WARS-NEUTRALIZING ANTIBODY AND USE THEREOF

Abstract

The present disclosure relates to an antibody binding specifically to tryptophanyl-tRNA synthetase (WARS) or a fragment thereof. The antibody or the fragment thereof of the present disclosure has an excellent ability to bind specifically to WARS with no cross-reactivity with other ARSs and has an excellent ability to detect WARS and inhibit the activity thereof. Therefore, it can be effectively used for diagnosing and/or treating an infectious disease.

Claims

1-28. (canceled)

29. An antibody binding specifically to tryptophanyl-tRNA synthetase (WARS), comprising a heavy chain variable domain (V.sub.H) comprising a heavy chain CDR1 of SEQ ID NO 3, a heavy chain CDR2 of SEQ ID NO 4 and a heavy chain CDR3 of SEQ ID NO 5 and a light chain variable domain (V.sub.L) comprising a light chain CDR1 of SEQ ID NO 6, a light chain CDR2 of SEQ ID NO 7 and a light chain CDR3 of SEQ ID NO 8, or an antigen binding fragment thereof.

30. The antibody or the antigen binding fragment thereof according to claim 29, wherein the antibody or the fragment thereof comprises a heavy chain variable domain (V.sub.H) of SEQ ID NO 1 and a light chain variable domain (V.sub.L) of SEQ ID NO 2 (V.sub.L), or a fragment thereof.

31. The antibody or the antigen binding fragment thereof according to claim 29, wherein the antibody or the antigen binding fragment thereof comprises one or more sequence selected from a group consisting of a C.sub.L sequence of SEQ ID NO 21, a C.sub.H1 sequence of SEQ ID NO 22, a hinge sequence of SEQ ID NO 23, a C.sub.H2 sequence of SEQ ID NO 24 and a C.sub.H3 sequence of SEQ ID NO 25.

32. The antibody or the antigen binding fragment thereof according to claim 29, wherein the fragment is a fragment selected from a group consisting of Fab, F(ab′).sub.2, Fd, sdFv, Fv, dAb, scFv, sdAb and a tetramer or the fragment bound to Fc.

33. A polynucleotide encoding the antibody or the antigen binding fragment thereof according to claim 29.

34. A vector comprising the polynucleotide according to claim 33.

35. A cell transformed with the vector according to claim 34.

36. A method for preparing an antibody binding specifically to WARS or an antigen binding fragment thereof, comprising a step of culturing the cell according to claim 35 under a condition where a polynucleotide is expressed and recovering the polypeptide.

37. A method for detecting WARS specifically, comprising a step of contacting the antibody or the antigen binding fragment thereof according to claim 29, a polynucleotide encoding the antibody or the antigen binding fragment thereof, a vector comprising the polynucleotide or a cell transformed with the vector with a sample.

38. A composition for detecting WARS specifically, comprising the antibody or the antigen binding fragment thereof according to claim 29, a polynucleotide encoding the antibody or the antigen binding fragment thereof, a vector comprising the polynucleotide or a cell transformed with the vector.

39. A composition for diagnosing an infectious disease, comprising the antibody or the antigen binding fragment thereof according to claim 29, a polynucleotide encoding the antibody or the antigen binding fragment thereof, a vector comprising the polynucleotide or a cell transformed with the vector.

40. The composition for diagnosing according to claim 39, wherein the infectious disease is an infectious inflammatory disease.

41. The composition for diagnosing according to claim 40, wherein the infectious inflammatory disease is sepsis or septic shock.

42. A method for treating a disease in a subject in need thereof, comprising a step of administering an effective amount of the antibody or the antigen binding fragment thereof according to claim 29, a polynucleotide encoding the antibody or the antigen binding fragment thereof, a vector comprising the polynucleotide, or a cell transformed with the vector to the subject.

43. The method for treating a disease according to claim 42, which further comprises administering an antibiotic.

44. The method for treating a disease according to claim 43, wherein the antibiotic is selected from a group consisting of gentamycin, ampicillin, kanamycin, chloramphenicol, streptomycin, tetracycline, erythromycin, vancomycin, penicillin, spectinomycin, sulfadiazine and trimethoprim.

