KR20230038626A - Bispecific antibody specifically targeting SARS-CoV-2 - Google Patents

Abstract

The present invention relates to a bispecific antibody specifically binding to SARS-CoV-2, and a pharmaceutical composition for treating SARS-CoV-2 infection comprising the same. The bispecific antibody against the SARS-CoV-2 of the present invention exhibits therapeutic efficacy against a wide range of SARS-CoV-2 variants to be usefully used as a therapeutic agent for SARS-CoV-2 infections.

Description

Bispecific antibody specifically targeting SARS-CoV-2 {Bispecific antibody specifically targeting SARS-CoV-2}

The present invention relates to bispecific antibodies that specifically bind to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel highly contagious pathogenic beta coronavirus that first appeared in late 2019. SARS-CoV-2 caused an acute respiratory illness called coronavirus disease 2019 (COVID-19), resulting in a global pandemic. As of early June 2022, approximately 532 million people worldwide have been infected with COVID-19, with 6.3 million confirmed deaths. The COVID-19 pandemic, coupled with a socioeconomic crisis, has caused an unprecedented level of stress on healthcare systems around the world. Although several vaccines and therapeutic methods have been developed, the rapid emergence of SARS-CoV-2 variants continues to be a challenge in controlling COVID-19. Therefore, the development of new therapeutic agents for the treatment of COVID-19 is urgently needed.

The present inventors have made intensive research efforts to develop a new therapeutic agent for SARS-CoV-2. As a result, the present invention was completed by developing a bispecific antibody against SARS-CoV-2 and identifying that the bispecific antibody exhibits therapeutic efficacy against a wide range of SARS-CoV-2 variants.

Accordingly, an object of the present invention is to provide a diabody against SARS-CoV-2.

Another object of the present invention is to provide a pharmaceutical composition for treating SARS-CoV-2 infection comprising a bispecific antibody against SARS-CoV-2.

According to one aspect of the present invention, the present invention provides a diabody against SARS-CoV-2.

In one embodiment of the present invention, the diabody is a diabody that specifically binds to SARS-CoV-2, including:

(a) heavy chain complementarity determining region 1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3 A heavy chain variable region comprising an H-CDR3 comprising; and light chain complementarity determining region 1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 4, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO: 6. An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an L-CDR3;

(b) a heavy chain variable region comprising H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; And an antibody comprising a light chain variable region comprising L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6 or an antigen-binding fragment thereof; or

(c) any combination thereof.

In one embodiment of the present invention, the present invention provides a diabody specifically binding to SARS-CoV-2 comprising:

(a) a heavy chain variable region comprising H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; And an antibody comprising a light chain variable region comprising L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6 or an antigen-binding fragment thereof; and

(b) a heavy chain variable region comprising H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; And an antibody comprising a light chain variable region comprising L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6 or an antigen-binding fragment thereof.

In one embodiment of the present invention, the antibodies (a) and (b) or antigen-binding fragments thereof are each derived from monoclonal antibody clones K102.1 and K102.2 selected in Examples of the present invention. .

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof specifically binding to SARS-CoV-2 of the present invention binds to the receptor binding domain (RBD) of SARS-CoV-2 Spike protein.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof specifically binding to SARS-CoV-2 of the present invention binds to the S1 domain of the SARS-CoV-2 S protein.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof specifically binding to SARS-CoV-2 of the present invention is a full-length spike protein of the SARS-CoV-2 S protein (full-length spike protein) combine

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2 of the present invention is specifically for a mutant virus in which the S protein of SARS-CoV-2 is mutated. combine

The double antibody according to one aspect of the present invention is in the form of a dimer or multimer in which each antibody or antigen-binding fragment is linked. The antibodies or antigen-binding fragments of the present invention are covalently linked to each other, and according to one embodiment of the present invention, the double antibody may be implemented in the form of a fused protein or conjugate. Thus, the antibody or antigen-binding fragment thereof may be chemically conjugated (known as organic chemistry methods) or other means (e.g., expressing the complex as a fusion protein, either directly or indirectly through a linker (e.g., an amino acid linker)). can be connected

According to a specific embodiment of the present invention, each antibody or antigen-binding fragment constituting the double antibody is connected by at least one linker. In this case, the linker may consist of an amino acid sequence represented by the general formula (GnSm)p or (SmGn)p:

wherein n, m and p are independently

n is an integer from 1 to 7;

m is an integer from 0 to 7;

the sum of n and m is an integer of 8 or less; and

p is an integer from 1 to 7;

According to another specific embodiment of the present invention, the linker is n = 1 to 5, m = 0 to 5. In a more specific embodiment, n = 4 and m = 1. In an even more specific embodiment, the linker is (GGGGS) ₃ . In another embodiment, the linker is GGGGS. In another specific embodiment, the linker is VDGS. In another specific embodiment, the linker is ASGS.

In one embodiment of the present invention, the heavy chain variable region of (a) includes the amino acid sequence of SEQ ID NO: 7.

In one embodiment of the present invention, the light chain variable region of (a) includes the amino acid sequence of SEQ ID NO: 8.

In one embodiment of the present invention, the heavy chain variable region of (b) includes the amino acid sequence of SEQ ID NO: 17.

In one embodiment of the present invention, the light chain variable region of (b) includes the amino acid sequence of SEQ ID NO: 18.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof of (a) includes a heavy chain comprising the amino acid sequence of SEQ ID NO: 9.

In one embodiment of the present invention, the antibody of (a) or an antigen-binding fragment thereof is a double antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 10.

In one embodiment of the present invention, the antibody of (b) or an antigen-binding fragment thereof is a scFv comprising the amino acid sequence of SEQ ID NO: 19.

In one embodiment of the present invention, the antibody of (a) or (b) or antigen-binding fragment thereof binds to the heavy chain C-terminus of the antibody of (b) or (a) or antigen-binding fragment thereof.

In one embodiment of the present invention, in the double antibody, the antibody or antigen-binding fragment thereof of (b) is bound to the C-terminus of the heavy chain of (a), but is not limited thereto.

In one embodiment of the present invention, the antibody of (a) or (b) or antigen-binding fragment thereof binds to the light chain N-terminus or light chain C-terminus of the antibody of (b) or (a) or antigen-binding fragment thereof. do.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof of (b) is bound to the N-terminus of the light chain of (a), but is not limited thereto.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof of (b) is bound to the C-terminus of the light chain of (a), a dual antibody.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof of (a) and the antibody or antigen-binding fragment thereof of (b) are each a receptor binding domain (RBD) of SARS-CoV-2 Spike protein. ) binds to different epitopes of

In one embodiment of the present invention, the double antibody inhibits the binding between RBD of SARS-CoV-2 Spike protein and angiotensin converting enzyme 2 (ACE2) of host cells.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof of (a) and the antibody or antigen-binding fragment thereof of (b) are in the form of IgG1 or IgG4, but are not limited thereto.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof of (a) is in the form of IgG1 or IgG4, but is not limited thereto.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof of (a) is in the form of IgG1 or IgG4, and the antibody or antigen-binding fragment thereof of (b) is scFv.

In this specification, the term “antibody” is a specific antibody against SARS-CoV S protein, and includes antigen binding fragments of antibody molecules as well as complete antibody forms.

A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses include gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50, W. B. Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

As used herein, the term "antigen-binding fragment" refers to a fragment having an antigen-binding function, such as Fab, F(ab'), F(ab') ₂ , chemically linked F(ab') ₂ and Fv, etc. includes Among antibody fragments, Fab has a structure having variable regions of light and heavy chains, constant regions of light chains, and a first constant region (CH1) of heavy chains, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab') ₂ antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv is a minimal antibody fragment having only the heavy chain variable region and the light chain variable region. Recombinant technology for generating Fv fragments is described in PCT International Publication Patent Applications WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344. In two-chain Fv, the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and in single-chain Fv, the heavy chain variable region and the short chain variable region are generally shared through a peptide linker. They are linked by bonds or directly linked at the C-terminus, so that they can form a dimer-like structure like double-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (eg, restriction digestion of whole antibodies with papain yields Fab and digestion with pepsin yields F(ab') ₂ fragments), or It can be produced through genetic recombination technology.

