Pharmaceutical composition comprising antibody binding specifically to lysyl-tRNA synthetase N-terminus as effective ingredient for preventing or treating immune cell migration-related disease
11965038 ยท 2024-04-23
Assignee
Inventors
Cpc classification
A61K39/395
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a novel use of an antibody biding specifically to the N-terminus of lysyl-tRNA synthetase and, more particularly, to a pharmaceutical composition comprising an antibody biding specifically to an epitope including the sequence of SEQ ID NO: 117 in the N-terminal domain of lysyl-tRNA synthetase (KRS) or a functional fragment thereof as an effective ingredient for preventing and treating an immune cell migration-related disease. A KRS N-terminus-specific antibody provided by the present invention can regulate the migration of immune cells, thereby exhibiting very remarkable effects in the prevention, alleviation, and treatment of immune cell migration-related diseases.
Claims
1. A method for treating an immune cell migration-related disease in a subject, the method comprising administering to the subject in need thereof an effective amount of a composition comprising an antibody binding specifically to an epitope containing the sequence of SEQ ID NO: 117 in the N-terminus of lysyl-tRNA synthetase (KRS) or an antigen binding fragment thereof as an effective ingredient, wherein the antibody or antigen binding fragment thereof comprises: (a) a heavy chain variable region comprising: a heavy chain complementarity determining region 1 containing the amino acid sequence defined by SEQ ID NO: 1, a heavy chain complementarity determining region 2 containing the amino acid sequence defined by SEQ ID NO: 3 or SEQ ID NO: 151, and a heavy chain complementarity determining region 3 containing the amino acid sequence defined by SEQ ID NO: 5; and a light chain variable region comprising: a light chain complementarity determining region 1 containing the amino acid sequence defined by SEQ ID NO: 7, a light chain complementarity determining region 2 containing the amino acid sequence defined by SEQ ID NO: 9, and a light chain complementarity determining region 3 containing the amino acid sequence defined by SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15; (b) a heavy chain variable region comprising: a heavy chain complementarity determining region 1 containing the amino acid sequence defined by SEQ ID NO: 1, a heavy chain complementarity determining region 2 containing the amino acid sequence defined by SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23, and a heavy chain complementarity determining region 3 containing the amino acid sequence defined by SEQ ID NO: 5; and a light chain variable region comprising: a light chain complementarity determining region 1 containing the amino acid sequence defined by SEQ ID NO: 7, a light chain complementarity determining region 2 containing the amino acid sequence defined by SEQ ID NO: 9, and a light chain complementarity determining region 3 containing the amino acid sequence defined by SEQ ID NO: 15: or (c) a heavy chain variable region comprising: a heavy chain complementarity determining region 1 containing the amino acid sequence defined by SEQ ID NO: 1, a heavy chain complementarity determining region 2 containing the amino acid sequence defined by SEQ ID NO: 21, and a heavy chain complementarity determining region 3 containing the amino acid sequence defined by SEQ ID NO: 5 or SEQ ID NO: 25; and a light chain variable region comprising: a light chain complementarity determining region 1 containing the amino acid sequence defined by SEQ ID NO: 7, a light chain complementarity determining region 2 containing the amino acid sequence defined by SEQ ID NO: 9, SEQ ID NO: 27, or SEQ ID NO: 29, and a light chain complementarity determining region 3 containing the amino acid sequence defined by SEQ ID NO: 15, wherein the immune cell migration-related disease is a cardiovascular disease, a fibrotic disease, an inflammatory disease, or Alport syndrome.
2. The method of claim 1, wherein the epitope is selected from the group consisting of SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150.
3. The method of claim 1, wherein the antibody or antigen binding fragment thereof decreases the level of KRS on a cell membrane.
4. The method of claim 1, wherein the antibody or antigen binding fragment thereof is a heavy chain variable region containing the amino acid sequence defined by SEQ ID NO: 31; and a light chain variable region containing the amino acid sequence defined by SEQ ID NO: 33.
5. The method of claim 1, wherein the antibody is selected from the group consisting of IgG, IgA, IgM, IgE and IgD, and the antigen binding fragment is selected from the group consisting of diabody, Fab, Fab, F (ab)2, F (ab)2, Fv and scFv.
6. The method of claim 5, wherein the scFv comprises an amino acid sequence defined by SEQ ID NO: 59.
7. The method of claim 1, wherein the cardiovascular disease is selected from the group consisting of hypertension, pulmonary arterial hypertension, atherosclerosis, angina pectoris, myocardial infarction, ischemic cerebrovascular disease, atherosclerosis and mesenteric sclerosis.
8. The method of claim 1, wherein the fibrotic disease is selected from the group consisting of scleroderma, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis, pulmonary fibrosis, hepatic fibrosis, liver cirrhosis, kidney fibrosis, glomerulosclerosis, myofibrosis, myofibrosis cordis, interstitial fibrosis, pancreatic fibrosis, splenic fibrosis, mediastinal fibrosis, vascular fibrosis, skin fibrosis, eye fibrosis, macular degeneration, joint fibrosis, thyroid fibrosis, endomyocardial fibrosis, peritoneal fibrosis, retroperitoneal fibrosis, progressive mass fibrosis, nephrogenic systemic fibrosis, systemic lupus erythematosus, hereditary fibrosis, infectious fibrosis, irritant fibrosis, fibrosis due to chronic autoimmunity, fibrosis due to antigen incompatibility during organ transplantation, fibrotic complications during surgery, fibrosis due to hyperlipidemia, fibrosis due to obesity, diabetic fibrosis, fibrosis due to hypertension, and occlusion due to fibrosis at the time of stent insertion.
9. The method of claim 1, wherein the inflammatory disease is selected from the group consisting of an autoimmune disease, inflammatory bowel disease, dermatitis, atopic dermatitis, eczema, psoriasis, diabetic eye disease, diabetic retinopathy, peritonitis, osteomyelitis, cellulites, meningitis, encephalitis, pancreatitis, trauma-induced shock, bronchial asthma, rhinitis, sinusitis, tympanitis, pneumonia, gastritis, enteritis, cystic fibrosis, apoplexy, stroke, bronchitis, bronchiolitis, hepatitis, cirrhosis, steatohepatitis, non-alcoholic steatohepatitis, nephritis, diabetic renal failure, proteinuria, arthritis, psoriatic arthritis, osteoarthritis, neuritis, diabetic neuropathy, multiple sclerosis, gout, spondylitis, Reiter's syndrome, polyarteritis nodosa, vasculitis, amyotrophic lateral sclerosis, Wegener's granulomatosis, hypercytokinemia, Polymyalgia rheumatica, articular cell arteritis, calcium crystalline arthritis, pseudogout, non-articular rheumatoid, bursitis, tendosynovitis, epicondylitis (tennis elbow), Charcot's joint, hemarthrosis, Henoch-Schonlein purpura, hypertrophic osteoarthritis, multicentric reticulocytoma, sarcoidosis, hemochromatosis, drepanocytosis, hyperlipoproteinemia, hypogammaglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, systemic lupus erythematosus, recurrent fever, psoriasis, multiple sclerosis, sepsis, septic shock, acute respiratory distress syndrome, multiple organs dysfunction, chronic obstructive pulmonary disease, acute lung injury, and broncho-pulmonary dysplasia.
10. The method of claim 9, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, psoriasis, asthma, ulcerative colitis, Behcet's disease, Crohn's disease, multiple sclerosis, dermatitis, collagen disease, vasculitis, arthritis, granulomatosis, organ specificity autoimmune diseases and GvHD (graft-versus-host disease).
11. The method of claim 5, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85 and SEQ ID NO: 87.
12. The method of claim 1, wherein the cardiovascular disease is pulmonary arterial hypertension.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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MODE FOR CARRYING OUT INVENTION
(29) Hereinafter, the present invention will be described in detail.
(30) However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
Example 1: Verification of Laminin Signal Role in Immune Cell Migration and Invasion
(31) In the extracellular matrix (ECM) that make up the blood vessels, it was identified which promotes the migration and invasion of monocytes/macrophages. Transwell migration assay was performed using collagen (collagen, Col), fibronectin (FN) and laminin (LN) as extracellular matrix. The specific experimental method is as follows. After transwells (Corning, #3421-5 mm) were coated with gelatin (0.5 mg/ml), RAW 264.7 cells (1?10.sup.5 cells/well) were seeded into the top chamber. Fibronectin or Collagen was placed in the bottom chamber. Serum Free DMEM (500 ?l) containing 10 ?g/ml of Laminin (laminin mixture, Biolamina), Fibronectin or Collagen, respectively, was placed in the bottom chamber. After 24 hours, the cells were treated with 70% methanol for 30 minutes and fixed, and then stained with 50% Hematoxylin for 30 minutes. After removing non-migrating cells from the top of the membrane with a cotton swab, the membrane was taken and mounted on the slide. The migrating cells on the underside of the membrane were observed and quantified under a high magnification microscope.
