Antibodies specifically binding to WRS protein and uses thereof

Abstract

The present invention relates to an antibody that specifically binds to a tryptophanyl-tRNA synthetase (WRS) protein, and more specifically to an antibody or a fragment of the antibody that specifically binds to a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 in the WRS protein; a polynucleotide encoding the antibody and a vector comprising the polynucleotide; a cell transformed with the vector; and uses thereof.

Description

Technical Field

[0001] This application claims priority from Korean Patent Application No. 10-2019-0087233, filed on Jul. 18, 2019, which is hereby incorporated by reference in its entirety.

[0002] The present invention relates to antibodies that specifically bind to WRS (tryptophanyl-tRNA synthetase) proteins and uses thereof, and more specifically to antibodies or fragments thereof that specifically bind to a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 in WRS (tryptophanyl-tRNA synthetase) proteins, polynucleotides encoding the antibodies, vectors containing the polynucleotides, cells transformed with the vectors, and uses thereof. Background Art

[0003] Aminoacyl-tRNA synthetases (ARSs) are enzymes that attach specific amino acids to corresponding tRNAs. Higher organisms contain 23 enzymes, including 20 enzymes that are specific to amino acids and three other enzymes that participate in the formation of multisynthetase complexes, such as AIMP1 (p43), (AIMP2) p38, and (AIMP3) p18. In addition to the enzymes that participate in the multisynthetase complex, some enzymes exist in free form. However, recent reports indicate that ARSs, in addition to their essential functions, possess various other active functions in specific environments, and WRS (tryptophanyl-tRNA synthetase) is one of these.

[0004] WRS was first reported among the ARS secreted by cells and exhibited cytokine activity, and to date, many papers have been published on the potential of WRS as an important biomarker for various types of cancer, including colorectal cancer (Ghanipour A. et al. The prognostic significance of tryptophanyl-tRNA synthetase in colorectal cancer (2009) Cancer Epidemiol. Biomarkers Prev. 18 (11), 2949-2955). In addition, it is reported that the level of WRS can be used as a marker for rapid and accurate diagnosis of infectious diseases and their complications as follows: when an infectious disease caused by bacteria, virus or fungus occurs, the level of WRS in the body increases rapidly from the early stage of infection, and in particular, when suffering from infectious inflammatory diseases, the level of WRS is greatly increased compared with the level of normal people, and in the case of non-infectious inflammatory diseases, the level of WRS is not related to this (Korean Patent Application Publication No. 10-2017-0027313).

[0005] These results suggest that WRS can be present in the serum of patients with cancer and infectious diseases and that WRS can be used as an important diagnostic biomarker for these diseases.

[0006] However, despite the importance of ARSs, including WRSs, as biomarkers, ARSs share many similarities in protein structure, so antibodies obtained from animal immune responses show cross-reactivity, i.e., the ability to bind to other ARSs, and in many cases do not generate highly sensitive antibodies at all. Summary of the Invention

[0007] Technical issues

[0008] Therefore, the inventors conducted intensive research to develop antibodies that specifically bind to WRS, and found that antibodies that specifically bind to polypeptides including specific amino acid sequences in the WRS protein and have specific CDR (complementarity determining region) sequences exhibit very high binding specificity and binding affinity to WRS, and therefore have very high utility, which ultimately led to the present invention.

[0009] Therefore, an object of the present invention is to provide an antibody or a fragment thereof that specifically binds to a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 in a WRS (tryptophanyl-tRNA synthetase) protein.

[0010] Another object of the present invention is to provide a polynucleotide encoding the antibody or a fragment thereof, a vector comprising the polynucleotide, and a cell transformed with the vector.

[0011] Yet another object of the present invention is to provide a method for producing an antibody or a fragment thereof that binds to human WRS, the method comprising producing a polypeptide comprising light chain and heavy chain variable regions by culturing cells under conditions allowing the expression of a polynucleotide, and recovering the polypeptide from the cells or the culture medium in which the cells are cultured.

[0012] Yet another object of the present invention is to provide a composition for diagnosing cancer or infectious diseases or infectious complications, which comprises the antibody or fragment thereof.

[0013] In addition, yet another object of the present invention is to provide a composition for diagnosing cancer or infectious diseases or infectious complications, wherein the composition is composed of the antibody or a fragment thereof.

[0014] In addition, yet another object of the present invention is to provide a composition for diagnosing cancer or infectious diseases or infectious complications, wherein the composition essentially consists of the antibody or fragment thereof.

[0015] Yet another object of the present invention is to provide use of the antibody or fragment thereof in the manufacture of a medicament for diagnosing cancer.

[0016] Another object of the present invention is to provide a method for diagnosing cancer, comprising:

[0017] a) obtaining a sample from a subject;

[0018] b) measuring the expression level of WRS protein in the sample using the antibody or fragment thereof; and

[0019] c) determining that the subject has cancer when the protein expression level measured in step b) is increased.

[0020] Still another object of the present invention is to provide use of the antibody or fragment thereof in the manufacture of a medicament for diagnosing infectious diseases or infectious complications.

[0021] Yet another object of the present invention is to provide a method for diagnosing an infectious disease or an infectious complication, the method comprising:

[0022] a) obtaining a sample from a subject;

[0023] b) measuring the expression level of WRS protein in the sample using the antibody or fragment thereof; and

[0024] c) determining that the subject suffers from an infectious disease or an infectious complication when the protein expression level measured in step b) increases.

[0025] Technical Solution

[0026] To achieve the above object of the present invention, the present invention provides an antibody or a fragment thereof that specifically binds to a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 in a WRS (tryptophanyl-tRNA synthetase) protein.

[0027] To achieve another object of the present invention, the present invention provides a polynucleotide encoding the antibody or a fragment thereof, a vector comprising the polynucleotide, and a cell transformed with the vector.

[0028] To achieve still another object of the present invention, the present invention provides a method for producing an antibody or a fragment thereof that binds to human WRS, the method comprising producing a polypeptide comprising light and heavy chain variable regions by culturing cells under conditions allowing the expression of a polynucleotide, and recovering the polypeptide from the cells or the culture medium of the cells.

[0029] To achieve yet another object of the present invention, the present invention provides a composition for diagnosing cancer or infectious diseases or infectious complications, wherein the composition comprises the antibody or fragment thereof.

[0030] In addition, the present invention provides a composition for diagnosing cancer or infectious diseases or infectious complications, which is composed of the antibody or a fragment thereof.

[0031] The present invention provides a composition for diagnosing cancer or infectious diseases or infectious complications, wherein the composition is substantially composed of the antibody or a fragment thereof.

[0032] In order to achieve still another object of the present invention, the present invention provides use of the antibody or fragment thereof in the manufacture of a medicament for diagnosing cancer.

[0033] In order to achieve another object of the present invention, the present invention provides a method for diagnosing cancer, comprising:

[0034] a) obtaining a sample from a subject;

[0035] b) measuring the expression level of WRS protein in the sample using the antibody or fragment thereof; and

[0036] c) determining that the subject has cancer when the protein expression level measured in step b) is increased.

[0037] In order to achieve yet another object of the present invention, the present invention provides use of the antibody or fragment thereof in the manufacture of a medicament for diagnosing infectious diseases or infectious complications.

[0038] In order to achieve yet another object of the present invention, the present invention provides a method for diagnosing an infectious disease or an infectious complication, the method comprising:

[0039] a) obtaining a sample from a subject;

[0040] b) measuring the expression level of WRS protein in the sample using the antibody or fragment thereof; and

[0041] c) determining that the subject suffers from an infectious disease or an infectious complication when the protein expression level measured in step b) increases.

[0042] Hereinafter, a detailed description will be given of the present invention.

