BINDING MOLECULE SPECIFIC TO LRIG-1 PROTEIN, AND USE THEREOF

Abstract

Provided herein is a binding molecule capable of specifically binding to LRIG-1 protein, which is located on the surface of a regulatory T cell. Also provided is pharmaceutical composition comprising the binding molecule. Further provided are methods of treating an immune-related disease using the binding molecule or pharmaceutical composition disclosed herein.

Claims

1-25. (canceled)

26. A binding molecule specific for leucine-rich and immunoglobulin like domains 1 (LRIG-1) protein, wherein the binding molecule is selected from the group consisting of: (i) a binding molecule comprising a heavy chain variable region having the amino acid sequence as set forth in SEQ ID NO: 11, and a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 12; (ii) a binding molecule comprising a heavy chain variable region having the amino acid sequence as set forth in SEQ ID NO: 19, and a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 20; (iii) a binding molecule comprising a heavy chain variable region having the amino acid sequence as set forth in SEQ ID NO: 27, and a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 28; and (iv) a binding molecule comprising a heavy chain variable region having the amino acid sequence as set forth in SEQ ID NO: 35, and a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 36.

27. The binding molecule of claim 26, wherein the LRIG-1 protein comprises the amino acid sequence as set forth in SEQ ID NO: 1 or 3.

28. The binding molecule of claim 27, wherein the LRIG-1 protein is encoded by a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 2 or 4.

29. The binding molecule of claim 26, further comprising an Fc region or a constant region.

30. The binding molecule of claim 29, wherein the Fc region is an Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody, or a hybrid Fc region.

31. The binding molecule of claim 26, further comprising a heavy chain constant region having the amino acid sequence as set forth in SEQ ID NOs: 37, 39, 41, 42, 43, 44, or 53.

32. The binding molecule of claim 26, further comprising a light chain constant region having the amino acid sequence as set forth in SEQ ID NO: 38 or 40.

33. The binding molecule of claim 26, further comprising a heavy chain constant region having the amino acid sequence as set forth in SEQ ID NO: 37, and a light chain constant region having the amino acid sequence as set forth in SEQ ID NO: 38.

34. The binding molecule of claim 26, further comprising a heavy chain constant region having the amino acid sequence as set forth in SEQ ID NO: 39, 41, 42, 43, or 53, and a light chain constant region having the amino acid sequence as set forth in SEQ ID NO: 40.

35. The binding molecule of claim 26, further comprising a heavy chain constant region having the amino acid sequence as set forth in SEQ ID NO: 44.

36. The binding molecule of claim 26, wherein the binding molecule is selected from the group consisting of: (i) a binding molecule comprising a heavy chain having the amino acid sequence as set forth in SEQ ID NO: 45, and a light chain having the amino acid sequence as set forth in SEQ ID NO: 46; (ii) a binding molecule comprising a heavy chain having the amino acid sequence as set forth in SEQ ID NO: 47, and a light chain having the amino acid sequence as set forth in SEQ ID NO: 48; (iii) a binding molecule comprising a heavy chain having the amino acid sequence as set forth in SEQ ID NO: 49, and a light chain having the amino acid sequence as set forth in SEQ ID NO: 50; and (iv) a binding molecule comprising a heavy chain having the amino acid sequence as set forth in SEQ ID NO: 51, and a light chain having the amino acid sequence as set forth in SEQ ID NO: 52.

37. The binding molecule of claim 26, wherein the binding molecule is an antibody or a fragment thereof.

38. The binding molecule of claim 37, wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a unibody, a diabody, a triabody, or a tetrabody, or a fragment thereof.

39. A pharmaceutical composition comprising the binding molecule of claim 26.

40. A method of preventing or treating an immune-related disease in a subject in need thereof, the method comprising administering a pharmaceutically effective amount of the binding molecule of claim 26 to the subject.

41. The method of claim 40, wherein the immune-related disease is an autoimmune disease, a graft versus host disease, an organ transplant rejection, asthma, atopy, or an acute or chronic inflammatory disease.

42. The method of claim 40, wherein the binding molecule is administered to the subject via oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal route.