In the present invention, the antibody is specifically scFv form or complete antibody form. In addition, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). Preferably, the constant regions are gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3) and gamma 4 (IgG4), most specifically the gamma 4 (IgG4) isotype. The light chain constant region may be of kappa or lambda type, preferably kappa type. Accordingly, specific antibodies of the present invention are scFv type or IgG4 type having kappa light chain and gamma 4 heavy chain, but are not limited thereto.

As used herein, the term "heavy chain" refers to a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2 and CH3 comprising an amino acid sequence having sufficient variable region sequence for conferring specificity to an antigen and a full-length heavy chain thereof I mean all fragments. In addition, the term "light chain" as used herein refers to both a full-length light chain and fragments thereof comprising a variable region domain V _L and a constant region domain _CL comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen. do.

As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of immunoglobulin heavy and light chains (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chains (CDR-H1, CDR-H2, and CDR-H3) and light chains (CDR-L1, CDR-L2, and CDR-L3) each contain three CDRs. CDRs provide key contact residues for antibody binding to an antigen or epitope.

In the present specification, the antibody or antigen-binding fragment thereof refers to a full-length or intact polyclonal or monoclonal antibody, as well as antigen-binding fragments thereof (eg, Fab, Fab', F(ab')2, Fab3 , Fv and variants thereof), fusion proteins comprising one or more antibody portions, human antibodies, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibodies (scFv) , scFv-Fc, bispecific antibodies, multispecific antibodies, glycosylation variants of antibodies, amino acid sequence variants of antibodies and other modified configurations of immunoglobulin molecules containing antigen recognition sites of the necessary specificity, and covalent modifications containing antibodies. Specific examples of modified antibodies and antigen-binding fragments thereof include nanobodies, AlbudAbs, dual affinity re-targeting (DARTs), bispecific T-cell engagers (BiTEs), tandem diabodies (TandAbs), dual acting Fabs (DAFs), two- These include in-one antibodies, small modular immunopharmaceuticals (SMIPs), fynomers fused to antibodies (FynomAbs), dual variable domain immunoglobulins (DVD-Igs), peptide modified antibodies (CovX-bodies), duobodies and triomAbs. The list of such antibodies and antigen-binding fragments thereof is not limited to the above.

As used herein, the term "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain generally consist of four FR domains FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences generally appear in the following order in VH (or VL/Vk):

(a) Framework region 1 of heavy chain (FRH1)-CDRH1 (complementarity determining region 1 of heavy chain)-FRH2-CDRH2-FRH3-CDRH3-FRH4; and

(b) Framework region 1 of light chain (FRL1)-complementarity determining region 1 of light chain (CDRL1)-FRL2-CDRL2-FRL3-CDRL3-FRL4.

As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, each domain having four conserved framework regions (FR) and three hypervariable regions (HVR) ). (Kindt et al., Kuby Immunology, 6th edition, W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, an antibody that binds to a particular antigen can be separated using a VH or VL domain from an antibody that binds the antigen and screens a library of complementary VL or VH domains, respectively.

As used herein, the term "specifically binds" or the like means that an antibody or antigen-binding fragment thereof, or other construct such as an scFv, forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 x 10 ^-6 M or less (eg, 9 x 10 ^-7 M, 8 x 10 ^-7 M, 7 x 10 -7 M, 6 x 10 ^{-7 M} , 5 x 10 ^-7 M , 4 x 10 ^-7 M, 3 x 10 ^-7 M, 2 x 10 ^-7 M, or 1 x 10 ^-7 M), preferably 1 x 10 ^-7 M or less (eg 9 x 10 ^-8 M , 8 x 10 ^-8 M, 7 x 10 ^-8 M, 6 x 10 ^-8 M, 5 x 10 ^-8 M, 4 x 10 -8 M, 3 x 10 ^-8 M, 2 x ^{10 -8 M} , or 1 x 10 ^-8 M), more preferably 1 x 10 ^-8 M or less (eg, 9 x 10 ^-9 M, 8 x 10 ^-9 M, 7 x 10 ^-9 M, 6 x 10 ^-9 M , 5 x 10 ^-9 M, 4 x 10 ^-9 M, 3 x 10 ^-9 M, 2 x 10 ^-9 M, or 1 x 10 ^-9 M) equilibrium dissociation constants (eg, K _D smaller than indicates a tighter bond). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

As used herein, the term "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg antibody) and its binding partner (eg antigen). As used herein, unless otherwise specified, “binding affinity” refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). indicates The affinity of a molecule Y with its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

Also used herein, the term “human antibody” refers to an amino acid sequence of an antibody produced by a human or a human cell, or an antibody derived from a non-human source that utilizes human antibody repertoires or other human antibody coding sequences. It has the corresponding amino acid sequence. This definition of a human antibody excludes humanized antibodies comprising non-human antigen-binding moieties.

As used herein, a "chimeric" antibody is one in which a portion of the heavy and/or light chain is from a particular source or species and the remainder of the heavy and/or light chain is from a different source or species. means antibody.

As used herein, the term "humanized antibody" refers to a chimeric immunoglobulin containing minimal sequence derived from a non-human immunoglobulin of a non-human (e.g., mouse) antibody, an immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequence of an antibody). In most cases, a humanized antibody is a CDR in which residues of a complementarity-determining region (CDR) of the recipient are from a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. is a human immunoglobulin (recipient antibody) replaced by residues of In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further improve and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to CDR regions of a non-human immunoglobulin; All or substantially all of the FR region has the sequence of the FR region of a human immunoglobulin. The humanized antibody includes at least a portion of an immunoglobulin constant region (Fc region) to substantially a human immunoglobulin constant region (Fc region) sequence.

Such variants have "substantial similarity", i.e. at least about 90% sequence identity, more preferably at least about It means sharing 95%, 98% or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functionality of the protein. Where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted up to correct for the conservative nature of the substitutions.

Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substituents revealed that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Accordingly, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is given a hydrophobicity index according to its hydrophobicity and charge: Isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); Tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); Lysine (-3.9); and arginine (-4.5).

The hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is a known fact that amino acids having similar hydrophobicity indexes should be substituted to retain similar biological activities. When a mutation is introduced with reference to the hydrophobicity index, substitution is made between amino acids showing a difference in hydrophobicity index, preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that substitution between amino acids having similar hydrophilicity values results in proteins having equivalent biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); Asphaltate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); Leucine (-1.8); isoleucine (-1.8); Tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4).

When a mutation is introduced by referring to the hydrophilicity value, substitution is preferably made between amino acids exhibiting a difference in hydrophilicity value within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

Amino acid exchanges in proteins that do not entirely alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

The antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2 of the present invention comprises a slight change in the above-described amino acid sequence, that is, a modification that has little effect on the tertiary structure and function of the antibody. Antibodies against the SARS-CoV-2 S protein or antigen-binding fragments thereof. Thus, in some embodiments, even if they do not match the sequence described above, they may have at least 100%, 93%, 95%, 96%, 97%, or 98% similarity.

According to one embodiment of the present invention, the SARS-CoV-2 S antibody or antigen-binding fragment thereof of the present invention is a monoclonal antibody, a bispecific antibody comprising a heavy chain variable region and a light chain variable region comprising the CDRs of the above-described sequence. Antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies (scFv), Fab fragments, F(ab')fragments, disulfide-linked Fvs (sdFV) and anti-idiotypic (anti-idiotype) Id) antibodies, and epitope-binding fragments of the antibodies, and the like, but are not limited thereto. In another embodiment of the present invention, the SARS-CoV-2 S protein antibody or antigen-binding fragment thereof of the present invention is a scFv.

