KR102662789B1 - Anti-SARS-CoV-2 S protein antibody or antigen binding fragment thereof, and pharmaceutical composition for preventing or treating SARS-CoV-2 infection comprising the same - Google Patents

Abstract

The present invention relates to an anti-SARS-CoV-2 S protein antibody or antigen-binding fragment thereof, and a pharmaceutical composition for treating COVID-19 comprising the same. The anti-SARS-CoV-2 S protein antibody or antigen-binding fragment thereof of the present invention binds specifically to the S protein, which plays an important role in the infiltration of SARS-CoV-2 into host cells, thereby causing SARS-CoV-2 infection. Since it can suppress, it can be useful as a treatment for COVID-19.

Description

Anti-SARS-CoV-2 S protein antibody or antigen binding fragment thereof, and pharmaceutical composition for preventing or treating SARS-CoV-2 infection comprising the same {Anti-SARS-CoV-2 S protein antibody or antigen binding fragment thereof , and pharmaceutical composition for preventing or treating SARS-CoV-2 infection comprising the same}

The present invention relates to an anti-SARS-CoV-2 S protein antibody or antigen-binding fragment thereof, and a pharmaceutical composition containing the same for preventing or treating SARS-CoV-2 infection.

Efforts are being made around the world to develop treatments for coronavirus disease-19 (COVID-19), which recently broke out in Wuhan, China and is spreading worldwide. In particular, when the spike protein (S protein) of SARS-CoV-2 binds to the receptor of the host cell, SARS-CoV-2 penetrates into the host cell through endocytosis. There are active attempts to treat COVID-19 using monoclonal antibodies against the spike protein of 2 and convalescent plasma from convalescent patients.

The present inventors have made extensive research efforts to develop a pharmaceutical composition for preventing or treating COVID-19. As a result, novel anti-SARS-CoV-2 S protein antibodies or antigen-binding fragments thereof were developed, it was confirmed that these antibodies or antigen-binding fragments exhibited neutralizing ability against SARS-CoV-2, and the present invention was completed. .

According to one aspect of the invention, the invention provides an antibody or antigen-binding fragment thereof of anti-SARS-CoV-2 S protein selected from:

(a) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

(b) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 17, HCDR2 of SEQ ID NO: 18, and HCDR3 of SEQ ID NO: 19; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 21, and LCDR3 of SEQ ID NO: 22;

(c) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 33, HCDR2 of SEQ ID NO: 34, and HCDR3 of SEQ ID NO: 35; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 38;

(d) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 49, HCDR2 of SEQ ID NO: 50, and HCDR3 of SEQ ID NO: 51; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 52, LCDR2 of SEQ ID NO: 53, and LCDR3 of SEQ ID NO: 54;

(e) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 65, HCDR2 of SEQ ID NO: 66, and HCDR3 of SEQ ID NO: 67; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 68, LCDR2 of SEQ ID NO: 69, and LCDR3 of SEQ ID NO: 70; or

(f) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 81, HCDR2 of SEQ ID NO: 82, and HCDR3 of SEQ ID NO: 83; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 84, LCDR2 of SEQ ID NO: 85, and LCDR3 of SEQ ID NO: 86.

In one embodiment of the present invention, the heavy chain variable region of (a) includes the amino acid sequence of SEQ ID NO: 7, and 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: 23, and the light chain variable region of (b) includes the amino acid sequence of SEQ ID NO: 24.

In one embodiment of the present invention, the heavy chain variable region of (c) includes the amino acid sequence of SEQ ID NO: 39, and the light chain variable region of (c) includes the amino acid sequence of SEQ ID NO: 40.

In one embodiment of the present invention, the heavy chain variable region of (d) includes the amino acid sequence of SEQ ID NO: 55, and the light chain variable region of (d) includes the amino acid sequence of SEQ ID NO: 56.

In one embodiment of the present invention, the heavy chain variable region of (e) includes the amino acid sequence of SEQ ID NO: 71, and the light chain variable region of (e) includes the amino acid sequence of SEQ ID NO: 72.

In one embodiment of the present invention, the heavy chain variable region of (f) includes the amino acid sequence of SEQ ID NO: 87, and the light chain variable region of (f) includes the amino acid sequence of SEQ ID NO: 88.

In one embodiment of the present invention, the anti-SARS-CoV-2 S protein antibody of the present invention binds to the receptor binding domain (RBD) of the SARS-CoV-2 S protein.

In a specific embodiment of the present invention, the RBD of the SARS-CoV-2 S protein includes the amino acid sequence of SEQ ID NO: 97.

In a specific embodiment of the present invention, the RBM (receptor binding motif) of the RBD of the SARS-CoV-2 S protein to which the anti-SARS-CoV-2 S protein antibody binds is 114 to 114 in the amino acid sequence of SEQ ID NO: 97. Includes amino acid residues 115, 117, 119-122, 128, 131-132, 134-136, 138, 146-154, 157-162, 166, 169-170, 174-175, 177, and 179.

In one embodiment of the present invention, the anti-SARS-CoV-2 S protein antibody of the present invention inhibits the binding between the RBD of SARS-CoV-2 S protein and human angiotensin converting enzyme 2 (ACE2).

As used herein, the term “antibody” refers to a specific antibody against the SARS-CoV S protein and includes not only the complete antibody form but also the antigen binding fragment of the antibody molecule.

A complete antibody has two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and is subclassed as gamma1 (γ1), gamma2 (γ2), and gamma3 (γ3). ), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

As used herein, the term “antigen binding fragment” refers to a fragment that possesses an antigen-binding function and includes Fab, F(ab'), F(ab') ₂ , and Fv. Among antibody fragments, Fab (fragment antigen binding) has one antigen binding site with a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain. 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 when the cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv is a minimal antibody fragment containing only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are known in the art. A two-chain Fv (two-chain Fv) connects the heavy chain variable region and the light chain variable region by a non-covalent bond, and a single-chain Fv (single-chin variable fragment, scFv) generally connects the heavy chain variable region and the short chain variable region through a peptide linker. The domains can be covalently linked or linked directly at the C-terminus to form a dimer-like structure, such as double-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab') ₂ fragment can be obtained by digestion with pepsin), or It can be produced through genetic recombination technology.

Therefore, in the present invention, antibodies specifically include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, and disulfide-linked antibodies. Includes, but is not limited to, Fvs (sdFv) and anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of these antibodies.

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 with sufficient variable region sequence to confer specificity to an antigen, and a full-length heavy chain thereof. It means all fragments. Also, as used herein, the term “light chain” refers to both a full-length light chain and fragments thereof including a variable region domain V _L and a constant region domain C _L containing an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. do.

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 (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain having four conserved framework regions (FR) and three hypervariable regions (HVR). ) includes. (Kindt et al., Kuby Immunology, 6th edition, W.H. Freeman and Co., page 91 (2007)).

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 chain (HCDR1, HCDR2, and HCDR3) and light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs. CDRs provide key contact residues for an antibody to bind to an antigen or epitope.

As used herein, the term “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. FRs in variable domains generally consist of four FR domains FR1, FR2, FR3 and FR4. Therefore, HVR and FR sequences generally appear in the VH in the following order:

FRH1 (Framework region 1 of Heavy chain)-HCDR1 (complementarity determining region 1 of Heavy chain)-FRH2-HCDR2-FRH3-HCDR3-FRH4;

Additionally, HVR and FR sequences typically appear in the VL (or Vk) in the following order:

FRL1(Framework region 1 of Light chain)-LCDR1(complementarity determining region 1 of Light chain)-FRL2-LCDR2-FRL3-LCDR3-FRL4.

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 can be characterized by an equilibrium dissociation constant of at least about 1 x 10 ^-6 M or less (e.g., smaller K _D indicates tighter binding). Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc.

