Anti-SFTS virus antibody and use thereof

Abstract

The present invention relates to: an anti-SFTS virus antibody or an antigen-binding fragment thereof; a nucleic acid encoding same; a vector comprising the nucleic acid; a cell transformed with the vector; a method for preparing the antibody or the antigen-binding fragment thereof; a composition for preventing or treating SFTS virus infection, comprising same; and a composition for diagnosing same.

Description

Anti-SFTS virus antibodies and uses thereof

[0001] The present invention relates to an anti-SFTS virus antibody or an antigen-binding fragment thereof, a nucleic acid encoding the same, a vector comprising the nucleic acid, a cell transformed with the vector, a method for producing the antibody or an antigen-binding fragment thereof, a composition comprising the same for preventing or treating SFTS virus infection, and a diagnostic composition thereof.

[0002]

[0003] Severe fever with thrombocytopenia syndrome (SFTS) is a febrile hemorrhagic illness in humans and animals caused by infection with the SFTS virus. It is primarily reported as a tick-borne disease. Infections are frequently reported in occupations such as agriculture and forestry, particularly in Gangwon Province, which has a high proportion of forests and farmland.

[0004] In South Korea, the first death was reported in 2012, and the number of deaths continues to increase annually. SFTS is a highly concerning emerging infectious disease with an average fatality rate of approximately 20%. Since 2020, SFTS has been designated as a zoonotic disease. It is known to be transmitted by the small, red tick, and is particularly transmitted to humans by companion animals. Human-to-human transmission is known to be possible through the blood of infected individuals. Viremia has been observed in animals such as goats, sheep, pigs, and dogs, which are believed to be intermediate hosts of the SFTS virus.

[0005] The SFTS virus is 80–100 nm in size and contains three segments of single-stranded negative-sense RNA: the large segment (L segment), the middle segment (M segment), and the small segment (S segment), each of which is synthesized by RNA polymerase, glycoprotein, and NP protein (Fig. 1, Vaccines. 2019; 7(4):125).

[0006] The main symptoms and signs of SFTS virus infection are fever, abdominal pain, nausea, vomiting, leukopenia, thrombocytopenia, systemic inflammatory response syndrome, coagulopathy, and multiple organ failure (Experimental & Molecular Medicine volume 53, pages 713-722 (2021)).

[0007] However, no clear vaccine or treatment has been developed, and research on post-disease treatment, rather than prevention, is currently underway, but is inadequate. In Korea, epidemiological investigations into the source of SFTS virus infection are underway, including analysis of virus positivity rates in wild and livestock animals.

[0008] In addition, animal models for the SFTS virus have been established in Korea, China, and the United States, and research on clinical symptoms and treatments caused by viral infection is being conducted. However, in vitro experiments are limited due to the lack of an appropriate animal model.

[0009] Against this backdrop, the inventors of the present application endeavored to develop a treatment for SFTS virus infection, a novel infectious disease with a very high mortality rate, averaging approximately 20% domestically and internationally. As a result, they confirmed the potential application of neutralizing antibodies against specific antigenic proteins of the SFTS virus as a therapeutic agent, and completed the present invention.

[0010]

[0011] Summary of the invention

[0012] The purpose of the present invention is to provide a novel antibody or antigen-binding fragment thereof against Severe Fever with Thrombocytopenia Syndrome (SFTS) virus.

[0013] Another object of the present invention is to provide a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[0014] Another object of the present invention is to provide a vector containing the nucleic acid, a cell transformed with the vector, and a method for producing the same.

[0015] Another object of the present invention is to provide a composition for preventing or treating SFTS virus infection comprising the antibody or an antigen-binding fragment thereof.

[0016] Another object of the present invention is to provide a composition for diagnosing SFTS virus infection comprising the antibody or an antigen-binding fragment thereof.

[0017] To achieve the above object, the present invention provides an antibody or an antigen-binding fragment thereof that binds to Severe Fever with Thrombocytopenia Syndrome (SFTS) virus, comprising a heavy chain variable region comprising a heavy chain CDR1 selected from the group consisting of sequences of SEQ ID NOs: 1 to 4, a heavy chain CDR2 selected from the group consisting of sequences of SEQ ID NOs: 5 to 11, and a heavy chain CDR3 selected from the group consisting of sequences of SEQ ID NOs: 12 to 19; and a light chain variable region comprising a light chain CDR1 selected from the group consisting of sequences of SEQ ID NOs: 28 to 32, a light chain CDR2 selected from the group consisting of sequences of SEQ ID NOs: 33 to 39, and a light chain CDR3 selected from the group consisting of sequences of SEQ ID NOs: 40 to 45.

