WO2014157229A1 - Tace peptide epitope, antihuman tace protein antibody and hybridoma producing antibody - Google Patents

Abstract

The present invention addresses the problem of providing an antibody that has a high ability to inhibit the function of TACE. Provided is an antihuman TACE protein antibody which specifically binds to an amino acid sequence region represented by VMYPIAVSGDHENNKMFSNCSKQ or MAKSYPNEEKDAW.

Description

TACE peptide epitope, anti-human TACE protein antibody, and hybridoma producing the antibody

[Cross-reference of related applications]
This application claims priority based on Japanese Patent Application No. 2013-069544 filed on Mar. 28, 2013, the entire disclosure of which is incorporated herein by reference.

The present invention relates to a TACE peptide epitope, an anti-human TACE protein antibody, and a hybridoma that produces the antibody.

Periodontal disease is one of the lifestyle-related diseases whose morbidity increases with age, and it develops when an excessive immune reaction is induced in the periodontal tissue by infection with periodontal pathogens. In severe periodontal disease, the alveolar bone fracture causes loss of tooth support tissue, resulting in masticatory disorders and markedly lower quality of life. It is known that this bone destruction is caused by excessive bone resorption promotion by osteoclasts, and RANKL (Non-patent Document 1), which is an essential factor for osteoclast formation and activation, is a periodontal tissue of periodontal disease. It has also been reported that expression is increased at the mRNA level and at the protein level (Non-patent Documents 2 and 3). In addition, regarding the type of cells that express RANKL in periodontal disease, it has been reported that lymphocytes (B cells and T cells) infiltrating the gingival tissue are the main source of this increased RANKL. (Non-Patent Document 2).

Briefly describing the progression of periodontal disease, plaque deposits in the groove (periodontal pocket) between the teeth and gums, and the bacteria and their products that make up the majority of the plaque are transferred from the periodontal pocket to the gingiva. It passes through the epithelium, enters the gingival tissue, and the invading bacteria and products stimulate immune system cells, causing infiltration of lymphocytes and activation of infiltrating lymphocytes. In this way, lymphocytes that have migrated from capillaries in the gingiva by being attracted by invading bacteria and products accumulate near the periodontal pocket, that is, near the epithelium far from the alveolar bone (non-patented). Reference 4)
RANKL (RANK ligand), an essential factor for osteoclast formation and activation, is usually membrane-bound on immune cells such as lymphocytes that support osteoclast formation and stromal cells such as osteoblasts. It is synthesized as a protein (Non-patent Document 1). Therefore, direct cell-cell contact between these osteoclast-forming support cells and osteoclast precursor cells / osteoclasts is considered essential for RANKL signal transmission (Non-patent Document 5). Membrane-bound RANKL is composed of three regions, an intracellular domain, a transmembrane domain, and an extracellular domain. Only a specific region of the extracellular domain is required for functioning as an osteoclast differentiation factor.

However, considering periodontal disease gingival tissue, as mentioned above, there are questions about RANKL-expressing cells (infiltrated activated lymphocytes) and osteoclasts that are present on the surface of the alveolar bone and actually resorb bone. There is a distance of several hundred micrometers (Non-patent Document 7), and it is unlikely that osteoclast-forming support cells (infiltrated activated lymphocytes) and osteoclasts transmit RANKL signals through cell-cell contact. As an explanation for the divergence of this phenomenon, reports have been made on the shedding of membrane-bound RANKL to free RANKL by enzymatic cleavage ( Non-Patent Documents 4, 6 to 8). The function of RANKL as an osteoclast differentiation factor requires only a specific region of the extracellular domain (Non-patent Document 9) and cleaves a specific amino acid sequence between that region (soluble RANKL domain) and the transmembrane domain Any enzyme capable of converting from membrane-bound to free RANKL while maintaining the osteoclast differentiation promoting function of RANKL can be achieved.

An object of the present invention is to provide an antibody having a high inhibitory ability of TACE function.

Among the enzymes reported as RANKL に よ る sheddase, the present inventor observed mRNA expression by RT-PCR in activated lymphocytes and expression at the protein level using flow cytometry. MMp-7, MMP114 , RANKL sheddase could be narrowed down to 4 enzymes, ADAM-10 and ADAM-17 (TACE: TNF-αconvertingenzyme). The present inventors have found that TACE plays an important role among these RANKLRANsheddases. Furthermore, as a result of earnest research, the present inventor has produced an antibody having the ability to inhibit TACE function by using a specific part of the amino acid sequence of TACE as an antigen in preparing an IgG anti-human TACE neutralizing antibody. I found it possible.