[0043] The present invention provides an antibody or a fragment thereof that specifically binds to a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 in a WRS (tryptophanyl-tRNA synthetase) protein.

[0044] In the present invention, term " WRS " refers to tryptophanyl-tRNA synthetase, also referred to as tryptophan-tRNA ligase, TrpRS, WARS etc.WRS is an enzyme that mediates the aminoacylation between amino acid tryptophan and tRNA.WRS is encoded by the WARS gene in the human body, and the amino acid sequence and mRNA nucleotide sequence of the protein are known to GenBank accession number NP_004175.2 (protein), GenBank accession number NM_004184.3 (mRNA nucleotide sequence) etc.WRS has two subtypes: cytoplasmic form (WARS or cytoplasmic tryptophanyl-tRNA synthetase) and mitochondrial form (WARS2 or mitochondrial tryptophanyl-tRNA synthetase).WRS in the present invention preferably adopts cytoplasmic form.

[0045] In the present invention, the term "antibody" refers to immunoglobulin (Ig), and is a general term for proteins that selectively act on antigens and participate in immunity in the body. Complete antibodies found in nature are generally composed of two pairs of light chains (LC) and one heavy chain (HC) (which are polypeptides composed of several domains), or these two pairs of HC / LC structures are composed as basic units. There are five types of heavy chains that make up mammalian antibodies, represented by the Greek letters α, δ, ε, γ and μ, and different types of antibodies are formed according to the different heavy chain types, such as IgA, IgD, IgE, IgG and IgM. There are two types of light chains that make up mammalian antibodies, represented by λ and κ.

[0046] Based on the variability of the amino acid sequence, the heavy and light chains of an antibody are structurally divided into a variable region and a constant region. The heavy chain constant region includes 3 or 4 heavy chain constant regions, namely CH1, CH2 and CH3 (IgA, IgD and IgG antibodies) and CH4 (IgE and IgM antibodies), depending on the antibody type, and the light chain includes CL as a constant region. The variable region of each heavy chain and light chain consists of a domain of the heavy chain variable region (VH) or the light chain variable region (VL). In each of the light and heavy chains, the variable region and the constant region are arranged side by side and connected by a covalent disulfide bond, and the heavy chains of the two molecules bound to the light chain are connected by two covalent disulfide bonds to form a complete antibody. The complete antibody specifically binds to the antigen through the variable regions of the heavy and light chains, and because the complete antibody includes two pairs of heavy and light chains (HC / LC), the complete antibody of one molecule has a bivalent monospecificity, that is, it can bind to the same two antigens through the two variable regions. The variable region of an antibody that binds to an antigen is called the antigen-binding site of the antibody, and the portion on the surface of the antigen recognized by the antibody is called the epitope.

[0047] The variable region of an antibody comprising an antigen-binding site is subdivided into a framework region (FR) with low sequence variability and a complementarity determining region (CDR) as a hypervariable region with high sequence variability. In each of VH and VL, three CDRs and four FRs are arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-terminus to C-terminus. The CDR with the highest sequence variability within the antibody variable region directly binds to the antigen and is the most important in determining the antigen specificity of the antibody.

[0048] In the present invention, the antibody or fragment thereof is an antibody or fragment thereof that specifically binds to the WRS protein or its variant protein, and specifically binds to a polypeptide comprising a sequence (SEQ ID NO: 2) of amino acids 48 to 104 of the WRS protein represented by SEQ ID NO: 1.

[0049] The "antibody" of the present invention may also be referred to as an "anti-WRS antibody," "humanized anti-WRS antibody," or "modified humanized anti-WRS antibody," and is used in the broadest sense throughout this specification. In particular, the antibodies include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., variable regions and other sites of antibodies that exhibit desired biological activity (e.g., binding to WRS)).

[0050] The antibodies of the present invention are antibodies comprising specific amino acid sequences in the light chain and heavy chain CDRs so that the antibodies can selectively bind to WRS, and include monoclonal antibodies and polyclonal antibodies, preferably monoclonal antibodies. In addition, the antibodies of the present invention include all of chimeric antibodies, humanized antibodies and human antibodies, and are preferably human antibodies.

[0051] The monoclonal antibodies of the present invention are antibodies obtained from a population of substantially homogeneous antibodies, wherein the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies bind very specifically to a single epitope.

[0052] In the present invention, the term "monoclonal" refers to the characteristic of the antibody obtained from a substantially homogeneous antibody population, and does not necessarily mean that the antibody must be produced by any ad hoc method. For example, the monoclonal antibody of the present invention can be produced by the hybridoma method (1975, Nature 256: 495) described first by the people such as Kohler or by recombinant DNA method (U.S. Patent number 4,816,567). It is also possible to use, for example, document (Clackson et al. (1991) Nature 352: 624-628, and Marks et al. (1991) J. Mol. Biol. 222: 581-597, and Presta (2005) J. Allergy Clin. Immunol. 116: 731) technology described in separating from phage antibody libraries.

[0053] The antibodies of the present invention specifically include chimeric antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class, while the remainder of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class, so long as the antibodies of the present invention exhibit the desired biological activity (e.g., selective binding to NRS) (U.S. Patent No. 4,816,567 and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).

[0054] Humanized antibodies are antibodies comprising human and non-human (e.g., mouse, rat) antibody sequences. Typically, except for the region (CDR) that binds to the epitope, the remainder belongs to human antibodies, and the region (CDR) that binds to the epitope can comprise sequences of non-human origin. Fully human antibodies are antibodies comprising only human immunoglobulin protein sequences, and can be produced from mice, mouse cells, or hybridomas derived from mouse cells, or can be produced by phage display methods.

[0055] The antibody or fragment thereof according to the present invention preferably comprises an antibody or fragment thereof comprising the following antibody light chain variable region (VL) and antibody heavy chain variable region (VH), wherein the antibody light chain variable region (VL) comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 3, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 4, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 5, and the antibody heavy chain variable region (VH) comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 6, a complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 7, and a complementarity determining region (CDR) H3 comprising the amino acid sequence represented by SEQ ID NO: 8.

[0056] In addition, the antibody or fragment thereof according to the present invention comprising the above-mentioned light chain and heavy chain CDRs may comprise a light chain variable region (VL) comprising the amino acid sequence represented by SEQ ID NO: 9 and a heavy chain variable region (VH) comprising the amino acid sequence represented by SEQ ID NO: 10.

[0057] The antibody or fragment thereof according to the present invention is not limited in its type, as long as it has the above-mentioned CDR, VH and VL, or light chain and heavy chain, and the antibody may be an IgG, IgA, IgM, IgE or IgD antibody. Preferably, the antibody is an IgG antibody.

[0058] In the present invention, the antibody fragment is an antibody fragment that retains WRS-specific binding affinity, and preferably, the fragment has at least 20%, 50%, 70%, 80%, 90%, 95%, 100% or higher affinity of the parent antibody for WRS protein. Specifically, the fragment can take the form of Fab, F(ab)2, Fab', F(ab')2, Fv, diabody, scFv, etc.

[0059] Fab (antigen binding fragment) is the antigen binding fragment of an antibody and includes a variable domain and a constant domain of each of the heavy and light chains. F(ab')2 is a fragment produced by hydrolyzing an antibody with pepsin and takes a form in which two Fabs are connected by a disulfide bond at the heavy chain hinge. F(ab') is a monomeric antibody fragment in which a heavy chain hinge is added to an isolated Fab by reducing the disulfide bonds of the F(ab')2 fragment. Fv (variable fragment) is an antibody fragment that only contains the variable regions of each of the heavy and light chains. ScFv (single-chain variable fragment) is a recombinant antibody fragment in which the heavy chain variable region (VH) and the light chain variable region (VL) are connected by a flexible peptide linker. Diabodies are a type of fragment in which the VH and VL of scFv are connected by a very short linker and cannot bind to each other, but form dimers by binding to the VL and VH of another scFv of the same type, respectively.