[0018] The present invention also provides a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[0019] The present invention also provides a vector comprising the nucleic acid.

[0020] The present invention also provides a cell transformed with the vector.

[0021] The present invention also provides a method for producing the antibody or antigen-binding fragment thereof, comprising the following steps: (a) culturing the cell; and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cell.

[0022] The present invention also provides a composition for preventing or treating SFTS virus infection, comprising the antibody or antigen-binding fragment thereof as an active ingredient. The present invention also provides a method for preventing or treating SFTS virus infection, comprising administering the antibody or antigen-binding fragment thereof. The present invention also provides a use of the antibody or antigen-binding fragment thereof in the manufacture of a composition for preventing or treating SFTS virus infection.

[0023] The present invention also provides a composition for diagnosing SFTS virus infection, comprising the antibody or antigen-binding fragment thereof as an active ingredient. The present invention also provides a method for diagnosing SFTS virus infection, comprising the step of contacting the antibody or antigen-binding fragment thereof with a sample derived from a patient infected with SFTS virus. The present invention further provides a use of the antibody or antigen-binding fragment thereof for diagnosing SFTS virus infection.

[0024]

[0025] Figure 1 illustrates the structure of the SFTS virus.

[0026] Figure 2 is a schematic diagram of the production of a clone expressing SFTS virus antigen protein.

[0027] Figure 3 shows the results of confirming the expression and purification of the SFTS virus antigen NP protein.

[0028] Figure 4 shows the results of titer measurement of the primary phage output by biopanning.

[0029] Figure 5 shows the results of titer measurement of secondary phage output by biopanning.

[0030] Figure 6 shows the results of titer measurement of the third phage output by biopanning.

[0031] Figure 7 shows the results of NP antigen protein binding scFv selection by indirect ELISA.

[0032] Figure 8 shows the results of scFv candidate discovery through CDR sequence analysis of the heavy chain and light chain of scFv candidate.

[0033] Figure 9 shows the results of confirming the antigen detection limit of scFv by indirect ELISA.

[0034] Figure 10 shows the results of confirming the expression and purification of NP target mouse IgG in CHO cells.

[0035] Figure 11 shows the results of confirming the expression and purification of NP target human IgG in CHO cells.

[0036] Figure 12 shows the antigen binding verification results of the purified NP target antibody.

[0037] Figure 13 shows the results of verification of the antigen detection limit of SFTSV NP target human IgG antibody.

[0038] Figure 14 shows the results of verification of the antigen detection limit of SFTSV NP target mouse IgG antibody.

[0039] Figure 15 shows the results of antigen binding assay of SFTSV NP target human IgG antibody.

[0040] Figure 16 shows the results of antigen binding assay of SFTSV NP target mouse IgG antibody.

[0041] Figure 17 shows the results of cross-reactivity verification of SFTSV NP targeting IgG antibodies with NP antigens of other similar viruses.

[0042]

[0043] Detailed description and specific implementation examples of the invention

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0045] Previously, antibodies recognizing the Gn protein of glycoprotein were discovered as therapeutic antibodies for SFTS virus, and antigen-antibody binding was confirmed (Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope PNAS 5, (2017) 114(36) E7564-E7573), and candidate antibodies were selected from SFTS infected patients and their neutralizing ability was confirmed in vitro / vivo (An anti-Gn glycoprotein antibody from a convalescent patient potently inhibits the infection of severe fever with thrombocytopenia syndrome virus PLOS pathgens, (2019) 1;15(2):e1007375).

[0046] The inventors of the present application identified an antibody or an antigen-binding fragment thereof that specifically binds to the nucleocapsid protein among the glycoproteins of the SFTS virus.

