebola outbreak and showed significant efficacy in preventing death. Given the significant efficacy of this intervention, the potent mAb became the first and, to date, the only drug recommended by the World Health Organization (WHO) for Ebola.

[0005] The Coronavirus Family comprises a large number of viral species, which are further divided into four genera. Seven species are human-associated and exhibit high diversity in adaptability, pathogenicity, and transmissibility. In fact, four species are endemic and highly adapted to humans; the recently emerging and currently circulating SARS-CoV-1 and SARS-CoV-2 belong to the same genus (β-coronavirus) and subgenus (Sarbecovirus), but differ phylogenetically and exhibit significantly different characteristics in transmissibility and clinical symptoms. These aspects suggest that comprehensive sampling of the entire Coronavirus Family is challenging to implement effective surveillance procedures, thus emphasizing the need to develop treatment strategies with broad activity against coronaviruses.

[0006] Given the crucial role of the transmembrane spike glycoprotein (S protein) of SARS-CoV-1 and SARS-CoV-2 in viral pathogenesis, it is considered a primary target for generating potent neutralizing antibodies and a focal point for developing therapeutic and preventative tools against the virus. Indeed, SARS-CoV-1 and SARS-CoV-2 entry 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 existing in a metastable pre-fusion conformation and a stable post-fusion conformation. Each S protein monomer consists of two distinct regions, the S1 and S2 subunits. When the receptor-binding domain (RBD) in the S1 subunit binds to the host cell membrane, structural rearrangement occurs. This interaction disrupts the pre-fusion state of the S protein, triggering its transition to the post-fusion conformation, thereby allowing viral particles to enter the host cell. Single-cell RNA sequencing analysis assessed ACE2 expression levels in different human organs, indicating that SARS-CoV-2, through S protein binding, can invade human cells in various major physiological systems, including the respiratory, cardiovascular, digestive, and urinary systems, thereby increasing the likelihood of transmission and infection. Therapeutic mAbs for treating COVID-19 have been developed at an unprecedented pace for any disease.

[0007] However, it is crucial to produce neutralizing mAbs or their antigen-binding portions that effectively block the entry of viruses and their variants, and which should be effective against a broader spectrum of neutralizable isolates / species. Therefore, there is an urgent need for a potent, broad-spectrum antibody therapeutic with cross-neutralizing activity against different isolates / species of the Coronaviridae family for the treatment, prevention, and diagnosis of coronaviruses, particularly SARS-CoV-2 and / or SARS-CoV-1-related diseases. Summary of the Invention

[0008] To identify potent mAbs against SARS-CoV-2, SARS-CoV-2 variants, and SARS-CoV-1, the inventors isolated thousands of S protein-specific memory B cells derived from several individuals previously infected with SARS-CoV-2 and vaccinated with either the COVID-19 BNT162b2 or mRNA-1273 mRNA vaccine. Using recombinant SARS-CoV-1 and SARS-CoV-2 S proteins as sorting decoys, the inventors isolated spike (S) protein-specific memory B cells. Naturally generated mAbs against SARS-CoV-1 or SARS-CoV-2 were then screened using a micro-neutralization assay. This screening strategy, detailed in the examples, identified ten human mAbs with potent neutralizing activity against coronaviruses, particularly SARS-CoV-2 and SARS-CoV-1.

[0009] In some aspects, the present invention provides a monoclonal antibody or antigen-binding moiety thereof capable of neutralizing the biological activity of at least one coronavirus, the antibody or antigen-binding moiety comprising a heavy chain variable domain (VH) and a light chain variable domain (VK) of a monoclonal antibody selected from the group consisting of: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13.

[0010] In some aspects, the present invention provides a monoclonal antibody or its antigen-binding portion thereof that specifically binds to the S protein of at least one virus selected from human severe acute respiratory syndrome (SARS) coronaviruses SARS-CoV-2, SARS-CoV-1, and their variants, wherein said antibody or its antigen-binding portion has neutralizing activity. Specifically, when tested in an in vitro neutralization assay against at least one virus selected from SARS-CoV-1, SARS-CoV-2, and their variants, such as against SARS-CoV-2 strains and Omeprón variants (BA.5, BA.2.75, BF.7, BQ.1.1, and XBB.1.5), such monoclonal antibodies or their antigen-binding portions exhibit a 100% inhibitory concentration (IC50) of less than 100 ng / ml. 100 ).

[0011] In some aspects, the present invention provides a monoclonal antibody or its antigen-binding portion thereof, which specifically comprises VK and VH domains, wherein the amino acid sequences of the VK and VH domains are at least 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequences of the VK and VH domains of a monoclonal antibody selected from the group consisting of: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

[0012] In some aspects, the present invention provides a monoclonal antibody or its antigen-binding portion thereof that competes with any antibody disclosed herein 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 their variants.

[0013] In some respects, the present invention provides a monoclonal antibody or antigen-binding portion as described in any of the embodiments disclosed herein for use in the prophylactic or therapeutic treatment of viral infections or symptoms or disorders caused by such infections.

[0014] In some aspects, the present invention provides a monoclonal antibody or antigen-binding portion as described in any of the embodiments disclosed herein for use in the prophylactic or therapeutic treatment of infection caused by at least one virus selected from SARS-CoV-2, SARS-CoV-1 and variants thereof, or symptoms or disorders caused by such infection, particularly COVID-19.

[0015] In some aspects, the present invention provides a method for preventing or treating infection caused by at least one virus selected from SARS-CoV-1, SARS-CoV-2 and variants thereof, or symptoms or disorders caused by such infection, particularly COVID-19, comprising administering to a subject in need a monoclonal antibody or antigen-binding portion according to any embodiment disclosed herein.

[0016] This invention also provides for the use of monoclonal antibodies or antigen-binding moieties according to any of the embodiments disclosed herein in the diagnosis, prevention, and / or treatment of subjects suffering from or at risk of developing a viral infection, particularly coronavirus infection, and more particularly infections caused by at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants. Furthermore, this invention relates to the use of the binding molecules and / or nucleic acid molecules of this invention in the diagnosis / detection of such viral infections.

[0017] In some aspects, the present invention provides a pharmaceutical composition comprising at least one or more monoclonal antibodies or antigen-binding portions thereof as described in any of the embodiments disclosed herein and a pharmaceutically acceptable carrier, and its use in the prevention and / or treatment of coronavirus infection or symptoms or disorders caused by such infection, preferably selected from infections caused by at least one virus selected from SARS-CoV-1, SARS-CoV-2 and variants thereof, particularly COVID-19.

[0018] In some aspects, the present invention provides an isolated cell line that produces an antibody or an antigen-binding portion thereof as described in any of the embodiments disclosed herein.

[0019] In some aspects, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding portion thereof as described in any of the embodiments disclosed herein.

[0020] In some aspects, the present invention provides a vector comprising a nucleic acid molecule encoding an antibody or an antigen-binding portion thereof as described in any of the embodiments disclosed herein, wherein the vector optionally comprises an expression control sequence operatively linked to the nucleic acid molecule.

[0021] In some aspects, the present invention provides a non-human transgenic animal or transgenic plant comprising the nucleic acid described in any of the foregoing embodiments, wherein the non-human transgenic animal or transgenic plant expresses the nucleic acid. In some embodiments, the non-human transgenic animal is a mammal.

[0022] In some respects, the present invention provides the use of monoclonal antibodies or antigen-binding portions thereof as described in any of the embodiments disclosed herein in the in vitro or ex vivo diagnosis of coronavirus infections, preferably infections caused by at least one virus selected from SARS-CoV-1, SARS-CoV-2 and variants thereof.

[0023] In some aspects, the present invention provides an in vitro method for determining the presence of at least one coronavirus in a sample, preferably selected from at least one virus selected from SARS-CoV-1, SARS-CoV-2, and their variants, the method comprising the following steps:

[0024] i) Contact with an antibody or its antigen-binding portion as described in any of the embodiments disclosed herein;

[0025] ii) Detect the binding of the antibody or its antigen-binding portion to the S protein of at least one coronavirus.

[0026] In some aspects, the present invention provides an in vitro method for diagnosing coronavirus infection in a subject, preferably caused by at least one virus selected from SARS-CoV-1, SARS-CoV-2, and their variants, the method comprising the following steps:

[0027] i) Contact the antibody or its antigen-binding portion as described in any of the embodiments disclosed herein with a biological sample of the subject;

[0028] ii) Detect the binding of the antibody or its antigen-binding portion to the S protein of at least one coronavirus.

[0029] In some aspects, the present invention provides a diagnostic kit comprising an antibody or its antigen-binding portion as described in any of the embodiments disclosed herein as a specific reagent, said kit being intended for use in methods for detecting or quantifying anti-coronavirus antibodies and / or coronavirus S protein in biological samples of patients, particularly anti-SARS-CoV-2 and / or anti-SARS-CoV-1 antibodies, and / or SARS-CoV-2 and / or SARS-CoV-1 S protein.

[0030] In some respects, the present invention provides the use of antibodies or antigen-binding portions thereof as described in any of the embodiments disclosed herein in the design of vaccines against at least one coronavirus, particularly against at least one virus selected from SARS-CoV-1, SARS-CoV-2 and variants thereof.

[0031] In some aspects, the present invention provides a mimic epitope that is specific to an idiotype of an antibody or its antigen-binding moiety as described in any of the embodiments disclosed herein.

[0032] In some aspects, the present invention provides an anti-idiotype antibody that is specific to an idiotype of the antibody or its antigen-binding portion as described in any of the embodiments disclosed herein.

[0033] This invention encompasses any combination of the foregoing aspects and embodiments.

[0034] Brief description of the attached figures

[0035] Figure 1 - Characteristics of neutralizing human coronavirus antibodies. The heatmap shows the binding of 10 nAbs screened according to the present invention, their neutralizing activity against SARS-CoV-1 and SARS-CoV-2, and the identified epitope regions. The titer legend only describes neutralizing activity. Detailed Implementation

[0036] Unless otherwise defined herein, scientific and technical terms related to this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural meanings, and plural terms shall include singular meanings. Generally, the terms and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of this invention are generally performed according to conventional methods well-known in the art and the methods and techniques described and discussed in the various general and more specific references cited and discussed in this specification. See Sambrook et al. Molecular Cloning: A Laboratory Manual, second ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); 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), all of which are incorporated herein by reference.

[0037] Unless otherwise stated, the following terms shall be understood to have the following meanings:

[0038] The term "peptide" encompasses natural or artificial proteins, protein fragments, and peptide analogs of protein sequences. Peptides can be monomers or polymers. The terms "isolated protein," "isolated peptide," or "isolated antibody" refer to a protein, peptide, or antibody that, due to its source or manner of derivation, is characterized by: (1) not being associated with any naturally related components in its native state; (2) not containing other proteins from the same species; (3) being expressed by cells of a different species; or (4) not existing in nature. Thus, a peptide synthesized chemically or in a cellular system different from the cells of its natural origin is "isolated" from its naturally related components. Isolation can also be achieved by using protein purification techniques well known in the art, making the protein substantially free of its naturally related components. Examples of antibodies for isolation include anti-SARS-CoV-2 and / or SARS-CoV-1 S protein antibodies affinity-purified using SARS-CoV-2 and / or SARS-CoV-1 S protein or portions thereof; anti-SARS-CoV-2 and / or SARS-CoV-1 S protein antibodies synthesized in vitro from hybridoma or other cell lines; and human anti-SARS-CoV-2 and / or SARS-CoV-1 S protein antibodies derived from transgenic animals. A protein or polypeptide is considered “substantially pure,” “substantially homogeneous,” or “substantially purified” when at least about 60% to 75% of the sample is presented as a single polypeptide. The polypeptide or protein can be a monomer or a polymer. Substantially pure polypeptides or proteins typically constitute about 50%, 60%, 70%, 80%, or 90% w / w of the protein sample, more commonly about 95%, and preferably more than 99% pure. Protein purity or homogeneity can be indicated by a variety of methods well known in the art, such as performing polyacrylamide gel electrophoresis on the protein sample followed by staining the gel with dyes well known in the art to observe individual polypeptide bands. For certain purposes, higher resolution can be provided by using high-performance liquid chromatography (HPLC) or other methods well known in the art for purification. The term "peptide fragment" as used herein refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion, but whose remaining amino acid sequence is identical to the corresponding position in the native sequence. In some embodiments, the fragment length is at least 5, 6, 8, or 10 amino acids. In other embodiments, the fragment length is at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150, or 200 amino acids. The term "peptide analog" as used herein refers to a polypeptide comprising a portion substantially identical to a portion of the amino acid sequence and having at least one of the following properties: (1) specifically binding to at least one coronavirus S protein under suitable binding conditions, and (2) being able to inhibit said at least one coronavirus S protein. Typically, peptide analogs contain conserved amino acid substitutions (or insertions or deletions) relative to the native sequence.Analogs are typically at least 20 or 25 amino acids in length, preferably at least 50, 60, 70, 80, 90, 100, 150, or 200 amino acids or longer, and can generally be as long as the full-length polypeptide. Some embodiments of the invention include polypeptide fragments or polypeptide analog antibodies having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 substitutions compared to the germline amino acid sequence. In some embodiments, amino acid substitutions against coronavirus S protein antibodies or their antigen-binding portions refer to those that: (1) reduce sensitivity to proteolysis, (2) reduce sensitivity to oxidation, (3) alter binding affinity to form a protein complex, and (4) impart or modify other physicochemical or functional properties to such analogs while still retaining specific binding to at least one coronavirus S protein. Analogs may include various mutants of sequences other than the native peptide sequence. For example, one or more amino acid substitutions, preferably conserved amino acid substitutions, can be made in the native sequence, preferably in the polypeptide portion outside the domains forming intermolecular contacts. Conserved amino acid substitutions should not significantly alter the structural characterization of the parental sequence; for example, the substituted amino acid should not alter the antiparallel [β]-sheet constituting the immunoglobulin-binding domain in the parental sequence, or disrupt other types of secondary structures characterizing the parental sequence. Generally, glycine and proline are not used for antiparallel [β]-sheets. Examples of recognized polypeptide secondary and tertiary structures in the art can be found in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature 354:105 (1991), which are incorporated herein by reference.

