Novel anti-glycoantibody and its use

Abstract

The present invention provides an antibody or a fragment thereof that specifically binds to a 6-sulfosiallyl Lewis X-type sugar chain, and the bond requires the fucose, the sulfate group, and the sialic acid that form the 6-sulfosiallyl Lewis X-type sugar chain.

Description

[Technical Field]

[0001] The present invention broadly relates to novel antibodies that target glycans, particularly antibodies or fragments thereof that specifically bind to 6-sulfosialyl Lewis X glycans and require the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X glycan for their binding, as well as uses thereof. [Background technology]

[0002] The pathogenesis of many immune-related diseases remains unknown, and autoimmune diseases, in particular, represent a significant area of unmet medical need. Natalizumab, an antibody targeting VLA-4 integrin, an adhesion molecule that mediates inflammatory cell infiltration into the central nervous system, has been shown to improve multiple sclerosis. However, it can also cause progressive multifocal leukoencephalopathy (PML) associated with JC virus infection. Furthermore, the small molecule drug fingolimod (FTY720) has been shown to be effective in treating multiple sclerosis, but its side effects, including infections and bradycardia, necessitate the development of novel therapeutics with different mechanisms of action. FTY720's mechanism of action is to retain antigen-sensitized lymphocytes within lymph nodes. However, inhibiting lymphocyte homing, the process of lymphocyte migration into lymph nodes, could potentially suppress antigen sensitization within lymph nodes and potentially lead to a fundamental cure for various immune-related diseases, including multiple sclerosis. However, it is unclear whether inhibiting lymphocyte homing to lymph nodes can treat immune-related diseases such as autoimmune diseases.

[0003] In the immune system, numerous immune cells circulate throughout the body via blood and lymph, monitoring for foreign antigens. The migration of lymphocytes circulating in peripheral blood to secondary lymphoid tissues is called lymphocyte homing, and is strictly controlled by a multi-step adhesion molecule cascade. Therefore, understanding these adhesion molecule cascades is important for understanding lymphocyte dynamics and tissue tropism.

[0004] Lymphocyte homing is a biological defense mechanism that efficiently induces immune responses. When lymphocytes migrate to secondary lymphoid tissues such as peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches, they adhere to high endothelial venules (HEVs), which are made up of endothelial cells with a special dice-shaped structure, and then infiltrate into the tissues.

[0005] If we could inhibit lymphocyte homing and thereby suppress the specific adhesion between lymphocytes and HEV, it would be possible to control the body's allergic response, for example, and this could be useful in treating various diseases. However, because various factors are involved in the development of immune-related diseases such as autoimmune diseases and allergic diseases, it is unclear whether inhibiting lymphocyte homing can be used to treat these immune-related diseases.

[0006] In the adhesion molecule cascade, 6-sulfosialyl Lewis X is a glycan structure essential for lymphocyte homing because it is responsible for the initial adhesion to high endothelial venules, which mediate lymphocyte infiltration. The present inventors generated mice lacking a gene encoding sulfotransferase, which transfers sulfate groups to glycans, and demonstrated that 6-sulfosialyl Lewis X, a sulfated glycan specifically expressed in HEV, plays an essential role in lymphocyte homing to secondary lymphoid tissues and the development of contact dermatitis (Non-Patent Document 1: Kawashima et al., Nat. Immunol., 11:1096-1104, 2005).

[0007] Although antibodies that recognize HEV are known, the amount of glycans expressed in vivo is small and they have diverse binding patterns, so the glycan structures recognized by each antibody vary. For example, MECA-79 (Streeter et al., J. Cell Biol., 107:1853-1862, 1988), which specifically stains HEV in peripheral lymph nodes, recognizes 6-sulfosialyl Lewis X only when present on a special O-glycan called the extended core 1 structure. Furthermore, while its recognition requires the sulfate group in 6-sulfosialyl Lewis X, it does not require the fucose or sialic acid in the glycans that make up 6-sulfosialyl Lewis X, which play important roles in lymphocyte homing. The S1 and S2 antibodies (Hirakawa et al., J. Biol. Chem., 285:40864-40878, 2010) also recognize 6-sulfosialyl Lewis X, but require the sulfate group and sialic acid in 6-sulfosialyl Lewis X for recognition, but not fucose. Therefore, these antibodies bind not only to HEV but also to tissues expressing the fucose-depleted 6-sulfosialylLacNAc glycan, potentially resulting in unexpected side effects in vivo other than the inhibition of lymphocyte homing. Furthermore, the anti-sialyl Lewis X monoclonal antibodies F1 and F2 (Matsumura et al., J. Biol. Chem., 290:15313-15326, 2015) also recognize 6-sulfosialyl Lewis X, but do not require the sulfate group in 6-sulfosialyl Lewis X for recognition, recognizing unsulfated sialyl Lewis X. Thus, in addition to binding to HEV and inhibiting lymphocyte homing, these antibodies also bind to leukocytes expressing sialyl Lewis X and inhibit P-selectin- and E-selectin-dependent leukocyte rolling.

[0008] Both the S1 and S2 antibodies are IgM class antibodies, making it difficult to prepare them in large quantities with high purity. This makes it difficult to verify their therapeutic effectiveness against autoimmune diseases and other immune-related diseases that require frequent administration, and their therapeutic effectiveness against autoimmune diseases and other immune-related diseases is unknown.

[0009] Furthermore, although antibodies other than the S1 and S2 antibodies that have binding affinity to 6-sulfosialyl Lewis X are known (Non-Patent Documents 2, 4, 5, and 6), the therapeutic effects of these antibodies on immune-related diseases such as autoimmune diseases are unknown. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Kawashima et al., Nat. Immunol., 11:1096-1104, 2005 [Non-patent document 2] Streeter et al., J. Cell Biol., 107:1853-1862, 1988 [Non-patent document 3] Hirakawa et al.,J.Biol.Chem.,285:40864-40878,2010 [Non-patent document 4] Matsumura et al.,J.Biol.Chem.,290:15313-15326,2015 [Non-patent document 5] Mitsuoka et al., J. Biol. Chem., 273:11225-11233, 1998 [Non-patent document 6] Hiroaki Matsuura et al., Abstracts of the 135th Annual Meeting of the Pharmaceutical Society of Japan (CD-ROM), Page: ROMBUNNO.27X-PM05S, 2015 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention aims to discover a glycan that is an effective therapeutic target for immune-related diseases such as multiple sclerosis, and to provide a novel antibody that targets the glycan, in particular, an antibody or fragment thereof that specifically binds to a 6-sulfosialyl Lewis X glycan and requires the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X glycan for its binding, as well as uses thereof. [Means for solving the problem]

[0012] The present inventors have generated several antibodies using a method devised by the inventors, in which glycosylase-deficient mice were immunized with cells overexpressing glycosylases. From these antibodies, an IgG1 class anti-glycan monoclonal antibody (SF1 antibody) that specifically binds to HEV expressing 6-sulfosialyl Lewis X glycans was selected by immunohistochemical analysis, and its binding specificity and sequence were analyzed. The results revealed that the obtained anti-glycan antibody has a novel sequence, recognizes 6-sulfosialyl Lewis X, and requires the simultaneous presence of three components of the 6-sulfosialyl Lewis X glycan: fucose, sulfate, and sialic acid. It was also found to strictly distinguish 6-sulfosialyl Lewis X from other glycan structures. Analysis by biolayer interferometry revealed that the K of binding between SF1 antibody and 6-sulfosialyl Lewis X was significantly higher than that of SF1 antibody. D The value is 6.09 x 10 -9 It was shown to be (M).

[0013] The inventors have demonstrated that the SF1 antibody inhibits the binding of HEV to the lymphocyte homing receptor L-selectin, and also inhibits the L-selectin-dependent rolling of lymphocytes on 6-sulfosialyl Lewis X-expressing cells, making it effective in inhibiting lymphocyte homing.

[0014] Next, we focused on the possibility that glycans involved in lymphocyte homing could be effective therapeutic targets for immune-mediated diseases such as multiple sclerosis, and analyzed experimental autoimmune encephalomyelitis using sulfotransferase GlcNAc6ST-1 / 2 double-deficient mice, in which lymphocyte homing is significantly impaired due to a deficiency in the synthesis of 6-sulfosialyl Lewis X glycans involved in lymphocyte homing. As a result, we discovered that 6-sulfosialyl Lewis X glycans synthesized by GlcNAc6ST-1 / 2 could be a therapeutic target for immune-mediated diseases such as multiple sclerosis.

[0015] Therefore, the present inventors conducted multifaceted non-clinical studies using animal models to examine the therapeutic effects of the SF1 antibody on immune-related diseases. As a result, they discovered that the antibody has significant preventive and therapeutic effects on experimental autoimmune encephalomyelitis. Furthermore, they demonstrated that the antibody also has disease-suppressing effects in rheumatoid arthritis and allergic rhinitis models. These findings demonstrate that the SF1 antibody specifically binds to 6-sulfosialyl Lewis X sugar chains and specifically inhibits lymphocyte homing to lymph nodes, thereby exerting significant therapeutic effects on immune-related diseases such as autoimmune and allergic diseases.

[0016] The present inventors performed immunohistochemical staining of human normal tissues using the SF1 antibody and demonstrated that the SF1 antibody specifically binds to HEV in human peripheral lymph nodes and tonsils.

[0017] Because the SF1 antibody is a murine antibody, humanization of the antibody is important for its use in the treatment of human immune-related diseases. Therefore, the inventors further constructed a humanized single-chain SF1 antibody by grafting the hypervariable region sequence of the SF1 antibody onto the frame sequence of a human antibody variable region, and demonstrated that it retained specific binding to HEV. Furthermore, they constructed a fully humanized SF1 antibody by replacing the variable region sequence with a human IgG1 heavy chain and a human κ light chain. The antibody exhibited sulfate- and fucose-dependent specific binding to HEV and retained its specific binding specificity for the 6-sulfosialyl Lewis X sugar chain.

[0018] Next, the present inventors demonstrated that humanized SF1 antibody, like mouse SF1 antibody, is effective in inhibiting lymphocyte homing and suppressing the onset of experimental autoimmune encephalomyelitis.

[0019] Furthermore, the present inventors have prepared humanized SF1 antibodies in which the sequence of the Fc portion has been altered to a sequence known to reduce effector activity mediated by the Fc portion of the antibody and reduce the risk of side effects, and have found that the antibodies retain specific tissue-binding properties. These humanized SF1 antibodies with altered Fc portion sequences are considered useful as therapeutic antibodies for immune-related diseases such as autoimmune diseases and allergic diseases.

[0020] That is, the present invention includes the following inventions. [1] An antibody or a fragment thereof that specifically binds to a 6-sulfosialyl Lewis X sugar chain, and the binding requires fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X sugar chain, comprising: (a) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:2; (c) CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; (d) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:4; (e) CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5; and (f) CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:6; or comprising at least one CDR selected from the group consisting of: At least one CDR consisting of an amino acid sequence having one or more amino acid deletions, substitutions or additions in the amino acid sequence of each CDR, or An antibody or fragment thereof comprising at least one CDR consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of each CDR. [2] below: (a) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2; and (c) CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; or a heavy chain variable region comprising a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, each of which has an amino acid sequence in which one or more amino acids have been deleted, substituted, or added; or The antibody or fragment thereof according to [1], comprising a heavy chain variable region including CDRH1, CDRH2 and CDRH3, each of which has an amino acid sequence having 80% or more identity with the amino acid sequence of each CDR. [3] The antibody or fragment thereof according to [2], wherein the heavy chain variable region comprises at least 90 consecutive amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 13, 15 to 17. [4] The antibody or fragment thereof according to [2] or [3], wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13, 15 to 17, an amino acid sequence set forth in any one of SEQ ID NOs: 13, 15 to 17 in which one or several amino acid deletions, substitutions or additions are present, or an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 13, 15 to 17. [5] The antibody or fragment thereof according to any one of [2] to [4], wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 25, 27 or 28, an amino acid sequence set forth in SEQ ID NO: 25, 27 or 28 in which one or several amino acid deletions, substitutions or additions are present, or an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 25, 27 or 28. [6] below: (d) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:4; (e) CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5; and (f) CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:6; or a light chain variable region comprising a light chain variable region comprising CDRL1, CDRL2, and CDRL3, each of which has an amino acid sequence with one or more amino acid deletions, substitutions, or additions in the amino acid sequence of each CDR; or The antibody or fragment thereof according to any one of [1] to [5], comprising a light chain variable region including CDRL1, CDRL2, and CDRL3, each of which has an amino acid sequence having 80% or more identity with the amino acid sequence of each CDR. [7] The antibody or fragment thereof according to [6], wherein the light chain variable region comprises at least 80 consecutive amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 14, 18 to 20. [8] The antibody or fragment thereof according to [6] or [7], wherein the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14, 18 to 20, an amino acid sequence set forth in any one of SEQ ID NOs: 14, 18 to 20 in which one or several amino acids have been deleted, substituted or added, or an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 14, 18 to 20. [9] The antibody or fragment thereof according to any one of [6] to [8], wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 26, an amino acid sequence set forth in SEQ ID NO: 26 with one or several amino acid deletions, substitutions or additions, or an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 26.

[10] below: (a) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:2; (c) CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; (d) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:4; (e) CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5; and (f) CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:6; or or CDRH1, CDRH2, and CDRH3, and CDRL1, CDRL2, and CDRL3, each consisting of an amino acid sequence having one or more amino acid deletions, substitutions, or additions in the amino acid sequence of each CDR; The antibody or fragment thereof according to any one of [1] to [9], comprising CDRH1, CDRH2 and CDRH3, and CDRL1, CDRL2 and CDRL3, each of which has an amino acid sequence having 80% or more identity with the amino acid sequence of each CDR.

[11] The antibody or fragment thereof according to any one of [1] to

[10] , which inhibits binding of L-selectin to high endothelial venules expressing 6-sulfosialyl Lewis X sugar chains.

[12] The antibody or fragment thereof according to any one of [1] to

[11] , wherein the constant region is derived from human.

[13] The antibody or fragment thereof according to any one of [1] to

[12] , which is humanized.

[14] The antibody or fragment thereof according to any one of [1] to

[13] , selected from the group consisting of Fab, F(ab')2, Fab', Fv and single-chain antibodies.

[15] The antibody or fragment thereof according to

[14] , wherein the single-chain antibody comprises an amino acid sequence of amino acids 1 to 117 and 133 to 238 in the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 24, an amino acid sequence having one or several amino acid deletions, substitutions or additions in the amino acid sequence of amino acids 1 to 117 and 133 to 238 in the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 24, or an amino acid sequence having 80% or more identity to the amino acid sequence of amino acids 1 to 117 and 133 to 238 in the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 24.

[16] The antibody or its fragment binds to the 6-sulfosialyl Lewis X sugar chain at a concentration of 1 × 10 -6 The dissociation constant (K D The antibody or fragment thereof according to any one of [1] to

[15] , wherein the antibody or fragment thereof exhibits a .alpha.-to-.alpha. value.