[0047] Based on this, the present invention relates to an antibody or an antigen-binding fragment thereof that binds to Severe Fever with Thrombocytopenia Syndrome (SFTS) virus, comprising a heavy chain variable region comprising a heavy chain CDR1 selected from the group consisting of sequences of SEQ ID NOs: 1 to 4, a heavy chain CDR2 selected from the group consisting of sequences of SEQ ID NOs: 5 to 11, and a heavy chain CDR3 selected from the group consisting of sequences of SEQ ID NOs: 12 to 19; and a light chain variable region comprising a light chain CDR1 selected from the group consisting of sequences of SEQ ID NOs: 28 to 32, a light chain CDR2 selected from the group consisting of sequences of SEQ ID NOs: 33 to 39, and a light chain CDR3 selected from the group consisting of sequences of SEQ ID NOs: 40 to 45.

[0048] The above antibody or antigen-binding fragment thereof may be characterized by specifically binding to the nucleocapsid protein among the glycoproteins of the SFTS virus. The nucleocapsid comprises the following sequence.

[0049]

[0050] As used herein, the term "antibody" refers to an anti-SFTS virus antibody that specifically binds to the nucleocapsid protein of the SFTS virus. The scope of the present invention includes not only complete antibody forms that specifically bind to the nucleocapsid protein of the SFTS virus, but also antigen-binding fragments of such antibody molecules.

[0051] A complete antibody has a structure with two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond.

[0052] As used herein, the term "heavy chain" refers to a full-length heavy chain and fragments thereof, which comprises a variable domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3. In addition, the term "light chain" as used herein refers to a full-length light chain and fragments thereof, which comprises a variable domain VL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant region domain CL.

[0053] The above whole antibodies include subtypes of IgA, IgD, IgE, IgM and IgG, and in particular, IgG includes IgG1, IgG2, IgG3 and IgG4. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and has subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1) and alpha2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0054] Antigen-binding fragments of antibodies, or antibody fragments, refer to fragments that possess antigen-binding function, and include Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure with variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 is generated when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond.

[0055] Fv is the smallest antibody fragment that contains only the heavy chain variable region and the light chain variable region. A two-chain Fv has a heavy chain variable region and a light chain variable region non-covalently linked, while a single-chain Fv (single-chain Fv, scFv) has a heavy chain variable region and a light chain variable region covalently linked, usually via a peptide linker, or directly linked at the C-terminus, so that it can form a dimer-like structure like a two-chain Fv. These antibody fragments can be produced using proteolytic enzymes (for example, restriction digestion of a complete antibody with papain yields Fab, and digestion with pepsin yields F(ab')2) or using genetic recombination technology.

[0056] An "Fv" fragment is an antibody fragment containing the complete antibody recognition and binding site. This region is a dimer composed of one heavy chain variable domain and one light chain variable domain.

[0057] The "Fab" fragment comprises the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. An F(ab')2 antibody fragment typically comprises a pair of Fab' fragments covalently linked by cysteines in the hinge region at the C-terminus of the Fab' fragment.

[0058] A "single-chain Fv (scFv)" antibody fragment is a structure composed of a single polypeptide chain comprising the VH and VL domains of an antibody. The scFv may additionally comprise a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. The linker may be a peptide linker and may be about 10-25 amino acids in length. For example, it may include, but is not limited to, hydrophilic amino acids such as glycine and / or serine.

[0059] In one embodiment, the antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFvs, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of the foregoing antibodies.

[0060] The heavy chain constant region can be selected from any one of the gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) isotypes. For example, the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region can be kappa or lambda type.

[0061] The term "monoclonal antibody" refers to antibodies obtained from a substantially homogeneous population of antibodies, i.e., antibodies that are identical except for possible naturally occurring mutations that may be present in trace amounts in individual antibodies within the population. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to conventional (polyclonal) antibodies, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0062] An "epitope" is a protein determinant to which an antibody can specifically bind. Epitopes typically consist of chemically active surface molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that binding to the former is lost in the presence of denaturing solvents, while binding to the latter is not.

[0063] Specifically, the antibody or antigen-binding fragment thereof according to the present invention can recognize or specifically bind to a polypeptide (epitope) comprising 5, 7, 10, 12 or more, or preferably 15 or more amino acids within 1-40, 1-42 of the tau protein.

[0064] The above "humanized" forms of non-human (e.g., mouse) antibodies are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region of the recipient antibody have been replaced with residues from the hypervariable region of a non-human species (donor antibody) that retains the desired specificity, affinity, and ability, such as mouse, rat, rabbit, or non-human primate.