Accordingly, the present invention provides the following sections:
Item 1. An anti-human TACE protein antibody that specifically binds to a region of the amino acid sequence represented by VMYPIAVSGDHENNKMFSNCSKQ or MAKSYPNEEKDAW.

Item 2. Item 2. The antibody according to Item 1, which specifically binds to a region of the amino acid sequence represented by YPIAVSGD.

Item 3. Item 3. The antibody according to Item 1 or 2, which is a monoclonal antibody.

Item 4. A TACE peptide epitope, which is a peptide consisting of at least 7 consecutive amino acids in the amino acid sequence represented by VMYPIAVSGDHENNKMFSNCSKQ, or a peptide consisting of an amino acid sequence represented by MAKSYPNEEKDAW.

Item 5 Item 5. The TACE peptide epitope according to Item 4, comprising the amino acid sequence YPIAVSG or PIAVSGD.

Item 6 Item 4. A hybridoma that produces the antibody according to any one of Items 1 to 3.

Item 7 A therapeutic or prophylactic agent for a disease caused by RANKL-RANK signaling, comprising an effective amount of the antibody according to any one of Items 1 to 3.

Item 8 Item 8. The therapeutic or prophylactic agent according to Item 7, wherein the disease caused by RANKL-RANK signaling is at least one selected from the group consisting of periodontal disease, rheumatoid arthritis, and metastatic bone tumor.

Item 9. A method for treating or preventing a disease caused by RANKL-RANK signaling, comprising a step of administering an effective amount of the antibody according to any one of items 1 to 3 to a subject.

Item 10. Item 10. The method according to Item 9, wherein the disease caused by RANKL-RANK signaling is at least one selected from the group consisting of periodontal disease, rheumatoid arthritis, and metastatic bone tumor.

Item 11. Item 4. The antibody according to any one of Items 1 to 3, for treating or preventing a disease caused by RANKL-RANK signaling.

Item 12. Item 12. The antibody according to Item 11, wherein the disease caused by RANKL-RANK signaling is at least one selected from the group consisting of periodontal disease, rheumatoid arthritis, and metastatic bone tumor.

Item 13. Item 4. Use of the antibody according to any one of Items 1 to 3 for producing a therapeutic or prophylactic agent for a disease caused by RANKL-RANK signaling.

Item 14. Item 14. The use according to Item 13, wherein the disease caused by RANKL-RANK signaling is at least one selected from the group consisting of periodontal disease, rheumatoid arthritis, and metastatic bone tumor.

The antibody according to the present invention has a very high ability to inhibit TACE function. Therefore, the antibody according to the present invention inhibits RANKL-RANK signaling and is very useful for the treatment or prevention of diseases caused by the signaling.

The result of the TACE inhibitory ability comparison of the affinity purified polyclonal antibody in Example 1 is shown. The result of the TACE inhibitory ability comparison of the affinity purified polyclonal antibody and the small molecule inhibitor TAPI-2 in Example 1 is shown. The result of the TACE inhibitory ability comparison of the monoclonal antibodies C6-30 and C6-62 and the small molecule inhibitor TAPI-2 in Example 2 is shown. The result of each test in Example 3 is shown. The measurement result of the osteoclast differentiation promotion cytokine from the activated T cell in Example 4 is shown. FIG. 5 (upper) shows the results of measuring TNF-α using a commercially available ELISA kit for the culture supernatant 2 hours after stimulation. FIG. 5 (bottom) shows the results of measurement of sRANKL using a commercially available ELISA kit for the culture supernatant on the third day after stimulation. The amino acid sequence of TACE is shown. The three-dimensional structure of TACE and the position of Peptide-1 are shown. The three-dimensional structure of TACE and the position of Peptide-2 are shown. The three-dimensional structure of TACE and the position of Peptide-3 are shown. The three-dimensional structure of TACE and the position of Peptide-4 are shown. The three-dimensional structure of TACE and the position of Peptide-5 are shown. The three-dimensional structure of TACE and the position of Peptide-6 are shown. The three-dimensional structure of TACE and the position of Peptide-7 are shown. The three-dimensional structure of TACE and the position of Peptide-8 are shown. The three-dimensional structure of TACE and the position of Peptide-9 are shown. The inhibitory effect of the monoclonal antibody with respect to the bone destruction in in-vivo in Example 5 is shown. *: P <0.05, NS: no significant difference between groups