(32) As shown in
Example 2: Immune Cell Migration and Invasion Effects by Laminin Subtypes
(33) The effects of laminin subtypes on immune cell migration and invasion were evaluated. A transwell cell migration assay was performed in the same manner as in the Example 1 using LN111, LN211, LN221, LN411, LN421, LN511, and LN521 at 1 ?g/ml as various laminin subtype proteins (purchased from Biolamina). The specific sequence of the laminin subtypes can be referenced according to the chains consisting of each laminin subtype, ?4 chain of SEQ ID NO: 120, ?2 chain of SEQ ID NO: 126, ?5 chain of SEQ ID NO: 127, ?2 chain of SEQ ID NO: 122, ?1 chain of SEQ ID NO: 128, ?1 chain of SEQ ID NO: 124
(34) As shown in
Example 3: Movement of Cytoplasmic KRS to Cell Membrane Following Laminin Treatment of Immune Cells Identification of Novel Pathology of Immune Cell Migration-Related Diseases
(35) After seeding RAW 264.7 cells (2?10.sup.6 cells) in 100 pie plate and incubating for 18 hr, the serum free-DMEM media was treated with 1 ?g/ml LN421 and the cells were harvested at 0 h, 12 h, and 24 h. RAW 264.7 cell proteins were separated into cytosol and membrane fractions using the ProteoExtract? Subcellular Proteome Extraction Kit (Calbiotech, cat #539790). The obtained protein was subjected to electrophoresis, transferred to PVDF membrane (Millipore) and blocked with 3% skim milk. KRS was then detected by the western blotting. Specifically, KRS polyclonal antibody (rabbit, Neomics, Co. Ltd. #NMS-01-0005) was added to bind for 1 hour. Unbound antibody was removed and then anti-rabbit secondary antibody (Thermo Fisher Scientific, #31460) was added. After reacting with the secondary antibody, the film was developed in the dark room using ECL reagent as a substrate. The detected bands were compared to standard molecular markers to identify the bands corresponding to the size of the KRS. Antibodies against Na+/K+ATPase (Abcam, ab76020) and tubulin (Santa cruz SC-5286) were used to identify plasma membrane and cytosol markers, respectively.
(36) As shown in
Example 4: Control of Immune Cell Migration/Invasion Through Reduction of KRS Levels in Cell Membrane Location and Confirmation of Therapeutic Effects of Immune Cell Migration-Related Diseases Use of Compounds that Inhibit KRS Migration to Cell Membrane
(37) The present inventors confirmed that the intracellular behavior of KRS significantly influences the migration of monocytes/macrophages from the results of the above-described examples. In particular, the phenomenon that KRS specifically increases in the area of the immune cell membrane as the KRS moves toward the cell membrane is considered to be an important pathology for diseases related to immune cell migration and invasion. Therefore, the present inventors have verified that suppressing this pathological phenomenon of KRS can be applied as one of the therapeutic strategies of diseases related to immune cell migration and invasion, which this has been disclosed in Korean applications 10-2017-0076718 and 10-2018-0069146.
(38) In the above document, the present inventors have discovered compounds that inhibit the movement of KRS to the cell membrane in order to inhibit the KRS increase in the immune cell membrane in immune cell migration-related diseases, and their therapeutic effect on immune cell migration-related diseases was confirmed and examined.
(39) Briefly, in order to suppress the KRS increase in immune cell membranes, compounds such as BC-KI-00053 (4-({(7-fluorobenzo [d] thiazol-2-yl) [2-(4-methoxyphenyl) ethyl] amino} methyl) benzoic acid) were found as a representative example of the inhibitor of migration of KRS to cell membranes. In inflammatory conditions such as acute inflammatory reactions (e.g., ear skin wound models) and ischemic immune responses (e.g., liver ischemia-reperfusion injury models), it was also confirmed that the migration and invasion of immune cells was inhibited by the administration of inhibitors of migration of KRS to the cell membrane (especially, BC-KI-00053) and the effect of alleviating disease appeared. In addition, in in vivo models of immune cell migration-related diseases such as hepathic fibrosis, pulmonary arterial hypertension (PAH), hypertension, proteinuria, glomerulosclerosis, kidney fibrosis and myofibrosis cordis, and Alport syndrome, the disease state caused by immune cell migration and invasion was confirmed, and here, the effect of treating the disease of the inhibitory compound of KRS migration to the cell membrane was confirmed.
Example 5: Construction of Antibody for Reducing Cellular Membrane KRS Level and Verification of Immune Cell Migration/Invasion Control Effect
(40) 5-1. Construction of KRS-N Term Specific Binding Antibody_N3 Antibody
(41) As the compound of Example 4 exhibited an effect of treating and improving immune cell migration/invasion-related diseases by inhibiting an increase in KRS level at the site of a cell membrane, the present inventors attempted to generate an antibody showing excellent therapeutic efficacy on a similar principle. On the other hand, the present inventors have identified that when KRS moves from the cytoplasm and is located in the cell membrane, some N-terminal regions are also exposed to the extracellular membranes (usually amino acid regions 1 to 72 of the KRS N-terminus). Therefore, among the anti-KRS antibodies, the antibody capable of binding to the KRS-N terminus was thought to have a more remarkable advantage in in vivo in the immune cell migration/invasion inhibition, and as a representative example, the present inventors confirmed these therapeutic advantages through the construction of N3 antibody as described below. N3 antibody was obtained by the following method.
(42) Specifically, to select scFv that specifically binds only to the human KRS N-terminal (SEQ ID NO: 148) region exposed to the outer membrane when moved to the cell membrane by laminin signal from the human KRS full length sequence (SEQ ID NO: 118), a phage display panning experiment was performed using scFv phage library derived from human B cells labeled with HA tag. The scFv display phage library (Library size: app.7.6?109, Library produced by prof. Hyunbo Shim) used in this experiment is described in Korean Patent No. 10-0961392. Human KRS full-length sequences and KRS fragments of different specific regions of the N-terminal site were used as antigenic proteins for a phage display panning experiment.
(43) The phage display panning experiment was specifically performed as follows. 1-10 ?g of the antigenic protein was added to an Immuno-tube containing 1 ml of 1?PBS solution, and the antigen was coated on the inner surface of the tube by reacting at 37? C. and 200 rpm for 1 hour. The antigen solution was drained, and uncoated antigens were removed by washing once with tap water. In order to prevent nonspecific binding between antigen protein and phage, immuno-tube and scFv library were reacted with 1?PBST (PBS containing 0.05% tween20) containing 3% skim milk at room temperature for 1 hour. After removing the skim milk from the immuno-tube, the scFv library was added and reacted at 37? C. and 150 rpm for 1 hour to bind the scFv phage to the antigen. The scFv phage specifically bound to each antigen was separated within 10 minutes by adding 1 ml of triethylamine (100 mM) at room temperature and neutralized with Tris (1 M, pH 7.4). The filtered phage scFv was added to ER2537 E. coli incubated with OD<1 and then infected it during incubating at 37? C. and 120 rpm for 1 hour and 30 minutes. E. coli infected with phage were centrifuged to remove some of the culture supernatant, and redispersed to spread to agarose plates with a diameter of 15 cm containing ampicillin and glucose (2%). The next day 5 ml SB medium was spread to obtain all the cells grown on the plate, and glycerol (50%) was added to 0.5 times the total volume, mixed and aliquoted with 1 ml each to storage them at ?80? C. (scFv panning stock). Twenty ?l of the prepared stock was inoculated in a 20 ml SB solution and cultured, and a helper phage was used to prepare a scFv phage library (1 ml) for phage panning in the next step. The above procedure was repeated 2-3 times to isolate phage expressing an antigen specific scFv.
(44) After the biopanning, the western blotting, and the immunoprecipitation were further performed to select scFv clones having high binding ability to the target protein.