43. A method of preventing or treating an immune-related disease in a subject in need thereof, the method comprising administering a pharmaceutically effective amount of the pharmaceutical composition of claim 39 to the subject.

44. The method of claim 43, wherein the immune-related disease is an autoimmune disease, a graft versus host disease, an organ transplant rejection, asthma, atopy, or an acute or chronic inflammatory disease.

45. The method of claim 43, wherein the pharmaceutical composition is administered to the subject via oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal route.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0145] FIG. 1 illustrates a structure of the Lrig-1 protein according to an embodiment of the present invention.

[0146] FIG. 2 illustrates a structure of the Lrig-1 protein according to an embodiment of the present invention.

[0147] FIG. 3 illustrates prediction results for epitopes of the Lrig-1 protein according to an embodiment of the present invention.

[0148] FIG. 4 illustrates prediction results for epitopes of the Lrig-1 protein according to an embodiment of the present invention.

[0149] FIG. 5 illustrates an expression level of Lrig-1 mRNA according to an embodiment of the present invention.

[0150] FIG. 6 illustrates an expression level of Lrig-1 mRNA according to an embodiment of the present invention.

[0151] FIG. 7 illustrates an expression level of Lrig-1 mRNA according to an embodiment of the present invention.

[0152] FIG. 8 illustrates expression levels of Lrig-1, Lrig-2, and Lrig-3 mRNAs according to an embodiment of the present invention.

[0153] FIG. 9 illustrates results obtained by comparing expression levels of Lrig-1 protein in regulatory T cells and non-regulated T cells according to an embodiment of the present invention.

[0154] FIG. 10 illustrates expression of the Lrig-1 protein on the surface of regulatory T cells according to an embodiment of the present invention.

[0155] FIG. 11 illustrates results obtained by analyzing binding capacity of Lrig-1 protein-specific monoclonal antibodies (A7, C8, E7, and G3) to the Lrig-1 protein according to an embodiment of the present invention.

[0156] FIG. 12 illustrates results obtained by analyzing the mechanism of regulating Lrig-1 protein-induced Stat3 phosphorylation, in regulatory T cells, of Lrig-1 protein-specific monoclonal antibodies (A7, C8, E7, and G3) according to an embodiment of the present invention.

[0157] FIG. 13 illustrates results obtained by analyzing therapeutic effects, on an autoimmune disease, of Lrig-1 protein-specific monoclonal antibodies (A7, C8, E7, and G3) according to an embodiment of the present invention.

DETAILED DESCRIPTION OF INVENTION

[0158] According to an embodiment of the present invention, there is provided a binding molecule selected from the group consisting of the following (1) to (4):

[0159] (1) a binding molecule comprising a heavy chain variable region that contains a heavy chain CDR1 represented by SEQ ID NO: 5, a heavy chain CDR2 represented by SEQ ID NO: 6, and a heavy chain CDR3 represented by SEQ ID NO: 7; and a light chain variable region that contains a light chain CDR1 represented by SEQ ID NO: 8, a light chain CDR2 represented by SEQ ID NO: 9, and a light chain CDR3 represented by SEQ ID NO: 10;

[0160] (2) a binding molecule comprising a heavy chain variable region that contains a heavy chain CDR1 represented by SEQ ID NO: 13, a heavy chain CDR2 represented by SEQ ID NO: 14, and a heavy chain CDR3 represented by SEQ ID NO: 15; and a light chain variable region that contains a light chain CDR1 represented by SEQ ID NO: 16, a light chain CDR2 represented by SEQ ID NO: 17, and a light chain CDR3 represented by SEQ ID NO: 18;

[0161] (3) a binding molecule comprising a heavy chain variable region that contains a heavy chain CDR1 represented by SEQ ID NO: 21, a heavy chain CDR2 represented by SEQ ID NO: 22, and a heavy chain CDR3 represented by SEQ ID NO: 23; and a light chain variable region that contains a light chain CDR1 represented by SEQ ID NO: 24, a light chain CDR2 represented by SEQ ID NO: 25, and a light chain CDR3 represented by SEQ ID NO: 26;

[0162] (4) a binding molecule comprising a heavy chain variable region that contains a heavy chain CDR1 represented by SEQ ID NO: 29, a heavy chain CDR2 represented by SEQ ID NO: 30, and a heavy chain CDR3 represented by SEQ ID NO: 31; and a light chain variable region that contains a light chain CDR1 represented by SEQ ID NO: 32, a light chain CDR2 represented by SEQ ID NO: 33, and a light chain CDR3 represented by SEQ ID NO: 34.