[0060] The antibodies or fragments thereof according to the present invention may include conservative amino acid substitutions that do not substantially alter their biological activity (referred to as conservative variants of the antibody).

[0061] In addition, the antibody or fragment thereof according to the present invention as described above can be conjugated with an enzyme, a fluorescent substance, a radioactive substance, a protein, etc., but the present invention is not limited thereto. In addition, methods for conjugating the above materials to antibodies are well known in the art.

[0062] The antibodies of the present invention can be derived from any animal, including mammals, including humans, birds, etc. Preferably, the antibodies are human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken antibodies, most preferably human or mouse antibodies.

[0063] Hybridoma cells can be produced using methods known in the art. Specifically, hybridoma cells can be produced by immunizing an animal with a polypeptide having the amino acid sequence of SEQ ID NO: 2 as an immunogen, fusing B cells, which are antibody-producing cells derived from the immunized animal, with myeloma cells to form hybridomas, and then selecting hybridomas that produce monoclonal antibodies that specifically bind to the polypeptide having the amino acid sequence of SEQ ID NO: 2. In addition to mice, animals to be immunized may also include animals such as goats, sheep, guinea pigs, rats, or rabbits.

[0064] The method for immunizing an animal to be immunized can be carried out by methods known in the art. For example, mice are immunized in the following manner: 1 to 100 μg of the immunogen is emulsified once with the same amount of saline and / or an antigen adjuvant such as Freund's adjuvant, and the immunogen is inoculated subcutaneously or intraperitoneally into the animal's abdomen 2-6 times every 2-5 weeks. After the animal is immunized, the spleen or lymph nodes are removed therefrom 3-5 days after the last immunization, and in the presence of a fusion promoter, the B cells contained in these tissues are fused with myeloma cells according to a cell fusion method known in the art. The example of the fusion promoter used can be materials such as polyethylene glycol (PEG). The example of myeloma cells can include cells derived from mice such as P3U1, NS-1, P3x63Ag8.653 and Sp2 / 0-Ag14, and cells derived from rats such as AG1 and AG2. In cell fusion methods known in the art, for example, B cells and myeloma cells are mixed at a ratio of 1:1-10:1, and PEG with a molecular weight of 1,000-6,000 is added thereto at a concentration of 10%-80%, followed by incubation at 30°C-37°C for 1-10 minutes. In addition, hybridomas that produce monoclonal antibodies that specifically bind to a polypeptide having the amino acid sequence of SEQ ID NO: 2 can be selected by culturing in a selective medium (such as HAT medium, etc.) in which only hybridoma cells can survive, and measuring the antibody activity in the hybridoma culture supernatant using a method such as ELISA. Finally, hybridomas that produce monoclonal antibodies that specifically bind to a polypeptide having the amino acid sequence of SEQ ID NO: 2 can be selected by repeatedly cloning hybridomas that produce monoclonal antibodies that specifically bind to a polypeptide having the amino acid sequence of SEQ ID NO: 2 by methods such as limiting dilution.

[0065] In addition, the present invention provides a polynucleotide encoding the antibody or a fragment thereof.

[0066] In the present invention, "polynucleotide" can be an oligonucleotide or a nucleic acid, and includes DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The polynucleotide can be single-stranded or double-stranded. The polynucleotide indicates a nucleotide sequence encoding an antibody consisting of a heavy chain and a light chain having a CDR configuration or a VH and VL configuration specific to the polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0067] The polynucleotides encoding the antibodies or fragments thereof according to the present invention can be obtained by methods well known in the art. For example, oligonucleotide synthesis techniques well known in the art, such as polymerase chain reaction (PCR) methods, can be used to synthesize based on DNA sequences encoding part or all of the heavy and light chains of the antibody or the corresponding amino acid sequences.

[0068] Furthermore, the present invention provides a vector comprising the polynucleotide.

[0069] The "vector" of the present invention is used for the purpose of replicating or expressing the polynucleotide of the present invention to recombinantly produce the antibody or fragment thereof according to the present invention, and generally includes at least one selected from the following: a signal sequence, an origin of replication, at least one marker gene, an enhancer element, a promoter, and a transcription termination sequence. The vector of the present invention is preferably an expression vector, and more preferably a vector comprising the polynucleotide of the present invention operably linked to a regulatory sequence, such as a promoter.

[0070] As a kind of plasmid of carrier, it is the linear or circular double-stranded DNA molecule that external polynucleotide fragment can be combined with it.Another kind of carrier is viral vector (for example replication defective retrovirus, adenovirus and adeno-associated virus), wherein other DNA fragmentation is introduced in viral genome.Some carrier can be autonomously replicated (for example bacterial vector and additional mammalian vector of bacterial origin) in the host cell that introduces them.Other carriers (for example, non-additional mammalian vector) are integrated in the genome of host cell by introducing host cell, and replicate thus together with host genome.

[0071] In the present invention, "vector" can be understood to have the same meaning as "expression vector", indicating a form of vector capable of expressing polynucleotides. When a regulatory sequence affects the expression of a polynucleotide sequence (e.g., the level, timing, or location of expression), the polynucleotide sequence is said to be "operably linked" to a regulatory sequence. A regulatory sequence is a sequence that affects the expression of a nucleic acid to which it is operably linked (e.g., the level, timing, or location of expression). For regulated nucleic acids, a regulatory sequence can produce an effect directly or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or the nucleic acid). Regulatory sequences include promoters, enhancers, and other expression control elements. The vector of the present invention preferably includes pOptiVEC TM -TOPO and pcDNA TM 3.3-TOPO.

[0072] Furthermore, the present invention provides cells transformed with the vector.

[0073] The cell of the present invention is not particularly limited in its type, as long as it can be used to express the polynucleotide encoding the antibody or its fragment contained in the expression vector of the present invention. Examples of cells (host cells) transformed with the expression vector according to the present invention may include prokaryotes (e.g., Escherichia coli), eukaryotes (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), and animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, insect cells, or hybridomas produced therefrom). Preferably, the cell is a cell derived from a mammal, including a human.

[0074] Suitable prokaryotes include gram-negative or gram-positive organisms, such as Enterobacteriaceae, including Escherichia (such as Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (such as Salmonella typhimurium), Serratia (such as Serratia marcescens), Shigella, Bacillus (such as Bacillus subtilis and Bacillus licheniformis), Pseudomonas (such as Pseudomonas aeruginosa), and Streptomyces. The cell of the present invention is not particularly limited, as long as it can express the vector of the present invention, but preferably Escherichia coli.

[0075] As an example of the cell of the present invention, the most common eukaryotic organism is Saccharomyces cerevisiae. However, many other genera, species, and strains may be used, examples of which include, but are not limited to, Schizosaccharomyces pombe, Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickerhamii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesei (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts (including A. nidulans and A. niger).

[0076] The term "transformation" refers to a change in the genotype of a host cell due to the introduction of an exogenous polynucleotide, and means the introduction of an exogenous polynucleotide into a host cell, regardless of the method used for transformation. An exogenous polynucleotide introduced into a host cell may be maintained after integration into the genome of the host cell, or may be maintained without integration, and the present invention encompasses both scenarios.

[0077] The recombinant expression vector capable of expressing the antibody or fragment thereof that specifically binds to the WRS protein according to the present invention can be introduced into cells for producing the antibody or fragment thereof by methods known in the art, thereby transforming the cells by methods known in the art, examples of which include but are not limited to transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun and other known methods for introducing nucleic acids into cells.