(45) The selected scFvs were converted to IgG (total antibody), and specific methods are as follows. First, polynucleotides encoding scFv in the genome of the scFv clone were amplified by PCR. The base sequences of the primers used to amplify the genes of the VH region of scFv are as follows: Forward (AGA GAG TGT ACA CTC C CA GGC GGC CGA GGT GCA G, SEQ ID NO: 129), and Reverse (CGC CGC TGG GCC CTT GGT GGA GGC) TGA GCT CAC GGT GAC CAG, SEQ ID NO: 130). The nucleotide sequence of the primers used to amplify the gene of the VL region of scFv is as follows: Forward (AAG CGG CCG CCA CCA TGG GAT GGA GCT GTA TCA TCC TCT TCT TGG TAG CAA CAG CTA CAG GTG TAC ACT CCC AGT CTG TGC TGA CTC AG, SEQ ID NO: 131), and Reverse (CGC CGC CGT ACG TAG GAC CGT CAG CTT GGT, SEQ ID NO: 132). PCR was performed with each phage DNA (50 ng) as a template by using the primers (10 pmol each) in conditions of: 95? C./3 min; 95? C./30 sec, 60? C./30 sec, 72? C./30 sec, 30 cycles; 72? C./5 min, thereby amplifying the VH or VL gene of scFv. The PCR product was inserted into the pcDNA3.4 vector (Thermo Fisher Scientific), a vector used for IgG production using restriction enzymes. The heavy and light chain proteins of IgG were individually expressed in plasmids respectively.
(46) The vector containing the DNA encoding the light and heavy chains of the IgG including the variable region of scFv prepared in this way was cotransformed into freestyle 293F cells (ATCC) to allow the light and heavy chains to be expressed together in the cells. Transformed 293F cells were incubated at 37? C. and 8% CO.sub.2 conditions for 7 days to obtain supernatants. The supernatants were filtered using a cellulose acetate membrane filter (0.22 ?m pore size, Corning) and purified using CaptivA? PriMAB protein A column (Repligen, USA). The obtained antibody concentration was measured using a BCA kit (Pierce, 23225), and the IgG antibody protein produced under reduced and non-reduced conditions was analyzed using a Bioanalyzer (Agilent 2100 Bioanalyzer).
(47) 5-2. Identification of the Inhibitory Efficacy on Immune Cell Migration and Invasion
(48) The effects on immune cell migration and invasion of the various candidate antibody generated in 5-1 were confirmed. The specific experimental method is as follows. Transwell (Corning #3421-5 mm) was coated with gelatin (0.5 mg/ml), and then RAW 264.7 cells (1?105 cells/well) were seeded into the top chamber. Serum Free DMEM (500 ?l) containing Laminin 421 (1 ?g/ml) was placed in the bottom chamber. Each antibody was treated at 100 nM concentration in the top chamber. After 24 hours, the cells were fixed with 70% methanol for 30 min and then stained with 50% Hematoxylin for 30 min. After removing the non-migrating cells in the upper part of the membrane with a cotton swab, the membrane was taken and mounted on the slide. The migrating cells on the underside of the membrane were observed under a high magnification microscope (
(49) In addition, the cells were incubated and harvested for 24 hours by treating Laminin 421 (1 ?g/ml) and antibody (100 nM) in RAW 264.7 cells. Subsequently, using the ProteoExtracttesubcellular proteom extraction kit (Calbiochem), the samples were divided into membrane and cytosol fractions, and then the western blotting was performed on KRS. The specific method is as described in the Example 2.
(50) As a result, it was confirmed that the N3 monoclonal antibody (an antibody specifically binding to KRS N-term) of the present invention effectively inhibits LN421-dependent monocyte/macrophage migration, which is shown in
(51) 5-3. N3 Antibody Sequencing
(52) The sequences of N3 scFv (SEQ ID NO: 59) and N3 IgG antibodies (SEQ ID NO: 89 and SEQ ID NO: 91) was confirmed using Omp primer according to the method described in Hye young Yang, et. al., 2009, Mol. Cells 27, 225-235. The sequence thus obtained was confirmed the sequence of the CDR region using the Bioedit program. The results of sequencing of the antigen binding site were shown in Table 1, and the scFv further included a linker of SEQ ID NO: 57.
(53) TABLE-US-00001 TABLE1 Aminoacid sequence DNAsequence VH CDR- SYDMS agttatgatatgagc H1 (SEQIDNO:1) (SEQIDNO:2) CDR- AISYDNGNTYYADSV gcgatctcttatgataatggtaatacatattacgctgatt H2 KG ctgtaaaaggt (SEQIDNO:3) (SEQIDNO:4) CDR- MALDFDY atggcgcttgatttcgactac H3 (SEQIDNO:5) (SEQIDNO:6) Full(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSAISYDNGNTYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYSARMALDFDYWGQGTLVTVSS (SEQIDNO:31) gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcct gtgcagcctctggattcacctttagcagttatgatatgagctgggtccgccaggctccagggaa ggggctggagtgggtctcagcgatctcttatgataatggtaatacatattacgctgattctgta aaaggtcggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcc tgagagccgaggacacggccgtgtattactctgcgagaatggcgcttgatttcgactactgggg ccagggtacactggtcaccgtgagctca (SEQIDNO:32) VL CDR- TGSSSNICSNYVT actggctcttcatctaatattggcagtaattatgtcacc L1 (SEQIDNO:7) (SEQIDNO:8) CDR- DNSNRPS gataatagtaatcggccaagc L2 (SEQIDNO:9) (SEQIDNO:10) CDR- ASWDDSLSAYV gcttcttgggatgatagcctgagtgcttatgtc L3 (SEQIDNO:11) (SEQIDNO:12) Full(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQLPGTAPKLLIYDNSNRPSGVPDRFS GSKSGTSASLAISGLRSEDEADYYCASWDDSLSAYVFGGGTKLTVL (SEQIDNO:33) cagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctctt gtactggctcttcatctaatattggcagtaattatgtcacctggtaccagcagctcccaggaac ggcccccaaactoctcatctatgataatagtaatcggccaagcggggtccctgaccgattctct ggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctg attattactgtgcttcttgggatgatagcctgagtgcttatgtcttcggcggaggcaccaagct gacggtccta (SEQIDNO:34)
5-4. Verification of Human KRS Binding Site of N3 Monoclonal Antibody
(54) In the N-terminal region of human KRS, the following experiment was performed to confirm the N3 monoclonal antibody binding site. First, as shown in
(55) TABLE-US-00002 TABLE2 Species MW F1(1-29) MAAVQAAEVKVDGSEPKLSK Human 3168 NELKRRLKA (SEQIDNO:133) F2(5-34) QAAEVKVDGSEPKLSKNELK Human 3351 RRLKAEKKVA (SEQIDNO:134) F3(10-38) KVDGSEPKLSKNELKRRLKA Human 3310 EKKVAEKEA (SEQIDNO:135) F4(15-42) EPKLSKNELKRRLKAEKKVA Human 3337 EKEAKQKE (SEQIDNO:136) F5(24-49) KRRLKAEKKVAEKEAKQKEL Human 3084 SEKQLS (SEQIDNO:137) mF1(1-28) MATLQESEVKVDGEQKLSKN Mouse 3230 ELKRRLKA (SEQIDNO:138) mF2(3-34) QESEVKVDGEQKLSKNELKR Mouse 3383 RLKAEKKLA (SEQIDNO:139) mF3(10-37) KVDGEQKLSKNELKRRLKAE Mouse 3268 KKLAEKEA (SEQIDNO:140) mF4(15-41) QKLSKNELKRRLKAEKKLAE Mouse 3253 KEAKQKE (SEQIDNO:141) mF5(24-48) RRLKAEKKLAEKEAKQKELS Mouse 2997 EKQLN (SEQIDNO:142) rF1(1-28) MATLREGEVKLDGEPKLSKN Rat 3211 ELKRRLKA (SEQIDNO:143) rF2(3-34) REGEVKLDGEPKLSKNELKR Rat 3364 RLKAEKKLA (SEQIDNO:144) rF3(10-37) KLDGEPKLSKNELKRRLKAE Rat 3251 KKLAEKEA (SEQIDNO:145) rF4(15-41) PKLSKNELKRRLKAEKKLAE Rat 3222 KEAKQKE (SEQIDNO:146) rF5(24-48) RRLKAEKKLAEKEAKQKELS Rat 2997 EKQLN (SEQIDNO:147)
(56) As shown in
(57) 5-5. Verification of Efficacy Evaluation of N3 Monoclonal Antibody in Other Animal Models
(58) In order to confirm whether the efficacy of the N3 antibody of the present invention can be evaluated by constructing disease animal models in mice and rats, sequence similarity across species in the KRS N-terminal sequences was analyzed. As shown in
(59) As shown in
Example 6 Efficacy Verification of KRS-N Term Specific Binding Antibodies in Immune Cell Migration-Related Diseases in In Vivo Models_In Vivo Pulmonary Hypertension Models
(60) Treatment of an antibody that specifically binds to the KRS N-terminus inhibits immune cell migration/invasion through a decrease in KRS levels (through endocytosis, etc.) at the site of a cell membrane, resulting in the same effect as the compound of Example 4 (decrease of KRS level in a cell membrane). Therefore, it is apparent that the KRS N-term specific antibody of the present invention (typically N3 antibody) will show a therapeutic effect against the same indication (diseases related to immune cell migration) of the compound of Example 4, which is further demonstrated through the following examples.
