Antibodies specifically binding to wrs proteins and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] This application claims priority to Korean Patent Application No. 10-2019-0087230, filed on July 18, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to antibodies that specifically bind to a WRS (tryptophanyl-tRNA synthetase) protein and uses thereof, and more particularly to an antibody or 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, a polynucleotide encoding the same, a vector comprising the same, a cell transformed with the vector, and uses thereof. BACKGROUND

[0003] Aminoacyl-tRNA synthetase (ARS) is an enzyme that functions to attach a specific amino acid to a corresponding tRNA. Higher organisms contain 23 enzymes, including 20 enzymes according to the type of amino acid and 3 other types of enzymes involved in the formation of a multisynthetase complex, such as AIMP1 (p43), (AIMP2) p38, and (AIMP3) p18, and in addition to the enzymes involved in the multisynthetase complex, some enzymes exist in a free form. However, recently, it has been reported that ARS has various other active functions in addition to its basic function in a specific environment, and WRS (tryptophanyl-tRNA synthetase) is one of them.

[0004] WRS was first reported in ARS secreted from 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 has been reported that the level of WRS can be used as a marker for rapidly and accurately diagnosing infectious diseases and their complications in the following manner: when an infectious disease caused by a bacterial, viral, or fungal infection occurs, the level of WRS in the body rapidly increases from the early stage of infection, and in particular, when an infectious inflammatory disease is contracted, the level of WRS greatly increases compared to that of a normal person, and in the case of a non-infectious inflammatory disease, the WRS level is not related thereto (Korean Patent Application Publication No. 10-2017-0027313).

[0005] These results indicate that WRS can exist 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, although ARS including WRS is important as a biomarker, ARS has many similarities in terms of protein structure, and thus antibodies obtained from an animal immune response show cross-reactivity, i.e., are able to bind other ARS, and in many cases do not produce a highly sensitive antibody at all. SUMMARY

[0007] TECHNICAL PROBLEM

[0008] Therefore, the inventors conducted intensive research to develop an antibody that specifically binds to WRS, and found that an antibody that specifically binds to a polypeptide including a specific amino acid sequence in a WRS protein and has a specific CDR (complementary determining region) sequence exhibits very high binding specificity and binding affinity for WRS, and thus has very high utility, thereby ultimately leading to the present invention.

[0009] Therefore, an object of the present invention is to provide an antibody or 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 fragment thereof, a vector comprising the polynucleotide, and a cell transformed with the vector.

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

[0012] Yet another object of the present invention is to provide a composition for diagnosing cancer or an infectious disease or an infectious complication, the composition comprising the antibody or fragment thereof.

[0013] Further, yet another object of the present invention is to provide a composition for diagnosing cancer or an infectious disease or an infectious complication, the composition consisting of the antibody or fragment thereof.

[0014] Further, yet another object of the present invention is to provide a composition for diagnosing cancer or an infectious disease or an infectious complication, the composition consisting essentially of the antibody or fragment thereof.

[0015] Still 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] It is another object of the present application to provide a method of diagnosing cancer, the method 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 expression level of protein measured in step b) is increased.

[0020] It is still another object of the present application to provide use of the antibody or fragment thereof in the manufacture of a medicament for diagnosing an infectious disease or an infection complication.

[0021] It is yet another object of the present application to provide a method of diagnosing an infectious disease or an infection 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 has an infectious disease or an infection complication when the expression level of protein measured in step b) is increased.

[0025] Technical Solution

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

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

[0028] To achieve another object of the present application, the present application provides a method of producing an antibody or fragment thereof that binds to human WRS, the method comprising producing a polypeptide comprising a light chain and a heavy chain variable region by culturing a cell under conditions in which a polynucleotide is expressed, and recovering the polypeptide from the cell or a culture medium in which the cell is cultured.

[0029] To achieve another object of the present application, the present application provides a composition for diagnosing cancer or an infectious disease or an infection complication, the composition comprising the antibody or fragment thereof.

[0030] Further, the present application provides a composition for diagnosing cancer or an infectious disease or an infection complication, the composition consisting of the antibody or the fragment thereof.

[0031] Further, the present application provides a composition for diagnosing cancer or an infectious disease or an infection complication, the composition consisting essentially of the antibody or the fragment thereof.

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

[0033] To achieve another object of the present application, the present application provides a method of diagnosing cancer, the method comprising:

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

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

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

[0037] To achieve still another object of the present application, the present application provides use of the antibody or the fragment thereof in the manufacture of a medicament for diagnosing an infectious disease or an infection complication.

[0038] To achieve yet another object of the present application, the present application provides a method of diagnosing an infectious disease or an infection complication, the method comprising:

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

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

[0041] c) determining that the subject has an infectious disease or an infection complication when the protein expression level measured in step b) is increased.

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

[0043] The present application 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 application, the term "WRS" refers to tryptophanyl-tRNA synthetase, also known as tryptophan-tRNA ligase, TrpRS, WARS, etc. WRS is an enzyme that mediates aminoacylation between the 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 as GenBank Accession No. NP_004175.2 (protein), GenBank Accession No. NM_004184.3 (mRNA nucleotide sequence), etc. WRS has two subtypes: a cytoplasmic form (WARS or cytoplasmic tryptophanyl-tRNA synthetase) and a mitochondrial form (WARS2 or mitochondrial tryptophanyl-tRNA synthetase). The WRS in the present application preferably adopts the cytoplasmic form.

[0045] In the present application, the term "antibody" refers to immunoglobulin (Ig), and is a general term for proteins that selectively act on antigens and are involved in immunity in the body. An intact antibody found in nature is usually composed of two pairs of light chains (LC) and one heavy chain (HC) which are polypeptides composed of several domains, or the two pairs of HC / LC structures as a basic unit. There are five types of heavy chains that make up mammalian antibodies, represented by the Greek letters α, δ, ε, γ, and μ, and depending on the type of heavy chain, different types of antibodies are formed, 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] According to the variability of the amino acid sequence, the heavy and light chains of the 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, i.e., CH1, CH2, and CH3 (IgA, IgD, and IgG antibodies) and CH4 (IgE and IgM antibodies), depending on the type of antibody, and the light chain includes CL as one constant region. The variable region of each of the heavy and light chains is composed of one 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 one covalent disulfide bond, and the two molecules of the heavy chain combined with the light chain are connected by two covalent disulfide bonds to form one complete antibody. The complete antibody specifically binds to an antigen through the variable regions of the heavy and light chains, and since the complete antibody includes two pairs of heavy and light chains (HC / LC), one molecule of the complete antibody has bivalent monospecificity, i.e., is capable of binding to the same two antigens through two variable regions. The antibody variable region that binds to an antigen is called an antigen binding site of the antibody, and the portion on the surface of the antigen that is recognized by the antibody is called an epitope.

[0047] The variable region of an antibody comprising an antigen-binding site is subdivided into regions of framelike sequence variability, termed framework regions (FRs) and into regions of more hypervariability, termed complementarity determining regions (CDRs). In each of the VH and VL, the three CDRs are arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in the direction from the N-terminus to the C-terminus. The CDRs having the highest sequence variability within the antibody variable region directly bind to an antigen, and are most important in determining the antigen specificity of the antibody.

[0048] In the present application, the antibody or fragment thereof is an antibody or fragment thereof that specifically binds to a WRS protein or a variant protein thereof, and specifically binds to a polypeptide comprising a sequence of the 1stto 47thamino acids of the WRS protein represented by SEQ ID NO: 1 (SEQ ID NO: 2).

[0049] The "antibody" of the present application can also be referred to as an "anti-WRS antibody", a "humanized anti-WRS antibody", or a "modified humanized anti-WRS antibody", and is used in the broadest sense in the present specification. In particular, the antibody includes a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, a multispecific antibody (e.g., a bispecific antibody), and an antibody fragment (e.g., a variable region and other sites of an antibody that exhibit a desired biological activity (e.g., binding to WRS)).

[0050] The antibody of the present application is an antibody comprising specific amino acid sequences in the light chain and heavy chain CDRs so as to enable the antibody to selectively bind to WRS, and includes a monoclonal antibody and a polyclonal antibody, preferably a monoclonal antibody. Furthermore, the antibody of the present application includes all of a chimeric antibody, a humanized antibody, and a human antibody, and is preferably a human antibody.

[0051] The monoclonal antibody of the present application is an antibody obtained from a population of substantially homogeneous antibodies, wherein each antibody comprising the population is identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, binding a single epitope.

[0052] In the present application, the term "monoclonal" refers to the property of the antibody being obtained from a population of substantially homogeneous antibodies, and does not necessarily imply that the antibody must be produced by any particular method. For example, the monoclonal antibodies of the present application can be made by the hybridoma method first described by Kohler et al. (1975, Nature 256:495) or by recombinant DNA methods (U.S. Patent No. 4,816,567). The techniques described in the literature, such as 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, can also be used to isolate antibodies from phage antibody libraries.