As used herein, the term “Affinity” refers to the strength of the sum of non-covalent interactions between a single binding site on a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless specified, “binding affinity” refers to the intrinsic binding affinity of a molecule X and its partner, reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of Y can generally be expressed in terms of the dissociation constant (K _D ). Affinity can be measured by conventional methods known in the art, including those described herein.

Also, as used herein, the term “human antibody” or “humanized antibody” refers to an antibody produced by a human or human cell, or a non-human antibody that utilizes human antibody repertoires or other human antibody coding sequences. It possesses an amino acid sequence corresponding to the amino acid sequence of the antibody from which it was derived.

As used herein, the term “chimeric antibody” refers to an antibody in which a portion of the heavy and/or light chain is derived from a specific source or species, and the remainder of the heavy and/or light chain is derived from a different source or species. It means antibody.

As recognized by a person skilled in the art, the anti-SARS-CoV-2 S antibody or antigen-binding fragment thereof of the present invention is an amino acid sequence variant within the range that can specifically recognize SARS-CoV-2 S protein. may include. For example, changes can be made to the amino acid sequence of an antibody to improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody.

These amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutions shows 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. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan, and tyrosine can be said to be biologically equivalent in function.

In introducing the amino acid mutation, the hydrophobic index of the amino acid may be considered. Each amino acid is assigned a hydrophobicity index based on 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 imparting interactive biological functions to proteins. It is a known fact that similar biological activity can be maintained only when substituted with an amino acid having a similar hydrophobicity index. When introducing a mutation with reference to the hydrophobicity index, substitution is made between amino acids showing a difference in hydrophobicity index of preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

Meanwhile, it is also well known that substitution between amino acids with similar hydrophilicity values results in proteins with equal biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values are 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 introducing a mutation with reference to the hydrophilicity value, substitution is made between amino acids showing a difference in hydrophilicity value of preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

Amino acid exchanges in proteins that do not overall 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/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

The anti-SARS-CoV-2 S antibody or antigen-binding fragment thereof of the present invention is an anti-SARS antibody containing minor changes to the above-described amino acid sequence, that is, modifications that have little effect on the tertiary structure and function of the antibody. -Includes CoV-2 S antibody or antigen-binding fragment thereof. Therefore, in some embodiments, even if it does not match the above-described sequence, it may have at least 90%, 93%, 95%, or 98% similarity.

According to one embodiment of the present invention, the anti-SARS-CoV-2 S antibody or antigen-binding fragment thereof of the present invention is a monoclonal antibody containing a heavy chain variable region and a light chain variable region including the CDRs of the above-described sequences. Specific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and anti-idiotypes (anti-Id) ) antibodies, and epitope-binding fragments of the above antibodies, but are not limited thereto.

In another embodiment of the present invention, the anti-SARS-CoV-2 S antibody or antigen-binding fragment thereof of the present invention is anti-SARS-CoV-2 S scFv. In a specific embodiment of the present invention, the heavy chain variable region and 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 ( It is connected by a linker such as Gly ₄ -Ser)n. Here, n is an integer from 1 to 6, specifically 3 to 4, but is not limited thereto. For example, the light chain variable region and heavy chain variable region of the scFv may exist in the following orientation: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

In one embodiment of the present invention, the antibody or antigen-binding fragment has neutralizing ability against SARS-CoV-2 of the S, V, or G clade.

In one embodiment of the present invention, the SARS-CoV-2 of the S clade is hCoV_19/South Korea/KCDC03/2020.

In one embodiment of the present invention, the SARS-CoV-2 of the V clade is hCoV_19/South Korea/KUMC15/2020.

In one embodiment of the present invention, the SARS-CoV-2 of the G clade is hCoV_19/South Korea/KUMC17/2020.

According to one aspect of the present invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-described anti-SARS-CoV-2 S antibody or antigen-binding fragment thereof.

As used herein, the term "nucleic acid molecule" is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also those with modified sugars or base sites. Also includes analogues (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

The nucleotide sequence encoding the antibody or antigen-binding fragment thereof, or the chimeric antigen receptor polypeptide of the present invention is sufficient to be a nucleotide sequence encoding the amino acid sequence constituting the chimeric antigen receptor molecule, and is not limited to any specific nucleotide sequence. It is obvious to those skilled in the art that this is not the case.

This is because even if a mutation in the nucleotide sequence occurs, there are cases in which no change occurs in the protein sequence when the mutated nucleotide sequence is expressed as a protein. This is called codon degeneracy. Accordingly, the nucleotide sequence can be either a functionally equivalent codon, or a codon encoding the same amino acid (e.g., due to codon degeneracy, there are six codons for arginine or serine), or a codon encoding a biologically equivalent amino acid. It contains a nucleotide sequence containing.

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 forming the light chain of the antibody or antigen-binding fragment thereof against the SARS-CoV-2 S protein of the present invention The sequences are listed in the sequence list attached to this specification.

The nucleic acid molecule of the present invention encoding an anti-SARS-CoV-2 S antibody or antigen-binding fragment thereof is interpreted to also include a nucleotide sequence showing substantial identity to the above-described nucleotide sequence. The above substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. refers to a nucleotide sequence that exhibits homology, more preferably at least 90% homology, and most preferably at least 95%, 97%, 98%, or 99% homology.

Considering the mutations having the above-mentioned bioequivalent activity, the antibody or antigen-binding fragment of the present invention; Alternatively, the nucleic acid molecule encoding the chimeric antigen receptor polypeptide is interpreted to also include sequences showing substantial identity to the sequences listed in the sequence listing. The above substantial identity is at least 61% when aligning the sequence of the present invention and any other sequence to correspond as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. It means a sequence showing homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are discussed 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 . BioScience . 8:155-65 (1992) and Pearson et al., Meth . Mol . Biol . 24:307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol . Biol . 215:403-10 (1990)) is accessible from NBCI (National Center for Biological Information), etc., and can be accessed on the Internet using blastp, blastn, It can be used in conjunction with sequence analysis programs such as blastx, tblastn, and tblastx. BLAST can be accessed 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 another aspect of the present invention, the present invention provides a recombinant vector containing a nucleic acid molecule encoding the above-described anti-SARS-CoV-2 S antibody or antigen-binding fragment thereof.

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 an RNA molecule in the host cell and a poly A signal sequence that acts in the host cell to cause polyadenylation of the 3'-end of the RNA molecule.

As used herein, the term “operatively linked” refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence. The regulatory sequence thereby regulates transcription and/or translation of the other nucleic acid sequences.

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

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. Additionally, the vector of the present invention can be constructed using prokaryotic cells or eukaryotic cells as hosts.

For example, when the vector of the present invention is an expression vector and uses a prokaryotic cell as a host, a strong promoter capable of advancing transcription (e.g., pL ^λ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) , a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. When E. coli is used as the host cell, the promoter and operator region of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024 (1984)) and the left promoter of phage λ (pL) ^{The λ} promoter, Herskowitz, I. and Hagen, D., Ann Rev., 14:399-445 (1980), can be used as a control region.

Meanwhile, vectors that can be used in the present invention include plasmids often used in the art (e.g., pSK349, pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt) ·Can be produced by manipulating viruses (e.g., λ4B, λ-Charon, λΔz1, and M13) or viruses (e.g., SV40, etc.).

The vector of the present invention may be fused with other sequences to facilitate purification of the polypeptide 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). Because of the additional sequences required for purification, proteins expressed in the host are quickly and easily purified via affinity chromatography.

The vector of the present invention may contain an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, and neomycin. and a resistance gene to tetracycline.

On the other hand, when the vector of the present invention is an expression vector and uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus) Viral late promoters, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and typically have a polyadenylation sequence as the transcription termination sequence.