[17] A polynucleotide encoding the antibody or fragment thereof according to any one of [1] to

[16] .

[18]

[17] An expression vector comprising the polynucleotide described in

[17] .

[19] A host cell transfected with the expression vector described in

[18] .

[20] The host cell according to

[19] , which is a eukaryotic cell. [twenty one] A hybridoma that produces the antibody according to any one of [1] to

[16] . [twenty two] A lymphocyte homing inhibitor comprising the antibody or fragment thereof according to any one of [1] to

[16] , the polynucleotide according to

[17] , or the expression vector according to

[18] . [twenty three] A pharmaceutical composition comprising the antibody or fragment thereof according to any one of [1] to

[16] , the polynucleotide according to

[17] , or the expression vector according to

[18] . [twenty four] The pharmaceutical composition according to

[23] for treating or preventing a disease caused by an excessive immune response brought about by lymphocyte homing. [twenty five] The pharmaceutical composition according to

[24] , wherein the disease caused by an excessive immune response brought about by lymphocyte homing is an immune-related disease.

[26] The pharmaceutical composition according to

[25] , wherein the immune-related disease is an allergic disease or an autoimmune disease.

[27] The pharmaceutical composition according to

[26] , wherein the immune-related disease is an allergic disease selected from the group consisting of allergic rhinitis, atopic dermatitis, food allergy, oral allergy syndrome, drug allergy, hay fever, allergic conjunctivitis, eosinophilic pneumonia, allergic gastroenteritis, urticaria, photosensitivity, metal allergy, cat allergy, dust mite allergy, and asthma.

[28] Immune-related diseases include multiple sclerosis, including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis; systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's disease; benign lymphocytic vasculitis; thrombocytopenic purpura; idiopathic thrombocytopenia; idiopathic autoimmune hemolytic anemia; pure red cell aplasia; Sjogren's syndrome; rheumatic diseases; connective tissue diseases; inflammatory rheumatoid arthritis; rheumatoid osteoarthritis; non-articular rheumatoid arthritis; juvenile rheumatoid arthritis; muscular rheumatism; chronic polyarthritis; cryoglobulinemic vasculitis; A NCA-associated vasculitis; antiphospholipid syndrome; myasthenia gravis; autoimmune hemolytic anemia; Guillain-Barré syndrome; chronic immune polyneuropathy; autoimmune thyroiditis; insulin-dependent diabetes mellitus; type 1 diabetes; Addison's disease; membranous glomerular nephropathy; Goodpasture's disease; autoimmune gastritis; autoimmune atrophic gastritis; pernicious anemia; pemphigus; pemphigus vulgaris; cirrhosis; primary biliary cirrhosis; dermatomyositis; polymyositis; fibromyositis; myosclerosis; celiac disease; immunoglobulin A nephropathy; Henoch-Schönlein purpura; Evans syndrome; psoriasis; psoriatic arthritis; Graves' disease ;Graves' ophthalmopathy;Scleroderma;Systemic sclerosis;Progressive systemic sclerosis;Primary biliary cirrhosis;Hashimoto's thyroiditis;Primary myxedema;Sympathetic ophthalmia;Autoimmune uveitis;Hepatitis;Chronic active hepatitis;Collagen disease;Ankylosing spondylitis;Scapular periarthritis;Panarteritis nodosa;Chondrocalcinosis;Wegener's granulomatosis;Microscopic polyangiitis;Chronic urticaria;Bullous skin diseases;Pemphigoid;Devic's disease;Pedictal autoimmune hemolytic anemia;Refractory or chronic autoimmune cytopenia;Acquired hemophilia A;Cold agglutinin disease;Neuromyelitis optica;Stiff-body syndrome;Pancreatitis;Myocarditis;Vasculitis;Gastritis;Gout ;Gouty arthritis;Psoriasis;Enocomplementemic urticarial vasculitis;Pericarditis;Myositis;Antisynthetase syndrome;Scleritis;Macrophage activation syndrome;Behçet's syndrome;PAPA syndrome;Blau syndrome;Adult and juvenile Still's disease;Cryopyrin-associated periodic syndrome;Muckle-Wells syndrome;Familial cold autoinflammatory syndrome;Neonatal-onset multisystem inflammatory disease;Familial Mediterranean fever;Chronic infantile-onset neurocutaneous-articular syndrome;Systemic juvenile idiopathic arthritis;Hyper IgD syndrome;Schnitzler's syndrome;Autoimmune retinopathy;Atherosclerosis;Chronic prostatitis;and TNF receptor-associated periodic syndrome (TRAPS).

[29] The pharmaceutical composition according to

[28] , wherein the autoimmune disease is multiple sclerosis or a collagen disease.

[30] The pharmaceutical composition according to

[28] or

[29] , wherein the collagen disease is one or more diseases selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, scleroderma, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, Sjogren's syndrome, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, hypersensitivity vasculitis, Behcet's disease, Cogan's syndrome, RS3PE, giant cell arteritis, adult Still's disease, polymyalgia rheumatica, fibromyalgia, and SAPHO syndrome.

[31] The pharmaceutical composition according to any one of

[24] to

[30] , further comprising a drug for treating or preventing a disease caused by an excessive immune response brought about by lymphocyte homing.

[32] A method for producing the antibody or fragment thereof according to any one of [1] to

[16] , comprising the step of culturing the host cell according to

[19] or

[20] , or the hybridoma according to

[21] .

[33] A pharmaceutical composition for treating or preventing a disease caused by an excessive immune response brought about by lymphocyte homing, comprising an antibody or a fragment thereof that specifically binds to a 6-sulfosialyl Lewis X sugar chain, the binding requiring the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X sugar chain, a polynucleotide encoding the antibody or the fragment thereof, or an expression vector containing the polynucleotide.

[34] The pharmaceutical composition according to

[33] , wherein the disease caused by an excessive immune response brought about by lymphocyte homing is an immune-related disease.

[35] The pharmaceutical composition according to

[34] , wherein the immune-related disease is an allergic disease or an autoimmune disease.

[36] The pharmaceutical composition according to

[35] , wherein the immune-related disease is an allergic disease selected from the group consisting of allergic rhinitis, atopic dermatitis, food allergy, oral allergy syndrome, drug allergy, hay fever, allergic conjunctivitis, eosinophilic pneumonia, allergic gastroenteritis, urticaria, photosensitivity, metal allergy, cat allergy, dust mite allergy, and asthma.

[37] Immune-related diseases include multiple sclerosis, including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis; systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's disease; benign lymphocytic vasculitis; thrombocytopenic purpura; idiopathic thrombocytopenia; idiopathic autoimmune hemolytic anemia; pure red cell aplasia; Sjogren's syndrome; rheumatic diseases; connective tissue diseases; inflammatory rheumatoid arthritis; rheumatoid osteoarthritis; non-articular rheumatoid arthritis; juvenile rheumatoid arthritis; muscular rheumatism; chronic polyarthritis; cryoglobulinemic vasculitis; A NCA-associated vasculitis; antiphospholipid syndrome; myasthenia gravis; autoimmune hemolytic anemia; Guillain-Barré syndrome; chronic immune polyneuropathy; autoimmune thyroiditis; insulin-dependent diabetes mellitus; type 1 diabetes; Addison's disease; membranous glomerular nephropathy; Goodpasture's disease; autoimmune gastritis; autoimmune atrophic gastritis; pernicious anemia; pemphigus; pemphigus vulgaris; cirrhosis; primary biliary cirrhosis; dermatomyositis; polymyositis; fibromyositis; myosclerosis; celiac disease; immunoglobulin A nephropathy; Henoch-Schönlein purpura; Evans syndrome; psoriasis; psoriatic arthritis; Graves' disease ;Graves' ophthalmopathy;Scleroderma;Systemic sclerosis;Progressive systemic sclerosis;Primary biliary cirrhosis;Hashimoto's thyroiditis;Primary myxedema;Sympathetic ophthalmia;Autoimmune uveitis;Hepatitis;Chronic active hepatitis;Collagen disease;Ankylosing spondylitis;Scapular periarthritis;Panarteritis nodosa;Chondrocalcinosis;Wegener's granulomatosis;Microscopic polyangiitis;Chronic urticaria;Bullous skin diseases;Pemphigoid;Devic's disease;Pedictal autoimmune hemolytic anemia;Refractory or chronic autoimmune cytopenia;Acquired hemophilia A;Cold agglutinin disease;Neuromyelitis optica;Stiff-body syndrome;Pancreatitis;Myocarditis;Vasculitis;Gastritis;Gout ;Gouty arthritis;Psoriasis;Enocomplementemic urticarial vasculitis;Pericarditis;Myositis;Antisynthetase syndrome;Scleritis;Macrophage activation syndrome;Behçet's syndrome;PAPA syndrome;Blau syndrome;Adult and juvenile Still's disease;Cryopyrin-associated periodic syndrome;Muckle-Wells syndrome;Familial cold autoinflammatory syndrome;Neonatal-onset multisystem inflammatory disease;Familial Mediterranean fever;Chronic infantile-onset neurocutaneous-articular syndrome;Systemic juvenile idiopathic arthritis;Hyper IgD syndrome;Schnitzler's syndrome;Autoimmune retinopathy;Atherosclerosis;Chronic prostatitis;and TNF receptor-associated periodic syndrome (TRAPS).

[38] The pharmaceutical composition according to

[37] , wherein the autoimmune disease is multiple sclerosis or a collagen disease.

[39]

[38] The pharmaceutical composition according to

[38] , wherein the collagen disease is one or more diseases selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, scleroderma, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, Sjogren's syndrome, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, hypersensitivity vasculitis, Behcet's disease, Cogan's syndrome, RS3PE, giant cell arteritis, adult Still's disease, polymyalgia rheumatica, fibromyalgia, and SAPHO syndrome.

[40] The pharmaceutical composition according to any one of

[33] to

[39] , wherein the antibody or a fragment thereof inhibits binding of L-selectin to high endothelial venules expressing 6-sulfosialyl Lewis X sugar chains.

[41] The pharmaceutical composition according to any one of

[33] to

[40] , wherein the antibody or fragment thereof is the antibody or fragment thereof according to any one of [1] to

[16] .

[42] A method for treating or preventing a disease caused by an excessive immune response brought about by lymphocyte homing in a subject, the method comprising the step of administering to the subject an effective amount of an antibody or fragment thereof that specifically binds to a 6-sulfosialyl Lewis X sugar chain and requires the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X sugar chain, a polynucleotide encoding the antibody or fragment thereof, or an expression vector containing the polynucleotide.

[43] The method according to

[42] , wherein the disease is an immune-related disease caused by an excessive immune response brought about by lymphocyte homing.

[44] The method according to

[43] , wherein the immune-related disease is an allergic disease or an autoimmune disease.

[45] The method according to

[44] , wherein the immune-related disease is an allergic disease selected from the group consisting of allergic rhinitis, atopic dermatitis, food allergy, oral allergy syndrome, drug allergy, hay fever, allergic conjunctivitis, eosinophilic pneumonia, allergic gastroenteritis, urticaria, photosensitivity, metal allergy, cat allergy, dust mite allergy, and asthma.

[46] Immune-related diseases include multiple sclerosis, including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis; systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's disease; benign lymphocytic vasculitis; thrombocytopenic purpura; idiopathic thrombocytopenia; idiopathic autoimmune hemolytic anemia; pure red cell aplasia; Sjogren's syndrome; rheumatic diseases; connective tissue diseases; inflammatory rheumatoid arthritis; rheumatoid osteoarthritis; non-articular rheumatoid arthritis; juvenile rheumatoid arthritis; muscular rheumatism; chronic polyarthritis; cryoglobulinemic vasculitis; A NCA-associated vasculitis; antiphospholipid syndrome; myasthenia gravis; autoimmune hemolytic anemia; Guillain-Barré syndrome; chronic immune polyneuropathy; autoimmune thyroiditis; insulin-dependent diabetes mellitus; type 1 diabetes; Addison's disease; membranous glomerular nephropathy; Goodpasture's disease; autoimmune gastritis; autoimmune atrophic gastritis; pernicious anemia; pemphigus; pemphigus vulgaris; cirrhosis; primary biliary cirrhosis; dermatomyositis; polymyositis; fibromyositis; myosclerosis; celiac disease; immunoglobulin A nephropathy; Henoch-Schönlein purpura; Evans syndrome; psoriasis; psoriatic arthritis; Graves' disease ;Graves' ophthalmopathy;Scleroderma;Systemic sclerosis;Progressive systemic sclerosis;Primary biliary cirrhosis;Hashimoto's thyroiditis;Primary myxedema;Sympathetic ophthalmia;Autoimmune uveitis;Hepatitis;Chronic active hepatitis;Collagen disease;Ankylosing spondylitis;Scapular periarthritis;Panarteritis nodosa;Chondrocalcinosis;Wegener's granulomatosis;Microscopic polyangiitis;Chronic urticaria;Bullous skin diseases;Pemphigoid;Devic's disease;Pedictal autoimmune hemolytic anemia;Refractory or chronic autoimmune cytopenia;Acquired hemophilia A;Cold agglutinin disease;Neuromyelitis optica;Stiff-body syndrome;Pancreatitis;Myocarditis;Vasculitis;Gastritis;Gout ;Gouty arthritis;Psoriasis;Enocomplementemic urticarial vasculitis;Pericarditis;Myositis;Antisynthetase syndrome;Scleritis;Macrophage activation syndrome;Behçet's syndrome;PAPA syndrome;Blau syndrome;Adult and juvenile Still's disease;Cryopyrin-associated periodic syndrome;Muckle-Wells syndrome;Familial cold autoinflammatory syndrome;Neonatal-onset multisystem inflammatory disease;Familial Mediterranean fever;Chronic infantile-onset neurocutaneous-articular syndrome;Systemic juvenile idiopathic arthritis;Hyper IgD syndrome;Schnitzler's syndrome;Autoimmune retinopathy;Atherosclerosis;Chronic prostatitis;and TNF receptor-associated periodic syndrome (TRAPS).

[47] The method according to

[46] , wherein the autoimmune disease is multiple sclerosis or a collagen disease.

[48] The method according to

[46] or

[47] , wherein the collagen disease is one or more diseases selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, scleroderma, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, Sjogren's syndrome, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, hypersensitivity vasculitis, Behcet's disease, Cogan's syndrome, RS3PE, giant cell arteritis, adult Still's disease, polymyalgia rheumatica, fibromyalgia, and SAPHO syndrome.

[49] The method according to any one of

[42] to

[48] , wherein the antibody or fragment thereof is the antibody or fragment thereof according to any one of [1] to

[16] .

[50] The method according to any one of

[42] to

[49] , further comprising the step of administering a drug for treating or preventing a disease caused by an excessive immune response brought about by lymphocyte homing.