[0053] The antibodies of the present application specifically include chimeric antibodies in which a portion of the heavy and / or light chain is identical with, or homologous to, a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class, while the remainder of the chain(s) is identical with, or homologous to, a corresponding sequence in an antibody derived from another species or belonging to another antibody class, so long as the antibodies of the present application exhibit the desired biological activity (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 that comprise sequences of both human and non-human (e.g., mouse, rat) antibodies. Typically, except for the regions that bind to the epitope (CDRs), the rest is of human antibodies, and the regions that bind to the epitope (CDRs) can comprise sequences of non-human origin. Fully human antibodies are antibodies that comprise 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] Hybridoma cells can be produced using methods known in the art. Specifically, the 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, and fusing B cells that are antibody-producing cells derived from the immunized animal with myeloma cells to form hybridomas, and then selecting hybridomas that produce a monoclonal antibody that specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 2 from among them. The animal to be immunized can include animals such as goats, sheep, guinea pigs, rats, or rabbits, in addition to mice.

[0056] The method of immunizing the immunized animal can be performed by a method known in the art. For example, mice are immunized by emulsifying 1 to 100 pg of the immunogen with the same amount of saline and / or an antigenic adjuvant such as Freund's adjuvant, and subcutaneously or intraperitoneally inoculating the immunogen to the abdomen of the animal 2 to 6 times every 2 to 5 weeks. After immunizing the animal, the spleen or lymph node is taken therefrom 3 to 5 days after the last immunization, and the B cells contained in these tissues are fused with myeloma cells in the presence of a fusion promoter according to a cell fusion method known in the art. An example of the fusion promoter used can be a material such as polyethylene glycol (PEG). Examples of the myeloma cells can include mouse-derived cells such as P3U1, NS-1, P3x63 Ag8.653, and Sp2 / 0-Ag14, and rat-derived cells such as AG1 and AG2. In the cell fusion method known in the art, for example, the B cells and the myeloma cells are mixed at a ratio of 1:1 to 10:1, and PEG having a molecular weight of 1,000 to 6,000 is added thereto at a concentration of 10% to 80%, followed by incubation at 30°C to 37°C for 1 to 10 minutes. Furthermore, the hybridoma producing the monoclonal antibody specifically binding to the polypeptide having the amino acid sequence of SEQ ID NO: 2 can be selected by culturing in a selective medium (e.g., HAT medium) in which only the hybridoma cells can survive, and measuring the antibody activity in the hybridoma culture supernatant using a method such as ELISA. Finally, the hybridoma producing the monoclonal antibody specifically binding to the polypeptide having the amino acid sequence of SEQ ID NO: 2 can be selected by repeatedly cloning the hybridoma producing the monoclonal antibody specifically binding to the polypeptide having the amino acid sequence of SEQ ID NO: 2 by a method such as limiting dilution.

[0057] For the monoclonal antibodies or fragments thereof provided in the present application, phage display technology can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable region gene repertoires from unimmunized donors (McCafferty et al., Nature 348:552-553 (1990)). According to this technique, antibody variable region genes are cloned palindromically into a phage (such as M13 or fd) major or minor coat protein and functional antibody fragments are displayed on the phage particle surface. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on antibody functional properties results in selection of genes encoding antibodies exhibiting these properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in various formats. In this regard, reference can be made to Johnson, Kevin S. and Chiswell, David J. [Current Opinions in Structural Biology 3:564-571 (1993)]. Several sources of variable region gene segments can be used for phage display. In Clackson et al. [Nature, 352:624-628 (1991)], a diverse array of anti-oxazone antibodies have been isolated from a small, randomly assembled library of variable region genes derived from the spleens of immunized mice. Libraries of variable region genes from unimmunized human donors can be constructed, and the techniques essentially described in the literature [e.g., Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993)] can be followed to isolate a diverse array of antibodies against antigens, including self-antigens [U.S. Patent Nos. 5,565,332 and 5,573,905].

[0058] The antibody or fragment thereof according to the present application is preferably selected from the group consisting of: (1) an antibody or fragment thereof comprising an antibody light chain variable region (VL) comprising a complementarity determining region (CDR) L1 comprising an amino acid sequence represented by SEQ ID NO: 5, a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 6, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 7, and an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 8, a complementarity determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 9, and a complementarity determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 10;

[0059] (2) an antibody or fragment thereof comprising an antibody light chain variable region (VL) comprising a complementarity determining region (CDR) LI comprising an amino acid sequence represented by SEQ ID NO: 13, a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 14, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 15, and an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) HI comprising an amino acid sequence represented by SEQ ID NO: 16, a complementarity determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 17, and a complementarity determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 18;

[0060] (3) an antibody or fragment thereof comprising an antibody light chain variable region (VL) comprising a complementarity determining region (CDR) LI comprising an amino acid sequence represented by SEQ ID NO: 21, a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 22, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 23, and an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) HI comprising an amino acid sequence represented by SEQ ID NO: 24, a complementarity determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 25, and a complementarity determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 26;

[0061] (4) an antibody or fragment thereof comprising an antibody light chain variable region (VL) comprising a complementarity determining region (CDR) LI comprising an amino acid sequence represented by SEQ ID NO: 29, a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 30, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 31, and an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) HI comprising an amino acid sequence represented by SEQ ID NO: 32, a complementarity determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 33, and a complementarity determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 34;

[0062] (5) an antibody or fragment thereof comprising an antibody light chain variable region (VL) comprising a complementarity determining region (CDR) L1 comprising an amino acid sequence represented by SEQ ID NO: 37, a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 38, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 39, and an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 40, a complementarity determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 41, and a complementarity determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 42; and

[0063] (6) an antibody or fragment thereof comprising an antibody light chain variable region (VL) comprising a complementarity determining region (CDR) L1 comprising an amino acid sequence represented by SEQ ID NO: 45, a complementarity determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 46, a complementarity determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 47, and an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 48, a complementarity determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 49, and a complementarity determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 50.

[0064] Further, the antibody or fragment thereof according to the present application comprising the CDRs of the light chain and the heavy chain described above is selected from:

[0065] (1) an antibody or fragment thereof comprising a light chain variable region (VL) comprising an amino acid sequence represented by SEQ ID NO: 3, and a heavy chain variable region (VH) comprising an amino acid sequence represented by SEQ ID NO: 4;

[0066] (2) an antibody or fragment thereof comprising a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12;

[0067] (3) an antibody or fragment thereof comprising a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 19 and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 20;

[0068] (4) an antibody or fragment thereof comprising a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 27 and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 28;

[0069] (5) an antibody or fragment thereof comprising a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 35 and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 36; and

[0070] (6) an antibody or fragment thereof comprising a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 43 and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 44.

[0071] The antibody or fragment thereof according to the present application is not limited in its type as long as it has the CDR, VH, and VL, or light chain and heavy chain described above, and the antibody can be an IgG, IgA, IgM, IgE, or IgD antibody. Preferably, the antibody is an IgG antibody.

[0072] In the present application, the fragment of the antibody is an antibody fragment that maintains the specific binding affinity of the WRS, and preferably, the fragment has an affinity of at least 20%, 50%, 70%, 80%, 90%, 95%, 100%, or more of the parent antibody to the WRS protein. Specifically, the fragment can take the form of, for example, Fab, F(ab)2, Fab', F(ab')2, Fv, diabody, scFv, etc.

[0073] Fab (antigen-binding fragment) is an antigen-binding fragment of an antibody, and includes one variable domain and one constant domain of each of a heavy chain and a light chain. F(ab')2 is a fragment produced by hydrolysis of an antibody with pepsin, and takes a form in which two Fab's are connected by a disulfide bond at the hinge of the heavy chain. F(ab') is a monomeric antibody fragment, which has a form in which the heavy chain hinge is added to a separated Fab by reducing the disulfide bond of the F(ab')2 fragment. Fv (fragment variable) is an antibody fragment including only the variable region of each of a heavy chain and a light chain. ScFv (single chain variable fragment) is a recombinant antibody fragment in which a variable region of a heavy chain (VH) and a variable region of a light chain (VL) are connected by a flexible peptide linker. A diabody is a fragment in a form in which VH and VL of an scFv are connected by a very short linker and cannot bind to each other, but form a dimer by binding to VL and VH of another scFv of the same type, respectively.

[0074] For the purpose of the present application, the fragment of the antibody is not limited in its structure or form as long as it maintains the binding specificity to the WRS, but is preferably an scFv. In the present application, the scFv has the above-described CDR configuration or VH and VL configuration specific to the WRS, and its sequence is not particularly limited as long as the C-terminal end of the VH and the N-terminal end of the VL are connected by a linker. The type of the linker is not particularly limited as long as it is a linker known in the art to be applied to an scFv.

[0075] The antibody or the fragment thereof according to the present application can include a conservative amino acid substitution which does not substantially change its biological activity (referred to as a conservative variant of the antibody).

[0076] In addition, the antibody or the fragment thereof according to the present application as described above can be conjugated with an enzyme, a fluorescent substance, a radioactive substance, a protein, etc., but the present application is not limited thereto. In addition, a method of conjugating the above-described materials with an antibody is well known in the art.

[0077] The antibody of the present application can be derived from any animal, including mammals, including humans, birds, etc. Preferably, the antibody is a human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken antibody, most preferably a human or mouse antibody.

[0078] In addition, the present application provides a polynucleotide encoding the antibody or the fragment thereof.

[0079] 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 specific to the polypeptide of SEQ ID NO: 2 or a VH and VL configuration.

[0080] The polynucleotide encoding the antibody or fragment thereof according to the present invention can be obtained by methods well known in the art. For example, it can be synthesized based on the DNA sequence or the corresponding amino acid sequence encoding part or all of the heavy and light chains of the antibody using oligonucleotide synthesis techniques well known in the art, such as polymerase chain reaction (PCR) methods, etc.