(61) Experiment Methods
(62) 1) Construction of Pulmonary Arterial Hypertension (PAH) Models and Administration of a Test Substance
(63) To induce PAH in 7-week-old SD rats (Oriental Bio), 60 mpk of MCT (monocrotaline) were subcutaneously injected. Thereafter, the rats were divided into four groups (tested with five animals in each group), and were administrated with 1 mpk of Mock human IgG (Thermo Fisher Scientific, negative control), 1mpk of N3 IgG antibody, 10mpk of N3 IgG antibody, and 25 mpk of sildenafil (positive control) for 3 weeks. All antibodies were i.v. injected twice a week and sildenafil was orally administered every day.
(64) 2) Blood Flow and Blood Pressure Measurement
(65) After three weeks, the rats were anesthetized with isoflurane, and blood flow and pressure were measured using an MPVS Cardiovascular Pressure and Volume system (model name: MPVS Ultra, manufacturer: Millar Instruments). The right ventricular end-systolic pressure (RVESP), right ventricular end-diastolic pressure, left ventricular end-systolic pressure, left ventricular end-diastolic pressure were measured using an exclusive catheter (Mikro-Tip rat pressure catheter, manufacturer: Millar Instruments). The cardiac output was measured using a perivascular blood flow probe (Transonic Flow probes, manufacturer: Millar Instruments), and experimental method thereof was performed by the same method as disclosed in the following literature: Pacher P, Nagayama T, Mukhopadhyay P, Batkai S, Kass D A. Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats. Nat Protoc 2008; 3(9):1422-34.
(66) 3) Immunohistochemistry (IHC)
(67) The collected lungs were fixed in PFA (paraformaldehyde) according to a conventional procedure, and then embedded in paraffin through washing, dehydration, and clearing. The paraffin blocks of Rat lung tissue were cut into 3 ?m thickness and a slide were manufactured. The sample was first treated with xylene for 5 min three times, treated with 100% ethanol, 95% ethanol, 90% ethanol, and 70% ethanol, and DW in that order for 2 min, and washed with PBS for 5 min. After 0.3% H.sub.2O.sub.2 treatment, the sample was washed with PBS for 5 min twice. After soaking in 0.01 M citrate buffer and heated, the sample washed with PBS-T (0.03% tween 20), and then blocking was performed at room temperature for 30 minutes (2% BSA & 2% goat serum in PBS). It was stained overnight at 4? C. with anti-CD68 antibody (1:200, ED1 clone, Abcam). After washing three times with PBS-T for 5 minutes, the sample was treated with a polymer-HRP anti-mouse envision kit (DAKO) for 1 hour at 4? C. After washing three times with PBS-T, the sample was developed by treatment with DAB substrate buffer and DAB chromogen 20. The stained tissue was treated with Mayer's hematoxylin (Sigma) for 1 minute, and then treated twice for 2 minutes in order of 70% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol. Finally, the tissue was treated with xylene three times for 5 min, and then observed under an optical microscope.
(68) Results
(69) 6-1. Verification of Blood Pressure and Cardiac Output Changes.
(70) The models of PAH, which is a disease having a close relation between immune cell invasion and pathological phenomena, were treated with N3 IgG antibody (1 mpk or 10 mpk) for 3 weeks (i.v., twice a week), and then measured for right ventricular end-systolic pressure (RVESP), right ventricular end-diastolic pressure (RVEDP), left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), and cardiac output (CO). The results thereof are shown in Table 3.
(71) TABLE-US-00003 TABLE3 MCT+ MCT+Mock MCT+ N3Ab MCT+ IgG N3Ab1mpk 10mpk Sildenafil (n=4) (n=5) (n=5) (n=5) RVESP 62.5?5.7 45.0?8.1 41.2?7.7 48.4?9.6 (mmHg) RVEDP 2.8?1.5 1.4?2.2 3.8?1.3 2.6?1.3 (mmHg) LVESP 81.5?11.4 95.8+4.8 93.4?11.3 83.2?4.7 (mmHg) LVEDP 1.0?0.8 2.6?1.9 4.6+3.9 3.6?2.3 (mmHg) CO(ml/min) 58?4.7 74.0?10.9 59.8?12.9 49.6?17.7 (n=4) (n=5) (n=5) (n=4)
(72) (CO was not measured in one animal of MCT+mock IgG group and one animal of sildenafil treatment group, since they died from anesthesia, and during surgery, respectively)
(73) Pulmonary hypertension causes the right ventricular pressure to rise due to narrowing of the pulmonary artery, resulting in right ventricular failure. In addition, if the reward mechanism is destroyed by persistent hypertension, right ventricular enlargement is followed by right ventricular enlargement. This causes the left ventricle compression due to the movement of the interventricular septum and a decrease in the left ventricular end diastolic volume and cardiac output (Lee Woo-seok et al., 2007; 37: 265-270). As a result, pulmonary hypertension is primarily associated with the right ventricle, but also with the function of the left ventricle.
(74) PAH patients showed a RVESP increase, which has also been confirmed in the PAH animal models of this experiment. In contrast, as shown in
(75) In addition, there was no decrease in the left ventricular end systolic pressure (LVESP) following administration of the N3 antibody (an antibody specifically binding to KRS N-term). Instead, LVESP was significantly increased in the group administered with the antibody of the present invention as shown in
(76) In summary, it was confirmed that administration of the KRS N-term binding antibody (particularly, N3 antibody) of the present invention reduced the risk of side effects of existing therapeutic drugs and showed PAH symptom alleviation and treatment effects.
(77) 6-2. Echocardiography
(78) The D-shaped left ventricle finding indicating pressure overload in the right ventricle was observed in three animals in the MCT alone administration group (i.e., test substance non-administration PAH models) and three animals in the MCT+sildenafil administration group, but was not observed in the therapeutic antibody administration groups.
(79) In addition, as shown in Table 4 below, the weight of each group was increased to a similar degree, with no significant difference. That is, the findings were not observed to indicate abnormal signs, including abnormal weight reduction, caused by the administration of the therapeutic antibody.
(80) TABLE-US-00004 TABLE 4 MCT + MCT + MCT + Mock Ab 1 Ab 10 MCT + IgG mpk mpk Sildenafil (n = 4) (n = 5) (n = 5) (n = 5) Absolute 101.4 ? 14.2 113.5 ? 14.6 104.1 ? 12.3 104.1 ? 26.4 change (g) Relative 48.8 ? 7.8 43.6 ? 5.2 40.7 ? 5.0 49.8 ? 10.5 change (%)
6-3. Verification of Monocyte/Macrophage Migration and Infiltration Degrees
(81) IHC staining was performed with respect to CD68, which is a monocyte/macrophage marker, by using the lung tissues of each experimental group. As shown in
Example 7: Verification of KRS-N Term Specific Binding Antibody Effect on Immune Cell Migration-Related Diseases In Vivo Models Acute Lung Injury Models
(82) Methods
(83) 1) Construction of LPS-Induced Acute Lung Injury Models and Administration of Test Substance
(84) Mouse models of acute lung injury were constructed by intratracheal injection of 2.5 mg/kg LPS (Sigma) into 7-week-old male C57BL/6 mice (duothermal bio). To investigate the effects of KRS inhibitors on acute lung injury, first, the intravenous injection of N3 IgG antibody to C57BL/6 mice was performed at 1 mg/kg or 10 mg/kg, respectively, followed by endotracheal injection of 2.5 mg/kg of LPS after 24 hours. Twenty-four hours after the LPS injection, each mouse was sacrificed to collect and analyze lung tissue and BALF (Bronchoalveolar lavage fluid).
(85) 2) Immune Cell Count in Bronchoalveolar Lavage Fluid (BALF)
(86) BALF obtained by washing the lungs with PBS was collected and pellets were collected by centrifugation at 800?g for 10 minutes at 4? C. After the cells were suspended, red blood cells were removed using RBC lysis buffer (eBioscience cat. no. 00-4333-57). After stopping the reaction with PBS, washed twice, and suspended in 400 ?l PBS to measure the number of cells by hemocytometer and neutrophil number through Hema3 staining.