45. The method for treating a disease according to claim 42, wherein the disease is an infectious disease.

46. The method for treating a disease according to claim 45, wherein the infectious disease is an infectious inflammatory disease.

47. The method for treating a disease according to claim 46, wherein the infectious inflammatory disease is sepsis or septic shock.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0089] FIGS. 1a-1b schematically show vectors for converting GKB101 to an IgG antibody. FIG. 1a shows a heavy chain vector, and FIG. 1B shows a light chain vector.

[0090] FIGS. 2a-2b show a result of measuring the target-binding ability of GKB101. FIG. 2a shows a result of measuring the binding ability of GKB101 for human and mouse WARS1 using GE Biacore T200, and FIG. 2b shows a result of measuring absorbance by ELISA.

[0091] FIGS. 3a-3b show a result of verifying the neutralizing ability of GKB101 in vitro. FIG. 3a shows decreased secretion of TNF-α, and FIG. 3b shows decreased secretion of mCXCL2 (homolog of human IL-8).

[0092] FIGS. 4a-4k show a result of verifying the effect of the WARS1-specific antibody on the survival rate of mice infected by bacteria. FIG. 4a shows a result of injecting PBS or GKB101 to mice in which severe sepsis was induced by intraperitoneal injection of a cecal slurry (CS) and then analyzing survival rate. FIGS. 4b and 4c show a result of verifying that the concentration of WARS1 is decreased by administration of GKB101 (FIG. 4b: plasma, FIG. 4c: peritoneal lavage fluid). FIGS. 4d-4g show a result of verifying that the concentration of IL-8 (FIG. 4e), IL-6 (FIG. 4f), MIP-1a (FIG. 4d) and TNF-α (FIG. 4g) secreted into a peritoneal lavage fluid is decreased by administration of GKB101. FIGS. 4h-4k show a result of verifying that the concentration of ALT (FIG. 4h), AST (FIG. 4i), BUN (FIG. 4j) and creatinine (FIG. 4k) is decreased relatively by administration of GKB101 (circles: normal group; squares: PBS group; triangles GKB101 group).

[0093] FIGS. 5a-5k show a result of verifying the effect of the WARS1-specific antibody on the survival rate of mice infected by bacteria. FIG. 5a shows a result of injecting isotype IgG or GKB101 to mice in which severe sepsis was induced by intraperitoneal injection of a cecal slurry (CS) and then analyzing survival rate. FIGS. and 5c show a result of verifying that the concentration of WARS1 is decreased by administration of GKB101 (FIG. 5b: plasma, FIG. 5c: peritoneal lavage fluid). FIGS. show a result of verifying that the concentration of IL-8 (FIG. 5e), IL-6 (FIG. 5f), MIP-1a (FIG. 5d) and TNF-α (FIG. 5g) secreted into a peritoneal lavage fluid is decreased by administration of GKB101. FIGS. 5i-5k show a result of verifying that the concentration of ALT (FIG. 5h), AST (FIG. 5i), BUN (FIG. 5j) and creatinine (FIG. is decreased relatively by administration of GKB101.

[0094] FIGS. 6a-6b show a result of verifying the effect of co-administration of GKB101 and an antibiotic in a severe sepsis mouse model. FIG. 6a shows an injection schedule of gentamicin and GKB101, and FIG. 6b shows survival rate over time after injection of gentamicin and GKB101.

[0095] FIGS. 7a-7g show a result of verifying the efficacy of GKB101 in a marmoset endotoxemia model. FIG. 7a shows an injection schedule of PBS and GKB101, and FIGS. 7b-7e show the change in body temperature (FIG. 7b), body weight (FIG. 7c), WBC in blood (FIG. 7d) and neutrophil (FIG. 7e) over time in a PBS or GKB101 administration group after LPS induction. FIG. 7f shows the level of WARS1 in plasma after administration of GKB101, and FIG. 7g shows a result of measuring the level of TNF-α.