[0081] In addition, the present invention provides a vector comprising the polynucleotide.

[0082] The "vector" of the present invention is used for the purpose of replication or expression of 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 group consisting of 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.

[0083] A plasmid as a vector is a linear or circular double-stranded DNA molecule to which an external polynucleotide fragment can be bound. Another form of a vector is a viral vector (e.g., replication-defective retrovirus, adenovirus, and adeno-associated virus), in which an additional DNA fragment is introduced into a viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors of bacterial origin and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell by introduction into the host cell, and are thereby replicated along with the host genome.

[0084] In the present application, "vector" can be understood to have the same meaning as "expression vector", indicating the form of a vector capable of expressing a polynucleotide. When a regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of a polynucleotide sequence, the polynucleotide sequence is said to be "operably linked" to the regulatory sequence. A regulatory sequence is a sequence that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid with which it is operably linked. For a regulated nucleic acid, the regulatory sequence can exert its influence 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 vectors of the present application preferably include pOptiVEC TM - TOPO and pcDNA TM 3.3-TOPO.

[0085] In addition, the present application provides a cell transformed with the vector.

[0086] The cell of the present application is not particularly limited in its type, as long as it can be used to express the polynucleotide encoding the antibody or fragment thereof contained in the expression vector of the present application. Examples of the cell (host cell) transformed with the expression vector according to the present application can include prokaryotes (e.g., E. 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 derived therefrom). Preferably, the cell is a cell derived from a mammal including a human.

[0087] Suitable prokaryotes include Gram-negative or Gram-positive organisms, such as Enterobacteriaceae including Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., S. typhimurium), Serratia (e.g., S. marcescens), Shigella, Bacillus (e.g., B. subtilis and B. licheniformis), Pseudomonas (e.g., P. aeruginosa), and Streptomyces. The cell of the present application is not particularly limited, as long as it can express the vector of the present application, but is preferably E. coli.

[0088] The most common eukaryotic example as a cell of the present application is S. cerevisiae. However, many other genera, species, and strains can 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 183,070); Candida; Trichoderma reesei (EP 244,234); Neurospora crassa; Schwanniomyces, such as S. occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, including A. nidulans and A. niger.

[0089] The term "transformation" refers to a change in genotype of a host cell as a result of the introduction of an exogenous polynucleotide and indicates introduction of an exogenous polynucleotide into a host cell, regardless of the method used for transformation. The exogenous polynucleotide introduced into a host cell can be maintained on integration into the genome of the host cell, or can be maintained without integration, and the present application includes both cases.

[0090] A recombinant expression vector capable of expressing an antibody or a fragment thereof according to the present application that specifically binds to a WRS protein can be introduced into a cell for producing the antibody or the fragment thereof by a method known in the art, thereby transforming the cell by a method 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, a gene gun, and other known methods of introducing nucleic acids into cells.

[0091] Further, the cell of the present application is a cultured cell that can be transformed or transfected with a polynucleotide of the present application or a vector comprising the same, which can then be expressed in a host cell. The recombinant cell is a cell that is transformed or transfected with a polynucleotide to be expressed. The cell of the present application can also be a cell comprising a polynucleotide of the present application, 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.

[0092] The cell of the present application can be cultured in various media. Commercially available 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 Medium (DMEM, Sigma-Aldrich Co.) are suitable for cell culture. If necessary, the medium can be supplemented with hormones and / or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements, and glucose or an equivalent energy source.

[0093] Further, the present application provides a method of producing an antibody or a fragment thereof that binds to WRS, the method comprising producing a polypeptide comprising a light chain and a heavy chain variable region by culturing a cell under conditions in which a polynucleotide is expressed, and recovering the polypeptide from the cell or a medium in which the cell is cultured.

[0094] The cell in the production method according to the present application is as described above, and comprises a polynucleotide encoding an antibody of the present application. The polypeptide in the production method described above can be an antibody or a fragment thereof according to the present application, or can be configured to comprise an antibody or a fragment thereof according to the present application and an additional amino acid sequence.

[0095] Accordingly, the antibody or the fragment thereof according to the present application can be recovered using methods well known to those skilled in the art. For culturing, the medium composition and culture conditions can vary depending on the type of cell, and can be appropriately selected and controlled by those skilled in the art.

[0096] 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 by an appropriate signal sequence, and preferably to the periplasm or supernatant. In addition, it is preferred to refold and assemble the produced antibody molecule into a functional conformation using methods well known to those skilled in the art. Depending on the nature of the polypeptide produced and the nature of the cell, the polypeptide can be recovered by various methods, which can be appropriately selected and controlled by those skilled in the art.

[0097] The polypeptide can be produced in the cell or periplasmic space, or can be directly secreted into the culture medium. If the polypeptide is produced in the cell, as a first step, the cell can be disrupted to release the protein. Particulate debris, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. When the antibody is secreted in the medium, the supernatant from the expression system is typically first concentrated using a commercially available protein concentration filter (such as an Amicon or Millipore Pellicon ultrafiltration unit). Proteinase inhibitors, such as PMSF, can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants. The antibody produced by the cell can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and the antibody of the present application is preferably purified by affinity chromatography.

[0098] Since the antibody or fragment thereof according to the present application specifically binds to WRS, it can be used in diagnostic assays for detecting and quantifying WRS protein (e.g., detecting WRS expression in certain cells, tissues, or serum).

[0099] Accordingly, the present application provides a method for specifically detecting WRS, the method comprising contacting the antibody or fragment thereof with a sample, and detecting the antibody or fragment thereof. To "detect" the antibody or fragment thereof, the antibody or fragment thereof can typically be labeled with a detectable moiety.

[0100] For example, labeling with a radioisotope or a fluorescent label can be performed using the techniques described in the literature [Current Protocols in Immunology, Vols. 1 and 2, 1991, Coligen et al., Eds. Wiley-Interscience, New York, N.Y., Pubs]. 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 examples of enzymatic labels include luciferases (firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456)), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase (such as horseradish peroxidase (HRPO)), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described by, for example, 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, N.Y., 73: 147-166].

[0101] Various known techniques can be used to conjugate labels indirectly to antibodies. For example, an antibody can be conjugated to biotin, and any of the labels belonging to the three general classes described above can be conjugated to avidin, and vice versa. Biotin selectively binds avidin, and thus the label can be conjugated to the antibody in an indirect fashion. Alternatively, to achieve indirect conjugation of a label to an antibody, the antibody can be conjugated to a small hapten (such as digoxin), and any of the different types of labels described above can be conjugated to an anti-hapten antibody (such as an anti-digoxin antibody). In this way, indirect conjugation of a label to an antibody can be achieved.

[0102] The antibodies or fragments thereof according to the present application can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.

[0103] The antibody or fragment thereof according to the present application can be used in a diagnostic kit, i.e., a diagnostic kit for performing a diagnostic assay, which includes a package combination of instructions for use and a predetermined amount of reagents. When the antibody is labeled with an enzyme, the kit can include substrates and cofactors required for the enzyme as a substrate precursor to provide a chromophore or fluorophore. In addition, other additives such as stabilizers, buffers (e.g., blocking buffer or lysis buffer), etc. can be included. The relative amounts of the various reagents can vary widely in order to provide a reagent solution concentration suitable for optimizing the sensitivity of the assay. The reagents can be provided in the form of a dry powder, typically freeze-dried, which includes an excipient that, upon dissolution, will provide a reagent solution having the appropriate concentration.

[0104] WRS detected by the antibody of the present application was first reported in 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 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).

[0105] Accordingly, WRS can be detected and used as a diagnostic marker for diagnosing certain types of cancer, disease progression, and evaluation of prognosis before and after treatment. Cancer diagnosis and its prognosis evaluation according to the present application can be performed by detecting WRS protein in a biological sample.

[0106] Accordingly, the present application provides a composition for diagnosing cancer, which comprises the antibody or fragment thereof according to the present application as an active ingredient.

[0107] The type of cancer is not particularly limited, and examples thereof can 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, cutaneous 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 bowel 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 cancer, 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.

[0108] Meanwhile, it has been reported that the expression level of WRS rapidly increases from the early stage of infection after bacterial, viral, or fungal infection, and in addition, the WRS level is greatly increased when symptoms such as pneumonia or sepsis occur as complications of infection, compared to normal controls. In addition, in sepsis patients, the expression level of WRS has a high correlation with the severity and prognosis of sepsis, and since the WRS level increases only in the case of infectious inflammation, infectious inflammatory diseases and non-infectious inflammatory diseases can be quickly and accurately distinguished from each other, and thus have a high value as diagnostic markers for the treatment of new infectious diseases and infection complications. In particular, the WRS level in the serum of patients with sepsis or septic shock caused by bacterial or fungal infection is greatly increased compared to the serum of healthy normal controls, and there is no statistically significant difference in the WRS elevation trend in patients with sepsis caused by gram-negative bacterial, gram-positive bacterial, or fungal infection, and thus WRS can be used to diagnose sepsis caused by all gram-negative bacterial, gram-positive bacterial, and fungal infections. In particular, it is known that the WRS level in the serum of patients with autoimmune diseases such as systemic inflammatory response syndrome (SIRS), non-infectious chronic inflammatory diseases such as asthma and rheumatoid arthritis, and Sjogren's syndrome does not have a statistically significant difference compared to normal controls. Therefore, the expression level of WRS does not increase in all inflammatory responses, but only specifically increases in inflammatory responses induced by bacterial, viral, or fungal infection. In addition, the WRS level of septic shock patients is more elevated than the WRS level of sepsis patients, and thus 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 a biological sample, infectious diseases or infection complications can be diagnosed and their prognosis can be predicted.

