Antibody for Specifically Binding to Lysyl-tRNA Synthetase N-Terminal Domain Exposed to Extracellular Membrane

Abstract

The present invention relates to an antibody that specifically binds to the lysyl-tRNA synthase N-terminal domain exposed to the extracellular membrane, more specifically, it specifically binds to the lysyl-tRNA synthetase (KRS, Lysyl-tRNA synthetase)N-terminal domain exposed to the extracellular membrane having a specific CDR (complementarity determining region) sequence described herein, and relates to the use for the prevention, treatment or diagnosis of cancer, cancer metastasis, or diseases related to immune cell migration of a composition comprising an antibody or fragment thereof having high affinity and stability or the antibody and fragment thereof as an active ingredient. The method of the present invention can be usefully used to prepare an antibody having a higher affinity for the KRS N-terminus than a conventional antibody.

Claims

1. An antibody or fragment thereof that specifically binds to an extracellularly exposed lysyl-tRNA synthetase (KRS)N-terminal region, the antibody or fragment thereof comprises: (a) a heavy chain variable region (VH) comprising (i) heavy chain complementarity determining region 1 (CDR1) containing an amino acid sequence SYDMS; (ii) heavy chain complementarity determining region 2 (CDR2) containing an amino acid sequence X.sub.1|X.sub.2X.sub.3X.sub.4X.sub.5GX.sub.6X.sub.7YYADSVKG, wherein X.sub.1 is A or V, X.sub.2 is S, D or G, X.sub.3 is Y, P, S or A, X.sub.4 is D, Q, L or Y, X.sub.5 is N, M, S, or G, X.sub.6 is N, R or P, X.sub.7 is T, V, I or S; and (iii) heavy chain complementarity determining region 3 (CDR3) containing an amino acid sequence X.sub.8ALDFDY, wherein X.sub.8 is M or L, and (b) a light chain variable region (VL) comprising (i) light chain complementarity determining region 1 (CDR1) containing an amino acid sequence TGSSSNIGSNYVT; (ii) light chain complementarity determining region 2 (CDR2) containing an amino acid sequence X.sub.9NX.sub.10X.sub.11RPS, wherein X.sub.9 is D, S or R, X.sub.10 is S or N, and X.sub.11 is N or Q; and (iii) light chain complementarity determining region 3 (CDR3) containing an amino acid sequence X.sub.12SFSDELGAYV, wherein X.sub.12 is A or S.

2. The antibody or fragment thereof of claim 1, wherein (a) the heavy chain variable region (VH) comprises a heavy chain complementarity determining region 1 (CDR1) containing an amino acid sequence defined by SEQ ID NO: 1; heavy chain complementarity determining region 2 (CDR2) containing at least one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 118; and heavy chain complementarity determining region 3 (CDR3) containing at least one amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 25.

3. The antibody or fragment thereof of claim 1, wherein (b) the light chain variable region (VL) comprises a light chain complementarity determining region 1 (CDR1) containing an amino acid sequence defined by SEQ ID NO: 7; a light chain complementarity determining regions 2 (CDR2) containing at least one amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 27 and SEQ ID NO: 29; a light chain complementarity determining region 3 (CDR3) containing at least one amino acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 15.

4. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises: i) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 3, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 5, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 9, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 13; ii) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 3, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 5, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 9, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 15; iii) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 118, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 5, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 9, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 13; iv) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 118, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 5, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 9, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 15; v) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 17, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 5, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 9, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 15; vi) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 19, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 5, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 9, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 15; vii) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 21, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 5, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 9, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 15; viii) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 23, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 5, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 9, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 15; ix) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 21, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 5, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 27, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 15; x) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 21, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 5, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 29, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 15; xi) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 21, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 25, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 9, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 15; xii) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 21, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 25, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 27, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 15; or xiii) an antibody comprising an antibody heavy variable region(VH) comprising heavy chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementary determining region 2(CDR2) containing the amino acid sequence defined by SEQ ID NO: 21, heavy chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 25, and an antibody light chain variable region (VL) comprising light chain complementary determining region 1(CDR1) containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence defined by SEQ ID NO: 29, and light chain complementary determining region 3(CDR3) containing the amino acid sequence defined by SEQ ID NO: 15.

5. The antibody or fragment thereof of claim 1, wherein antibody or fragment thereof contains a heavy chain (HC) comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, and SEQ ID NO: 105; and a light chain (LC) comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, and SEQ ID NO: 115.

6. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises: a heavy chain containing the amino acid sequence defined by SEQ ID NO: 89, and a light chain containing the amino acid sequence defined by SEQ ID NO: 107; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 89, and a light chain containing the amino acid sequence defined by SEQ ID NO: 109; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 93, and a light chain containing the amino acid sequence defined by SEQ ID NO: 107; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 93, and a light chain containing the amino acid sequence defined by SEQ ID NO: 109; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 95, and a light chain containing the amino acid sequence defined by SEQ ID NO: 109; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 97, and a light chain containing the amino acid sequence defined by SEQ ID NO: 109; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 99, and a light chain containing the amino acid sequence defined by SEQ ID NO: 109; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 101, and a light chain containing the amino acid sequence defined by SEQ ID NO: 109; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 103, and a light chain containing the amino acid sequence defined by SEQ ID NO: 111; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 103, and a light chain containing the amino acid sequence defined by SEQ ID NO: 113; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 103, and a light chain containing the amino acid sequence defined by SEQ ID NO: 115; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 105, and a light chain containing the amino acid sequence defined by SEQ ID NO: 111; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 105, and a light chain containing the amino acid sequence defined by SEQ ID NO: 113; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 105, and a light chain containing the amino acid sequence defined by SEQ ID NO: 115; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 99, and a light chain containing the amino acid sequence defined by SEQ ID NO: 111; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 103, and a light chain containing the amino acid sequence defined by SEQ ID NO: 109.

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

8. The antibody or fragment thereof of claim 1, wherein the fragment contains the one or more amino acid sequences 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.

9. A polynucleotide encoding the antibody or fragment thereof of claim 1.

10. (canceled)

11. (canceled)

12. A method for producing an antibody or fragment thereof specifically binding to an extracellularly exposed lysyl-tRNA synthetase (KRS)N-terminal region, the method comprising: (a) transforming host cells with a recombinant expression vector comprising the polynucleotide of claim 9; (b) incubating the transformed host cells to produce an antibody or fragment thereof; and (c) collecting the antibody or fragment thereof produced in the host cells.

13. (canceled)

14. (canceled)

15. The antibody or fragment thereof of claim 1, wherein the antibody is a polypeptide containing an Fc variant of a wild-type human IgG Fc region, and wherein the Fc variant comprises L117A, L118A, T182A, P212G of a wild-type human IgG1 Fc region defined by SEQ ID NO: 126 or at least one additional amino acid substitution which is T179A of the human IgG4 Fc region defined by SEQ ID NO: 138, and wherein the polypeptide has a reduced ADCC/CDC function compared to a polypeptide comprising the wild-type IgG Fc region.

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

20. A method for preventing or inhibiting cancer and cancer metastasis, administering an effective amount of a composition comprising the antibody or fragment thereof of claim 1 to a subject in need thereof.

21. (canceled)

22. A method for diagnosing cancer or cancer metastasis, the method comprising: a) obtaining a biological sample from an individual (subject) suspected of cancer metastasis; b) administering a composition comprising the antibody or a fragment thereof of claim 1 to the sample or subject; c) detecting the expression level of the KRS protein in the sample or subject of step b); and d) comparing the expression level of the KRS protein with a normal control group, and diagnosing that cancer and cancer metastasis have occurred when the expression level of KRS is increased.

23. (canceled)

24. A method for treating an immune cell migration-related disease, administering an effective amount of a composition comprising the antibody or fragment thereof of claim 1 to a subject in need thereof.

25. (canceled)

26. A method for diagnosing an immune cell migration-related disease, the method comprising: a) obtaining a biological sample from a subject suspected of an immune cell migration-related disease; b) administering a composition comprising the antibody or a fragment thereof of claim 1 to the sample or subject; c) detecting the expression level of the KRS protein in the sample or subject of step b); and d) comparing the expression level of the KRS protein with a normal control group, and diagnosing as an immune cell migration-related disease when the expression level of KRS is increased.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0255] FIG. 1 is shown in a schematic diagram of selection and construction strategy of two libraries constructed based on each of the heavy chain variable region (VH) and light chain variable region (VL) of N3 in order to improve the affinity of the KRS N-terminal target antibody N3 to the KRS N-terminus.