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

According to one aspect of the present invention, the present invention provides a host cell transformed with a recombinant vector.

Host cells capable of stably and continuously cloning and expressing the vector of the present invention can be any host cell known in the art, such as E. coli Origami2, E. coli JM109, E. coli BL21 (DE3) ), E. coli strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli Enterobacteriaceae strains such as Salmonella Typhimurium, Serratia marcescens and various Pseudomonas species.

In addition, when the vector of the present invention is transformed into eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, and animal cells (e.g., CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293) are used as host cells. , HepG2, 3T3, RIN and MDCK cell lines), etc. can be used.

The method of transporting the vector of the present invention into a host cell is, when the host cell is a prokaryotic cell, the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973) ), Hanahan method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166:557-580 (1983)) and electroporation (Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145 (1988)). Additionally, when the host cell is a eukaryotic cell, microinjection method (Capecchi, MR, Cell, 22:479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52:456 (1973)), Electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, TK 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., the vector can be injected into the host cell.

In the present invention, the recombinant vector injected into the host cell can express the above recombinant polypeptide or polypeptide complex within the host cell, and in this case, a large amount of polypeptide or polypeptide complex is obtained. For example, if the expression vector contains the lac promoter, gene expression can be induced by treating the host cell with IPTG.

Transformed host cells can be cultured using known host cell culture methods or modified methods. For example, if the host cell is E. coli , the medium for culturing the transformed host cells can be a natural medium or a synthetic medium as long as it contains a carbon source, nitrogen source, inorganic salt, etc. that E. coli can efficiently utilize. there is. Carbon sources that can be used include carbohydrates such as glucose, fructose, and sucrose; Starch, hydrolyzate of starch; Organic acids such as acetic acid and propionic acid; Includes alcohols such as ethanol, propanol, and glycerol. The nitrogen source is ammonia; ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate; Peptone, meat extract, yeast extract, corn steeping liquid, casein hydrolyzate, soybean extract, soybean hydrolyzate; Includes various fermented cells and their decomposition products. Inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, manganese sulfate, copper sulfate, calcium carbonate, etc.

The culture is usually carried out under aerobic conditions such as shaking culture or rotation by rotator. The culture temperature is preferably in the range of 10 to 40°C, and the culture 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, etc. During culture, if necessary, antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline may be added for maintenance and expression of the recombinant vector. When cultivating host cells transformed with a recombinant expression vector having an inducible promoter, a suitable inducer can be added to the medium if necessary. For example, if the expression vector contains the lac promoter, IPTG (isopropyl-beta-D-thiogalactopyranoside) can be added, and if the expression vector contains the trp promoter, indoleacrylic acid can be added to the medium.

According to another aspect of the present invention, the present invention provides a polypeptide complex in which the above-described anti-SARS-CoV-2 S antibody or antigen-binding fragment thereof and an additional polypeptide are linked.

In this case, the polypeptide complex is in the form of a multimer in which monomers of each polypeptide or antigen or antigen-binding fragment are linked. The polypeptide complex of the present invention is covalently linked to each other, and according to one embodiment of the present invention, the polypeptide complex may be implemented in the form of a fused protein or conjugate.

In one embodiment of the present invention, the polypeptide complex may be implemented in the form of a fused protein or conjugate. Accordingly, the antibodies or antigen-binding fragments thereof and affibody molecules may be conjugated via chemical conjugation (known as organic chemistry methods) or other means (e.g., expressing the complex as a fusion protein, directly or via linkers (e.g., amino acid linkers). It can be indirectly connected through.

According to a specific embodiment of the present invention, each polypeptide monomer forming the polypeptide complex 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:

where 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 less than or equal to 8; and

p is an integer from 1 to 7.

According to another specific embodiment of the present invention, n = 1 to 5 and m = 0 to 5 for the linker. For a more specific embodiment, n = 4 and m = 1. In a 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.

According to another aspect of the invention, the invention provides a nucleic acid molecule encoding the polypeptide complex.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating SARS-CoV-2 infection, comprising the following antibodies or antigen-binding fragments thereof and a pharmaceutically acceptable carrier:

(a) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

(b) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 17, HCDR2 of SEQ ID NO: 18, and HCDR3 of SEQ ID NO: 19; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 21, and LCDR3 of SEQ ID NO: 22;

(c) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 33, HCDR2 of SEQ ID NO: 34, and HCDR3 of SEQ ID NO: 35; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 38;

(d) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 49, HCDR2 of SEQ ID NO: 50, and HCDR3 of SEQ ID NO: 51; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 52, LCDR2 of SEQ ID NO: 53, and LCDR3 of SEQ ID NO: 54;

(e) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 65, HCDR2 of SEQ ID NO: 66, and HCDR3 of SEQ ID NO: 67; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 68, LCDR2 of SEQ ID NO: 69, and LCDR3 of SEQ ID NO: 70; or

(f) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 81, HCDR2 of SEQ ID NO: 82, and HCDR3 of SEQ ID NO: 83; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 84, LCDR2 of SEQ ID NO: 85, and LCDR3 of SEQ ID NO: 86.

In one embodiment of the present invention, the SARS-CoV-2 is SARS-CoV-2 of the S, V, or G clade.

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

As demonstrated in the examples below, the anti-SARS-CoV-2 S antibody or antigen-binding fragment thereof of the present invention binds to the SARS-CoV-2 virus belonging to the SARS-CoV-2 S, V, or G clade. This neutralizes the virus and prevents intracellular infection by the virus. Therefore, the pharmaceutical composition containing the anti-SARS-CoV-2 S antibody or antigen-binding fragment thereof of the present invention as an active ingredient can be usefully used for the prevention or treatment of SARS-CoV-2 infection.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Includes, but is limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It doesn't work. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations 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, intratumoral injection, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration. It can be administered, etc.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. Usually, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. 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” refers to an amount sufficient to prevent or treat the above-mentioned disease.

As used herein, the term “prophylaxis” refers to the prevention or protective treatment of a disease or disease state. As used herein, the term “treatment” means reducing, suppressing, calming, or eradicating a disease state.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art. Alternatively, it can be manufactured by placing it in a multi-capacity 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 dispersant or stabilizer.

The anti-SARS-CoV-2 S protein antibody or antigen-binding fragment thereof of the present invention binds specifically to the S protein, which plays an important role in the infiltration of SARS-CoV-2 into host cells, thereby causing SARS-CoV-2 infection. Since it can suppress, it can be useful as a treatment for COVID-19.

Figure 1 is a diagram showing SDS-PAGE analysis of SARS-CoV-2 RBD (receptor binding domain) and NTD recombinant protein.
Figures 2 and 3 are diagrams showing the binding activity of the anti-SARS-CoV-2 S protein antibody or antigen-binding fragment thereof of the present invention.
Figure 4 shows the PRNT measurement (plaque) of six antibodies (R1, R3, R4, R9, R15, and R17) among the anti-SARS-CoV-2 S protein antibody or antigen-binding fragment thereof of the present invention against SARS-CoV. This diagram shows the results of measuring the number of viral plaques using a reduction neutralization test.
Figure 5 is a PRNT measurement method for SARS-CoV of the S, V, and G clades of three antibodies (R1, R3, and R4) among the anti-SARS-CoV-2 S protein antibody or antigen-binding fragment thereof of the present invention. This diagram shows the results of measuring neutralization ability (%) from the plaque reduction neutralization test.
Figure 6a is a diagram showing the binding characteristics of the RBD of the SARS-CoV-2 S protein and the human ACE2 protein, and Figure 6b is a diagram showing the binding characteristics of the anti-SARS-CoV-2 S protein antibody or antigen-binding fragment (R1, R3) thereof of the present invention. , R4, and R15) inhibit the binding between the RBD of the SARS-CoV-2 S protein and the human ACE2 protein.
Figures 7a and 7b show the mechanism by which the anti-SARS-CoV-2 S protein antibody or antigen-binding fragment (R1, R3, R4, and R15) thereof exhibits neutralizing ability against the virus. Antibodies of the anti-SARS-CoV-2 S protein of the invention or antigen-binding fragments (R1, R3, R4, and R15) thereof bind to the RBM of SARS-CoV-2 (Figure 7a: SARS-CoV-2-RBD ), does not bind to the RBM of SARS-CoV (Figure 7b: SARS-CoV-2-chRBD).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating 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, “%” used to indicate the concentration of a specific substance means (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.