BEST MODE

[0096] Hereinafter, the present disclosure will be described more specifically through examples. The following examples are provided only to describe the present disclosure more specifically and it will be obvious to those having ordinary knowledge in the art that the scope of the present disclosure is not limited by the examples.

Examples

Example 1. Conversion of scFv Antibody to IgG1 Antibody

[0097] As an scFv antibody binding specifically to WARS1 (SEQ ID NO 26), an scFv antibody including a heavy chain variable region (SEQ ID NO 1) including heavy chain CDR1 (SEQ ID NO 3), heavy chain CDR2 (SEQ ID NO 4) and heavy chain CDR3 (SEQ ID NO 5) and a light chain variable region (V.sub.L) of SEQ ID NO 2 including light chain CDR1 (SEQ ID NO 6), light chain CDR2 (SEQ ID NO 7) and light chain CDR3 (SEQ ID NO 8) was selected. The selected scFv antibody was cloned into a plasmid DNA vector by inserting into a heavy chain vector (FIG. 1a) and a light chain vector (FIG. 1b). First, a polynucleotide encoding the scFv was amplified by PCR. The base sequences of the primers used to amplify the CDR regions of the scFv are as follows: forward (gtggccacagcggccgatgtccactcggaagtacagttggtcgaaagtggc, SEQ ID NO 9), reverse (gaagaccgatgggcccttggtgctagccgatgagacggtcactaaagtgcc, SEQ ID NO 10).

[0098] The base sequences of the primers used to amplify the V.sub.L region of the scFv are as follows: forward (gccacagcggccgatgtccactcggacattcaaatgacgcagagtccctc, SEQ ID NO 11), reverse (gaagacagatggtgcagccacagatcttttaatttccactttagttccctgcc, SEQ ID NO 12).

[0099] A monoclonal antibody (hereinafter, referred to as GKB101) was prepared using the scFv antibody by IgG conversion as follows:

[0100] A heavy chain and a light chain for IgG conversion of GKB101 were amplified using pOptiVEC and pcDNA 3.3, which are parent vectors expressing herceptin (FIGS. 1a and 1b). The base sequences of the primers used to amplify C.sub.H (V.sub.H3-23) and C.sub.L regions are as follows: C.sub.H forward (ggcactttagtgaccgtctcatcggctagcaccaagggcccatcggtcttc, SEQ ID NO 13), C.sub.H reverse (gccactttcgaccaactgtacttccgagtggacatcggccgctgtggccac, SEQ ID NO 14), C.sub.L forward (ggcagggaactaaagtggaaattaaaagatctgtggctgcaccatctgtcttc, SEQ ID NO 15), C.sub.L reverse (gagggactctgcgtcatttgaatgtccgagtggacatcggccgctgtggc, SEQ ID NO 16).

[0101] Fab.sub.2 (V.sub.H-1-69) for IgG conversion of GKB101 was amplified by PCR using an insert V.sub.H1-69_Forward (gtggccacagcggccgatgtccactcgcaagttcagctggtccagagcggc, SEQ ID NO 17), V.sub.H1-69_Reverse (gaagaccgatgggcccttggtgctagccgatgagacggtaaccagagtaccc, SEQ ID NO 18) and a vector C.sub.H1-69_Forward (gccgctctggaccagctgaacttgcgagtggacatcggccgctgtggccacc, SEQ ID NO 19), C.sub.H1-69_Reverse (gggtactctggttaccgtctcatcggctagcaccaagggcccatcggtcttc, SEQ ID NO 20).