[0163] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only for describing the present invention in more detail, and it will be apparent to those skilled in the art that according to the gist of the present invention, the scope of the present invention is not limited by these examples.

EXAMPLES

[Preparation Example 1] T Cell Subset Cell Culture

[0164] In order to identify whether the Lrig-1 protein is expressed only in regulatory T cells (Treg), the subsets of T cells, Th0, Th1, Th2, Th17, and iTreg, were prepared. The iTreg refers to cells whose differentiation has been artificially induced in a medium containing the following composition, unlike nTreg which has been naturally isolated.

[0165] The subsets of the T cells were induced to differentiate into respective cells by first isolating naive T cells obtained from the spleen of mice, causing RPMI1640 (Invitrogen Gibco, Grand Island, N.Y.) nutrient medium that contains 10% fetal bovine serum (FBS; HyClone, Logan, Utah) to further contain the respective ingredients of Table 1 below, and performing 72-hour incubation in an incubator at 37° C., 5% CO.sub.2.

TABLE-US-00001 TABLE 1 Differentiated cell Composition Th0 anti-CD3, anti-CD28 Th1 IL-12, anti-IL-4 antibody Th2 IL-4, anti-IFNβ Th17 IL-6, TGFβ, anti-IFNβ, anti-IL-4 iTreg IL-2, TGFβ

[Example 1] Structural Analysis of Lrig-1

[0166] A three-dimensional steric structure of the extracellular domain of the Lrig-1 protein was predicted to produce antibodies specific for the Lrig-1 protein, a surface protein of regulatory T cells.

[0167] First, in order to predict base sequences of epitopes (epitopes), tools of Uniprot (http://www.uniprot.org) and RCSB Protein Data Bank (http://www.rcsb.org/pdb) were used to predict a three-dimensional steric structure of the extracellular domain (ECD) of the Lrig-1 protein so that the structure of ECD is identified. Then, the results are illustrated in FIGS. 1 and 2.

[0168] As illustrated in FIG. 1, a total of 15 leucine-rich regions of LRR1 to LRR15 existed in the Lrig-LRR domain (amino acid sequence at positions 41 to 494) in the extracellular domain of the Lrig-1 protein. Each of the LRR domains is composed of 23 to 27 amino acids, with 3 to 5 leucine being present.

[0169] In addition, as illustrated in FIG. 2, three immunoglobulin-like domains exist in amino acid sequences at positions 494 to 781 of the Lrig-1 protein in the extracellular domain of the Lrig-1 protein.

[Example 2] Prediction of Lrig-1 Epitope Amino Acid Sequence

[0170] Prediction of the above base sequence was performed using Ellipro server (http://tools.iedb.org/ellipro/) which is an epitope prediction software based on a structure of the Lrig-1 protein. The Ellipro search engine was used because it corresponds to a search engine known to be the most reliable among the existing algorithms for predicting an epitope.

[0171] The extracellular domain analyzed in Example 1 was entered into the epitope prediction software, and then predicted contiguous or discontiguous amino acid sequences of the predicted epitopes are illustrated in FIGS. 3 and 4.

[0172] As illustrated in FIGS. 3 and 4, a total of 22 contiguous epitope amino acid sequences were predicted, and a total of 8 discontiguous epitope amino acid sequences were predicted.

[Production Examples 1 to 4] Production of Monoclonal Antibodies Specific to Lrig-1 Protein

[0173] Antibodies specific for the Lrig-1 protein according to the present invention were produced. The present antibodies were not produced by specifying a certain epitope, but were produced as antibodies capable of binding to any site on the Lrig-1 protein.

[0174] In order to produce the antibodies, cells expressing the Lrig-1 protein were produced. More specifically, a DNA fragment corresponding to SEQ ID NO: 2 and pcDNA (hygro) were cleaved with a cleavage enzyme, incubated at 37° C., and ligated to produce pcDNA into which a DNA sequence of the Lrig-1 protein is inserted. The thus produced pcDNA into which SEQ ID NO: 2 is inserted was introduced, through transfection, into L cells, so that the Lrig-1 protein is allowed to be expressed on the surface of the L cells.