[0078] In addition, the cells of the present invention are cultured cells that can be transformed or transfected with a polynucleotide of the present invention or a vector comprising the polynucleotide, which can then be expressed in a host cell. A recombinant cell is a cell transformed or transfected with a polynucleotide to be expressed. The cells of the present invention can also be cells that comprise a polynucleotide of the present invention but in which the polynucleotide is not expressed to a desired level (unless a regulatory sequence is introduced into the cell such that the regulatory sequence is operably linked to the polynucleotide).

[0079] The cells of the present invention can be cultured in a variety of culture media. Commercially available culture media, such as Ham's F10 (Sigma-Aldrich Co., St. Louis, MO), Minimal Essential Medium (MEM, Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich Co.), are suitable for cell culture. If necessary, the culture medium can be supplemented with hormones and / or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements, and glucose or an equivalent energy source.

[0080] Furthermore, the present invention provides a method for producing an antibody or a fragment thereof that binds to WRS, the method comprising producing a polypeptide comprising light and heavy chain variable regions by culturing cells under conditions allowing expression of a polynucleotide, and recovering the polypeptide from the cells or a medium in which the cells are cultured.

[0081] The cell in the production method according to the present invention is as described above and comprises a polynucleotide encoding an antibody of the present invention. The polypeptide in the above-mentioned production method can be an antibody according to the present invention or a fragment thereof, or can be configured to comprise an antibody according to the present invention or a fragment thereof and another amino acid sequence.

[0082] Therefore, the antibodies or fragments thereof according to the present invention can be recovered using methods well known to those skilled in the art.For cultivation, the medium composition and culture conditions may vary depending on the type of cells and may be appropriately selected and controlled by those skilled in the art.

[0083] The antibody molecule can accumulate in the cytoplasm of the cell, can be secreted from the cell, or can be targeted to the periplasm or supernatant via an appropriate signal sequence, and is preferably targeted to the periplasm or supernatant. In addition, it is preferred that the antibody molecule produced be refolded and assembled into a functional conformation using methods well known to those skilled in the art. Depending on the properties of the polypeptide produced and the properties of the cell, the polypeptide can be recovered by various methods, which can be appropriately selected and controlled by those skilled in the art.

[0084] Polypeptide can be produced in cell or periplasmic space, or can be directly secreted in substratum.If polypeptide is produced in cell, then as the first step, can destroy cell thereby release protein.By for example centrifugal or ultrafiltration removal particulate debris, host cell or cracking fragment.When antibody is secreted in substratum, usually first use commercially available protein concentration filter (as Amicon or Millipore Pellicon ultrafiltration unit) to concentrate the supernatant from expression system.Can comprise protease inhibitor such as PMSF to suppress proteolysis in any preceding step, and can comprise antibiotic to prevent the growth of external pollutants.Can use for example hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography to carry out purification by the antibody produced by cell, and antibody of the present invention is preferably carried out purification by affinity chromatography.

[0085] Since the antibody or fragment thereof according to the present invention specifically binds to WRS, it can be used in diagnostic assays for detecting and quantifying WRS protein (eg, detecting WRS expression in certain cells, tissues or serum).

[0086] Therefore, the present invention provides a WRS-specific detection method, comprising contacting the antibody or fragment thereof with a sample, and detecting the antibody or fragment thereof. In order to "detect" the antibody or fragment thereof, the antibody or fragment thereof can generally be labeled with a detectable moiety.

[0087] For example, the technology described in document [Current Protocols in Immunology, Vol. 1 and 2, 1991, Coligen et al., ed. Wiley-Interscience, New York, New York, Pubs] can be used to carry out labeling with radioisotopes or fluorescent labels. Radioactivity can be measured by, for example, scintillation counting, and fluorescence can be quantified using a fluorometer. Alternatively, various enzyme-substrate labels are available, and the example of enzyme labeling includes luciferase (such as fruit fly luciferase and bacterial luciferase (U.S. Patent number 4,737,456)), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase (such as horseradish peroxidase (HRPO)), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase etc. Techniques for conjugating enzymes to antibodies are described, for example, by O'Sullivan et al. [1981, Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J. Langone and H. Van Vunakis, eds.), Academic press, New York, 73: 147-166].

[0088] The label can be indirectly conjugated to the antibody using various known techniques. For example, the antibody can be conjugated to biotin, and any label belonging to the above three categories can be conjugated to avidin, and vice versa. Biotin selectively binds to avidin, so the label can be conjugated to the antibody in an indirect manner. Alternatively, in order to achieve indirect conjugation of the label to the antibody, the antibody can be conjugated to a small hapten (e.g., digoxin), and any of the above different types of labels can be conjugated to an anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect conjugation of the label to the antibody can be achieved.

[0089] The antibodies or fragments thereof according to the present invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays and immunoprecipitation assays.

[0090] Antibody according to the present invention or its fragment can be used for diagnostic kit, i.e., for carrying out diagnostic assay, the diagnostic kit comprising a packaged combination of instructions for use and a predetermined amount of reagent. When the antibody is labeled with an enzyme, the kit may comprise substrate and cofactor required for the enzyme as a substrate precursor providing a chromophore or fluorophore. In addition, other additives may be included, such as stabilizers, buffer (such as blocking buffer or lysis buffer) etc. The relative amount of various reagents can vary widely, so as to provide a reagent solution concentration suitable for optimizing assay sensitivity. Reagent can be provided in the form of conventional lyophilized dry powder, which is included in a solution containing an excipient providing a reagent solution with appropriate concentration when dissolved.

[0091] WRS detected by the antibody of the present invention was first reported among cell-secreted ARS and exhibited cytokine activity, and to date, many papers have been published on the potential of WRS as an important biomarker in various types of cancer, including colorectal cancer (Ghanipour A. et al. The prognostic significance of tryptophanyl-tRNA synthetase in colorectal cancer (2009) Cancer Epidemiol Biomarkers Prev. 18 (11), 2949-2955).

[0092] Therefore, WRS can be detected and used as a diagnostic marker for diagnosing certain types of cancer, disease progression, and evaluating prognosis before and after treatment. Cancer diagnosis and prognosis evaluation according to the present invention can be performed by detecting WRS protein in biological samples.

[0093] Therefore, the present invention provides a composition for diagnosing cancer, comprising the antibody or fragment thereof according to the present invention as an active ingredient.

[0094] The type of cancer is not particularly limited, and examples thereof may include breast cancer, colorectal cancer, lung cancer, small cell lung cancer, bone cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestinal cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, CNS tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, and pituitary adenoma, and preferred examples of the type of cancer include colorectal cancer or pancreatic cancer.

[0095] It has also been reported that WRS expression levels increase rapidly from the initial stages of infection following bacterial, viral, or fungal infection, and that WRS levels are significantly increased compared to normal controls when symptoms such as pneumonia or sepsis develop as complications of infection. Furthermore, WRS expression levels in patients with sepsis are highly correlated with sepsis severity and prognosis. Since WRS levels increase only in cases of infectious inflammation, it can quickly and accurately distinguish infectious inflammatory diseases from non-infectious inflammatory diseases, making it highly valuable as a diagnostic marker for the treatment of emerging infectious diseases and infectious complications. In particular, WRS levels are significantly elevated in the serum of patients with sepsis or septic shock caused by bacterial or fungal infections compared to those in healthy controls. There is no statistically significant difference in the elevated WRS levels between patients with sepsis caused by Gram-negative bacteria, Gram-positive bacteria, or fungi. Therefore, WRS can be used to diagnose sepsis caused by all Gram-negative and Gram-positive bacteria, as well as fungal infections. In particular, it is known that there is no statistically significant difference in the serum levels of WRS in patients with autoimmune diseases such as systemic inflammatory response symptoms (SIRS), non-infectious chronic inflammatory diseases (such as asthma and rheumatoid arthritis) and Sjögren's syndrome compared with normal controls. Therefore, the expression level of WRS does not increase in all inflammatory reactions, but only specifically increases in inflammatory reactions induced by bacterial, viral or fungal infections. In addition, the WRS level of septic shock patients is more elevated than that of sepsis patients, so the expression level of WRS is also related to the severity of sepsis. It can be determined that the higher the expression level of WRS, the more severe the symptoms of sepsis (Korean Patent Application Publication No. 10-2017-0027313). By detecting the expression level of WRS in biological samples, infectious diseases or infectious complications can be diagnosed and their prognosis can be predicted.