(87) 3) FACS on Immune Cells in Lung Tissue
(88) Lung tissues were collected and rotated for 45 min at 37? C. using gentleMACS Octo Dissociator (MACS Miltenyi Biotec, Order no. 130-095-937) to crush tissue. After filtering using a cell strainer (40 ?m) was centrifuged at room temperature for 5 minutes at 1500 rpm. The pellet was collected and red blood cells were removed using RBC lysis buffer (eBioscience cat. no. 00-4333-57). The cells were collected and suspended in FACS buffer (PBS containing 1% NaN3 and 3% FBS), 50 ?l were placed in a tube, mixed well with the same amount of antibody mxiture, and stained by blocking light at 4? C. for 1 hour. FITC Rat Anti-CD11 b (BD Pharmingen) and PE Rat Anti-Mouse F4/80 (BD Pharmingen) antibodies were used for analysis of interstitial macrophage (IM) moving to the lungs. After washing twice at 400?g for 5 minutes using FACS buffer, it was analyzed by Navios Flow Cytometer (Beckman).
(89) 4) Masson's Trichrome Staining for Lung Tissue
(90) Lung tissue was embedded in paraffin in the original manner and then cut out. Thereafter, the tissue slide from which paraffin was removed using xylene was washed with DW, and then treated with Bouin Fluid at 56-60? C. for 1 hour. After stained with Weigert's iron hematoxylin solution for 10 minutes, the tissue slide was washed. After stained again with Biebrich scarlet-acid fuchsin solution for 10-15 minutes, the silde was washed. Phosphomolybdic-phosphotungstic acid solution was treated to the slide for 10-15 minutes, and then the slide was transferred to aniline blue solution and stained for 5-10 minutes. After washing, the slide was treated with 1% acetic acid solution for 2-5 minutes. After washing and dehydration, the slide was treated with xylene and mounted.
(91) Results
(92) 7-1. Verification of the Inhibitory Effect on Immune Cell Migration in Bronchoalveolar Lavage Fluid (BALF)
(93) As shown in
(94) 7-2. Verification of the Antibody Inhibitory Effect on Immune Cell Migration in Lung Tissue
(95)
Example 8: Construction of N3 Modified Antibody with Increased Affinity for the N-Terminus of KRS
(96) The present inventors attempted to obtain an antibody having better affinity for the N-terminal region of KRS by modifying the N3 antibody in order to produce an antibody that shows better performance as a therapeutic antibody. Therefore, the light chain variable region and heavy chain variable region of the N3 antibody were improved by the following series of processes.
(97) 8-1. Construction of the scFab Library in N3 Antibody-Based Sequence Variants (Yeast Cell Surface Expression Library)_Primary Library
(98) Using the Homology modeling method, the rough structure of the N3 antibody was predicted, and through this, a random mutation was introduced into a CDR region predicted to play an important role in antigen binding to construct a library. Specifically, in the library based on the heavy chain variable region, NNK, which is a degenerated codon that can include all 20 amino acid sequences for residues in the heavy chain CDR2 or CDR3, was used. In the light chain variable region library, NNK, a degenerated codon that can contain all 20 amino acid sequences for residues in the light chain CDR2 or CDR3 of the N3 antibody, was used. The DNA encoding the designed library was amplified using a PCR technique, and then concentrated using an ethanol precipitation method.
(99) Thereafter, a scFab library expressed on surface of yeast containing various variations of the light chain variable region was constructed with reference to the method described in the following literature: Baek D S and Kim Y S, Construction of a large synthetic human Fab antibody library on yeast cell surface by optimized yeast mating, J Microbiol Biotechnol. 2014 Mar. 28; 24(3):408-20.
(100) Briefly, yeast surface expression vectors (C-aga2: pYDS) expressing aga2 protein at the C-terminus for homologous recombination were treated with Nhel and MIul restriction enzymes, purified using agarose gel extraction, and concentrated using ethanol precipitation method.
(101) A 4 ?g of vector treated with the restriction enzyme in 12 ?g of each library-coding DNA was transformed by electroporation into yeast EBY100 (yeast for surface expression), and the library size was confirmed by measuring the number of colonies grown in the selection medium, SD-CAA (20 g/L Glucose, 6.7 g/L Yeast nitrogen base without amino acids, 5.4 g/L Na.sub.2HPO.sub.4, 8.6 g/L NaH.sub.2PO.sub.4, 5 g/L casamino acids) through serial dilution.
(102) 8-2. Screening scFab with Improved Affinity for KRS (Residues 1-72 Aa) Peptide First Library Selection
(103) Fabs with increased affinity were selected for the library constructed in the Example 8-1 using GST-conjugated KRS (residues 1-72 aa, N-term) peptide as an antigen.
(104) Specifically, 10 nM of GST-conjugated KRS peptide (residues 1-72 aa, purified state) using SG-CAA medium (20 g/L Galactose, 6.7 g/L Yeast nitrogen base without amino acids, 5.4 g/L Na.sub.2HPO.sub.4, 8.6 g/L NaH.sub.2PO.sub.4, 5 g/L casamino acids) for primary FACS screening was reacted with yeast inducing scFab library expression on the cell surface in Example 8-1 for 1 hour at room temperature. Thereafter, GST-conjugated KRS (residues 1-72) peptide and yeast expressing the library were reacted with PE-conjugated Streptavidin-R-phycoerythrin conjugate (SA-PE) at 4? C. for 20 minutes and were suspended by FACS (Fluorescence activated cell sorting, FACS Caliber; BD biosciences). Subsequently, a second FACS screening was performed with KRS (residues 1-72) peptide conjugated with 1 nM GST, and a third FACS screening was performed with KRS (residues 1-72) peptide conjugated with 0.5 nM GST.
(105) Through the selection process using the FACS, clones with high affinity for the KRS (residues 1-72) peptide were selected compared to the N3 antibody. In this way, three excellent clones (N3-1, N3-3, N3-4) having high affinity and specificity for KRS (residues 1-72) peptide were selected through analysis of binding ability for individual clones, and another light chain variable region and heavy chain variable region were combined with each other to construct another unique clone N3-5. The N3-5 clone was also confirmed to have excellent affinity and specificity for the KRS (residues 1-72) peptide, and finally a total of four unique (magnetic activated cell sorting) clones, N3-1, N3-3, N3-4, and N3-5, was selected.
(106) The CDR sequences of the light chain variable region and the heavy chain variable region of four individual clones, N3-1, N3-3, N3-4, and N3-5, showing high binding ability to the KRS (residues 1-72) peptide were shown in Table 5, and Table 6 showed the full sequences of heavy chain variable region and light chain variable region.
(107) TABLE-US-00005 TABLE5 Heavy Light CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 N3 SYDMS AISYDNGNTYYADSV MALDFDY TGSSSNIGSNYVT DNSNRPS ASWDDSLSAYV (SEQID KG (SEQID (SEQIDNO: (SEQID (SEQIDNO: NO:1) (SEQIDNO:3) NO:5) 7) NO:9) 11) N3-1 SYDMS AISYDNGNTYYADSV MALDFDY TGSSSNIGSNYVT DNSNRPS ASFSDELGAYV (SEQID KG (SEQID (SEQIDNO: (SEQID (SEQIDNO: NO:1) (SEQIDNO:3) NO:5) 7 NO:9) 13) N-3 SYDMS AISYDNGNTYYADSV MALDFDY TGSSSNIGSNYVT DNSNRPS SSFSDELGAYV (SEQID KG (SEQID (SEQIDNO: (SEQID (SEQIDNO: NO:1) (SEQIDNO:3) NO:5) 7) NO:9) 15) N3-4 SYDMS VISSDGGNTYYADSV MALDFDY TGSSSNIGSNYVT DNSNRPS ASFSDELGAYV (SEQID KG (SEQID (SEQIDNO: (SEQID (SEQIDNO: NO:1) (SEQIDNO: NO:5) 7) NO:9) 13) 151) N3-5 SYDMS VISSDGGNTYYADSV MALDFDY TGSSSNIGSNYVT DNSNRPS SSFSDELGAYV (SEQID KG (SEQID (SEQIDNO: (SEQID (SEQIDNO: NO:1) (SEQIDNO: NO:5) 7) NO:9) 15) 151)
(108) TABLE-US-00006 TABLE6 SEQIDNO: Sequence (Sequencename) N3 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQA SEQIDNO:31 PGKGIEWVSAISYDNGNTYYADSVKGRFTISRDNSKNTLY (N3VH) LQMNSLRAEDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQL SEQIDNO:33 PGTAPKLLIYDNSNRPSGVPDRFSGSKSGTSASLAISGLR (N3VL) SEDEADYYCASWDDSLSAYVFGGGTKLTVL N3-1 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQA SEQIDNO:31 PGKGLEWVSAISYDNGNTYYADSVKGRFTISRDNSKNTLY (N3VH) LQMNSLRAEDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQL SEQIDNO:49 PGTAPKLLIYDNSNRPSGVPDRFSGSKSGTSASLAISGLQ (N3VLmutant1) SEDEADYYCASFSDELGAYVFGGGTKLTVL N3-3 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQA SEQIDNO:31 PGKGLEWVSAISYDNGNTYYADSVKGRFTISRDNSKNILY (N3VH) LQMNSLRAEDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQL SEQIDNO:51 PGTAPKLLIYDNSNRPSGVPDRFSGSKEGTSASLAISGLQ (N3VLmutant2) SEDEADYYCSSFSDELGAYVFGGGTKLTVL N3-4 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQA SEQIDNO:35 PGKGLEWVSVISSDGGNTYYADSVKGRFTISRDNSKNTLY (N3VHmutant1) LQMNSLRAEDTAVYYSARMALDFDYWGQGTLVTVSS QSVITQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQL SEQIDNO:49 VL PGTAPKLLIYDNSNRPSGVPDRFSGSKSGTSASLAISGLQ (N3VLmutant1) SEDEADYYCASFSDELGAYVFGGGTKLTVL N3-5 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQA SEQIDNO:35 PGKGLEWVSVISSDGGNTYYADSVKGRFTISRDNSKNTLY (N3VHmutant1) LQMNSLRAEDTAVYYSARMALDFDYWGQGTIVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQL SEQIDNO:51 PGTAPKLLIYDNSNRPSGVPDRFSGSKSGTSASLAISGLQ (N3VLmutant2) SEDEADYYCSSFSDELGAYVFGGGTKLTVL
(109) In addition, the affinity of the selected individual clones, N3-1, N3-3, N3-4, and N3-5, for the KRS N-terminus was reconfirmed using the ELISA method. In this experiment, the clones converted to IgG antibody were tested. The conversion to an IgG antibody refers to the method of Example 5-1 above.