Antibody TACE (TNF-α converting enzyme, ADAM-17) targeted by the antibody of the present invention is a 70 kDa (824 amino acid residue) enzyme that recognizes and cleaves a specific amino acid sequence (VGPQRJFSGAPAMME). Belongs. The amino acid sequence of human TACE is shown in FIG. 6 (SEQ ID NO: 10).
. Expression in various cells such as T cells and B cells has been reported. TACE is a membrane-bound enzyme that is composed of three domains: the extracellular domain (215-671), the transmembrane domain (672-692), and the intracellular domain (693-824). The catalytic domain (223-474) is present in the extracellular domain (Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, CastnerBJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP. 1997 A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature. 385 (6618): 729- 33.; Moss ML, Jin SL, MiIIa ME, Bickett DM. Burkhart W, Carter HL, Chen WJ, Clay WC. Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH, Leesnitzer MA, McCauley P, McGreehan G , Mitchell J, Moyer M, Pahel G Rocque W, Overton LK, Schoenen F, Seaton T, Su JL, Becherer JD, et al. 1997 Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alp ha. Nature. 385 (6618): 73316). Furthermore, high homology of amino acid sequences is maintained across species, with 95% sequence matching between human-rat / human-mouse and 87% sequence matching between human-rabbit.

The antibody according to the present invention specifically binds to a region of the amino acid sequence represented by VMYPIAVSGDHENNKMFSNCSKQ (SEQ ID NO: 9) or MAKSYPNEEKDAW (SEQ ID NO: 4) in the amino acid sequence of TACE. Among the antibodies according to the present invention, those that specifically bind to the region of the amino acid sequence represented by VMYPIAVSGDHENNKMFSNCSKQ (SEQ ID NO: 9) are preferable, and among the amino acid regions, the amino acid region represented by YPIAVSGD (SEQ ID NO: 12) Those that specifically bind to are more preferred.

The specific binding of the antibody according to the present invention to the region of the amino acid sequence derived from TACE shown above can be visualized by a known method. Examples include immunoassay methods (for example, ELISA method) for samples in vitro, immunoelectrophoresis methods (for example, Western blot method), and immunohistochemistry for tissues or cells. It is not limited to. For visualization, the antibody of the present invention is a fluorescent substance (for example, fluorescein isothiocyanate), a radioisotope (for example, iodine 125), an enzyme (for example, alkaline phosphatase, horseradish peroxidase), and other proteins (for example, avidin). It may be labeled with a molecule such as Alternatively, a secondary antibody that specifically recognizes the antibody of the present invention may be used.

The antibody of the present invention is not limited as long as it can specifically bind to the above-mentioned TACE-derived amino acid sequence region. For example, IgG, IgM, IgA, IgD, IgE, IgY and the like can be used. Can be mentioned. Furthermore, the subclass is not limited. Preferably, it is IgG.

The antibody of the present invention is derived from a mammal (for example, human, mouse, rat, rabbit) or bird (for example, chicken or ostrich) capable of producing the antibody.

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. Furthermore, among monoclonal antibodies, a chimeric antibody, a humanized antibody, or a human antibody may be used. Here, the chimeric antibody and the humanized antibody refer to an antibody derived from a human other than a site capable of binding to an antigen among immunoglobulin proteins. A human antibody refers to a human-derived antibody or an antibody derived from a human antibody-producing mouse having a complete human-derived immunoglobulin gene. Chimeric antibodies, humanized antibodies, and human antibodies have low immunogenicity in humans and are useful in using antibodies in medical preparations and the like. Therefore, when the antibody of the present invention is used for medical preparations, monoclonal antibodies are preferable, and humanized antibodies or human antibodies are particularly preferable.

The antibody of the present invention may be a full-length immunoglobulin protein or a fragment thereof containing a site capable of binding to an antigen. It may also be a composition containing an antibody, such as antiserum.

The antibody of the present invention can be prepared by administering an antigen to an immunized animal. As the antigen, the TACE peptide epitope of the present invention described later can be used. When an antigen is administered to an immunized animal, since the peptide has a small molecular weight and may not cause an immune response sufficiently, the peptide is bound to an appropriate protein (eg, albumin, thyroglobulin hemocyanin, etc.) It may be used as

Alternatively, the antibody of the present invention can also be obtained by searching for an antibody that binds to an antigen or a fragment thereof from a library such as a cell or phage that can produce various antibodies or fragments thereof. In this case, the TACE peptide epitope of the present invention described later can be used as the antigen.

The method for obtaining a peptide used as an antigen is not particularly limited, and can be a known method. For example, it may be naturally derived, chemically synthesized, cell-free synthesized or recombinant peptide.

As for general antibody production methods, for example, Harlow et al., `` Using Antibodies: A laboratory manual '' (Cold Spring Harbor Laboratory Press, New York (1998)), Iwasaki et al. `` Monoclonal Antibody Hybridoma and ELISA, Kodansha ( 1991) ". Hereinafter, a method for producing each of a polyclonal antibody, a monoclonal antibody, and a humanized antibody will be briefly described.