Example 1. SARS using animal cells CoV -2 RBD and N.T.D. protein production

Recombinant proteins for the RBD (receptor binding domain) and NTD (N-terminal domain) regions of the SARS-CoV-2 virus spike protein were produced in the form of a mouse Fc (mFc) fusion protein using HEK293F cells.

Expression vectors for SARS-CoV-2 RBD and NTD recombinant proteins were prepared as follows. SARS-CoV-2 RBD (Genbank: MN908947.3) and NTD (Genbank: MN908947.3) genes and mouse Fc gene (UniProtKB: P01863) were prepared by requesting synthesis from Bionics. The synthesized genes were produced in the form of RBD-mFc and NTD-mFc through overlapping PCR, and then KpnI (NEB, Cat. #. R0142L) and XhoI (NEB) were added to pcDNA3.3 vector (Invitrogen, cat. #. K8300-01). , cat. #. R0146L), gene cloning was performed to create an expression vector.

HEK293F cells were grown in a volume of ¹⁰⁰ mL at _a concentration of 1 Add 5 mL of culture medium to a sterilized 15 mL tube, add RBD and NTD recombinant protein expression vector DNA, and mix well to prepare DNA. PEI (Polysciences, cat. #. 23966) was added to a 15 mL tube containing culture medium and DNA, mixed well, added to HEK293F cells, and cultured for 7 days. The recombinant protein in the culture medium was purified using Protein A resin (GE healthcare, cat. #. 17-5438-03), and the buffer was replaced using the diafiltation method (Satorius, cat. #. VS2002). The purified recombinant protein was quantified by measuring the absorbance at 280. The purified recombinant protein was analyzed on 12% SDS-PAGE (Figure 1).

Example 2.SARS- CoV -2 Selection of human antibodies specific to spike protein

As a result of screening human antibodies that specifically bind to the spike protein, which plays a key role in the process of SARS-CoV-2 virus entering human cells, using a bio-panning method, 59 types of antibodies that specifically bind to the spike protein were identified. Selected.

Bio-panning screening to select human antibodies that specifically bind to the spike protein was conducted as follows. SARS-CoV S1 (Sino Biological, Cat. #. 40150-V08B1) and SARS-CoV-2 S1 (Sino Biological, Cat. #. 40591-V08H) antigens used for bio-panning were purchased from Sino Biological. . NTD and RBD antigens were produced and used as recombinant proteins in the form of mouse Fc fusion as described in Example-1. The human antibody library (patent application number: 10-2014-0195243) was rescued in phage form using VCSM13 helper phage and used for panning. The number of library phage bound to the first antigen was 10 ¹³ . Panning rounds were conducted up to 4 rounds, and as a panning strategy that allows phages with high affinity to be selectively selected, a method of reducing the amount of antigen and increasing the number of washes was applied as the number of panning rounds increases. The number of phages bound to the target antigen was titrated using ER2537 E. coli as follows. The binder phage obtained from each round of bio-panning was eluted with Glycine-HCl buffer (pH 2.5). ER2537 cultured overnight in SB media (MOPS 10 g/L, Bacto YEAST extract 20 g/L, Trypton 30 g/L) was passaged in new SB media at a 1/200 dilution, and then further cultured at 37°C for 3 hours. This resulted in reaching log phage. Mix 100 μL of fresh ER2537 and 10 μL of diluted phage in a 1.5 mL tube, incubate for 30 minutes, spread on an ampicillin LB plate, and incubate overnight at 37°C. Then, apply the number of colonies produced and the dilution factor. The number of phages was measured. The binder phage obtained from each round of bio-panning was infected with ER2537 and maintained in colony form, and binding to each antigen was confirmed using the ELISA method as follows. Colonies obtained by infecting binder phage were inoculated into SB media and cultured at OD ₆₀₀ until 0.5. Then, 0.5 mM IPTG (LPS solution) was added and shaken incubation was performed at 30°C to overexpress the scFv protein. Periplasmic extraction was performed using BBS buffer (200mM Boric acid, 150mM NaCl, 1mM EDTA). The extracted scFv antibody was treated on a plate coated with SARS-CoV-2 S1 recombinant protein, ant-HA mouse HRP (Roche, cat. #. 12013819001) was treated as a secondary antibody, and Absignal (AbClon, cat. #. After performing a color reaction with AbC3001), the OD ₄₅₀ value was measured and confirmed using an ELISA reader (Victor X3 PerkinElmer). The antibody clone selected to specifically bind to the SARS-CoV-2 S1 protein was obtained from a phagemid plasmid, and the base sequence of the variable region was confirmed through sequencing analysis.

Example 3.SARS- CoV -2 To RBD antigen About Binding capacity Confirmatory ELISA assay

The binder phage obtained from each round of panning was infected with ER2537 and the colonies obtained were confirmed for binding to the antigen using ELISA. Colonies obtained by infecting binder phage were inoculated into SB media (MOPS 10 g/L, Bacto YEAST extract 20 g/L, Trypton 30 g/L), incubated until OD600 reached 0.8, and then incubated with 1 mM IPTG ( LPS solution) was added and shaken incubation was performed at 30°C to ensure scFv overexpression. scFv was subjected to periplasmic extraction using BBS buffer (200mM Boric acid, 150mM NaCl, 1mM EDTA). Using this, the binding ability to SARS-CoV-2 antigen was confirmed through ELISA method. For ELISA, scFv periplasmic extract was processed on a plate coated with SARS-CoV-2 RBD-mFc, SARS-CoV-2 S1, and BSA proteins at a concentration of 2 ug/mL, and then secondary antibody (anti-HA-HRP (anti-HA-HRP) was used. After treatment with Roche, 12013819001), a color reaction was generated with TMB (biofx, TMBC-1000-01), and the OD450 value was measured using an ELISA reader (Victor X3, Perkinelmer). As a result of ELISA, 13 types of antibodies binding to SARS-CoV-2 RBD and S1 antigen protein were initially identified (Figure 2), and additional ELISA analysis was performed to identify 34 types of antibodies binding to SARS-CoV-2 RBD and S1 antigen protein. and 12 types of antibodies binding to SARS-CoV-2 S1 were identified (Figure 3).

Example 4. Antibodies to SARS- CoV -2 against viruses Neutralizing ability check

The SARS-CoV-2 virus neutralizing ability of the antibodies developed in Examples 1 to 3 of the present invention was evaluated using the PRNT assay (plaque reduction neutralization test). The SARS-CoV-2 virus of the S clade (hCoV_19/South Korea/KCDC03/2020) was used for the neutralization ability analysis. For the PRNT measurement, the antibody sample stock solution is diluted 1:10 in PBS, then diluted 3-fold, mixed with an equal amount of virus (100 PFU/100 μl), and reacted at 37°C for 1 hour. After 1 hour of reaction, the entire amount was infected into Vero cells, and the neutralizing antibody titer was analyzed using plaque assay. When the virus-infected plate (NEST Scientific, #703003) was cultured in an incubator at 37°C and 5% CO ₂ for 3 days, it was seen that round-shaped plaques were formed due to virus infection. To measure the number of plaques formed, staining was performed using crystal violet (Georgiachem, #548-6-29). After staining, the neutralizing ability (PRNT ₅₀ ) was analyzed by calculating the maximum antibody dilution that reduces the number of plaques by more than 50% compared to the control group that was not treated with antibodies. The results are shown in Figure 4. From the results in Figure 4, six types of antibodies (R1, R3, R4, R9, R15, and R17) with SARS-CoV-2 neutralizing ability were identified.