(110) Specifically, the N-terminal region (residues 1-72) peptide of KRS was treated in a 96-well EIA/RIA plate (COSTAR Corning) and bound at 25? C. for 1 hour, followed by washing 3 times with PBS (pH 7.4, 137 mM NaCl, 12 mM phosphate, 2.7 mM KCl) (SIGMA) for 10 minutes. Thereafter, 4% BSA PBS (4% Bovine Serum Albumin, pH7.4, 137 mM NaCl, 12 mM phosphate, 2.7 mM KCl) (SIGMA) was treated for 1 hour, and then washed 3 times with PBS for 10 minutes. Then, the N3 antibody, N3-1 antibody, N3-3 antibody, N3-4 antibody, and N3-5 antibody of IgG-type were treated and bound, respectively, and then washed three times with 0.1% PBST for 10 minutes. As a labeled antibody, Horseradish peroxidase-conjugated anti-human mAb (SIGMA) was used. Then, it was reacted with TMB (3,3, 5,5-Tetramethylbenzidine) (Sigma) and measured at 450 nm absorbance to quantify.
(111) As a result, as shown in
(112) 8-3. Comparison of Affinity Between N3 Antibody and Primary Improved Antibody and Verification of their Inhibitory Effect on Cell Migration
(113) There was no significant difference in binding ability between KRS and the improved antibodies, N3-1, N3-3, N3-4, and N3-5. Thus, using the representative N3-1 IgG antibody among the improved antibodies selected in the Example 8-2, the affinity of the parent antibody, N3 antibody and KRS N-terminus was more specifically compared. Using KRS fragment (1-207 aa) purified protein as an antigen, the binding ability between N3 antibody and N3-1 antibody (IgG) was analyzed through Surface Plasmon Resonance (SPR). The SPR experiment was performed using a Biacore T200 (GE Healthcare) equipped with a Series S sensor chip CM5 (GE Healthcare) at 25? C. After the antibody was immobilized on the chip using an amine coupling kit (GE Healthcare), the antigen was diluted 4 times in PBS solution in the range of 4.8 nM-1250 nM and flowed for 60 seconds. Thereafter, PBS was flowed for 300 seconds. The obtained data was analyzed with Biacore T200 Evaluation software v2.0 (GE Healthcare).
(114) As a result, as shown in
(115) The light chain variable region sequence was fixed with the sequence of the N3-3 antibody (SEQ ID NO: 51), and a library was constructed with various variable sequences in the heavy chain variable region. First, the homology modeling method was used to predict the approximate modeling structure of the N3-3 antibody, and through this, a random mutation was introduced into the CDR predicted to play an important role in antigen binding. Specifically, NNK, a degenerated codon capable of containing all 20 amino acid sequences, was used for the residues of CDR2 and CDR3 of the heavy chain variable region in the N3-3 antibody, and the scFab library was constructed in the same manner as in the Example 8-1.
(116) 8-5. Screening ScFab with improved affinity for KRS (residues 1-72) Secondary library screening
(117) Using the GST-conjugated KRS (residues 1-72) peptide as an antigen, Fabs with increased affinity were selected in the library constructed in the Example 8-4. Since the affinity of N3-3 and N3-1 was determined to be almost the same and the sequences were almost similar, the comparative experiment was performed with N3-1.
(118) First, GTP-conjugated KRS (residues 1-72) peptide was treated with the library-expressing yeast constructed in the Example 8-4. Subsequently, the yeast expressing the library bound with the GTP-conjugated KRS (resides 1-72) peptide was reacted with Streptavidin Microbead? (Miltenyi Biotec) at 4? C. for 20 minutes, and yeast expressing scFab having high affinity to the KRS (1-72 aa) peptide was suspended using magnetic activated cell sorting (MACS). The yeast expressing the library selected through the MACS was cultured in SG-CAA (20 g/L Galactose, 6.7 g/L Yeast nitrogen base without amino acids, 5.4 g/L Na.sub.2HPO.sub.4, 8.6 g/L NaH.sub.2PO.sub.4, 5 g/L casamino acids) medium to induce library expression. Subsequently, in the same manner as in the Example 8-2, sequential screening was performed using FACS. Briefly, the primary FACS screening was performed with KRS (1-72) peptide bound with 10 nM GST, the secondary FACS screening with KRS (1-72) peptide bound with 1 nM GST, the third FACS screening was performed with the KRS (1-72) peptide bound with 0.5 nM GST, and the forth FACS screening was performed with the KRS (1-72) peptide bound with 0.1 nM GST.
(119) Through the screening process using the magnetic activated cell sorting (MACS) and Fluorescence Activated Cell Sorting (FACS), clones with high affinity dependent on the heavy chain variable region (VH) for the KRS (1-72 aa) peptide compared to the N3-1 antibody (showing similar affinity to N3-3 antibody) were selected. In this way, four excellent clones, N3-6, N3-7, N3-8, and N3-9, with high affinity and specificity for KRS (residues 1-72) peptide were selected through binding ability analysis for individual clones.
(120) The CDR sequences of the light chain variable region and heavy chain variable region of four individual clones, N3-6, N3-7, N3-8, and N3-9, which show high binding ability to the KRS (1-72 aa) peptide, were shown in Table 7, and Table 8 showed the full sequences of heavy chain variable region sequence and light chain variable region.