[0102] The C.sub.H, C.sub.L and V.sub.H 1-69 genes of V.sub.H, V.sub.L and herceptin expression vectors of GKB101 for GKB101 conversion was amplified by PCR using each vector template (pcDNA3.3-herceptin_C.sub.L, pOptiVEC-herceptin_C.sub.H) and insert template (scFv, Fab phage vector) and the primers described above (10 pmol each) under the condition of ° C./3 min; 95° C./30 sec, 60° C./30 sec, 72° C./30 sec, 30 cycles; 72° C./5 min. The PCR product was inserted into a vector used for IgG production using a Dpnl restriction enzyme. The prepared vector including a DNA which encodes the light chain and heavy chain of IgG including the variable region of the scFv was co-transfected into Expi293F cells so that the light chain and the heavy chain were co-expressed in the cells.

Example 2. Transient Transfection and Purification Using Column

[0103] ExpiHEK293 cells (Thermofisher) and a Freestype293 expression medium (Thermofisher) were used for purification of proteins using a transient expression system. Transfection was conducted using an Expifectamine 293 reagent (Thermofisher) and a plasmid DNA with the heavy chain and the light chain of GKB101 at 1:1. After the transfection, the cells were cultured for about 5 days under shaking. After centrifuging the cell culture, only the supernatant was recovered and used for isolation and purification of proteins. The recovered supernatant was loaded in a Hitrap MabselectSure column (GE Healthcare Life Sciences). After removing nonspecifically bound proteins by washing with PBS, proteins specifically bound to the column were separated using a 100 mM citrate buffer (pH 3.0)+50 mM NaCl. High-purity GKB101 was obtained by loading an eluate obtained by FPLC in a size-exclusion chromatography system (GE Healthcare Life Sciences). The final yield of GKB101 obtained by optimizing the condition of the transient expression system was 37 mg for 500 mL of the cell culture and the yield of the endotoxin was about 0.002 EU/g.

[0104] The sequences of the prepared GKB101 antibody are as follows:

TABLE-US-00001 TABLE 1 Sequences of GKB101 antibody Amino acid sequence V.sub.H (SEQ ID NO 1) EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYDMSWVRQA PGKGLEWVSA ISSGGSSIYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDV AWDMLGDFDY WGQGTLVTVS S V.sub.L (SEQ ID NO 2) QSVLTQPPSA SGTPGQRVTI SCTGSSSNIG SNYVYWYQQL PGTAPKLLIY ANSHRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYCG AWDDSLSAYV FGGGTKLTV C.sub.L (SEQ ID NO 21) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC C.sub.H1 (SEQ ID NO 22) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKV Hinge (SEQ ID NO 23) EPKSCDKTHTCPPCPAPELLGGP C.sub.H2 (SEQ ID NO 24) SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKG C.sub.H3 (SEQ ID NO 25) REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSL

Example 3. Measurement of Target-Binding Ability of GKB101

[0105] 1) SPR: Binding ability was measured using Series S Sensor CM5 Chip (GE Healthcare Life Sciences) and GE Bioacore T200. A HBS-EP buffer (GE Healthcare Life Sciences) was used. After injecting serially diluted GKB101 or buffer for 1-10 minutes at a flow rate of 30 μL/min, the analyte was washed for 20 minutes.

[0106] 2) ELISA: After coating the GKB101 on a 96-well plate (Maxisorp, Thermofisher) at a concentration of 1 μg/mL, it was blocked at room temperature for 1 hour with PBS containing 1% BSA. After diluting the recombinant WARS1 protein to different concentrations and adding to each coated well, reaction was conducted at room temperature for 1 hour and each well was washed 4 times with PBST (PBS+Tween 20). After adding anti-WARS1 antibody (Abfrontier) to each well and conducting reaction at room temperature for 1 hour, each well was washed 4 times with PBST. After reacting with HRP-conjugated secondary antibody (anti-Human IgG, Cell Signaling) for 1 hour, followed by washing with PBST, absorbance was measured at 490 nm (VersaMax microplate reader) after reacting with a TMB substrate (BD).