[0039] The term "SARS-CoV-1" refers to the severe acute respiratory syndrome coronavirus (also known as SARS-CoV or SARS-CoV-1) (World Health Organization, 2020). SARS coronavirus (SARS-CoV) is a member of the Coronaviridae family, an encapsulated, positive-sense RNA virus, and a group of viruses with a wide host range. It contains three major structural proteins: the spike protein (S), the membrane protein (M), and the nucleocapsid protein (N). Although passive protection against murine hepatitis virus (MHV, a well-studied coronavirus) infection has been demonstrated through the administration of mAbs targeting all the major structural proteins of the virus, the spike protein (S) is the major antigenic determinant of coronaviruses.

[0040] The term “SARS-CoV-2” refers to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the type of coronavirus that causes COVID-19 (World Health Organization, 2020).

[0041] As used herein, the term “variant” in relation to SARS-CoV-1 and / or SARS-CoV-2 viruses is intended to cover any known variants of the viruses, such as SARS-CoV-2 strain variants and Omeprón variants, such as BA.5, BA.2.75, BF.7, BQ.1.1, and XBB.1.5.

[0042] When “antibody” is used in this invention, it is generally understood that its antigen-binding portion may also be used. The antigen-binding portion competes with the intact antibody for specific binding. See Fundamental Immunology, Ch. 7 (Paul, W., ed., second ed. Raven Press, NY (1989) (the entire text of which is incorporated herein by reference for full understanding). The antigen-binding portion may be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. In some embodiments, the antigen-binding portion includes Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, biantibodies, nanobodies, and any polypeptide containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0043] From the N-terminus to the C-terminus, both the mature light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. The amino acid assignments for each domain in this paper follow the definitions of the IMGT convention described in Lefranc et al. (2003), Developmental & Comparative Immunology 27.1 (2003): 55-77.

[0044] As used herein, antibodies represented by numbers are the same as monoclonal antibodies obtained from wells of a specific sorted B cell plate; for example, antibody 01J19 was obtained from well J19 of a plate with ID 01.

[0045] As used in this article, the Fd fragment refers to an antibody fragment composed of the VH and CH1 domains; the Fv fragment is composed of the VK or VL and VH domains of the antibody single arm; and the dAb fragment (Ward et al, Nature 341:544-546 (1989)) is composed of the VH domain.

[0046] In some embodiments, the antibody is a single-chain antibody (scFv) in which the VL or VK domain pairs with the VH domain via a synthetic linker to form a monovalent molecule, which enables them to be prepared as a single protein chain (Bird et al, Science 242:423-426 (1988) and Huston et al, Proc. Natl Acad. ScL USA 85:5879-5883 (1988)). In some embodiments, the antibody is a bivalent antibody, where the VH and VL or VK domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain. This forces these domains to pair with complementary domains on another chain, forming two antigen-binding sites (see, for example, Holliger P. et al, Proc. Natl. Acad. ScL USA 90:6444-6448 (1993), and Poljak RJ et al, Structure 2:1121-1123 (1994)). In such embodiments, the CDR may be incorporated as part of a larger polypeptide chain, covalently linked to another polypeptide chain, or non-covalently incorporated. In embodiments with one or more binding sites, the binding sites may be identical or different.

[0047] In certain preferred aspects of the invention, the monoclonal antibody in any embodiment disclosed in this specification and claims is a human antibody, i.e., a human monoclonal antibody, or its antigen-binding portion.

[0048] As used herein, the term "human antibody" refers to any antibody whose variable and constant domain sequences are human-derived sequences, or any CDR of the variable domain sequence is a human-derived sequence. The term encompasses antibodies whose sequences are derived from human genes but have been modified (e.g., to reduce potential immunogenicity, increase affinity, eliminate cysteine ​​residues that may cause undesirable folding, etc.). The term also encompasses antibodies recombined in non-human cells that may possess glycosylation features specific to non-human cells. As used herein, the term "chimeric antibody" refers to an antibody comprising regions derived from two or more different antibodies.

[0049] The term "epitaph" refers to any protein determinant capable of specifically binding to immunoglobulins or T-cell receptors or otherwise interacting with molecules. Epitopes, or antigenic determinants, are typically composed of surface groups on chemically active molecules, such as amino acids or carbohydrate or sugar side chains, and usually possess specific three-dimensional structural features as well as specific charge characteristics. Epitopes can be "linear" or "conformal." In a linear epitope, all interaction sites between the protein and the interacting molecule (such as an antibody) are arranged linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites are distributed on amino acid residues on the protein that are separated from each other.

[0050] "Neutralizing antibody" (also referred to herein as nAb), antibody with "neutralizing activity," or antibody "capable of neutralizing biological activity" as used herein refers to an antibody that neutralizes the biological effects that its target (such as a pathogen or infectious particle) may have. For example, as used herein, "neutralizing antibody," antibody with "neutralizing activity," or antibody "capable of neutralizing biological activity" refers to an antibody or its antigen-binding moiety that, 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-2, and variants thereof, shows a 100% inhibitory concentration (IC100) of at least 100 ng / ml, preferably less than 50 ng / ml, more preferably less than 25 ng / ml, as performed, for example, as in the examples herein.

[0051] An antibody is considered to specifically bind to an antigen when the dissociation constant is, for example, ≤1 mM, preferably <100 nM, and most preferably <10 nM. The dissociation constant can be measured by any method available in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, or surface plasmon resonance, such as BIACORE™. For example, the expression “specifically binds to the region of the coronavirus spike (S) protein” in this document means that the antibody or its antigen-binding portion induces more than 50% inhibition of the interaction between the human ACE2 receptor and the viral spike protein, as measured by the NOB assay described in the examples.

[0052] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides with a length of at least 10 bases, which is a ribonucleotide or deoxyribonucleotide, or a modified form of any of these nucleotides. This term includes both single-stranded and double-stranded forms.

[0053] As used herein, the term “isolated polynucleotide” refers to a polynucleotide derived from a genome, cDNA, or synthesized, or a combination thereof. Due to its origin, the “isolated polynucleotide”: (1) is not associated with all or part of a polynucleotide found in nature that coexists with the “isolated polynucleotide”; (2) is operatively linked to a polynucleotide not associated with it in nature; or (3) is not present in nature as part of a larger sequence.

[0054] As used herein, the term "naturally occurring nucleotide" includes deoxyribonucleotides and ribonucleotides. As used herein, the term "modified nucleotide" includes nucleotides having modified or replaced sugar groups. The term "oligonucleotide link" as used herein includes oligonucleotide links such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, aniline thiophosphates, phoshoraniladates, and phosphoramide esters. See, for example, LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984); Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design 6:539 (1991); Zon et al.. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); U.S. Patent No. 5,151,510; Uhlmann and Peyman, ChemicalReviews 90:543 (1990), the disclosures of which are incorporated herein by reference in their entirety. If desired, oligonucleotides may contain detection markers. "Operable linker" sequences include expression control sequences adjacent to the target gene and expression control sequences that act at a distance or in a trans manner to control the target gene. As used herein, the term "expression control sequence" refers to a polynucleotide sequence essential to the expression and processing of the coding sequence to which it is linked. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and sequences that enhance protein secretion when needed. The nature of such control sequences varies from host organism to host organism; in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may also include other components that are beneficial in their presence, such as leader sequences and fusion chaperone sequences. The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.In some embodiments, the vector is a plasmid, i.e., a circular double-stranded DNA segment therein to which other DNA fragments can be ligated. In some embodiments, the vector is a viral vector in which other DNA fragments can be ligated into a viral genome. In some embodiments, the vector is capable of autonomous replication in the host cell to which it is introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). In other embodiments, the vector (e.g., a non-free mammalian vector) is capable of integrating into the host cell's genome after being introduced into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0055] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It should be understood that "recombinant host cell" and "host cell" refer not only to the specific test cell but also to the progeny of such cells. Due to mutations or environmental influences, certain modifications may occur during successive passages, and therefore these progeny cells may not actually be completely identical to the parent cells, but they still fall within the scope of the term "host cell" as used herein.

[0056] In the context of nucleotide or amino acid sequences, the term "percentage of sequence identity" refers to the number of identical residues in two sequences when aligned with maximum correspondence. The length of a sequence identity comparison can exceed at least about 9 nucleotides, typically at least about 18 nucleotides, more commonly 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. Several different algorithms are known in the art for measuring nucleotide sequence identity. For example, FASTA, Gap, or Bestfit can be used to compare polynucleotide sequences, which are programmatically available and provide alignments of the best overlapping regions between the query and search sequences and percentage sequence identity (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J. Mol. Biol. 276:71-84 (1998); incorporated herein by reference). When the terms "substantially similar" or "substantially sequence similar" are used with nucleic acids or fragments thereof or amino acids, they mean that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, the nucleotide sequence identity is at least about 85%, preferably at least about 90%, 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 described above. When applied to peptides, the term "substantially identical" means that two peptide sequences, when optimally aligned (e.g., by the GAP or BESTFIT procedure with the default gap weights provided by the procedure), share at least 70%, 75%, or 80% sequence identity, preferably at least 90% or 95%, more preferably at least 97%, 98%, or 99% sequence identity. In some embodiments, the positions of non-identical residues differ by conserved amino acid substitutions. A "conserved amino acid substitution" means that one amino acid residue is replaced by another amino acid residue with a side chain R group having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not significantly alter the functional properties of proteins. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the percentage of sequence identity can be upregulated to correct for the conservation of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, for example, Pearson, Methods Mol. Biol. 243:307-31 (1994).Examples of amino acid groups with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy 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; 7) sulfur-containing side chains: cysteine ​​and methionine. Groups of conserved amino acid substitutions include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conserved substitution refers to any change with a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al, Science 256:1443-45 (1992), which is incorporated herein by reference. "Moderately conserved" substitution refers to any change that has a non-negative value in the PAM250 log-likelihood matrix. Peptide sequence identity is typically measured using sequence analysis software. Protein analysis software uses similarity measures assigned to different substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match sequences. For example, GCG includes programs such as "Gap" and "Bestfit," which can use default parameters to determine sequence homology or sequence identity between closely related peptides, such as homologous peptides from different biological species or between wild-type proteins and their mutants.

[0057] As used herein, the terms "labeled" or "labeled" refer to the incorporation of an additional molecule into an antibody. In one embodiment, the label is a detectable marker, such as a polypeptide incorporating a radiolabeled amino acid or attached to a biotinylate group detectable by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable 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 known in the art are available.

[0058] Anti-coronavirus antibodies and their characteristics

[0059] In one embodiment, the present invention provides a monoclonal antibody or antigen-binding portion thereof capable of neutralizing the biological activity of at least one coronavirus, the antibody or antigen-binding portion thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VK) of a monoclonal antibody selected from the group consisting of: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13.

[0060] The nucleic acids containing the variable domains (VH and VL) in the heavy and light chains of the antibodies numbered 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13 in this specification, along with their corresponding deduced amino acid sequences, can be found in the sequence listing attached to this specification.