The CDR sequences, amino acid sequences constituting the heavy chain variable region and light chain variable region of the six antibodies, and the nucleotide sequences encoding them are shown in Tables 1 to 6 below.

antibodies Heavy chain Light chain note R1 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC GGTTATTCTATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA TACATCTCTTCTTACTACTACTCTACGTATTACGCTGATTCTGTAAAAGGT CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC TGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGCGT TACGGTTACTCTGGTTAC TTCGACTACTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA

EVQLLESGGGLVQPGGSLRLSCAASGFTFS GYSMS WVRQAPGKGLEWVS YISSYYYSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR YGYSGY FDYWGQGTLVTVSS CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGT AGTGGCTCTTCATCTAATATTGGCTCTAATTATGTCTAC TGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT AGAAATAACCAGCGGGCCAAGC GGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAG GCCGATTATTACTGT GCTGCTTTCTCCGGCTTACTACTACTATGTC TTCGGCGGAGGCACCAAGCTGACGGTCCTAGGT

QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC AASPAYYYYV FGGGTKLTVLG Under bar:
CDR regions

antibody Heavy chain Light chain note R3 GAGGTGCAGTTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC TCTTATGGTATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA TACATCGGTGGTTACGGTGGTACGTATTACGCTGATTCTGTAAAAGGT CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC AGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGCGT TACGGTGGTTCTGCTTAC TTCGACTACTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA
EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLEWVS YIGGGYGGTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR YGGSAY FDYWGQGTLVTVSS CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGT AGTGGCTCTTCATCTAATATTGGCTCTAATTATGTCTAC TGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT AGAAATAACCAGCGGGCCAAGC GGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAG GCCGATTATTACTGT GCTGCTTACTACCGTTACAACCGTTATGTC TTCGGCGGAGGCACCAAGCTGACGGTCCTAGGT

QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC AAYYRYNRYV FGGGTKLTVLG Under bar:
CDR regions

antibody Heavy chain Light chain note R4 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC AATTATTCTATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA TACATCTCTTCTTCTTACTACTCTACGTATTACGCTGATTCTGTAAAAGGT CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC TGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGCGT TACGGTTACAACGAC TTCGACTACTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA
EVQLLESGGGLVQPGGSLRLSCAASGFTFS NYSMS WVRQAPGKGLEWVS YISSSYYSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR YGYND FDYWGQGTLVTVSS CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGT AGTGGCTCTTCATCTAATATTGGCTCTAATTATGTCTAC TGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT AGAAATAACCAGCGGGCCAAGC GGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAG GCCGATTATTACTGT GCTGCTGCTCCGTCTCCTTCTTACTATGTC TTCGGCGGAGGCACCAAGCTGACGGTCCTAGGT

QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC AAAPSPSYYV FGGGTKLTVLG Under bar:
CDR regions

antibody Heavy chain Light chain note R9 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC TACTATGGTATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA TACATCTCTTCTTACTACTCTTACACGTATTACGCTGATTCTGTAAAAGGT CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAG CCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGCGT TACGGTGGTTCTTACCCGTACGACTAC TTCGACTACTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA
EVQLLESGGGLVQPGGSLRLSCAASGFTFS YYGMS WVRQAPGKGLEWVS YISSYYSYTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR YGGSYPYDY FDYWGQGTLVTVSS CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGT AGTGGCTCTTCATCTAATATTGGCTCTAATTATGTCTAC TGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT AGAAATAACCAGCGGGCCAAGC GGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAG GCCGATTATTACTGT GCTGCTGCTCCGGACTACGCTTCTTATGTC TTCGGCGGAGGCACCAAGCTGACGGTCCTAGGT

QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC AAAPDYASYV FGGGTKLTVLG Under bar:
CDR regions

antibody Heavy chain Light chain note R15 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC TCTTATGGTATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA TACATCTCTGGTTACTACTACTCTACGTATTACGCTGATTCTGTAAAAGGT CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC TGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGCGT TACTCTTACGACTACTCTTCT TTCGACTACTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA
EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLEWVS YISGYYYSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR YSYDYSS FDYWGQGTLVTVSS CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGT AGTGGCTCTTCATCTAATATTGGCTCTAATTATGTCTAC TGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT AGAAATAACCAGCGGGCCAAGC GGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAG GCCGATTATTACTGT GCTGCTTCTGCTAACGACTACTATGTC TTCGGCGGAGGCACCAAGCTGACGGTCCTAGGT


QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC AASANDYYV FGGGTKLTVLG Under bar:
CDR regions

antibody Heavy chain Light chain note R17 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC AATTATTCTATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA TACATCTCTGGTCACTACGGTTACACGTATTACGCTGATTCTGTAAAAGGT CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC TGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGCGT TACTCTTACTCTAAC TTCGACTACTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA
EVQLLESGGGLVQPGGSLRLSCAASGFTFS NYSMS WVRQAPGKGLEWVS YISGHYGYTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR YSYSN FDYWGQGTLVTVSS CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGT AGTGGCTCTTCATCTAATATTGGCTCTAATTATGTCTAC TGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT AGAAATAACCAGCGGGCCAAGC GGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAG GCCGATTATTACTGT GCTGCTTCTGGTAACTACCGTTCTTATGTC TTCGGCGGAGGCACCAAGCTGACGGTCCTAGGT

QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC AASGNYRSYV FGGGTKLTVLG Under bar:
CDR regions

In addition, among the above six types of antibodies, the three types of antibodies R1, R3, and R4, which have particularly excellent neutralizing ability, are S clade (hCoV_19/South Korea/KCDC03/2020), V clade (hCoV_19/South Korea/KUMC15/2020), and G clade. The neutralizing ability of (hCoV_19/South Korea/KUMC17/2020) against the SARS-CoV-2 virus was analyzed using the PRNT measurement method. Neutralization ability (%) was calculated based on the number of plaques in wells treated with antibodies compared to the number of plaques in wells not treated with antibodies (Figure 5), and the EC ₅₀ value was derived using the Prism program (GraphPad). (Table 7).

EC ₅₀ value (μg/mL) clade Antibody R1 R3 R4 S 0.044 0.013 1.072 G 0.091 0.017 1.445 V 0.095 0.007 0.355

From the results in Table 7 and Figure 5, all three antibodies (R1, R3, and R4) of the present invention have neutralizing ability against SARS-CoV-2 viruses of the S, G, and V clades, and therefore have neutralizing ability against COVID-19. It was confirmed that it can be usefully used as a therapeutic agent.

Example 5. Mechanism of action and binding site analysis

The present inventors also confirmed by ELISA test whether the antibodies showing virus neutralizing ability inhibited the binding to ACE2, known as the cellular receptor of SARS-CoV-2. RBD-mFc and humanACE2-His (Bioss antibodies, bs-46001P) recombinant proteins produced using animal cells were used in the test.