[0096] Therefore, the present invention provides a composition for diagnosing infectious diseases or infectious complications, comprising the antibody or fragment thereof according to the present invention as an active ingredient.

[0097] The biological sample includes blood and other liquid samples of biological origin, biopsy samples, solid tissue samples (such as tissue culture), or cells derived therefrom. More specific examples may include, but are not limited to, tissues, extracts, cell lysates, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites, synovial fluid, peritoneal fluid, etc. The sample can be obtained from a subject. The subject includes animals, preferably mammals, and most preferably humans. The pretreatment of the sample can be carried out before being used for detection. Examples thereof may include filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc. In addition, nucleic acids and proteins can be separated from the sample and used for detection.

[0098] Detection was as described above.

[0099] In the present invention, infection means that one or more types of exogenous bacteria (all bacteria, including Gram-negative bacteria and Gram-positive bacteria), viruses and fungi enter the body and settle, reproduce and parasitize. Infectious diseases can be any disease caused by pathogen infection in a living body causing a reaction. The reaction of an infectious disease can include inflammation, pain, fever, fatigue, edema, hypotension, etc. Preferably, the infectious disease of the present invention includes salmonellosis, food poisoning, typhoid fever, paratyphoid fever, pneumonia, tuberculosis, tuberculosis, sepsis, septic shock, urinary tract infection, cystitis, pyelonephritis, urethritis, prostatitis, upper respiratory tract infection and otitis media, more preferably salmonellosis, food poisoning, pneumonia, sepsis and septic shock, and most preferably sepsis or septic shock.

[0100] In the present invention, sepsis is a systemic inflammatory response syndrome that occurs as a complication of an infectious disease. If the cause of sepsis cannot be diagnosed promptly and accurately at an early stage, sepsis is a fatal disease that progresses to severe sepsis or septic shock, multiple organ dysfunction syndrome (MODS) (which causes dysfunction of the lungs, kidneys, liver, circulatory system, etc.), disseminated intravascular coagulation syndrome (DIC), acute respiratory distress syndrome (ARDS), or acute renal failure (AKI), leading to death.

[0101] Sepsis as used herein includes, but is not limited to, sepsis associated with the final stages of sepsis, severe sepsis, septic shock, and sepsis complications such as multiple organ dysfunction syndrome (MODS), disseminated intravascular coagulation syndrome (DIC), acute respiratory distress syndrome (ARDS), or acute renal failure (AKI), and includes sepsis at any stage.

[0102] In addition, the present invention provides use of the antibody or fragment thereof in the manufacture of a medicament for diagnosing cancer.

[0103] Furthermore, the present invention provides a method for diagnosing cancer, comprising:

[0104] a) obtaining a sample from a subject;

[0105] b) measuring the expression level of WRS protein in the sample using the antibody or fragment thereof; and

[0106] c) determining that the subject has cancer when the protein expression level measured in step b) is increased.

[0107] In one embodiment, the present invention provides a method for diagnosing and treating cancer in a subject (to be tested), the method comprising:

[0108] i) obtaining a sample from a subject;

[0109] ii) measuring the expression level of the WRS protein in the sample;

[0110] iii) determining that the subject has cancer when the protein measured in step ii) is fully expressed; and iv) treating the cancer by administering a therapeutic drug (anticancer drug, etc.), radiotherapy, or surgery to the determined subject.

[0111] The method comprising steps i) to iv) should be understood based on the method comprising the above-mentioned steps a) to c).

[0112] Step iv) is treating the disease by administering a therapeutic drug (such as an anticancer drug), radiotherapy or surgery to the subject diagnosed with the disease in step iii).

[0113] In addition, the present invention provides use of the antibody or fragment thereof in the manufacture of a medicament for diagnosing infectious diseases or infectious complications.

[0114] In addition, the present invention provides a method for diagnosing an infectious disease or an infectious complication, the method comprising:

[0115] a) obtaining a sample from a subject;

[0116] b) measuring the expression level of WRS protein in the sample using the antibody or fragment thereof; and

[0117] c) determining that the subject suffers from an infectious disease or an infectious complication when the protein expression level measured in step b) increases.

[0118] In one embodiment, the present invention provides a method for diagnosing and treating an infectious disease or infectious complication in a subject (to be tested), the method comprising:

[0119] i) obtaining a sample from a subject;

[0120] ii) measuring the expression level of the WRS protein in the sample;

[0121] iii) determining that the subject suffers from an infectious disease or an infectious complication when the protein measured in step ii) is fully expressed; and

[0122] iv) treating an infectious disease or an infectious complication by administering to the identified subject a therapeutic drug or surgery for treating the infectious disease or an infectious complication.

[0123] The method comprising steps i) to iv) should be understood based on the method comprising the above-mentioned steps a) to c).

[0124] Step iv) is to treat the disease by administering a therapeutic drug, performing surgery, etc. to the subject diagnosed with the disease in step iii).

[0125] "Treatment" of the present invention generally refers to improving cancer or cancer symptoms, or infectious diseases or infectious complications or symptoms thereof, and may include eliminating, substantially preventing or improving the condition of the disease, as well as alleviating, eliminating or preventing one symptom or most symptoms caused by the disease, but the present invention is not limited thereto.

[0126] In the present invention, the terms "comprising" or "including" are used synonymously with "containing" or "characterized by" and mean that other components or method steps not mentioned are not excluded in the composition or method. The term "consisting of excludes other unmentioned elements, steps or ingredients. The term "consisting essentially of" means that the composition or method includes the described components or steps within its scope, as well as components or steps that do not materially affect its basic characteristics.

[0127] Beneficial effects

[0128] The antibodies or fragments thereof according to the present invention specifically bind to WRS and have no cross-reactivity with other proteins included in the same ARS family, and thus can detect and inhibit WRS. The antibodies or fragments thereof according to the present invention can be effectively used to detect WRS and diagnose WRS-related diseases, such as cancer, inflammatory diseases or infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 Shown are the amino acid sequences of the light chain variable region and the heavy chain variable region of the monoclonal antibody specifically binding to WRS selected in the present invention, and the nucleotide sequences encoding the amino acid sequences;

[0130] Figure 2 The figure shows the results of confirming the approximate molecular weight and band position by electrophoresis after constructing the WRS protein (1-471) represented by the amino acid sequence of SEQ ID NO: 1 and its fragment peptides (48-471, 1-104, 1-154, and 48-154);

[0131] Figure 3 The results of Western blotting detection of WRS protein (1-471) and its fragment peptides (48-471, 1-104, 1-154, and 48-154) using the antibodies of the present invention are shown to identify the polypeptide sequences in WRS specifically recognized by the monoclonal antibodies produced in the examples of the present invention;

[0132] Figure 4 Schematically shows the Figure 3The Western blot results confirmed in the experimental results show that the monoclonal antibodies produced in the examples of the present invention specifically recognize the polypeptides in WRS;

[0133] Figure 5 The results of comparing the WRS binding specificity of the monoclonal antibody produced in the embodiment of the present invention and two commercial antibodies are shown; and

[0134] Figure 6 The results of indirect ELISA assays regarding the cross-reactivity of the monoclonal antibodies produced in Examples of the present invention are shown. DETAILED DESCRIPTION

[0135] The present invention can be better understood by the following examples. These examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0136] Example 1: Production of monoclonal antibodies

[0137] (1) Production of hybridoma cells

[0138] 1) Animal immunization and cell fusion

[0139] - Preparation of immunogen: 1.5 to 2 mg WRS protein (purity > 75%, concentration > 0.4 mg / ml)

[0140] -Animal immunization: Antibody production was induced by inoculating the immunogen into Balb / c mice.