[0061] In some aspects, the present invention provides a monoclonal antibody or its antigen-binding portion 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 sequence of the heavy chain variable domain 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 amino acid sequence of the light chain variable domain of an antibody selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13; (c) (a) the heavy chain variable domain and (b) the light chain variable domain; or (d) the amino acid sequences of the heavy chain and light chain variable domains, respectively containing the heavy chain and light chain variable domain amino acid sequences of the same antibody selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13.

[0062] In some aspects, the present invention provides a monoclonal antibody or its antigen-binding portion 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 CDR1, CDR2, and CDR3 amino acid sequences of antibodies 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 antibodies selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13; (c) the heavy chain variable domain of (a) and the light chain variable domain of (b); or (d) (c) The heavy chain variable domain and light chain variable domain contain the heavy chain and light chain CDR amino acid sequences of the same antibody selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13.

[0063] In some aspects, the present invention provides a monoclonal antibody or its antigen-binding portion thereof that specifically binds to the S protein of at least one coronavirus, wherein the antibody comprises a VH and VL amino acid sequence selected from the following antibodies: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13.

[0064] In some aspects, the present invention provides a monoclonal antibody capable of neutralizing the biological activity of at least one coronavirus, wherein the 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 present invention provides a monoclonal antibody capable of neutralizing the biological activity of at least one coronavirus, wherein the 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 present invention provides a monoclonal antibody capable of neutralizing the biological activity of at least one coronavirus, wherein the antibody comprises the heavy and light chains of the same antibody selected from the group consisting of: O1J19, O2K18, O5F22, O1J18, O2K05, O1B20, O2G11, O3O07, O5N18 and O2N13.

[0065] In some aspects, the present invention provides a monoclonal antibody or its antigen-binding portion thereof capable of neutralizing the biological activity of at least one coronavirus, the monoclonal antibody or its antigen-binding portion comprising VL and VH domains, the VL and VH domains having amino acid sequence identity of at least 85%, 90%, 95%, 97%, 98%, or 99% with the VL and VH domains of a monoclonal antibody selected from the group consisting of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13, respectively.

[0066] In some aspects, the present invention provides a monoclonal antibody or its antigen-binding moiety capable of neutralizing the biological activity of at least one coronavirus, the monoclonal antibody or its antigen-binding moiety comprising a light chain and a heavy chain, the light chain and the heavy chain having an amino acid sequence identity of at least 85%, 90%, 95%, 97%, 98% or 99% with the light chain and the heavy chain of the monoclonal antibody selected from the group consisting of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13, respectively.

[0067] Preferably, the monoclonal antibody described in any embodiment disclosed in this specification and claims is a human antibody or a human monoclonal antibody.

[0068] According to a preferred aspect, the monoclonal antibody or its antigen-binding portion as described in any embodiment disclosed in this specification and claims is capable of neutralizing the biological activity of at least one coronavirus 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 selected from SARS-CoV-1, SARS-CoV-2 and variants thereof.

[0069] In another preferred aspect, the monoclonal antibody or its antigen-binding portion as described in any embodiment disclosed in this specification and claims is capable of neutralizing the biological activity of at least two, at least three, or at least four species belonging to the Coronaviridae family, or exhibits neutralizing activity against at least two, at least three, or at least four species belonging to the Coronaviridae family, particularly at least against SARS-CoV-2 and SARS-CoV-1.

[0070] In some respects, the monoclonal antibody or its antigen-binding portion as described in any embodiment disclosed in this specification and claims specifically binds to the spike (S) protein region of at least one coronavirus, particularly the S protein of SARS-CoV-2 and / or SARS-CoV-1, and at least partially inhibits the binding of the S protein to the receptor.

[0071] In one embodiment, the region is i) the S1 domain of the S protein; or (ii) the S2 domain of the S protein; or (iii) the S protein trimer of at least one coronavirus, in its pre-fusion or post-fusion conformation, or a combination of i) and ii), or a combination of i) and iii), or a combination of ii) and iii), or a combination of i) and iv), or a combination of ii) and iv).

[0072] Preferably, the region is a trimer of the S protein of SARS-CoV-2 and / or SARS-CoV-1 and / or any variant thereof. Alternatively, the region is an NTD domain of the S protein of SARS-CoV-2 and / or SARS-CoV-1 and / or any variant thereof.

[0073] In one embodiment, the present invention provides a monoclonal antibody or antigen-binding portion thereof that specifically binds to at least one coronavirus, wherein the antibody or antigen-binding portion thereof produces an inhibition of 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or greater against the interaction between the viral spike protein of the at least one coronavirus and the human ACE2 receptor, as determined by a binding neutralization (NOB) assay.

[0074] In some respects, a monoclonal antibody or its antigen-binding portion according to any embodiment disclosed herein specifically binds to the S protein of at least one coronavirus, wherein said antibody or its antigen-binding portion has neutralizing activity. Specifically, when subjected to an in vitro neutralization assay against said at least one coronavirus at 100 TCID50... 50 When viral doses were tested, these monoclonal antibodies or their antigen-binding moieties showed an inhibitory concentration (IC50) of less than 100 ng / ml, preferably less than 50, 25, 20, 10, 8, 6, 5, 4, 3, 2 or 1 ng / ml. 100 For example, targeting at least one virus selected from SARS-CoV-1, SARS-CoV-2 and their variants.

[0075] One possible amino acid substitution involves changing one or more cysteine ​​residues (potentially chemically reactive) in the antibody to another residue, such as, but not limited to, alanine or serine. In one embodiment, a non-classical cysteine ​​substitution is performed. The substitution can be performed in the CDR or frame region of the antibody's variable domains, or in constant domains. In some embodiments, the cysteine ​​is classical.

[0076] Another possible amino acid substitution is altering any potential proteolytic sites in the antibody. Such sites can be located in the CDR or frame region of the antibody's variable domains, or in constant domains. Replacing cysteine ​​residues and removing proteolytic sites can reduce the risk of any heterogeneity in the antibody product, thereby improving its homogeneity. Another amino acid substitution is eliminating the asparagine-glycine pair that forms a potential deamidation site by altering one or two residues. In some embodiments, the C-terminal lysine of the heavy chain of the anti-coronavirus S protein antibody of the present invention is cleaved. In various embodiments of the present invention, the heavy and light chains of the anti-coronavirus S protein antibody may optionally contain a signal sequence.

[0077] The class and subclass of the antibody according to the invention can be determined by any method known in the art. Typically, the class and subclass of the antibody can be determined using antibodies specific to a particular class and subclass. Such antibodies are commercially available. The class and subclass can be determined by ELISA or Western blotting (immunoblotting) and other techniques. Alternatively, the class and subclass can be determined by sequencing all or part of the constant domains of the antibody's heavy chain and / or light chain; comparing its amino acid sequence with the amino acid sequences of various known classes and subclasses of immunoglobulins; and determining the class and subclass of the antibody.

[0078] In some embodiments, the antibody described according to any of the embodiments disclosed herein is an IgG, IgM, IgE, IgA, or IgD molecule. In one embodiment, the antibody is IgG, and is a subclass of IgG1, IgG2, IgG3, or IgG4. In another embodiment, the human antibody subclass is IgG1.

[0079] In some embodiments of the invention, the antibody described in any of the embodiments disclosed herein binds with high affinity to the S protein of at least one coronavirus, particularly at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants. In some embodiments, the antibody described in any of the embodiments disclosed herein binds with high affinity to the SARS-CoV-2 and / or SARS-CoV-1 S protein trimer. In some embodiments, the antibody binds to the N-terminal domain or receptor-binding domain (RBD) of the SARS-CoV-2 and / or SARS-CoV-1 S protein. In another embodiment, the antibody binds to the S1 or S2 domain of the SARS-CoV-2 and / or SARS-CoV-1 S protein. The binding affinity and dissociation rate of the antibody to the coronavirus S protein can be determined by methods known in the art. Binding affinity can be measured by ELISA, RIA, flow cytometry, or surface plasmon resonance (e.g., BIACORE™). Dissociation rate can be measured by surface plasmon resonance. Preferably, binding affinity and dissociation rate are measured using surface plasmon resonance. More preferably, affinity and dissociation rate are measured using BIACORE™. A person skilled in the art can determine whether an antibody has substantially the same KD as an anti-coronavirus S protein antibody using methods known in the art.

[0080] The present invention also provides a monoclonal antibody that binds to the S protein of at least one coronavirus, particularly at least one virus selected from SARS-CoV-1, SARS-CoV-2, and their variants, and competes or cross-competes with and / or binds to the same epitope with antibodies selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13. If two antibodies compete with each other when binding to the S protein of said at least one coronavirus, they are said to be cross-competing.

[0081] Those skilled in the art can determine whether an antibody binds to the same epitope as, or competes with, the anti-S protein antibody according to the present invention using methods known in the art. In one embodiment, those skilled in the art allow the antibody of the present invention to bind to the S protein of at least one coronavirus under saturation conditions, and then measure the binding ability of the test antibody to the S protein. If the test antibody can bind to the S protein simultaneously with the antibody of the present invention, the epitope bound by the test antibody is different from the epitope bound by the antibody of the present invention. However, if the test antibody cannot bind to the S protein of at least one coronavirus simultaneously, the epitope bound by the test antibody is the same as, overlaps with, or is close to the epitope bound by the antibody of the present invention, or the binding of the antibody according to the present invention can induce a conformational change in the S protein of at least one coronavirus, which prevents or reduces the binding of the test antibody. This experiment can be performed using ELISA, RIA, BIACORE™, flow cytometry, or other methods known in the art.

[0082] To test whether the antibody according to the invention cross-competes with another antibody capable of binding to at least one coronavirus S protein, a person skilled in the art can use the above-described competitive method in two directions: determining whether the reference antibody blocks the test antibody, and vice versa. In one embodiment, the experiment is performed using an ELISA. The method for determining KD will be discussed further below.

[0083] In another embodiment, the present invention provides an antibody that inhibits, blocks, or reduces the binding of the coronavirus S protein to a receptor, particularly to angiotensin-converting enzyme 2 (ACE2). In another embodiment, the present invention provides an antibody that inhibits, blocks, or reduces coronavirus S protein-mediated viral entry into cells. In another embodiment, the present invention provides an antibody against the SARS-CoV-2 S protein and / or against SARS-CoV-1 or its variants, which inhibits, blocks, or reduces viral fusion with the cell membrane. In another embodiment, the present invention provides an antibody that reduces viral load. In another embodiment, the present invention provides an antibody that inhibits, blocks, or reduces the severity of symptoms or illness caused by SARS-CoV-2 and / or SARS-CoV-1 infection at any time period. In some embodiments, the present invention provides an antibody that inhibits, blocks, or reduces the severity of symptoms or illness caused by 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% over one day, one week, one month, six months, one year, or the remaining lifespan of a subject. In some embodiments, the present invention provides an antibody that can perform any combination of the foregoing embodiments.

[0084] In some implementations, the constant region of the antibody's mAb is modified to prolong its half-life and reduce the risk of antibody-dependent enhancement (ADE) of the disease. For example, to enhance the therapeutic activity of the mAb, two different sets of substitution mutations (M252Y / S254T / T256E, according to Dall'Acqua et al., 2006; M428L / N434S, as reported by Zalevsky et al., 2010) can be applied to its constant domain.

[0085] In some implementations, to reduce the risk of antibody-dependent enhancement (ADE) of the disease, mutations eliminating binding to the Fc receptor are introduced into the Fc region of the IgG1 molecule, as previously described (L234A / L235A, Hezareh et al., 2001; Beltramello et al., 2010; P329G LALA, Schlothauer et al., 2016). All of these modifications can be performed via site-directed mutagenesis, for example using the Agilent Quick-Change II site-directed mutagenesis kit, following the manufacturer's recommendations.

[0086] In some embodiments, the antibody comprises a variable region and a mutated IgG1 constant region backbone selected from antibodies of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13, which includes one or more mutations from the following mutation groups: L234A / L235A (Hezareh et al., 2001; Beltramello et al., 2010), P329G (Schlothauer et al., 2016); M428L / N434S (Zalevsky et al., 2010). Preferably, the antibody comprises a variable region and a mutated IgG1 constant region backbone selected from antibodies of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13, which contains all three groups of such mutations.