[0175] Light and heavy chain amino acid sequences capable of binding to Lrig-1 expressed on the cell surface were selected from the Human scFv library so that a total of eight heavy and light chains were selected.

[0176] The selected heavy and light chain amino acid sequences were fused with the mlgG2a Fc region, to produce monoclonal antibodies. The sequences of the monoclonal antibodies are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Sequence Classification Clone Location Amino acid sequence information Production A7 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYD SEQ ID NO: 45 Example 1 clone chain MSWVRQAPGKGLEWVSLIYPDSGNKYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DAGLSWAGAFDYWGQGTLVTVSSTTAPSVYPL APVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSG SLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQS ITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPA PNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVS EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTL RVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDS DGSYFMYSKLRVEKKNWVERNSYSCSVVHEGL HNHHTTKSFSRTPGK Light QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVT SEQ ID NO: 46 chain WYQQLPGTAPKLLIYSDSHRPSGVPDRFSGSKSG TSASLAISGLQSEDEADYYCGSWDYSLSAYVFG GGTKLTVLRTVAAPTVSIFPPSSEQLTSGGASVV CFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC Production C8 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYY SEQ ID NO: 47 Example 2 clone chain MSWVRQAPGKGLEWVSGISPGDSSTYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GLYSNPNEPFDYWGQGTLVTVSSTTAPSVYPLAP VCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLS SGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITC NVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNL LGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSED DPDVQISWFVNNVEVHTAQTQTHREDYNSTLRV VSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI SKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMV TDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDG SYFMYSKLRVEKKNWVERNSYSCSVVHEGLHN HHTTKSFSRTPGK Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYVS SEQ ID NO: 48 chain WYQQLPGTAPKLLIYDDSQRPSGVPDRFSGSKSG TSASLAISGLRSEDEADYYCGTWDYSLNGYVFG GGTKLTVLRTVAAPTVSIFPPSSEQLTSGGASVV CFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC Production E7 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDM SEQ ID NO: 49 Example 3 clone chain SWVRQAPGKGLEWVSGISPDGSNIYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVG LRCRYEACSYAYGMDVWGQGTLVTVSSTTAPS VYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSST WPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPP CKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCV VVDVSEDDPDVQISWFVNNVEVHTAQTQTHRED YNSTLRVVSALPIQHQDWMSGKEFKCKVNNKD LPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTE PVLDSDGSYFMYSKLRVEKKNWVERNSYSCSV VHEGLHNHHTTKSFSRTPGK Light QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVS SEQ ID NO: 50 chain WYQQLPGTAPKLLIYSDSHRPSGVPDRFSGSKSG TSASLAISGLRSEDEADYYCATWDSSLNGYVFG GGTKLTVLRTVAAPTVSIFPPSSEQLTSGGASVV CFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC Production G3 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYD SEQ ID NO: 51 Example 4 clone chain MSWVRQAPGKGLEWVSSISPSSGSIYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKD LDAFWRPSFDYWGQGTLVTVSSTTAPSVYPLAP VCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLS SGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITC NVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNL LGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSED DPDVQISWFVNNVEVHTAQTQTHREDYNSTLRV VSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI SKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMV TDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDG SYFMYSKLRVEKKNWVERNSYSCSVVHEGLHN