In the ELISA method, blocking was performed with 3% skim milk dissolved in PBS on a plate coated with RBD-mFc and protein at a concentration of 1 μg/mL. On the plate where blocking was completed, humanACE2-His protein was diluted from 10 μg/mL to 1/4 and treated at 7 levels of concentration, followed by secondary antibody (anti-His-HRP (R&D systems, MAB050H)). . Between each step, washing was performed three times with PBST (0.05% Tween in PBS). Finally, a color reaction was generated with TMB (biofx, TMBC-1000-01) and confirmed by measuring the OD ₄₅₀ value using an ELISA reader (Victor X3 PerkinElmer) (FIG. 6a). Through this, it was confirmed that the RBD-mFc protein coated on the plate and the processed humanACE2-His protein bound.

Next, the extent to which the binding between SARS-CoV-2-RBD and ACE2 was inhibited by the antibody was confirmed using the ELISA method. Among the antibodies used in the test, in addition to the developed antibodies (R1, R3, R4, R15), the CR3022 antibody is known to bind to the SARS-CoV-2 RBD but has no ACE2 neutralizing ability (Science. 2020 May 8; 368(6491 ): 630-633). Blocking was performed with 3% skim milk dissolved in PBS on a plate coated with RBD-mFc protein at a concentration of 1 μg/mL. On the blocked plate, humanACE2-his protein at a concentration of 0.15 μg/mL and antibodies (Negative Ab, CR3022, R1, R3, R4, and R15) diluted by 1/3 from 7.5 μg/mL were added in 9 steps each. After processing the diluted mixture, secondary antibody (anti-his-HRP (R&D systems, MAB050H)) was applied. Between each step, washing was performed three times with PBST (0.05% Tween in PBS). A color reaction was generated with TMB (biofx, TMBC-1000-01) and confirmed by measuring the OD ₄₅₀ value using an ELISA reader (Victor X3 PerkinElmer) (FIG. 6b). Through this, it was confirmed that the developed antibodies inhibit the binding between ACE2 and RBD.

The present inventors also analyzed the binding of the developed antibodies to the chimeric RBD protein to confirm the binding site within the SARS-CoV-2-RBD protein. First, it was confirmed that all developed antibodies did not cross-react with the S protein of SARS-CoV. Based on this, a protein expressing only the RBD part of the spike protein of the SARS-CoV-2 virus (SARS-CoV-2-RBD) and a receptor (RBM) known to be directly involved in binding to ACE2 in SARS-CoV-2-RBD were used. The binding degree of the selected antibody was confirmed for a protein (SARS-CoV-2-chRBD) in which the sequence of the binding motif region was replaced with the RBM sequence of SARS-CoV. The amino acid sequences of SARS-CoV-2-RBD, SARS-CoV-RBD, and SARS-CoV-2-chRBD are shown in Table 8. Specifically, blocking was performed with 3% skim milk dissolved in PBS on plates coated with RBD-mFc and chRBD-mFC proteins at a concentration of 1 μg/mL. On the plate where blocking was completed, the purified antibody was diluted to 7 levels of concentration from 9 μg/mL to 1/5, and then secondary antibody (anti-hIgG-Fab-HRP (Jackson, JAC-109- 035-097)) was processed. Between each step, washing was performed three times with PBST (0.05% Tween in PBS). Finally, a color reaction was generated with TMB (biofx, TMBC-1000-01) and confirmed by measuring the OD ₄₅₀ value using an ELISA reader (Victor X3 PerkinElmer). The results are shown in Figures 7A and 7B.

RBD region sequences of SARS-CoV-2-RBD and SARS-CoV-2-chRBD proteins division Sequence of RBD region sequence number SARS-CoV-2-RBD SIVRFPNITN LCPFGEVFNA TRFASVYAWN RKRISNCVAD YSVLYNSASF STFKCYGVSP TKLNDLCFTN VYADSFVIRG DEVRQIAPGQ TGKIADYNYK LPDDFTGCVI AWN SN N L D SK VG GNYNY L YR LF R KSN L K PF ERDIS TEIYQ AGST PC NGVE GF NCY F PL QS YGF QP T N G V G YQPYRVVVLS FELLHAPATV CGPKK

Under bar: ACE2 Receptor binding motif 97 SARS-CoV-RBD RVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWDYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKL STDLIKNQCVNF 98 SARS-CoV-2-chRBD SIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLHAPATVCGPKK 99

As shown in Figures 7a and 7b, the developed antibodies (R1, R3, R4, and R15) bind to the RBD region of the S protein of SARS-CoV-2, which directly binds to humanACE2, thereby creating a barrier between the S protein and the ACE2 protein. It was confirmed that it exhibits neutralizing ability against the SARS-CoV-2 virus by inhibiting its binding.