[0087] Recombinant methods for nucleic acids, vectors, host cells, and antibody preparation

[0088] Nucleic acid

[0089] This invention also covers nucleic acid molecules encoding antibodies or antigen-binding moieties thereof as described in any of the embodiments disclosed herein. In some embodiments, different nucleic acid molecules encode the heavy and light chains of antibodies according to the invention. In other embodiments, the same nucleic acid molecule encodes the heavy and light chains of antibodies according to the invention. In one embodiment, the nucleic acid encodes an antibody or antigen-binding moieties thereof of the invention. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a VK amino acid sequence that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions and / or 1, 2, or 3 non-conserved substitutions compared to the germline. Substitutions may be in CDR regions, frame regions, or constant domains. In some embodiments, the nucleic acid molecule encodes a VK amino acid sequence containing one or more variations compared to the germline sequence, which 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.

[0090] In some embodiments, the nucleic acid molecule encodes at least three amino acid substitutions compared to the germline sequence found in the VK of an antibody selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13.

[0091] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a VK amino acid sequence of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13, or variants thereof, or portions thereof. In some embodiments, the nucleic acid encodes an amino acid sequence of a light chain CDR of an antibody comprising one of the antibodies listed above. In some embodiments, the portion is a continuous portion comprising CDR1-CDR3. In some embodiments, the nucleic acid encodes an amino acid sequence of the light chain CDR of the antibody. In some embodiments, the portion encodes a continuous region of the light chain CDR1-CDR3 of the antibody according to the invention.

[0092] In some embodiments, the nucleic acid molecule encodes a VK amino acid sequence whose identity with the VK region of any one of the antibodies 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. The nucleic acid molecules of the present invention include nucleic acids (as described above) that hybridize under highly stringent conditions to a nucleotide sequence encoding a VK region amino acid sequence.

[0093] In another embodiment, the nucleic acid encodes the full-length light chain of an antibody selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13, or a light chain containing a mutation (such as those disclosed herein).

[0094] In another embodiment, the nucleic acid molecule encodes a heavy chain variable domain (VH) comprising a human VH1, VH3, or VH4 family gene sequence or a sequence derived therefrom. In some embodiments, compared to the germline sequence, the nucleic acid molecule encodes one or more amino acid mutations that are identical to amino acid mutations found in the VH of one of the monoclonal antibodies 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

[0095] In some embodiments, the nucleic acid molecule comprises at least a portion, all three CDR regions, a continuous portion of CDR1-CDR3, or the entire VH region of a monoclonal antibody selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13, 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 the antibodies selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13, or the sequence lacking a signal sequence. In some preferred embodiments, the nucleic acid molecule comprises at least a portion of the nucleotide sequence of one of the antibodies selected from 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13, or the sequence lacking the signal sequence. In some embodiments, the portion encodes a VH region (with or without the signal sequence), a CDR3 region, all three CDR regions, or a contiguous region comprising CDR1-CDR3.

[0096] 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 sequence of any of the antibodies 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13.

[0097] The nucleic acid molecules of the present invention include nucleic acids (as described above) that hybridize under highly stringent conditions with nucleotide sequences encoding amino acid sequences of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13 or encoding their VH regions.

[0098] 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 (with or without a signal sequence) encoding an amino acid sequence of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13, or a heavy chain containing a mutation (such as one of the variants described herein). In addition, nucleic acids may contain nucleotide sequences of 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18 and 02N13 (with or without a signal sequence), or contain nucleic acid molecules that encode a heavy chain containing a mutation (such as one of the variants described herein).

[0099] Nucleic acid molecules encoding the heavy or light chain of an antibody or a portion thereof according to the invention can be isolated from any source that produces such antibodies. In various embodiments, the nucleic acid molecules are isolated from B cells derived from animals immunized with the S protein of at least one coronavirus (such as SARS-CoV-2 and / or SARS-CoV-1 or variants thereof); or from immortalized cells derived from a class of B cells expressing or encoding antibodies against SARS-CoV-2 and / or SARS-CoV-1 S proteins. Methods for isolating the mRNA encoding the antibody are well known in the art. See, for example, Sambrook et al. mRNA can be used to generate cDNA for polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In one embodiment, the nucleic acid molecules are isolated from hybridomas having human immunoglobulin-producing cells from non-human transgenic animals as one of their fusion partners. In a more preferred embodiment, the human immunoglobulin-producing cells are isolated from XENOMOUSE animals. In another embodiment, the human immunoglobulin-producing cells are derived from non-human, non-mouse transgenic animals as described above. In another embodiment, the nucleic acid is isolated from a non-human, non-GMO animal. Nucleic acid molecules isolated from non-human, non-GMO animals can be used, for example, for humanized antibodies. In some embodiments, the nucleic acid encoding the heavy chain of the antibody of the present invention may contain a nucleotide sequence encoding the VH domain of the present invention, which is in-frame linked with a nucleotide sequence encoding a constant domain of the heavy chain from any source. Similarly, the nucleic acid molecule encoding the light chain of the antibody of the present invention may contain a nucleotide sequence encoding the VK domain of the present invention, which is in-frame linked with a nucleotide sequence encoding a constant domain of the light chain from any source. In another aspect of the invention, nucleic acid molecules encoding variable domains of the heavy chain (VH) and / or the light chain (VL or VK) are “converted” into a full-length antibody gene. In one embodiment, a nucleic acid molecule encoding a VH, VL, or VK domain is converted into a full-length antibody gene by inserting a nucleic acid molecule encoding a heavy chain constant (CH) or light chain constant (CL) domain into an expression vector, such that the VH fragment is operatively linked to the CH fragment within the vector, and / or the VL or VK fragment is operatively linked to the CL fragment within the vector. In another embodiment, a nucleic acid molecule encoding a VH and / or VL or VK domain is converted into a full-length antibody gene by using standard molecular biology techniques to link (e.g., ligate) a nucleic acid molecule encoding a CH and / or CL domain. The nucleotide sequences of human heavy chain and light chain immunoglobulin constant domain genes are known in the art.See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publ. No. 91-3242, 1991. The nucleic acid molecules encoding the full-length heavy and / or light chains can then be expressed by the cells to which they are introduced, and antibodies can be isolated.

[0100] Nucleic acid molecules can be used for recombinant expression of large quantities of the antibodies of this invention. Nucleic acid molecules can also be used to produce chimeric antibodies, bispecific antibodies, single-chain antibodies, immunoadhesins, biantibodies, mutant antibodies, and antibody derivatives, as further described below. If the nucleic acid molecule is derived from a non-human, non-transgenic animal, it can be used for antibody humanization, also as described below.

[0101] In another embodiment, the nucleic acid molecule of the present invention is used as a probe or PCR primer targeting a specific antibody sequence. For example, the nucleic acid can be used as a probe in a diagnostic method or as a PCR primer to amplify a DNA region, including but not limited to other nucleic acid molecules that can be used to isolate variable domains encoding antibodies. In some embodiments, the nucleic acid molecule is an oligonucleotide. In some embodiments, the oligonucleotide is derived from highly variable domains of the heavy and light chains of the target antibody. In some embodiments, the oligonucleotide encodes all or part of one or more CDRs of the antibodies described herein, such as 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13, or variants thereof.

[0102] carrier

[0103] This invention provides a vector comprising a nucleic acid molecule encoding the heavy chain of an antibody of the present invention or its antigen-binding portion thereof. This invention also provides a vector comprising a nucleic acid molecule encoding the light chain of such an antibody or its antigen-binding portion thereof. This invention further provides a vector comprising a nucleic acid molecule encoding a fusion protein, a modified antibody, an antibody fragment, and a probe thereof. In some embodiments, the gene is operatively linked to necessary expression control sequences (such as transcription and translation control sequences) to express the antibody or antigen-binding portion of the present invention by inserting DNA encoding the portion or full-length light and heavy chains obtained as described above into an expression vector. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses (such as cauliflower mosaic virus, tobacco mosaic virus), granules, YACs, EBV-derived episomes, etc. The antibody gene is linked to the vector such that the transcription and translation control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. An expression vector and expression control sequences compatible with the host cell used for expression are selected. The antibody light chain gene and the antibody heavy chain gene can be inserted into different vectors. In one embodiment, both genes are inserted into the same expression vector. Antibody genes are inserted into expression vectors using standard methods, such as linking antibody gene fragments to complementary restriction sites on the vector, or blunt-end ligation if no restriction sites are available. Convenient vectors are those encoding fully functional human CH or CL immunoglobulin sequences with engineered appropriate restriction sites to facilitate the insertion and expression of any VH, VL, or VK sequence, as described above. In these vectors, splicing typically occurs between the splice donor site of the inserted J region and the splice acceptor site preceding the human C domain, and also within the splice region of the human CH exon. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding region. Recombinant expression vectors can also encode signal peptides that promote the secretion of antibody chains from host cells. Antibody chain genes can be cloned into the vector such that the signal peptide frame is linked 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 derived from a non-immunoglobulin protein). In addition to the antibody chain gene, the recombinant expression vectors of this invention also carry regulatory sequences controlling antibody chain gene expression in host cells. Those skilled in the art should understand that the design of expression vectors, including the selection of regulatory sequences, can depend on factors such as the selection of host cells to be transformed and the desired protein expression level.Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), polyomaviruses, and strong mammalian promoters, such as innate immunoglobulin and actin promoters. For further description of viral regulatory elements and their sequences, see, for example, U.S. Patent Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing antibodies in plants, including descriptions of promoters and vectors and methods of plant transformation, are known in the art. See, for example, U.S. Patent No. 6,517,529, which is incorporated herein by reference. Methods for expressing peptides in bacterial or fungal cells (e.g., yeast cells) are also well known in the art. In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the present invention may also carry other sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selective marker genes. Selective marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017, which are incorporated herein by reference). For example, selective marker genes typically confer resistance to drugs (such as G418, hygromycin, or methotrexate) in host cells into which the vector has been introduced. Preferred selective marker genes include the dihydrofolate reductase (DHFR) gene (for dhfr-host cells with methotrexate selectivity / amplification), the neo gene (for G418 selection), and the glutamate synthase gene.

[0104] Methods for producing proteins using non-hybridoma host cells and recombinant proteins

[0105] Nucleic acid molecules encoding antibodies and vectors according to the invention containing these nucleic acid molecules can be used to transfect or transform suitable mammalian, plant, bacterial, or yeast host cells. Transfection or transformation can be performed by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the cell nucleus. Additionally, nucleic acid molecules can be introduced into mammalian cells via viral vectors. Methods for cell transformation 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, which are incorporated herein by reference). Methods for plant cell transformation are well known in the art, including, for example, Agrobacterium-mediated transformation, gene gun transformation, direct injection, electroporation, and viral transformation. Methods for bacterial and yeast cell transformation are also well known in the art. Mammalian cell lines suitable as expression hosts are well known in the art, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These cell lines include, but are not limited to, Chinese hamster ovary (CHO) cells, N50 cells, SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, young hamster kidney (BHK) cells, African green monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (such as Hep G2), A549 cells, and many other cell lines. Particularly preferred cell lines are selected by identifying which cell lines exhibit high expression levels. Other usable cell lines are insect cell lines, such as Sf9 or Sf21 cells. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient for the antibody to be expressed in the host cells, or more preferably, for a period sufficient for the antibody to be secreted into the culture medium in which the host cells grow. The antibody can be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, tobacco, Arabidopsis thaliana, duckweed, maize, wheat, and potato. Bacterial host cells include *Escherichia coli* and *Streptomyces* species. Yeast host cells include *Schizosaccharomyces pombe*, *Saccharomyces cerevisiae*, and *Pichia pastoris*. Furthermore, various known techniques can be used to enhance the expression of the antibodies of this invention in production cell lines. For example, the glutamine synthase gene expression system (GS system) is a commonly used method for enhancing expression under certain conditions. The GS system is discussed in whole or in part in European patent numbers 0 216846, 0 256 055, 0 323 997, and 0 338 841. Antibodies expressed in different cell lines or in transgenic animals are likely to have different glycosylations.However, antibodies encoded entirely by the nucleic acid molecules provided herein, or antibodies containing the amino acid sequences provided herein, are all part of this invention, regardless of how the antibody is glycosylated.

[0106] Genetically modified plants and animals

[0107] The monoclonal antibodies of the present invention can also be produced transgenic—by generating mammals or plants with transgenic heavy and light chain sequences of the target immunoglobulin, and producing antibodies therefrom in a recyclable form. In terms of mammalian transgenic production, the antibodies of the present invention can be produced and recovered from 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, which are incorporated herein by reference. In some embodiments, a non-human transgenic animal containing a human immunoglobulin locus is immunized with the S protein of at least one coronavirus, such as at least one variant or immunogenic portion thereof disclosed herein, as described above. Methods for preparing antibodies in plants are described, for example, U.S. Patents 6,046,037 and 5,959,177, which are incorporated herein by reference.