[51] The antibody or fragment thereof according to any one of [1] to

[16] , for use in the treatment or prevention of a disease caused by an excessive immune response brought about by lymphocyte homing.

[52] The antibody or fragment thereof according to

[51] , wherein the disease is an immune-related disease caused by an excessive immune response brought about by lymphocyte homing.

[53] The antibody or fragment thereof according to

[52] , wherein the immune-related disease is an allergic disease or an autoimmune disease.

[54] The antibody or fragment thereof according to

[53] , wherein the immune-related disease is an allergic disease selected from the group consisting of allergic rhinitis, atopic dermatitis, food allergy, oral allergy syndrome, drug allergy, hay fever, allergic conjunctivitis, eosinophilic pneumonia, allergic gastroenteritis, urticaria, photosensitivity, metal allergy, cat allergy, dust mite allergy, and asthma.

[55] Immune-related diseases include multiple sclerosis, including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis; systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's disease; benign lymphocytic vasculitis; thrombocytopenic purpura; idiopathic thrombocytopenia; idiopathic autoimmune hemolytic anemia; pure red cell aplasia; Sjogren's syndrome; rheumatic diseases; connective tissue diseases; inflammatory rheumatoid arthritis; rheumatoid osteoarthritis; non-articular rheumatoid arthritis; juvenile rheumatoid arthritis; muscular rheumatism; chronic polyarthritis; cryoglobulinemic vasculitis; A NCA-associated vasculitis; antiphospholipid syndrome; myasthenia gravis; autoimmune hemolytic anemia; Guillain-Barré syndrome; chronic immune polyneuropathy; autoimmune thyroiditis; insulin-dependent diabetes mellitus; type 1 diabetes; Addison's disease; membranous glomerular nephropathy; Goodpasture's disease; autoimmune gastritis; autoimmune atrophic gastritis; pernicious anemia; pemphigus; pemphigus vulgaris; cirrhosis; primary biliary cirrhosis; dermatomyositis; polymyositis; fibromyositis; myosclerosis; celiac disease; immunoglobulin A nephropathy; Henoch-Schönlein purpura; Evans syndrome; psoriasis; psoriatic arthritis; Graves' disease ;Graves' ophthalmopathy;Scleroderma;Systemic sclerosis;Progressive systemic sclerosis;Primary biliary cirrhosis;Hashimoto's thyroiditis;Primary myxedema;Sympathetic ophthalmia;Autoimmune uveitis;Hepatitis;Chronic active hepatitis;Collagen disease;Ankylosing spondylitis;Scapular periarthritis;Panarteritis nodosa;Chondrocalcinosis;Wegener's granulomatosis;Microscopic polyangiitis;Chronic urticaria;Bullous skin diseases;Pemphigoid;Devic's disease;Pedictal autoimmune hemolytic anemia;Refractory or chronic autoimmune cytopenia;Acquired hemophilia A;Cold agglutinin disease;Neuromyelitis optica;Stiff-body syndrome;Pancreatitis;Myocarditis;Vasculitis;Gastritis;Gout ;Gouty arthritis;Psoriasis;Enocomplementemic urticarial vasculitis;Pericarditis;Myositis;Antisynthetase syndrome;Scleritis;Macrophage activation syndrome;Behçet's syndrome;PAPA syndrome;Blau syndrome;Adult and juvenile Still's disease;Cryopyrin-associated periodic syndrome;Muckle-Wells syndrome;Familial cold autoinflammatory syndrome;Neonatal-onset multisystem inflammatory disease;Familial Mediterranean fever;Chronic infantile-onset neurocutaneous-articular syndrome;Systemic juvenile idiopathic arthritis;Hyper IgD syndrome;Schnitzler's syndrome;Autoimmune retinopathy;Atherosclerosis;Chronic prostatitis;and TNF receptor-associated periodic syndrome (TRAPS).

[56] The antibody or fragment thereof according to

[55] , wherein the autoimmune disease is multiple sclerosis or a collagen disease.

[57] The antibody or fragment thereof according to

[55] or

[56] , wherein the collagen disease is one or more diseases selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, scleroderma, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, Sjogren's syndrome, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, hypersensitivity vasculitis, Behcet's disease, Cogan's syndrome, RS3PE, giant cell arteritis, adult Still's disease, polymyalgia rheumatica, fibromyalgia, and SAPHO syndrome.

[58] Use of the antibody or fragment thereof according to any one of [1] to

[16] for the manufacture of a pharmaceutical for treating or preventing a disease caused by an excessive immune response brought about by lymphocyte homing.

[59] The use according to

[58] , wherein the disease caused by an excessive immune response brought about by lymphocyte homing is an immune-related disease.

[60] The use according to

[59] , wherein the immune-related disease is an allergic disease or an autoimmune disease.

[61] The use according to

[59] , wherein the immune-related disease is an allergic disease selected from the group consisting of allergic rhinitis, atopic dermatitis, food allergy, oral allergy syndrome, drug allergy, hay fever, allergic conjunctivitis, eosinophilic pneumonia, allergic gastroenteritis, urticaria, photosensitivity, metal allergy, cat allergy, dust mite allergy, and asthma.

[62] Immune-related diseases include multiple sclerosis, including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis; systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's disease; benign lymphocytic vasculitis; thrombocytopenic purpura; idiopathic thrombocytopenia; idiopathic autoimmune hemolytic anemia; pure red cell aplasia; Sjogren's syndrome; rheumatic diseases; connective tissue diseases; inflammatory rheumatoid arthritis; rheumatoid osteoarthritis; non-articular rheumatoid arthritis; juvenile rheumatoid arthritis; muscular rheumatism; chronic polyarthritis; cryoglobulinemic vasculitis; A NCA-associated vasculitis; antiphospholipid syndrome; myasthenia gravis; autoimmune hemolytic anemia; Guillain-Barré syndrome; chronic immune polyneuropathy; autoimmune thyroiditis; insulin-dependent diabetes mellitus; type 1 diabetes; Addison's disease; membranous glomerular nephropathy; Goodpasture's disease; autoimmune gastritis; autoimmune atrophic gastritis; pernicious anemia; pemphigus; pemphigus vulgaris; cirrhosis; primary biliary cirrhosis; dermatomyositis; polymyositis; fibromyositis; myosclerosis; celiac disease; immunoglobulin A nephropathy; Henoch-Schönlein purpura; Evans syndrome; psoriasis; psoriatic arthritis; Graves' disease ;Graves' ophthalmopathy;Scleroderma;Systemic sclerosis;Progressive systemic sclerosis;Primary biliary cirrhosis;Hashimoto's thyroiditis;Primary myxedema;Sympathetic ophthalmia;Autoimmune uveitis;Hepatitis;Chronic active hepatitis;Collagen disease;Ankylosing spondylitis;Scapular periarthritis;Panarteritis nodosa;Chondrocalcinosis;Wegener's granulomatosis;Microscopic polyangiitis;Chronic urticaria;Bullous skin diseases;Pemphigoid;Devic's disease;Pedictal autoimmune hemolytic anemia;Refractory or chronic autoimmune cytopenia;Acquired hemophilia A;Cold agglutinin disease;Neuromyelitis optica;Stiff-body syndrome;Pancreatitis;Myocarditis;Vasculitis;Gastritis;Gout ;Gouty arthritis;Psoriasis;Enocomplementemic urticarial vasculitis;Pericarditis;Myositis;Antisynthetase syndrome;Scleritis;Macrophage activation syndrome;Behçet's syndrome;PAPA syndrome;Blau syndrome;Adult and juvenile Still's disease;Cryopyrin-associated periodic syndrome;Muckle-Wells syndrome;Familial cold autoinflammatory syndrome;Neonatal-onset multisystem inflammatory disease;Familial Mediterranean fever;Chronic infantile-onset neurocutaneous-articular syndrome;Systemic juvenile idiopathic arthritis;Hyper IgD syndrome;Schnitzler's syndrome;Autoimmune retinopathy;Atherosclerosis;Chronic prostatitis;and TNF receptor-associated periodic syndrome (TRAPS).

[63] The use according to

[62] , wherein the autoimmune disease is multiple sclerosis or a collagen disease.

[64] The use according to

[62] or

[63] , wherein the collagen disease is one or more diseases selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, scleroderma, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, Sjogren's syndrome, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, hypersensitivity vasculitis, Behcet's disease, Cogan's syndrome, RS3PE, giant cell arteritis, adult Still's disease, polymyalgia rheumatica, fibromyalgia, and SAPHO syndrome. [Effects of the Invention]

[0021] The anti-glycoprotein antibodies of the present invention inhibit the binding of L-selectin expressed on lymphocytes to glycans on high endothelial venules (HEVs), making it possible to treat immune-related diseases (e.g., autoimmune diseases such as multiple sclerosis) by inhibiting lymphocyte homing. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram of the structure of the 6-sulfosialyl Lewis X sugar chain. [Figure 2] Figure 2 shows the results of immunofluorescence staining analyzing the reactivity of SF1 antibody and MECA-79 antibody to mouse lymph node high endothelial venules (HEV). [Figure 3] FIG. 3 shows the results of immunostaining using fluorescent immunostaining to analyze the reactivity of SF1 antibody, F2 antibody, and S2 antibody to mouse lymph node high endothelial venules (HEV). [Figure 4] FIG. 4 shows the results of immunostaining analyzing the effect of sialidase treatment on the reactivity of SF1 antibody to mouse lymph node HEV. [Figure 5] FIG. 5 shows the results of analyzing the reactivity of SF1 antibody to various sugar chains using a glycan array. [Figure 6] FIG. 6 shows the results of measuring the dissociation constant of the binding between SF1 antibody and 6-sulfosialyl Lewis X by biolayer interferometry. [Figure 7] FIG. 7 shows the amino acid sequences of the hypervariable regions of the heavy and light chain variable regions of the SF1 antibody. [Figure 8] FIG. 8 shows the amino acid sequence of the heavy chain variable region of the SF1 antibody. [Figure 9] FIG. 9 shows the amino acid sequence of the light chain variable region of the SF1 antibody. [Figure 10] FIG. 10 shows the inhibitory effect of SF1 antibody on the binding of L-selectin-Fc to mouse lymph node HEV. [Figure 11] FIG. 11 shows the inhibitory effect of SF1 antibody on L-selectin-dependent lymphocyte rolling. [Figure 12] FIG. 12 shows the results of analyzing the inhibitory effect of SF1 antibody on lymphocyte homing. [Figure 13] FIG. 13 is a graph comparing the EAE clinical scores of GlcNAc6ST-1 / 2 double-deficient mice and wild-type mice. [Figure 14] FIG. 14 is a graph showing the effect of administration of SF1 antibody at the time of antigen sensitization on EAE clinical scores. [Figure 15] FIG. 15 is a graph showing the effect of administration of SF1 antibody after the onset of EAE (administration of SF1 antibody from day 8 after the initial antigen sensitization) on EAE clinical scores. [Figure 16] FIG. 16 is a graph showing the effect of administration of SF1 antibody after the onset of EAE (administration of SF1 antibody from day 21 after the initial antigen sensitization) on EAE clinical scores. [Figure 17] FIG. 17 is a graph showing the effect of administration of SF1 antibody on the incidence rate (left) and clinical score (right) of collagen-induced arthritis (CIA). [Figure 18] FIG. 18 is a graph showing the inhibitory effect of SF1 antibody on sneezing (left panel) and nose-scratching behavior (right panel) in a mouse allergic rhinitis model. [Figure 19]FIG. 19 shows the results of immunostaining normal human tissues with SF1 antibody. [Figure 20] FIG. 20 shows an enlarged view of the immunostaining results of lymph nodes and tonsils in the photographs of FIG. [Figure 21] FIG. 21 shows the amino acid sequences of the heavy and light chain variable regions of the humanized SF1 antibody. [Figure 22] FIG. 22 shows the amino acid sequence of the humanized SF1 single chain antibody (scFv). [Figure 23] FIG. 23 shows the results of fluorescent immunostaining of mouse lymph nodes using humanized SF1 single-chain antibody (scFv). [Figure 24] FIG. 24 shows the full-length amino acid sequence of the heavy chain of the humanized SF1 antibody. [Figure 25] FIG. 25 shows the full-length amino acid sequence of the light chain of the humanized SF1 antibody. [Figure 26] FIG. 26 shows the results of fluorescent immunostaining of mouse lymph nodes using humanized SF1 antibody composed of the heavy and light chains of the humanized SF1 antibody shown in FIGS. [Figure 27] FIG. 27 shows the results of analyzing the inhibitory effect of humanized SF1 antibody (HSF1 antibody) on lymphocyte homing. [Figure 28] FIG. 28 is a graph showing the effect of administration of humanized SF1 antibody (HSF1 antibody) at the time of antigen sensitization on EAE clinical scores. [Figure 29] FIG. 29 shows the full-length amino acid sequence of the heavy chain IgG1-LALA form of the humanized SF1 antibody. [Figure 30] FIG. 30 shows the full-length amino acid sequence of the heavy chain IgG4-SPLE form of the humanized SF1 antibody. [Figure 31] Figure 31 shows the results of fluorescent immunostaining of mouse lymph nodes using humanized SF1 antibody-IgG1-LALA (HSF1-LALA) and humanized SF1 antibody-IgG4-SPLE (HSF1-G4PE), which are composed of the heavy chain variants of the humanized SF1 antibody shown in Figures 29 and 30 and the light chain shown in Figure 25. DETAILED DESCRIPTION OF THE INVENTION

[0023] (Anti-sugar chain antibody) In a first aspect, an antibody or a fragment thereof is provided that specifically binds to a 6-sulfosialyl Lewis X sugar chain, and that the binding requires the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X sugar chain. Hereinafter, this antibody will also be referred to simply as an anti-sugar chain antibody. As used herein, the phrase "requires the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X sugar chain" for binding to or recognizing 6-sulfosialyl Lewis X means that the antibody requires all three elements, fucose, sulfate group, and sialic acid, to bind to 6-sulfosialyl Lewis X, and that the binding activity to a sugar chain lacking any one of fucose, sulfate group, or sialic acid from the 6-sulfosialyl Lewis X sugar chain is reduced compared to the binding activity to 6-sulfosialyl Lewis X.

[0024] The reactivity of 6-sulfosialyl Lewis X glycans with antibodies can be measured by standard methods, for example, using a glycan array in which glycans are immobilized on a substrate. Glycan arrays can be performed using, for example, the Glycan Array from the US Consortium for Functional Glycomics. The reactivity of 6-sulfosialyl Lewis X glycans with antibodies can also be evaluated using surface plasmon resonance or isothermal titration calorimetry.

[0025] In one embodiment, the dissociation constant (K D value) is usually 1×10 -6 M or less (e.g., 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, 1×10 -11 M or less).