<110> AbClon Inc. <120> Anti-SARS-CoV-2 S protein antibody or antigen binding fragment thereof, and pharmaceutical composition for preventing or treating SARS-CoV-2 infection comprising the same <130> PN200074P <150> KR 10-2020-0041801 <151 > 2020-04-06 <160> 99 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 of R1 <400> 1 Gly Tyr Ser Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 of R1 <400> 2 Tyr Ile Ser Ser Tyr Tyr Tyr Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 of R1 <400> 3 Tyr Gly Tyr Ser Gly Tyr 1 5 <210> 4 <211> 13 < 212> PRT <213> Artificial Sequence <220> <223> LCDR1 of R1 <400> 4 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr 1 5 10 <210> 5 <211> 7 <212> PRT < 213> Artificial Sequence <220> <223> LCDR2 of R1 <400> 5 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 of R1 <400> 6 Ala Ala Ser Pro Ala Tyr Tyr Tyr Tyr Val 1 5 10 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of R1 <400 > 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Tyr Tyr Tyr Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Tyr Ser Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 8 <211 > 110 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of R1 <400> 8 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Ser Pro Ala Tyr Tyr 85 90 95 Tyr Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 9 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HCDR1 of R1 <400> 9 ggttatcta tgagc 15 <210> 10 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> HCDR2 of R1 <400> 10 tacatctctt cttactacta ctctacgtat tacgctgatt ctgtaaaagg t 51 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HCDR3 of R1 <400> 11 tacggttact ctggttac 18 <210> 12 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> LCDR1 of R1 <400> 12 agtggctctt catctaatat tggctctaat tatgtctac 39 <210 > 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LCDR2 of R1 <400> 13 agaaataacc agcggccaag c 21 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence < 220> <223> LCDR3 of R1 <400> 14 gctgcttctc cggcttacta ctactatgtc 30 <210> 15 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region of R1 <400> 15 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctgg cctgagactc 60 tcctgtgcag cctctggatt cacctttagc ggttattcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatac atctcttctt actactactc tacgtattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gcg ttacggt 300 tactctggtt acttcgacta ctggggccag ggtacactgg tcaccgtgag ctca 354 <210> 16 <211> 330 <212> DNA <213> Artificial Sequence <220> < 223> Light Chain Variable of R1 <400> CTATTATGGTA CCAGCTC 120 CCAGGAACGGGGGGGGGGGGGTCCT 180 GACCGAAG CTGCTCAA GTCTGGCACCCT CCC TGGCATCAG TGGCTCGG 240 TCCGAGAGAGAGAGAGTA TTACTGTGTGTGCTGTCTCGG CTTACTA CTACTACTTC 330 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 of R3 <400> 17 Ser Tyr Gly Met Ser 1 5 <210> 18 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 of R3 <400> 18 Tyr Ile Gly Gly Gly Tyr Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 19 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 of R3 <400> 19 Tyr Gly Gly Ser Ala Tyr 1 5 <210> 20 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 of R3 <400> 20 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr 1 5 10 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 of R3 < 400> 21 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 22 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 of R3 <400> 22 Ala Ala Tyr Tyr Arg Tyr Asn Arg Tyr Val 1 5 10 <210> 23 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of R3 <400> 23 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Gly Gly Gly Tyr Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Gly Ser Ala Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 24 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of R3 <400> 24 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Tyr Tyr Arg Tyr Asn 85 90 95 Arg Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 25 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HCDR1 of R3 <400> 25 tcttatggta tgagc 15 <210> 26 <211> 51 <212> DNA <213> Artificial Sequence <220> < 223> HCDR2 of R3 <400> 26 tacatcggtg gtggttacgg tggtacgtat tacgctgatt ctgtaaaagg t 51 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HCDR3 of R3 <400> 27 tacggtggtt ct gcttac 18 < 210> 28 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> LCDR1 of R3 <400> 28 agtggctctt catctaatat tggctctaat tatgtctac 39 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LCDR2 of R3 <400> 29 agaaataacc agcggccaag c 21 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LCDR3 of R3 <400> 30 gctgcttact accgttacaa ccgttatgtc 30 <210> 31 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region of R3 <400> 31 gaggtgcagt tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag c ctctggatt cacctttagc tcttatggta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatac atcggtggtg gttacggtgg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gcgttacggt 30 0 ggttctgctt acttcgacta ctggggccag ggtacactgg tcaccgtgag ctca 354 <210> 32 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Light Chain Variable of R3 <400> 32 TATGGTGTA CCAGCTC 120 CCAGAACGGGGGGGGGTCAAG CCCTATAACC AGCCTAACCCT 180 GACCGATCT CTGCAA GTCCAA GGCCATCAG TGGCTCGG 240 TCCGAGAGAGAGTA TTACTGTGTGCT GCTTACCCGTACCGTCGTC 300 GGCGGAGCACACTGAC GGTCTAGT 330 < 210> 33 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 of R4 <400> 33 Asn Tyr Ser Met Ser 1 5 <210> 34 <211> 17 <212> PRT <213 > Artificial Sequence <220> <223> HCDR2 of R4 <400> 34 Tyr Ile Ser Ser Ser Tyr Tyr Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 35 <211> 5 <212> PRT < 213> Artificial Sequence <220> <223> HCDR3 of R4 <400> 35 Tyr Gly Tyr Asn Asp 1 5 <210> 36 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 of R4 <400> 36 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr 1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 of R4 <400> 37 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 of R4 <400> 38 Ala Ala Ala Pro Ser Pro Ser Tyr Tyr Val 1 5 10 <210> 39 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of R4 <400> 39 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Tyr Tyr Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Tyr Asn Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 40 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of R4 <400> 40 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Ala Pro Ser Pro Ser 85 90 95 Tyr Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 41 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HCDR1 of R4 <400> 41 aattattcta tgagc 15 <210> 42 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> HCDR2 of R4 <400> 42 tacatctctt cttcttacta ctctacgtat tacgctgatt ctgtaaaagg t 51 <210> 43 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HCDR3 of R4 <400> 43 tacggttaca acgac 15 <210> 4 4 < 211> 39 <212> DNA <213> Artificial Sequence <220> <223> LCDR1 of R4 <400> 44 agtggctctt catctaatat tggctctaat tatgtctac 39 <210> 45 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LCDR2 of R4 <400> 45 agaaataacc agcggccaag c 21 <210> 46 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LCDR3 of R4 <400> 46 gctgctgctc cgtctccttc ttactatgtc 30 <210 > 47 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region of R4 <400> 47 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattattcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatac atctcttctt cttactactc tacgtattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gcgttacggt 300 tacaacgact tcgactactg gggccagggt acactggtca cc gtgagctc a 351 <210> 48 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> light chain variable region of R4 <400> 48 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc taatattggc tctaattatg tctactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat agaaataacc caag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggccgatta ttactgtgct gctgctccgt ctccttctta ctatgtcttc 300 ggcggaggca ccaagctgac ggtcctaggt 330 <21 0> 49 < 211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 of R9 <400> 49 Tyr Tyr Gly Met Ser 1 5 <210> 50 <211> 17 <212> PRT <213> Artificial Sequence < 220> <223> HCDR2 of R9 <400> 50 Tyr Ile Ser Ser Tyr Tyr Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 51 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 of R9 <400> 51 Tyr Gly Gly Ser Tyr Pro Tyr Asp Tyr 1 5 <210> 52 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 of R9 <400> 52 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr 1 5 10 <210> 53 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 of R9 <400> 53 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 54 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 of R9 <400> 54 Ala Ala Ala Pro Asp Tyr Ala Ser Tyr Val 1 5 10 <210> 55 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of R9 <400> 55 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Tyr Tyr Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Gly Ser Tyr Pro Tyr Asp Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 56 <211> 110 <212> PRT <213> Artificial Sequence <220 > <223> light chain variable region of R9 <400> 56 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Ala Pro Asp Tyr Ala 85 90 95 Ser Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 57 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HCDR1 of R9 <400> 57 tactatggta tgagc 15 <210> 58 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> HCDR2 of R9 <400> 58 tacatctctt cttactactc ttacacgtat tacgctgatt ctgtaaaagg t 51 <210> 59 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HCDR3 of R9 <400> 59 tacggtggtt cttacc cgta cgactac 27 <210> 60 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> LCDR1 of R9 <400> 60 agtggctctt catctaatat tggctctaat tatgtctac 39 <210> 61 <211> 21 <212> DNA <213 > Artificial Sequence <220> <223> LCDR2 of R9 <400> 61 agaaataacc agcggccaag c 21 <210> 62 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LCDR3 of R9 <400> 62 gctgctgctc cggactacgc ttcttatgtc 30 <210> 63 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region of R9 <400> 63 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagact c 60 tcctgtgcag cctctggatt cacctttagc tactatggta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatac atctcttctt actactctta cacgtattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gcg ttacggt 300 ggttcttacc cgtacgacta cttcgactac tggggccagg gtacactggt caccgtgagc 360 tca 363 <210> 64 <211> 330 <212> DNA <213> Artificial Sequence <220 > <223> light chain variable region of R9 <400> 64 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc taatattggc tctaattatg tctactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat a gaaataacc agcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggccgatta ttactgtgct gctgctccgg actacgcttc ttatgtcttc 300 ggcggaggca ccaagctgac ggtcctaggt 330 <210> 65 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 of R15 <400> 65 Ser Tyr Gly Met Ser 1 5 <210> 66 <211> 17 <212> PRT < 213> Artificial Sequence <220> <223> HCDR2 of R15 <400> 66 Tyr Ile Ser Gly Tyr Tyr Tyr Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 67 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 of R15 <400> 67 Tyr Ser Tyr Asp Tyr Ser Ser 1 5 <210> 68 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 of R15 <400> 68 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr 1 5 10 <210> 69 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 of R15 < 400> 69 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 70 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 of R15 <400> 70 Ala Ala Ser Ala Asn Asp Tyr Tyr Val 1 5 <210> 71 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of R15 <400> 71 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Gly Tyr Tyr Tyr Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Ser Tyr Asp Tyr Ser Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 72 <211> 109 <212> PRT <213> Artificial Sequence <220> < 223> light chain variable region of R15 <400> 72 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Ser Ala Asn Asp Tyr 85 90 95 Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 <210> 73 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HCDR1 of R15 <400> 73 tcttatggta tgagc 15 <210> 74 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> HCDR2 of R15 <400> 74 tacatctctg gttactacta ctctacgtat tacgctgatt ctgtaaaagg t 51 <210> 75 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HCDR3 of R15 <400> 75 tactcttacg actactcttc t 21 <21 0> 76 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> LCDR1 of R15 <400> 76 agtggctctt catctaatat tggctctaat tatgtctac 39 <210> 77 <211> 21 <212> DNA <213> Artificial Sequence < 220> <223> LCDR2 of R15 <400> 77 agaaataacc agcggccaag c 21 <210> 78 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> LCDR3 of R15 <400> 78 gctgcttctg ctaacgacta ctatgtc 27 <210> 79 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region of R15 <400> 79 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc tcttatggta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatac atctctggtt actactactc tacgtattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gcgttactct 300 tacgactact cttctttcga ctact ggggc cagggtacac tggtcaccgt gagctca 357 <210> 80 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> light chain variable region of R15 <400> 80 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc taatattggc tctaattatg tctactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat agaaataacc ag cggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggccgatta ttactgtgct gcttctgcta acgactacta tgtcttcggc 300 ggaggcacca agctgacggt cctaggt 327 <210 > 81 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 of R17 <400> 81 Asn Tyr Ser Met Ser 1 5 <210> 82 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 of R17 <400> 82 Tyr Ile Ser Gly His Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 83 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 of R17 <400> 83 Tyr Ser Tyr Ser Asn 1 5 <210> 84 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 of R17 <400 > 84 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr 1 5 10 <210> 85 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 of R17 <400> 85 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 86 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 of R17 <400> 86 Ala Ala Ser Gly Asn Tyr Arg Ser Tyr Val 1 5 10 <210> 87 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of R17 <400> 87 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Gly His Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Ser Tyr Ser Asn Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 88 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of R17 <400> 88 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Ser Gly Asn Tyr Arg 85 90 95 Ser Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 89 <211> 15 <212 > DNA <213> Artificial Sequence <220> <223> HCDR1 of R17 <400> 89 aattattcta tgagc 15 <210> 90 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> HCDR2 of R17 < 400> 90 tacatctctg gtcactacgg ttacacgtat tacgctgatt ctgtaaaagg t 51 <210> 91 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HCDR3 of R17 <400> 91 tactcttact ctaac 15 <210> 92 11> 39 <212> DNA <213> Artificial Sequence <220> <223> LCDR1 of R17 <400> 92 agtggctctt catctaatat tggctctaat tatgtctac 39 <210> 93 <211> 21 <212> DNA <213> Artificial Sequence <220> <223 > LCDR2 of R17 <400> 93 agaaataacc agcggccaag c 21 <210> 94 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LCDR3 of R17 <400> 94 gctgcttctg gtaactaccg ttcttatgtc 30 <210> 95 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region of R17 <400> 95 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc actc 60 tcctgtgcag cctctggatt cacctttagc aattattcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatac atctctggtc actacggtta cacgtattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gcg ttactct 300 tactctaact tcgactactg gggccagggt acactggtca ccgtgagctc a 351 <210> 96 <211> 330 <212> DNA <213> Artificial Sequence <220> < 223> light chain variable region of R17 <400> 96 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc taatattggc tctaattatg tctactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat a gaaataacc agcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggccgatta ttactgtgct gcttctggta actaccgttc ttatgtcttc 300 ggcggaggca ccaagctgac ggtccta ggt 330 <210> 97 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2-RBD <400> 97 Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu 1 5 10 15 Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys 20 25 30 Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala 35 40 45 Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn 50 55 60 Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly 65 70 75 80 Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp 85 90 95 Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp 100 105 110 Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu 115 120 125 Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile 130 135 140 Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu 145 150 155 160 Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr 165 170 175 Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu 180 185 190 Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys 195 200 205 <210> 98 <211> 212 <212> PRT <213> Artificial Sequence < 220> <223> SARS-CoV-RBD <400> 98 Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val 20 25 30 Tyr Ala Trp Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr 35 40 45 Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe 50 55 60 Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg 65 70 75 80 Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys 85 90 95 Leu Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn 100 105 110 Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu 115 120 125 Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro 130 135 140 Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr 145 150 155 160 Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr 165 170 175 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro 180 185 190 Ala Thr Val Cys Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln 195 200 205 Cys Val Asn Phe 210 <210> 99 <211> 204 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2-chRBD < 400> 99 Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu 1 5 10 15 Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys 20 25 30 Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala 35 40 45 Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn 50 55 60 Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly 65 70 75 80 Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp 85 90 95 Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp 100 105 110 Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys 115 120 125 Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile 130 135 140 Ser Asn Val Pro Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala 145 150 155 160 Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr 165 170 175 Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu 180 185 190Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys 195 200