[0108] In some embodiments, one or more nucleic acid molecules encoding the antibodies of the present invention are introduced into an animal or plant using standard transgenic techniques to produce a non-human transgenic animal or plant. See Hogan and U.S. Patent 6,417,429, as described above. The transgenic cells used to prepare the transgenic animal may be embryonic stem cells, somatic cells, or fertilized eggs. The transgenic non-human organism may be a chimera, a non-chimeric heterozygote, or a non-chimeric homozygote. See, for example, Hofian et al. Manipulating the Mouse Embryo: A Laboratory Manual second ed., Cold SpringHarbor 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 of which are incorporated herein by reference. In some embodiments, the transgenic non-human animal has targeted disruption and substitution achieved through a targeted construct that encodes a target heavy chain and / or light chain. In one embodiment, the transgenic animal comprises and expresses nucleic acid molecules encoding heavy and light chains that specifically bind to the S protein of at least one coronavirus, and preferably specifically bind to (i) the S1 domain of the SARS-CoV-2 and / or SARS-CoV-1 S protein; (ii) the S2 domain of the SARS-CoV-2 and / or SARS-CoV-1 S protein; or (iii) both (i) and (ii). In one embodiment, the transgenic animal comprises and expresses nucleic acid molecules encoding heavy and light chains that specifically bind to human SARS-CoV-2 and / or SARS-CoV-1 S protein or variants thereof. In some embodiments, the transgenic animal comprises nucleic acid molecules encoding modified antibodies (such as single-chain antibodies, chimeric antibodies, or humanized antibodies). The antibodies of the present invention can be prepared in any transgenic animal. In one embodiment, the non-human animal is a mouse, rat, sheep, pig, goat, cow, or horse. The non-human transgenic animal expresses the encoded polypeptide in blood, milk, urine, saliva, tears, mucus, and other bodily fluids.

[0109] Class conversion

[0110] Another aspect of the present invention provides a method for converting the class or subclass of the antibody of the present invention into another class or subclass. In some embodiments, a nucleic acid molecule encoding VL, VK, or VH (which does not contain a sequence encoding CL or CH) is isolated using methods well known in the art. This nucleic acid molecule is then operatively linked to a nucleotide sequence encoding CL or CH from the desired immunoglobulin class or subclass. This can be achieved by using a vector or nucleic acid molecule containing a CL or CH chain, as described above. For example, an anti-SARS-CoV-2 S protein antibody originally IgM can be class-converted to IgG. Furthermore, class conversion can be used to convert one IgG subclass into another, for example, from IgG1 to IgG2. Another method for producing the antibody of the present invention containing the desired isotype includes the steps of: isolating a nucleic acid encoding the heavy chain of the antibody of the present invention and a nucleic acid encoding the light chain of the antibody of the present invention; isolating a sequence encoding the VH region; linking the VH sequence to a sequence encoding the heavy chain constant region of the desired isotype; expressing the light chain gene and the heavy chain construct in cells; and collecting the antibody having the desired isotype.

[0111] Modified antibodies

[0112] In another embodiment, a fusion antibody or immunoadhesin comprising all or part of the antibody of the present invention and linked to an additional polypeptide can be prepared. In one embodiment, only the variable domains of the antibody as described in any of the embodiments disclosed herein are linked to the polypeptide. In another embodiment, the VH domain of the antibody of the present invention is linked to a first polypeptide, while the VK domain of the antibody of the present invention is linked to a second polypeptide, which binds to the first polypeptide in a manner such that the VH and VK domains can interact to form an antigen-binding site. In another embodiment, the VH and VK domains are separated by a linker, such that the VH and VL domains can interact (see “Single-chain antibody” below). The VH-linker-VK antibody is then linked to the target polypeptide. This fusion antibody is useful for directing the polypeptide to cells or tissues expressing the coronavirus S protein, such as cells or tissues expressing the S protein of SARS-CoV-2 and / or SARS-CoV-1. The polypeptide can be a therapeutic agent, such as a toxin, chemokine, or other regulatory protein, or a diagnostic agent, such as an easily visualized enzyme, such as horseradish peroxidase. In addition, fusion antibodies can be prepared by linking two (or more) single-chain antibodies together. This is useful if the technician wants to prepare bivalent or multivalent antibodies on a single polypeptide chain, or if the technician wants to prepare bispecific antibodies or nanobodies. To prepare single-chain antibodies (scFv), DNA fragments encoding VH and VK are operatively linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (GIy4-Ser)3), such that the VH and VK sequences can be expressed as a continuous single-chain protein, wherein the VK and VH domains are linked by a 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). If only a single VH and VK are used, the single-chain antibody can be monovalent; if two VH and VK are used, it is a bivalent antibody; if more than two VH and VK are used, it is a multivalent antibody. Bispecific or multivalent antibodies can be generated that specifically bind to the S protein of SARS-CoV-2 and / or SARS-CoV-1 or its variants, as well as other molecules. Bispecific antibodies or antigen-binding fragments can be prepared by a variety of methods, including hybridoma fusion or Fab' fragment ligation.See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al, J. Immunol. 148:1547-1553 (1992). Furthermore, bispecific antibodies can be formed as “biantibodies” or “Janusins.” In some embodiments, the bispecific antibody binds to two distinct epitopes of the SARS-CoV-2 and / or SARS-CoV-1 S protein. In some embodiments, the bispecific antibody has a first heavy chain and a first light chain derived from monoclonal antibodies 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13, and additional antibody heavy and light chains. In some embodiments, the additional light and heavy chains are also derived from any of the aforementioned monoclonal antibodies, 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 of the monoclonal antibodies provided herein.

[0113] Derived and labeled antibodies

[0114] The antibody or antigen-binding portion of the present invention can be derivatized or linked to other molecules, such as other peptides or proteins. Typically, the antibody or a portion thereof is derivatized such that derivatization or labeling does not adversely affect neutralizing activity against at least one coronavirus. Therefore, the antibodies and antibody portions of the present invention are intended to include both the full and modified forms of the antibodies described herein. For example, the antibody or antibody portion of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent binding, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a double-stranded antibody), a detection agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the binding of the antibody or antibody portion to another molecule, such as a streptavidin core region or a polyhistidine tag. One type of derivatized antibody is generated by crosslinking two or more antibodies (of the same or different types, such as those used to prepare bispecific antibodies). Suitable crosslinking agents include: heterobifunctional crosslinking agents having two distinct reactive groups separated by a suitable spacer group (e.g., m-maleimide benzoyl-N-hydroxysuccinimide ester); or homobifunctional crosslinking agents (e.g., bissuccinimide octanoate). Such crosslinking agents are available from Pierce Chemical Company, Rockford, II.

[0179] . Another type of derivatized antibody is a labeled antibody. Useful detection agents that can be used to derivatize the antibody or antigen-binding moiety of the present invention include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, phycoerythrin, 5-dimethylamino-1-naphthalenesulfonyl chloride, lanthanide phosphors, etc. Antibodies can also be labeled with enzymes useful for detection, such as horseradish peroxidase, [β]-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When an antibody is labeled with a detectable enzyme, detection is achieved by adding an additional reagent that the enzyme uses to produce a recognizable reaction product. For example, in the presence of horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine produces a colored reaction product that is detectable. Antibodies can also be labeled with biotin and detected by indirectly measuring the binding of avidin or streptoavidin. Antibodies can also be labeled with predetermined peptide epitopes that are recognized by secondary reporter sequences (such as leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, or epitope tags). In some embodiments, the label is attached via spacer arms of varying lengths to reduce potential steric hindrance. Antibodies can also be labeled with radiolabeled amino acids. This radiolabeling can be used for diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect cells or tumors expressing SARS-CoV-2 and / or SARS-CoV-1 S proteins via X-rays or other diagnostic techniques. Furthermore, radiolabeling can also be used therapeutically as a toxin against cancer cells or tumors.In some embodiments, the antibodies may be labeled with paramagnetic, radioactive, or florigenic ions that are detectable during imaging. In some embodiments, the paramagnetic ions are chromium (III), manganese (II), iron (III), 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 ions are 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 antibodies of the present invention are labeled with X-ray imaging agents such as lanthanum (III), gold (III), lead (II), and bismuth (III).

[0115] Composition and kit

[0116] This invention relates to compositions comprising any antibody of the present invention or its antigen-binding portion and one or more pharmaceutically acceptable excipients and / or carriers.