[0109] Accordingly, the present application provides a composition for diagnosing infectious diseases or infection complications, the composition comprising the antibody or fragment thereof according to the present application as an active ingredient.

[0110] The biological sample includes blood and other liquid samples, biopsy samples, solid tissue samples (such as tissue cultures), or cells derived therefrom of biological origin. More specific examples can 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, and the like. The sample can be obtained from a subject. The subject includes animals, preferably mammals, most preferably humans. Pretreatment of the sample can be performed prior to detection. Examples thereof can include filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. In addition, nucleic acids and proteins can be isolated from the sample and used for detection.

[0111] detecting as described above.

[0112] In the present invention, infection means the entry and colonization, multiplication and parasitism of one or more types of exogenous bacteria (all bacteria, including gram-negative bacteria and gram-positive bacteria), viruses and fungi into the body. Infectious diseases can be any disease that occurs in a living body due to a reaction caused by infection of a pathogen. The reaction of infectious diseases can include inflammation, pain, fever, fatigue, edema, decreased blood pressure, etc. Preferably, the infectious diseases of the present invention include salmonellosis, food poisoning, typhoid fever, paratyphoid fever, pneumonia, pulmonary 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.

[0113] In the present invention, sepsis is a systemic inflammatory response syndrome that occurs as a complication of infectious diseases. In the case where the cause of sepsis cannot be diagnosed early in a timely and accurate manner, sepsis is a fatal disease that causes death due to progression 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 kidney injury (AKI).

[0114] Sepsis as used herein includes, but is not limited to, 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 kidney injury (AKI)) associated with the final stage of sepsis, and includes sepsis at any stage.

[0115] In addition, the present invention provides use of the antibody or fragment thereof in the manufacture of a medicament for diagnosing cancer.

[0116] In addition, the present invention provides a method of diagnosing cancer, the method comprising:

[0117] a) obtaining a sample from a subject;

[0118] b) measuring the expression level of WRS protein in the sample using the antibody or fragment thereof; and

[0119] c) determining that the subject has cancer when the protein expression level measured in step b) is increased.

[0120] In one embodiment, the present invention provides a method of diagnosing and treating cancer in a subject (to be tested), the method comprising:

[0121] i) obtaining a sample from a subject;

[0122] ii) measuring the expression level of the WRS protein in the sample;

[0123] iii) determining that the subject has cancer when the protein measured in step ii) is completely expressed; and

[0124] iv) treating cancer by administering to the determined subject a therapeutic drug (anti-cancer drug, etc.) for treating cancer, radiation therapy, or surgery.

[0125] The method comprising steps i) to iv) should be understood based on the method comprising steps a) to c) described above.

[0126] Step iv) is the treatment of a disease by administering to a subject diagnosed with the disease in step iii) a therapeutic drug (e.g., an anti-cancer drug), radiation therapy, or surgery.

[0127] Further, the present application provides use of the antibody or fragment thereof in the manufacture of a medicament for diagnosing an infectious disease or an infection complication.

[0128] Further, the present application provides a method of diagnosing an infectious disease or an infection complication, the method comprising:

[0129] a) obtaining a sample from a subject;

[0130] b) measuring the expression level of the WRS protein in the sample using the antibody or fragment thereof; and

[0131] c) determining that the subject has an infectious disease or an infection complication when the expression level of the protein measured in step b) is increased.

[0132] In one embodiment, the present application provides a method of diagnosing and treating an infectious disease or an infection complication in a subject (to be tested), the method comprising:

[0133] i) obtaining a sample from a subject;

[0134] ii) measuring the expression level of the WRS protein in the sample;

[0135] iii) determining that the subject has an infectious disease or an infection complication when the protein measured in step ii) is completely expressed; and

[0136] iv) treating an infectious disease or an infection complication by administering to the determined subject a therapeutic drug or surgery for treating an infectious disease or an infection complication.

[0137] The method including steps i) to iv) should be understood based on the method including steps a) to c) above.

[0138] Step iv) is to treat the disease by administering therapeutic drugs, surgery, etc., to the subject who was diagnosed with the disease in step iii).

[0139] The “treatment” of this invention generally refers to the improvement of cancer or cancer symptoms, or infectious diseases or infectious complications or their symptoms, and may include the elimination, substantial prevention or improvement of the condition of the disease and the reduction, elimination or prevention of one or most symptoms caused by the disease, but the invention is not limited thereto.

[0140] In this invention, the terms "comprising" or "including" are used synonymously with "containing" or "characterized in" and mean that other unmentioned constituent elements or method steps are not excluded from the composition or method. The term "consisting of" excludes other unmentioned elements, steps, or ingredients. The term "substantially consisting of" means that, within the scope of the composition or method, it includes the described constituent elements or steps, as well as constituent elements or steps that do not substantially affect its essential characteristics.

[0141] Beneficial effects

[0142] 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, thus enabling the detection and inhibition of 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. Attached Figure Description

[0143] Figures 1 to 6 The amino acid sequences of the light chain variable region and heavy chain variable region of the monoclonal antibody specifically binding to WRS selected in Examples 1 and 2 of the present invention are shown, along with the nucleotide sequences encoding the amino acid sequences.

[0144] Figure 7 The results show the approximate molecular weight and band positions confirmed by electrophoresis after constructing the WRS protein (1-471) and its fragment peptides (48-471, 1-104, 1-154 and 48-154) represented by the amino acid sequence of SEQ ID NO:1.

[0145] Figure 8 The results of Western blot analysis of WRS protein (1-471) and its fragment peptides (48-471, 1-104, 1-154 and 48-154) using each antibody are shown to identify polypeptide sequences in WRS specifically recognized by the six monoclonal antibodies generated in the embodiments of the present invention.

[0146] Figure 9 Western blot results confirmed in the experimental results based on Figure 8 polypeptides in WRS specifically recognized by each monoclonal antibody produced in the examples of the present application;

[0147] Figure 10 showing results comparing the WRS binding specificity of the six monoclonal antibodies produced in the examples of the present application to two commercial antibodies; and

[0148] Figure 11 showing indirect ELISA assay results regarding the cross-reactivity of the six monoclonal antibodies produced in the examples of the present application. DETAILED DESCRIPTION

[0149] The present application can be better understood by following the examples. These examples are intended to be illustrative only and should not be construed as limiting the scope of the present application.

[0150] Example 1: Production of monoclonal antibodies using hybridoma cells

[0151] (1) Production of hybridoma cells

[0152] 1) Animal immunization and cell fusion

[0153] - Preparation of immunogen: 1.5 to 2 mg of WRS protein (purity > 75%, concentration > 0.4 mg / ml)

[0154] - Animal immunization: antibody production was induced by inoculating the immunogen into Balb / c mice.

[0155] - Cell fusion: at least 10,000 hybridoma cells were obtained by electrofusion of mouse B cells and mouse myeloma cells.

[0156] 2) Selection of hybridoma cells

[0157] - Primary selection: hybridoma cells producing antigen-binding antibodies were selected by indirect ELISA.

[0158] - Secondary selection: three hybridoma cell lines binding to the antigen were selected by Western blot using positive clones obtained in the primary selection.

[0159] - Typing: five clones with the best results in the selection process were typed.

[0160] 3) Subcloning, cell expansion, cryopreservation and antibody production

[0161] - Subcloning, cell expansion, and cryopreservation: clones with good results were subcloned, expanded, and cryopreserved.

[0162] - Antibody production: Antibodies in an amount of at least 2 mg were produced from the hybridoma cell lines having the best results in the selection process.

[0163] 2. Ascites formation

[0164] 1) After the mice were adapted for at least 3 days, pristane adjuvant was administered to the mice in an amount of 100 μl / mouse. The hybridoma cell lines were cultured so that they could be injected 5 to 7 days after the administration of the pristane adjuvant.

[0165] 2) The cultured hybridoma cell lines were collected in 50 ml tubes, washed three times with 10 ml of PBS, and centrifuged.

[0166] 3) After centrifugation, the supernatant was removed, and then 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.

[0167] 4) The above solution was placed in a 1 ml syringe, and air in the syringe was removed by turning the syringe needle upward.

[0168] 5) Each Balb / c mouse was injected intraperitoneally with 100 μl of the solution, and then the mice were placed in cages and observed for ascites.

[0169] 6) From 5 days after the injection of the hybridoma cell lines into the mice, abdominal distension was observed every day.

[0170] 7) When abdominal distension was noted, ascites was collected from the abdominal cavity of the mice using a product having an injection needle of 23G or less (using a 3 ml or 5 ml syringe).

[0171] 8) The ascites collected and placed in a tube was incubated at room temperature for 10 min to allow red blood cells to aggregate, and then centrifuged.

[0172] 9) After centrifugation, only the supernatant was placed in a new 1.5 ml tube and stored at -70°C.