Claims (10)

Antibodies or antigen-binding fragments thereof of anti-SARS-CoV S protein selected from:
(a) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;
(b) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 17, HCDR2 of SEQ ID NO: 18, and HCDR3 of SEQ ID NO: 19; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 21, and LCDR3 of SEQ ID NO: 22;
(c) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 33, HCDR2 of SEQ ID NO: 34, and HCDR3 of SEQ ID NO: 35; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 38;
(d) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 49, HCDR2 of SEQ ID NO: 50, and HCDR3 of SEQ ID NO: 51; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 52, LCDR2 of SEQ ID NO: 53, and LCDR3 of SEQ ID NO: 54;
(e) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 65, HCDR2 of SEQ ID NO: 66, and HCDR3 of SEQ ID NO: 67; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 68, LCDR2 of SEQ ID NO: 69, and LCDR3 of SEQ ID NO: 70; or
(f) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 81, HCDR2 of SEQ ID NO: 82, and HCDR3 of SEQ ID NO: 83; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 84, LCDR2 of SEQ ID NO: 85, and LCDR3 of SEQ ID NO: 86.
The method of claim 1, wherein the antibodies or antigen-binding fragments of (a), (b), and (c) have neutralizing ability against SARS-CoV-2 of the S, V, or G clade, and the (d) ), (e), and (f), the antibodies or antigen-binding fragments thereof have neutralizing ability against SARS-CoV-2 of the S clade.
The antibody or antigen-binding fragment thereof according to claim 2, wherein the SARS-CoV-2 of the S clade is hCoV_19/South Korea/KCDC03/2020.
The antibody or antigen-binding fragment thereof according to claim 2, wherein the SARS-CoV-2 of the V clade is hCoV_19/South Korea/KUMC15/2020.
The antibody or antigen-binding fragment thereof according to claim 2, wherein the SARS-CoV-2 of the G clade is hCoV_19/South Korea/KUMC17/2020.
A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of claim 1.
A recombinant vector containing the nucleic acid molecule of claim 6.
A host cell transformed with the recombinant vector of claim 7.
A pharmaceutical composition for preventing or treating SARS-CoV-2 infection, comprising the following antibodies or antigen-binding fragments thereof and a pharmaceutically acceptable carrier:
(a) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;
(b) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 17, HCDR2 of SEQ ID NO: 18, and HCDR3 of SEQ ID NO: 19; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 21, and LCDR3 of SEQ ID NO: 22;
(c) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 33, HCDR2 of SEQ ID NO: 34, and HCDR3 of SEQ ID NO: 35; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 38;
(d) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 49, HCDR2 of SEQ ID NO: 50, and HCDR3 of SEQ ID NO: 51; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 52, LCDR2 of SEQ ID NO: 53, and LCDR3 of SEQ ID NO: 54;
(e) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 65, HCDR2 of SEQ ID NO: 66, and HCDR3 of SEQ ID NO: 67; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 68, LCDR2 of SEQ ID NO: 69, and LCDR3 of SEQ ID NO: 70; or
(f) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 81, HCDR2 of SEQ ID NO: 82, and HCDR3 of SEQ ID NO: 83; and an antibody or antigen-binding fragment thereof comprising the light chain variable region of LCDR1 of SEQ ID NO: 84, LCDR2 of SEQ ID NO: 85, and LCDR3 of SEQ ID NO: 86,
However, the antibodies or antigen-binding fragments thereof of (a), (b), and (c) are for prevention or treatment of SARS-CoV-2 of the S, V, or G clade, and (d), ( The antibodies or antigen-binding fragments of e), and (f) are for prevention or treatment of SARS-CoV-2 of the S clade.
The pharmaceutical composition of claim 9, wherein the SARS-CoV-2 is SARS-CoV-2 of the S, V, or G clade.