(1) Polyclonal antibody A polyclonal antibody can be obtained by immunizing an immunized animal (eg, mouse, rat, rabbit, chicken) by a usual method, preparing serum, and purifying the antibody from antiserum. Immunization can be performed, for example, by inoculating an immunogen solution emulsified with an equal volume of Freund's complete or incomplete adjuvant (primary immunization) and then immunizing several times at intervals of 2 to 4 weeks. it can. Purification of the polyclonal antibody from the antiserum can be performed by DEAE ion exchange chromatography or protein G affinity chromatography.

(2) Monoclonal antibody A monoclonal antibody can be prepared by preparing a hybridoma producing a monoclonal antibody and purifying the antibody produced by the hybridoma. Alternatively, it can also be prepared by a phage display method, that is, a method of searching for an antibody or antibody fragment that specifically binds to an antigen from a phage library that expresses various antibodies or antibody fragments.

Methods for separating hybridomas are known per se (for example, Kohler et al., Nature, 256: 495 (1975)), and can be obtained, for example, by the following method. The spleen is aseptically removed from the immunized animal (eg, mouse) several days after the final immunization, and spleen cells are prepared from the spleen. Spleen cells are used in the cell fusion step together with myeloma cells (myeloma cells). For example, NS-1, P3U1, SP2 / 0 and the like can be used as myeloma cells. As a method of cell fusion, for example, spleen cells and myeloma cells are mixed in a medium containing a fusion promoter such as PEG. It can be carried out by mixing at a conventional mixing ratio (a ratio of about 1/5 to 1/10). After the fusion, only the hybridoma is grown using a selection medium (for example, HAT medium). Among them, the hybridoma secreting the target monoclonal antibody in the culture supernatant is confirmed, for example, by screening the reactivity of the culture supernatant with the TACE peptide epitope by enzyme linked immunoassay (ELISA). Can be selected.

Monoclonal antibodies produced by hybridomas can be easily prepared by culturing them. The culture can be performed in an appropriate medium (for example, Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum) or in vivo (for example, in the peritoneal cavity of a mouse). The antibody can also be purified using the culture supernatant or ascites as a starting material. For antibody purification, a general method for protein purification, such as ammonium sulfate salting-out, ion exchange chromatography, gel filtration chromatography, affinity chromatography using protein A or protein G binding polymer, or dialysis, is used. They can be used in appropriate combinations.

The phage display method is also known per se (for example, Clackson et al., Nature 352: 624-8 (1991)), and will be briefly described below. A phage that displays an antigen recognition site that binds to a TACE peptide epitope is selected from a phage library that displays antigen recognition sites of various antibodies prepared in advance. The antigen recognition site displayed by the selected phage may be used as it is, or a full-length antibody can be obtained by incorporating the full-length antibody into a CHO cell capable of producing the full-length antibody by a genetic engineering technique.

(3) Humanized antibody, human antibody A humanized antibody is an antibody protein whose origin is other than the minimum necessary region for antigen recognition (complementarity-determining region: CDR). is there. A method for producing a humanized antibody is described in, for example, EP0239400, US5585089, and the like.

A human antibody can be obtained from a human antibody-producing mouse having a complete human-derived immunoglobulin gene. Human antibody-producing mice are known and described in WO 97/07671.

The antibody of the present invention has a very high ability to neutralize TACE activity. Since TACE plays an important role in RANK-RANKL signaling, the antibody of the present invention is useful for the treatment and prevention of diseases caused by RANK-RANKL signaling.

Epitope The present invention is a TACE peptide, which is a peptide consisting of at least 7 consecutive amino acids of the amino acid sequence represented by VMYPIAVSGDHENNKMFSNCSKQ (SEQ ID NO: 9) or a peptide consisting of an amino acid sequence represented by MAKSYPNEEKDAW (SEQ ID NO: 4). Provide an epitope.