[0173] 3. Antibody production

[0174] 1) The ascites produced at -70°C was taken out and thawed at 4°C, and the type of beads to be used was determined by confirming the subtype of the antibody to be purified. The amount of beads used was 0.5 times the volume of the ascites.

[0175] 2) The mixed protein A beads or G beads were placed in a 5 ml chromatography column in the calculated amount, and bead washing was performed by flowing 5 ml of 1X PBS into the column.

[0176] 3) After the washing was completed, the thawed ascites was placed in the column, and the column was capped.

[0177] 4) The beads and antibodies were bound to each other by rotating the combination at 4°C for 1 hour.

[0178] 5) After the rotation combination, a flow process was performed on the entire solution.

[0179] 6) Column washing was performed using 100 ml of 1X PBS.

[0180] 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 immediate neutralization after IgG elution. Ten fractions were obtained under the same conditions.

[0181] 8) A portion of each fraction was loaded onto a 12% SDS-PAGE gel, and a band was confirmed by gel staining. The fractions were stored at 4°C during the staining process.

[0182] 9) The fraction having a clear band was collected, placed in a dialysis tube, and sealed with a clip to prevent leakage. The dialysis tube and stir bar were placed in a beaker containing 1 L of 1X PBS, and dialysis was performed at 4°C for 1 hour using a stirrer.

[0183] 10) Dialysis was performed overnight (15 hours) using 1 L of fresh 1X PBS under the same conditions as described in 9) above.

[0184] 11) The next day, the solution was collected from the dialysis tube and immediately quantified using a BCA assay kit.

[0185] According to the above-described method, three hybridoma cell lines producing monoclonal antibodies specifically binding to WRS were selected. The antibodies produced from the selected hybridoma cell lines were designated 3B10H5, 6A3B4, and 1D4C3.

[0186] Example 2: Selection of monoclonal antibodies by phage display

[0187] (1) ScFv phage display biopanning

[0188] 1) 1 ml of 1X PBS and 10 μg of WRS antigen were placed in an immunization tube and vortexed, followed by coating at 37°C and 200 rpm for 1 hour in a state sealed at the tube inlet with a tape.

[0189] 2) 50 μl of ER2537 E. coli cells were inoculated into 20 ml of SB medium, followed by incubation at 200 rpm and 37°C until OD600 = 0.5.

[0190] 3) Discard the coating solution, then wash once with tap water, then block with 5 ml of 3% skim milk for 1 hour at room temperature. Here, 600 1 of 3% skim milk is put into the Ag-coated tubes (400 1 per tube), then block for 1 hour at room temperature.

[0191] 4) After blocking, place the blocked phage into the Ag-coated tubes (leave about 5 μl for input testing), then incubate for 1 hour and 30 minutes in a shaking incubator at 150 rpm and 37°C.

[0192] 5) After phage binding, remove the solution from the Ag-coated tubes and wash twice with tap water. After this, wash twice with 0.05% PBST. Here, wash with 0.05% PBST by adding about 1 ml of PBST to the sample, vortexing, then adding an excess of PBST, after which the PBST is discarded (after the 2nd round, the washing is done five times).

[0193] 6) To elute the phage bound to the antigen, add 1 ml of 100 mM triethylamine (TEA solution) to it, then incubate for 8 min at room temperature.

[0194] 7) During the incubation, place 0.5 ml of 1 M Tris-HCl (pH 7.4) in a 50 ml tube, and after the incubation, add the eluted phage solution to it, mix by pipetting and neutralize.

[0195] 8) Add the 8.5 ml of E. coli cells incubated in 2) above to the neutralized phage, then infect for 1 hour at 120 rpm and 37°C (output).

[0196] 9) Centrifuge the output sample at 3,000 rpm and 4°C for 5 min, after which discard the supernatant and use 100 1 of SB medium to aspirate the pellet and spread it on a 150 mm Petri dish (LB agar plate, Amp+).

[0197] 10) Input and output titration

[0198] - Input titer: 2.32 μl of the library stock left from step 6 in 500 μl of SB → 2.32 μl in 500 μl of SB → 2.32 μl in 500 μl of SB → 1 μl in 100 μl of E. coli cells → 100 μl plated → colony count (n) the next day → input = n*10 -10

[0199] - Output titer: 1 μl of the output sample in 1 ml of SB (output 1) → 10 μl in 100 μl of SB (output 2) → all of output 1, 2 (n1, n2) plated in 100 μl → output 1 = n1*10-5 Output 2 = n2*10 -6

[0200] 11) The next day, 5 ml of SB medium was placed in a 150 mm output Petri dish, said output Petri dish being coated with the output sample and all the colonies were scraped with a spatula.

[0201] 12) Afterwards, 3 ml of the scraped bacterial solution was placed in a 15 ml tube, to which 1.5 ml of 50% glycerol (autoclaved) was added and mixed, and the resulting mixture was divided into 3 vials and stored as stock at -70°C.

[0202] 13) 50 μl of the panning output stock was placed in a 50 ml tube containing 20 ml of SB-ampicillin and grown at 200 rpm and 37°C until OD600 < 1.

[0203] 14) 1 ml of helper phage was added, followed by infection at 120 rpm and 37°C for 1 hour.

[0204] 15) Kanamycin was added at a final concentration of 70 μg / ml, followed by incubation at 200 rpm and 30°C overnight (15 hours).

[0205] 16) The next day, the overnight culture of the output phage solution was centrifuged at 4°C and 12,000 rpm for 20 min. The supernatant was placed in a 50 ml tube containing 5 ml of 5X PEG buffer, inverted and incubated on ice for 30 min.

[0206] 17) The incubated output phage solution was centrifuged at 4°C and 12,000 rpm for 20 min. The supernatant was discarded and the pellet was resuspended in 400 μl of PBS, transferred to a 1.5 ml microtube and centrifuged at 14,000 rpm and 4°C for 2 min. The supernatant was transferred to a new tube and used as the input library for the next round of panning.

[0207] 18) The stock of each round was made and stored at -70°C.

[0208] - From the 2nd round, the WRS antigen was used in reduced amounts.

[0209] - When the 2nd library was used, TG1 E. coli cells were used instead of ER2537 E. coli cells.

[0210] 2,112 candidates were selected from the phage library by biopanning according to the method described above.

[0211] (2) ELISA screening

[0212] 1) Take the enriched wheel stock at -70°C and thaw.

[0213] 2) To obtain single colonies, dilute the thawed stock 1 / 1,000 or 1 / 10,000, spread on 90 mm LB (+Amp) plates and incubate overnight (15 hours) in a 37°C incubator.

[0214] 3) The next day, add 200 μl SB (+Amp.) to each well of a 96 well cell culture plate (experimental plate) and 150 μl SB (+Amp.) to each well of a duplicate 96 well plate for storage.

[0215] 4) Store the duplicate plate at 4°C for a period of time and use a sterile toothpick to pick a single colony from the 90 mm LB plate and add to each well of the experimental plate.

[0216] 5) Place the experimental plate in a plate shaker and then incubate with shaking at a speed of 2.5 until at least 80% confluence is achieved.

[0217] 6) After removing the duplicate plate, use a 96 needle replicator to copy the cells growing on the experimental plate to the duplicate plate.

[0218] 7) Add IPTG to the experimental plate to a final concentration of 1 mM (11 μl of 1 M IPTG for 1 ml of SB (+Amp.)) and then incubate overnight (15 hours) at 30°C with the duplicate plate using a plate shaker at a speed of 2.

[0219] 8) The next day, add 75 μl of 50% glycerol to each well of the overnight incubated duplicate plate and store at -70°C. Remove the induced overnight experimental plate and centrifuge at 4°C and 3,500 rpm for 20 min.

[0220] 9) During centrifugation, coat a 96 well half plate with 4 μg / ml of the desired antigen for which the screen is being performed for 1 hour at 37°C. Use a plate that has not been coated with antigen as a negative control.

[0221] 10) Discard the supernatant from the centrifuged experimental plate and gently blot the plate with a paper towel, then add 60 μl of IX TES buffer to each well and then shake at 37°C for 20 min using a plate shaker at a speed of 4.

[0222] 11) Add an additional 90 μl of 0.2X TES buffer to each well of the experimental plate and then shake at 37°C for 5 min using a plate shaker at a speed of 2.5.

[0223] 12) Incubate the experimental plate on ice for 30 min or longer.

[0224] 13) Discard the solution from the antigen coated plate and wash the plate once with tap water and wipe with a paper towel.

[0225] 14) Add 130 μΐ of 3% skim milk to each well of the antigen coated plate and block for 1 hour at room temperature.

[0226] 15) About 20 min before the completion of the blocking, centrifuge the experimental plate incubated on ice at 3,500 rpm and 4°C for 20 min.

[0227] 16) Remove the blocking solution from the antigen coated plate and wash the plate once with tap water and wipe with a paper towel.

[0228] 17) In the experimental plate, add 25 μΐ of supernatant to each well of the antigen coated plate and negative control plate. Here, add in the same well position of both plates, followed by a 1 hour binding process at room temperature.

[0229] 18) During the binding process, prepare the secondary antibody (anti-HA HRP, 1 :2,000) in advance and store at 4°C.

[0230] 19) Discard the binding solution from the antigen coated plate and add 130 μΐ of 1X PBS-T to each well, then discard (washing process). This process is repeated 2 more times (total of 3 times). Finally, gently wipe using a dry paper towel.