[0065] The above "human antibody" means a molecule derived from human immunoglobulin, and the entire amino acid sequence constituting the antibody, including the complementarity determining region and structural region, is composed of human immunoglobulin.

[0066] "Chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0067] The "variable region" of an antibody used in the present invention refers to the light chain and heavy chain portions of an antibody molecule, including the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and the framework regions (FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0068] "Complementary determining region (CDR)" refers to the amino acid residues in an antibody variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3.

[0069] In the present invention, the antibody or antigen-binding fragment thereof that binds to the nucleocapsid protein of the SFTS virus may include a heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 selected from the group consisting of the following, and a light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3.

[0070] [heavy chain variable region]

[0071]

[0072] [Light chain variable region]

[0073]

[0074] The "framework regions" (FRs) are the variable domain residues other than the CDR residues. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4.

[0075] Antibodies that bind to the nucleocapsid protein of SFTS virus may be single-chain or double-chain. Functionally, the binding affinity of antibodies that bind to the nucleocapsid protein of SFTS virus is 10 -5 M to 10 -12 It is within the M range. For example, the binding affinity of an antibody that binds to the nucleocapsid protein of SFTS virus is 10 -6　 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12M, 10 -5　 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9　 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5　 M to 10 -10 M, 10 -6 M to 10 -10　 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9　 M to 10 -10 M, 10 -5　 M to 10 -9 M, 10 -6 M to 10 -9　 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5　 M to 10 -8 M, 10 -6 M to 10 -8　 M, 10 -7 M to 10 -8 M, 10 -5　 M to 10 -7 M, 10 -6 M to 10 -7　 M or 10 -5 M to 10 -6 It's M.

[0076] The antibody or antigen-binding fragment thereof that binds to the nucleocapsid protein of the SFTS virus may include a heavy chain variable region of a sequence selected from the group consisting of sequences of SEQ ID NOs: 20 to 27 or a sequence having 90% or more homology thereto. The antibody or antigen-binding fragment thereof may include a light chain variable region of a sequence selected from the group consisting of sequences of SEQ ID NOs: 47 to 54 or a sequence having 90% or more homology thereto. The antibody or antigen-binding fragment thereof may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology with a sequence selected from the group consisting of sequences of SEQ ID NOs: 20 to 27 or a sequence selected from the group consisting of sequences of SEQ ID NOs: 47 to 54.

[0077] In a specific embodiment according to the present invention, a heavy chain variable region of SEQ ID NO: 20 and a light chain variable region of SEQ ID NO: 47;

[0078] A heavy chain variable region of SEQ ID NO: 21 and a light chain variable region of SEQ ID NO: 48;

[0079] A heavy chain variable region of SEQ ID NO: 22 and a light chain variable region of SEQ ID NO: 49;

[0080] A heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 50;

[0081] A heavy chain variable region of SEQ ID NO: 24 and a light chain variable region of SEQ ID NO: 51;

[0082] A heavy chain variable region of SEQ ID NO: 25 and a light chain variable region of SEQ ID NO: 52;

[0083] A heavy chain variable region of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 53; or

[0084] It may include a heavy chain variable region of sequence number 27 and a light chain variable region of sequence number 54.

[0085] The antibody or antibody fragment of the present invention may include not only the sequence of the anti-SFTS virus antibody of the present invention described herein, but also biological equivalents thereof, as long as it can specifically recognize the nucleocapsid protein of the SFTS virus. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; Alanine, glycine and serine; phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

[0086] Considering the mutations having the above-described biological equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same is interpreted to also include a sequence showing substantial identity with the sequence described in the sequence number. The substantial identity means a sequence showing at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST is accessible at www.ncbi.nlm.nih.gov / BLAST / . Instructions for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_ help.html.

[0087] Based on this, the antibody or antigen-binding fragment thereof of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology to the specified sequences or the entirety of the sequences described in the specification. Such homology may be determined by sequence comparison and / or alignment using methods known in the art. For example, the percent sequence homology of the nucleic acids or proteins of the present invention may be determined using sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0088] In another aspect, the present invention relates to a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[0089] The nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention can be isolated and recombinantly produced. The nucleic acid can be further cloned (DNA amplification) or expressed by inserting it into a replicable vector. Based on this, the present invention relates to a vector comprising the nucleic acid from another perspective.