[0117] In some embodiments, the composition may comprise the antibody or its binding portion as described in any of the foregoing embodiments. In some embodiments, the subject of treatment is a human. In other embodiments, the subject is an animal subject. In some embodiments, an antagonistic anti-SARS-CoV-2 and / or SARS-CoV-1 S protein antibody binding to the S1 domain and an antagonistic anti-SARS-CoV-2 and / or SARS-CoV-1 S protein antibody binding to the S2 domain (or the antigen-binding portion of one or both) are administered to the subject together or separately. In some embodiments, the antibody is present in a composition comprising a pharmaceutically acceptable carrier. In another embodiment, one or more antagonistic antibodies of the present invention are administered in combination with one or more antagonistic antibodies binding to different epitopes on at least one coronavirus S protein, antagonistic antibodies binding to the S protein of different isolates of SARS-CoV-2 and / or SARS-CoV-1, and / or antagonistic antibodies binding to different stages (i.e., early, intermediate, or late stages of SARS-CoV-2 and / or SARS-CoV-1 or its variants). As used herein, "pharmaceutically acceptable carrier" refers to any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic agent, and absorption delay agent. Some examples of pharmaceutically acceptable carriers are water, physiological saline, phosphate-buffered saline, glucose, glycerol, ethanol, and combinations thereof. In many cases, the composition preferably contains an isotonic agent, such as sugar, polyol (e.g., mannitol, sorbitol), or sodium chloride. Other examples of pharmaceutically acceptable substances are humectants or small amounts of excipients, such as humectants or emulsifiers, preservatives, or buffers, which improve the shelf life or efficacy of the antibody. The compositions of the present invention can be in various forms, such as liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injections and infusions), dispersants or suspensions, tablets, pills, powders, liposomes, and suppositories. Preferred forms depend on the intended method of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusionable solutions, such as those similar to those used for human passive immunization. Preferred administration methods include parenteral administration, such as intravenous, subcutaneous, intraperitoneal, and intramuscular. In one embodiment, the antibody is administered via intravenous infusion or injection. In another embodiment, the antibody is administered via intramuscular or subcutaneous injection. The therapeutic composition is generally sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, dispersant, liposome, or other ordered structure suitable for high drug concentrations. A sterile injectable solution can be prepared by incorporating the antibody according to any embodiment of the invention in a desired amount into a suitable solvent containing one or more of the desired components listed above, followed by filtration sterilization. Typically, dispersants are prepared by incorporating the active compound into a sterile carrier containing a base dispersion medium and the other desired components listed above.In the case of using sterile powders to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield the powder of the active ingredient and any other desired components from a previously sterile filtered solution. For example, the appropriate flowability of the solution can be maintained by using a coating such as lecithin, maintaining the desired particle size in the case of a dispersant, and using a surfactant. The absorption of the injectable composition can be prolonged by including agents that delay absorption (such as monostearate and gelatin) in the composition. The antibodies of the present invention can be administered by various methods known in the art, although for many therapeutic applications, the preferred route / method of administration is subcutaneous, intramuscular, or intravenous infusion. Those skilled in the art will understand that the route and / or method of administration will vary depending on the desired outcome. Other routes of administration include intraperitoneal, intrabronchial, transmucosal, intraspinal, intrasynovial, intraaortic, intranasal, ocular, ear, topical, and buccal administration. In some embodiments, the active compound of the antibody composition can be prepared with a carrier that protects the antibody from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems (JR Robinson, ed., Marcel Dekker, Inc., New York, 1978). The present invention also provides compositions suitable for inhalation administration comprising any one or more antibodies described herein. Any antibody of the present invention can be conveniently delivered to a subject in the form of an aerosol via pressurized packaging or nebulizer, using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of pressurized aerosols, a valve can be provided to determine the unit of measurement for delivery of a quantitative amount. Capsules and cartridges (e.g., gelatin) for inhalers or blow-offs can be formulated as powder mixtures containing the compound and a suitable powder matrix (e.g., lactose or starch). Dellamary et al. (2004) J Control Release.;95(3): 489-500 describes a pulmonary delivery formulation of antibodies. The present invention also provides compositions suitable for administration via the oral mucosa, comprising any one or more antibodies described herein. Oral mucosal delivery refers to the delivery of a carrier through the mucosa of the oral cavity, pharynx, or esophagus, and contrasts with, for example, conventional oral delivery, in which drug absorption occurs in the intestine.Therefore, the administration routes of antibodies via absorption through the buccal, sublingual, gingival, pharyngeal, and / or esophageal mucosa are all encompassed within the term "oral mucosal delivery" as used herein. For mucosal administration, any antibody of the present invention can be formulated, for example, into chewing gum (see U.S. Patent No. 5,711,961) or buccal patches (see, for example, U.S. Patent No. 5,298,256). The present invention also provides compositions suitable for vaginal mucosal administration comprising any one or more antibodies described herein. The antibodies of the present invention can be formulated into vaginal suppositories, foams, creams, tablets, capsules, ointments, or gels. In some embodiments, the antibody-containing compositions are formulated with a permeabilizer suitable for penetrating across the mucosal barrier. Such permeabilizers are known in the art and include, for example, bile salts and fusidic acid derivatives for mucosal administration. In some embodiments, the antibodies of the present invention can be administered orally, for example, with an inert diluent or an absorbable, edible carrier. The compounds (and other ingredients, if desired) may also be encapsulated in hard or soft-shell gelatin capsules, compressed into tablets, or directly incorporated into the subject's diet. For oral therapeutic administration, the antibodies may be mixed with excipients and administered in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. To administer the compounds of the invention by means other than gastrointestinal administration, it may be necessary to coat the compounds with materials or administer them co-with the compounds to prevent their inactivation. Other active compounds may also be incorporated into the composition. In some embodiments, the neutralizing antibodies of the invention are formulated and / or co-administered with one or more other 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 drugs, antihypertensive agents, analgesics, antidepressants, antibiotics, anticancer agents, anesthetics, antiemetics, anti-infectives, contraceptives, antidiabetic agents, steroids, antiallergic agents, chemotherapeutic agents, antimigraine agents, smoking cessation agents, antiviral agents, immunosuppressants, thrombolytics, cholesterol-lowering agents, and anti-obesity agents. Therapeutic agents also include peptide analogs that inhibit the activity of the S protein of at least one coronavirus (such as SARS-CoV-1 and / or SARS-CoV-2 or variants thereof); antibodies or other molecules that prevent said coronaviruses from entering cells, including but not limited to preventing the S protein from binding to receptors (such as the ACE2 receptor); and agents that inhibit coronavirus S protein expression. In one embodiment, additional agents for inhibiting coronavirus S protein expression include antisense nucleic acids capable of hybridizing with coronavirus S protein mRNA, such as hairpin RNA or siRNA, locked nucleic acids (LNA), 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 neutralizing antibodies and co-administered agents to avoid the potential toxicities or complications associated with various monotherapy treatments.In some specific embodiments, the therapeutic agent co-formulated and / or co-administered with the neutralizing antibody of the present invention is an antibacterial agent. Antibacterial agents include antibiotics (such as antimicrobial agents), antiviral agents, antifungal agents, and antigenic agents. Non-limiting examples of antibacterial agents include sulfonamides, trimethoprim-sulfamethoxazole, quinolones, penicillins, and cephalosporins. The compositions of the present invention may comprise a “therapeuticly effective amount” or a “preventatively effective amount” of the antibody or antigen-binding portion of the present invention. A “therapeuticly effective amount” refers to an amount that effectively achieves the desired therapeutic effect at the necessary dose and for the necessary time period. The therapeutically effective amount of an antibody or antibody portion may vary due to factors such as individual disease state, age, sex, and weight, as well as the ability of the antibody or antibody portion to elicit the desired response in the individual. A therapeutically effective amount is also the amount at which any toxic or harmful effects of the antibody or antibody portion are offset by its beneficial therapeutic effect. A “preventatively effective amount” refers to an amount that effectively achieves the intended preventative effect at the necessary dose and for the necessary time period. Typically, because the preventative dose is administered to the subject early in or before the onset of disease, the effective preventative dose may be less than the effective therapeutic dose. Dosing regimens can be adjusted to provide the optimal expected response (e.g., therapeutic or preventative response). For example, the dose may be administered as a single bolus, or in divided doses over time, or the dose may be proportionally reduced or increased according to indications of the urgency of the treatment situation. For ease of administration and uniform dosage, it is particularly advantageous to formulate the parenteral composition in unit dosage form. As used herein, “unit dosage form” refers to a physically discrete unit suitable as a single dose to a mammalian subject being treated; each unit contains a predetermined amount of the active compound, which is calculated to bind with the desired drug carrier to produce the intended therapeutic effect. The specifications of the unit dosage form of this invention are determined by and directly depend on the following factors: (a) the unique characteristics of the antibody or a portion thereof and the specific therapeutic or preventative effect to be achieved, and (b) the inherent limitations in the field of formulating such antibodies to treat individual sensitivities. Exemplary and non-limiting therapeutic or preventative effective doses of the antibody or antibody portion of the present invention range from 0.025 to 50 mg / kg, more preferably from 0.1 to 50 mg / kg, even more preferably from 0.1 to 25 mg / kg, 0.1 to 10 mg / kg, or 0.1 to 3 mg / kg. In some embodiments, the formulation comprises 5 mg / ml of antibody in 20 mM sodium citrate buffer, 140 mM NaCl, and 0.2 mg / ml polysorbate 80 at pH 5.5. It should be noted that dose values ​​may vary depending on the type and severity of the condition to be alleviated. It should also be further understood that, for any particular subject, the specific dosing regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the dose ranges described herein are merely examples and are not intended to limit the scope or practice of the claimed compositions.Another aspect of the invention provides a kit comprising the antibody or antigen-binding portion of the invention, or a composition comprising such an antibody or antigen-binding fragment. In addition to the antibody or composition, the kit may also include diagnostic or therapeutic agents. The kit may also include instructions for use in diagnostic or therapeutic methods, and packaging materials, such as, but not limited to, ice, dry ice, polystyrene foam, foam, plastic, cellophane, shrink film, bubble wrap, cardboard, and peanut starch. In one embodiment, the kit comprises an antibody or an antibody-containing composition and a diagnostic agent that can be used in the methods described below. In another embodiment, the kit comprises an antibody or an antibody-containing composition and one or more therapeutic agents that can be used in the methods described below.

[0118] In one embodiment, an antibody or a binding portion thereof, or a composition comprising such an antibody, according to any embodiment disclosed herein, is used for the prevention or treatment of patients infected with coronaviruses, particularly those infected with SARS-CoV-2 and / or SARS-CoV-1 or variants thereof. Uses of such antibodies and compositions thereof include, but are not limited to, passive immunization of populations at risk of infection (e.g., occupationally exposed individuals, residents of epidemic areas) and treatment of acute cases, whether or not hospitalized. The invention also relates to compositions for inhibiting viral infection in mammals, particularly coronavirus infection, more specifically SARS-CoV-2 and / or SARS-CoV-1 or variants thereof, comprising a specific amount of the antibody of the invention in combination with a specific amount of an antiviral agent, wherein the amounts of the antibody and the antiviral agent together effectively inhibit viral replication, viral infection of new cells, or viral load.

[0119] Application of diagnostic methods

[0120] The antibodies of the present invention can also be used as a diagnostic tool for rapid detection of at least one coronavirus infection. In another aspect, the present invention provides a diagnostic method. The antibodies according to any embodiment of the present invention can be used to detect the S protein of at least one coronavirus, particularly the S protein of viruses selected from SARS-CoV-2, SARS-CoV-1, and their variants, in in vitro, ex vivo, or in vivo biological samples. In one embodiment, the present invention provides a method for diagnosing the presence or location of one or more coronaviruses in a subject with this need. The antibodies of the present invention can be used for routine immunoassays, including but not limited to ELISA, RIA, flow cytometry, tissue immunohistochemistry, Western blotting (immunoblotting), or immunoprecipitation. The antibodies of the present invention can be used to detect coronavirus S proteins from humans. The present invention provides a method for detecting coronavirus S proteins in biological samples, particularly the S protein of at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants, comprising contacting the biological sample with the antibody of the present invention and detecting the binding of the antibody. In one embodiment, the antibody of the present invention is directly labeled with a detectable marker. In another embodiment, the antibody (first antibody) is unlabeled, while a second antibody or other molecule capable of binding the S protein antibody is labeled. As is well known to those skilled in the art, the selected second antibody is capable of specifically binding to a specific species and class of the first antibody. For example, if the selected antibody is human IgG, the secondary antibody can be anti-human IgG. Other molecules that can bind to the antibody include, but are not limited to, protein A and protein G, both of which are commercially available, such as those from Pierce Chemical Co. Examples of biological samples used in the diagnostic methods disclosed herein include urine, feces, blood, saliva, biopsy tissue, cerebrospinal fluid, nasopharyngeal and oropharyngeal lavage fluid, sputum, tracheal aspirate, bronchoalveolar lavage fluid, or other biological samples available from human subjects.

[0121] Suitable labeling of antibodies or secondary antibodies has been disclosed above, including various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, [β]-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, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol. In other embodiments, the S protein can be determined in a biological sample by a competitive immunoassay using an S protein standard labeled with a detectable substance and the unlabeled antibody of the present invention. In this assay, the biological sample, the labeled S protein standard, and the antibody are mixed, and the amount of labeled S protein standard bound to the unlabeled antibody is measured. The amount of coronavirus S protein in the biological sample is inversely proportional to the amount of labeled S protein standard bound to the antibody. Technicians can use the above-described immunoassay for a variety of purposes. For example, the antibody can be used to detect the S protein in cultured cells or as a diagnostic assay in subject samples, particularly to detect the S protein of at least one virus selected from SARS-CoV-2, SARS-CoV-1, and their variants. Following a diagnostic method according to any embodiment disclosed herein, further steps may be performed, such as administering at least the antibody of the present invention to a positive subject, for example, according to any treatment method disclosed herein.

[0122] Application of treatment methods

[0123] In another embodiment, the present invention provides a method for neutralizing at least one coronavirus, particularly at least one virus selected from SARS-CoV-2, SARS-CoV-1, and variants thereof, by administering an antibody according to any embodiment disclosed herein to a patient in need of treatment. Any type of antibody described herein may be used for treatment. In various embodiments, the antibody is a human antibody. In some embodiments, the antibody or its antigen-binding portion binds to the S1 domain of the coronavirus S protein. In some embodiments, the patient is a human patient. Alternatively, the patient may be a mammal infected with at least one coronavirus, for example, infected with at least one virus selected from SARS-CoV-2, SARS-CoV-1, and variants thereof. In one embodiment, the present invention provides a method for treating, adjunctive treating, preventing, or adjunctive preventing infection with at least one coronavirus (preferably selected from SARS-CoV-2, SARS-CoV-1, and variants thereof) and symptoms or disorders caused by such infection by administering a therapeutically or prophylactically effective amount of the antibody of the present invention to a subject. The antibody may be administered locally or systemically. The antibody, as an antagonist of the coronavirus S protein, and its antigen-binding fragment may be used as a therapeutic agent for such infection. The antibody may be administered locally or systemically. Therapeutic compositions comprising one or more antibodies according to any embodiment disclosed herein can be administered to subjects in a variety of pharmaceutically acceptable dosage forms, such as oral, nasal, vaginal, buccal, rectal, ocular, or pulmonary routes of administration, as is known to those skilled in the art. For example, antibodies can be administered via the nasal route using a nasal inhaler device. Antibodies can also be administered ocularly in gel form. For example, prior to administration, a formulation containing one or more antibodies according to any embodiment disclosed herein can be conveniently packaged into a dual-compartment unit-dose container, one compartment containing the lyophilized antibody formulation and the other containing physiological saline. The antibody dose is typically in the range of 0.1-100 mg / kg, more preferably 0.5-50 mg / kg, further preferably 1-20 mg / kg, and even more preferably 1-10 mg / kg. The serum concentration of the antibody can be measured by any method known in the art.

[0124] In another embodiment, the antibody of the present invention is administered to a subject in combination with other therapeutic agents. In one embodiment, the additional therapeutic agent may alone treat the symptoms of coronavirus infection and may optionally synergize with the action of the antibody. The additional therapeutic agent administered may be selected by those skilled in the art for treating the infection. Combined administration of the antibody and the additional therapeutic agent (combined therapy) encompasses the administration of a composition comprising an antibody and an additional therapeutic agent, as well as the administration of two or more separate compositions, one comprising an antibody and the others comprising an additional therapeutic agent. Furthermore, while combined administration or combined therapy generally means that the antibody and the additional therapeutic agent are administered simultaneously with each other, this also covers situations where the antibody and the additional therapeutic agent are administered at different times. For example, the antibody may be administered every three days, while the additional therapeutic agent is administered daily. Alternatively, the antibody may be administered before or after treatment with the additional therapeutic agent, for example, after the patient has failed treatment with the additional therapeutic agent. Similarly, any antibody of the present invention may be administered before or after other treatments.