In a specific embodiment of the present invention, the heavy chain variable region and the light chain variable region included in the antibody or antigen-binding fragment thereof are (Gly-Ser)n, (Gly ₂ -Ser)n, (Gly ₃ -Ser)n or ( linked by a linker such as Gly ₄ -Ser)n. Here, n is an integer of 1 to 6, specifically 3 to 4, but is not limited thereto. The light chain variable region and heavy chain variable region of the scFv may exist, for example, in the following orientation: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-described double antibody specifically binding to SARS-CoV-2.

As used herein, the term "nucleic acid molecule" has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, are not only natural nucleotides, but also analogs having modified sugar or base sites. (analogue) is also included (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

It is apparent to those skilled in the art that the nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention is sufficient to be a nucleotide sequence encoding the amino acid sequence constituting the antibody or antigen-binding fragment thereof, and is not limited to any specific nucleotide sequence. .

This is because, even if a nucleotide sequence is mutated, expression of the mutated nucleotide sequence as a protein may not result in a change in the protein sequence. This is called codon degeneracy. Thus, the nucleotide sequence may be a functionally equivalent codon or a codon encoding the same amino acid (e.g., by codon degeneracy, there are six codons for arginine or serine), or a codon encoding a biologically equivalent amino acid. It includes a nucleotide sequence comprising a.

Considering the mutations having the above-described biologically equivalent activity, the nucleic acid molecule of the present invention encoding the amino acid sequence constituting the antibody or antigen-binding fragment thereof is interpreted to include a sequence exhibiting substantial identity therewith. The above substantial identity is at least when the sequence of the present invention and any other sequence described above are aligned so as to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. 60% or greater homology (eg 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), more specifically 70% or greater homology (eg 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%), even more specifically greater than or equal to 80% homology (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), even more specifically greater than or equal to 90% homology (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), most specifically a sequence that exhibits at least 95% homology (eg, 95%, 96%, 97%, 98%, or 99%). All integers of 60% or more and 100% or less and minor numbers existing therebetween are included within the scope of the present invention with respect to % homology.

Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981) ; Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) is accessible from the National Center for Biological Information (NBCI), etc., and blastp, blastn, It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLAST is accessible through the BLAST page on the ncbi website. The sequence homology comparison method using this program can be found on the BLAST help page of the ncbi website.

According to a specific embodiment of the present invention, the heavy chain CDR, light chain CDR, heavy chain variable region, light chain variable region, heavy chain, or polypeptide constituting the light chain of the antibody or antigen-binding fragment thereof against the SARS-CoV-2 S protein of the present invention. And the nucleotide sequence encoding it is listed in the sequence listing attached to this specification.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule described above.

As used herein, the term "vector" refers to a plasmid vector as a means for expressing a gene of interest in a host cell; cosmid vector; and viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors, but are not limited thereto.

According to one embodiment of the present invention, in the vector of the present invention, a nucleic acid molecule encoding a heavy chain variable region and a nucleic acid molecule encoding a light chain variable region are operatively linked to a promoter.

As used herein, the term "operatively linked" refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, and , whereby the regulatory sequence controls the transcription and/or translation of the other nucleic acid sequence.

The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), , this document is incorporated herein by reference.

Vectors of the present invention may typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

For example, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., metallotionine promoter, beta-actin promoter, human hemoglobin promoter, human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g. adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, The LTR promoter of HIV, the promoter of Moloney virus, the promoter of Epstein Barr virus (EBV) and the promoter of Rouss sarcoma virus (RSV) can be used, and usually have a polyadenylation sequence as a transcription termination sequence.

Vectors of the present invention may be fused with other sequences to facilitate purification of antibodies expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

In addition, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column or the like without an additional sequence for purification.

On the other hand, the expression vector of the present invention contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neo There are genes for resistance to mycin and tetracyclines.

Optionally, the vector may additionally carry genes encoding reporter molecules (eg, luciferase and -glucuronidase).

According to one embodiment of the present invention, the expression vector is a vector into which a nucleic acid molecule encoding the anti-SARS-CoV-2 S antibody or antigen-binding fragment thereof is inserted, and operably binds to the nucleotide sequence of the nucleic acid molecule It is a recombinant vector for host cell expression that is operatively linked and contains a promoter that forms RNA molecules in host cells and a poly A signal sequence that acts in host cells to cause polyadenylation at the 3'-end of RNA molecules.

According to another aspect of the present invention, an isolated host cell comprising a recombinant vector is provided.

Any host cell capable of stably and continuously cloning and expressing the vector of the present invention can be used as any host cell known in the art. For example, suitable eukaryotic host cells for the vector are yeast (Saccharomyce cerevisiae), insect cells , monkey kidney cells 7 (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line , HuT 78 cells and HEK-293 cells.

As used herein, the terms "transformed", "transduced" or "transfected" refer to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. "Transformed", "transduced" or "transfected" An "infected" cell is a cell that has been transformed, transduced or transfected with an exogenous nucleic acid, including the cell and progeny cells resulting from its passage.

Methods for delivering the vector of the present invention into a host cell, when the host cell is a eukaryotic cell, include a microinjection method (Capecchi, M.R., Cell, 22:479 (1980)), a calcium phosphate precipitation method (Graham, F.L. et al. , Virology, 52:456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, T.K. et al., Gene , 10:87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)), etc., vectors can be injected into host cells.

In the present invention, the recombinant vector injected into the host cell can express the recombinant double antibody in the host cell, and in this case, a large amount of double antibody is obtained. For example, when the expression vector includes a lac promoter, gene expression can be induced by treating host cells with IPTG.

The culturing is usually carried out under aerobic conditions, such as by shaking culturing or rotation by a rotator. The incubation temperature is preferably in the range of 10 to 40°C, and the incubation time is generally 5 hours to 7 days. The pH of the medium is preferably maintained in the range of 3.0 to 9.0 during culture. The pH of the medium can be adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like. During cultivation, antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline may be added to maintain and express the recombinant vector, if necessary. When culturing a host cell transformed with a recombinant expression vector having an inducible promoter, a suitable inducer may be added to the medium if necessary. For example, if the expression vector contains the lac promoter, IPTG (isopropyl-beta-D-thiogalactopyranoside) may be added, and if the expression vector contains the trp promoter, indoleacrylic acid may be added to the medium.

According to another aspect of the present invention, a pharmaceutical composition for treating SARS-CoV-2 infection comprising the above-described bispecific antibody and a pharmaceutically acceptable carrier thereof is provided.

In one embodiment of the present invention, the SARS-CoV-2 has a mutation consisting of N354D / D364Y, V367F, W436R, R408I, G476S, V483A, V341I, F342L, A435S, or a combination thereof in the amino acid sequence of RBD. However, it is not limited to this.

In one embodiment of the present invention, the SARS-CoV-2 is selected from the group consisting of wild type, alpha mutant, beta mutant, gamma mutant, delta mutant, and kappa mutant, but is not limited thereto.

Since the pharmaceutical composition of the present invention uses the above-described antibody or antigen-binding fragment thereof against SARS-CoV-2 of the present invention as an active ingredient, the common content between the two is to avoid excessive complexity of the present specification, omit the description.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, including, but not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; it is not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, such as intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration. can

The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, route of administration, excretion rate and reaction sensitivity, A ordinarily skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to prevent or treat the above-mentioned diseases.

As used herein, the term "prophylaxis" refers to preventive or protective treatment of a disease or condition. As used herein, the term "treatment" means reduction, suppression, sedation or eradication of a disease state.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a bispecific antibody against SARS-CoV-2.

(b) The present invention provides a pharmaceutical composition for treating SARS-CoV-2 infection comprising a bispecific antibody against SARS-CoV-2.