(121) TABLE-US-00007 TABLE7 Heavy Light CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 N3-6 SYDMS AISPQMGR MALDFDY TGSSSNI DNSNRPS SSFSDELGA (SEQID VYYADSVK (SEQID GSNYVT (SEQID YV NO:1) G NO:5) (SEQID NO:9) (SEQID (SEQID NO:7) NO:15) NO:17) N3-7 SYDMS AIDPLGGN MALDFDY TGSSSNI DNSNRPS SSFSDELGA (SEQID IYYADSVK (SEQID GSNYVT (SEQID YV NO:1) G NO:5) (SEQID NO:9) (SEQID (SEQID NO:7) NO:15) NO:19) N3-8 SYDMS AISPYSGR MALDFDY TGSSSNI DNSNRPS SSFSDELGA (SEQID IYYADSVK (SEQID GSNYVT (SEQID YV NO:1) G NO:5) (SEQID NO:9) (SEQID (SEQID NO:7) NO:15) NO:21) N3-9 SYDMS AIGADGGP MALDFDY TGSSSNI DNSNRPS SSFSDELGA (SEQID SYYADSVK (SEQID GSNYVT (SEQID YV NO:1) G NO:5) (SEQID NO:9) (SEQID (SEQID NO:7) NO:15) NO:23)
(122) TABLE-US-00008 TABLE8 SEQIDNO: Sequence (Sequencename) N3-6 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVR SEQIDNO:37 QAPGKGLEWVSAISPQMGRVYYADSVKGRFTISRDNSK (N3VHmutant2) NTLYLQMNSLRAEDTAVYYSARMALDFDYWGQGTLVTV SS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQ SEQIDNO:51 QLPGTAPKLLIYDNSNRPSGVPDRFSGSKSGTSASLAI (N3VLmutant2) SGLQSEDEADYYCSSFSDELGAYVFGGGTKLTVL N3-7 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVR SEQIDNO:39 QAPGKGLEWVSAIDPLGGNIYYADSVKGRFTISRDNSK (N3VHmutant3) NTLYLQMNSLRAEDTAVYYSARMALDFDYWGQGTLVTV SS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQ SEQIDNO:51 QLPGTAPKLLIYDNSNRPSGVPDRFSGSKSGTSASLAI (N3VLmutant2) SGLQSEDEADYYCSSFSDELGAYVFGGGTKLTVL N3-8 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVR SEQIDNO:41 QAPGKGLEWVSAISPYSGRIYYADSVKGRFTISRDNSK (N3VHmutant4) NTLYLQMNSLRAEDTAVYYSARMALDFDYWGQGTLVTV SS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQ SEQIDNO:51 QLPGTAPKLLIYDNSNRPSGVPDRFSGSKSGTSASLAI (N3VLmutant2) SGLQSEDEADYYCSSFSDELGAYVFGGGTKLTVL N3-9 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVR SEQIDNO:43 QAPGKGLEWVSAIGADGGPSYYADSVKGRFTISRDNSK (N3VHmutant5) NTLYLQMNSLRAEDTAVYYSARMALDFDYWGQGTLVTV SS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQ SEQIDNO:51 QLPGTAPKLLIYDNSNRPSGVPDRFSGSKSGTSASLAI (N3VLmutant2) SGLQSEDEADYYCSSFSDELGAYVFGGGTKLTVL
(123) In addition, the affinity of the selected individual clones, N3-6, N3-7, N3-8, and N3-9, for the KRS N-terminus was reconfirmed using the ELISA method. After converting the clones to IgG antibodies in this experiment, they were used in ELISA experiments, and the method is referred to the Example 5-1 above.
(124) As a result, as shown in
(125) In addition, the result of inhibitory effects on cell migration by treating N3-6 antibody, N3-7 antibody, N3-8 antibody, and N3-9 antibody (IgG) as improved antibodies (see the above-described Examples for methods) was confirmed that the improved antibodies significantly inhibit cell migration than the N3-1 antibody. There was no significant difference in the inhibitory effect of cell migration among N3-6 antibody, N3-7 antibody, N3-8 antibody, and N3-9 antibody.
(126) 8-6. Verification of Epitopes and Affinity of Improved Antibodies N3-6, N3-7, N3-8, and N3-9
(127) Using KRS epitope peptide F4 (EPKLSKNELKRRLKAEKKVAEKEAKQKE: SEQ ID NO: 136) as an antigen epitope, the binding ability of N3 antibody, N3-6 antibody, N3-7 antibody, N3-8 antibody, and N3-9 antibody was analyzed through Surface Plasmon Resonance (SPR). SPR experiment was carried out in the same manner as in the Example 5-4. The epitope was diluted in PBS solution and diluted 2-fold in the range of 15.7 nM-4000 nM, and allowed to flow for 90 seconds. Thereafter, PBS was flowed for 2400 seconds. The obtained data was analyzed with Biacore T200 Evaluation software v2.0 (GE Healthcare).
(128) As a result, as shown in
(129) Also, as shown in
(130) 8-7. Production of Improved Antibody with Productivity and Stability Based on N3-8 Antibody (N3-8 Antibody Sequence Refinement)
(131) In the above example, it was confirmed that the N3-8 antibody had the best affinity to KRS (especially N-term). Thus, in order to confirm the properties such as productivity and stability of the N3-8 antibody and to make these properties even better, N3-8 derivatives were produced by inducing mutations in sequences expected to affect stability in the N3-8 antibody sequence.
(132) As a result, two heavy chain sequences (SEQ ID NO: 45, SEQ ID NO: 47) in which mutations were introduced into the heavy chain variable region of the N3-8 antibody were obtained. In addition, three light chain sequences (SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55) in which mutations were introduced into the light chain variable region were obtained. The seven types of sequences of N3-8 derivatives according to the combination of heavy and light chain sequences are shown in Tables 9 and 10 below, and they maintain the affinity properties of N3-8 antibodies.
(133) TABLE-US-00009 TABLE9 Heavy Light CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 N3- SYDMS AISPYSGRIYY MALDFDY TGSSSNIGSN DNSNRPS SSFSDELG 8-1 (SEQID ADSVKG (SEQID YVT (SEQID AYV NO:1) (SEQIDNO: NO:5) (SEQID NO:9) (SEQID 21) NO:7) NO:15) N3- SYDMS AISPYSGRIYY MALDFDY TGSSSNIGSN SNNQRPS SSFSDELG 8-2 (SEQID ADSVKG (SEQID YVT (SEQID AYV NO:1) (SEQIDNO: NO:5) (SEQID NO:27) (SEQID 21) NO:7) NO:15) N3- SYDMS AISPYSGRIYY MALDFDY TGSSSNIGSN RNNQRPS SSFSDELG 8-3 (SEQID ADSVKG (SEQID YVT (SEQID AYV NO:1) (SEQIDNO: NO:5) (SEQID NO:29) (SEQID 21) NO:7) NO:15) N3- SYDMS AISPYSGRIYY LALDFDY TGSSSNGSN DNSNRPS SSFSDELG 8-4 (SEQID ADSVKG (SEQID YVT (SEQID AYV NO:1) (SEQIDNO: NO:25) (SEQID NO:9) (SEQID 21) NO:7) NO:15) N3- SYDMS AISPYSGRIYY LALDFDY TGSSSNIGSN SNNQRPS SSFSDELG 8-5 (SEQID ADSVKG (SEQID YVT (SEQID AYV NO:1) (SEQIDNO: NO:25) (SEQID NO:27) (SEQID 21) NO:7) NO:15) N3- SYDMS AISPYSGRIYY LALDFDY TGSSSNIGSN RNNQRPS SSFSDELG 8-6 (SEQID ADSVKG (SEQID YVT (SEQID AYV NO:1) (SEQIDNO: NO:25) (SEQID NO:29) (SEQID 21) NO:7) NO:15) N3- SYDMS AISPYSGRIYY MALDFDY TGSSSNIGSN DNSNRPS SSFSDELG 8-7 (SEQID ADSVKG (SEQID YVT (SEQID AYV NO:1) (SEQIDNO: NO:5) (SEQID NO:9) (SEQID 21) NO:7) NO:15)
(134) TABLE-US-00010 TABLE10 SEQIDNO: Sequence (Sequencename) N3-8-1 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPG SEQIDNO:45 KGLEWVSAISPYSGRIYYADSVKGRFTISRDNSKNTLYLQMN (N3VHmutant SLRAEDTAVYYCARMALDFDYWGQGTLVTVSS 6) VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQLPG SEQIDNO:51 TAPKLLIYDNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDE (N3VLmutant: ADYYCSSFSDELGAYVFGGGTKLTVL 2) N3-8-2 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPG SEQIDNO:45 KGLEWVSAISPYSGRIYIADSVKGRFTISRDNSKNTLYLQMN (N3VHmutant SLRAEDTAVYYCARMALDFDYWGQGTLVTVSS 6) VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQLPG SEQIDNO:53 TAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDE (N3VLmutant ADYYCSSFSDELGAYVFGGGTKLTVL 3) N3-8-3 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPG SEQIDNO:45 KGLEWVSAISPYSGRIYYADSVKGRFTISRDNSKNTLYLQMN (N3VHmutant SLRAEDTAVYYCARMALDFDYWGQGTLVTVSS 6) VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQLPG SEQIDNO:55 TAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDE (N3VLmutant: ADYYCSSFSDELGAYVFGGGTKLTVL 4) N3-8-4 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPG SEQIDNO:47 KGLEWVSAISPYSGRIYYADSVKGRFTISRDNSKNTLYLQMN (N3VHmutant SLRAEDTAVYYCARLALDFDYWGQGTLVTVSS 7) VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQLPG SEQIDNO:51 TAPKLLIYDNSNRPSGVPDRFSGSKSGTSASLAISGLQEDE (N3VLmutant ADYYCSSFSDELGAYVFGGGTKLTVL 2) N3-8-5 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPG SEQIDNO:47 KGLEWVSAISPYSGRIYYADSVKGRFTISRDNSKNTLYLQMN (N3VHmutant SLRAEDTAVYYCARLALDFDYWGQGTLVTVSS V) VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQLPG SEQIDNO:53 TAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDE (N3VLmutant: ADYYCSSFSDELGAYVFGGGTKLTVL 3) N3-8-6 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPG SEQIDNO:47 KGLEWVSAISPYSGRIYYADSVKGPFTISRDNSKNTLYLQMN (N3VHmutant SLRAEDTAVYYCARLALDFDYWGQGTLVTVSS 7) VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQLPG SEQIDNO:55 TAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDE (N3VLmutant ADYYCSSFSDELGAYVFGGGTKLTVL 4) N3-8-7 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPG SEQIDNO:41 KGLEWVSAISPYSGRIYYADSVKGRFTISRDNSKNTLYLQMN (N3VHmutant SLRAEDTAVYYSARMALDFDYWGQGTLVTVSS 4) VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVTWYQQLPG SEQIDNO:51 TAPKLLIYDNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDE (N3VLmutant ADYYCSSFSDELGAYVFGGGTKLTVL 2)
(135) Table 11 shows the heavy chain (HC) and light chain (LC) sequences of the entire IgG antibodies used in the above-described examples.