[0090] "Nucleic acid" is a comprehensive term encompassing DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic structural units of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base moieties. The sequence of the nucleic acid encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, non-conservative substitutions, or conservative substitutions of nucleotides.

[0091] DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to DNA encoding the heavy and light chains of the antibody). Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0092] As used herein, the term "vector" refers to a means for expressing a target gene in a host cell, including plasmid vectors; cosmid vectors; viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. In the vector, a nucleic acid encoding an antibody is operably linked to a promoter.

[0093] “Operably linked” means a functional association between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0094] In the case of a prokaryotic cell as a host, it is common to include a strong promoter that can drive transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. In addition, for example, when a eukaryotic cell is used as a host, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

[0095] In some cases, the vector may be fused with other sequences to facilitate purification of the antibody 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).

[0096] The above vector contains antibiotic resistance genes commonly used in the art as selectable markers, for example, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0097] In another aspect, the present invention relates to cells transformed with the aforementioned vector. The cells used to produce the antibodies of the present invention may be, but are not limited to, prokaryotic, yeast, or higher eukaryotic cells.

[0098] Prokaryotic host cells can be used, such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, and Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis and Staphylococcus (e.g., Staphylococcus carnosus).

[0099] However, animal cells are of greatest interest, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0100] In another aspect, the present invention relates to a method for producing an antibody or an antigen-binding fragment thereof, comprising the steps of (a) culturing the cells; and (b) recovering the antibody or an antigen-binding fragment thereof from the cultured cells.

[0101] The above cells can be cultured in various media. Any commercially available medium can be used as a culture medium. Any other essential supplements known to those skilled in the art may also be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., are already used with the host cells selected for expression and will be readily apparent to those skilled in the art.

[0102] The antibody or antigen-binding fragment thereof can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and the resultant product can be purified, for example, by using affinity chromatography. Additional purification techniques, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can be used.

[0103] In another aspect, the present invention relates to a composition for preventing or treating SFTS virus infection, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0104] The antibody or antigen-binding fragment thereof according to the present invention exhibits neutralizing ability by binding to an SFTS virus antigen protein, particularly a nucleocapsid protein, thereby enabling prevention or treatment of SFTS virus infection.

[0105] “Prevention” means any act of suppressing or delaying the progression of various symptoms related to infection by administering a composition according to the present invention, and “treatment” means alleviating or eliminating various symptoms related to infection.

[0106] Pharmaceutically acceptable carriers included in the composition of the present invention are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0107] The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0108] Since proteins or peptides are digested upon oral administration, oral compositions must be formulated to coat the active agent or protect it from degradation in the stomach. Furthermore, the pharmaceutical composition can be administered by any device capable of transporting the active agent to the target cell.

[0109] The appropriate dosage of the composition according to the present invention varies depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. A skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg.

[0110] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0111] In another aspect, the present invention relates to a composition for diagnosing SFTS virus infection comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0112] The present invention relates to a composition for diagnosing a degenerative brain disease, comprising the antibody as an active ingredient. SFTS virus infection can be diagnosed using a diagnostic composition comprising the antibody of the present invention or an antigen-binding fragment thereof.

[0113] The above composition may be included in a diagnostic kit. The kit may further comprise a composition, solution, or device having one or more other components suitable for the analytical method.

[0114] In one embodiment, the kit may include a bottle, vial, bag, syringe, or syringe. The container may be formed from a variety of materials, such as glass, plastic, or metal. A label included with the container may indicate directions for use. Additionally, other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, etc., may be included.

[0115]

[0116] Example

[0117] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0118]

[0119] Example 1. scFv selection

[0120] 1-1. Production of SFTS NP virus antigen protein expression clone

[0121] To discover antibodies for the purpose of developing a diagnostic agent for SFTS virus, pcDNA3.1(-) was used as a vector for antigen protein production in mammalian cells. The base sequence of the SFTSV NP antigen gene of SFTS virus genotype B type was optimized for mammalian expression (codon optimized), and a leader sequence was added to the N-terminus for antigen protein extraction, and 6X His was added to the C-terminus for protein purification. After amplifying the SFTSV NP antigen gene by PCR, the SFTSV NP gene and pcDNA3.1(-) vector were digested with restriction enzymes AgeI and AfiII, and then ligated (Fig. 2).