[0141] -Cell fusion: At least 10,000 hybridoma cells were obtained by electrofusion of mouse B cells and mouse myeloma cells.

[0142] 2) Selection of hybridoma cells

[0143] -Primary selection: Hybridoma cells producing antigen-binding antibodies are selected by indirect ELISA.

[0144] - Secondary selection: One hybridoma cell line that binds to the antigen was selected by Western blotting using the positive clones obtained in the primary selection, and the antibody produced from the selected hybridoma cell line was named 4D10G6.

[0145] - Typing: The five clones with the best results during the selection process are typed.

[0146] 3) Subcloning, cell expansion, cryopreservation, and antibody production

[0147] -Subcloning, cell expansion, and cryopreservation: clones with good results are subcloned, expanded, and cryopreserved.

[0148] - Antibody production: Antibodies were produced in an amount of at least 2 mg from the hybridoma cell line with the best results in the selection process.

[0149] 2. Formation of ascites

[0150] 1) After the mice have been acclimated for at least 3 days, pristane adjuvant is administered to the mice at a dose of 100 μl / mouse. Hybridoma cell lines are cultured so that they can be injected 5 to 7 days after administration of pristane adjuvant.

[0151] 2) The cultured hybridoma cell line was collected in a 50 ml tube, washed three times with 10 ml of PBS, and centrifuged.

[0152] 3) After centrifugation, the supernatant was removed by aspiration, and the number of cells required per 100 μl was calculated, 1X PBS was added, mixed well, and then transferred to a 1.5 ml tube.

[0153] 4) Place the above solution in a 1 ml syringe and remove the air from the syringe by turning the syringe needle upward.

[0154] 5) Each Balb / c mouse was intraperitoneally injected with 100 μl of the solution, and then the mouse was placed in a cage and observed for the presence of ascites.

[0155] 6) Starting from day 5 after the hybridoma cell lines were injected into the mice, abdominal distension was observed daily.

[0156] 7) When abdominal distension was noted, ascites was collected from the abdominal cavity of the mouse using a product with an injection needle of 23G or smaller (using a 3 ml or 5 ml syringe).

[0157] 8) The collected ascites in a tube was incubated at room temperature for 10 minutes to allow red blood cells to aggregate, followed by centrifugation.

[0158] 9) After centrifugation, place only the supernatant in a new 1.5 ml tube and store at -70°C.

[0159] 3. Antibody Production

[0160] 1) Take out the generated ascites at -70°C and thaw it at 4°C, and determine the type of beads to be used by confirming the subtype of the antibody to be purified. The amount of beads used is 0.5 times the volume of the ascites.

[0161] 2) Place the calculated amount of mixed protein A beads or G beads into a 5 ml chromatography column, and wash the beads by flowing 5 ml of 1X PBS into the column.

[0162] 3) After washing, the thawed ascites was placed in the column and the column was capped.

[0163] 4) The beads and the antibody were allowed to bind to each other by rotation at 4°C for 1 hour.

[0164] 5) After rotation and binding, the entire solution is subjected to a flow process.

[0165] 6) Wash the column with 100 ml of 1X PBS.

[0166] 7) 100 μl of neutralization buffer was added to a 1.5 ml tube, and 1 ml of IgG elution buffer was added to the column to enable neutralization immediately after elution of IgG. A total of 10 fractions were obtained under the same conditions.

[0167] 8) Load a portion of each fraction onto a 12% SDS-PAGE gel and confirm the bands by gel staining. During the staining process, store the fractions at 4°C.

[0168] 9) Collect the fractions with distinct bands, place them in dialysis tubing, and seal with a clamp to prevent leakage. Place the dialysis tubing and a stirring bar in a beaker containing 1 L of 1X PBS and dialyze at 4°C for 1 hour using a stirrer.

[0169] 10) Under the same conditions as in 9) above, dialyze overnight (15 hours) using 1 L of fresh 1X PBS.

[0170] 11) The next day, the solution was collected from the dialysis tubing and immediately quantified using a BCA assay kit.

[0171] Example 2: Sequencing

[0172] Total RNA was isolated from hybridoma cells according to the technical manual of the TRIzol reagent. Total RNA was reverse transcribed into cDNA using universal primers according to the technical manual of the PrimeScript 1st Strand cDNA Synthesis Kit. Antibody fragments of the heavy chain variable region (VH) and light chain variable region (VL) were amplified by RACD (rapid amplification of cDNA ends). The amplified antibody fragments were individually cloned into a standard cloning vector. Colony PCR was performed to screen for clones with inserts of the correct size. For each fragment, at least 5 colonies with inserts of the correct size were sequenced. The sequences of different clones were compared, and a consensus sequence for these clones was provided.

[0173] The amino acid sequences of the light and heavy chain variable regions of the sequenced monoclonal antibodies of the present invention and the polynucleotide sequences encoding the amino acid sequences are shown in Figure 1 middle.

[0174] Example 3: Identification of polypeptides in WRS specifically bound by monoclonal antibodies

[0175] To identify the polypeptide regions recognized by the monoclonal antibody generated in Example 1, the WRS protein (1 - 471) of SEQ ID NO:1 consisting of 471 amino acids and protein fragments (48 - 471, 1 - 104, 1 - 154, and 45 - 154) were prepared.

[0176] 1) To purify the recombinant WRS protein and its fragment peptides, the competent cells for protein expression were transformed with a plasmid in which the WRS protein and its fragment genes were cloned into the pET28a vector.

[0177] 2) The transformed cells were spread on LB (+ kanamycin) plates and then cultured at 37 °C for 15 hours.

[0178] 3) The next day, a single colony was inoculated into 3 ml of LB (+ Kan) and then cultured at 200 rpm and 37 °C for 3 hours.

[0179] 4) All the cells from the small cultures were placed into 500 ml of LB (+ Kan) and then cultured at 37 °C and 200 rpm for 4 hours.

[0180] 5) When the OD value was measured to be 0.8 < OD value < 1, 250 μl of 1 M IPTG stock solution (final 0.5 mM IPTG) was added thereto, and then induced overnight (15 hours) at 18 °C and 200 rpm.

[0181] 6) The next day, the induced cells were centrifuged at 4,000 rpm for 10 min.

[0182] 7) The supernatant was removed, and the pellet was suspended in 10 ml of washing buffer 1.

[0183] 8) The cells were lysed using an ultrasonic processor. Treated with 35% AMPL for 2 seconds and stored on ice for 1 min. This process was repeated 14 times (a total of 15 ultrasonic treatments).

[0184] 9) Centrifuged at 15,000 rpm and 4 °C for 30 min to separate the pellet and the supernatant from each other.

[0185] 10) 200 μl of Ni - NTA beads were placed into a poly - prep chromatography column, and 5 ml of washing buffer 1 was added to achieve equilibrium.

[0186] 11) After centrifugation, the supernatant was filtered through a 0.45 μm filter in a 50 ml tube and allowed to flow into the column containing the beads. This procedure was performed again.

[0187] 12) Washed with washing buffer 1.

[0188] 13) Washed with washing buffer 2.

[0189] 14) Wash with Wash Buffer 3.

[0190] 15) Wash with Wash Buffer 4.