[0256] FIG. 2a shows the results of analysis of the KRS (1-72) peptide bound to 10 nM, 1 nM or 0.1 nM GST and the analyzed binding capacity by flow cytometry (FACS) for each step library-expressing yeast selected using FACS (Fluorescence Activated Cell Sorting).

[0257] FIG. 2b shows the results of analyzing the KRS (1-72) peptide bound to 0.1 nM GST and the binding ability with a flow cytometer for yeasts expressing 47 individual clones in the final selected library.

[0258] FIGS. 3a and 3b show ELISA results for measuring affinity for the N-terminus of KRS of the N3-1 antibody, N3-3 antibody, N3-4 antibody and N3-5 antibody selected as having high affinity and specificity for the peptide of KRS N-term (residues 1-72).

[0259] FIG. 4 shows the results of confirming the cell migration inhibitory effect of the N3 antibody and the N3-1 antibody.

[0260] FIG. 5 shows the result of comparing the affinity for KRS of N3 antibody and N3-1 antibody by SPR (surface plasmon resonance) method.

[0261] FIG. 6 shows the results of analysis of the KRS (1-72) peptide bound to 10 nM, 1 nM or 0.1 nM GST and binding ability analyzed with by flow cytometer for each step library-expressing yeast selected using MACS and FACS (Fluorescence Activated Cell Sorting) FIG. 7 shows the ELISA results for measuring the affinity of the N3-1 antibody, N3-6 antibody, N3-7 antibody, N3-8 antibody and N3-9 antibody for the N-terminus of KRS.

[0262] FIG. 8 shows the results of comparing the affinity of N3-6 antibody, N3-7 antibody, N3-8 antibody and N3-9 antibody for KRS by the surface plasmon resonance (SPR) method.

[0263] FIG. 9 shows the results of confirming the cell migration inhibitory effect of the N3 antibody, N3-1 antibody, N3-6 antibody, N3-7 antibody, N3-8 antibody, and N3-9 antibody.

[0264] FIG. 10 shows the results of confirming the endocytosis of the N3 antibody and the N3-8 antibody in breast cancer cells by the IHC (immunohistochemistry) method.

[0265] FIG. 11 shows the change of right ventricular end-systolic pressure (RVESP) in the pulmonary arterial hypertension (PAH) models by administration of the N3 antibody of the present invention (Mock IgG: negative control, Ab 1 mpk: N3 antibody 1 mpk, Ab 10 mpk: N3 antibody 10 mpk, sildenafil: positive control).

[0266] FIG. 12 is a result of confirming by IHC staining that immune cell migration and invasion are reduced by administration of the N3 antibody of the present invention in the pulmonary arterial hypertension (PAH) models.

[0267] FIG. 13 shows the result of confirming that the total number of immune cells increased in the BALF (Bronchoalveolar lavage fluid) in the mouse models of acute lung injury were reduced depending on the treatment concentration of N3 antibody (antibody binding to the N-terminus of KRS).

[0268] FIG. 14 shows the result of confirming that neutrophils which are particularly increased in bronchoalveolar lavage fluid (BALF) of the mouse models of acute lung injury were reduced depending on the treatment concentration of N3 antibody (antibody binding to the N-terminus of KRS).

[0269] FIG. 15 shows the results of confirming by FACS that increased macrophage (IM, CD11 b+/F4/80+) migration and invasion in the lung tissue of the mouse models of acute lung injury was reduced depending on the treatment concentration of N3 antibody (antibody binding to the N-terminus of KRS).

[0270] FIG. 16 is a graph quantifying the results of FIG. 15.

[0271] FIG. 17 is a tissue image showing that the tissue fibrosis advanced in lung tissue of the mouse models of acute lung injury mouse models is inhibited by treatment with an N3 antibody (KRS N-terminal binding antibody). Tissues of each experimental group and control group were observed under a microscope after Masson's trichrome staining.

[0272] FIG. 18 shows the results confirming that cell migration was inhibited by treatment with N3-8, N3-8-1 antibodies, and N3-8-1 derivative antibodies from which ADCC/CDC functions were removed.

[0273] FIG. 19 shows the results confirming that cell migration of cancer cells was inhibited by treatment with N3-8, N3-8-1 antibodies, and N3-8-1 derivative antibodies from which ADCC/CDC functions have been removed.

MODE FOR CARRYING OUT INVENTION

[0274] Hereinafter, the present invention will be described in detail.

[0275] However, the following examples are merely for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1: Construction of Yeast Cell Surface Expression Library for Increasing Affinity

[0276] The affinity for the N-terminus of the antibody N3 (application number: 10-2018-0035446) targeting the N-terminus of the existing KRS is about 150 nM, which is lower than that of various antibodies in the complete IgG form. Accordingly, in order to increase the affinity to prepare an antibody having a better effect, the light chain variable region and the heavy chain variable region of the N3 antibody were improved.

[0277] Homology model was used to predict the approximate structure of N3, through which random mutations were introduced into the CDR regions predicted to play an important role in antigen binding. Specifically, in the library based on the heavy chain variable region, NNK, a degenerated codon, which can contain all 20 amino acid sequences for CDR3 residues was used. In the library based on the light chain variable region, NNK, a degenerated codon that can contain all 20 amino acid sequences, was used for CDR3 residues.

[0278] Specifically, the DNA encoding the designed library was amplified using a PCR technique and then concentrated using an ethanol precipitation method.

[0279] Yeast surface expression vector (C-aga2), which expresses aga2 protein at the C-terminus for homologous recombination, is treated with Nhel and MIul restriction enzymes and purified using agarose gel extraction method and concentrated ethanol precipitation method.

[0280] Restriction enzyme-treated 4 μg vectors for 12 μg of each library-encoding DNA were transformed into yeast EBY100 for expression on the yeast surface by electroporation, and the library size was confirmed by measuring the number of colonies grown in the selective 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.

[0281] This process is shown in FIG. 1.

Example 2: Selection of Light Chain Variable Region (VL) and Heavy Chain Variable Region (VH) with Improved Affinity to GST-Conjugated KRS (1-72) Peptide

[0282] Two types of N3-based affinity improving libraries constructed in Example 1 were selected using GST-conjugated KRS (1-72) peptide as an antigen.

[0283] Specifically, a 10 nM level of purified GST-conjugated KRS (1-72) peptide was incubated with yeasts expressing a single-chain Fab (scFab)-type light chain variable region library on the cell surface 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 1 hour at room temperature for primary FACS screening.

[0284] Thereafter, the GST-conjugated KRS (residues 1-72) peptide and yeasts 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 1 nM KRS (residues 1-72) peptide conjugated with GST, and a third FACS screening was performed with 0.5 nM KRS (residues 1-72) peptide conjugated with GST.

[0285] As a result, as shown in FIGS. 2a and 2b, through the selection process using FACS, compared with the N3 antibody, it was confirmed that clones having high affinity for the GST-conjugated KRS (1-72) peptide were selected and the affinity was dependent to a heavy chain variable region (VH) or light chain variable region (VL). Three unique clones (N3-1, N3-3, and N3-4) having high affinity and specificity for GST-conjugated KRS (1-72) peptide were selected through individual clone binding ability analysis. In addition, another unique clone (N3-5) was constructed by combining the light chain variable region and the heavy chain variable region with each other. That is, a total of four unique clones (N3-1, N3-3, N3-4, N3-5) were selected.

[0286] Table 1 shows the CDR sequences of the light chain variable region and the heavy chain variable region of four individual clones showing high binding ability to GST-conjugated KRS (1-72) peptide. Table 2 shows the heavy chain variable region sequence and the light chain variable region sequence.