Since the bispecific antibody against SARS-CoV-2 of the present invention exhibits therapeutic efficacy against a wide range of SARS-CoV-2 variants, it can be usefully used as a treatment for SARS-CoV-2 infection.

1 is a diagram showing the results of performing competitive ELISA analysis to confirm that the isolated antibodies recognize SARS-CoV-2 RBD epitopes independent of each other.
2 to 4 are schematic diagrams showing the structures of three types of bispecific antibodies that can be prepared with the antibodies K102.1 and K102.2 of the present invention and the structures of vectors for expressing them.
Figure 5 is a diagram showing the measurement of the expression level in cells of the antibodies (K102.1 and K102.2) and double antibodies (K202.A, K202.B and K202.C) of the present invention.
Figure 6 is a diagram showing through Western blot that the antibodies (K102.1 and K102.2) and the double antibodies (K202.A, K202.B and K202.C) of the present invention are expressed with high purity.
7 shows the results of SPR analysis to confirm the binding affinity of the antibodies and diabodies of the present invention to wild type and alpha, beta, gamma, delta and kappa variants of SARS-CoV-2.
8 is a result of competition analysis using SPR to confirm that the double antibody of the present invention recognizes two different independent epitopes.
Figure 9 is a diagram showing the binding inhibitory effect of each antibody concentration in order to confirm the neutralizing activity for the interaction between hACE2 of the double antibody of the present invention and the RBD of SARS-CoV-2 wild type and mutant.
Figure 10 shows the antibody concentration to confirm the neutralizing activity of the interaction between hACE2 of the diabodies of the present invention and RBDs with N354D/D364Y, V367F, W436R, R408I, G476S, V483A, V341I, F342L, or A435S mutations. It is a diagram showing the star binding inhibitory effect.
11 is a diagram showing that a 293T stable cell line (293T/hACE2 cells) overexpressing hACE2 was established.
12 is a diagram showing the neutralizing activity of antibodies against pseudovirus infection of SARS-CoV-2 wild-type and variants.
13 is a diagram showing changes in pseudovirus infectivity of various SARS-CoV-2 wild-type and variants according to the presence or absence of the double antibody K202.B of the present invention.
14 is a diagram showing the results of pharmacokinetic analysis of the double antibody K202.B of the present invention.
15 is a diagram showing the outline of an experiment to analyze the in vivo efficacy of the double antibody K202.B of the present invention against SARS-CoV-2 wild type.
16 to 20 are daily weight changes (FIG. 16) and clinical scores (FIG. 17) according to treatment of SARS-CoV-2 wild-type virus and antibody, in order to analyze the in vivo efficacy of the bispecific antibody K202.B of the present invention. , the expression level of virus-derived genes (FIGS. 18 and 19), and the lung pathology test results (FIG. 20).
21 and 22 are diagrams showing the cell viability (FIG. 21) and the expression level of cell adhesion molecules (FIG. 22) when endothelial cells were treated in order to analyze the in vitro toxicity of the double antibody K202.B of the present invention.
23 is a diagram showing the results of biochemical tests showing liver and kidney toxicity from serum after K202.B was administered to mice in order to analyze the in vivo toxicity of the double antibody K202.B of the present invention.
24 is a diagram showing the outline of an experiment to analyze the in vivo efficacy of the double antibody K202.B of the present invention against the SARS-CoV-2 delta variant.
25 to 29 are daily weight changes according to treatment of SARS-CoV-2 delta variant virus and antibody (FIG. 25), clinical scores (FIG. 26), expression levels of virus-derived genes (FIGS. 27 and 28), and lung pathology test results (FIG. 29).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, unless otherwise specified, "%" used to indicate the concentration of a particular substance is (weight/weight) % for solids/solids, (weight/volume) % for solids/liquids, and Liquid/liquid is (volume/volume) %.

Example 1: Design, generation and characterization of bsAbs

We isolated four SARS-CoV-2 RBD-specific human scFvs with distinct complementarity determining region (CDR) sequences from a human synthetic scFv library using phage display technology. To prevent Fab arm exchange resulting in unwanted heterogeneous mixtures of antibodies by half molecules exchanged for endogenous IgG4, we constructed an IgG4-based mAb with the S228P mutation [IgG4(S228P)].

Among these antibodies, the present inventors identified K102.1 and K102.2, which are non-competitive mAb pairs that recognize independent epitopes of SARS-CoV-2 RBD, using a competitive ELISA assay (FIG. 1).

Based on these parental mAbs, three IgG4(S228P)-(scFv)2 bispecific antibodies with the same structure as K202.A (Fig. 2), K202.B (Fig. 3) and K202.C (Fig. 4) , bsAb) was designed. As a result of expressing the bispecific antibody of the above three structures in cells, the expression rate of the bispecific antibody of the K202.C structure was quite low (FIG. 5).

Among the structures of the designed double antibody, two types of IgG4(S228P)-(scFv)2 bsAbs, K202.A and K202.B, were produced with a purity of 90% or more (FIG. 6). Then, surface plasmon resonance (SPR) analysis was performed to compare the binding affinity of bsAbs to purified RBDs of wild-type and alpha, beta, gamma, delta, and kappa variants of SARS-CoV-2 and the binding affinity of mAbs. The results are shown in FIG. 7 .

As shown in Figure 7 and Table 1, K202.B exhibited strong binding to the RBD of all tested SARS-CoV-2 variants in the sub-nanomolar range, comparable to the parental mAb against SARS-CoV-2 wild type. showed

We performed a competition assay using SPR to further confirm whether K202.B can recognize two independent epitopes of SARS-CoV-2 RBD. Results are shown in FIG. 8 .

As shown in Figure 8, K202.B was able to bind to RBD even after being saturated with K102.1 or K102.2 (Figure 8, A and B). In contrast, both K102.1 and K102.2 did not bind to RBD after being saturated with K202.B (Fig. 8, C and D), indicating that bsAb K202.B has two independent binding sites specific. indicates recognition.

Equilibrium dissociation constants of parental mAbs and bsAbs for RBDs of wild-type and variants of SARS-CoV-2 RBD type K102.1 K102.2 K _a (M ^-1 ) K _d (M ^-1 S ^-1 ) K _D (nM) K _a (M ^-1 ) K _d (M ^-1 S ^-1 ) K _D (nM) Wild-type 1.61 × 10 ⁵ 2.76 × 10 ^-4 1.72 1.97 × 10 ⁵ 4.34 × 10 ^-4 2.20 B.1.1.7 2.06 × 10 ⁵ 1.93 × 10 ^-4 0.94 1.54 × 10 ⁵ 5.57 × 10 ^-4 3.62 B.1.351 0.93 × 10 ⁵ 2.95 × 10 ^-4 3.18 4.92 × 10 ⁵ 7.88 × 10 ^-4 1.60 P.1 0.70 × 10 ⁵ 3.15 × 10 ^-4 4.51 4.54 × 10 ⁵ 9.39 × 10 ^-4 2.07 B.1.617.2 2.11 × 10 ⁵ 2.98 × 10 ^-4 1.41 3.33 × 10 ⁵ 5.11 × 10 ^-4 1.53 B.1.617.1 0.95 × 10 ⁵ 3.17 × 10 ^-4 3.34 4.07 × 10 ⁵ 6.83 × 10 ^-4 1.68 RBD type K202.A K202.B K _a (M ^-1 ) K _d (M ^-1 S ^-1 ) K _D (nM) K _a (M ^-1 ) K _d (M ^-1 S ^-1 ) K _D (nM) Wild-type 1.65 × 10 ⁵ 1.29 × 10 ^-4 0.81 1.47 × 10 ⁵ 9.98 × 10 ^-4 0.68 B.1.1.7 1.89 × 10 ⁵ 1.08 × 10 ^-4 0.57 8.31 × 10 ⁵ 7.93 × 10 ^-4 0.95 B.1.351 0.78 × 10 ⁵ 2.49 × 10 ^-4 3.17 2.22 × 10 ⁵ 2.00 × 10 ^-4 0.90 P.1 2.03 × 10 ⁵ 3.81 × 10 ^-4 1.66 0.87 × 10 ⁵ 1.86 × 10 ^-4 2.15 B.1.617.2 2.47 × 10 ⁵ 2.26 × 10 ^-4 0.92 1.50 × 10 ⁵ 1.17 × 10 ^-4 0.78 B.1.617.1 2.48 × 10 ⁵ 2.82 × 10 ^-4 1.14 2.78 × 10 ⁵ 1.60 × 10 ^-4 0.58