[0122]

[0123] 1-2. Expression and purification of SFTS virus antigen NP protein

[0124] SFTSV NP antigen protein expression vector was transfected with 80 µg of the antigen expression vector into 6 × 106 Chinese hamster ovary (CHO) cells, and cultured for 10 days to collect the cell culture medium. The culture medium was centrifuged to collect the supernatant, and Ni-NTA beads were added, followed by rotation incubation overnight at 4°C. The cultured Ni-NTA beads were washed in the column with 10 mM, 20 mM, and 40 mM imidazole, and then eluted with 250 mM imidazole.

[0125] The eluted and fractionated antigen protein was run on an SDS-PAGE gel and confirmed by commassie blue staining (Fig. 3). Furthermore, Western blot analysis using an anti-His antibody confirmed the expression of a 28-kDa NP protein. The purified NP protein was buffer-changed to PBS to remove imidazole.

[0126] The amino acid sequence of the NP protein is represented by sequence number 55.

[0127]

[0128] 1-3. Rescue of Phage Library for Phage Display Screening

[0129] OPAL library of 4 strains was cultured in 2xYT / carbenicillin 50ug / ml / 2% glucose media, infected with helper phage at 20MOI, and cultured. Then, the phage library was precipitated using NaCl and PEG8000, and the titer was measured.

[0130]

[0131] 1-4. Screening of NP antigen-targeting scFv by biopanning of phage display library

[0132] To identify diagnostic antibody candidates that bind to the SFTSV NP antigen protein, three rounds of phage display library screening were performed. The NP antigen protein was coated onto an immunotube at 10 μg / ml overnight at 4°C, and the phage library was incubated with rotation at 4°C for 1 h. After incubation, the unbound phage library was washed, and the antigen-bound phages were eluted. After infection with the ER2738 strain, the titer was measured to be 1.78×10^7 cfu / ml (1st phage output) (Fig. 4).

[0133] The eluted phage was infected into the ER2738 strain, and after infection and culture with helper phage, the phage was amplified and the titer was measured (primary phage output amplification) (Fig. 4).

[0134] The primary output phage was incubated with rotation at 4°C for 1 hour in an immunotube coated with 10 μg / ml of NP protein. After washing the unbound phage library, the antigen-bound phage was eluted, and then infected with the ER2738 strain. The titer was measured to be 1×10^5 cfu / ml (secondary phage output) (Fig. 5).

[0135] The eluted phage was infected into the ER2738 strain, and after infection and culture with helper phage, the phage was amplified and the titer was measured (secondary phage output amplification) (Fig. 5).

[0136] The secondary output phage was incubated with rotation at 4°C for 1 hour in an immunotube coated with 1 μg / ml of NP protein. After washing the unbound phage library, the antigen-bound phage was eluted, and then infected with the ER2738 strain. The titer was measured to be 5×10^9 cfu / ml (tertiary output) (Fig. 6).

[0137]

[0138] 1-5. scFv induction and extraction

[0139] After the third output was infected with ER2738, it was spread on a LB plate containing 2xYT / carbenicillin 50ug / ml / 2% glucose and incubated overnight at 37℃. 600 colonies were picked and cultured in 600ul of 2xYT / carbenicillin 50ug / ml / 2% glucose. The cultured cells were treated with 0.1 mM IPTG and shaken overnight at 30℃. The cultured cells were centrifuged to remove the culture medium. The cells were lysed with Tris (pH 8.0) / EDTA (pH 8.0) / Sucrose) solution, centrifuged, and the supernatant was collected.

[0140]

[0141] 1-6. Verification of antigen binding of scFv by indirect ELISA

[0142] NP antigen was coated on an immunocoated plate at 1 μg / ml and incubated overnight at 4°C. The NP-coated immunoplate was washed three times with PBS-T (0.1% tween) and blocked with 3% skim milk at 37°C for 1 h. The blocked immunoplate was washed three times with PBS-T (0.1% tween), and the extracted scFv was added and incubated at 37°C for 1 h. The immunoplate was washed three times, and the secondary anti-HA antibody was added and incubated at 37°C for 1 h. The cultured immunoplate was washed three times, and the TMB substrate was added and reacted for 5 min. After the stop solution was added, the OD at 450 nm was measured using a microreader. The resulting OD value was calculated as the OD value of NP / OD value of BSA, and 200 scFv candidates with high binding were selected (Fig. 7).