[0026] In one embodiment, the K of the antibody or fragment thereof of the present invention is used to measure the binding affinity of the antibody or fragment thereof to a sugar chain obtained by deleting at least one of fucose, sulfate group, or sialic acid from a 6-sulfosialyl Lewis X sugar chain (e.g., 6-sulfo Lewis X, 6-sulfosialyl LacNAc, sialyl Lewis X). D The value is the K for 6-sulfosialyl Lewis X glycans. D Typically 10 times or more (e.g., 10 2 more than twice, 10 3 more than twice, 10 4 more than twice, 10 5 This is a value that is (more than twice) larger.

[0027] In one embodiment, the K for the binding affinity of the antibody or fragment thereof of the present invention to 6-sulfo Lewis X, 6-sulfosialyl LacNAc, and sialyl Lewis X is D All values are K for 6-sulfosialyl Lewis X sugar chains. D Typically 10 times or more (e.g., 10 2 more than twice, 10 3 more than twice, 10 4 more than twice, 10 5 This is a value that is (more than twice) larger.

[0028] K for the binding affinity of an antibody or its fragment to a carbohydrate antigen D The value can be measured by analyzing the binding properties of an antibody or its fragment to a biotinylated glycan bound to a streptavidin biosensor by biolayer interferometry using Octet RED96 (Pall-Fortebio), in accordance with the Examples described below.

[0029] 6-Sulfosialyl Lewis X is a sugar chain consisting of an N-acetyllactosamine (LacNAc) structure composed of galactose and N-acetylglucosamine (GlcNAc), to which sialic acid, fucose, and sulfate groups are attached (Figure 1). Biosynthesis of 6-sulfosialyl Lewis X requires sulfation at the 6th position of N-acetylglucosamine, and the sulfotransferases (STs) that catalyze this sulfation have been cloned in mice: N-acetylglucosamine-6-O-sulfotransferases (GlcNAc6ST)-1 to GlcNAc6ST-4.

[0030] In lymphocyte homing, lymphocytes infiltrate into lymph nodes via rolling on high endothelial venules (HEVs) (lymphocyte rolling) through specific binding between L-selectin expressed on lymphocytes and 6-sulfosialyl Lewis X expressed in the lumen of HEVs. Thus, the sulfated glycan 6-sulfosialyl Lewis X is essential for lymphocyte homing, as it is responsible for the initial adhesion to HEVs that mediate lymphocyte infiltration.

[0031] The binding of L-selectin to 6-sulfosialyl Lewis X sugar chains involves fucose, sulfate groups, and sialic acid. Because fucose, sulfate groups, and sialic acid play important roles in lymphocyte homing, an anti-sugar chain antibody or a fragment thereof that simultaneously recognizes fucose, sulfate groups, and sialic acid is thought to highly efficiently inhibit lymphocyte homing, particularly lymphocyte homing from cells expressing 6-sulfosialyl Lewis X sugar chains, such as lymph nodes (e.g., peripheral lymph nodes, mesenteric lymph nodes), or high endothelial venules of tonsils, and thus to be effective in treating or preventing diseases caused by excessive immune responses induced by lymphocyte homing. In a preferred embodiment, the anti-sugar chain antibody or a fragment thereof inhibits L-selectin-dependent lymphocyte rolling during the homing process.

[0032] Antibodies that specifically bind to 6-sulfosialyl Lewis X sugar chains and require the fucose, sulfate, and sialic acid that make up the 6-sulfosialyl Lewis X sugar chain for their binding can be produced by, for example, immunizing mice lacking GlcNAc6ST-1 and GlcNAc6ST-2, sulfotransferases responsible for sulfation of sialyl Lewis X expressed in HEV (hereinafter also referred to as sulfotransferase double-deficient mice or DKO mice) with cells expressing sulfated sugar chains through gene transfer of fucosyltransferase and sulfotransferase, and fusing the antibody-producing cells with myelomas to obtain hybridomas. Sulfotransferase double-deficient mice can be generated using techniques known to those skilled in the art, such as the technique described in Kawashima et al. (supra). The produced antibodies may be subjected to additional steps, such as purification, before recovery. For example, to increase purity, salting out, filtration, clarification using centrifugation, affinity recovery, intermediate purification by ion exchange, and final purification by gel filtration are preferred.

[0033] Examples of anti-glycan antibodies obtained in this manner include antibodies having a complementarity-determining region (CDR) that recognizes 6-sulfosialyl Lewis X, such as the following: (a) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1 (GFSLTSYA); (b) CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2 (IWGGGST); (c) CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3 (AKHEKLGRFPY); (d) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4 (SSVSY); (e) CDRL2 consisting of the amino acid sequence (EIS) set forth in SEQ ID NO: 5; and (f) CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:6 (QQWNYPLAT); Examples include antibodies containing at least one or more CDRs, preferably all six CDRs, selected from the group consisting of: (a) CDR1, (b) CDR2, (c) CDR3, and (d). Antibodies typically have a structure in which two heavy chains and two light chains are covalently linked by disulfide bonds, with each heavy chain being divided into a heavy chain variable region and a heavy chain constant region, and each light chain being divided into a light chain variable region and a light chain constant region. Each variable region contains three CDRs. Here, CDRH1, CDRH2, and CDRH3 represent the heavy chain complementarity-determining regions in order from the amino terminus of the heavy chain amino acid sequence, while CDRL1, CDRL2, and CDRL3 represent the light chain complementarity-determining regions in order from the amino terminus of the light chain amino acid sequence.

[0034] Therefore, the anti-glycan antibody or fragment thereof preferably comprises: (a) CDRH1 consisting of the amino acid sequence (GFSLTSYA) set forth in SEQ ID NO: 1; (b) CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2 (IWGGGST), and (c) a heavy chain variable region comprising a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3 (AKHEKLGRFPY); and / or (d) CDRL1 consisting of the amino acid sequence (SSVSY) set forth in SEQ ID NO: 4; (e) CDRL2 consisting of the amino acid sequence (EIS) set forth in SEQ ID NO: 5, and (f) A light chain variable region comprising CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6 (QQWNYPLAT).

[0035] The amino acid sequence of each CDR may have one or several amino acid deletions, substitutions, or additions, as long as the anti-glycan antibody or fragment thereof specifically binds to a 6-sulfosialyl Lewis X glycan and the binding requires the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X glycan. As used herein, "several" refers to 2 to 10, preferably 2 to 5, and more preferably 2 or 3, depending on the length of the sequence.

[0036] The substituted amino acid is preferably an amino acid that conserves the properties of the original amino acid side chain as classified below: However, amino acid substitution is not limited to conservative substitution. hydrophobic amino acids (A, I, L, M, F, P, W, Y, V); Hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T); amino acids with aliphatic side chains (G, A, V, L, I, P); Amino acids with hydroxyl-containing side chains (S, T, Y); Amino acids with sulfur atom-containing side chains (C, M); Amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q); amino acids with base-containing side chains (R, K, H); Amino acids with aromatic-containing side chains (H, F, Y, W)

[0037] In one embodiment, the amino acid substitution may be an amino acid that occurs at the corresponding position in a naturally occurring human antibody.

[0038] In one embodiment, the amino acid substitutions may occur outside of the complementarity determining regions (CDRs).

[0039] In one embodiment, the amino acid substitutions may be in the heavy chain variable region or the light chain variable region.

[0040] Alternatively, the amino acid sequence of each CDR may be composed of an amino acid sequence having 80% or more homology, preferably 80% or more identity, to the sequence set forth in each SEQ ID NO, as long as it specifically binds to a 6-sulfosialyl Lewis X sugar chain and the binding requires the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X sugar chain. The sequence homology or identity is preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, more preferably 99% or more.

[0041] In one embodiment, the anti-glycan antibody is one of the following: (a) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:2; (c) CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; (d) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:4; (e) CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5; and (f) CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:6; or a variable region comprising at least one CDR selected from the group consisting of At least one variable region containing a CDR consisting of an amino acid sequence having one or more amino acid deletions, substitutions or additions in the amino acid sequence of each CDR; or The antibody or fragment thereof may contain at least one variable region containing a CDR consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of each CDR, and binds to an epitope to which the antibody or fragment thereof binds.

[0042] As used herein, "epitope" refers to a partial structure of a 6-sulfosialyl Lewis X sugar chain to which an antibody or a fragment thereof binds, which specifically binds to the 6-sulfosialyl Lewis X sugar chain and requires the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X sugar chain. When the epitope is a conformational epitope, it can be identified by analyzing the structure of the 6-sulfosialyl Lewis X sugar chain bound to the antibody using X-ray crystallography or the like.

[0043] In one embodiment, the anti-glycan antibody is one of the following: (a) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2; and (c) CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; or a heavy chain variable region comprising a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, each of which has an amino acid sequence in which one or more amino acids have been deleted, substituted, or added; or The antibody or fragment thereof may comprise a heavy chain variable region including CDRH1, CDRH2, and CDRH3, each of which has an amino acid sequence that is 80% or more identical to the amino acid sequence of each CDR.

[0044] In one embodiment, the heavy chain variable region of the anti-glycan antibody or a fragment thereof may comprise at least 90 consecutive amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 13, 15 to 17.

[0045] As used herein, "at least 90 consecutive amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 13, 15 to 17" means 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, or 117 consecutive amino acids in the amino acid sequence set forth in any one of SEQ ID NOs: 13, 15 to 17. Preferably, such consecutive amino acids include CDRH1, CDRH2, and CDRH3, which are represented by amino acids 26 to 33, 51 to 57, and 96 to 106, respectively.

[0046] In one embodiment, the heavy chain variable region of an anti-glycan antibody or a fragment thereof may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 15 to 17, an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence set forth in any one of SEQ ID NOs: 15 to 17, or an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in any one of SEQ ID NOs: 15 to 17.

[0047] In one embodiment, the heavy chain of the anti-glycan antibody or a fragment thereof may comprise the amino acid sequence set forth in SEQ ID NO: 25, 27, or 28, an amino acid sequence having one or several amino acid deletions, substitutions, or additions in the amino acid sequence set forth in SEQ ID NO: 25, 27, or 28, or an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 25.

[0048] In one embodiment, the anti-glycan antibody is one of the following: (d) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:4; (e) CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5; and (f) CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:6; or a light chain variable region comprising a light chain variable region comprising CDRL1, CDRL2, and CDRL3, each of which has an amino acid sequence with one or more amino acid deletions, substitutions, or additions in the amino acid sequence of each CDR; or The antibody or fragment thereof may comprise a light chain variable region including CDRL1, CDRL2, and CDRL3, each of which has an amino acid sequence that is 80% or more identical to the amino acid sequence of each CDR.

[0049] In one embodiment, the anti-glycan antibody is one of the following: (a) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:2; (c) CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; (d) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:4; (e) CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5; and (f) CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:6; or a variable region comprising a variable region comprising CDRH1, CDRH2 and CDRH3, and CDRL1, CDRL2 and CDRL3, each of which has an amino acid sequence with one or more amino acid deletions, substitutions or additions in the amino acid sequence of each CDR; or The antibody or fragment thereof may comprise a variable region including CDRH1, CDRH2, CDRH3, and CDRL1, CDRL2, and CDRL3, each of which has an amino acid sequence that is 80% or more identical to the amino acid sequence of each CDR.

[0050] In one embodiment, the light chain variable region of the anti-glycan antibody or a fragment thereof may comprise at least 80 consecutive amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 14, 18 to 20.

[0051] As used herein, "at least 80 consecutive amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 14, 18 to 20" means 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106 consecutive amino acids in the amino acid sequence set forth in any one of SEQ ID NOs: 14, 18 to 20. Preferably, such consecutive amino acids include CDRL1, CDRL2, and CDRL3, which are represented by amino acids 27 to 31, 49 to 51, and 88 to 96, respectively.

[0052] In one embodiment, the light chain variable region of an anti-glycan antibody or a fragment thereof may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 14, 18 to 20, an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence set forth in any one of SEQ ID NOs: 14, 18 to 20, or an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in any one of SEQ ID NOs: 14, 18 to 20.

[0053] In one embodiment, the light chain of the anti-glycan antibody or a fragment thereof may comprise the amino acid sequence set forth in SEQ ID NO: 26, an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 26, or an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 26.

[0054] As long as the anti-glycan antibody has the above-mentioned CDRs, other structures, such as the structures of the variable region and constant region other than the CDRs, are not particularly limited. Examples of amino acid sequences encoding the heavy chain variable region containing the above-mentioned CDRH1, CDRH2, and CDRH3, and the light chain variable region containing CDRL1, CDRL2, and CDRL3, respectively, include the amino acid sequences of SEQ ID NOs: 13, 15, 16, and 17, and the amino acid sequences of SEQ ID NOs: 14, 18, 19, and 20, respectively.

[0055] In a preferred embodiment, the anti-glycan antibody or fragment thereof may comprise a heavy chain variable region having the amino acid sequence of any of SEQ ID NOs: 13, 15, 16, or 17, and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 14, 18, 19, or 20.

[0056] In a preferred embodiment, the humanized anti-glycan antibody or fragment thereof may comprise a heavy chain variable region having the amino acid sequence of any of SEQ ID NOs: 15, 16, or 17, and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 18, 19, or 20. (Examples of combinations: SEQ ID NO: 15 and SEQ ID NO: 18, SEQ ID NO: 15 and SEQ ID NO: 19, SEQ ID NO: 15 and SEQ ID NO: 20, SEQ ID NO: 16 and SEQ ID NO: 18, SEQ ID NO: 16 and SEQ ID NO: 19, SEQ ID NO: 16 and SEQ ID NO: 20, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 17 and SEQ ID NO: 19, SEQ ID NO: 17 and SEQ ID NO: 20)

[0057] In a more preferred embodiment, the anti-glycan antibody or fragment thereof may comprise any of the following combinations of heavy and light chain variable regions: a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14 (SEQ ID NO: 4) (SF1); a heavy chain variable region (H1) comprising the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region (L1) comprising the amino acid sequence represented by SEQ ID NO: 18; a heavy chain variable region (H1) comprising the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region (L2) comprising the amino acid sequence represented by SEQ ID NO: 19; a heavy chain variable region (H2) comprising the amino acid sequence represented by SEQ ID NO: 16 and a light chain variable region (L1) comprising the amino acid sequence represented by SEQ ID NO: 18; A heavy chain variable region (H3) comprising the amino acid sequence represented by SEQ ID NO: 17 and a light chain variable region (L3) comprising the amino acid sequence represented by SEQ ID NO: 20.

[0058] As used herein, "antibody" refers to a natural or wholly or partially synthetically produced artificial immunoglobulin or fragment thereof. Antibodies may be of any isotype: IgG, IgM, IgA, IgE, or IgD. Nine known classes (isotypes) of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM.