TABLE-US-00001 TABLE 1 Heavy Light CDR H1 CDR H2 CDR H3 CDR L1 CDR L2 CDR L3 N3 SYDMS AISYDNGNTY MALDFDY TGSSSNIGSN DNSNRPS ASWDDSLSAY (SEQ ID NO: 1) YADSVKG (SEQ ID NO: 5) YVT (SEQ ID NO: 9) V (SEQ ID NO: 3) (SEQ ID NO: 7) (SEQ ID NO: 11) N3-1 SYDMS AISYDNGNTY MALDFDY TGSSSNIGSN DNSNRPS ASFSDELGAY (SEQ ID NO: 1) YADSVKG (SEQ ID NO: 5) YVT (SEQ ID NO: 9) V (SEQ ID NO: 3) (SEQ ID NO: 7) (SEQ ID NO: 13) N3-3 SYDMS AISYDNGNTY MALDFDY TGSSSNIGSN DNSNRPS SSFSDELGAY (SEQ ID NO: 1) YADSVKG (SEQ ID NO: 5) YVT (SEQ ID NO: 9) V (SEQ ID NO: 3) (SEQ ID NO: 7) (SEQ ID NO: 15) N3-4 SYDMS VISSDGGNTY MALDFDY TGSSSNIGSN DNSNRPS ASFSDELGAY (SEQ ID NO: 1) YADSVKG (SEQ ID NO: 5) YVT (SEQ ID NO: 9) V (SEQ ID (SEQ ID NO: 7) (SEQ ID NO: 13) NO: 118) N3-5 SYDMS VISSDGGNTY MALDFDY TGSSSNIGSN DNSNRPS SSFSDELGAY (SEQ ID NO: 1) YADSVKG (SEQ ID NO: 5) YVT (SEQ ID NO: 9) V (SEQ ID (SEQ ID NO: 7) (SEQ ID NO: 15) NO: 118)

TABLE-US-00002 TABLE 2 SEQ ID NO:  Sequence (Sequence name) N3 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 31 (N3 SSYDMSWVRQAPGKGLEWVSAISYDNGN VH) TYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 33 (N3 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV VL) PDRFSGSKSGTSASLAISGLRSEDEADYYC ASWDDSLSAYVFGGGTKLTVL N3-1 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 31 (N3 SSYDMSWVRQAPGKGLEWVSAISYDNGN VH) TYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 49 (N3 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV VL mutant 1) PDRFSGSKSGTSASLAISGLQSEDEADYYC ASFSDELgAYVFGGGTKLTVL N3-3 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 31 (N3 SSYDMSWVRQAPGKGLEWVSAISYDNGN VH) TYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 51 (N3 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV VL mutant 2) PDRFSGSKSGTSASLAISGLQSEDEADYYC SSFSDELgAYVFGGGTKLTVL N3-4 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 35 (N3 SSYDMSWVRQAPGKGLEWVSVISSDGGN VH mutant 1) TYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 49 (N3 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV VL mutant 1) PDRFSGSKSGTSASLAISGLQSEDEADYYC ASFSDELgAYVFGGGTKLTVL N3-5 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 35 (N3 SSYDMSWVRQAPGKGLEWVSVISSDGGN VH mutant 1) TYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 51 (N3 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV VL mutant 2) PDRFSGSKSGTSASLAISGLQSEDEADYYC SSFSDELgAYVFGGGTKLTVL

[0287] In addition, ELISA was performed to measure the affinity for the N-terminus of KRS to confirm whether the affinity for the N-terminus of KRS was increased.

[0288] Specifically, the N-terminal region (residues 1-72) peptide of KRS was coated in a 96-well EIA/RIA plate (COSTAR Corning)at 25° C. for 1 hour, and the plate was washed 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 incubated, respectively, and then the plate was 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 the absorbance was measured at 450 nm to quantify the antibody binding.

[0289] As a result, as shown in FIGS. 3a and 3b, it was confirmed that the affinity of the mutant N3-1, N3-3, N3-4, and N3-5 antibodies was increased compared to the wild-type N3 antibody. It was found that all clones did not bind to GST or NRP1-b1b2 used as a negative control. There was no significant difference in KRS binding ability between mutant antibodies N3-1, N3-3, N3-4, and N3-5.

Example 3: Comparison of Affinity Between N3 Antibody and N3-1 Antibody

[0290] 3-1. Cell Migration Inhibitory Effect of Antibody

[0291] Among the N3 mutant antibodies of Example 2, the N3-1 antibody was converted to an IgG antibody using a conventional method. The following experiment was performed using the converted IgG antibody.

[0292] Cell migration was measured using a 24-well transwell chamber having a commonly used polycarbonate membrane (8.0 μm pore size, Costar). The lower well was coated with 10 μg Laminin In the transwell chamber. Then, A549 cells were suspended in serum-free RPMI medium, and placed in the upper chamber at a concentration of 1×10.sup.5 cells per well. N3, N3-1 IgG, and human mock IgG (control) were treated in the chamber at 10 nM or 100 nM, respectively, and incubated for 24 hours. The non-migrating cells present above the membrane were removed with a cotton swab. Then, it was washed twice with PBS and treated with 70% MeOH (in PBS) for 30 minutes. After washing twice with PBS, hematoxylin solution was treated for 30 minutes. Then, after washing the chamber three times with DW, the membrane in the chamber was cut and mounted on a slide glass to observe.

[0293] As a result, as shown in FIG. 4, it was confirmed that the N3-1 antibody significantly inhibited the migration of A549 cells compared to the N3 antibody.

[0294] 3-2. KRS Affinity Effect of Antibody

[0295] Using the purified protein of the KRS fragment (1-207aa) as an antigen, the binding ability to N3 and N3-1 antibodies was analyzed via Surface Plasmon Resonance (SPR).

[0296] 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).

[0297] As a result, as shown in FIG. 5, the KD value of the N3-1 antibody was measured to be 31 nM, indicating that the binding ability to the KRS protein was increased compared to the N3 antibody.

Example 4: Construction of Yeast Cell Surface Expression Library for Affinity Enhancement (N 3-1 Antibody)

[0298] The N3-1, N3-3, N3-4, and N3-5 antibodies targeting the KRS N-terminus derived in the Example 2 have similar affinity to KRS, and as shown in the result of the N3-1 antibody, it was determined to have an affinity of about 31 nM. This is still low compared to the affinity of the various antibodies in the complete IgG form. In order to increase the affinity and obtain a more effective antibody, it was attempted to intensively improve the heavy chain variable region of the antibody.

[0299] The light chain variable region sequence was fixed to N3-3, and the approximate modeling structure of N3-3 was predicted using homology modeling. Through this, a random mutation was introduced into the CDR predicted to play an important role in antigen binding.

[0300] Specifically, the residues of the CDR2 and CDR3 of the heavy chain variable region used NNK, a degenerated codon that can contain all 20 amino acid sequences, and a library was constructed in the same manner as in Example 1.

Example 5. Selection of Light Chain Variable Region (VL) and Heavy Chain Variable Region (VH) with Improved Affinity to GST-Conjugated KRS (1-72) Peptide

[0301] Using the GST-conjugated KRS (residues 1-72) peptide as an antigen, two types of N3-3-based affinity improving libraries constructed in the Example 4 were selected. Since the affinity of N3-3 and N3-1 was expected to be almost the same and the sequences were almost similar, the comparative experiment was performed with N3-1.

[0302] Specifically, the yeasts expressing the library bound with the GTP-conjugated KRS (resides 1-72) peptide were reacted with Streptavidin Microbead™ (Miltenyi Biotec) at 4° C. for 20 minutes, and yeasts expressing the heavy chain variable region with high affinity to the KRS (1-72 aa) peptide were suspended using magnetic activated cell sorting (MACS). The yeasts 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 2, sequential screening was performed using FACS.

[0303] The primary FACS screening was performed with 10 nM KRS (1-72) peptide conjugated with GST, the secondary FACS screening with 1 nM KRS (1-72) peptide conjugated with GST, the third FACS screening was performed with 0.5 nM KRS (1-72) peptide conjugated with GST, and the forth FACS screening was performed with 0.1 nM KRS (1-72) peptide conjugated with GST.

[0304] As a result, as shown in FIG. 6, through the selection process using MACS and FACS (Fluorescence Activated Cell Sorting), it was confirmed that clones having high affinity depending on the heavy chain variable region (VH) for the GST-conjugated KRS (1-72) peptide were selected compared with the N3-1 antibody, and four unique clones (N3-6, N3-7, N3-8, N3-9) having high affinity and specificity for GST-conjugated KRS (1-72) peptide were selected through individual clone binding ability analysis.

[0305] The CDR sequences of the light chain variable region and heavy chain variable region of four individual clones, which show high binding ability to the KRS (1-72 aa) peptide, were shown in Table 3, and Table 4 shows the sequences of heavy chain variable region sequence and light chain variable region.