[0143]

[0144] 1-7. Mini-prep and base sequence analysis for sequence analysis of the excavated scFv.

[0145] Phage that was confirmed to bind to the NP antigen protein by indirect ELISA was cultured, and phagemid DNA was mini-prepared. After analyzing the scFv base sequence of the phagemid DNA, the amino acid sequences of CDR1-3 of the heavy and light chains were analyzed, respectively, to identify a total of 20 scFv candidates (Fig. 8).

[0146]

[0147] 1-8. Concentration-dependent binding affinity of the scFv identified by indirect ELISA

[0148] After performing indirect ELISA by coating NP antigen in a concentration-dependent manner on the 20 scFvs discovered, the detection limit was confirmed, and 11 final scFv candidates were selected (Fig. 9).

[0149]

[0150] 1-9. IgG conversion of NP target scFv candidates

[0151] To convert scFv of 11 Capture and Detector antibody candidates for the development of a rapid antigen diagnostic kit into Mouse IgG and Human IgG forms, heavy chain and light chain genes were amplified by PCR, and the heavy chain or light chain was cloned into the Mouse IgG backbone pTGEX vector and the Human IgG backbone pcDNA vector, respectively.

[0152]

[0153] 1-10. Expression and purification of NP targeting antibodies

[0154] Mouse or human antibody expression vectors were transfected into 6×106 Chinese hamster overy (CHO) cells with 40 µg each of heavy chain and light chain, and cultured for 10 days to harvest the cell culture. The culture was centrifuged to collect the supernatant, and Protein A beads were added, followed by overnight rotation incubation at 4°C. The cultured beads were washed in a column with PBS and eluted with 0.1 M glycin-HCl.

[0155] The fractionated antibodies were run on an SDS PAGE gel and confirmed by commassie blue staining (Figs. 10 and 11). Purification of nine mouse IgG antibodies and eight human IgG antibodies was confirmed, and the purified antibodies were buffer-changed to PBS to remove glycine-HCl.

[0156] Example 2. Verification of concentration-dependent binding affinity of the discovered scFv by indirect ELISA

[0157]

[0158] The antibodies converted to IgG were tested for binding to the NP antigen by indirect ELISA. The NP antigen was coated at 1 μg / ml and incubated overnight at 4°C. After blocking with 3% skim milk, the plates were incubated with 1 μg / ml of IgG antibody at 37°C for 1 h. After washing three times with PBS, the plates were reacted with anti-secondary mouse-HRP or human IgG-HRP at 37°C for 1 h. After washing three times, the plates were reacted with TBS substrate for 5 min. After adding stop solution, the OD450 nm wavelength was measured. Binding was confirmed for eight antibodies targeting NP mouse IgG and five antibodies targeting human IgG (Figure 12). The sequences of the identified antibodies are presented in Tables 1 to 4.

[0159]

[0160]

[0161]

[0162]

[0163] Example 3. Verification of the antigen detection limit of the discovered antibody by indirect ELISA

[0164] NP antigen was diluted 10-fold from 1 pg / ml to 10 ug / ml in a concentration-dependent manner and coated on an immunoplate. The plate was then incubated overnight at 4°C. After blocking with 3% skim milk, the plate was incubated with 1 ug / ml of NP target human IgG or NP target mouse IgG antibody at 37°C for 1 h. After washing three times with PBS, the plate was reacted with anti-secondary mouse-HRP or human IgG-HRP at 37°C for 1 h, washed three times, reacted with TBS substrate for 5 minutes, and measured at OD450 nm wavelength after adding stop solution. In the case of NP target human IgG, the antigen detection limit of P01C09 antibody was confirmed to be 100 pg / ml to 1 ng / ml, and that of P01F07 antibody was confirmed to be 1 ng / ml to 10 ng / ml. P04D08, P01A05, and P01B10 antibodies detected concentrations ranging from 10 ng / ml to 100 ng / ml (Fig. 13). For NP target mouse IgG antibodies, the detection limits for P02E04 and P01F08 were 100 pg / ml to 1 ng / ml, for P01F07 antibody, 1 ng / ml to 10 ng / ml, and for P01A05, P01B10, P04D08, P05B01, and P05D07 antibodies, 10 ng / ml to 100 ng / ml (Fig. 14).