[0059] The antibody may be derived from any mammal, such as a human or a non-human animal, such as a mouse, rat, guinea pig, hamster, rabbit, goat, sheep, or camel. Not only human antibodies, but also recombinant antibodies such as humanized antibodies and chimeric antibodies can be used. As used herein, a "humanized antibody" refers to an antibody in which the CDRs of a human antibody are replaced with CDRs derived from a non-human animal antibody. In addition to the CDRs, some framework amino acid residues may also be grafted onto a human antibody. When a CDR derived from a non-human animal antibody is used in a human, it is preferable to combine it with the constant region of a human antibody. The antibody may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody, particularly a monoclonal antibody derived from a mammal, is preferred.

[0060] Anti-glycan antibodies may be chemically or biologically modified as long as the desired effect is achieved. Chemical modifications include the attachment of a chemical moiety to the amino acid backbone, chemical modifications of N- or O-linked carbohydrate chains, etc. Biological modifications may include post-translational modifications (e.g., N- or O-linked glycosylation, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation), and those in which a methionine residue is added to the N-terminus by expression in a prokaryotic host cell.

[0061] Further modifications may include deletions of one or two amino acids at the carboxyl terminus of the heavy chain, and amidation of such deletions (for example, a heavy chain in which the proline residue at the carboxyl terminus is amidated).

[0062] The amino acids Leu234-Leu235 of IgG1, as determined by Eu numbering (Kabat Eu numbering), are known to be a site that influences the expression of effector activity through antibody binding to Fc receptors and complement. Substituting Leu in this region of IgG1 with Ala (the resulting mutant is referred to as an "IgG1-LALA mutant") can sometimes reduce the effector activity mediated by the Fc portion of the antibody and reduce the risk of side effects (U.S. Patent Publication US5885573). Such modifications can also be made to the Fc portion of the antibody, if necessary. In addition to these mutations, substituting Pro329 with Gly (the resulting mutant is referred to as an "IgG1-P329G LALA mutant") can sometimes reduce the effector activity mediated by the Fc portion of the antibody and further reduce the risk of side effects (U.S. Patent Publication US8969526; Protein Eng. Des Sel., 29:457-466, 2016). Such modifications can also be made to the Fc portion of the antibody, if necessary. An example of a heavy chain of an IgG1-LALA body is a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:27.

[0063] It is known that substituting Ser228 of IgG4 with Pro in terms of EU numbering prevents IgG4 from becoming a half antibody. It is also known that substituting Leu235 of IgG4 with Glu reduces the expression of effector activity mediated by antibody binding to Fc receptors. Substituting Ser228 with Pro and Leu235 with Glu in this region of IgG4 (the resulting mutant is called an "IgG4-SPLE body") may reduce the effector activity mediated by the Fc portion of the antibody and reduce the risk of side effects (Newman et al., Clin. Immunol., 98:164-174, 2001). Such modifications may also be made to the Fc portion of an antibody, if necessary. An example of a heavy chain of an IgG4-SPLE body is a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 28.

[0064] In another embodiment, other amino acid substitutions or deletions known to reduce the expression of effector activity mediated by the Fc portion of an antibody (e.g., in EU numbering, substitution of Asp265 with Ala, deletion of Glu294, substitution of Asn297 with Ala, substitution of Lys322 with Ala, substitution of Ala330 with Leu, and substitution of Pro331 with Ser) may be made in the Fc portion of the antibody.

[0065] The two heavy chains constituting an anti-glycan antibody may be a combination of any one heavy chain selected from the group consisting of full-length and the above-mentioned deletion forms, or a combination of any two of them. The quantitative ratio of each deletion form may be affected by the type of cultured mammalian cells producing the anti-glycan antibody and the culture conditions, but an example of a major component of an anti-glycan antibody is one in which one amino acid residue is deleted at the carboxyl terminus in both of the two heavy chains.

[0066] As used herein, the term "fragment" of an antibody refers to a functional fragment that retains at least some of the functions of the antibody before fragmentation, and examples thereof include Fab, F(ab')2, scFv, Fab', and single-chain antibodies (scFv). The fragment is not limited to these molecules, and may be any fragment that specifically binds to a 6-sulfosialyl Lewis X sugar chain and requires the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X sugar chain for this binding. For example, an anti-glycoprotein antibody may be an antibody that has a single heavy chain variable region and does not have a light chain sequence, such as a single-domain antibody or nanobody.

[0067] Examples of expected functions of anti-glycan antibodies include recognition of 6-sulfosialyl Lewis X glycans and inhibition of lymphocyte homing. The antibody or a fragment thereof may be a multispecific antibody having specificity for one or more antigens other than 6-sulfosialyl Lewis X glycans. In addition to imparting multiple specificities, anti-glycan antibodies can also be modified with anticancer drugs or multivalently modified to improve binding affinity depending on the desired application.

[0068] Depending on the purpose, anti-glycoprotein antibodies may contain a linker such as a triple repeat sequence of Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 29). For example, when multiple variable regions constituting the antigen-binding site of an antibody are linked together, such as in a single-chain antibody, a linker may be interposed between them. For example, a single-chain antibody can be obtained by linking a heavy chain variable region and a light chain variable region via a linker. The sequence of the linker can be determined appropriately by those skilled in the art.

[0069] Examples of single-chain antibodies comprising amino acid sequences encoding the above-mentioned heavy-chain variable region comprising CDRH1, CDRH2, and CDRH3, and light-chain variable region comprising CDRL1, CDRL2, and CDRL3 include antibodies comprising the amino acid sequences of positions 1 to 117 and positions 133 to 238 in the amino acid sequence set forth in SEQ ID NO: 21, 22, 23, or 24. Such single-chain antibodies comprise, for example, the amino acid sequence set forth in SEQ ID NO: 21, 22, 23, or 24.

[0070] Anti-glycoprotein antibody fragments can be obtained by treating the full-length antibody protein molecule with enzymes such as papain and pepsin. Alternatively, they may be produced by expressing genes encoding the desired fragments in appropriate host cells. When transforming host cells, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or they can be inserted into separate expression vectors. Host cells may be either prokaryotic or eukaryotic. Examples of prokaryotic cells include bacterial cells such as Escherichia coli and Bacillus subtilis. When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. Animal cells, plant cells, or fungal cells can be used. Examples of animal cells include the following cells: (1) Mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, HEK293, Ba / F3, HL-60, Jurkat, SK-HEP1, etc. (2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0071] In preferred embodiments, the anti-glycan antibody may comprise any of the following heavy and light chain combinations: a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 25 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 26; a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 27 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 26; and A heavy chain comprising the amino acid sequence set forth in SEQ ID NO:28 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:26.

[0072] (Polynucleotide) In a second aspect, a polynucleotide encoding the anti-sugar chain antibody of the present invention or a fragment thereof (polynucleotide of the present invention) is provided.

[0073] The polynucleotide of the present invention may be a single-stranded or double-stranded polynucleotide (DNA (cDNA, etc.), RNA (mRNA, cRNA, etc.)) comprising a nucleotide sequence encoding the amino acid sequence of an anti-glycan antibody or a fragment thereof and / or its complementary strand.

[0074] The heavy chain variable region (or heavy chain) and light chain variable region (or light chain) of an anti-glycan antibody or a fragment thereof may be encoded on the same polynucleotide, or may be encoded on separate polynucleotides and provided as a combination thereof. A combination of a polynucleotide encoding a heavy chain variable region (or heavy chain) and a polynucleotide encoding a light chain variable region (or light chain) is also encompassed by the term "polynucleotide encoding an anti-glycan antibody or a fragment thereof."

[0075] Examples of polynucleotides encoding anti-glycan antibodies or fragments thereof include: below: (a) a nucleotide sequence encoding CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence encoding CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2; (c) a nucleotide sequence encoding CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; (d) a nucleotide sequence encoding CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4; (e) a nucleotide sequence encoding CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5; and (f) a nucleotide sequence encoding CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; Examples include those having a base sequence encoding at least one or more CDRs selected from the group consisting of:

[0076] Examples of the nucleotide sequences of (a) to (f) are represented, respectively, by SEQ ID NOs: 7 to 12. A polynucleotide encoding the gene for an antibody or a fragment thereof may have a nucleotide sequence substantially identical to any of the nucleotide sequences represented by SEQ ID NOs: 7 to 12. A nucleotide sequence substantially identical to a nucleotide sequence represented by any one of SEQ ID NOs: 7 to 12 refers to a polynucleotide consisting of a sequence having 80% or more homology, preferably 80% or more identity, to a nucleotide sequence represented by any one of SEQ ID NOs: 7 to 12, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, preferably 99% or more identity, or a polynucleotide that can hybridize under stringent conditions to a polynucleotide consisting of a sequence complementary to a nucleotide sequence represented by any one of SEQ ID NOs: 7 to 12, and wherein the protein encoded by the polynucleotide has a desired function, for example, a protein that specifically binds to a 6-sulfosialyl Lewis X sugar chain, and the binding requires the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X sugar chain.

[0077] Here, stringent conditions refer to hybridization conditions that can be easily determined by those skilled in the art, and are generally empirical experimental conditions that depend on the base length of the nucleic acid, washing temperature, and salt concentration. Generally, the longer the base, the higher the temperature for proper annealing, and the shorter the base, the lower the temperature. Hybridization generally depends on the ability of complementary strands to reanneal in an environment slightly lower than their melting point.

[0078] In one embodiment, the polynucleotide encoding the anti-glycan antibody or fragment thereof comprises: (a) a nucleotide sequence encoding CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence encoding CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2; and (c) a nucleotide sequence encoding CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; have or or having a nucleotide sequence encoding CDRH1, CDRH2, and CDRH3, each of which is composed of a nucleotide sequence having one or more base deletions, substitutions, or additions in the nucleotide sequence encoding each CDR; or It may have nucleotide sequences encoding CDRH1, CDRH2, and CDRH3, each of which has 80% or more identity to the nucleotide sequence encoding each CDR.

[0079] In one embodiment, the polynucleotide encoding the anti-glycan antibody or fragment thereof comprises: (d) a nucleotide sequence encoding CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4; (e) a nucleotide sequence encoding CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5; and (f) a nucleotide sequence encoding CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; have or or a nucleotide sequence encoding CDRL1, CDRL2, and CDRL3, each of which has a deletion, substitution, or addition of one or several nucleotides in the nucleotide sequence encoding each CDR; It may have a nucleotide sequence encoding CDRL1, CDRL2, and CDRL3, which consists of a nucleotide sequence having 80% or more identity with the nucleotide sequence encoding each CDR.

[0080] In one embodiment, the polynucleotide encoding the gene for the anti-glycan antibody or a fragment thereof is one of the following: (a) a nucleotide sequence encoding CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence encoding CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2; (c) a nucleotide sequence encoding CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; (d) a nucleotide sequence encoding CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4; (e) a nucleotide sequence encoding CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5; and (f) a nucleotide sequence encoding CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; Contains or or having a nucleotide sequence encoding CDRH1, CDRH2, and CDRH3, and CDRL1, CDRL2, and CDRL3, each of which consists of a nucleotide sequence having one or more base deletions, substitutions, or additions in the nucleotide sequence encoding each CDR; It may have nucleotide sequences encoding CDRH1, CDRH2 and CDRH3, and CDRL1, CDRL2 and CDRL3, each of which has 80% or more identity to the nucleotide sequence encoding each CDR.

[0081] (Vector and host cell) In a third aspect, there is provided an expression vector comprising a polynucleotide encoding an anti-glycan antibody or a fragment thereof, and a host cell transfected with the expression vector, which is capable of producing the anti-glycan antibody or a fragment thereof.

[0082] A polynucleotide encoding the heavy chain variable region (or heavy chain) of an anti-glycan antibody or a fragment thereof, and a polynucleotide encoding the light chain variable region (or light chain) of an anti-glycan antibody or a fragment thereof may be contained in the same expression vector, or may be incorporated into separate expression vectors and provided as a combination thereof. A combination of an expression vector containing a polynucleotide encoding a heavy chain variable region (or heavy chain) (heavy chain variable region (or heavy chain) expression vector) and an expression vector containing a polynucleotide encoding a light chain variable region (or light chain) (light chain variable region (or light chain) expression vector) is also encompassed by the term "expression vector containing a polynucleotide encoding an anti-glycan antibody or a fragment thereof."

[0083] Expression vectors containing polynucleotides encoding anti-glycan antibodies or fragments thereof and host cells transfected with the expression vectors can be obtained by techniques known to those skilled in the art. For example, a polynucleotide encoding a single-chain anti-glycan antibody is incorporated into a vector suitable for its expression, and this is then introduced into a host, thereby expressing the anti-glycan antibody or a fragment thereof. The expression vector preferably contains a promoter, terminator, etc. suitable for the expression of the anti-glycan antibody or a fragment thereof in host cells. The expression vector can further contain other appropriate control sequences, such as an enhancer.

[0084] The vector can be appropriately selected from expression vectors well known to those skilled in the art, taking into consideration the type of host cell, the expression efficiency of the anti-glycan antibody or a fragment thereof, etc., and the type of vector may be a plasmid vector or a viral vector derived from a retrovirus or adenovirus.

[0085] The host cells are not limited as long as they are capable of expressing an anti-glycan antibody or a fragment thereof from an expression vector, and may be prokaryotic cells such as Escherichia coli, mammalian cells such as CHO, COS, NIH3T3, HEK293, HEK293T, and COS-7, insect cells such as F9, and eukaryotic cells such as yeast cells. Mammals include primates such as humans and chimpanzees, and rodents such as mice, rats, and hamsters.

[0086] Transfection of an expression vector into a host cell can be carried out using methods well known to those skilled in the art, such as electroporation, calcium phosphate, lipofection, or DEAE-dextran, or methods using commercially available reagents.

[0087] A host cell (transfectant) transfected with an expression vector containing a polynucleotide encoding an anti-glycan antibody or a fragment thereof includes: a transfectant containing the expression vector and expressing an anti-glycoprotein antibody or a fragment thereof; and A transfectant (stable transfectant) that expresses an anti-glycan antibody or a fragment thereof by incorporating a polynucleotide encoding the anti-glycan antibody or a fragment thereof into the chromosome of a host cell in an expressible manner. The stable transfectant may or may not contain an expression vector containing a polynucleotide encoding an anti-glycan antibody or a fragment thereof. The transfectant is preferably a mammalian cell such as CHO, COS, NIH3T3, HEK293, HEK293T, or COS-7.

[0088] (hybridoma) In a fourth aspect, a hybridoma producing an anti-sugar chain antibody is provided.

[0089] Hybridomas producing anti-glycan antibodies can be obtained by immunizing an animal by injecting a glycoprotein having a 6-sulfosialyl Lewis X sugar chain attached thereto, or by administering a vector encoding a 6-sulfosialyl Lewis X sugar chain and designed to express the 6-sulfosialyl Lewis X sugar chain in the immunized animal, and then fusing the immune cells obtained from the immunized animal with known myeloma cells by a conventional cell fusion method. Cells producing anti-glycan antibodies are screened from such hybridomas. Examples of immunized animals include rodents such as mice, rats, hamsters, and rabbits, and mammals such as monkeys.