HHTTKSFSRTPGK Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNNVN SEQ ID NO: 52 chain WYQQLPGTAPKLLIYSDSHRPSGVPDRFSGSKSG TSASLAISGLRSEDEADYYCGSWDDSLSAYVFGG GTKLTVLRTVAAPTVSIFPPSSEQLTSGGASVVCF LNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTST SPIVKSFNRNEC Production A8 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYD — Example 5 clone chain MSWVRQVPGKGLEWVSWISHGGGSIYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GLGLCKTGLCYYYDAMDVWGQGTLVTVSSTTA PSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTL TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSS TWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP PCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTC VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHRE DYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK DLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKK QVTLTCMVTDFMPEDIYVEWTNNGKTELNYKN TEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS VVHEGLHNHHTTKSFSRTPGK Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNSVT — chain WYQQLPGTAPKLLIYADNNRPSGVPDRFSGSKS GTSASLAISGLRSEDEADYYCAAWDSSLSAYVFG GGTKLTVLRTVAAPTVSIFPPSSEQLTSGGASVV CFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC Production B8 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYY — Example 6 clone chain MSWVRQAPGKGLEWVSGISHDSGSKYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR HWTTFDYWGQGTLVTVSSTTAPSVYPLAPVCGD TTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVH TFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAH PASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGP SVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDV QISWFVNNVEVHTAQTQTHREDYNSTLRVVSAL PIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPK GSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFM PEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM YSKLRVEKKNWVERNSYSCSVVHEGLHNHHTT KSFSRTPGK Light QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNNVT — chain WYQQLPGTAPKLLIYANSNRPSGVPDRFSGSKSG TSASLAISGLRSEDEADYYCGAWDYSLSAYVFG GGTKLTVLRTVAAPTVSIFPPSSEQLTSGGASVV CFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC Production D9 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYA — Example 7 clone chain MSWVRQAPGKGLEWVSAIYPGGGSIYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DILPCPWGRCYYDYAMDVWGQGTLVTVSSTTA PSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTL TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSS TWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP PCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTC VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHRE DYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK DLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKK QVTLTCMVTDFMPEDIYVEWTNNGKTELNYKN TEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS VVHEGLHNHHTTKSFSRTPGK Light QSVLTQPPSASGTPGQRVTISCSDSSSNIGSNTVS — chain WYQQLPGTAPKLLIYADNNRPSGVPDRFSGSKS GTSASLAISGLRSEDEADYYCGTWDYSLSGYVF GGGTKLTVLRTVAAPTVSIFPPSSEQLTSGGASV VCFLNNFYPKDINVKWKIDGSERQNGVLNSWTD QDSKDSTYSMSSTLTLTKDEYERHNSYTCEATH KTSTSPIVKSFNRNEC Production H6 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYA — Example 8 clone chain MSWVRQAPGKGLEWVSVISHGGGSTYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR VISNCHLGVCYYSNGMDVWGQGTLVTVSSTTAP SVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSST WPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPP CKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCV VVDVSEDDPDVQISWFVNNVEVHTAQTQTHRED YNSTLRVVSALPIQHQDWMSGKEFKCKVNNKD LPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTE PVLDSDGSYFMYSKLRVEKKNWVERNSYSCSV VHEGLHNHHTTKSFSRTPGK Light QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNDVY — chain WYQQLPGTAPKLLIYSDSQRPSGVPDRFSGSKSG TSASLAISGLRSEDEADYYCGTWDYSLSGYVFG GGTKLTVLRTVAAPTVSIFPPSSEQLTSGGASVV CFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC

[Example 3] Identification of Specific Expression of Lrig-1 mRNA in Regulatory T Cells

[0177] Verification was made of whether the Lrig-1 protein can act as a biomarker specific for regulatory T cells.

[0178] For the verification, CD4.sup.+ T cells were isolated using magnet-activated cell sorting (MACS), through CD4 beads, from the spleen of rats. Subsequently, regulatory T (CD4.sup.+CD25.sup.+ T) cells and non-regulatory T (CD4.sup.+CD25.sup.− T) cells were isolated with a fluorescence-activated cell sorter (FACS) using a CD25 antibody. For the respective cells and the cells differentiated in Preparation Example 1, mRNA was extracted using Trizol, and gDNA was removed from genomic RNA using gDNA extraction kit (Qiagen) according to the protocol provided by the manufacturer. The gDNA-removed mRNA was synthesized into cDNA through the BDsprint cDNA Synthesis Kit (Clonetech).

[0179] Real-time polymerase chain reaction (RT PCR) was performed to quantitatively identify an expression level of Lrig-1 mRNA in the cDNA.

[0180] The real-time polymerase chain reaction was performed with primers shown in Table 3 below using SYBR Green (Molecular Probes) by the protocol provided by the manufacturer under conditions of 40 cycles consisting of 95° C. for 3 minutes, 61° C. for 15 seconds, 72° C. for 30 seconds, a relative gene expression level was calculated using the ΔΔCT method, and normalized using HPRT. The results are illustrated in FIGS. 5 to 8.

TABLE-US-00003 TABLE 3 Primer Sequence Mouse Lrig-1 Forward 5′-GAC GGA ATT CAG TGA GGA GAA CCT-3′ Reverse 5′-CAA CTG GTA GTG GCA GCT TGT AGG-3′ Mouse Lrig-2 forward 5′-TCA CAA GGA ACA TTG TCT GAA CCA-3′ reverse 5′-GCC TGA TCT AAC ACA TCC TCC TCA-3′ Mouse Lrig-3 forward 5′-CAG CAC CTT GAG CTG AAC AGA AAC-3′ reverse 5′-CCA GCC TTT GGT AAT CTC GGT TAG-3′ Mouse FOXP3 forward 5′-CTT TCA CCT ATC CCA CCC TTA TCC-3′ reverse 5′-ATT CAT CTA CGG TCC ACA CTG CTC-3′ ACTG1 forward 5′-GGC GTC ATG GTG GGC ATG GG-3′ reverse 5′-ATG GCG TGG GGA AGG GCG TA-3′

[0181] As illustrated in FIG. 5, it can be seen that the expression of Lrig-1 in regulatory T (CD4.sup.+CD25.sup.+ T) cells is 18.1 times higher than non-regulatory T (CD4.sup.+CD25.sup.− T) cells. This was about 10 times higher expression level than Lag3 and Ikzf4, which are previously known markers for regulatory T cells.

[0182] In addition, as illustrated in FIGS. 6 and 7, the expression of Lrig-1 mRNA was remarkably high in regulatory T cells as compared with other types of immune cells, and in particular, was remarkably high in naturally isolated regulatory T cells (nTreg) as compared with induced regulatory T cells (iTreg cells).

[0183] In addition, as illustrated in FIG. 8, expression of Lrig-1 was the highest among Lrig-1, Lrig-2, and Lrig-3 which correspond to the Lrig family.

[0184] From the above results, it can be seen that the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells, in particular, naturally-occurring regulatory T cells.

[Example 4] Identification of Specific Expression of Lrig-1 Protein in Regulatory T Cells

[0185] It was identified whether the Lrig-1 protein expressed from Lrig-1 mRNA is specifically expressed only in regulatory T cells.

[0186] Using FOXP3-RFP-knocked-in mice, the FOXP3-RFP obtained by coupling red fluorescence protein (RFP) to FOXP3 promoter, a transcription factor specific for regulatory T cells, CD4.sup.+ T cells were isolated using magnet-activated cell sorting (MACS), through CD4 beads, from the spleen of the mice. Subsequently, using RFP protein, regulatory T (CD4.sup.+RFP.sup.+ T) cells and non-regulatory T (CD4.sup.+RFP.sup.− T) cells were obtained by performing isolation through a fluorescence-activated cell sorter (FACS). The respective cells were stained with the purchased Lrig-1 antibody and a negative control was stained with an isotype-matched control antibody, to measure an expression level of Lrig-1 with the fluorescence-activated cell sorter. The results are illustrated in FIG. 9.

[0187] As illustrated in FIG. 9, the non-regulatory T cells indicated by a dotted line showed almost the same expression level of Lrig-1 as the negative control, whereas there were a large number of cells with high expression level of Lrig-1 in the regulatory T cells.

[0188] From the above results, it can be seen that the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells.

[Example 5] Identification of Specific Expression of Lrig-1 Protein on Surface of Regulatory T Cells

[0189] From the viewpoint that in order to be a target of cell therapy, the Lrig-1 protein must be expressed on the surface of regulatory T cells, which in turn allows a more effective target therapy, it was identified whether the Lrig-1 protein is expressed on the surface of the regulatory T cells.

[0190] The respective differentiated T cell subsets of Preparation Example 1 were stained with anti-CD4-APC and anti-Lrig-1-PE antibodies, and expression levels of Lrig-1 were measured at the respective cell surfaces using a fluorescence-activated cell sorter (FACS). The results are illustrated in FIG. 10.