K _a , Association constant

K _d , Dissociation constant

K _D , Equilibrium dissociation constant

Example 2: Neutralizing activity of bsAbs in hACE2-RBD interaction in vitro, SARS-CoV-2 pseudovirus, and live virus infection

2-1. Neutralizing activity of bsAbs in hACE2-RBD interaction

In order to evaluate the neutralizing activity of bsAbs in the hACE2-RBD interaction, the present inventors immobilized each recombinant RBD protein of SARS-CoV-2 wild type and Alpha, Beta, Gamma, Delta and Kappa mutants on a microtiter plate and performed ELISA Based neutralization assay was performed. Microtiter plates were individually incubated with recombinant hACE2 in the presence or absence of parental mAbs, mAb cocktails containing parental mAbs together, or bsAbs. The results are shown in Figure 9 and Table 2.

IC ₅₀ values of the antibodies of the present invention in the direct interaction between SARS-CoV-2 wild-type and variant RBD and hACE2 RBD type IC ₅₀ (nM) K102.1 K102.2 K102.1
+ K102.2 K202.A K202.B Wild-type 1.33 ± 0.04 4.94 ± 0.06 1.55 ± 0.07 1.57 ± 0.20 1.85 ± 0.09 B.1.1.7 23.10 ± 0.06 ND 16.79 ± 0.16 2.04 ± 0.07 1.21 ± 0.10 B.1.351 ND 0.86 ± 0.06 1.94±0.06 0.75 ± 0.03 0.45 ± 0.15 P.1 ND 3.23 ± 0.16 5.52 ± 0.13 1.19 ± 0.12 1.73±0.10 B.1.617.2 1.83 ± 0.09 1.14 ± 0.11 1.08 ± 0.08 0.73 ± 0.09 0.18 ± 0.12 B.1.617.1 24.32 ± 0.09 0.40 ± 0.10 0.71 ± 0.06 0.34 ± 0.08 0.19 ± 0.11

ND, not determined

As shown in FIG. 9 and Table 2, in the case of hACE2 binding to wild-type RBD, all antibodies showed similar inhibitory effects. However, for all variants of the RBD, the K202.B diabody inhibited hACE2 binding more strongly than either the mAb or mAb cocktail. In particular, the IC ₅₀ of K202.B on hACE2 binding to RBD of the beta, delta and kappa variants was shown to have a slightly advantageous inhibitory effect compared to that of K202.A in the sub-nanomolar range.

In addition, K202.B showed a strong inhibitory effect on hACE2 binding to RBDs with N354D/D364Y, V367F, W436R, R408I, G476S, V483A, V341I, F342L, or A435S mutations (see FIG. 10).

2-2. SARS-CoV-2 pseudovirus Neutralizing activity of bsAbs against infection

To evaluate the neutralizing ability of the bsAbs of the present invention in SARS-CoV-2 pseudovirus infection, we tested a 293T stable cell line (293T/hACE2 cells) overexpressing hACE2 in the presence or absence of parental mAb, mAb cocktail and bsAb ( SARS-CoV-2 pseudovirus neutralization assay was performed for FIG. 11).

The results are shown in Figure 12 and Table 3.

IC ₅₀ values of antibodies against pseudovirus infection of SARS-CoV-2 wild-type and variants Pseudovirus
type IC ₅₀ (nM) K102.1 K102.2 K102.1
+ K102.2 K202.A K202.B Wild-type 1.94±0.07 ND 1.96 ± 0.08 0.27 ± 0.07 0.16 ± 0.04 B.1 2.22 ± 0.09 ND 2.05 ± 0.14 0.39 ± 0.14 0.12 ± 0.11 B.1.1.7 6.16 ± 0.08 ND 3.12 ± 0.07 0.18 ± 0.03 0.15 ± 0.03 B.1.351 ND ND ND 0.46 ± 0.04 0.10 ± 0.04 P.1 ND ND ND 1.74±0.10 1.04 ± 0.09 B.1.617.2 4.48 ± 0.10 ND 3.04 ± 0.08 1.83 ± 0.13 0.13 ± 0.09 B.1.617.1 ND ND 15.63 ± 0.08 0.24 ± 0.04 0.05 ± 0.04

ND, not determined

As shown in Figure 12, we found that the parental antibody K102.1 of the present invention significantly inhibited the infection of wild-type, B.1 (D614G), alpha and delta variant pseudoviruses in the nanomolar range, but inhibited other variants. observed that it did not. On the other hand, K102.2 showed no effect on all pseudoviruses tested.

On the other hand, the diabody K202.B showed a stronger inhibitory effect against all pseudoviruses than the parental mAb or mAb cocktail with IC ₅₀ values of mostly subnanomolar or nanomolar concentrations. The diabody K202.A showed a similar or slightly weaker inhibitory effect to that of the diabody K202.B (FIG. 12, Table 3).

2-3. Neutralizing activity of bsAbs in live viral infection

Next, the present inventors used permissive cells (293T/hACE2) and Fc gamma receptor-bearing cells (including 293T, K562 and THP-1 cells) to demonstrate that the diabody K202.B of the present invention enhances antibody-dependent enhancement. , ADE) were evaluated. The present inventors monitored changes in pseudovirus infectivity of various SARS-CoV-2 wild-type and variants according to the presence or absence of K202.B.

Results are shown in FIG. 13 .

As shown in Figure 13, no significant change was observed in the pseudovirus infection rate depending on the presence or absence of K202.B. This indicates that the double antibody K202.B of the present invention does not induce ADE in vivo.

Example 3: In vivo efficacy and toxicity evaluation of bispecific antibody K202.B in wild-type SARS-CoV-2 infected animal model

3-1. Serum pharmacokinetic analysis in vivo

In order to investigate the pharmacokinetics of the double antibody K202.B, the present inventors intravenously injected 5 mg/kg K202.B into ICR mice, collected blood samples over time, and measured the serum level of K202.B using ELISA. analyzed.

Results are shown in FIG. 14 .

As shown in Figure 14, K202.B showed an in vivo half-life of about 78 hours in mice.

3-2. In vivo efficacy assay

Next, the present inventors analyzed the in vivo efficacy of the double antibody K202.B against wild-type SARS-CoV-2.

Specifically, wild-type SARS-CoV-2 virus was intranasally administered to K18-hACE2 transgenic (TG) mice. After 3 hours, mice were intravenously injected with two doses (5 and 30 mg/kg) of K202.B. As a parental mAb control, 30 mg/kg of K102.1 was intravenously injected. After 6 days, the mice were sacrificed and the following analysis was performed (FIG. 15).

Results are shown in FIGS. 16 and 17 .

As shown in FIG. 16, wild-type SARS-CoV-2-infected mice administered with the double antibody K202.B did not show significant weight loss throughout the observation period, whereas PBS and K102.1-treated mice 6 days after infection (dpi ) showed significant weight loss.

Also, as shown in Figure 17, at 6 dpi, each K202.B-treated group showed a clinical severity score of ≤1 (mostly 0), whereas the PBS or K102.1-treated groups had very coarse coat, slightly closed eyes and and/or a score greater than 3 characterizing moribund state.