Of these TACE peptide epitopes, a peptide consisting of at least 7 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 9 is preferred. The TACE peptide epitope is preferably composed of 20 amino acids or less, more preferably 15 amino acids or less, further preferably 10 amino acids or less, and 9 amino acids or less. More preferably. The epitope of the present invention preferably includes the amino acid sequence YPIAVSG (SEQ ID NO: 19) or PIAVSGD (SEQ ID NO: 20). Preferred peptide epitopes include, for example, VMYPIAVSGDHE (SEQ ID NO: 21), MYPIAVSGDHE (SEQ ID NO: 22), VMYPIAVSGDH (SEQ ID NO: 23), YPIAVSGDHE (SEQ ID NO: 24), VMYPIAVSGD (SEQ ID NO: 25), MYPIAVSGDH (SEQ ID NO: 26), YPIAVSGDH (SEQ ID NO: 27), MYPIAVSGD (SEQ ID NO: 28), PIAVSGDHE (SEQ ID NO: 29), VMYPIAVSG (SEQ ID NO: 30), PIAVSGDH (SEQ ID NO: 31), MYPIAVSG (SEQ ID NO: 32), YPIAVSGD (SEQ ID NO: 33), YPIAVSG (SEQ ID NO: 34), PIAVSGD (SEQ ID NO: 35), and the like. YPIAVSGDHE (SEQ ID NO: 24), VMYPIAVSGD (SEQ ID NO: 25, MYPIAVSGDH (SEQ ID NO: 26), YPIAVSGDH (SEQ ID NO: 27), MYPIAVSGD (SEQ ID NO: 28) , YPIAVSGD (SEQ ID NO: 12) and the like are more preferable, and YPIAVSGD (SEQ ID NO: 12) is most preferable.

The peptide epitope of the present invention can be used for preparing an antibody having the ability to inhibit TACE function. When the peptide epitope of the present invention is used as an antigen, it may be bound to an appropriate protein and used as an antigen as described above.

Hybridoma The present invention also provides a hybridoma that produces an antibody. The method for producing a hybridoma can be carried out according to a method known per se, such as the method of Examples of the present application and the method described above for antibodies.

Therapeutic or preventive agent The present invention provides a therapeutic or prophylactic agent for a disease caused by RANKL-RANK signaling, comprising the aforementioned antibody.

The therapeutic or prophylactic agent for diseases caused by RANKL-RANK signaling of the present invention is formulated with various pharmaceutically acceptable carriers (including vaginal excipients, binders, disintegrants, lubricants, etc.) in the antibody. It may be a prepared formulation. The formulation may contain conventional additives.

The form of the preparation is not particularly limited. For example, tablets, pills, capsules, powders, granules, syrups and the like orally administered; vaginal injections (intravenous injection, intramuscular injection, local injection, etc.), mouthwashes, drops And various preparation forms such as external preparations and parenteral administration agents such as suppositories. Preferable pharmaceutical forms include, for example, topical injections, coating agents for gingival mucosa, gargles and the like.

The content of the antibody of the present invention in the preparation varies depending on the administration route, patient age, body weight, symptoms, etc., and cannot be defined unconditionally. However, the daily dose of the polypeptide is usually about 10 to 1000 mg, more preferably 100 to 100 mg. The amount may be about 500mg. When it is administered once a day, it is sufficient that this amount is contained in one preparation, and when it is administered three times a day, it is sufficient that this one-third amount is contained in one preparation.

The therapeutic or prophylactic agent of the present invention is administered to a patient such as a mammal. Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, sheep and the like.

The disease caused by RANKL-RANK signaling is not particularly limited, and examples thereof include periodontal disease, rheumatoid arthritis (chronic rheumatoid arthritis), osteoarthritis, and metastatic bone tumor.

関 連 The association between periodontal disease and RANKL-RANK signaling is as described above. In addition, it has been reported that TACE expression is increased in periodontal disease in an advanced state compared with normal (J Dent Res. 2008 Mar; 87 (3): 273-7. Tumor necrosis factor- alpha-converting enzyme (TACE) levels in periodontal diseases. Bostanci N, Emingil G, Afacan B, Han B, Ilgenli T, Atilla G, Hughes FJ, Belibasakis GN.).