[0191] As illustrated in FIG. 10, Lrig-1 was expressed in an amount of 0.77 to 15.3 in activated T cells, Th1 cells, Th2 cells, Th17 cells, and naive T cells, whereas Lrig-1 was expressed as high as 83.9 in differentiation-induced T cells (iTreg cells).

[0192] From the above results, it can be seen that the Lrig-1 protein according to the present invention is not only specifically expressed in regulatory T (Treg) cells, but also is, in particular, expressed at a higher level on the surface of the Treg cells.

[Example 6] Evaluation of Binding Capacity of Antibody According to Present Invention to Lrig-1 Protein

[0193] In order to identify whether the monoclonal antibodies according to the present invention produced in Production Examples 1 to 8 well recognize Lrig-1, each of the antibodies of Production Examples 1 to 8 was bound to L cells that stably express Lrig-1. Then, a secondary antibody which is conjugated with eFlour 670 and is capable of recognizing the mouse antibodies was added thereto, and then binding capacity of the monoclonal antibodies to the Lrig-1 protein was analyzed using FACS. The results are illustrated in FIG. 11.

[0194] As illustrated in FIG. 11, it was found that all Lrig-1 protein-specific monoclonal antibodies (A7, C8, E7, and G3) according to the present invention effectively recognize and bind to the Lrig-1 protein present on the surface of L cells.

[Example 7] Regulation of Signal Transduction Pathway in Treg Cells, by Antibody According to Present Invention

[0195] In order to analyze how the monoclonal antibodies according to the present invention produced in Production Examples 1 to 8 affect the signal transduction pathway in Treg cells through the Lrig-1 protein, Lrig-1 present on the surface of the Treg cells was stimulated by treating the Treg cells with the antibodies of Production Examples 1 to 8, and then a level of tyrosine phosphorylation of Stat3 protein present in the stimulated Treg cells was analyzed through phosphotyrosine immunoblot. The results are illustrated in FIG. 12.

[0196] As illustrated in FIG. 12, it was found that the Lrig-1 protein-specific monoclonal antibodies (A7, C8, E7, and G3) according to the present invention increase phosphorylation of Stat3 at the same level as Th17 cells.

[Example 8] Therapeutic Effects of Antibody According to the Present Invention on Autoimmune Disease

[0197] In order to identify therapeutic effects of the monoclonal antibodies (A7, C8, E7, and G3) according to the present invention produced in Production Examples 1 to 4 on an autoimmune disease, RAG-1.sup.−/− mice were subjected to adoptive transfer with CD45RB (high) cells so that inflammatory bowel disease (IBD), which is an autoimmune disease, was induced. Then, the antibodies of Production Examples 1 to 4 were intraperitoneally injected in an amount of 200 μg/mouse, and then therapeutic effects thereof on the autoimmune disease were analyzed. The results are illustrated in FIG. 13.

[0198] As illustrated in FIG. 13, it was found that the Lrig-1 protein-specific monoclonal antibodies (A7, C8, E7, and G3) according to the present invention remarkably inhibit a body weight-decreasing effect in inflammatory bowel disease-induced mice.

[0199] From this, it can be seen that the Lrig-1 protein-specific monoclonal antibody according to the present invention are capable of effectively preventing, ameliorating, or treating immune-related diseases, such as autoimmune diseases, graft-versus-host diseases, organ transplant rejection, asthma, atopy, or acute or chronic inflammatory disease, which are induced by excessive activation and expression of various immune cells and inflammatory cells.

[0200] Although the present invention has been described in detail above, the scope of the present invention is not limited thereto. It will be obvious to those skilled in the art that various modifications and changes can be made without departing from the technical spirit of the present invention described in the claims.

INDUSTRIAL APPLICABILITY

[0201] The present invention relates to a binding molecule capable of specifically binding to leucine-rich and immunoglobulin-like domains 1 (Lrig-1) protein, which is a protein present on the surface of regulatory T cells (Treg cells), and a use thereof, specifically, prevention or treatment of immune-related diseases, such as autoimmune diseases, graft-versus-host diseases, organ transplant rejection, asthma, atopy, or acute or chronic inflammatory diseases.