TABLE-US-00003 TABLE 3 Heavy Light CDR H1 CDR H2 CDR H3 CDR L1 CDR L2 CDR L3 N3-6 SYDMS AISPQMGRV MALDFDY TGSSSNIG DNSNRPS SSFSDELGA (SEQ ID YYADSVKG (SEQ ID SNYVT (SEQ ID YV NO: 1) (SEQ ID NO:  NO: 5) (SEQ ID NO: 9) (SEQ ID NO:  17) NO: 7) 15) N3-7 SYDMS AIDPLGGNIY MALDFDY TGSSSNIG DNSNRPS SSFSDELGA (SEQ ID YADSVKG (SEQ ID SNYVT (SEQ ID YV NO: 1) (SEQ ID NO:  NO: 5) (SEQ ID NO: 9) (SEQ ID NO:  19) NO: 7) 15) N3-8 SYDMS AISPYSGRIY MALDFDY TGSSSNIG DNSNRPS SSFSDELGA (SEQ ID YADSVKG (SEQ ID SNYVT (SEQ ID YV NO: 1) (SEQ ID NO:  NO: 5) (SEQ ID NO: 9) (SEQ ID NO:  21) NO: 7) 15) N3-9 SYDMS AIGADGGPS MALDFDY TGSSSNIG DNSNRPS SSFSDELGA (SEQ ID YYADSVKG (SEQ ID SNYVT (SEQ ID YV NO: 1) (SEQ ID NO:  NO: 5) (SEQ ID NO: 9) (SEQ ID NO:  23) NO: 7) 15)

TABLE-US-00004 TABLE 4 SEQ ID NO:  Sequence (Sequence name) N3-6 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO:  37 SSYDMSWVRQAPGKGLEWVSAISPQMGR (N3 VH mutant 2) VYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 51 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV (N3 VL mutant 2) PDRFSGSKSGTSASLAISGLQSEDEADYYC SSFSDELgAYVFGGGTKLTVL N3-7 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO:  39 SSYDMSWVRQAPGKGLEWVSAIDPLGGNI (N3 VH mutant 3) YYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 51 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV (N3 VL mutant 2) VPDRFSGSKSGTSASLAISGLQSEDEADYYC SSFSDELgAYVFGGGTKLTVL N3-8 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 45 SSYDMSWVRQAPGKGLEWVSAISPYSGRI (N3 VH mutant 6) YYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 51 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV (N3 VL mutant 2) PDRFSGSKSGTSASLAISGLQSEDEADYYC SSFSDELgAYVFGGGTKLTVL N3-9 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 43 SSYDMSWVRQAPGKGLEWVSAIGADGGP (N3 VH mutant 5) SYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 51 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV (N3 VL mutant 2) PDRFSGSKSGTSASLAISGLQSEDEADYYC SSFSDELgAYVFGGGTKLTVL

[0306] In addition, ELISA was performed in the same manner as in the Example 2 in order to measure the affinity for the N-terminus of KRS to confirm whether the affinity for the N-terminus of KRS was increased.

[0307] Specifically, the N-terminal portion (1-72) of KRS was coated in a 96-well EIA/RIA plate (COSTAR Corning) at 25° C. for 1 hour, and then the plate was washed three times with PBS for 10 minutes. Then, the plate was incubated with 4% BSA PBS for 1 hour, and then washed 3 times with PBS for 10 minutes. Then, the IgG-type KRS N-terminal target antibodies N3-1, N3-6, N3-7, N3-8, and N3-9 were treated and incubated and the plate was washed three times for 10 minutes with 0.1% PBST. An anti-human antibody conjugated with HRP was used, and it was reacted with TMB (3,3′,5,5′-Tetramethylbenzidine) and absorbance was measured at 450 nm and the binding was quantified.

[0308] As a result, as shown in FIG. 7, it was confirmed that the affinity of the mutant antibodies N3-6, N3-7, N3-8, and N3-9 was increased compared to N3-1 antibody. All the antibodies did not interact with NRP1-b1 b2, which was used as a negative control.

Example 6: Comparison of Affinity Between the N3-1 Antibody and the N3-6, N3-7, N3-8, N3-9 Antibodies

[0309] 6-1. Comparison of Antibody Binding to KRS

[0310] Using KRS epitope peptide F4 (EPKLSKNELKRRLKAEKKVAEKEAKQKE: SEQ ID NO: 117) 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 via Surface Plasmon Resonance (SPR). SPR experiment was carried out in the same manner as in the Example 3-2. 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).

[0311] As a result, as shown in FIG. 8, the KD of the N3-8 antibody was exhibited to be excellent, the KD of the N3-9 and N3-6 antibodies were similar, and the KD value of the N3-7 antibody was the largest. The dissociation time of N3-6 antibody was longer than that of N3-7 and N3-9, and showed a sensorgram with longer binding.

[0312] Also, ELISA was performed to identify residues that are important for antibody-epitope binding, using peptides in which the single amino acids of KRS epitope peptide F4 (SEQ ID NO: 117) were substituted with alanine (A), respectively. As a result, the residues in KRS epitope peptide F4 that are important in binding to each antibody was able to be identified.

[0313] 6-2. Cell Migration Inhibitory Effect of Antibody

[0314] Experiments were performed in the same manner as in Example 3-1. N3-6, N3-7, N3-8, N3-9 antibodies prepared in the above Example were converted to IgG by a conventional method. The following experiment was performed using the converted IgG antibodies.

[0315] Cells were put into the upper chamber at a concentration of 1×10.sup.5, and then N3 IgG was treated at 100 nM, and N3-1, N3-6, N3-7, N3-8 and N3-9 IgG, and human mock IgG (control) were each treated in the chamber at 10 nM and cultured for 24 hours. The non-migrating cells present above the membrane were removed with a cotton swab.

[0316] Then, the membrane was washed twice with PBS and treated with 70% MeOH (in PBS) for 30 minutes. After washing twice with PBS, hematoxylin solution was treated for 30 minutes. Then, after washing the chamber with DW, the membrane in the chamber was mounted on a slide glass and observed.

[0317] As a result, as shown in FIG. 9, it was confirmed that N3-6, N3-7, N3-8, and N3-9 antibodies significantly inhibited cell migration compared to the N3-1 antibody. In addition, there was no significant difference in the effect of inhibiting cell migration among N3-6, N3-7, N3-8, and N3-9 antibodies.

Example 7: Sequence Refinement of N3-8 Antibody

[0318] 7-1. Mutation Production of N3-8 Antibody Sequence

[0319] In the above example, it was confirmed that N3-8 antibody has the best affinity. Thus, experiments were conducted as follows to confirm physical properties such as productivity and stability of N3-8 antibody.

[0320] A mutation was induced in the sequence expected to affect stability in the N3-8 antibody sequence. As a result, two additional heavy chain sequences in which mutations were introduced into the heavy chain sequence (HC) of N3-8 antibody were obtained. In addition, it was possible to obtain three additional light chain sequences into which the mutation was introduced. Accordingly, 7 kinds of antibody sequences (N3-8 derivatives) in which the sequence of N3-8 was changed are shown in Tables 5 and 6 below.

TABLE-US-00005 TABLE 5 Heavy Light CDR H1 CDR H2 CDR H3 CDR L1 CDR L2 CDR L3 N3-8-1 SYDMS AISPYSGR MALDFDY TGSSSNI DNSNRPS SSFSDELG (SEQ ID IYYADSVK (SEQ ID GSNYVT (SEQ ID AYV NO: 1) G NO: 5) (SEQ ID NO: 9) (SEQ ID (SEQ ID NO: 7) NO: 15) NO: 21) N3-8-2 SYDMS AISPYSGR MALDFDY TGSSSNI SNNQRPS SSFSDELG (SEQ ID IYYADSVK (SEQ ID GSNYVT (SEQ ID AYV NO: 1) G NO: 5) (SEQ ID NO: 27) (SEQ ID (SEQ ID NO: 7) NO: 15) NO: 21) N3-8-3 SYDMS AISPYSGR MALDFDY TGSSSNI RNNQRPS SSFSDELG (SEQ ID IYYADSVK (SEQ ID GSNYVT (SEQ ID AYV NO: 1) G NO: 5) (SEQ ID NO: 29) (SEQ ID (SEQ ID NO: 7) NO: 15) NO: 21) N3-8-4 SYDMS AISPYSGR LALDFDY TGSSSNI DNSNRPS SSFSDELG (SEQ ID IYYADSVK (SEQ ID GSNYVT (SEQ ID AYV NO: 1) G NO: 25) (SEQ ID NO: 9) (SEQ ID (SEQ ID NO: 7) NO: 15) NO: 21) N3-8-5 SYDMS AISPYSGR LALDFDY TGSSSNI SNNQRPS SSFSDELG (SEQ ID IYYADSVK GSNYVT (SEQ ID  (SEQ ID AYV NO: 1) G (SEQ ID NO: 7) NO: 27) (SEQ ID (SEQ ID NO: 25) NO: 15) NO: 21) N3-8-6 SYDMS AISPYSGR LALDFDY TGSSSNI RNNQRPS SSFSDELG (SEQ ID IYYADSVK (SEQ ID GSNYVT (SEQ ID AYV NO: 1) G NO: 25) (SEQ ID NO: 29) (SEQ ID (SEQ ID NO: 7) NO: 15) NO: 21) N3-8-7 SYDMS AISPYSGR MALDFDY TGSSSNI DNSNRPS SSFSDELG (SEQ ID IYYADSVK (SEQ ID GSNYVT (SEQ ID AYV NO: 1) G NO: 5) (SEQ ID NO: 9) (SEQ ID (SEQ ID NO: 7) NO: 15) NO: 21)