In addition, the present inventors subjected lung samples of all mice sacrificed at 6 dpi to RT-qPCR to confirm the relative expression of virus E and RNA-dependent RNA polymerase (RdRp) genes.

Results are shown in Figures 18 and 19.

As shown in FIGS. 18 and 19 , it was observed that the expression of two viral genes was significantly decreased in a dose-dependent manner in each of the diabody K202.B-treated groups when compared to the PBS-treated group. However, only a slight decrease was shown in the K102.1 treated group (Figures 16 and 17).

In addition, pathological examination was performed on the lungs of the infected mice at 6 dpi and is shown in FIG. 20 and Table 4.

Lung pathology score analysis of wild-type SARS-CoV-2 infected mice Pathological score Number of specimens (n = 7) PBS K102.1 (30 mg/kg) K202.B (5 mg/kg) K202.B (30 mg/kg) 0 0 0 2 (28.57%) 3 (42.86%) 0.5 0 0 0 0 One 2 (28.57%) 3 (42.86%) 1 (14.29%) 4 (57.14%) 1.5 3 (42.86%) 4 (57.14%) 0 0 2 2 (28.57%) 0 4 (57.14%) 0 Mean* 1.43 1.07 1.29 0.57

*Mean = (pathological score Х numbers of specimen)/total numbers of specimenPathological score = (0, 0%; 1, ≤ 10%; 2, 10%-50%; 3, ≥ 50%; +0.5, pulmonary edema or alveolar hemorrhage)

As shown in Table 4, PBS and K102.1 treated mice appeared to score ≧1 or 2 due to significant lung lesions. On the other hand, in the case of the K202.B treatment group, 0 points were shown at both 5 and 30 mg/kg doses at a high rate.

As also shown in Figure 20, further histopathological analysis revealed normal features in the K202.B treated lungs, but PBS and K102.1 treated mice showed severe pulmonary edema or alveolar hemorrhage.

3-3. In vitro toxicity assay

To analyze the in vitro toxicity of the double antibody of the present invention, endothelial cells were treated with 20 μg/ml K202.B or 36 μg/ml 5-fluorouracil, and cell viability was measured. Results are shown in FIG. 21 .

As shown in Figure 21, there was no significant effect on endothelial cell survival and activation in the bispecific antibody K202.B-treated group compared to the control group, resulting in no serious endothelial toxicity.

In addition, in order to confirm the endothelial cell activation effect of the double antibody, the present inventors treated inflammatory cytokine (hTNF-α) or double antibody K202.B and measured the expression of cell adhesion molecules (ICAM-1, VCAM-1). Confirmed. Results are shown in FIG. 22 .

As shown in FIG. 22, the expression of cell adhesion molecules (ICAM-1, VCAM-1) was activated in the group treated with hTNF-α, but in the group treated with the double antibody K202.B of the present invention, cell adhesion molecules ( There was no change in the expression of ICAM-1 and VCAM-1). Therefore, it was confirmed that the double antibody K202.B of the present invention did not cause endothelial toxicity due to no endothelial cell activating effect.

3-4. In vivo toxicity assay

In order to confirm the in vivo toxicity of the double antibody K202.B, the present inventors intravenously injected K202.B into mice and evaluated liver and kidney toxicity from serum.

Results are shown in FIG. 23 .

As shown in FIG. 23, there was no change in body weight and no change was observed in biochemical indicators representing liver and kidney toxicity. From the above results, it can be seen that the double antibody K202.B of the present invention does not cause toxicity in vivo.

Example 4: In vivo efficacy and toxicity evaluation of bispecific antibody K202.B in an animal model infected with SARS-CoV-2 delta variant

4-1. In vivo efficacy assay

The present inventors analyzed the in vivo efficacy of the double antibody K202.B against the delta variant of SARS-CoV-2. In order to evaluate the therapeutic efficacy of K202.B against SARS-CoV-2 Delta variant, 5 or 30 mg/kg of K18-hACE2 TG mice were challenged intranasally with SARS-CoV-2 Delta variant virus and 3 hours later. K202.B was injected intravenously (FIG. 24).

As shown in FIG. 25, no dramatic weight loss was observed in the double antibody K202.B-treated group compared to the PBS-treated group, which showed a statistically significant rapid weight loss at 6 dpi.

In addition, as shown in Fig. 26, the mice treated with the double antibody K202.B showed an average score of 1 point or less (0 point for most mice) for clinical severity.

In addition, the present inventors subjected lung samples of all mice sacrificed at 6 dpi to RT-qPCR to confirm the relative expression of virus E and RNA-dependent RNA polymerase (RdRp) genes. As shown in FIGS. 27 and 28 , RT-qPCR results in the K202.B treated group compared to the PBS treated group showed that the expression of both virus E and RdRp genes was effectively reduced. In particular, in the 30 mg/kg K202.B treated group, viral gene expression was hardly detected at 6 dpi.

In addition, the results of pathology examination on the lungs of infected mice at 6 dpi are shown in Table 5 and FIG. 29. As shown in Table 5, in the K202.B-treated group, the pathological score of 0 was increased in a dose-dependent manner, whereas in the PBS-treated group, scores of 1-2 (mostly 2) with lung lesions were observed.

As shown in FIG. 29 , histopathological analysis showed normal findings in the K202.B-treated lungs, whereas clear pulmonary edema or alveolar hemorrhage was observed in PBS-treated mice.

Analysis of pathological scores in the lungs of SARS-CoV-2 delta variant-infected mice Pathological score Numbers of specimen (n = 7) PBS K202.B (5 mg/kg) K202.B (30 mg/kg) 0 0 2 (28.57%) 5 (71.43%) 0.5 0 0 0 One 2 (28.57%) 5 (71.43%) 2 (28.58%) 1.5 1 (14.29%) 0 0 2 4 (57.14%) 0 0 Mean* 1.57 0.71 0.29

*Mean = (pathological score Х numbers of specimen)/total numbers of specimenPathological score = (0, 0%; 1, ≤ 10%; 2, 10%-50%; 3, ≥ 50%; +0.5, pulmonary edema or alveolar hemorrhage)

Experiment materials and methods

1. Cell culture

293T, K562 and THP-1 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA), and CHOZN-GS cells (derived from CHO-K1 and adapted to serum-free and suspension culture conditions) were purchased from Merck ( Merck, Whitehouse Station, NJ, USA). Expi293 cells were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Basel, Switzerland). 293T cells were cultured in DMEM medium (Thermo Fisher Scientific), whereas K562 and THP-1 cells were cultured in 10% (v/v) FBS (Thermo Fisher Scientific) and 100 U/ml penicillin-streptomycin (Thermo Fisher Scientific). cultured at 37° C. and 5% CO ₂ in this supplemented RPMI medium (Thermo Fisher Scientific). HUVECs were maintained in EGM-2 medium (Lonza). Expi293 cells were cultured at 37° C., 125 rpm and 8% CO ₂ using Expi293 expression medium in a shaking incubator. CHOZN-GS cells were cultured in EX-CELL Advanced CHO Fed-batch medium (Sigma-Aldrich, Burlington, MA, USA) at 37 °C in a microscale bioreactor Ambr ^® 15 (Sartorius, Gφttingen, Germany).

2. Surface plasmon resonance (SPR)

The binding kinetics of antibodies to SARS-CoV-2 RBD were measured at room temperature on an iMSPR-mini instrument (iCLUEBIO, Seongnam, Korea) in 10 mM HEPES pH 7.4, 700 mM NaCl, 2 mM CaCl ₂ , and 1 mM MnCl. ₂ , and 0.005% (v/v) Tween-20 as running buffer. Recombinant SARS-CoV-2 RBD [B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta) or B. 1.617 (kappa) for wild-type and variants with up to 500 response units were covalently immobilized on the surface of a COOH-Au chip (iCLUEBIO) via standard amine coupling. Antibodies (8, 16, 32, 64, 128 nM) were injected onto the surface of the sensor chip at a flow rate of 50 μl/min. Kinetic evaluation data were obtained using a 1:1 binding model.