[0191] 16) The washed column was placed in a 1.5 ml tube, and then the elution buffer was passed therethrough, and the eluate was collected therefrom.

[0192] 17) 5X sample buffer and DW were placed in a 5 ml tube and flow-through was performed, and wash buffer and eluate were added thereto, followed by boiling in a heating block for 5 min.

[0193] 18) Assemble the precast 15-well comb and 15% SDS-PAGE gel in a cassette, place the cassette in a well, and fill the gel and well with 1X running buffer.

[0194] 19) Load protein marker and samples sequentially.

[0195] 20) During gel loading, heat the dialysis tubing in a DW bath at 100°C for 10 min. Replace the DW with fresh DW and repeat the DW bath heating process twice more, followed by cooling with 200 ml of cold 1X PBS.

[0196] 21) After loading, separate the gel from the cassette and stain by pouring Quick Blue until the gel is submerged ( Figure 2 ).

[0197] Western blotting was performed according to a typical method using the WRS protein produced by the above method, its fragment, and the monoclonal antibody produced in Example 1 as primary antibodies.

[0198] The results are as follows Figure 3 As shown in , it was confirmed that the monoclonal antibody specifically recognized a fragment consisting of amino acids 48 to 104 among amino acids 1 to 471 of the WRS protein consisting of the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 2). Example 4: Antibody Binding Affinity Analysis

[0199] In order to evaluate the binding affinity of the monoclonal antibody generated in Example 1 and two commercial antibodies (Abnova, anti-WRS antibody (Cat. No. H00007453-M02) and Novus biological, anti-WRS antibody (Cat. No. NBP2-32186)) to the full-length WRS protein of SEQ ID NO: 1, an indirect ELISA assay was performed.

[0200] Briefly, the binding affinity of antibodies was evaluated according to the following method.

[0201] 1) WRS protein was diluted to 1 μg / ml in PBS, loaded into a 96-well plate at 100 μl / well, and reacted at room temperature for 1 hour, thereby coating the wells with the protein.

[0202] 2) After coating, the membrane was washed once with PBST (0.05% Tween-20) buffer, and 3% BSA and PBST (0.1% Tween-20) were dispensed, followed by blocking reaction at room temperature for 1 hour.

[0203] 3) The biotin-attached antibody was diluted with blocking buffer according to each concentration, and then reacted at room temperature for 1 hour.

[0204] 4) Wash with PBST (0.05% Tween-20).

[0205] 5) Streptavidin-HRP was diluted with blocking buffer, followed by reaction at room temperature for 1 hour.

[0206] 6) Wash five times with PBST (0.05% Tween-20) to remove any unattached residues.

[0207] 7) 50 μl / well of TMB was added thereto, followed by reaction at room temperature for 5 minutes, and then the same amount of 2M H 2 SO 4 was added to terminate the reaction.

[0208] 8) The absorbance was measured using a spectrophotometer (Sunrise, Tecan) (450 nm).

[0209] 9)EC 50 The value is calculated from the result of 8) above.

[0210] The results are shown in Table 1 below.

[0211] [Table 1]

[0212] 4D10G6 Abnova Novus <![CDATA[EC 50 ]]> 40.5 1655.6 532.8

[0213] As is apparent from Table 1, it was confirmed that the antibody according to the present invention exhibited a very high affinity to the WRS protein compared with the two commercial antibodies.

[0214] Example 5: Antibody Binding Specificity Analysis

[0215] To evaluate the binding specificity of the monoclonal antibody produced in Example 1 and two commercial antibodies (Abnova, anti-WRS antibody (Catalog No. H00007453-M02) and Novus biological, anti-WRS antibody (Catalog No. NBP2-32186)), 20 μg of HCT116 cell lysate was treated with each of the primary and secondary antibodies under the following conditions and subjected to Western blotting according to typical methods.

[0216] *Primary antibody (room temperature, 1 hour)

[0217] 4D10G6: 1 μg / ml

[0218] Abnova Ab: 1:5,000 dilution

[0219] Novus Ab: 1:10,000 dilution

[0220] *Secondary antibody (room temperature, 1 hour)

[0221] Anti-mouse HRP (Millipore, AP181P): 1:10,000 dilution: Abnova, 4D10G6

[0222] Anti-rabbit HRP (Millipore, AP187P): 1:10,000 dilution: Novus

[0223] The results are shown in Figure 5 middle.

[0224] like Figure 5 As shown in , it was confirmed that the antibodies according to the present invention all showed a single band, while several bands appeared in the two commercial antibodies.

[0225] Therefore, it was confirmed that the antibody according to the present invention exhibited very high binding specificity compared with commercial antibodies.

[0226] Example 6: Verification of cross-reactivity

[0227] In order to evaluate whether the monoclonal antibodies produced in Example 1 exhibit cross-reactivity with other ARS (aminoacyl-tRNA synthetase) proteins secreted by cells other than WRS, CRS (cysteinyl-tRNA synthetase), AIMP1 (aminoacyl-tRNA synthetase complex multifunctional interacting protein 1), GRS (glycyl-tRNA synthetase), and KRS (lysyl-tRNA synthetase), an indirect ELISA assay was performed according to the following method.

[0228] 1) Antigen coating: 1 μg / ml in PBS, 100 μl / well, 4°C, overnight coating

[0229] 2) Washing: 0.05% PBST (0.05% Tween 20), 200 μl / well, 3 times

[0230] 3) Blocking: 0.5% BSA in 0.05% PBST, 200 μl / well, RT, 1 hour

[0231] 4) Primary antibody binding: 500 ng / ml in 0.05% PBST, 100 μl / well, RT, 1 hour

[0232] 5) Secondary antibody binding: anti-mouse HRP (AP160P) 1:10,000 in 0.05% PBST, 100 μl / well, RT, 1 hour 6) TMB detection

[0233] 7) Reaction termination (2M H2SO4)

[0234] 8) Absorbance measurement: 450nm

[0235] The results are shown in Figure 6 middle.

[0236] like Figure 6 As shown in , it was confirmed that the antibody according to the present invention does not bind to ARS proteins other than WRS.