TABLE-US-00006 TABLE 6 SEQ ID NO:  Sequence (Sequence name) N3-8-1 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 45 SSYDMSWVRQAPGKGLEWVSAISPYSGR (N3 VH mutant 6) IYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 51 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV (N3 VL mutant 2) PDRFSGSKSGTSASLAISGLQSEDEADYY CSSFSDELGAYVFGGGTKLTVL N3-8-2 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 45 SSYDMSWVRQAPGKGLEWVSAISPYSGR (N3 VH mutant 6) IYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 53 SNYVTWYQQLPGTAPKLLIYSNNQRPSGV (N3 VL mutant 3) PDRFSGSKSGTSASLAISGLQSEDEADYY CSSFSDELGAYVFGGGTKLTVL N3-8-3 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 45 SSYDMSWVRQAPGKGLEWVSAISPYSGR (N3 VH mutant 6) IYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 55 SNYVTWYQQLPGTAPKLLIYRNNQRPSGV (N3 VL mutant 4) PDRFSGSKSGTSASLAISGLQSEDEADYY CSSFSDELGAYVFGGGTKLTVL N3-8-4 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 47 SSYDMSWVRQAPGKGLEWVSAISPYSGR (N3 VH mutant 7) IYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARLALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 51 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV (N3 VL mutant 2) PDRFSGSKSGTSASLAISGLQSEDEADYY CSSFSDELGAYVFGGGTKLTVL N3-8-5 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 47 SSYDMSWVRQAPGKGLEWVSAISPYSGR (N3 VH mutant 7) IYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARLALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 53 SNYVTWYQQLPGTAPKLLIYSNNQRPSGV (N3 VL mutant 3) PDRFSGSKSGTSASLAISGLQSEDEADYY CSSFSDELGAYVFGGGTKLTVL N3-8-6 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 47 SSYDMSWVRQAPGKGLEWVSAISPYSGR (N3 VH mutant 7) IYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARLALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 55 SNYVTWYQQLPGTAPKLLIYRNNQRPSGV (N3 VL mutant 4) PDRFSGSKSGTSASLAISGLQSEDEADYY CSSFSDELGAYVFGGGTKLTVL N3-8-7 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF SEQ ID NO: 41 SSYDMSWVRQAPGKGLEWVSAISPYSGR (N3 VH mutant 4) IYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYSARMALDFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNIG SEQ ID NO: 51 SNYVTWYQQLPGTAPKLLIYDNSNRPSGV (N3 VL mutant 2) PDRFSGSKSGTSASLAISGLQSEDEADYY CSSFSDELGAYVFGGGTKLTVL

[0321] 7-2. Measurement of Productivity and Stability of N3-8 Antibody Derivatives and Tm Measurement

[0322] The vector expressing the N3-8 antibody derivative obtained in Example 7-1 was expressed and purified using transient transfection.

[0323] To 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. 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% C02.

[0324] To produce each monoclonal antibody, suitable heavy and light chain plasmids were transfected into 10 ml FreeStyle 293 expression medium (Invitrogen) at a ratio of 1:1 or 1:2 of heavy chain: light chain DNA. When heavy chain: light chain DNA is used in a 1:1 ratio, 125 μg heavy chain and 125 μg light chain, a total of 250 μg (2.5 μg/ml) DNA is mixed with 10 ml of medium containing PEI 750 μg (7.5 μg/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 cells previously aliquoted with 100 ml, and incubated at 150 rpm and 37° C., with 8% C02 for 4 hours, and then additional 100 ml of FreeStyle 293 expression medium was added and cultured for 6 days.

[0325] 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 antibody 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 based on absorbance measurement and absorption coefficient at a wavelength of 280 nm.

[0326] In addition, the thermal stability of the antibody was measured using 100 μl of the purified antibodies at a concentration of 1 mg/ml. Thermostability was investigated 4 times using protein thermal shift dye kit (Thermofisher) and Quant Studio 3 Real-time PCR equipment (Thermofisher).

[0327] As a result, as shown in Table 7 below, the yield of all the N3-8 antibody derivatives tested was improved or showed a high yield at a similar level of the N3-8 antibody. Further, as shown in Table 7, a Tm value was obtained. The thermal transition was observed to be 1-2 depending on the antibody, but the Tm value was increased in all N3-8 antibody derivatives.

[0328] Through this, it was confirmed that the N3-8 antibody derivatives had higher yield and their thermal stability was improved compared to that of N3-8 antibody.

TABLE-US-00007 TABLE 7 Yield (mg/L) 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

[0329] Table 8 shows the heavy chain (HC) and light chain (LC) sequences of the entire IgG antibodies used in the above-described examples.

TABLE-US-00008 TABLE 8 Amino acid sequence DNA sequence N3 HC SEQ ID NO: 89 SEQ ID NO: 90 LC SEQ ID NO: 91 SEQ ID NO: 92 N3-1 HC SEQ ID NO: 89 SEQ ID NO: 90 LC SEQ ID NO: 107 SEQ 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: 96 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: 103 SEQ ID NO: 104 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

[0330] 7-3. Affinity Comparison of N3-8-1 and N3-8-4 Antibodies

[0331] As described in Example 6, the KRS epitope peptide F4(EPKLSKNELKRRLKAEKKVAEKEAKQKE: SEQ ID NO: 117) was used as an antigen epitope, and the binding strength to N3-8-1 and N3-8-4 antibodies was analyzed via Surface Plasmon Resonance (SPR).

[0332] The SPR experiment was carried out in the same manner as in the Example 3-2, and epitope was diluted in PBS solution, diluted twice in a range of 15.7 nM-4000 nM, and flowed for 90 seconds. After that, PBS was flowed for 2400 seconds. The obtained data was analyzed with Biacore T200 Evaluation software v2.0 (GE Healthcare).

[0333] As a result, as shown in Table 9 below, it was found that the KD of the N3-8-1 antibody was the most excellent.

TABLE-US-00009 TABLE 9 Peptide Ab Ka (1/Ms) Kd (1/s) KD (nM) F4 N3-8-1 267900 0.000215 0.8025 N3-8-4 89480 0.00090 10.06

Example 8: Confirmation of Mechanism of Antibody

[0334] After conjugation of a fluorescent probe with antibody (Ab) and treatment on 4T1 breast cancer cells, it was confirmed that the anti-KRS antibodies (N3, N3-8) were endocytosed.

[0335] Anti-KRS antibodies (N3, N3-8) labeled with Alexa fluor 488 (Thermofisher) fluorescent probe and 1 μM of Mock IgG (Thermofisher) as a control were treated to the cells, and the localization of antibodies was monitored after 4 hours. At this time, Lysotracker (Thermofisher) was used as a lysosome marker and DAPI was used for nucleus staining. Unlike Mock IgG, N3 and N3-8 antibodies were present within the cells at 4 hours.

[0336] As a result, as shown in FIG. 10, it was confirmed that the anti-KRS antibody recognizes the cell membrane KRS and is endocytosed rapidly, thereby lowering the cell membrane KRS level.

Example 9: Sequence and Purification of Antibody from which ADCC/CDC Function has been Removed

[0337] 9-1. Mutation Introduction into Antibody Sequence to Remove ADCC/CDC Function

[0338] In order to remove the ADCC/CDC function from the antibody, an experiment was performed as follows. In each of the above antibody sequences, mutations were introduced into the portion expected to function for ADCC/CDC in a constant region of the IgG1 heavy chain. Five additional heavy chain sequences where mutations were introduced were obtained. In addition, IgG4 heavy chain sequence and additional one into which a mutation was introduced were generated. Accordingly, the mutant antibody sequences from which the ADCC/CDC function has been removed from each antibody are shown in Table 10, respectively.