Competition experiments were performed to evaluate the ability of K202.B to bind to different regions of the RBD. After immobilizing 5 nM WT-RBD-His on the surface of the COOH-Au chip, a high concentration (512 nM) of K102.1 or K102.2 antibody was added to saturate the corresponding binding site of RBD. Then 128 nM K202.B was added. Conversely, after adding 512 nM K202.B to the surface of the recombinant WT-RBD-His-immobilized sensor chip, 256 nM K102.1 or K102.2 was subsequently added. Curve fitting and data analysis were performed using iMSPR analysis software (Tracedrawer; iCLUEBIO).

3. SARS-CoV-2 RBD-human ACE2 neutralization assay

The ability of the antibody to inhibit the interaction of SARS-CoV-2 RBD with hACE2 was investigated using ELISA. 50 ng of purified Fc-tagged hACE2 (hACE2-Fc) (R&D Systems, Minneapolis, Minn., USA) was coated on each well of a 96-well plate and incubated for 2 hours at room temperature. After washing with immunobuffer (BPS Bioscience, San Diego, CA, USA), the plate was blocked with blocking buffer (BPS bioscience) for 1 hour at room temperature. Simultaneously, 25 nM of purified SARS-CoV-2 WT- or variant-RBD-His (alpha, beta, gamma, delta and kappa) (Sino Biological) was tested in the presence of mAbs or bsAbs (0.006, 0.024, 0.097, 0.39, 1.56, 6.25, 25 and 100 nM) or without pre-incubation for 1 hour at RT.

4. Establishment of hACE2 overexpressing 293T stable cell line (293T/hACE2 cell line)

To generate a stable 293T/hACE2 cell line, the pUNO1-hACE2 plasmid (InvivoGen, San Diego, CA, USA) was transfected into 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After transfection for 48 hours, cells were cultured in medium containing 20 μg/ml blasticidin (InvivoGen) to select positive cell populations. Expression of hACE2 was determined using immunoblot and immunocytochemical analysis. For immunoblot analysis, 293T and 293T/hACE2 cell lines were lysed with SDS sample buffer and subjected to immunoblot analysis using a polyclonal anti-hACE2 antibody (R&D Systems). The distribution of hACE2 in cell membranes was studied by immunocytochemical analysis. Briefly, 293T and 293T/hACE2 cells were plated on Nunc ^® Lab-Tek ^® II chamber slides (Thermo Fisher Scientific) coated with poly-L-lysine (0.1 mg/ml) (Sigma-Aldrich, St. Louis, MO, USA). ) were plated on. After 24 hours, cells were fixed with 4% formaldehyde for 10 minutes and washed twice with PBS.

Cells were blocked using PBS containing 1% (w/v) BSA and incubated overnight at 4° C. with a polyclonal anti-hACE2 antibody. After washing three times with PBS, cells were incubated with Alexa Fluor 488-labeled anti-goat secondary antibody (Invitrogen) for 1 hour at room temperature, followed by mounting with mounting solution (Dako North America, Carpinteria, CA, USA). did Stained cells were imaged using a confocal microscope (LSM510; Carl Zeiss, Oberkochen, Germany).

5. SARS-CoV-2 pseudovirus neutralization assay

SARS-CoV-2 spike (S) protein carrying the wild-type or D614G variant and firefly luciferase reporter gene was isolated using Lenti-X ^TM SARS-CoV-2 packaging mix according to the instructions of the manufacturer (Takara Bio, Kusatsu, Japan). Pseudotyped replication-deficient lentiviral particles with Briefly, the packaging mix was transiently transfected into Expi293 cells using ExpiFectamine 293 reagent. After incubation for 72 hours, the supernatant containing the pseudovirus was collected and briefly centrifuged (500 g for 10 minutes) to remove cell debris. Virus titers were determined using Lenti-X GoStix Plus (Takara Bio) according to the manufacturer's instructions. A pseudotyped replication-deficient Moloney murine leukemia virus (MLV) particle with a SARS-CoV-2 S protein that has alpha, beta, gamma, delta or kappa variants and expresses a firefly luciferase reporter gene was generated from eEnzyme (Gaithersburg, MD, USA). obtained.

To determine the neutralizing activity of mAbs or bsAbs against pseudovirus infection, 1×10 ⁴ 293T/hACE2 cells in 50 μl culture medium were seeded in 96-well tissue culture plates overnight. Serial dilutions of antibody were pre-incubated with 50 μl of pseudovirus (1×10 ⁷ PFU/ml) for 10 min at RT and then the mixture was incubated with cells for 24 h. Firefly luciferase reporter gene expression (indicating viral presence) was measured using ONE-Glo luciferase substrate (Promega, Madison, WI, USA). Next, the culture medium was removed and incubated with 100 μl of ONE-Glo substrate. After 5 minutes, 70 μl supernatant was transferred to a white flat-bottom 96-well assay plate (Corning) and the luminescence signal was measured using a Synergy H1 microplate reader. Relative luminescence units recorded were normalized to units derived from cells infected with SARS-CoV-2 pseudovirus in the absence of antibody. Dose-response curves for IC ₅₀ values were determined by non-linear regression (GraphPad Prism 8).

6. In vivo mouse studies

For in vivo efficacy studies, 8-week-old female B6.Cg-Tg(K18-ACE2)2Prlmn/J(hACE2) mice (The Jackson Laboratory, CA, USA) were used in a certified A/BSL3 facility (Korea Zoonosis Research Institute, Iksan, Korea). Republic of Korea). All procedures were approved by the KOTUS Institutional Animal Care Committee (No. 22-KE-0076), and all experimental protocols requiring biosafety were approved by the Institutional Biosafety Committee of Chonbuk National University (approval number: JBNU 2020-11-003-003). approved, and performed in a biosafety cabinet at the BL3 and ABL3 facilities of the Korea Institute of Animal Infectious Diseases, Chonbuk National University.

hACE2-transgenic (hACE2-TG) mice (n = 7) were inoculated intranasally with 30 μl of wild-type or delta mutant virus (1Х10 ⁴ PFU) under anesthesia. Three hours after infection, PBS, mAb or bsAb was injected intravenously.

Mice were monitored daily for weight change and clinical severity based on the following criteria:

Score Description Appearance & Mobility 0 Healthy No observable sign of disease One Slightly ruffled Slightly ruffled coat 2 Ruffled Ruffled coat throughout the body and a wet appearance 3 Sick Very ruffled coat and slightly closed, inset eyes 4 very sick Very ruffled coat; closed, inset eyes; and moribund state

SARS-CoV-2 burden in lung tissue was determined via RT-qPCR. Lung tissues were collected from hACE2-TG mice 6 days after SARS-CoV-2 wild type or Delta mutant infection, and total RNA was extracted from the collected tissues using Wizol Reagent (Wizbiosolutions, Seongnam, Korea). Samples were subjected to RT-qPCR using the CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA).