[0125] The 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, relieved, or cured. Preferably, the combination therapy is a multiple-dose regimen. The combination therapy may be administered three times daily to once every six months. Dosing may be scheduled, 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 micropump. The combination therapy may be administered orally, via mucous membranes, buccally, intranasally, by inhalation, intravenously, subcutaneously, intramuscularly, or parenterally. In some aspects, the present invention provides a method for treating, preventing, or alleviating symptoms of at least one coronavirus-mediated disorder in a subject with such need, particularly a disorder mediated by a virus selected from SARS-CoV-1 and / or SARS-CoV-1 or its variants, comprising the step of administering to the subject an antibody or antigen-binding portion according to any one of the foregoing embodiments, further comprising at least one additional therapeutic agent selected from the group consisting of: (a) one or more antibodies selected from the group consisting of: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13:

[0126] and

[0127] (b) One or more antibodies that specifically bind to the S protein of multiple coronavirus strains; and / or

[0128] (c) One or more neutralizing antibodies that do not bind to the coronavirus S protein; and / or

[0129] (d) One or more agents that bind to the coronavirus S protein receptor; and / or

[0130] (e) One or more antiviral agents.

[0131] In some aspects, the present invention provides a kit for treating, preventing, or alleviating symptoms of at least one coronavirus-mediated disorder in a subject with such need, particularly a disorder mediated by a virus selected from SARS-CoV-1 and / or SARS-CoV-2 or variants thereof, the kit comprising: a) one or more antibodies selected from the group consisting of: 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13; and

[0132] (b) One or more antibodies that specifically bind to the S protein of multiple coronavirus strains; and / or

[0133] (c) One or more neutralizing antibodies that do not bind to the S protein; and / or

[0134] (d) One or more agents that bind to the coronavirus S protein receptor; and / or

[0135] (e) One or more antiviral agents.

[0136] The human monoclonal antibodies or antigen-binding portions thereof disclosed herein can also be advantageously used as diagnostic reagents in in vitro methods for detecting anti-coronavirus antibodies, particularly SARS-CoV-2 and / or SARS-CoV-1 antibodies, in biological samples previously obtained from patients (e.g., serum, plasma, blood samples, or any other suitable biological material obtained from patients, preferably humans). These antibodies may be found in biological samples obtained from patients, for example, due to prior exposure to the virus, or because the monoclonal antibodies of the present invention have been previously administered to patients for therapeutic, preventative, or research purposes. Therefore, diagnostic kits containing the human monoclonal antibodies or antigen-binding portions thereof disclosed herein as specific reagents are also within the scope of the present invention, said kits being specifically designed to detect and / or quantify anti-coronavirus antibodies in biological samples previously obtained from patients.

[0137] The human monoclonal antibodies or their antigen-binding moieties disclosed herein can also be advantageously used to design vaccines against coronaviruses. 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 mAbs can be used to recognize protective antigens / epitopes. The structural characteristics of the Ab-antigen complex can be used to guide antigen design. Therefore, the methods or uses of the human monoclonal antibodies or their antigen-binding moieties disclosed herein for designing vaccines against coronaviruses, particularly against SARS-CoV-2, SARS-CoV-1, and / or their variants, also fall within the scope of this invention.

[0138] The human monoclonal antibodies or their antigen-binding portions disclosed herein can be used to prepare mimicry epitopes, such as anti-idiotype antibodies, peptides, truncated S proteins, or artificial forms, which have the ability to activate the antibodies disclosed herein. Anti-idiotype antibodies are preferred. Anti-idiotype antibodies are antibodies specifically targeting the idiotype of the neutralizing antibodies used to prepare them, and therefore can mimic the key epitopes they recognize. The preparation of anti-idiotype antibodies is carried out by methods known per se and does not require further explanation here. Therefore, anti-idiotype antibodies are preferred for mimicry epitopes of the antibodies of the present invention and are also within the scope of the present invention. The human monoclonal antibodies or their antigen-binding portions disclosed herein can be used to prepare anti-idiotype antibodies according to methods known per se. Anti-idiotype antibodies are antibodies specifically targeting the idiotype of the broad-spectrum neutralizing antibodies used to prepare them, and therefore can mimic the key epitopes they recognize. Therefore, anti-idiotype antibodies against the monoclonal antibodies of the present invention are also within the scope of the present invention.

[0139] The following experimental section is provided illustratively and not restrictively 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.

[0140] Example

[0141] 1. Materials and Methods

[0142] Donor recruitment and human peripheral blood mononuclear cell (PBMC) isolation

[0143] In this study, six donors were recruited who had received at least three doses of the mRNA vaccine and had been infected with SARS-CoV-2 at least twice. This cohort was named Super Mixed Immunization (SHI). Human peripheral blood mononuclear cells (PBMCs) were collected from these donors for single-cell sorting of spike (S) protein-specific transition memory B cells (MBCs). To identify broadly reactive human monoclonal antibodies (mAbs), the S proteins of SARS-CoV-1 and SARS-CoV-2 were used as sorting decoys. A total of 4,505 S protein-specific MBCs were isolated from all donors and subjected to a cytopathic effect-based neutralization assay (CPE-MN) to identify SARS-CoV-2 neutralizing antibodies (nAbs) and SARS-CoV-1 nAbs using our mimicry platform. A total of 365 mAbs were identified, exhibiting varying degrees of neutralizing titers and broad-spectrum coverage against SARS-CoV-2 strains and variants, as well as SARS-CoV-1. Of all the identified nAbs, ten antibodies were selected and named 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13. To evaluate the broad binding spectrum of the selected nAbs, their binding affinity to the SARS-CoV-2 S protein trimer, receptor-binding domain (RBD), N-terminal domain (NTD), and S2 domain, as well as to the S proteins of other coronaviruses including SARS-CoV-1, OC43, HKU1, 229E, and NL63, was analyzed. Furthermore, nAbs targeting the SARS-CoV-2 S protein RBD were further characterized using competition assays to identify the epitope regions targeted by these antibodies. This paper reports the specific characteristics of these newly discovered antibodies.

[0144] Functional characteristics of neutralizing human coronavirus nAb

[0145] Ten nAbs exhibiting broad functional activity were initially characterized by their binding activity to the SARS-CoV-2 S protein trimer, receptor-binding domain (RBD), N-terminal domain (NTD), and S2 domain, as well as to other coronavirus S proteins, 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 (…). Figure 1 (Left figure). Among these 10 antibodies, antibodies 02G11 and 03O07 also showed recognition of the SARS-CoV-1 S protein ( Figure 1(See figure in the middle). None of our antibodies recognized the S protein of OC43, HKU1, 229E, and NL63. Subsequently, the neutralizing titers of these nAbs against SARS-CoV-1 and SARS-CoV-2 strains and the Omeprón variant were evaluated. Of the two antibodies that could bind to the SARS-CoV-1 S protein, 03O07 exhibited extremely high neutralizing titer against SARS-CoV-1, with a 50% inhibitory concentration (IC50) of [missing value]. 50 The concentration was 56.0 ng / ml. -1 ( Figure 1 (See right figure). Conversely, all nAbs were able to neutralize SARS-CoV-2 strains and Omeprón variants (BA.5, BA.2.75, BF.7, BQ.1.1, and XBB.1.5), and exhibited varying ranges of neutralizing titers. Figure 1 (See right figure). 100% inhibitory concentration (IC50) of 01J19 100 The range was 9.4 to 37.7 ng / ml. -1 02K18 ranged from 15.5 to 110.8 ng / ml -1 05F22 ranged from 9.5 to 167.4 ng / ml. -1 01J18 ranged from 15.2 to 107.6 ng / ml. -1 02K05 ranges from 3.0 to 53.5 ng / ml -1 01B20 ranged from 4.7 to 3767.5 ng / ml. -1 02G11 ranged from 126.8 to 405.8 ng / ml -1 03O07 ranged from 67.9 to 479.9 ng / ml -1 05N18 ranged from 21.0 to 627.5 ng / ml. -1 The concentrations of 02N13 ranged from 11.1 to 97.8 ng / ml. -1 ( Figure 1 (See right image).

[0146] Finally, according to class 1 / 2 J08 1 Antibodies, Class 3 S309 2 Antibodies and Class 4 CR3022 3 The competitive ability of antibodies was assessed by classifying nine nAbs targeting the RBD. The majority of antibodies (5 / 9; 55.7%), namely 01J19, 02K18, 05F22, 01J18, and 02K05, competed with J08, thus targeting the class 1 / 2 region (…). Figure 1(See right figure). Next, antibodies 02G11 and 03O07 (2 / 9; 22.2%) and antibody 01B20 (1 / 9; 11.1%) targeted class 4 and class 3 epitope regions, respectively. Finally, one antibody (1 / 9; 11.1%), 05N18, did not compete with any of the three antibodies, therefore we could not identify its targeted epitope region ( Figure 1 (See right image).

[0147] Genetic characteristics of neutralizing human coronavirus nAb

[0148] The heavy and light chain encoding genes of antibodies 01J19, 02K18, 05F22, 01J18, 02K05, 01B20, 02G11, 03O07, 05N18, and 02N13 were characterized. Table 1 lists the full-length nucleotide and amino acid sequences of the heavy and light chains of these antibodies. Table 2 shows the gene rearrangements of immunoglobulin heavy chain V, D, and J (IGHV; IGHD; IGHJ) and light chain V and J (IGLV; IGLJ), the lengths of the complementarity-determining region 3 (CDR3) of the heavy chain (H-CDR3) and light chain (L-CDR3), and the mutation frequencies (%) of the V gene in the heavy and light chains. Overall, the mutation level of the V gene in the heavy chain (mean 8.35%) was observed to be higher than that in the light chain (mean 4.08%). Similarly, H-CDR3 (mean 16.4 amino acids) was observed to be longer than L-CDR3 (mean 9.70 amino acids) (Table 2). The current nAb groups employ different heavy and light chain rearrangements. Most nAbs (4 / 10; 40.0%) are encoded by IGHV3-66 germline paired with different heavy chain D and J genes and light chain genes (Table 2). 02K18 and 05F22 use the same heavy chain V, D, and J (IGHV3-66; IGHD1-26; IGHJ4-1) and light chain V and J (IGKV3-15; IGKJ2-1) gene rearrangements, but there are significant differences in the CDR3 lengths of the two chains (Table 2). Similarly, 02G11 and 03O07 have the same heavy chain (IGHV1-46; IGHD5-5; IGHJ4-1) and light chain (IGKV3-D15; IGKJ1-1) rearrangements, with almost identical CDR3 lengths and V gene mutation levels. In our group, four antibodies (01J19, 02K05, 01B20, and 02N13) use a heavy chain V gene that differs from other nAbs. These antibodies include IGHV4-31 (01J19), IGHV3-53 (02K05), IGHV1-3 (01B20), and IGHV3-48 (02N13) (Table 2).

[0149] Table 1. Sequences of neutralizing human coronavirus nAb

[0150]

[0151]

[0152]

[0153]

[0154] Table 2. Genetic characteristics of neutralizing human coronavirus nAb

[0155]

[0156] Declaration made pursuant to Article 170bis

[0157] In accordance with Article 170bis of the Italian Industrial Property Law, the applicant of this patent application declares that: 5 - with respect to biological materials, objects, or such materials used in this patent application, including microorganisms or genetically modified organisms, compliance has been maintained with national or community regulations, particularly with respect to the obligations arising from the amendments in Legislative Decree No. 206 of April 12, 2001 and Legislative Decree No. 224 of July 8, 2003; - written informed consent has been obtained from the donors of the human blood samples used in this patent application. This research has been approved by the local ethics committee.