TABLE-US-00010 TABLE 10 Amino acid sequence DNA sequence N3 HC IgG1 SEQ ID NO: 89 SEQ ID NO: 90 IgG1 mutant TA SEQ ID NO: 140 SEQ ID NO: 141 IgG1 mutant LALA SEQ ID NO: 142 SEQ ID NO: 143 IgG1 mutant LALATA SEQ ID NO: 144 SEQ ID NO: 145 IgG1 mutant LALAPG SEQ ID NO: 146 SEQ ID NO: 147 IgG1 mutant LALAPGTA SEQ ID NO: 148 SEQ ID NO: 149 IgG4 SEQ ID NO: 150 SEQ ID NO: 151 IgG4 mutant TA SEQ ID NO: 152 SEQ ID NO: 153 LC SEQ ID NO: 91 SEQ ID NO: 92 N3-1 HC IgG1 SEQ ID NO: 89 SEQ ID NO: 90 IgG1 mutant TA SEQ ID NO: 140 SEQ ID NO: 141 IgG1 mutant LALA SEQ ID NO: 142 SEQ ID NO: 143 IgG1 mutant LALATA SEQ ID NO: 144 SEQ ID NO: 145 IgG1 mutant LALAPG SEQ ID NO: 146 SEQ ID NO: 147 IgG1 mutant LALAPGTA SEQ ID NO: 148 SEQ ID NO: 149 IgG4 SEQ ID NO: 150 SEQ ID NO: 151 IgG4 mutant TA SEQ ID NO: 152 SEQ ID NO: 153 LC SEQ ID NO: 107 SEQ ID NO: 108 N3-3 HC IgG1 SEQ ID NO: 89 SEQ ID NO: 90 IgG1 mutant TA SEQ ID NO: 140 SEQ ID NO: 141 IgG1 mutant LALA SEQ ID NO: 142 SEQ ID NO: 143 IgG1 mutant LALATA SEQ ID NO: 144 SEQ ID NO: 145 IgG1 mutant LALAPG SEQ ID NO: 146 SEQ ID NO: 147 IgG1 mutant LALAPGTA SEQ ID NO: 148 SEQ ID NO: 149 IgG4 SEQ ID NO: 150 SEQ ID NO: 151 IgG4 mutant TA SEQ ID NO: 152 SEQ ID NO: 153 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-4 HC IgG1 SEQ ID NO: 93 SEQ ID NO: 94 IgG1 mutant TA SEQ ID NO: 154 SEQ ID NO: 155 IgG1 mutant LALA SEQ ID NO: 156 SEQ ID NO: 157 IgG1 mutant LALATA SEQ ID NO: 158 SEQ ID NO: 159 IgG1 mutant LALAPG SEQ ID NO: 160 SEQ ID NO: 161 IgG1 mutant LALAPGTA SEQ ID NO: 162 SEQ ID NO: 163 IgG4 SEQ ID NO: 164 SEQ ID NO: 165 IgG4 mutant TA SEQ ID NO: 166 SEQ ID NO: 167 LC SEQ ID NO: 107 SEQ ID NO: 108 N3-5 HC IgG1 SEQ ID NO: 93 SEQ ID NO: 94 IgG1 mutant TA SEQ ID NO: 154 SEQ ID NO: 155 IgG1 mutant LALA SEQ ID NO: 156 SEQ ID NO: 157 IgG1 mutant LALATA SEQ ID NO: 158 SEQ ID NO: 159 IgG1 mutant LALAPG SEQ ID NO: 160 SEQ ID NO: 161 IgG1 mutant LALAPGTA SEQ ID NO: 162 SEQ ID NO: 163 IgG4 SEQ ID NO: 164 SEQ ID NO: 165 IgG4 mutant TA SEQ ID NO: 166 SEQ ID NO: 167 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-6 HC IgG1 SEQ ID NO: 95 SEQ ID NO: 96 IgG1 mutant TA SEQ ID NO: 168 SEQ ID NO: 169 IgG1 mutant LALA SEQ ID NO: 170 SEQ ID NO: 171 IgG1 mutant LALATA SEQ ID NO: 172 SEQ ID NO: 173 IgG1 mutant LALAPG SEQ ID NO: 174 SEQ ID NO: 175 IgG1 mutant LALAPGTA SEQ ID NO: 176 SEQ ID NO: 177 IgG4 SEQ ID NO: 178 SEQ ID NO: 179 IgG4 mutant TA SEQ ID NO: 180 SEQ ID NO: 181 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-7 HC IgG1 SEQ ID NO: 97 SEQ ID NO: 98 IgG1 mutant TA SEQ ID NO: 182 SEQ ID NO: 183 IgG1 mutant LALA SEQ ID NO: 184 SEQ ID NO: 185 IgG1 mutant LALATA SEQ ID NO: 186 SEQ ID NO: 187 IgG1 mutant LALAPG SEQ ID NO: 188 SEQ ID NO: 189 IgG1 mutant LALAPGTA SEQ ID NO: 190 SEQ ID NO: 191 IgG4 SEQ ID NO: 192 SEQ ID NO: 193 IgG4 mutant TA SEQ ID NO: 194 SEQ ID NO: 195 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-8 HC IgG1 SEQ ID NO: 103 SEQ ID NO: 104 IgG1 mutant TA SEQ ID NO: 224 SEQ ID NO: 225 IgG1 mutant LALA SEQ ID NO: 226 SEQ ID NO: 227 IgG1 mutant LALATA SEQ ID NO: 228 SEQ ID NO: 229 IgG1 mutant LALAPG SEQ ID NO: 230 SEQ ID NO: 231 IgG1 mutant LALAPGTA SEQ ID NO: 232 SEQ ID NO: 233 IgG4 SEQ ID NO: 234 SEQ ID NO: 235 IgG4 mutant TA SEQ ID NO: 236 SEQ ID NO: 237 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-9 HC IgG1 SEQ ID NO: 101 SEQ ID NO: 102 IgG1 mutant TA SEQ ID NO: 210 SEQ ID NO: 211 IgG1 mutant LALA SEQ ID NO: 212 SEQ ID NO: 213 IgG1 mutant LALATA SEQ ID NO: 214 SEQ ID NO: 215 IgG1 mutant LALAPG SEQ ID NO: 216 SEQ ID NO: 217 IgG1 mutant LALAPGTA SEQ ID NO: 218 SEQ ID NO: 219 IgG4 SEQ ID NO: 220 SEQ ID NO: 221 IgG4 mutant TA SEQ ID NO: 222 SEQ ID NO: 223 LC SEQ ID NO: 109 SEQ ID NO: 110 N3-8-1 HC IgG1 SEQ ID NO: 103 SEQ ID NO: 104 IgG1 mutant TA SEQ ID NO: 224 SEQ ID NO: 225 IgG1 mutant LALA SEQ ID NO: 226 SEQ ID NO: 227 IgG1 mutant LALATA SEQ ID NO: 228 SEQ ID NO: 229 IgG1 mutant LALAPG SEQ ID NO: 230 SEQ ID NO: 231 IgG1 mutant LALAPGTA SEQ ID NO: 232 SEQ ID NO: 233 IgG4 SEQ ID NO: 234 SEQ ID NO: 235 IgG4 mutant TA SEQ ID NO: 236 SEQ ID NO: 237 LC SEQ ID NO: 111 SEQ ID NO: 112 N3-8-2 HC IgG1 SEQ ID NO: 103 SEQ ID NO: 104 IgG1 mutant TA SEQ ID NO: 224 SEQ ID NO: 225 IgG1 mutant LALA SEQ ID NO: 226 SEQ ID NO: 227 IgG1 mutant LALATA SEQ ID NO: 228 SEQ ID NO: 229 IgG1 mutant LALAPG SEQ ID NO: 230 SEQ ID NO: 231 IgG1 mutant LALAPGTA SEQ ID NO: 232 SEQ ID NO: 233 IgG4 SEQ ID NO: 234 SEQ ID NO: 235 IgG4 mutant TA SEQ ID NO: 236 SEQ ID NO: 237 LC SEQ ID NO: 113 SEQ ID NO: 114 N3-8-3 HC IgG1 SEQ ID NO: 103 SEQ ID NO: 104 IgG1 mutant TA SEQ ID NO: 224 SEQ ID NO: 225 IgG1 mutant LALA SEQ ID NO: 226 SEQ ID NO: 227 IgG1 mutant LALATA SEQ ID NO: 228 SEQ ID NO: 229 IgG1 mutant LALAPG SEQ ID NO: 230 SEQ ID NO: 231 IgG1 mutant LALAPGTA SEQ ID NO: 232 SEQ ID NO: 233 IgG4 SEQ ID NO: 234 SEQ ID NO: 235 IgG4 mutant TA SEQ ID NO: 236 SEQ ID NO: 237 LC SEQ ID NO: 115 SEQ ID NO: 116 N3-8-4 HC IgG1 SEQ ID NO: 105 SEQ ID NO: 106 IgG1 mutant TA SEQ ID NO: 238 SEQ ID NO: 239 IgG1 mutant LALA SEQ ID NO: 240 SEQ ID NO: 241 IgG1 mutant LALATA SEQ ID NO: 242 SEQ ID NO: 243 IgG1 mutant LALAPG SEQ ID NO: 244 SEQ ID NO: 245 IgG1 mutant LALAPGTA SEQ ID NO: 246 SEQ ID NO: 247 IgG4 SEQ ID NO: 248 SEQ ID NO: 249 IgG4 mutant TA SEQ ID NO: 250 SEQ ID NO: 251 LC SEQ ID NO: 111 SEQ ID NO: 112 N3-8-5 HC IgG1 SEQ ID NO: 105 SEQ ID NO: 106 IgG1 mutant TA SEQ ID NO: 238 SEQ ID NO: 239 IgG1 mutant LALA SEQ ID NO: 240 SEQ ID NO: 241 IgG1 mutant LALATA SEQ ID NO: 242 SEQ ID NO: 243 IgG1 mutant LALAPG SEQ ID NO: 244 SEQ ID NO: 245 IgG1 mutant LALAPGTA SEQ ID NO: 246 SEQ ID NO: 247 IgG4 SEQ ID NO: 248 SEQ ID NO: 249 IgG4 mutant TA SEQ ID NO: 250 SEQ ID NO: 251 LC SEQ ID NO: 113 SEQ ID NO: 114 N3-8-6 HC IgG1 SEQ ID NO: 105 SEQ ID NO: 106 IgG1 mutant TA SEQ ID NO: 238 SEQ ID NO: 239 IgG1 mutant LALA SEQ ID NO: 240 SEQ ID NO: 241 IgG1 mutant LALATA SEQ ID NO: 242 SEQ ID NO: 243 IgG1 mutant LALAPG SEQ ID NO: 244 SEQ ID NO: 245 IgG1 mutant LALAPGTA SEQ ID NO: 246 SEQ ID NO: 247 IgG4 SEQ ID NO: 248 SEQ ID NO: 249 IgG4 mutant TA SEQ ID NO: 250 SEQ ID NO: 251 LC SEQ ID NO: 115 SEQ ID NO: 116 N3-8-7 HC IgG1 SEQ ID NO: 99 SEQ ID NO: 100 IgG1 mutant TA SEQ ID NO: 196 SEQ ID NO: 197 IgG1 mutant LALA SEQ ID NO: 198 SEQ ID NO: 199 IgG1 mutant LALATA SEQ ID NO: 200 SEQ ID NO: 201 IgG1 mutant LALAPG SEQ ID NO: 202 SEQ ID NO: 203 IgG1 mutant LALAPGTA SEQ ID NO: 204 SEQ ID NO: 205 IgG4 SEQ ID NO: 206 SEQ ID NO: 207 IgG4 mutant TA SEQ ID NO: 208 SEQ ID NO: 209 LC SEQ ID NO: 111 SEQ ID NO: 112