[0090] In one embodiment, hybridomas producing anti-glycan antibodies are obtained by immunizing mice lacking GlcNAc6ST-1 and GlcNAc6ST-2, sulfotransferases responsible for the sulfation of 6-sulfosialyl Lewis X sugar chains expressed in HEV, with cells expressing 6-sulfosialyl Lewis X sugar chains through gene transfer of fucosyltransferase and sulfotransferase, preparing splenocytes from the mice, fusing the cells with myelomas, and then immunofluorescently staining mouse peripheral lymph nodes using the culture supernatant to select hybridomas secreting IgG class antibodies that specifically bind to wild-type mouse HEV.

[0091] (Method of producing anti-glycoantibody) In a fifth aspect, there is provided a method for producing an anti-glycan antibody or a fragment thereof. The anti-glycan antibody or a fragment thereof may be produced by culturing the above-mentioned host cells, transfectants, or hybridomas, or by using recombinant DNA technology.

[0092] Antibodies produced as recombinant proteins using recombinant DNA technology include, for example, chimeric antibodies, humanized antibodies, and human antibodies. These antibodies can be produced, for example, by culturing host cells transformed with a recombinant vector encoding the antibody or its heavy or light chain. Recombinant antibodies can also be produced using transgenic animals into which a gene encoding the desired antibody has been introduced.

[0093] The culture conditions for host cells, transfectants, and hybridomas can be appropriately determined by those skilled in the art depending on the type of desired anti-glycan antibody or fragment thereof and host cell. The production method may further comprise a step of recovering, from the culture obtained in the culturing step, an antibody or fragment thereof that specifically binds to a 6-sulfosialyl Lewis X glycan and that requires the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X glycan.

[0094] The produced antibody or fragment thereof may be subjected to additional steps such as a purification step before recovery. For example, to increase purity, it is preferable to perform salting out, filtration, clarification using centrifugation, affinity recovery, intermediate purification by ion exchange, and final purification by gel filtration.

[0095] (lymphocyte homing inhibitor) In a sixth aspect, there is provided a lymphocyte homing inhibitor comprising an anti-sugar chain antibody or a fragment thereof.

[0096] Lymphocyte homing to lymph nodes is a host defense mechanism for efficiently inducing immune responses. Lymphocyte homing to lymph nodes is regulated by the following three steps: step 1: lymphocyte rolling mediated by binding of L-selectin expressed on lymphocytes to the sulfated glycan 6-sulfosialyl Lewis X expressed in the HEV lumen; step 2: activation of adhesion molecules, integrins, via signal transduction mediated by binding of chemokines displayed on heparan sulfate in the HEV lumen to chemokine receptors; and step 3: strong adhesion of activated integrins to immunoglobulin superfamily adhesion molecules, ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1), resulting in lymphoid tissue infiltration. Through these three steps, lymphocytes migrate into the lymphoid tissue parenchyma.

[0097] Of these, the sulfated sugar chain 6-sulfosialyl Lewis X expressed in the lumen of HEVs plays an important role in Step 1, while heparan sulfate plays an important role in Step 2. Therefore, a lymphocyte homing inhibitor can be prepared by using an antibody or a fragment thereof that specifically binds to the fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X sugar chain as an active ingredient. The lymphocyte homing inhibitor particularly inhibits lymphocyte homing from lymph nodes that express the 6-sulfosialyl Lewis X sugar chain, such as peripheral lymph nodes, mesenteric lymph nodes, or high endothelial venules of tonsils, and more specifically, preferably inhibits L-selectin-dependent lymphocyte rolling.

[0098] (Pharmaceutical composition) In a seventh aspect, there is provided a pharmaceutical use of an anti-glycan antibody or a fragment thereof, for example, a pharmaceutical composition comprising an anti-glycan antibody or a fragment thereof.

[0099] Pharmaceutical uses of anti-glycan antibodies or fragments thereof include the treatment or prevention of diseases caused by excessive immune responses induced by lymphocyte homing. Specifically, administration of anti-glycan antibodies or fragments thereof before the onset of a disease can have a preventive effect, administration of anti-glycan antibodies after the onset of a disease can have a therapeutic effect, and administration of anti-glycan antibodies after the onset of a disease can prevent the recurrence of chronic diseases that undergo repeated relapse and remission. Among the excessive immune responses caused by lymphocyte homing, tissues in which administration of anti-glycan antibodies is thought to be particularly effective include peripheral lymph nodes, mesenteric lymph nodes, and tonsils. Examples of diseases caused by excessive immune responses caused by lymphocyte homing, particularly those induced by specific adhesion between lymphocytes and HEV, exceeding normal levels include immune-related diseases such as autoimmune diseases. Immune-related diseases include, for example, multiple sclerosis, including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis; systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's disease; benign lymphocytic vasculitis; thrombocytopenic purpura; idiopathic thrombocytopenia; idiopathic autoimmune hemolytic anemia; pure red cell aplasia; Sjogren's syndrome; rheumatic diseases; connective tissue diseases; inflammatory rheumatoid arthritis; rheumatoid osteoarthritis; non-articular rheumatoid arthritis; juvenile rheumatoid arthritis; muscular rheumatism; chronic polyarthritis; cryoglobulinemic vasculitis; ANCA-associated vasculitis; antiphospholipid syndrome; myasthenia gravis; autoimmune hemolytic anemia; Guillain-Barré syndrome; chronic immune-mediated polyneuropathy; autoimmune thyroiditis; insulin-dependent Diabetes mellitus; Type 1 diabetes; Addison's disease; Membranous glomerulonephropathy; Goodpasture's disease; Autoimmune gastritis; Autoimmune atrophic gastritis; Pernicious anemia; Pemphigus; Pemphigus vulgaris; Liver cirrhosis; Primary biliary cirrhosis; Dermatomyositis; Polymyositis; Fibromyositis; Myosclerosis; Celiac disease; Immunoglobulin A nephropathy; Henoch-Schönlein purpura; Evans syndrome; Psoriasis; Psoriatic arthritis; Graves' disease ;Graves' ophthalmopathy;Scleroderma;Systemic sclerosis;Progressive systemic sclerosis;Primary biliary cirrhosis;Hashimoto's thyroiditis;Primary myxedema;Sympathetic ophthalmia;Autoimmune uveitis;Hepatitis;Chronic active hepatitis;Collagen disease;Ankylosing spondylitis;Periarthritis of the shoulder;Panarteritis nodosa;Chondrocalcinosis;Wegener's granulomatosis;Microscopic polyangiitis;Chronic urticaria;Bullous skin diseases;Pemphigoid;Devik's disease;The autoimmune disease is selected from the group consisting of pediatric autoimmune hemolytic anemia, refractory or chronic autoimmune cytopenia, acquired hemophilia A, cold agglutinin disease, neuromyelitis optica, stiff-person syndrome, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis, psoriasis, normocomplementemic urticarial vasculitis, pericarditis, myositis, antisynthetase syndrome, scleritis, macrophage activation syndrome, Behçet's syndrome, PAPA syndrome, Blau syndrome, adult and juvenile Still's disease, cryopyrin-associated periodic syndrome, Muckle-Wells syndrome, familial cold autoinflammatory syndrome, neonatal-onset multisystem inflammatory disease, familial Mediterranean fever, chronic infantile-onset neurocutaneous-articular syndrome, systemic juvenile idiopathic arthritis, hyper-IgD syndrome, Schnitzler syndrome, autoimmune retinopathy, atherosclerosis, chronic prostatitis, and TNF receptor-associated periodic syndrome (TRAPS).

[0100] The autoimmune disease may be multiple sclerosis or a collagen vascular disease. Examples of the collagen vascular disease include rheumatoid arthritis, systemic lupus erythematosus, scleroderma, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, Sjögren's syndrome, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, hypersensitivity vasculitis, Behçet's disease, Cogan's syndrome, RS3PE, giant cell arteritis, adult Still's disease, polymyalgia rheumatica, fibromyalgia, and SAPHO syndrome. In addition to the above diseases, the autoimmune disease may be one or more diseases selected from the group consisting of other diseases in which an excessive immune response causes self-harm.

[0101] Other examples of diseases caused by excessive immune responses mediated by lymphocyte homing include immune-related diseases such as allergic diseases, which may be one or more diseases selected from the group consisting of allergic rhinitis, atopic dermatitis, food allergies, oral allergy syndrome, drug allergies, hay fever, allergic conjunctivitis, eosinophilic pneumonia, allergic gastroenteritis, urticaria, photosensitivity, metal allergies, cat allergies, dust mite allergies, and asthma.

[0102] In addition to the anti-glycan antibody or a fragment thereof as an active ingredient, the pharmaceutical composition may further contain additional ingredients such as pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers, preservatives, adjuvants, etc. depending on the route of administration, the disease to be treated, etc. The dosage of the anti-glycan antibody can be determined appropriately depending on the type of disease, the age, sex, and weight of the patient, the degree of disease and treatment, etc. The pharmaceutical composition may further contain a drug other than the anti-glycan antibody that is known to be effective in treating or preventing diseases caused by excessive immune responses induced by lymphocyte homing.

[0103] The dosage form of the pharmaceutical composition may be a liquid, a freeze-dried preparation, or the like, and examples of routes of administration of the pharmaceutical composition include, but are not limited to, intravenous drip infusion, subcutaneous injection, or intramuscular injection.

[0104] In another aspect, there is provided a method for treating or preventing a disease caused by an excessive immune response induced by lymphocyte homing, particularly specific adhesion between lymphocytes and HEV, comprising the step of administering an anti-glycan antibody or a fragment thereof to a subject in need of such treatment or prevention. The anti-glycan antibody or a fragment thereof may be used in combination with other drugs known to be effective in treating or preventing a disease caused by an excessive immune response induced by lymphocyte homing.

[0105] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples. [Example]

[0106] 1. Preparation of anti-glycan antibodies We generated a monoclonal antibody against 6-sulfosialyl Lewis X glycans. First, we expressed 6-sulfosialyl Lewis X glycans in Chinese hamster ovary (CHO) cells by transfecting them with genes encoding the fucosyltransferase FucT-VII and the sulfotransferase GlcNAc6ST-2. Sulfotransferase double-deficient mice (Kawashima et al., supra) lacking both the sulfotransferases GlcNAc6ST-1 and GlcNAc6ST-2 were immunized with these CHO cells. Their spleen cells were fused with P3X63Ag8.653 myeloma cells using polyethylene glycol (PEG). A hybridoma producing the anti-sulfated glycan monoclonal antibody SF1 (mouse IgG1, kappa) was established by immunohistochemical screening, which was reactive with wild-type (WT) mouse high endothelial venules (HEV). The obtained antibody was named SF1 antibody because it was reactive to sulfated and fucosylated glycans, as described below (S means that it is a clone that recognizes sulfate groups, and F means that it is a clone that recognizes fucose).

[0107] 2. Analysis of the reactivity of SF1 antibody to HEV The SF1 antibody was purified and biotinylated according to standard methods. Lymph node cryosections from WT mice, mice doubly deficient in the fucosyltransferase (FucT)-IV and FucT-VII genes (FucT DKO), and mice doubly deficient in the sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 genes (G6ST DKO) were incubated overnight at 4°C. After washing the sections with PBS, they were further incubated with streptavidin labeled with AlexaFluor 594, a fluorescent substance, at room temperature for 1 hour, followed by fluorescent immunostaining (Figure 2).

[0108] The SF1 antibody reacted strongly with lymph node HEV from WT mice, but not with HEV from FucT DKO or G6ST DKO mice. This indicates that both fucose and sulfate groups are involved in the reactivity of the SF1 antibody to mouse lymph nodes. On the other hand, the known antibody MECA-79 (Streeter et al., supra) reacted with mouse lymph node HEV, but the reactivity was not abolished in FucT DKO mice, suggesting that fucose, which plays an important role in lymphocyte homing, is not involved in the reactivity.

[0109] We also performed immunofluorescence staining using antibodies that recognize other glycans on HEV. As described above, the SF1 antibody reacted strongly with lymph node HEV from WT mice but not with HEV from FucT DKO or G6ST DKO mice. On the other hand, the F2 antibody (Matsumura et al., supra) reacted with lymph node HEV from WT mice but not with FucT DKO mice. However, its reactivity was not abolished by G6ST DKO mice, suggesting that sulfate groups, which play an important role in lymphocyte homing, are not involved in the reactivity. In contrast, the S2 antibody (Hirakawa et al., supra) reacted with lymph node HEV from WT mice but not with G6ST DKO mice. However, its reactivity was not abolished by FucT DKO mice, suggesting that fucose, which plays an important role in lymphocyte homing, is not involved in the reactivity. The results are shown in Figure 3.

[0110] Next, we examined the involvement of sialic acid in the reactivity to HEV by treating frozen sections of lymph nodes from WT mice with sialidase. The results are shown in Figure 4.

[0111] The reactivity of the SF1 antibody disappeared after sialidase treatment, indicating that the reactivity of the SF1 antibody to mouse lymph nodes involves not only fucose and sulfate groups, but also sialic acid, which plays an important role in lymphocyte homing. On the other hand, the reactivity of the known anti-glycan monoclonal antibody MECA-79 does not involve sialic acid. These immunofluorescence staining results demonstrate that the SF1 antibody obtained in this study is an anti-glycan monoclonal antibody that specifically recognizes glycan structures containing fucose, sulfate groups, and sialic acid on mouse lymph node HEV.

[0112] 3. Analysis of the reactivity of SF1 antibody to synthetic sugar chains Next, the reactivity of the SF1 antibody with various glycans was analyzed using the Glycan Array of the US Consortium for Functional Glycomics. The results showed that the SF1 antibody specifically bound only to the 6-sulfosialyl Lewis X glycan among the 611 types of glycans on the glycan array. Figure 5 shows excerpts from the results for the 6-sulfosialyl Lewis X glycan present on the glycan array and nine glycans with partial structural similarities to this glycan. The numbers in parentheses in the figure indicate the ID numbers of each glycan on the glycan array. Figure 5 shows that the SF1 antibody does not bind at all to LacNAc (ID number 170) or sialyl LacNAc (ID number 261). Furthermore, the SF1 antibody did not bind to 6-sulfosialylLacNAc (ID number 252), which is a 6-sulfosialyl Lewis X sugar chain with the fucose removed, sialyl Lewis X (ID number 255), which is a 6-sulfosialyl Lewis X sugar chain with the sulfate group removed, or 6-sulfosialyl Lewis X (ID number 291), which is a 6-sulfosialyl Lewis X sugar chain with the sialic acid removed, but rather specifically bound to 6-sulfosialyl Lewis X (ID number 253). Furthermore, the SF1 antibody did not bind to any of the following: 6'-sulfosialyl Lewis X (ID #231), in which the sulfate group is attached to galactose instead of GlcNAc; 6-sulfosialyl Lewis X (ID #220), in which a sulfate group is attached to the 3-position of galactose instead of the α2-3-linked sialic acid (Neu5Ac) at the terminal; 6,6'-disulfoLacNAc (ID #445), in which both the 6-positions of galactose and GlcNAc are sulfated. These results demonstrate that the SF1 antibody specifically binds to the 6-sulfosialyl Lewis X glycan.