[0107] 3) Immunoblot: After loading 20 ng of each of WARS1 human and mouse recombinant proteins and 30 μg of each of THP-1 human monocytes and J774.1A mouse macrophages, stimulated with LPS for 24 hours, on 8% SDS-PAGE, the cells were transferred to a PVDF membrane (Millipore). The membrane was reacted with 1 μg/mL GKB101 for 1 hour and then washed three times with TBST (TBS+Tween After reacting with a secondary antibody (anti-human IgG HRP, Millipore) for an hour, followed by washing 3 times with TBST, the membrane was reacted with ECL (West® bright ECL, Advanta) (LAS-4000, Fujifilm).

[0108] 4) Result: As a result of measuring the binding ability of GKB101 for the recombinant WARS1 proteins, it showed strong binding ability for human WARS1 in both ELISA and SPR. The binding ability was about 0.2 nM for the human recombinant protein and about 3.76 nM for the mouse recombinant protein (FIG. 2a). In addition, WARS1 in the culture of human monocytes and mouse macrophages could also be detected effectively with GKB101 in the immunoblot experiment (FIG. 2b).

Example 4. Verification of Neutralizing Ability of WARS1-Specific Antibody In Vitro

[0109] The neutralizing ability of the WARS1-specific antibody GKB101 in vitro was investigated using J774.1A mouse macrophages. The mouse macrophages were cultured using high-glucose DMEM (GIBCO) supplemented with 10% FBS and 1% penicillin/streptomycin. 8×10.sup.4 cells were seeded on a 48-well cell culture plate (Nunc) and the medium was replaced with plain DMEM 2 hours before treating the cells with an antigen-antibody mixture. After mixing 200 nM recombinant protein WARS1 with GKB101 of different concentrations and reacting in a 37° C. incubator for 2 hours, the mixture was added to the cells seeded on each well. About 15 hours later, the cell culture was centrifuged and the supernatant was used to measure CXCL2 (R&D) and TNF-α (Biolegend). ELISA was conducted according to the manufacturers' instructions.

[0110] In the mouse macrophages, GKB101 resulted in significantly decreased secretion of TNF-α in a concentration-dependent manner with respect to 200 nM WARS1 at 1:2.5 and 1:5 (FIG. 3a). mCXCL2 (mouse IL-8 homolog) was decreased at 1:5 and 1:2.5, and statistically significant difference was observed at 1:2.5 (FIG. 3b). All the results were analyzed statistically by ANOVA.

Example 5. Effect of WARS1-Specific Antibody on Survival Rate of Mice Infected by Bacteria

[0111] After inducing severe sepsis by injecting CS into mice, 2.5 mg/kg PBS or GKB101 was injected i.v twice at 4 hours or 20 hours after the intraperitoneal injection of the CS. Then, survival rate was monitored until 60 hours.

[0112] WARS1 and cytokine assay: In order to investigate the neutralizing ability of GKB101 for WARS1, peritoneal lavage fluid and blood were taken at 5 hours, 11 hour, 18 hours and 20 hours, and the level of WARS1 (in-house), TNF-α (Biolegend), MIP-1a (R&D), mIL-6 (Biolegend) and mCXCL2 (IL-8 homolog, R&D) was measured by ELISA. The ELSIA for the cytokines was conducted according to the manufacturers' instructions except for WARS1. WARS1 level was measured as follows. After coating GKB101 onto each well of a 96-well plate (Maxisorp, Thermofisher) at 1 μg/mL, the well was blocked at room temperature for 1 hour with PBS containing 1% BSA. After adding diluted standard plasma, peritoneal lavage fluid sample and recombinant mouse recombinant protein to the blocked well, reaction was conducted at room temperature for 1 hour and the well was washed 4 times with PBST (PBS+Tween After adding anti-WARS1 antibody (Abfrontier) and conducting reaction at room temperature for 1 hour, each well was washed 4 times with PBST. After reacting with HRP-conjugated secondary antibody (anti-human IgG, Cell Signaling) for 1 hour, the well was washed with PBST. After reacting with a TMB substrate (BD), absorbance was measured at 490 nm (VersaMax microplate reader).