[0339] 9-2. Purification of Mutant Antibodies with ADCC/CDC Function Removed

[0340] The vector expressing the mutant N3-8-1 antibody among the antibodies from which the ADCC/CDC function had been removed as described in the Example 9-1 was transiently transfected into cells to express and purify the protein.

[0341] HEK293-F cells (Invitrogen) were transfected in a shake flask according to the method described in the Example 7-2 above. Then, HEK293-F cells were seeded in a medium at a density of 2×10 6 cells/ml, and cultured at 150 rpm, 8% CO2, and 37° C.

[0342] In order to produce each monoclonal antibody, suitable heavy and light chain plasmids were transfected into 10 ml FreeStyle 293 expression medium (Invitrogen) at a ratio of 1:1 or 1:2 of heavy chain: light chain DNA. In the case of 1:1, the heavy chain 125 μg and the light chain 125 μg, a total of 250 μg (2.5 μg/ml), were mixed with 10 ml medium containing 750 μg of PEI (7.5 μg/ml) and reacted at room temperature for 10 minutes. In the case of 1:2, the concentration of the light chain DNA was doubled. Thereafter, the reacted mixed medium was put into 100 ml of the cells and incubated for 4 hours at 150 rpm, 8% CO2, and 37° C., and the additional 100 ml of FreeStyle 293 expression medium was added and cultured for 6 days.

[0343] Then, the cell culture solution was transferred to 50 ml tubes and centrifuged at 3000 rpm for 5 minutes. Subsequently, the protein was purified from the collected cell culture supernatant. The antibody was applied to a Protein A Sepharose column and washed with PBS (pH 7.4). After eluting the antibody at pH 3.0 using 0.1 M glycine buffer, the sample was immediately neutralized using 1 M Tris buffer. The eluted antibody fraction was concentrated by exchanging the buffer with PBS (pH 7.4) through a dialysis method. The purified protein was quantified based on absorbance measurement and extinction coefficient at a wavelength of 280 nm.

[0344] The purity of the purified antibody was measured, and thermostability was investigated 4 times using a Quant Studio 3 Real-time PCR equipment (Thermofisher) and a protein thermal shift dye kit (Thermofisher).

[0345] As a result, as shown in Table 11 below, the yield of all tested N3-8-1 antibody mutants was similar to or higher than that of the wild type, of which LALAPGTA mutations were found to have the highest yield. In addition, all of the N3-8-1 antibody mutants showed high purity at a level similar to that of wild type antibody.

[0346] Through this, it was confirmed that the antibodies from which the ADCC/CDC function of N3-8-1 has been removed have similar or higher yields and have similar purity compared to the N3-8-1 antibody.

TABLE-US-00011 TABLE 11 Antibody Yield (mg/ml) Purity (%) N3-8-1 wild type 78.72 99.47 mutant LALA 70.7 99.87 mutant LALAPG 73.78 99.86 mutant LALATA 77.7 99.91 mutant LALAPGTA 146.37 99.9

Example 10: Efficacy Verification of KRS-N Term Specific Antibodies in Immune Cell Migration-Related In Vivo Disease Models_In Vivo Pulmonary Arterial Hypertension Models

[0347] When treated with an antibody that specifically binds to the KRS-N terminal end, immune cell migration/invasion is inhibited due to internalization of KRS at the site of the cell membrane (through endocytosis, etc.), and as a result, it can be seen that it has the effect of reducing the cell membrane KRS level. Therefore, it is believed that the KRS N-term specific antibody of the present invention (typically N3 antibody) will have a therapeutic effect against diseases related to immune cell migration, which is further demonstrated through the examples described below.

[0348] Experiment Methods

[0349] 1) Construction of pulmonary arterial hypertension (PAH) models and administration of a test substance

[0350] 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), 1 mpk 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.

[0351] 2) Blood Flow and Blood Pressure Measurement

[0352] 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 DA. Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats. Nat Protoc 2008; 3(9):1422-34.

[0353] 3) Immunohistochemistry (IHC)

[0354] 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 μm 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.

[0355] Results

[0356] 10-1. Verification of Blood Pressure and Cardiac Output Changes.

[0357] The animals with 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 12.

TABLE-US-00012 TABLE 12 MCT + Mock MCT + N3 MCT + N3 MCT + IgG Ab 1 mpk Ab 10 mpk 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) (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)

[0358] Pulmonary arterial 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 hypertrophy. 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., Clinical characteristics and prognostic factors of patients with severe pulmonary hypertension, Korean Circulation J. 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.

[0359] PAH patients showed a RVESP increase, which has also been confirmed in the PAH animal models of this experiment. In contrast, as shown in FIG. 11, N3 antibody (an antibody specifically binding to KRS N-term) significantly reduced RVESP at both concentrations, and especially decreased RVESP better than Sildenafil, a positive control drug.

[0360] 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 FIG. 13. This is in contrast to the risk of lowering the systemic blood pressure by causing the expansion of the pulmonary artery, as well as the expansion of the systemic artery in the case of Sildenafil, which is used as a conventional treatment for pulmonary hypertension. That is, it was confirmed that the antibody of the present invention showed a tendency of having a low effect on systemic artery pressure compared with sildenafil, and this effect is thought to be a favorable characteristic of a therapeutic agent considering that sildenafil administration may be a risk of developing hypotension in clinical sites. Moreover, severe pulmonary arterial hypertension causes systolic RV failure, which may be accompanied by low cardiac output and systemic hypotension. Whereas, a treatment to alleviate pulmonary arterial hypertension by the N3 antibody of the present invention is expected to increase the cardiac output and systemic blood pressure, thereby normalizing the blood pressure.

