Antibody for specifically binding to lysyl-tRNA synthetase N-terminal domain exposed to extracellular membrane
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.
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 the amino acid sequence SYDMS (SEQ ID NO: 1);
(ii) heavy chain complementarity determining region 2 (CDR2) containing the amino acid sequence X 1 |X 2 X 3 X 4 X 5 GX 6 X 7 YYADSVKG (SEQ ID NO: 252), wherein X 1 is A or V, X 2 is S, D or G, X 3 is Y, P, S or A, X 4 is D, Q, L or Y, X 5 is N, M, S, or G, X 6 is N, R or P, X 7 is T, V, I or S; and
(iii) heavy chain complementarity determining region 3 (CDR3) containing the amino acid sequence X 8 ALDFDY (SEQ ID NO: 253), wherein X 8 is M or L, and
(b) a light chain variable region (VL) comprising
(i) light chain complementarity determining region 1 (CDR1) containing the amino acid sequence TGSSSNIGSNYVT (SEQ ID NO: 7);
(ii) light chain complementarity determining region 2 (CDR2) containing the amino acid sequence X 9 NX 10 X 11 RPS (SEQ ID NO: 254), wherein X 9 is D, S or R, X 10 is S or N, and X 11 is N or Q; and
(iii) light chain complementarity determining region 3 (CDR3) containing the amino acid sequence X 12 SFSDELGAYV (SEQ ID NO: 255), wherein X 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 the amino acid sequence of SEQ ID NO: 1; heavy chain complementarity determining region 2 (CDR2) containing one of the amino acid sequences 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 one of the amino acid sequences 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 the amino acid sequence of SEQ ID NO: 7; a light chain complementarity determining regions 2 (CDR2) containing one of the amino acid sequences 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 one of the amino acid sequences 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 3, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 9, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 3, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 9, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 118, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 9, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 118, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 9, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 17, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 9, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 19, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 9, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 21, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 9, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 23, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 9, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 21, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 27, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 21, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 29, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 21, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 9, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 21, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 27, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 1, heavy chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 21, heavy chain complementary determining region 3 (CDR3) containing the amino acid sequence of 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 of SEQ ID NO: 7, light chain complementary determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 29, and light chain complementary determining region 3 (CDR3) containing the amino acid sequence of SEQ ID NO: 15.
5 . The antibody or fragment thereof of claim 1 , wherein the antibody or fragment thereof contains a heavy chain (HC) comprising one of the 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 of the 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 of SEQ ID NO: 89, and a light chain containing the amino acid sequence of SEQ ID NO: 107;
a heavy chain containing the amino acid sequence of SEQ ID NO: 89, and a light chain containing the amino acid sequence of SEQ ID NO: 109;
a heavy chain containing the amino acid sequence of SEQ ID NO: 93, and a light chain containing the amino acid sequence of SEQ ID NO: 107;
a heavy chain containing the amino acid sequence of SEQ ID NO: 93, and a light chain containing the amino acid sequence of SEQ ID NO: 109;
a heavy chain containing the amino acid sequence of SEQ ID NO: 95, and a light chain containing the amino acid sequence of SEQ ID NO: 109;
a heavy chain containing the amino acid sequence of SEQ ID NO: 97, and a light chain containing the amino acid sequence of SEQ ID NO: 109;
a heavy chain containing the amino acid sequence of SEQ ID NO: 99, and a light chain containing the amino acid sequence of SEQ ID NO: 109;
a heavy chain containing the amino acid sequence of SEQ ID NO: 101, and a light chain containing the amino acid sequence of SEQ ID NO: 109;
a heavy chain containing the amino acid sequence of SEQ ID NO: 103, and a light chain containing the amino acid sequence of SEQ ID NO: 111;
a heavy chain containing the amino acid sequence of SEQ ID NO: 103, and a light chain containing the amino acid sequence of SEQ ID NO: 113;
a heavy chain containing the amino acid sequence of SEQ ID NO: 103, and a light chain containing the amino acid sequence of SEQ ID NO: 115;
a heavy chain containing the amino acid sequence of SEQ ID NO: 105, and a light chain containing the amino acid sequence of SEQ ID NO: 111;
a heavy chain containing the amino acid sequence of SEQ ID NO: 105, and a light chain containing the amino acid sequence of SEQ ID NO: 113;
a heavy chain containing the amino acid sequence of SEQ ID NO: 105, and a light chain containing the amino acid sequence of SEQ ID NO: 115;
a heavy chain containing the amino acid sequence of SEQ ID NO: 99, and a light chain containing the amino acid sequence of SEQ ID NO: 111;
a heavy chain containing the amino acid sequence of SEQ ID NO: 103, and a light chain containing the amino acid sequence of 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 one of the 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 . 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 of SEQ ID NO: 126 or at least one additional amino acid substitution which is T179A of the human IgG4 Fc region of 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.
10 . A polynucleotide encoding the antibody or fragment thereof of claim 1 .
11 . A method for producing an antibody or fragment thereof that specifically binds 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 10 ;
(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.
12 . A method for inhibiting cancer and/or cancer metastasis, the method comprising:
administering an effective amount of a composition comprising the antibody or fragment thereof of claim 1 to a subject in need thereof,
wherein the cancer is a cancer that overexpresses KRS N-terminal region on the cell membrane.
13 . 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,
wherein the cancer is a cancer that overexpresses KRS N-terminal region on the cell membrane.
14 . A method for treating an immune cell migration-related disease, the method comprising:
administering an effective amount of a composition comprising the antibody or fragment thereof of claim 1 to a subject in need thereof,
wherein the immune cell migration-related disease is a disease that overexpresses KRS N-terminal region on the cell membrane, and is selected from the group consisting of a cardiovascular disease, a fibrotic disease, an inflammatory disease, and Alport syndrome.
15 . 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;
c) detecting the expression level of the KRS protein in the sample 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,
wherein the immune cell migration-related disease is a disease that overexpresses KRS N-terminal region on the cell membrane, and is selected from the group consisting of a cardiovascular disease, a fibrotic disease, an inflammatory disease, and Alport syndrome.