[0231] 20) Add 100 μΐ of secondary antibody to each well, followed by a 1 hour binding process at room temperature.

[0232] 21) Repeat the washing process of 19) above.

[0233] 22) Add 50 μΐ of TMB solution to each well, followed by incubation at room temperature for up to 15 min, after which 50 μΐ of stop solution (H2SO4) is added to each well to stop the reaction, and the value is measured at OD 450 nm using an ELISA reader.

[0234] By the above method, 209 scFv candidates were again selected.

[0235] (3) Purification of scFv clones

[0236] 1) Take 5 μΐ of the selected monoclonal phage from the stock solution stored at -70°C, then place in a 14 ml tube containing 3 ml of SB.

[0237] 2) Incubate at 200 rpm and 37°C for 2 hours. After this, place all the cells in a flask containing 100 ml of SB and grow at 200 rpm and 37°C until OD600 < 1.

[0238] 3) Add 100 μΐ of 1 M IPTG to a final concentration of 1 mM, followed by induction at 200 rpm and 30°C overnight (15 hours).

[0239] 4) The next day, collect the cells in two 50 ml tubes and centrifuge at 3,500 rpm and 4°C for 15 min, after which discard the supernatant and use only the pellet.

[0240] 5) Resuspend the pellet in 3 ml of 1X TES, add 4.5 ml of 0.2X TES and incubate on ice for 30 min or more.

[0241] 6) Centrifuge at 12,000 rpm and 4°C for 30 min and filter the supernatant (crude extract) using a 0.45 μιη filter.

[0242] 7) Place 200 μΐ of Ni-NTA beads in a column and wash the beads using 5 ml of 1X PBS.

[0243] 8) Thereafter, filter the extract twice more, followed by binding to the Ni-NTA beads.

[0244] 9) After binding, wash using 30 ml of wash buffer (5 mM imidazole in PBS).

[0245] 10) Add 5 ml of elution buffer (200 mM imidazole in PBS), after which collect 1 ml of eluate in one 1.5 ml tube and 0.5 ml of eluate in four 1.5 ml tubes.

[0246] 11) Add 4 μΐ of 5X sample buffer to each of five new 1.5 ml tubes, add 16 μΐ of eluate to each and boil the tubes at 100°C for 7 min.

[0247] 12) Assemble a 10% SDS-PAGE gel in a cassette, place the cassette in a tank and fill the gel and tank with 1X running buffer.

[0248] 13) Load 5 μΐ of protein marker and 20 μΐ of the five samples in that order.

[0249] 14) Run the electrophoresis.

[0250] 15) After loading, detach the gel from the cassette and stain using instant blue staining solution until the gel is flooded.

[0251] 16) After staining, measure the thickness of the bands. Place the solution containing the banded fractions into a dialysis tube and dialyze with 1XPBS at 4°C for 1 hour.

[0252] 17) After 1 hour, replace with 1L of fresh 1X PBS, then dialyze overnight at 4°C (15 hours).

[0253] 18) On the second day, remove the sample, open the upper inlet of the dialysis tube, and harvest the sample.

[0254] 19) Use the BCA test for protein quantification.

[0255] 20) After quantification, the purified scFv was stored at -70℃.

[0256] After purification, human cell lysates were used, and based on these, three scFv clones were ultimately selected through Western blotting, immunoprecipitation, and IgG engineering.

[0257] Example 3: Sequencing

[0258] Total RNA was isolated from the hybridoma cells selected in Example 1 according to the TRIzol reagent technical manual. Total RNA was reverse transcribed into cDNA using universal primers according to the PrimeScript First-Strand cDNA Synthesis Kit technical manual. 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 standard cloning vectors. Colony PCR was performed to screen clones with inserts of the correct size. For each fragment, at least five colonies with inserts of the correct size were sequenced. Sequences of different clones were compared, and common sequences among these clones were identified.

[0259] After extracting plasmid DNA using the HiYield Plasmid Mini Kit (Real Biotech Corporation, YPD100) according to the manufacturer's instructions, each scFv monoclonal phage selected in Example 2 was sequenced.

[0260] The amino acid sequences of the light chain and heavy chain variable regions and the polynucleotide sequences encoding the amino acid sequences of the six monoclonal antibodies selected in Examples 1 and 2 according to the above method were identified and presented. Figures 1 to 6 middle( Figures 1 to 3 The monoclonal antibody selected according to the method in Example 1, and Figures 4 to 6 (The monoclonal antibody selected according to the method in Example 2).

[0261] Example 4: Identification of peptides in WRS specifically bound by monoclonal antibodies

[0262] To identify the polypeptide region recognized by the monoclonal antibodies produced in Examples 1 and 2, WRS proteins (1-471) and protein fragments (1-104, 1-154, 48-154, and 48-471) consisting of SEQ ID NO: 1 of 471 amino acids were prepared as follows.

[0263] 1) To purify the WRS fragment proteins, competent cells for protein expression were transformed with WRS fragment genes into plasmids in a pET28a vector.

[0264] 2) The transformed cells were spread on LB (+ kanamycin) plates and then incubated at 37°C for 15 hours.

[0265] 3) The next day, single colonies were inoculated into 3 ml of LB (+ Kan) and then incubated at 200 rpm and 37°C for 3 hours.

[0266] 4) All of the small culture cells were placed in 500 ml of LB (+ Kan) and then incubated at 37°C and 200 rpm for 4 hours.

[0267] 5) When the OD value was measured to be 0.8 < OD value < 1, 250 μl of 1M IPTG stock solution (final 0.5 mM IPTG) was added thereto and then induced at 18°C and 200 rpm overnight (15 hours).

[0268] 6) The next day, the induced cells were centrifuged at 4,000 rpm for 10 min.

[0269] 7) The supernatant was removed, and the precipitate was suspended in 10 ml of wash buffer 1.

[0270] 8) The cells were lysed using an ultrasonicator. Treatment with 35% AMPL was performed for 2 seconds and stored on ice for 1 min. This process was repeated 14 times (total of 15 times of ultrasonication).

[0271] 9) Centrifugation was performed at 15,000 rpm and 4°C for 30 min to separate the precipitate and the supernatant from each other.

[0272] 10) 200 μl of Ni-NTA beads were placed in a poly-prep chromatography column, and 5 ml of wash buffer 1 was added to reach equilibrium.

[0273] 11) After centrifugation, the supernatant was filtered in a 50 ml tube using a 0.45 μm filter and flowed into the column containing the beads. This procedure was performed again.

[0274] 12) Washing was performed using wash buffer 1.

[0275] 13) Washing was performed using wash buffer 2.

[0276] 14) Wash using Wash Buffer 3.

[0277] 15) Wash using Wash Buffer 4.

[0278] 16) Place the washed column in a 1.5 ml tube and then pass the Elution Buffer through it and collect the eluate therefrom.

[0279] 17) Place 5X Sample Buffer and DW in a 5 ml tube and perform a flow through process and add Wash Buffer and eluate thereto and then boil in a heating block for 5 min.

[0280] 18) Assemble a pre-made 15 well comb and 15% SDS-PAGE gel in a cassette, place the cassette in a tank and fill the gel and tank with IX Running Buffer.

[0281] 19) Load protein markers and samples in order.

[0282] 20) During the gel loading process, heat the dialysis tubing in a DW bath at 100°C for 10 min. Replace the DW with fresh DW and repeat the heating process in a DW bath two more times followed by cooling using 200 ml of cold IX PBS.

[0283] 21) After loading, separate the gel from the cassette and stain by pouring in blue stain until the gel is submerged. Figure 7 ).

[0284] Western blots were performed according to the typical method using the WRS protein produced by the above method, fragments thereof, and the six monoclonal antibodies produced in Examples 1 and 2 as primary antibodies.

[0285] The results, as shown in Figure 8 and Figure 9 , confirmed that the monoclonal antibodies specifically recognized a fragment consisting of the first to 47th amino acids in the first to 471st amino acids of the WRS protein consisting of the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 2).

[0286] Example 5: Antibody binding affinity analysis

[0287] In order to evaluate the binding affinity of the six monoclonal antibodies produced in Examples 1 and 2 and two commercial antibodies (Abnova, anti-WRS antibody (catalog number H00007453-M02) and Novus biological, anti-WRS antibody (catalog number NBP2-32186)) to the full-length WRS protein of SEQ ID NO: 1, an indirect ELISA assay was performed.

[0288] Briefly, the binding affinity of the antibodies was evaluated according to the following method.

[0289] 1) The WRS protein was diluted in PBS to 1 μg / ml, loaded in a 96-well plate in an amount of 100 μl / well and allowed to react for 1 hour at room temperature, whereby the wells were coated with the protein.

[0290] 2) After coating was complete, washing was performed once with PBST (0.05% Tween-20) buffer and 3% BSA and PBST (0.1% Tween-20) was dispensed, followed by a blocking reaction for 1 hour at room temperature.

[0291] 3) The biotin-attached antibodies were diluted according to each concentration with blocking buffer and then allowed to react for 1 hour at room temperature.

[0292] 4) Washing was performed with PBST (0.05% Tween-20).

[0293] 5) Streptavidin-HRP was diluted with blocking buffer and then allowed to react for 1 hour at room temperature.