In addition, similarities between bone destruction and periodontal disease in rheumatoid arthritis and osteoarthritis have been pointed out (Inflamm Res. 2004 Nov; 53 (11): 596-600. Bone lysis and inflammation. Haynes DR.) In rheumatoid arthritis, activated lymphocytes express RANKL and RANKL and TNF-alpha play an important role in joint destruction (Bone. 2002 Feb; 30 (2): 340-6. Involvement of receptor activator of NFkappaB ligand and tumor necrosis factor-alpha in bone destruction in rheumatoid arthritis. Romas E, Gillespie MT, Martin TJ. and TACE plays an important role in the production of TNF-alpha in rheumatoid arthritis J Rheumatol. 2001 Aug; 28 (8): 1756-63. Tumor necrosis factor-alpha (TNF-alpha) converting enzyme contributes to production of TNF-alpha in synovial tissues from patients with rheumatoid arthritis. Ohta, M, Kawaguchi Y, Sugiura T, Takagi K, Fukasawa C, Hara M, Kamatani N.) have been reported. Furthermore, the expression of TNF-alpha in osteoarthritis (Osteoarthritis Cartilage. 1999 Jul; 7 (4): 392-4. Regulation of tumor necrosis factor-alpha and tumor necrosis factor converting enzyme in human osteoarthritis. Amin AR.), TACE (J Immunol. 1998 May 1; 160 (9): 4570-9. TNF-alpha convertase enzyme from human arthritis-affected cartilage: isolation of cDNA by differential display, expression of the active enzyme, and regulation of TNF-alpha. Patel IR, Attur MG, Patel RN, Stuchin SA, Abagyan RA, Abramson SB, Amin AR.) and TACE is released from the osteoclast differentiation-promoting cytokines RANKL and TNF-alpha・ It is assumed that it plays an important role in activation. Therefore, the application of TACE inhibitors to rheumatoid arthritis has been attempted to date (Bioorg Med Chem. 2009 Jan 15; 17 (2): 444-59. Doi: 10.1016 / j.bmc.2008.11.067. Epub 2008 Dec 3. Current perspective of TACE inhibitors: a review. DasGupta S, Murumkar PR, Giridhar R, Yadav MR.).

Regarding the inhibition of tumor bone metastasis, 1) TACE is upregulated in various tumors (Eur J Cell Biol. 2011; 90 (6-7): 527-35, Curr Pharm Des. 2009 ; 15 (20): 2319-35, Semin Cell Dev Biol. 2009; 20 (2): 164-74), 2) Interferon gamma (IFNG) interferes with intracellular signal of osteoclast differentiation factor RANKL It has been reported to have the ability to block osteoclast differentiation (Nature 2000, 408 (6812): 600-5.). The inventor has also found that TACE can decompose IFNG. It is also known that tumor cells express RANKL and promote osteoclast differentiation and activation, thereby causing bone metastasis. From the above, TACE, whose expression of tumor cells is increased, degrades IFNG produced from peritumoral immune system cells, and further enhances antitumor immunity reduction and osteoclast differentiation activation by host IFNG. Furthermore, it is considered that the bone metastasis of tumor cells is enhanced by further promoting osteoclast differentiation / activation. It has also been reported that TACE expression is increased in tumors (Eur J Cell Biol. 2011; 90 (6-7): 527-35, Curr Pharm Des. 2009; 15 (20): 2319-35, and Semin Cell Dev Biol. 2009; 20 (2): 164-74). Therefore, it is considered that inhibiting TACE expressed by tumor cells can prevent IFNG degradation and thereby suppress reduction of host antitumor immunity and activation of osteoclast differentiation.

Diagnostic agent Since the above-mentioned expression of TACE correlates with the progression of the disease state, the progression of the disease state is predicted by detecting an increase in TACE in the pathological tissue specimen, serum sample, tissue fluid sample, etc. using the antibody of the present invention. be able to. Therefore, the present invention provides a diagnostic agent (for example, an in vitro diagnostic agent) for a disease caused by RANKL-RANK signaling, comprising the above-mentioned anti-human TACE protein antibody. If human TACE protein is detected in a test sample such as serum of a patient suspected of suffering from a disease caused by the above-mentioned RANKL-RANK signaling (for example, the human TACE protein exceeds a preset threshold value) And the like) can be diagnosed as suffering from the disease or in an advanced stage. As a method for detecting human TACE protein, a method known per se, such as ELISA, can be appropriately used.

Hereinafter, the present invention will be described in detail based on examples and the like, but the present invention is not limited thereto.

In this example, the following animals, cells, reagents, etc. were used:
Animal 6-week-old male BALB / C mice (18 animals in total) were purchased from Japan SLC (Hamamatsu) and used for experiments. This experimental protocol was planned in compliance with the Tohoku University Animal Experiment Guidelines, and the experiment was conducted after obtaining approval from the University Committee before the experiment was started.

Cell mouse T cell cell line EL4-TK
EL4-TK established from mouse T cell lymphoma was used as received from Tohoku University Institute of Aging Medicine, Medical Cell Resource Center.
(Http://www2.idac.tohoku.ac.jp/dep/ccr/TKGdate/TKGvo101/0192.html)
Mouse myeloma cell line SP-2
Mouse myeloma cell line SP-2 was used as received from Tohoku University Institute of Aging Medicine, Medical Cell Resource Center.
(Http://www2.idac.tohoku.ac.jp/dep/ccr/TKGdate/TKGvo102/0248.html)
Reagents Recombinant human TACE (930-ADB-010) and fluorescent peptide substrate (ES010) were purchased from R & D Systems, Inc. (Minneapolis, Minn.) And used for experiments. An anti-mouse TNF-alpha ELISA kit (430902) was purchased from BioLegend, Inc. (San Diego, Calif.) And used for experiments. The anti-rodent sRANKL ELISA kit (900-K233) was purchased from PeproTech, Inc. (Rocky Hill, NJ) and used for experiments.
HRP-labeled anti-mouse IgG antibody (710-1332) was purchased from Rockland Immunochemicals Inc. (Gilbertsville, PA) and used in the experiments. TMB (860336) was purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO) and used for experiments. TACE extracellular domain short polypeptide was synthesized from Life Technologies Japan (Tokyo). The sequence of the synthesized TACE extracellular domain short polypeptide is shown below.