[0113] 4. Determination of the dissociation constant of the binding between SF1 antibody and 6-sulfosialyl Lewis X Next, the dissociation constant (K ) of the binding between SF1 antibody and 6-sulfosialyl Lewis X was determined by biolayer interferometry using Octet RED96 (Pall-Fortebio). D) was measured. After biotin-labeled 6-sulfosialyl Lewis X was bound to a streptavidin biosensor, the binding and dissociation of the SF1 antibody were analyzed in real time. The results are shown in Figure 6.

[0114] As a result, the K of the binding between SF1 antibody and 6-sulfosialyl Lewis X D The value is 6.09 x 10 -9 When biotin-labeled sialyl-LacNAc was used as a negative control, no binding was observed.

[0115] 5. Determination of the amino acid sequence of anti-glycan antibodies Next, the antibody gene was obtained from the hybridoma producing the SF1 antibody, and its nucleotide sequence was analyzed to determine the amino acid sequences of the hypervariable regions of the heavy and light chain variable regions of the SF1 antibody. The heavy chain CDR1, CDR2, and CDR3 of the SF1 antibody are shown as SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3), respectively, and the light chain CDR1, CDR2, and CDR3 of the SF1 antibody are shown as SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3), respectively, in Figure 7. Because no antibody with such a hypervariable region has been reported to date, this antibody has a novel sequence.

[0116] The complete amino acid sequence of the heavy chain variable region of the SF1 antibody is set forth as SEQ ID NO: 13 and is shown in Figure 8. The sequences of CDRH1, CDRH2, and CDRH3 are shown in bold.

[0117] The complete amino acid sequence of the light chain variable region of the SF1 antibody is set forth as SEQ ID NO: 14 and is shown in Figure 9. The sequences of CDRL1, CDRL2, and CDRL3 are shown in bold.

[0118] 6. Analysis of the inhibitory effect of SF1 antibody on the binding of L-selectin to HEV Mouse lymph node frozen sections were incubated for 2 hours at room temperature in buffer A (0.1% BSA, 20 mM HEPES-NaOH, 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.4) alone or with SF1 antibody dissolved in buffer A for 2 hours at room temperature, followed by incubation overnight at 4°C with L-selectin and human IgG fusion protein (L-selectin-Fc) dissolved in buffer A. After washing the sections with buffer A, they were incubated with biotin-labeled anti-human IgG antibody for 2 hours at room temperature. After washing the sections with buffer A, they were further incubated with AlexaFluor 594-labeled streptavidin, a fluorescent substance, for 1 hour at room temperature for fluorescent immunostaining (Figure 10).

[0119] The SF1 antibody strongly inhibited the binding of L-selectin-Fc to HEV, indicating that the SF1 antibody inhibits the interaction between the lymphocyte homing receptor L-selectin and its ligand expressed on HEV.

[0120] 7. Analysis of the inhibitory effect of SF1 antibody on L-selectin-dependent lymphocyte rolling CHO cells stably expressing the core protein CD34 and 6-sulfosialyl Lewis X glycan, which are ligands for L-selectin expressed on HEVs, were cultured in a petri dish and placed in a flow chamber. Lymphocytes prepared from mouse spleens were then subjected to various shear stresses (0.5, 1.0, 1.5, and 2.0 dynes / cm). 2 ) and counted the number of rolling cells on the cells reconstituted with L-selectin ligand. The cells were divided into three groups: untreated (None in the figure), a group in which L-selectin ligand-reconstituted cells were pre-incubated with SF1 antibody (SF1 in the figure), and a group in which lymphocytes were pre-incubated with anti-L-selectin antibody (MEL-14 in the figure). The results are shown in Figure 11.

[0121] As a result, the observed rolling was inhibited by anti-L-selectin antibody, confirming that it was L-selectin dependent, and it was found that the SF1 antibody has the effect of suppressing L-selectin-dependent rolling.

[0122] 8. Analysis of the lymphocyte homing inhibitory effect of SF1 antibody WT mice were injected intravenously with SF1 antibody or PBS, followed by intravenous injection of lymphocytes (derived from mesenteric lymph nodes and spleens) labeled with the fluorescent substance CFSE. Two hours later, each lymphoid tissue was collected and the number of CFSE-labeled lymphocytes that had infiltrated (homed) to each tissue was determined (Figure 12).

[0123] The SF1 antibody was found to inhibit lymphocyte homing to mouse peripheral lymph nodes by over 90%. It also inhibited homing to mesenteric lymph nodes by approximately 60%. However, it was found to be a highly specific antibody, showing no significant inhibitory effect on homing to Peyer's patches, which express the glycan recognized by the SF1 antibody in less than 5% of HEVs, or to the spleen, which does not express the same glycan at all. Because the Peyer's patches are responsible for the intestinal immune response to intestinal microorganisms and food-derived antigens, and the spleen is responsible for the immune response to foreign substances invaded by the blood, the SF1 antibody is expected to be useful for the treatment or prevention of immune-related diseases caused by excessive immune responses in peripheral and mesenteric lymph nodes, without causing the side effects associated with suppressing normal immune responses in Peyer's patches and the spleen.

[0124] 9. Analysis of the effect of SF1 antibody in EAE model We analyzed the effects of the SF1 antibody on immune-mediated diseases using an experimental autoimmune encephalomyelitis (EAE) model, which is a known animal model of multiple sclerosis, a type of intractable autoimmune disease. In this model, mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide, which induces an immune response via lymphocyte homing to lymph nodes, resulting in the development of autoimmune encephalomyelitis. First, we compared the symptoms of EAE in WT and G6ST DKO mice (Figure 13).

[0125] As a result, delayed onset, reduced peak symptoms, and reduced symptoms at relapse were observed in G6ST DKO mice, indicating that the 6-sulfosialyl Lewis X glycan synthesized by GlcNAc6ST-1 and GlcNAc6ST-2 in HEVs is a therapeutic target for multiple sclerosis.

[0126] Therefore, we performed an experiment to suppress the onset of EAE using the SF1 antibody, which specifically binds to the same glycan. The SF1 antibody was administered twice to EAE model mice on days 0 and 2 after sensitization with the MOG peptide. Similar to the DKO mice described above, the onset of EAE was delayed, and symptoms at the peak and at the time of relapse were reduced (Figure 14).

[0127] This indicates that the onset of EAE is suppressed by SF1 antibody.

[0128] Furthermore, administration of the SF1 antibody every 3 days for 12 doses starting on day 8 after the initial antigen sensitization, when EAE symptoms began to appear, significantly suppressed recurrence observed from day 30 onwards, and this suppression of recurrence was maintained even after day 42, when antibody administration was discontinued (Figure 15). Similarly, administration of the SF1 antibody every 3 days for 12 doses starting on day 21 after the initial peak of EAE, significantly suppressed recurrence observed from day 30 onwards, and this suppression of recurrence was maintained even after day 54, when antibody administration was discontinued (Figure 16).

[0129] Multiple sclerosis is known to undergo repeated relapses and remissions. Based on the above results, the SF1 antibody has the effect of suppressing relapses, and is therefore considered to be an antibody that can be used to treat multiple sclerosis.

[0130] 10. Analysis of the effect of SF1 antibody in a mouse rheumatoid arthritis model Next, we examined the effect of the SF1 antibody in autoimmune disease models other than EAE. Specifically, we used a murine collagen-induced rheumatoid arthritis model. PBS or SF1 was administered on Day 0 (the first subcutaneous administration of antigen), Day 21 (the second subcutaneous administration of antigen), and Days 24, 27, 30, and 33 (Figure 17).

[0131] The results showed that both the incidence of arthritis symptoms and clinical scores were inhibited in the SF1 antibody-administered group.

[0132] 11. Analysis of the effect of SF1 antibody in a mouse allergic rhinitis model Next, we examined the effect of the SF1 antibody in a mouse allergic rhinitis model. Specifically, the allergic rhinitis model was induced by intranasal administration of the antigen ovalbumin (OVA) and the adjuvant cholera toxin (CT) four times every other week to mice. PBS or the SF1 antibody was then administered intraperitoneally twice a week (Fig. 18).

[0133] As a result, the number of sneezes and nose-scratching behaviors observed in mice for 12 minutes after the fourth intranasal administration of OVA and CT was significantly inhibited in the SF1 antibody-administered group.

[0134] 12. Immunostaining of normal human tissues with SF1 antibody Immunohistochemical staining of normal human tissues using the SF1 antibody revealed no staining in the aorta, mammary gland, cerebellum, cerebrum, heart, lung, small intestine, large intestine, skin, or spleen, but staining was observed in the lymph nodes and tonsils (Figures 19 and 20).

[0135] The enlarged photograph in Figure 20 shows that the SF1 antibody specifically binds to high endothelial venules HEV (arrows in the figure), which are blood vessels through which lymphocytes pass when migrating to these lymphatic tissues.

[0136] 13. Preparation of humanized single-chain SF1 antibody and immunostaining using it From among human antibody heavy chain variable region frame sequences highly homologous to the heavy chain variable region frame sequence of the mouse SF1 antibody shown in Figure 8, trial and error revealed that activity was well maintained when IGHV4-59 was used. Specifically, the heavy chain hypervariable region sequence of the SF1 antibody shown in Figure 7 was grafted into IGHV4-59 to design humanized SF1 heavy chain variable region sequences H1 and H2, which were used for activity evaluation. The H1 sequence was a sequence in which two amino acids in the human heavy chain variable region frame sequence portion of the H2 sequence were backmutated to those of the mouse SF1 antibody. Further trial and error revealed that activity was well maintained when IGHV3-43 was used as an example of a different sequence from the human antibody heavy chain variable region frame sequences highly homologous to the heavy chain variable region frame sequence of the SF1 antibody shown in Figure 8, and the two amino acids were backmutated to those of the mouse SF1 antibody. Specifically, the heavy chain hypervariable region sequence shown in Figure 7 was grafted into IGHV3-43 having such a heavy chain variable region frame sequence to design a humanized sequence, SF1 heavy chain variable region sequence H3, which was used for activity evaluation.

[0137] Similarly, trial and error revealed that activity was well maintained when IGKV1-39 was used among human antibody light chain variable region frame sequences highly homologous to the light chain variable region frame sequence of the mouse SF1 antibody shown in Figure 9. Specifically, humanized SF1 light chain variable region sequences L1 and L2 were designed by grafting the light chain hypervariable region sequence of the SF1 antibody shown in Figure 7 into IGKV1-39, and these sequences were used for activity evaluation. The L1 sequence was a sequence in which one amino acid in the human light chain variable region frame sequence portion of the L2 sequence was backmutated to that of the mouse SF1 antibody. Further trial and error revealed that activity was well maintained when IGKV6-21, an example of a different sequence, was used among human antibody light chain variable region frame sequences highly homologous to the light chain variable region frame sequence of the SF1 antibody shown in Figure 9, and the single amino acid was backmutated to that of the mouse SF1 antibody. Specifically, the humanized SF1 light chain variable region sequence L3 was designed by grafting the light chain hypervariable region sequence shown in Figure 7 into IGKV6-21, which has such a light chain variable region frame sequence, and used for activity evaluation.

[0138] The amino acid sequences of the humanized SF1 heavy chain variable regions H1, H2, and H3 and the humanized SF1 light chain variable regions L1, L2, and L3 are shown in Figure 21. The sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are shown in bold.

[0139] Next, using a cell-free protein synthesis system with wheat germ extract, we produced humanized SF1 single-chain antibodies (scFv) containing various combinations of the humanized SF1 heavy chain variable regions H1-H3 and the humanized SF1 light chain variable regions L1-L3 shown in Figure 21. The full amino acid sequences of the various humanized SF1 scFvs produced are shown in Figure 22. The underlined triplet of Gly-Gly-Gly-Gly-Ser (GGGGS) (G4S linker) is the linker sequence between the heavy and light chains, and the double-underlined Gly-Leu-Gln-Gln-Gly-Gly-Thr-His-His-His-His-His-His (GLQQGGTHHHHHH) sequence at the C-terminus is the tag sequence for binding to biotinylated anti-(His)6 antibody. The sequences in bold indicate, from the N-terminus, the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively. Humanized SF1-scFv-H1L1 is a sequence containing a humanized SF1 heavy chain variable region H1 and a humanized SF1 light chain variable region L1, humanized SF1-scFv-H1L2 is a sequence containing a humanized SF1 heavy chain variable region H1 and a humanized SF1 light chain variable region L2, humanized SF1-scFv-H2L1 is a sequence containing a humanized SF1 heavy chain variable region H2 and a humanized SF1 light chain variable region L1, and humanized SF1-scFv-H3L3 is a sequence containing a humanized SF1 heavy chain variable region H3 and a humanized SF1 light chain variable region L3.

[0140] Humanized SF1-scFv, which had been previously incubated with biotinylated anti-(His)6 antibody to form a complex, was then incubated with mouse lymph node frozen sections overnight at 4°C. After washing the sections with PBS, they were further incubated with AlexaFluor 594-labeled streptavidin, a fluorescent substance, at room temperature for 1 hour, and subjected to fluorescent immunostaining. All humanized SF1-scFv combinations demonstrated specific staining for HEV (Fig. 23). These results demonstrate that the hypervariable region sequence of the SF1 antibody shown in Fig. 7 is a versatile sequence that retains activity when linked to various variable region framework sequences of human and mouse antibodies.

[0141] 14. Preparation of humanized SF1 antibody and immunostaining using it The humanized SF1 heavy chain variable region sequence H1 shown in Figure 21 was linked to the IgG1 constant region of a human antibody heavy chain to design the full-length amino acid sequence of a humanized SF1 heavy chain (Figure 24). Similarly, the humanized SF1 light chain variable region sequence L1 shown in Figure 21 was linked to the κ chain constant region of a human antibody light chain to design the full-length amino acid sequence of a humanized SF1 light chain (Figure 25). Genes encoding these full-length amino acid sequences of the humanized SF1 heavy chain and the humanized SF1 light chain were introduced into HEK293T cells, a human-derived cell line, to produce a humanized SF1 antibody. The complete amino acid sequence of the heavy chain of the humanized SF1 antibody is set forth as SEQ ID NO: 25 and is shown in Figure 24. The sequences of CDRH1, CDRH2, and CDRH3 are shown in bold.

[0142] The complete amino acid sequence of the light chain variable region of the humanized SF1 antibody is set forth as SEQ ID NO: 26 and is shown in Figure 25. The sequences of CDRL1, CDRL2, and CDRL3 are shown in bold.