[0294] 6) Washing was performed five times with PBST (0.05% Tween-20) to remove all unattached residues.

[0295] 7) 50 μl / well of TMB was added thereto and then allowed to react for 5 min at room temperature, after which the same amount of 2 M H2SO4 was added to stop the reaction.

[0296] 8) The absorbance was measured using a spectrophotometer (Sunrise, Tecan) at 450 nm.

[0297] 9) EC 50 values were calculated from the results of 8) above.

[0298] The results thereof are shown in Table 1 below.

[0299] [Table 1]

[0300] 3B6 4G4 4H9 6A 3B 4 1D 4C 3 3B 10H 5 Abnova Novus EC 50 ]]> 508.1 95.3 55.4 78.4 59.8 64.7 1655.6 532.8

[0301] As is evident from Table 1, it was confirmed that the six antibodies according to the present application exhibit very high affinity for the WRS protein compared to the two commercial antibodies.

[0302] Example 6: Analysis of antibody binding specificity

[0303] To evaluate the binding specificity of the six monoclonal antibodies produced in Examples 1 and 2 and two commercial antibodies (Abnova, anti-WRS antibody (catalog number H00007453-M02) and Novus biological, anti-WRS antibody (catalog number NBP2-32186)), 20 μg of HCT116 cell lysate was treated with each of the primary and secondary antibodies under the following conditions, and Western blotting was performed according to the typical method.

[0304] *Primary antibody (room temperature, 1 hour)

[0305] 3B6, 4G4, 4H9, 1D4C4, 3B10H5, 6A3B4: 1 μg / ml

[0306] Abnova Ab: 1:5,000 dilution

[0307] Novus Ab: 1:10,000 dilution

[0308] *Secondary antibody (room temperature, 1 hour)

[0309] Anti-human HRP (GenScript, A00166): 1:5,000 dilution: 4H9, 3B6, 4G4

[0310] Anti-mouse HRP (Millipore, AP181P): 1:10,000 dilution: Abnova, 1D4C3, 3B10H5, 6A3B4 Anti-rabbit HRP (Millipore, AP187P): 1:10,000 dilution: Novus

[0311] The results thereof are shown in Figure 10 .

[0312] As shown in Figure 10 , it was confirmed that all six antibodies according to the present application showed a single band, while several bands appeared in the two commercial antibodies.

[0313] Thus, it was confirmed that the antibodies according to the present application exhibit very high binding specificity compared to the commercial antibodies.

[0314] Example 7: Verification of cross-reactivity

[0315] To evaluate whether the six monoclonal antibodies produced in Examples 1 and 2 exhibit cross-reactivity with other ARS (aminoacyl-tRNA synthetase) proteins CRS (cysteinyl-tRNA synthetase), AIMP1 (aminoacyl tRNA synthetase complex multifunctional interacting protein 1), GRS (glycyl-tRNA synthetase), and KRS (lysyl-tRNA synthetase) secreted by cells other than WRS, indirect ELISA assays were performed according to the following method.

[0316] 1) Antigen coating: 1 μg / ml in PBS, 100 μl / well, coating at 4°C overnight

[0317] 2) Washing: 0.05% PBST (0.05% Tween 20), 200 μl / well, 3 times

[0318] 3) Blocking: 0.5% BSA in 0.05% PBST, 200 μl / well, RT, 1 hour

[0319] 4) Primary antibody binding: 500 ng / ml in 0.05% PBST, 100 μl / well, RT, 1 hour

[0320] 5) Secondary antibody binding: anti-mouse HRP (AP160P) 1:10,000 in 0.05% PBST, 100 μl / well, RT, 1 hour

[0321] 6) TMB detection

[0322] 7) Reaction termination (2 M H2SO4)

[0323] 8) Absorbance measurement: 450 nm

[0324] The results thereof are shown in Figure 11 .

[0325] As shown in Figure 11 , it was confirmed that the six antibodies according to the present application do not bind to ARS proteins other than WRS.