[0361] 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.

[0362] 10-2. Echocardiography

[0363] The D-shaped left ventricle indicating pressure overload in the right ventricle was observed in three animals in the MCT alone administration group (i.e., animals without antibody treatment) and three animals in the MCT+sildenafil administration group, but was not observed in the therapeutic antibody administration groups.

[0364] In addition, as shown in Table 13 below, the weight of each group was increased to a similar degree, with no significant difference. That is, no abnormal signs including abnormal weight reduction were observed in the animals treated with the therapeutic antibody.

TABLE-US-00013 TABLE 13 MCT + Mock MCT + Ab 1 MCT + 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 (%)

[0365] 10-3. Verification of Monocyte/Macrophage Migration and Infiltration

[0366] IHC staining was performed with the lung tissues of each experimental group to detect CD68, which is a monocyte/macrophage marker. As shown in FIG. 12, it was confirmed that the N3 antibody (KRS N-term binding antibody) treatment group of the present invention explicitly reduced the monocyte/macrophage infiltration into lung tissues, and such effect was significantly excellent than that of sildenafil.

Example 11: Efficacy Verification of KRS-N Term Specific Antibodies in Immune Cell Migration-Related In Vivo Disease Models_Acute Lung Injury Models

[0367] Methods

[0368] 1) Construction of LPS-Induced Acute Lung Injury Models and Administration of Test Substance

[0369] Acute lung injury was introduced into mice 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).

[0370] 2) Immune Cell Count in Bronchoalveolar Lavage Fluid (BALF)

[0371] BALF obtained by washing the lungs with PBS was harvested and cell 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, cells were washed twice, and suspended in 400 μl PBS to measure the number of cells by hemocytometer and neutrophil number through Hema3 staining.

[0372] 3) FACS to Analyze Immune Cells in Lung Tissue

[0373] 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 μm), cells were 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). Cells (50 μl) were placed in a tube, mixed well with the same amount of antibody mixture, 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) infiltrated to the lungs. After washing twice at 400×g for 5 minutes using FACS buffer, cells were analyzed by Navios Flow Cytometer (Beckman).

[0374] 4) Masson's Trichrome Staining for Lung Tissue

[0375] 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.

[0376] Results

[0377] 11-1. Verification of the Inhibitory Effect on Immune Cell Migration in Bronchoalveolar Lavage Fluid (BALF)

[0378] As shown in FIG. 13, it was confirmed that the total number of immune cells in BALF was increased in mice where acute lung injury was induced by LPS treatment. The number of infiltrated immune cells was reduced by N3 antibody (KRS N-term binding antibody) treatment in a concentration dependent manner.

[0379] In particular, as shown in FIG. 14, it was confirmed that neutrophils were increased in mice with acute lung injury by LPS treatment, and N3 antibody (KRS N-term binding antibody) treatment reduced these neutrophil levels. As a result, it was confirmed that infiltration of immune cells, particularly neutrophils, into lungs of BALF was significantly inhibited by treating the antibody specifically binding to KRS N-term.

[0380] 11-2. Verification of the Antibody Inhibitory Effect on Immune Cell Migration in Lung Tissue

[0381] FIGS. 15 and 16 show the results of FACS analysis of macrophages migrated to lung tissue with acute lung injury. Interstitial macrophage (IM) is CD11b+/F4/80+cells, which are migrating macrophages that do not reside in the lung but migrate to the lung in certain situations. LPS treatment increased the infiltration of IM into the lung, but N3 antibody treatment reduced the migration of IM to the lung in a concentration dependent manner. Through this, it was confirmed that the migration and invasion of immune cells such as macrophages/monocytes into lung tissues were inhibited by the treatment of antibodies (typically, N3 antibody) that specifically bind to KRS N-term.

[0382] The excessive migration and invasion of immune cells, such as macrophages/monocytes, are important pathological phenomena in tissues of fibrotic disease. As a result of observation of Masson's trichrome staining of lung tissue with respect to the acute lung injury model (FIG. 17), it was confirmed that fibrosis in the lung tissue proceeded considerably. In contrast, it was confirmed that the treatment of the N3 antibody (an antibody that specifically binds to KRS N-term) inhibited such fibrosis.

Example 12: Immune Cell Migration Analysis of Mutant Antibodies with ADCC/CDC Function Removed

[0383] In order to confirm the effect of the mutant antibody from which the ADCC/CDC function had been removed on immune cell migration, a cell migration assay was performed according to the method described in the prior literature (Park, S. G. et al. Human lysyl-tRNA synthetase is secreted to trigger pro-inflammatory response, Proc. Natl. Acad. Sci. USA 102, 6356-6361 (2005)).

[0384] Measurements were made in a trans well chamber with a polycarbonate membrane (5.0 μm pore size, Costar). LN421 was put into the lower chamber at a concentration of 2.5 μg/ml in the trans well chamber. Then, RAW264.7 cells were placed in the upper chamber at a concentration of 5×10.sup.4 cells per well. Then, each antibody was put into the chamber at a concentration of 10 nM, and then incubated for 24 hours. Then, it was washed twice with PBS and treated with 70% MeOH (in PBS). After washing twice with PBS again, the transferred cells were stained with crystal violet (Sigma) and dried. Then, the upper chamber was put in 33% acetic acid (Merck) and stirred for 10 minutes. Crystal violet-dissolved acetic acid solution was transferred to a 96-well plate, and absorbance was measured at 590 nm in a microplate reader (Tecan).

[0385] As a result, as shown in FIG. 18, it was found that all of the N3-8, N3-8-1, N3-8-1 mutant LALA, N3-8-1 mutant LALATA, N3-8-1 mutant LALAPG, N3-8-1 mutant LALAPGTA antibodies inhibited LN421-dependent cell migration at a level similar to that of the control group (Control, C) that was not treated with anything.

Example 13: Analysis for the Effect of Mutant Antibodies with ADCC/CDC Function Removed on Cancer Cell Migration

[0386] In order to confirm the effect of the mutant antibody from which the ADCC/CDC function had been removed on immune cell migration, a cell migration assay was performed according to the method described in Example 12.

[0387] Measurements were made in a 24-well trans well chamber with a polycarbonate membrane (8.0 μm pore size, Costar). Laminin was added to the lower chamber at a concentration of 1 mg/ml, and a migration assay was performed using stable MDA-MB-231 cells overexpressing T52D KRS, which mimics a phosphorylated KRS. To induce T52D KRS expression, T52D KRS-MDA-MB-231 stable cells were treated with doxycycline (0.1 μg/ml) for one day, and then seeded in the upper chamber at a concentration of 4×10.sup.4 cells after suspended in serum-free RPMI medium. Then, each 10 nM of N3-8, N3-8-1, N3-8-1 mutant LALA, N3-8-1 mutant LALATA, N3-8-1 mutant LALAPG, N3-8-1 mutant LALAPGTA antibody was added in the chamber and incubated for 7 hours. The non-migrating cells existing above the membrane were removed with a cotton swab. The membrane was washed twice with PBS and treated with 70% MeOH (in PBS) for 30 minutes. Again washed twice with PBS, the membrane was stained using crystal violet (Sigma), and dried. Then, the upper chamber was put in 33% acetic acid (Merck) and stirred. Crystal violet-dissolved acetic acid solution was transferred to a 96-well plate, and absorbance was measured at 590 nm in a microplate reader (Tecan).

[0388] As shown in FIG. 19, all of the N3-8, N3-8-1, N3-8-1 mutant LALA, N3-8-1 mutant LALATA, N3-8-1 mutant LALAPG, N3-8-1 mutant LALAPGTA antibodies inhibited the laminin-dependent migration of cancer cells.

INDUSTRIAL APPLICABILITY

[0389] As described above, the antibodies or fragments thereof of the present invention have a specific CDR (complementarity determining region) sequence described herein, and have very excellent specific binding capacity and affinity to the KRS N-terminal region exposed to the extracellular membrane. Therefore, it can be used for the diagnosis of diseases accompanying the specific behavior of KRS, such as cancer or immune cell migration-related diseases. And they have excellent productivity and stability, and excellent cancer metastasis inhibitory effect. Therefore, it can be usefully used as cancer therapeutics as well as preventor or inhibitor of cancer metastasis, and can be very useful in the prevention, improvement and treatment of diseases related to immune cell migration.