[0158] Serial list in the instruction manual

[0159] >Amino acid sequence of the heavy chain variable domain of the antibody defined in this paper as SEQ ID NO:1 (01J19_VH)

[0160]

[0161] >Amino acid sequence of the heavy chain variable domain of the antibody defined in this article as SEQ ID NO:2 (02K18_VH)

[0162]

[0163] >Amino acid sequence of the heavy chain variable domain of the antibody defined in this article as SEQ ID NO:3 (05F22_VH)

[0164]

[0165] >Amino acid sequence of the heavy chain variable domain of the antibody defined in this article as SEQ ID NO:4 (01J18_VH)

[0166]

[0167] >Amino acid sequence of the heavy chain variable domain of the antibody defined in this article as SEQ ID NO:5 (02K05_VH)

[0168]

[0169] >Amino acid sequence of the heavy chain variable domain of the antibody defined in this article as SEQ ID NO:6 01B20 (01B20_VH)

[0170]

[0171] >Amino acid sequence of the heavy chain variable domain of the antibody defined in this article as SEQ ID NO:7 02G11 (02G11_VH)

[0172]

[0173] >The amino acid sequence of the heavy chain variable domain of the antibody defined in this article as SEQ ID NO:8 (03O07_VH)

[0174]

[0175] >SEQ ID NO:9 Amino acid sequence of the heavy chain variable domain of the antibody defined in this article as 05N18 (05N18_VH)

[0176]

[0177] >SEQ ID NO:10 Amino acid sequence of the heavy chain variable domain of the antibody defined in this article as 02N13 (02N13_VH)

[0178]

[0179] >SEQ ID NO:11 Nucleotide sequence of the heavy chain variable domain of the antibody defined in this paper as 01J19 (01J19_VH)

[0180]

[0181] >SEQ ID NO:12 Nucleotide sequence of the heavy chain variable domain of the antibody defined in this article as 02K18 (02K18_VH)

[0182]

[0183] >SEQ ID NO:13 Nucleotide sequence of the heavy chain variable domain of the antibody defined in this article as 05F22 (05F22_VH)

[0184]

[0185] >SEQ ID NO:14 Nucleotide sequence of the heavy chain variable domain of the antibody defined in this article as 01J18 (01J18_VH)

[0186]

[0187] >SEQ ID NO:15 Nucleotide sequence of the heavy chain variable domain of the antibody defined in this article as 02K05 (02K05_VH)

[0188]

[0189]

[0190] >SEQ ID NO:16 Nucleotide sequence of the heavy chain variable domain of the antibody defined in this article as 01B20 (01B20_VH)

[0191]

[0192] >SEQ ID NO:17 Nucleotide sequence of the heavy chain variable domain of the antibody defined in this article as 02G11 (02G11_VH)

[0193]

[0194] >SEQ ID NO:18 Nucleotide sequence of the heavy chain variable domain of the antibody defined in this article as 03O07 (03O07_VH)

[0195]

[0196] >SEQ ID NO:19 Nucleotide sequence of the heavy chain variable domain of the antibody defined in this article as 05N18 (05N18_VH)

[0197]

[0198] >SEQ ID NO:20 Nucleotide sequence of the heavy chain variable domain of the antibody defined in this article as 02N13 (02N13_VH)

[0199]

[0200] >SEQ ID NO:21 Amino acid sequence of the light chain variable domain of the antibody defined in this article as 01J19 (01J19_Vk)

[0201]

[0202] >SEQ ID NO:22 Amino acid sequence of the light chain variable domain of the antibody defined in this article as 02K18 (02K18_Vk)

[0203]

[0204] >Amino acid sequence of the light chain variable domain of the antibody SEQ ID NO:23, defined in this article as 05F22 (05F22_Vk).

[0205]

[0206] >SEQ ID NO:24 Amino acid sequence of the light chain variable domain of the antibody defined in this article as 01J18 (01J18_Vk)

[0207]

[0208] >SEQ ID NO:25 Amino acid sequence of the light chain variable domain of the antibody defined in this article as 02K05 (02K05_Vk)

[0209]

[0210] >Amino acid sequence of the light chain variable domain of the antibody SEQ ID NO:26, defined in this article as 01B20 (01B20_Vk).

[0211]

[0212] >SEQ ID NO:27 Amino acid sequence of the light chain variable domain of the antibody defined in this article as 02G11 (02G11_Vk)

[0213]

[0214] >SEQ ID NO:28 Amino acid sequence of the light chain variable domain of the antibody defined in this article as 03O07 (03O07_Vk)

[0215]

[0216] >SEQ ID NO:29 Amino acid sequence of the light chain variable domain of the antibody defined in this article as 05N18 (05N18_Vk)

[0217]

[0218] >SEQ ID NO:30 Amino acid sequence of the light chain variable domain of the antibody defined in this article as 02N13 (02N13_Vk)

[0219]

[0220] >SEQ ID NO:31 Nucleotide sequence of the light chain variable domain of the antibody defined in this article as 01J19 (01J19_Vk)

[0221]

[0222] >SEQ ID NO:32 Nucleotide sequence of the light chain variable domain of the antibody defined in this article as 02K18 (02K18_Vk)

[0223]

[0224] >nucleotide sequence of the light chain variable domain of the antibody defined in this article as SEQ ID NO:33 (05F22_Vk)

[0225]

[0226] >SEQ ID NO:34 Nucleotide sequence of the light chain variable domain of the antibody defined in this article as 01J18 (01J18_Vk)

[0227]

[0228] >SEQ ID NO:35 Nucleotide sequence of the light chain variable domain of the antibody defined in this article as 02K05 (02K05_Vk)

[0229]

[0230] >nucleotide sequence of the light chain variable domain of the antibody SEQ ID NO:36, defined in this article as 01B20 (01B20_Vk).

[0231]

[0232] >SEQ ID NO:37 Nucleotide sequence of the light chain variable domain of the antibody defined in this article as 02G11 (02G11_Vk).

[0233]

[0234] >SEQ ID NO:38 Nucleotide sequence of the light chain variable domain of the antibody defined in this article as 03O07 (03O07_Vk)

[0235]

[0236] >SEQ ID NO:39 Nucleotide sequence of the light chain variable domain of the antibody defined in this article as 05N18 (05N18_Vk).

[0237]

[0238] >SEQ ID NO:40 Nucleotide sequence of the light chain variable domain of the antibody defined in this article as 02N13 (02N13_Vk).

[0239]

[0240] S protein sequence

[0241] The sequence of the surface glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known in the art. The sequences of the S1 (“Spike_rec_bind”) and S2 (“Coronavirus S2 glycoprotein”) domains are in GenBank, ID number QHD43416.1.

Claims

1. A monoclonal antibody or its antigen-binding moiety thereof, capable of neutralizing the biological activity of at least one coronavirus, preferably selected from SARS-CoV-2, SARS-CoV-1, and their variants, wherein the antibody or its antigen-binding moiety comprises a heavy chain variable domain (VH) and a light chain variable domain (VK), wherein: The VH has the amino acid sequence shown in SEQ ID NO:1, and the VK has the amino acid sequence shown in SEQ ID NO:21; or The VH has the amino acid sequence shown in SEQ ID NO:2, and the VK has the amino acid sequence shown in SEQ ID NO:22; or The VH has the amino acid sequence shown in SEQ ID NO:3, and the VK has the amino acid sequence shown in SEQ ID NO:23; or The VH has the amino acid sequence shown in SEQ ID NO:4, and the VK has the amino acid sequence shown in SEQ ID NO:24; or The VH has the amino acid sequence shown in SEQ ID NO:5, and the VK has the amino acid sequence shown in SEQ ID NO:25; or The VH has the amino acid sequence shown in SEQ ID NO:6, and the VK has the amino acid sequence shown in SEQ ID NO:26; or The VH has the amino acid sequence shown in SEQ ID NO:7, and the VK has the amino acid sequence shown in SEQ ID NO:27; or The VH has the amino acid sequence shown in SEQ ID NO:8, and the VK has the amino acid sequence shown in SEQ ID NO:28; or The VH has the amino acid sequence shown in SEQ ID NO:9, and the VK has the amino acid sequence shown in SEQ ID NO:29; or The VH has the amino acid sequence shown in SEQ ID NO:10, and the VK has the amino acid sequence shown in SEQ ID NO:

30.

2. The monoclonal antibody or its antigen-binding portion according to claim 1, wherein the VL and VK have at least 85% identity with the following amino acid sequence, preferably at least 95% identity with the following amino acid sequence, more preferably at least 98% or at least 99% identity with the following amino acid sequence: VH with SED ID NO:1 and VK with SED ID NO:21; or VH with SED ID NO:2 and VK with SED ID NO:22; or VH with SED ID NO:3 and VK with SED ID NO:23; or VH with SED ID NO:4 and VK with SED ID NO:24; or VH with SED ID NO:5 and VK with SED ID NO:25; or VH with SED ID NO:6 and VK with SED ID NO:26; or VH with SED ID NO:7 and VK with SED ID NO:27; or VH with SED ID NO:8 and VK with SED ID NO:28; or VH with SED ID NO:9 and VK with SED ID NO:29; or VH has SED ID NO:10 and VK has SED ID NO:

30.

3. The monoclonal antibody or its antigen-binding portion according to claim 1 or 2, wherein the antibody or its antigen-binding portion is a human monoclonal antibody.

4. The monoclonal antibody or its antigen-binding moiety according to any one of claims 1 to 3, wherein, When tested in an in vitro neutralization assay against at least one virus selected from SARS-CoV-2, SARS-CoV-1 and their variants, the antibody or its antigen-binding moiety showed a 100% inhibitory concentration (IC100) of less than 100 ng / ml.

5. A monoclonal antibody or its antigen-binding portion thereof, which competes with any antibody or antigen-binding portion according to any one of claims 1 to 4 for binding to the S protein of at least one coronavirus, preferably selected from at least one coronavirus of SARS-CoV-1, SARS-CoV-2 and its variants.

6. The monoclonal antibody or its antigen-binding portion according to any one of claims 1 to 5, for use in the prophylactic or therapeutic treatment of viral infection or symptoms or disorders caused by such infection, particularly for use in the prevention and / or treatment of infection caused by at least one coronavirus.

7. The monoclonal antibody or antigen-binding portion according to any one of claims 1 to 6, for use in the prophylactic or therapeutic treatment of infection caused by at least one virus selected from SARS-CoV-2, SARS-CoV-1 and variants thereof, or symptoms or disorders caused by such infection, particularly COVID-19.

8. A pharmaceutical composition comprising one or more monoclonal antibodies or antigen-binding portions thereof according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. The composition according to claim 8, for use in the preventive or therapeutic treatment of viral infection or symptoms or disorders caused by such infection, particularly for use in the prevention and / or treatment of coronavirus infection, more particularly for use in the preventive or therapeutic treatment of infection caused by at least one virus selected from SARS-CoV-2, SARS-CoV-1 and its variants, or symptoms or disorders caused by such infection, particularly for use in the prevention and / or treatment of SARS-CoV-2 and / or SARS-CoV-1 infection.

10. Use of the monoclonal antibody or its antigen-binding portion according to any one of claims 1 to 7 in in vitro or ex vivo diagnosis of infection caused by at least one coronavirus, preferably caused by at least one virus selected from SARS-CoV-2, SARS-CoV-1 and its variants.

11. An in vitro method for determining the presence of at least one coronavirus in a sample, comprising the following steps: i) Contact the sample with the antibody or its antigen-binding portion according to any one of claims 1 to 7; ii) Detect the binding of the antibody or its antigen-binding portion to the S protein of at least one coronavirus, preferably wherein the coronavirus is at least one virus selected from SARS-CoV-2, SARS-CoV-1 and their variants.

12. An in vitro method for diagnosing infection of a subject with at least one coronavirus, comprising the following steps: i) Contacting the antibody or its antigen-binding portion according to any one of claims 1 to 7 with the biological sample of the subject; ii) Detect the binding of the antibody or its antigen-binding portion to the S protein of at least one coronavirus, preferably wherein the coronavirus is at least one virus selected from SARS-CoV-2, SARS-CoV-1 and their variants.

13. A diagnostic kit comprising an antibody or antigen-binding moiety thereof according to any one of claims 1 to 7 as a specific reagent, said kit being intended for use in methods for detecting or quantifying anti-coronavirus antibodies and / or coronavirus S protein, particularly anti-SARS-CoV-2 and / or anti-SARS-CoV-1 antibodies, and / or SARS-CoV-2 and / or SARS-CoV-1 S protein, in biological samples of patients.

14. Use of the antibody or antigen-binding portion thereof according to any one of claims 1 to 7 in designing a vaccine against at least one coronavirus, particularly against at least one virus selected from SARS-CoV-2, SARS-CoV-1 and its variants.

15. A mimic epitope, said mimic epitope being specific to a unique type of an antibody or its antigen-binding moiety according to any one of claims 1 to 7.

16. An anti-idiotype antibody, specifically targeting the idiotype of the antibody or its antigen-binding moiety according to any one of claims 1 to 7.

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