[0326] Utility

[0327] The antibody or fragment thereof according to the present application specifically binds to WRS and is not cross-reactive with other proteins included in the same ARS family, and thus can detect and inhibit WRS, and thus can be effectively used for detecting WRS and diagnosing WRS-related diseases (such as cancer, inflammatory diseases, or infectious diseases), thereby exhibiting very high industrial utility. <110> JW Biosciences Co., Ltd. Theracycle Co., Ltd. <120> Antibodies specifically binding to WRS protein and uses thereof <130> OP20-0031 / PCT <150> KR 10-2019-0087230 <151> 2019-07-18 <160> 50 <170> KoPatentIn 3.0 <210> 1 <211> 471 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> human tryptophanyl-tRNA synthetase (WRS) full length <400> 1 Met Pro Asn Ser Glu Pro Ala Ser Leu Leu Glu Leu Phe Asn Ser Ile 1 5 10 15 Ala Thr Gin 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 Gin 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 Gin Val Lys Gly lie Phe Gly Phe Thr Asp Ser 260 265 270 Asp Cys lie Gly Lys lie Ser Phe Pro Ala lie Gin Ala Ala Pro Ser 275 280 285 Phe Ser Asn Ser Phe Pro Gin lie Phe Arg Asp Arg Thr Asp lie Gin 290 295 300 Cys Leu lie Pro Cys Ala lie Asp Gin Asp Pro Tyr Phe Arg Met Thr 305 310 315 320 Arg Asp Val Ala Pro Arg lie Gly Tyr Pro Lys Pro Ala Leu Leu His 325 330 335 Ser Thr Phe Phe Pro Ala Leu Gin Gly Ala Gin Thr Lys Met Ser Ala 340 345 350 Ser Asp Pro Asn Ser Ser lie Phe Leu Thr Asp Thr Ala Lys Gin lie 355 360 365 Lys Thr Lys Val Asn Lys His Ala Phe Ser Gly Gly Arg Asp Thr lie 370 375 380 Glu Glu His Arg Gin 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 Gin lie 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> 47 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment of human tryptophanyl-tRNA synthetase <400> 2 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 35 40 45 <210> 3 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region of anti-WRS monoclonal antibody 3B10H5 <400> 3 Met Ser Ser Ala Gin Phe Leu Gly Leu Leu Leu Leu Cys Phe Gin Gly 1 5 10 15 Thr Arg Cys Asp He Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala 20 25 30 Ser Leu Gly Asp Arg Val Thr He Ser Cys Arg Ala Ser Gin Asp He 35 40 45 Ser Asn Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Thr Val Lys 50 55 60 Leu Leu He Ser Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr He Ser Asn 85 90 95 Leu Glu Gin Glu Asp He Ala Thr Tyr Phe Cys Gin Gin Gly Tyr Thr 100 105 110 Leu Pro His Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys 115 120 125 <210> 4 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody 3B10H5 heavy chain variable region <400> 4 Met Gly Phe Ser Arg lie Phe Leu Phe Leu Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Leu Val Arg 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Asn Leu His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu 50 55 60 Lys Trp lie Gly Ala lie Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Trp His Tyr Gly Ser Ser Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser 130 135 140 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of the light chain of the anti-WRS monoclonal antibody 3B10H5 <400> 5 Arg Ala Ser Gin Asp He Ser Asn Tyr Leu Asn 1 5 10 <210> 6 <211 > 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of the light chain of the anti-WRS monoclonal antibody 3B10H5 <400> 6 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 7 <211 > 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of the light chain of the anti-WRS monoclonal antibody 3B10H5 <400> 7 Gln Gin Gly Tyr Thr Leu Pro His Thr 1 5 <210> 8 <211 > 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of the heavy chain of the anti-WRS monoclonal antibody 3B10H5 <400> 8 Ser Tyr Asn Leu His 1 5 <210> 9 <211 > 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of heavy chain of anti-WRS monoclonal antibody 3B10H5 <400> 9 Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of heavy chain of anti-WRS monoclonal antibody 3B10H5 <400> 10 Trp His Tyr Gly Ser Ser Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 11 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region of anti-WRS monoclonal antibody 6A3B4 <400> 11 Met Glu Lys Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala 20 25 30 Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser 35 40 45 Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser 65 70 75 80 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser 85 90 95 Leu Asn Ile His Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys 100 105 110 Gln Gln Ser Lys Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Lys 130 <210> 12 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of anti-WRS monoclonal antibody 6A3B4 <400> 12 Met Arg Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Trp Met His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu 50 55 60 Glu Trp He Gly Arg He His Pro Ser Ala Ser Asp Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Asn Ala Asp Tyr Trp Gly Gin Gly Thr Thr Leu Thr 115 120 125 Val Ser Ser 130 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> CDR1 domain of the light chain of the anti-WRS monoclonal antibody 6A3B4 <400> 13 Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly He Ser Phe Met Asn 1 5 10 15 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> CDR2 domain of the light chain of the anti-WRS monoclonal antibody 6A3B4 <400> 14 Ala Ala Ser Asn Gin Gly Ser 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 domain of the light chain of the anti-WRS monoclonal antibody 6A3B4 <400> 15 Gln Gin Ser Lys Glu Val Pro Trp Thr 1 5 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 domain of the heavy chain of the anti-WRS monoclonal antibody 6A3B4 <400> 16 Asn Tyr Trp Met His 1 5 <210> 17 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 domain of the heavy chain of the anti-WRS monoclonal antibody 6A3B4 <400> 17 Arg lie His Pro Ser Ala Ser Asp Thr Asn Tyr Asn Gin Lys Phe Lys 1 5 10 15 Gly <210> 18 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of anti-WRS monoclonal antibody 6A3B4 <400> 18 Ala Asp Tyr 1 <210> 19 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region of anti-WRS monoclonal antibody 1D4C3 <400> 19 Met Ser Ser Ala Gin Phe Leu Gly Leu Leu Leu Leu Cys Phe Gin Gly 1 5 10 15 Thr Arg Cys Asp He Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala 20 25 30 Ser Leu Gly Asp Arg Val Thr He Ser Cys Arg Ala Ser Gin Asp He 35 40 45 Ser Asn Tyr Leu Asn Trp Phe Gin Gin Lys Pro Asp Gly Thr Val Lys 50 55 60 Leu Leu He Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr He Ser Asn 85 90 95 Leu Glu Gin Glu Asp Phe Ala Thr Tyr Phe Cys Gin Gin Gly Lys Thr 100 105 110 Leu Pro His Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 125 <210> 20 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of anti-WRS monoclonal antibody 1D4C3 <400> 20 Met Gly Phe Ser Arg Ile Phe Leu Phe Leu Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu 50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Ser Ser Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Gly Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Trp His Tyr Gly Ser Thr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 140 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> CDR1 of the light chain of the anti-WRS monoclonal antibody 1D4C3 <400> 21 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> CDR2 of the light chain of the anti-WRS monoclonal antibody 1D4C3 <400> 22 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> CDR3 of the light chain of the anti-WRS monoclonal antibody 1D4C3 <400> 23 Gln Gln Gly Lys Thr Leu Pro His Thr 1 5 <210> 24 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of heavy chain of anti-WRS monoclonal antibody 1D4C3 <400> 24 Ser Tyr Asn Met His 1 5 <210> 25 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of heavy chain of anti-WRS monoclonal antibody 1D4C3 <400> 25 Ala Ile Tyr Pro Gly Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of heavy chain of anti-WRS monoclonal antibody 1D4C3 <400> 26 Trp His Tyr Gly Ser Thr Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 27 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region of anti-WRS monoclonal antibody 3B6 <400> 27 Gln Ser Val Leu Thr Gin Pro Pro Ser Ala Ser Gly Thr Pro Gly Gin 1 5 10 15 Arg Val Thr lie Ser Cys Ser Gly Ser Ser Ser Asn lie Gly Asn Asn 20 25 30 Asn Val Ser Trp Tyr Gin Gin Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asn Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala lie Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ala Ser Leu 85 90 95 Ser Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 28 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of anti-WRS monoclonal antibody 3B6 <400> 28 Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala He Ser Tyr Asp Asn Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Ala Thr Thr Gin Pro His Thr Ser Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Asn Ser 115 120 <210> 29 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR1 of Anti-WRS Monoclonal Antibody 3B6 <400> 29 Ser Gly Ser Ser Ser Asn He Gly Asn Asn Asn Val Ser 1 5 10 <210> 30 <211> 4 <212> PRT <213> Artificial Sequence<213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody 3B6 heavy chain CDR2 <400> 30 Tyr Asn Ser His 1 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody 3B6 light chain CDR3 <400> 31 Gly Thr Trp Asp Ala Ser Leu Ser Ala 1 5 <210> 32 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody 3B6 heavy chain CDR1 <400> 32 Ser Tyr Ser Met Ser 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Anti-WRS monoclonal antibody 3B6 heavy chain CDR2 <400> 33 Ala Ile Ser Tyr Asp Asn Gly Asn Thr 1 5 <210> 34 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of heavy chain of anti-WRS monoclonal antibody 3B6 <400> 34 Lys Gly Ala Thr Thr Gin Pro His Thr Ser Phe Asp Tyr 1 5 10 <210> 35 <211> 111 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Variable region of light chain of anti-WRS monoclonal antibody 4G4 <400> 35 Gln Ser Val Leu Thr Gin Pro Pro Ser Ala Ser Gly Thr Pro Gly Gin 1 5 10 15 Arg Val Thr lie Ser Cys Ser Gly Ser Ser Ser Asn lie Gly Ser Asn 20 25 30 Ser Val Thr Trp Tyr Gin Gin Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr His Asp Ser His Pro Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala lie Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 36 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Variable region of heavy chain of anti-WRS monoclonal antibody 4G4 <400> 36 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Ser Pro Gly Asp Gly Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Thr Ile Pro Cys Arg Arg Thr Thr Cys Tyr Ser Ala Asn 100 105 110 Gly Met Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 37 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of light chain of anti-WRS monoclonal antibody 4G4 <400> 37 Ser Gly Ser Ser Ser Asn He Gly Ser Asn Ser Val Thr 1 5 10 <210> 38 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of light chain of anti-WRS monoclonal antibody 4G4 <400> 38 His Asp Ser His 1 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of light chain of anti-WRS monoclonal antibody 4G4 <400> 39 Ala Ala Trp Asp Asp Ser Leu Ser Gly 1 5 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of heavy chain of anti-WRS monoclonal antibody 4G4 <400> 40 Asn Tyr Ala Met Ser 1 5 <210> 41 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of the heavy chain of the anti-WRS monoclonal antibody 4G4 <400> 41 Trp lie Ser Pro Gly Asp Gly Asn Lys 1 5 <210> 42 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of the heavy chain of the anti-WRS monoclonal antibody 4G4 <400> 42 Arg Val Thr lie Pro Cys Arg Arg Thr Thr Cys Tyr Ser Ala Asn Gly 1 5 10 15 Met Asp Val <210> 43 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Variable region of the light chain of the anti-WRS monoclonal antibody 4H9 <400> 43 Gln Ser Val Leu Thr Gin Pro Pro Ser Ala Ser Gly Thr Pro Gly Gin 1 5 10 15 Arg Val Thr lie Ser Cys Ser Gly Ser Ser Ser Asn lie Gly Ser Asn 20 25 30 Asp Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 44 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Variable region of heavy chain of anti-WRS monoclonal antibody 4H9 <400> 44 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser lie Tyr Pro Asp Gly Gly Gly lie Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Pro Phe Gly Pro Asp Thr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 45 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR1 domain of the light chain of the anti-WRS monoclonal antibody 4H9 <400> 45 Ser Gly Ser Ser Ser Asn lie Gly Ser Asn Asp Val Thr 1 5 10 <210> 46 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> CDR2 domain of the light chain of the anti-WRS monoclonal antibody 4H9 <400> 46 Asp Asn Ser Lys 1 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of light chain of anti-WRS monoclonal antibody 4H9 <400> 47 Gly Ala Trp Asp Asp Ser Leu Ser Gly 1 5 <210> 48 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of heavy chain of anti-WRS monoclonal antibody 4H9 <400> 48 Asn Tyr Ala Met Ser 1 5 <210> 49 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of heavy chain of anti-WRS monoclonal antibody 4H9 <400> 49 Ser Ile Tyr Pro Asp Gly Gly Gly Ile 1 5 <210> 50 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of heavy chain of anti-WRS monoclonal antibody 4H9 <400> 50 Arg Asp Leu Tyr Pro Phe Gly Pro Asp Thr Phe Asp Tyr 1 5 10

Claims

1. An antibody or fragment thereof that specifically binds to a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 in a WRS (tryptophanyl-tRNA synthetase) protein, wherein the antibody or fragment thereof 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 of the amino acid sequence represented by SEQ ID NO: 13, a complementarity-determining region (CDR) L2 of the amino acid sequence represented by SEQ ID NO: 14, and a complementarity-determining region (CDR) L3 of the amino acid sequence represented by SEQ ID NO: 15, and wherein the antibody heavy chain variable region (VH) comprises a complementarity-determining region (CDR) H1 of the amino acid sequence represented by SEQ ID NO: 16, a complementarity-determining region (CDR) H2 of the amino acid sequence represented by SEQ ID NO: 17, and a complementarity-determining region (CDR) H3 of the amino acid sequence represented by SEQ ID NO:

18.

2. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof is an antibody or fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is represented by SEQ ID NO: 11 and the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO:

12.

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

4. The antibody or a fragment thereof according to claim 1, wherein the antibody is selected from IgG, IgA, IgM, IgE and IgD.

5. The antibody or a fragment thereof according to claim 1, wherein the fragment of the antibody is selected from biantibody, Fab, Fab', F(ab)2, F(ab')2, Fv and scFv.

6. A polynucleotide encoding an antibody or a fragment thereof according to any one of claims 1 to 5.

7. A vector comprising the polynucleotide according to claim 6.

8. A cell transformed using the vector according to claim 7.

9. A method for generating an antibody or fragment thereof that binds to WRS, the method comprising: A polypeptide comprising a light chain variable region and a heavy chain variable region is produced by culturing cells according to claim 8 under conditions that enable polynucleotide expression; as well as The polypeptide is recovered from the cells or the culture medium in which the cells are cultured.

10. A composition for diagnosing cancer, said composition comprising an antibody or a fragment thereof according to any one of claims 1 to 5.

11. A composition for diagnosing infectious diseases or infectious complications, said composition comprising an antibody or a fragment thereof according to any one of claims 1 to 5.

12. The composition according to claim 11, wherein the infectious disease is an infectious inflammatory disease.

13. The composition of claim 12, wherein the infectious inflammatory disease is sepsis or septic shock.

Images