[0113] Biochemical assay using blood: After injecting some of blood into a vacutainer holding a clot activator, the blood was coagulated by leaving alone at room temperature for 10-15 minutes. After centrifuging at 3,000 rpm for 10 minutes, the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine in the obtained serum was measured using a biochemical blood analyzer (7180 Hitachi, Japan).

[0114] In order to investigate the effect of GKB101 on the survival rate of mice, animal experiment was conducted as described schematically in FIG. 4a. PBS or GKB101 was injected i.v at 4 hours and 20 hours after induction of severe sepsis by intraperitoneal injection of a cecal slurry (CS). The survival rate of the mice was monitored until 60 hours after the infection. As a result, for CS+PBS (PBS was injected after induction of severe sepsis), the survival rate was 50% at 29 hours and 20% at 41 hours. For CS+GKB101 (GKB101 was injected after induction of severe sepsis), the survival rate was increased as compared to the PBS group, as 80% at 29 hours and 50% at 41 hours. The concentration of WARS1 in the peritoneal lavage fluid (FIG. 4c) and plasma (FIG. 4b) was decreased significantly by GKB101 at 11 hours and 18 hours, and the concentration of IL-8 (FIG. 4e), IL-6 (FIG. 4f), MIP-1a (FIG. 4d) and TNF-α (FIG. 4g) secreted into the peritoneal lavage fluid was also decreased significantly at 11 hours and 18 hours. The concentration of ALT (FIG. 4h), AST (FIG. 4i), BUN (FIG. 4j) and creatinine (FIG. 4k) was decreased relatively, and the concentration of ALT and BUN was decreased significantly at 11 hours in the GKB101 injection group as compared to the PBS group. All the results were analyzed statistically by ANOVA (*, p<0.05; **, p<0.01; ***, p<0.001).

Example 6. Effect of WARS1-Specific Antibody on Survival Rate of Mice Infected by Bacteria 2

[0115] After inducing severe sepsis by injecting CS to mice, 10 mg/kg isotype IgG or GKB101 was injected i.p three times at 4 hours, 8 hours and 12 hours as 5 mg/kg, 2.5 mg/kg and 2.5 mg/kg, respectively. Then, survival rate was monitored until 72 hours.

[0116] WARS1 and cytokine assay: In order to investigate the neutralizing ability of GKB101 for WARS1, peritoneal lavage fluid and blood were taken at 6 hours, 10 hours and 14 hours, and the level of WARS1 (in-house), TNF-α (Biolegend), MIP-1a (R&D), mIL-6 (Biolegend) and mCXCL2 (IL-8 homolog, R&D) was measured by ELISA. The ELSIA for the cytokines was conducted according to the manufacturers' instructions except for WARS1. WARS1 level was measured as follows. After coating GKB101 onto each well of a 96-well plate (Maxisorp, Thermofisher) at 1 μg/mL, the well was blocked at room temperature for 1 hour with PBS containing 1% BSA. After adding diluted standard plasma, peritoneal lavage fluid sample and recombinant mouse recombinant protein to the blocked well, reaction was conducted at room temperature for 1 hour and the well was washed 4 times with PBST (PBS+Tween 20). After adding anti-WARS1 antibody (Abfrontier) and conducting reaction at room temperature for 1 hour, each well was washed 4 times with PBST. After reacting with HRP-conjugated secondary antibody (anti-human IgG, Cell Signaling) for 1 hour, the well was washed with PBST. After reacting with a TMB substrate (BD), absorbance was measured at 490 nm (VersaMax microplate reader).