Cell culture Unless otherwise stated, 10% FBS (HyClone: Thermoscience, Rockford, IL) and penicillin / streptomycin-containing α-MEM were used as the culture solution. The culture was performed at 37 ° C. and 5% CO ₂ .

Example 1
Nine TACE extracellular domain short polypeptides synthesized by peptide immunosynthesis in mice were respectively dissociated into Hemocyanin, from Keyhole Limpet (KLH: Wako Pure Chemicals, Tokyo, Japan) using Disuccinimidyl suberate (DSS: Thermoscience, Rockford, IL) And crosslinked. KLH-binding polypeptide was injected subcutaneously with Floyd's adjuvant according to standard procedures. Booster immunizations were performed every 2 weeks. Blood was collected immediately before immunization and during booster immunization, and the antibody titer in the serum was measured by the following method using ELISA.

Antibody titer measurement in serum ELISA plate coated with TACE extracellular domain short polypeptide or recombinant human TACE (R & D Systems, Inc. (Minneapolis, Minn.)) At Block Ace (DS Pharma Biomedical, Osaka, Japan) Serum samples diluted 1000-fold after masking non-specific binding sites were added to 100 microL of each well. After washing the wells, an HRP-labeled anti-mouse IgG antibody (Rockland Immunochemicals Inc., Gilbertsville, PA) was reacted as a detection antibody. Further, after washing the wells, TMB (Sigma-Aldrich Co. LLC., St. The absorbance at 650 nm was measured using Louis, MO), and the amount of serum IgG bound to each antigen was relatively evaluated.

Affinity purification from mouse serum of polyclonal antibody When the serum antibody titer sufficiently increased, about 5 ml of blood was stored, and IgG was purified using Ab-Rapid (Protenova, Takamatsu, Japan) according to the recommended protocol. Each polypeptide is then specifically bound to each TACE extracellular domain short polypeptide in purified IgG using an agarose gel column cross-linked to NHS-Activated agarose gel (Thermoscience, Rockford, IL) IgG was affinity purified. After purification, the solvent was replaced with Phosphate buffered Saline (PBS) using a Vivaspin 500 centrifugal filtration unit (MWCO 10 kDa; Sartorius AG, Goettingen, Germany).

Purified IgG concentration measurement Normal mouse IgG dilution series with known concentration and sample IgG are coated on ELISA plate, non-specific binding sites are masked with Block Ace, and HRP-labeled anti-mouse IgG antibody is reacted as a detection antibody. After washing, the absorbance at 650 nm was measured using TMB as a reaction substrate for HRP. Using the IgG concentration-absorbance standard curve of a normal mouse IgG dilution series, the IgG concentration of the sample was measured from the absorbance of the sample, and the concentration of each antibody solution was made constant.

TACE activity inhibition measurement Recombinant human TACE (R & D Systems, Inc., Minneapolis, MN) 0.4 μmol (when measuring polyclonal antibody inhibition ability) or 4 μmol (when measuring monoclonal antibody inhibition ability), 100 μL of reaction solution (2.5 microM chloride) Dilute in zinc, PBS containing 0.005% Brij35), add small molecule inhibitor TAPI-2 (Santa Cruz, Inc., Santa Cruz, CA) or purified antibody, and add fluorescent peptide substrate (R & D Systems, Inc., Minneapolis) , MN) After adding 0.5 nmol, the fluorescence generated by cleaving the fluorescent peptide substrate by TACE was performed using a SpectraMax M2e fluorescence plate reader (Molecular Device Japan, Tokyo) with excitation light of 320 nm and fluorescence of 405 nm. Measurement and comparison of inhibition of TACE enzyme activity from the fluorescence value. The results are as follows.

Comparison of TACE inhibitory ability of each affinity purified polyclonal antibody In order to evaluate the TACE neutralizing ability of the affinity purified polyclonal antibody obtained , TACE activity was achieved using recombinant human TACE, fluorescently labeled TACE substrate and equal amounts of affinity purified polyclonal antibody. Was measured.