[0143] Fluorescent immunostaining of mouse lymph nodes using the humanized SF1 antibody showed that the humanized SF1 antibody specifically bound to high endothelial venules in wild-type (WT) mice, but did not react at all with G6ST DKO mice, which lack the sulfate group of 6-sulfosialyl Lewis X, or FucT DKO mice, which lack fucose (Fig. 26). This indicates the high specificity of the humanized SF1 antibody for 6-sulfosialyl Lewis X.

[0144] The above results confirmed that the humanized antibody having the hypervariable region amino acid sequences of the heavy and light chain variable regions of the SF1 antibody retains the specific sugar-binding specificity for 6-sulfosialyl Lewis X, similar to that of the mouse SF1 antibody.

[0145] 15. Analysis of the lymphocyte homing inhibitory effect of humanized SF1 antibody Next, humanized SF1 antibody or PBS was injected intravenously into the tail vein of WT mice, followed by intravenous injection of fluorescently labeled CFSE lymphocytes (derived from mesenteric lymph nodes and spleens). Two hours later, each lymphoid tissue was harvested and the number of CFSE-labeled lymphocytes homing to each tissue was counted (Fig. 27).

[0146] The results showed that the humanized SF1 antibody, like the mouse SF1 antibody, inhibited lymphocyte homing to mouse peripheral lymph nodes by more than 90%. It also inhibited homing to mesenteric lymph nodes by approximately 70%. However, it did not significantly inhibit homing to Peyer's patches or the spleen.

[0147] 16. Analysis of the effect of humanized SF1 antibody in EAE model We performed an experiment to evaluate whether humanized SF1 antibody can suppress the onset of EAE. Humanized SF1 antibody was administered twice to EAE model mice on days 0 and 2 after sensitization with the MOG peptide. Similar to the mouse SF1 antibody, the humanized SF1 antibody delayed the onset of EAE, reduced symptoms at the peak, and reduced symptoms at relapse (Figure 28).

[0148] This demonstrates that the humanized SF1 antibody suppresses the onset of EAE.

[0149] 17. Preparation of humanized SF1 antibody with modified Fc sequence Furthermore, we prepared a humanized SF1 antibody with altered Fc region sequences. Specifically, we designed the full-length amino acid sequence of a humanized SF1 heavy chain IgG1-LALA antibody by substituting Ala234-Ala235 for Leu234-Leu235 in the humanized SF1 heavy chain IgG1 constant region shown in Figure 24 (Figure 29). Similarly, we substituted the humanized SF1 heavy chain IgG1 constant region shown in Figure 24 with an IgG4 constant region, and then substituted Ser228 with Pro and Leu235 with Glu in the EU (Figure 30). The genes encoding the full-length amino acid sequences of the humanized SF1 heavy chains with modified Fc sequences were each introduced into HEK293T cells together with the gene encoding the full-length amino acid sequence of the humanized SF1 light chain shown in Figure 25 to produce humanized SF1 antibody-IgG1-LALA antibody (HSF1-LALA) and humanized SF1 antibody-IgG4-SPLE antibody (HSF1-G4PE).

[0150] The full amino acid sequence of the IgG1-LALA heavy chain of the humanized SF1 antibody is set forth as SEQ ID NO: 27 and is shown in Figure 29. The sequences of CDRH1, CDRH2, and CDRH3 are shown in bold.

[0151] The full amino acid sequence of the heavy chain IgG4-SPLE form of the humanized SF1 antibody is set forth as SEQ ID NO: 28 and is shown in Figure 30. The sequences of CDRH1, CDRH2, and CDRH3 are shown in bold.

[0152] Next, mouse lymph nodes were subjected to fluorescent immunostaining using culture supernatants from 293T cells containing humanized SF1 antibody-IgG1-LALA (HSF1-LALA) and humanized SF1 antibody-IgG4-SPLE (HSF1-G4PE), which are composed of the heavy chain variants of the humanized SF1 antibody shown in Figures 29 and 30 and the light chain shown in Figure 25. As a result, no staining was observed with the culture supernatant from untransfected 293T cells alone, but specific binding to mouse lymph node high endothelial venules was observed with the culture supernatants containing HSF1-LALA and HSF1-G4PE (Figure 31).

[0153] The above results confirmed that humanized SF1 antibodies with altered Fc region sequences also retain specific tissue binding. Humanized antibodies with altered Fc region sequences such as these are known to reduce Fc-mediated effector activity of antibodies and reduce the risk of side effects. Based on these findings, humanized SF1 antibodies and humanized SF1 antibodies with altered Fc region sequences, like mouse SF1 antibodies, possess highly efficient lymphocyte homing inhibitory activity due to their specific tissue binding, and are considered useful as therapeutic antibodies that exert therapeutic effects against immune-related diseases such as autoimmune diseases and allergic diseases.

[0154] This application is based on Japanese Patent Application No. 2020-169966, the contents of which are incorporated in their entirety into this specification. [Sequence List Free Text]

[0155] SEQ ID NO: 1: Shows the amino acid sequence of CDRH1 of SF1 antibody. SEQ ID NO: 2: Amino acid sequence of CDRH2 of SF1 antibody. SEQ ID NO: 3: Amino acid sequence of CDRH3 of SF1 antibody. SEQ ID NO: 4: Shows the amino acid sequence of CDRL1 of SF1 antibody. SEQ ID NO: 5: Shows the amino acid sequence of CDRL2 of SF1 antibody. SEQ ID NO: 6: Shows the amino acid sequence of CDRL3 of SF1 antibody. SEQ ID NO: 7: Shows the base sequence of SF1 antibody CDRH1. SEQ ID NO: 8: Shows the base sequence of SF1 antibody CDRH2. SEQ ID NO: 9: shows the base sequence of SF1 antibody CDRH3. SEQ ID NO: 10: Shows the base sequence of SF1 antibody CDRL1. SEQ ID NO: 11: shows the base sequence of SF1 antibody CDRL2. SEQ ID NO: 12: shows the base sequence of SF1 antibody CDRL3. SEQ ID NO: 13: Amino acid sequence of the heavy chain variable region of SF1 antibody. SEQ ID NO: 14: Amino acid sequence of the light chain variable region of SF1 antibody. Sequence number 15: Amino acid sequence of the heavy chain variable region H1 of the humanized SF1 antibody. Sequence number 16: Amino acid sequence of the heavy chain variable region H2 of the humanized SF1 antibody. Sequence number 17: Amino acid sequence of the heavy chain variable region H3 of the humanized SF1 antibody. Sequence number 18: Amino acid sequence of the light chain variable region L1 of the humanized SF1 antibody. Sequence number 19: Amino acid sequence of the light chain variable region L2 of the humanized SF1 antibody. Sequence number 20: Amino acid sequence of the light chain variable region L3 of the humanized SF1 antibody. SEQ ID NO: 21: shows the amino acid sequence of humanized SF1-scFv-H1L1. SEQ ID NO: 22: Amino acid sequence of humanized SF1-scFv-H1L2. SEQ ID NO: 23: Shows the amino acid sequence of humanized SF1-scFv-H2L1. SEQ ID NO: 24: Amino acid sequence of humanized SF1-scFv-H3L3. Sequence number 25: shows the amino acid sequence of the full-length heavy chain of humanized SF1 antibody. Sequence number 26: shows the amino acid sequence of the full-length light chain of humanized SF1 antibody. Sequence number 27: shows the amino acid sequence of the full-length IgG1-LALA heavy chain of humanized SF1 antibody. SEQ ID NO: 28: Full-length amino acid sequence of the heavy chain IgG4-SPLE form of humanized SF1 antibody. SEQ ID NO: 29: Shows the amino acid sequence of the G4S linker.

Claims

1. An antibody or a fragment thereof that specifically binds to a 6-sulfosialyl Lewis X sugar chain, and the binding requires fucose, sulfate group, and sialic acid that constitute the 6-sulfosialyl Lewis X sugar chain, comprising: (a) a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:2; and (c) a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; and a heavy chain variable region comprising: (d) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:4; (e) a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:5; and (f) a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:6; and a light chain variable region comprising:

2. The antibody or fragment thereof according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 15 to 17, and the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 18 to 20.

3. Any of the following combinations of heavy chain variable regions and light chain variable regions: (1) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 18; (2) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19; (3) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 18; or (4) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 20; The antibody or fragment thereof of claim 1, comprising:

4. An antibody or fragment thereof described in claim 1, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:

14.

5. Any of the following combinations of heavy chains and light chains: (1) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 25 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 26; (2) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 27 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 26; or (3) A heavy chain comprising the amino acid sequence represented by SEQ ID NO: 28 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 26; The antibody or fragment thereof of claim 1, comprising:

6. The antibody or fragment thereof according to any one of claims 1 to 5, which inhibits the binding of L-selectin to high endothelial venules expressing 6-sulfosialyl Lewis X sugar chains.

7. The antibody or fragment thereof according to any one of claims 1 to 6, wherein the constant region is derived from a human.

8. The antibody or fragment thereof according to any one of claims 1 to 7, which is humanized.

9. The antibody or fragment thereof according to any one of claims 1 to 8, which is selected from the group consisting of Fab, F(ab')2, Fab', Fv and single-chain antibodies.

10. The antibody or fragment thereof according to claim 9, wherein the single-chain antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 21 to 24.

11. The antibody or its fragment binds to the 6-sulfosialyl Lewis X sugar chain at a concentration of 1 × 10 -6 Dissociation constant (K D The antibody or fragment thereof according to any one of claims 1 to 10, wherein the antibody or fragment thereof exhibits a .alpha.-to-.alpha. value.

12. A polynucleotide encoding the antibody or fragment thereof according to any one of claims 1 to 11.

13. An expression vector comprising the polynucleotide of claim 12.

14. A host cell transfected with the expression vector of claim 13.

15. The host cell of claim 14, which is a eukaryotic cell.

16. A hybridoma producing the antibody according to any one of claims 1 to 11.

17. A lymphocyte homing inhibitor comprising the antibody or fragment thereof according to any one of claims 1 to 11, the polynucleotide according to claim 12, or the expression vector according to claim 13.

18. A pharmaceutical composition comprising the antibody or fragment thereof according to any one of claims 1 to 11, the polynucleotide according to claim 12, or the expression vector according to claim 13.

19. The pharmaceutical composition according to claim 18, for treating or preventing a disease caused by an excessive immune response induced by lymphocyte homing.

20. The pharmaceutical composition according to claim 19, wherein the disease caused by an excessive immune response induced by lymphocyte homing is an immune-related disease.

21. The pharmaceutical composition according to claim 20, wherein the immune-related disease is an allergic disease or an autoimmune disease.

22. 22. The pharmaceutical composition of claim 21, wherein the immune-related disease is an allergic disease selected from the group consisting of allergic rhinitis, atopic dermatitis, food allergy, oral allergy syndrome, drug allergy, hay fever, allergic conjunctivitis, eosinophilic pneumonia, allergic gastroenteritis, urticaria, photosensitivity, metal allergy, cat allergy, dust mite allergy, and asthma.

23. immune-related diseases include multiple sclerosis, including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis; systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's disease; benign lymphocytic vasculitis; thrombocytopenic purpura; idiopathic thrombocytopenia; idiopathic autoimmune hemolytic anemia; pure red cell aplasia; Sjogren's syndrome; rheumatic diseases; connective tissue diseases; Inflammatory rheumatism; Rheumatoid osteoarthritis; Non-articular rheumatoid arthritis; Juvenile rheumatoid arthritis; Muscular rheumatoid arthritis; Chronic polyarthritis; Cryoglobulinemic vasculitis; ANCA-associated vasculitis; Antiphospholipid syndrome; Myasthenia gravis; Autoimmune hemolytic anemia; Guillain-Barré syndrome; Chronic immune polyneuropathy; Autoimmune thyroiditis; Insulin-dependent diabetes mellitus; Type 1 diabetes; Addison's disease; Membranous glomerular nephropathy; Goodpasture's disease; Autoimmune Gastritis; Autoimmune atrophic gastritis; Pernicious anemia; Pemphigus; Pemphigus vulgaris; Cirrhosis; Primary biliary cirrhosis; Dermatomyositis; Polymyositis; Fibromyositis; Myoschisis; Celiac disease; Immunoglobulin A nephropathy; Henoch-Schönlein purpura; Evans syndrome; Psoriasis; Psoriatic arthritis; Graves' disease; Graves' ophthalmopathy; Scleroderma; Systemic sclerosis; Progressive systemic sclerosis; Primary biliary cirrhosis; Hashimoto's thyroiditis; Primary myxedema; Sympathetic ophthalmia; Autoimmune uveitis; Hepatitis; Chronic active hepatitis; Collagen vascular diseases; Ankylosing spondylitis; Periarthritis of the shoulder; Panarteritis nodosa; Chondrocalcinosis; Wegener's granulomatosis; Microscopic polyangiitis; Chronic urticaria; Bullous skin diseases; Pemphigoid; Devic's disease; Pediatric autoimmune hemolytic anemia; Refractory or chronic autoimmune cytopenia; Acquired hemophilia A; Cold agglutinin disease; Neuromyelitis optica; Stiff-person syndrome; Pancreatitis; Myocarditis; Vasculitis; Gastritis; Gout; Gouty arthritis; Psoriasis; Eucomplementemic urticarial vasculitis; Pericarditis; Myositis; Antisynthetase syndrome; Scleritis; Macrophage activation syndrome; Behçet's syndrome; PAPA syndrome; Blau syndrome; Adult and juvenile Still's disease; Cryopyrin-associated periodic syndrome; Muckle-Wells syndrome; Familial cold autoinflammatory syndrome; neonatal-onset multisystem inflammatory disease; The pharmaceutical composition of claim 21, wherein the autoimmune disease is selected from the group consisting of familial Mediterranean fever; chronic infantile-onset neurological cutaneous and articular syndrome; systemic juvenile idiopathic arthritis; hyper-IgD syndrome; Schnitzler syndrome; autoimmune retinopathy; atherosclerosis; chronic prostatitis; and TNF receptor-associated periodic syndromes (TRAPS).

24. The pharmaceutical composition according to claim 23, wherein the autoimmune disease is multiple sclerosis or a collagen disease.

25. 25. The pharmaceutical composition of claim 23 or 24, wherein the collagen disease is one or more diseases selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, scleroderma, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, Sjogren's syndrome, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, hypersensitivity vasculitis, Behcet's disease, Cogan's syndrome, RS3PE, giant cell arteritis, adult Still's disease, polymyalgia rheumatica, fibromyalgia, and SAPHO syndrome.

26. The pharmaceutical composition according to any one of claims 19 to 25, further comprising an agent for treating or preventing a disease caused by an excessive immune response brought about by lymphocyte homing.

27. A method for producing the antibody or fragment thereof according to any one of claims 1 to 11, comprising the step of culturing the host cell according to claim 14 or 15, or the hybridoma according to claim 16.

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