After reverse transcription of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA, USA), the reaction mixture (20 μl total) contained 2 μl of template cDNA, 10 μl of 2X Premix Ex Taq, 200 nM primers and probes. ( E gene : forward primer 5'-ACAGGTACGTTAATAGTTAATAGCGT-3', reverse primer 5'-ATATTGCAGCAGTACGCACACA-3', probe 5'-FAM-ACACTAGCCATCCTTACTGCGC TTCG-BHQ1-3'; RdRp gene : forward primer 5'-ATGAGCTTAGTCCTGTTG-3' , reverse primer 5'-CTCCCTTTGTTGTGTTGT-3', probe 5'-HEX-AGATTGTCTTGTGCTGCCGGTA-BHQ1-3'). The reaction was denatured at 95 °C for 30 sec followed by 45 cycles of 95 °C for 5 sec and 60 °C for 20 sec. After the reaction cycle was complete, the temperature was increased from 65 °C to 95 °C at a rate of 0.2 °C/15 sec, and the fluorescence was measured every 5 sec to construct a melting curve. A control sample without template DNA was run with each assay. All measurements were performed in duplicate to ensure reproducibility. The authenticity of the amplified products was determined using melting curve analysis. All data were analyzed using Bio-Rad CFX Manager analysis software version 2.1 (Bio-Rad Laboratories). Viral burden was expressed as the number of copies of viral RNA per nanogram of total RNA after calculating the absolute copy number of viral RNA compared to a standard cDNA template.

histology

Excised mouse lung tissue was fixed with 4% (v/v) paraformaldehyde (PFA) in PBS and processed for paraffin embedding. Paraffin blocks were cut to 3 μm thickness using a microtome (HistoCore MULTICUT R; Leica, Germany) and mounted on silane-coated glass slides (5116-20F; Muto, Tokyo, Japan). Histopathological changes in all organs were confirmed using hematoxylin, eosin, periodic acid-Schiff, and modified Masson's trichrome stain. Histopathology of lung tissue was observed using an optical microscope (Axio Scope A1; Carl Zeiss). Pathological scores were determined based on the percentage of inflammatory area for each section in each group using the following scoring system. 0, no pathological changes; 1, affected area (≤ 10%); 2, area affected (10% to 50%); 3, affected area (≥50%); If pulmonary edema and/or alveolar hemorrhage were observed, 0.5 points were added.

7. In vitro antibody-dependent enhancement assay

50 microliters of SARS-CoV-2 pseudovirus (1 x 10 ⁷ PFU/ml) was pre-incubated with various concentrations of K202.B (0.044, 0.138, 0.42, 1.24, 3.7, 11.1, 33.3, 100 mM) in culture medium. did After incubation at room temperature for 30 minutes, the mixture was added to 293T, 293T/hACE2, K562 or THP-1 cells (1×10 ⁴ cells in a 96-well plate). The cells were cultured for 24 hours and the luciferase activity of the infected cells was measured as described in "SARS-CoV-2 Pseudovirus Neutralization Assay".

8. Endothelial cell viability assay

A total of 5 x 10 ³ HUVECs were plated in 96-well plates and incubated for 24 hours at 37°C in the presence or absence of 20 μg/ml K202.B or 36 μg/ml 5-fluorouracil. Cell viability was determined using the Cell Counting Kit-8 (Sigma) according to the manufacturer's instructions. Final absorbance was measured at 450 nm using a spectrophotometer (BioTek).

9. Flow Cytometry

The effect of K202.B on endothelial cell activation was investigated by treating the cells with 20 ng/ml of human tumor necrosis factor-α (hTNF-α; Millipore), 20 μg/ml of K202.B or PBS for 24 hours and 4% (v/v) fixed with PFA. Next, 10 μg/well of intercellular cell adhesion molecule-1 (ICAM-1; Abcam, Cambridge, MA, USA) or vascular cell adhesion molecule-1 (VCAM-1; Abcam) antibody was incubated at 25° C. for 1 hour. did Then, Alexa Fluor 647-conjugated anti-mouse IgG or anti-rabbit IgG (1:1000; Invitrogen) was incubated at 25°C for 1 hour. All samples were analyzed using flow cytometry with the help of FlowJo software (TreeStar, Ashland, OR, USA).

10. In vivo toxicity and serum pharmacokinetic analysis

In vivo toxicity and serum pharmacokinetic studies using animals were approved by the IACUC (approval number NCC-21-693) of the National Cancer Center of Korea. 8-week-old female ICR mice (Orient Bio Inc., Seongnam, Republic of Korea) were intravenously injected with 5 or 30 mg/kg of K202.B (n = 3 per group). Blood samples (50 μl) were collected from each mouse at 4, 8, 24, 72, 120, 168, 264, 384 and 504 hours after injection and centrifuged at 5000 x g for 20 minutes at 4°C. Serum was stored at -80°C for evaluation of biochemical parameters. Serum levels of glutamine oxaloacetic transaminase (GOT), glutamine pyruvate transaminase (GPT), creatinine (CRE) and blood urea nitrogen (BUN) were measured using a Fuji Dri-Chem 3500 Biochemistry Analyzer (Fujifilm, Tokyo, Japan). was measured using

Serum levels of K202.2B were determined using a human IgG ELISA kit (Abcam) according to the manufacturer's instructions. Optical densities were measured using a Synergy H1 microplate reader and values were compared to those of a standard curve analyzed in parallel.

11. Statistical Analysis

Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA) with two-tailed Student's t-test for comparisons between two groups and one-way analysis of variance with Bonferroni's correction for multiple comparisons. of variance, ANOVA) was used for analysis. All data represent the mean ± standard deviation of the mean (SEM). P values less than 0.05 are considered statistically significant (*P < 0.05, ** P < 0.01, *** P < 0.001).

Claims (18)

Bispecific antibodies that specifically bind to SARS-CoV-2, including:
(a) heavy chain complementarity determining region 1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3 A heavy chain variable region comprising an H-CDR3 comprising; and light chain complementarity determining region 1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 4, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO: 6. An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an L-CDR3;
(b) a heavy chain variable region comprising H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; And an antibody comprising a light chain variable region comprising L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6 or an antigen-binding fragment thereof; or
(c) any combination thereof. The double antibody according to claim 1, wherein the heavy chain variable region of (a) comprises the amino acid sequence of SEQ ID NO: 7. The double antibody according to claim 1, wherein the light chain variable region of (a) comprises the amino acid sequence of SEQ ID NO: 8. The double antibody according to claim 1, wherein the heavy chain variable region of (b) comprises the amino acid sequence of SEQ ID NO: 17. The double antibody according to claim 1, wherein the light chain variable region of (b) comprises the amino acid sequence of SEQ ID NO: 18. The double antibody according to claim 1, wherein the antibody or antigen-binding fragment thereof of (a) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9. The dual antibody according to claim 1, wherein the antibody or antigen-binding fragment thereof of (a) comprises a light chain comprising the amino acid sequence of SEQ ID NO: 10. The double antibody according to claim 1, wherein the antibody or antigen-binding fragment thereof of (b) is an scFv comprising the amino acid sequence of SEQ ID NO: 19. The diabody according to claim 1, wherein the antibody of (a) or (b) or an antigen-binding fragment thereof binds to the heavy chain C-terminus of the antibody of (b) or (a) or an antigen-binding fragment thereof. The method of claim 1, wherein the antibody or antigen-binding fragment thereof of (a) and the antibody or antigen-binding fragment thereof of (b) are each of the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein. Bispecific antibodies that bind to different epitopes. The dual antibody according to claim 1, wherein the dual antibody inhibits the binding between RBD of SARS-CoV-2 Spike protein and angiotensin converting enzyme 2 (ACE2) of host cells. The double antibody according to claim 1, wherein the antibody or antigen-binding fragment thereof of (a) and the antibody or antigen-binding fragment thereof of (b) are in the form of IgG1 or IgG4. A nucleic acid molecule comprising a nucleotide sequence encoding the diabody of any one of claims 1 to 12. A recombinant vector comprising the nucleic acid molecule of claim 13 . An isolated host cell comprising the recombinant vector of claim 14 . A pharmaceutical composition for treating SARS-CoV-2 infection comprising the double antibody of any one of claims 1 to 12 and a pharmaceutically acceptable carrier. The method of claim 16, wherein the SARS-CoV-2 has a mutation consisting of N354D / D364Y, V367F, W436R, R408I, G476S, V483A, V341I, F342L, A435S, or a combination thereof in the amino acid sequence of RBD. Pharmaceutical composition. The pharmaceutical composition according to claim 16, wherein the SARS-CoV-2 is selected from the group consisting of wild type, alpha variant, beta variant, gamma variant, delta variant, and kappa variant.

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