(136) TABLE-US-00011 TABLE 11 Amino acid sequence DNA sequence N3 HC SEQ ID NO: 89 SEO ID NO: 90 LC SEO ID NO: 91 SEQ ID NO: 92 N3-1 HC SEQ ID NO: 89 SEO ID NO: 90 LC SEQ ID NO: 107 SEO ID NO: 108 N3-3 HC SEQ ID NO: 89 SEQ ID NO: 90 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-4 HC SEQ ID NO: 93 SEQ ID NO: 94 LC SEQ ID NO: 107 SEQ ID NO: 108 N3-5 HC SEQ ID NO: 93 SEQ ID NO: 94 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-6 HC SEQ ID NO: 95 SEQ ID NO: 90 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-7 HC SEQ ID NO: 97 SEQ ID NO: 98 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-8 HC SEQ ID NO: 99 SEQ ID NO: 100 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-9 HC SEQ ID NO: 101 SEQ ID NO: 102 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-8-1 HC SEQ ID NO: 103 SEQ ID NO: 104 LC SEQ ID NO: 111 SEQ ID NO: 112 N3-8-2 HC SEQ ID NO: 103 SEQ ID NO: 104 LC SEQ ID NO: 113 SEQ ID NO: 114 N3-8-3 HC SEQ ID NO: 103 SEQ ID NO: 104 LC SEQ ID NO: 115 SEQ ID NO: 116 N3-8-4 HC SEQ ID NO: 105 SEQ ID NO: 106 LC SEQ ID NO: 111 SEQ ID NO: 112 N3-8-5 HC SEQ ID NO: 105 SEQ ID NO: 106 LC SEQ ID NO: 113 SEQ ID NO: 114 N3-8-6 HC SEQ ID NO: 105 SEQ ID NO: 106 LC SEQ ID NO: 115 SEQ ID NO: 116 N3-8-7 HC SEQ ID NO: 99 SEQ ID NO: 100 LC SEQ ID NO: 111 SEQ ID NO: 112
8-8. Measurement of Productivity and Stability of N3-8 Derivatives (Tm Measurement)
(137) Each antibody protein was expressed and purified by a transient transfection method using plasmids expressing the light and heavy chains of the N3-8 derivative antibodies obtained in the Example 8-7. In HEK293-F cells (Invitrogen) suspended in serum-free FreeStyle 293 expression medium (Invitrogen) in a shake flask, the plasmid and polyethylenimine (Polyethylenimine, Polyscience) were transfected.
(138) Specifically, during transfection into a 200 mL shake flask, HEK293-F cells were seeded in 100 ml of medium at a density of 2?10.sup.6 cells/ml and cultured at 150 rpm and 37? C. with 8% 002. The heavy chain plasmid and light chain plasmid suitable for the production of each monoclonal antibody were transfected by treating them in a ratio of heavy chain: light chain DNA 1:1 or 1:2 (mixed treatment in 10 ml FreeStyle 293 expression medium). First, when heavy chain: light chain DNA is used in a 1: 1 ratio, 125 ?g heavy chain, 125 ?g light chain, and 250 ?g (2.5 ?g/ml) in total are mixed with 10 ml of medium diluted with PEI 750 ?g (7.5 ?g/ml) at room temperature. The reaction was carried out for 10 minutes. In the case of the ratio 1: 2, the concentration of the light chain DNA was doubled. Thereafter, the mixed medium was treated with HEK293-F cells previously aliquoted with 100 ml, and incubated at 150 rpm and 37? C., with 8% CO.sub.2, and for 4 hours. And the remaining 100 ml of FreeStyle 293 expression medium was added and cultured for 6 days.
(139) Then, the cell culture solution was transferred to 50 ml tubes and centrifuged for 5 minutes at 3000 rpm. Protein was then purified from the collected cell culture supernatant. The supernatant was applied to a Protein A Sepharose column, and then washed with PBS (pH 7.4). After eluting the antibody at pH 3.0 with 0.1 M glycine buffer, the sample was immediately neutralized with 1 M Tris buffer. The eluted antibody fraction was concentrated by exchanging buffer with PBS (pH 7.4) through a dialysis method. The purified protein was quantified using absorbance and absorption coefficient at a wavelength of 280 nm.
(140) In addition, the thermal stability of the antibody was measured using 100 ?l of the purified antibodies at a concentration of 1 mg/ml. Protein thermal shift Dye kit (Thermofisher) was performed 4 times with Quant Studio 3 Real-time PCR equipment (Thermofisher).
(141) As a result, as shown in Table 12 below, it was confirmed that the yield of all N3-8 derivative antibodies tested was improved over that of the N3-8 antibody. In addition, as shown in Table 12, Tm values were obtained, and thermal transition was observed differently according to the antibody as Tm1-Tm2, but it was found that the Tm value was increased in all N3-8 derivative antibodies.
(142) Through this, it was confirmed that the N3-8 antibody derivatives had higher yield and improved thermal stability than the N3-8 antibody.
(143) TABLE-US-00012 TABLE 12 Yield (mg/L) Heavy chain DNA: Heavy chain DNA: Light chain DNA Light chain DNA Thermal stability Antibody 1:1 1:2 Tm1 Tm2 N3-8 69.9 104.61 67.37 N3-8-1 87.13 109.9 69.94 N3-8-2 96.76 109.68 72.41 N3-8-3 93.44 93.53 71.02 76.31 N3-8-4 86.14 89.23 70.31 N3-8-5 84.31 107.37 72.9 N3-8-6 105.95 92.9 71.0 76.97 (In the above table, refers to the same value as Tm1)
Example 9: Confirmation of Increased Therapeutic Efficacy of N3 Improved Antibody
(144) With respect to the N3 improved antibodies produced in the above example, it was confirmed whether the therapeutic effect is remarkable for immune cell migration-related diseases even in vivo. Pulmonary arterial hypertension model was used as a disease model, and N3-6 antibody and N3-8 antibody were typically used as improved antibodies.
(145) To induce pulmonary arterial hypertension in 7-week-old SD rats (Orient Bio), 60 mpk of MCT (monocrotaline) was injected subcutaneously. After inducing disease for 2 weeks, it was divided into four groups (five experiments per group), and PBS, 1 mpk of N3 antibody 1 mpk, 1 mpk of N3-6 antibody, and 1 mpk of N3-8 antibody were administered for 3 weeks, respectively. All antibodies were injected with i.v. twice a week. The measurement of blood flow and blood pressure, and immunohistochemical staining (IHC) were performed for each experimental group in the same manner as described in the Example 6 above. In addition, the lung tissue of each experimental group was stained with Masson's trichrome staining method to measure the degree of lung fibrosis, and Masson's trichrome staining method was performed in the same manner as described in the Example 7.
(146) In the lungs of animals treated with MCT alone, endothelitis of the pulmonary vessels, smooth muscle proliferation of the arterioles, intimal hyperplasia, and vascular occlusion were observed. Therefore, it was confirmed that an animal model of pulmonary arterial hypertension disease was well produced.
(147) The phenomena exhibited by the pulmonary arterial hypertension were significantly reduced by treating the antibody of the present invention. Specifically, as shown in
(148)
(149) In addition, as shown in
INDUSTRIAL APPLICABILITY
(150) The present invention relates to a novel use of an antibody biding specifically to the N-terminus of lysyl-tRNA synthetase and, more particularly, to a pharmaceutical composition comprising an antibody biding specifically to an epitope including the sequence of SEQ ID NO: 117 in the N-terminal domain of lysyl-tRNA synthetase (KRS) or a functional fragment thereof as an effective ingredient for preventing and treating an immune cell migration-related disease. A KRS N-terminus-specific antibody provided by the present invention can regulate the migration of immune cells, and thus can be very usefully used in the prevention, alleviation, and treatment of immune cell migration-related diseases, and therefore have high industrial applicability.