[0117] In order to investigate the effect of GKB101 on the survival rate of mice, animal experiment was conducted as described schematically in FIG. 5a. Isotype IgG or GKB101 was injected i.p at 4 hours, 8 hours and 12 hours after induction of severe sepsis by intraperitoneal injection of a cecal slurry (CS). The survival rate of the mice was monitored until 72 hours after the infection. As a result, for CS+isotype IgG (isotype IgG was injected after induction of severe sepsis), the survival rate was 20% at 28 hours and 10% at 36 hours. For CS+GKB101 (GKB101 was injected after induction of severe sepsis), the survival rate was increased remarkably as compared to the isotype IgG group, as 80% at 28 hours and 70% at 36 hours (FIG. 5a). The concentration of WARS1 in the peritoneal lavage fluid (FIG. 5c) and plasma (FIG. 5b) was decreased significantly by GKB101 at 6 hours, 10 hours and 14 hours, and the concentration of IL-8 (FIG. 5e), IL-6 (FIG. 5f), MIP-1a (FIG. 5d) and TNF-α (FIG. 5g) secreted into the peritoneal lavage fluid was also decreased after the injection of GKB101. In particular, the concentration of IL-8, MIP-1a and TNF-α was decreased significantly at 14 hours. The concentration of ALT (FIG. 5h), AST (FIG. 5i), BUN (FIG. 5j) and creatinine (FIG. 5k) was decreased relatively, and the concentration of ALT and BUN was decreased significantly at 14 hours in the GKB101 injection group as compared to the isotype IgG group. All the results were analyzed statistically by ANOVA (*, p<0.05; **, p<0.01; ***, p<0.001).

Example 7. Effect of WARS1-Specific Antibody and Antibiotic on Survival Rate of Mice Infected by Bacteria

[0118] After administering an antibiotic (gentamicin) to mice in which severe sepsis had been induced by injecting CS, 10 mg/kg isotype IgG or GKB101 was injected i.v at 4 hours, 8 hours and 20 hours as 5 mg/kg, 2.5 mg/kg and 2.5 mg/kg, respectively. Then, survival rate was monitored until 72 hours.

[0119] As a result of analyzing the survival rate in the CS-induced mice to which GKB101 and the antibiotic were co-administered, significantly difference in survival rate was observed with 80% for the GKB101 administration group at 24 hours and 70% for the isotype IgG administration group at 72 hours. All the results were analyzed statistically by ANOVA (FIG. 6).

Example 8. Verification of Efficacy of GKB101 Using Marmoset Endotoxemia Model

[0120] Marmosets (Callithrix jacchus) were acquired from the Laboratory Animal Center of Osong Medical Innovation Foundation (Chungbuk, Korea). All the common marmosets were housed in stainless steel cages. They were maintained in 12-hour dawn and 12-hour dusk conditions at a temperature of 27±2° C. and a humidity of 30%-70% in the SPF animal facility. The common marmosets were provided with a standard marmoset diet (#0639; Altromin, Lage, Germany) and water ad libitum. All protocols were approved by the Committee on Animal Care of the Laboratory Animal Center of Osong Medical Innovation Foundation. After injecting LPS to the marmosets and inducing endotoxemia, 2 mg/kg PBS or GKB101 was injected i.v twice at 0.2 hours and 2 hours and the animals were monitored for 24 hours.

[0121] As a result of investigating the number of immune cells in the blood of the marmoset endotoxemia model, the group to which the anti-WARS1 antibody was administered showed significant increase of WBCs and neutrophils at 12 hours (*, p<**, p<0.01). In addition, as a result of comparing the effect of the administration of GKB101 on the expression of WARS1, cytokines and chemokines in blood, WARS1 was decreased significantly at 0.5, 1, 2, 4 and 8 hours (****, p<0.0001) and TNF-α was decreased significantly at 1 and 2 hours (2 hours, ****, p<0.0001) in the GKB101 administration group. All the results were analyzed statistically by ANOVA (FIGS. 7a-7g).

[0122] The results of the above-described examples verify that GKB101 has a strong binding ability to WARS1 and has the neutralizing ability of decreasing the expression of cytokines expressed by WARS1 in vivo and in vitro. As described above, it is expected that the composition of the present disclosure is industrially applicable as an antibody drug which specifically detects WARS1, which induces hyperinflammation in the early stage of infection, and inhibits the same.

[0123] Although the examples of the present disclosure have been described above, those having ordinary knowledge in the art can change and modify the present disclosure variously through addition, change or deletion without departing from the scope of the present disclosure defined by the appended claims.