[0165]

[0166] Example 4. Verification of antigen binding affinity of the discovered antibody by indirect ELISA

[0167] NP antigen was diluted to 1 ug / ml and coated on an immunoplate, followed by overnight incubation at 4°C. After blocking with 3% skim milk, NP target human IgG antibody or NP target mouse IgG antibody was diluted concentration-dependently to 0.001 pM to 1 μM and incubated at 37°C for 1 h. After washing three times with PBS, the plate was reacted with anti-secondary mouse-HRP or human IgG-HRP at 37°C for 1 h, washed three times, reacted with TBS substrate for 5 minutes, and measured at OD450 nm wavelength after adding stop solution. NP target human IgG bound at a concentration of 1 pM to 100 pM (Fig. 15), and NP target mouse IgG bound at a concentration of 100 pM to 1 nM (Fig. 16).

[0168]

[0169] Example 5. Verification of cross-reactivity of discovered antibodies by indirect ELISA

[0170] To verify the cross-reactivity of the NP targeting antibody with other pseudoviruses, the NP antigen proteins of Rift Valley fever virus (RVFV) belonging to the Phenuiviridae family and Hantaan virus (HTNV) belonging to the Hantaviridae family, among viruses belonging to the Bunyavirale family, were coated on an immunoplate at 1 μg / ml and incubated overnight at 4°C. After blocking with 3% skim milk, the identified mouse or human IgG was incubated at a concentration of 1 μg / ml at 37°C for 1 h. After washing three times with PBS, the plate was reacted with anti-secondary mouse-HRP or human IgG-HRP at 37°C for 1 h, washed three times, reacted with TBS substrate for 5 min, and measured at OD450 nm after adding stop solution. The NP targeting identified antibody did not bind to RVFV or HTNV antibodies (Fig. 17).

[0171]

[0172] The antibody or antigen-binding fragment thereof according to the present invention exhibits the desired binding affinity to the nucleocapsid protein among the glycoproteins of the SFTS virus, and can be usefully used for the prevention, treatment, or diagnosis of the desired SFTS virus infection.

[0173]

[0174] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0175]

[0176] Electronic file attached.

Claims

1. A heavy chain variable region comprising a heavy chain CDR1 selected from the group consisting of sequences of SEQ ID NOs: 1 to 4, a heavy chain CDR2 selected from the group consisting of sequences of SEQ ID NOs: 5 to 11, and a heavy chain CDR3 selected from the group consisting of sequences of SEQ ID NOs: 12 to 19; and An antibody or an antigen-binding fragment thereof that binds to Severe Fever with Thrombocytopenia Syndrome (SFTS) virus, comprising a light chain variable region comprising a light chain CDR1 selected from the group consisting of sequences of SEQ ID NOs: 28 to 32, a light chain CDR2 selected from the group consisting of sequences of SEQ ID NOs: 33 to 39, and a light chain CDR3 selected from the group consisting of sequences of SEQ ID NOs: 40 to 45.

2. In the first paragraph, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof, characterized in that it specifically binds to the nucleocapsid protein among the glycoproteins of the SFTS virus.

3. An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region selected from the group consisting of sequences of SEQ ID NOs: 20 to 27 in the first paragraph.

4. An antibody or an antigen-binding fragment thereof comprising a light chain variable region selected from the group consisting of sequences of SEQ ID NOs: 47 to 54 in the first paragraph.

5. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

6. An expression vector containing the nucleic acid of clause 5.

7. Cells transformed with the expression vector of Article 6.

8. A method for producing an antibody or an antigen-binding fragment thereof that binds to a tau protein comprising the following steps: (a) a step of culturing the cell of clause 7 to produce an antibody or an antigen-binding fragment thereof that binds to tau protein; and (b) a step of recovering the generated antibody or antigen-binding fragment thereof.

9. A composition for preventing or treating SFTS virus infection, comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

10. A composition for diagnosing SFTS virus infection, comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

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