[0237] Practicality

[0238] The antibodies or fragments thereof according to the present invention specifically bind to WRS and have no cross-reactivity with other proteins included in the same ARS family, thereby being able to detect and inhibit WRS, and are therefore effectively used for detecting WRS and diagnosing WRS-related diseases (such as cancer, inflammatory diseases or infectious diseases), thereby exhibiting high industrial applicability. <110> JW Biosciences Co., Ltd. Therapy Co., Ltd. <120> Antibodies specifically binding to WRS protein and uses thereof <130> OP20-0032 / PCT <150> KR 10-2019-0087233 <151> 2019-07-18 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Full-length human tryptophanyl-tRNA synthetase (WRS) <400> 1 Met Pro Asn Ser Glu Pro Ala Ser Leu Leu Glu Leu Phe Asn Ser Ile 1 5 10 15 Ala Thr Gln Gly Glu Leu Val Arg Ser Leu Lys Ala Gly Asn Ala Ser 20 25 30 Lys Asp Glu Ile Asp Ser Ala Val Lys Met Leu Val Ser Leu Lys Met 35 40 45 Ser Tyr Lys Ala Ala Ala Gly Glu Asp Tyr Lys Ala Asp Cys Pro Pro 50 55 60 Gly Asn Pro Ala Pro Thr Ser Asn His Gly Pro Asp Ala Thr Glu Ala 65 70 75 80 Glu Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr Ser Ser Ala Lys 85 90 95 Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly Ser Ser Lys Ile 100 105 110 Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg Pro 115 120 125 His His Phe Leu Arg Arg Gly Ile Phe Phe Ser His Arg Asp Met Asn 130 135 140 Gln Val Leu Asp Ala Tyr Glu Asn Lys Lys Pro Phe Tyr Leu Tyr Thr 145 150 155 160 Gly Arg Gly Pro Ser Ser Glu Ala Met His Val Gly His Leu Ile Pro 165 170 175 Phe Ile Phe Thr Lys Trp Leu Gln Asp Val Phe Asn Val Pro Leu Val 180 185 190 Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu Trp Lys Asp Leu Thr Leu 195 200 205 Asp Gln Ala Tyr Ser Tyr Ala Val Glu Asn Ala Lys Asp Ile Ile Ala 210 215 220 Cys Gly Phe Asp Ile Asn Lys Thr Phe Ile Phe Ser Asp Leu Asp Tyr 225 230 235 240 Met Gly Met Ser Ser Gly Phe Tyr Lys Asn Val Val Lys Ile Gln Lys 245 250 255 His Val Thr Phe Asn Gln Val Lys Gly Ile Phe Gly Phe Thr Asp Ser 260 265 270 Asp Cys Ile Gly Lys Ile Ser Phe Pro Ala Ile Gln Ala Ala Pro Ser 275 280 285 Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg Asp Arg Thr Asp Ile Gln 290 295 300 Cys Leu Ile Pro Cys Ala Ile Asp Gln Asp Pro Tyr Phe Arg Met Thr 305 310 315 320 Arg Asp Val Ala Pro Arg Ile Gly Tyr Pro Lys Pro Ala Leu Leu His 325 330 335 Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala Gln Thr Lys Met Ser Ala 340 345 350 Ser Asp Pro Asn Ser Ser Ile Phe Leu Thr Asp Thr Ala Lys Gln Ile 355 360 365 Lys Thr Lys Val Asn Lys His Ala Phe Ser Gly Gly Arg Asp Thr Ile 370 375 380 Glu Glu His Arg Gln Phe Gly Gly Asn Cys Asp Val Asp Val Ser Phe 385 390 395 400 Met Tyr Leu Thr Phe Phe Leu Glu Asp Asp Asp Lys Leu Glu Gln Ile 405 410 415 Arg Lys Asp Tyr Thr Ser Gly Ala Met Leu Thr Gly Glu Leu Lys Lys 420 425 430 Ala Leu Ile Glu Val Leu Gln Pro Leu Ile Ala Glu His Gln Ala Arg 435 440 445 Arg Lys Glu Val Thr Asp Glu Ile Val Lys Glu Phe Met Thr Pro Arg 450 455 460 Lys Leu Ser Phe Asp Phe Gln 465 470 <210> 2 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment of human tryptophanyl-tRNA synthetase <400> 2 Met Ser Tyr Lys Ala Ala Ala Gly Glu Asp Tyr Lys Ala Asp Cys Pro 1 5 10 15 Pro Gly Asn Pro Ala Pro Thr Ser Asn His Gly Pro Asp Ala Thr Glu 20 25 30 Ala Glu Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr Ser Ser Ala 35 40 45 Lys Gly Ile Asp Tyr Asp Lys Leu Ile 50 55 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody light chain CDR1 <400> 3 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody light chain CDR2 <400> 4 Gly Thr Asn Asn Arg Ala Pro 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody light chain CDR3 <400> 5 Val Leu Trp Tyr Ser Asn His Trp Val 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody heavy chain CDR1 <400> 6 Asp Tyr Asn Met Asn 1 5 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody heavy chain CDR2 <400> 7 Val Ile Asn Pro Asn Tyr Gly Thr Ile Arg Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody heavy chain CDR3 <400> 8 Leu Leu Arg Gly Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 9 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody light chain variable region <400> 9 Met Ala Trp Ile Ser Leu Ile Leu Ser Leu Leu Ala Leu Ser Ser Gly 1 5 10 15 Ala Ile Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser 20 25 30 Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val 35 40 45 Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu 50 55 60 Phe Thr Gly Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile 85 90 95 Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Val Leu Trp 100 105 110 Tyr Ser Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 115 120 125 <210> 10 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of anti-WRS monoclonal antibody <400> 10 Met Gly Trp Ser Trp Ile Phe Leu Leu Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Ser Glu Phe Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe 35 40 45 Ile Asp Tyr Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu 50 55 60 Glu Trp Ile Gly Val Ile Asn Pro Asn Tyr Gly Thr Ile Arg Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Gly Leu Leu Arg Gly Tyr Tyr Ala Met Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135

Claims

1. An antibody or fragment thereof that specifically binds to a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 in a WRS (tryptophanyl-tRNA synthetase) protein, wherein the antibody comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein the antibody light chain variable region (VL) comprises a complementarity determining region (CDR) L1, a complementarity determining region (CDR) L2, and a complementarity determining region (CDR) L3, and the antibody heavy chain variable region (VH) comprises a complementarity determining region (CDR) H1, a complementarity determining region (CDR) H2, and a complementarity determining region (CDR) H3, wherein the amino acid sequence of the complementarity determining region (CDR) L1 is represented by SEQ ID NO: 3, the amino acid sequence of the complementarity determining region (CDR) L2 is represented by SEQ ID NO: 4, and the amino acid sequence of the complementarity determining region (CDR) L3 is represented by SEQ ID NO: 5; the amino acid sequence of the complementarity determining region (CDR) H1 is represented by SEQ ID NO: 6, the complementarity determining region (CDR) The amino acid sequence of H2 is represented by SEQ ID NO: 7, and the amino acid sequence of the complementarity determining region (CDR) H3 is represented by SEQ ID NO: 8; and The antibody fragment is selected from the group consisting of Fab, F(ab)2, Fab', F(ab')2, Fv and diabodies.

2. The antibody or fragment thereof according to claim 1, wherein the antibody fragment is a scFv.

3. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9 and a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:

10. The antibody or fragment thereof according to claim 1 , wherein the antibody is a monoclonal antibody.

5. The antibody or fragment thereof according to claim 1, wherein the antibody is selected from the group consisting of IgG, IgA, IgM, IgE and IgD. The antibody or fragment thereof according to claim 1 , wherein the antibody fragment is selected from the group consisting of Fab, Fab′, F(ab) 2 , F(ab′) 2 and Fv.

7. The antibody or fragment thereof according to claim 6, wherein the Fv is a scFv.

8. A polynucleotide encoding the antibody or fragment thereof according to any one of claims 1 to 7.

9. A vector comprising the polynucleotide according to claim 8.

10. A cell transformed with the vector according to claim 9.

11. A method for producing an antibody or fragment thereof that binds to WRS, the method comprising: producing a polypeptide comprising a light chain variable region and a heavy chain variable region by culturing the cell of claim 10 under conditions that allow expression of the polynucleotide; as well as The polypeptide is recovered from the cells or from the medium in which the cells are cultured. 12 . A composition for diagnosing cancer, comprising the antibody or fragment thereof according to claim 1 .

13. A composition for diagnosing an infectious disease or infectious complication, comprising the antibody or fragment thereof according to any one of claims 1 to 7.

14. Use of the antibody or fragment thereof according to any one of claims 1 to 7 in the preparation of a reagent for a method for diagnosing sepsis, the method comprising: a) obtaining a sample from a subject; b) measuring the expression level of WRS protein in the sample using the antibody or fragment thereof according to any one of claims 1 to 7; and c) determining that the subject has sepsis when the protein expression level measured in step b) is increased.

15. Use of the antibody or fragment thereof according to any one of claims 1 to 7 in the preparation of a reagent for a method for diagnosing septic shock, the method comprising: a) obtaining a sample from a subject; b) measuring the expression level of WRS protein in the sample using the antibody or fragment thereof according to any one of claims 1 to 7; and c) determining that the subject suffers from septic shock when the protein expression level measured in step b) is increased.

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