Specifically, after reacting recombinant human TACE and affinity purified antibody for 30 minutes, a fluorescently labeled TACE substrate was added to the reaction system, and an increase in fluorescence associated with the degradation of the fluorescently labeled TACE substrate was observed with a fluorescent plate reader. The results are shown in FIG. The graph shows that the higher the value, the lower the ability of the purified antibody to inhibit TACE enzyme activity, and the lower the value of the graph, the higher the ability to inhibit TACE enzyme activity.
Data show mean values as well as SD bars.
*: Significant difference between groups with p <0.05.

As shown in FIG. 1, when the intact TACE activity is 100% and complete TACE activity inhibition is 0%, the purified antibody obtained by immunizing polypeptide 7 containing the enzyme activity center is When it was allowed to act, the neutralizing ability was not so high (82.1%). Among the obtained purified antibodies, affinity purified polyclonal antibodies obtained from animals immunized with polypeptide 9 at the three-dimensional proximity to the enzyme activity center showed the highest neutralizing ability of TACE activity (42.5%). (FIGS. 7 to 15 show the three-dimensional structure of TACE and the positions of Peptide-1 to Peptide-9). However, the affinity-purified polyclonal antibody obtained by immunizing polypeptide 5 at a site close to the enzyme activity center three-dimensionally like polypeptide 9 showed almost no neutralizing ability (94.1%). The affinity-purified polyclonal antibody obtained by immunizing polypeptide 4 which is a three-dimensionally distant site from the enzyme activity center showed the second highest TACE activity neutralizing ability after polypeptide 9 (46.6%).

Comparison of TACE inhibitory ability of affinity purified polyclonal antibody and small molecule inhibitor TAPI-2 Compared to TACE small molecule inhibitor (TAPI-2), the affinity purified polyclonal antibody obtained from polypeptide 9 shows how neutralizing activity is. Concentration-response curves were prepared to evaluate the neutralization activity in order to clarify whether they had the same. The comparison was made by comparing the antibody or inhibitor molar concentration ND50 required to neutralize recombinant human TACE activity by 50%.

Specifically, human recombinant TACE and affinity purified antibody or TACE small molecule inhibitor (TAPI-2) are reacted for 30 minutes, then fluorescently labeled TACE substrate is added to the reaction system, and fluorescent labeled TACE substrate is decomposed with a fluorescent plate reader. The accompanying increase in fluorescence was observed.

5) Five points of 5-fold dilution systems were prepared for both antibodies and small molecule inhibitors, and the ability to inhibit TACE enzyme activity was evaluated. The results are shown in FIG. The red horizontal line indicates 50% inhibition of TACE activity, and the intersection with each graph indicates ND50. The more the curve of the graph is on the left, the higher the neutralization ability at a lower concentration. Compared with TAPI-2 (3500 nM), the affinity purified polyclonal antibody (0.02 nM) obtained this time showed an ND50 value 6 digits lower.

Example 2
Hybridoma preparation TACE extracellular domain short polypeptide 4 days after the final immunization, the immunized mice were sacrificed, and the spleen was removed. After preparing the spleen cell suspension, the erythrocytes were hemolyzed using 0.017M Tris buffer (pH = 7.65) containing 0.75% ammonium chloride, washed with PBS and suspended in a serum-free medium to prepare a cell suspension. Spleen cells and mouse myeloma SP-2 cells were fused using the cell fusion reagent GenomOne-CF (Ishihara Sangyo, Osaka) according to the recommended protocol. The cells were seeded in a 96-well plate, and the culture was started on the medium containing HT media supplement Hyb Max (Sigma-Aldrich Co. LLC., St. Louis, MO) the day after cell fusion.

Selection of hybridomas When colonies of hybridomas were confirmed to be formed, the culture supernatant was collected and the reactivity of IgG produced by each clone was confirmed using an ELISA plate. TACE extracellular domain short polypeptide, KLH binding TACE extracellular domain short polypeptide, KLH, and recombinant human TACE were used as antigens. Among these, clones showing a high value on the KLH-binding polypeptide-coated plate were primarily selected, and clones showing a high value on the KLH-coated plate were excluded. Next, from those selected clones, clones showing high values with TACE extracellular domain short polypeptide and recombinant human TACE coated plate were selected, and TACE enzyme activity inhibition test was performed using an equal amount of purified monoclonal IgG antibody. It was.

Comparison of monoclonal antibody C6-30, C6-62 and small molecule inhibitor TAPI-2 TACE inhibitory ability Polypeptide 9 compared to TACE small molecule inhibitor (TAPI-2) In order to clarify whether it has a sum activity, a concentration-response curve was prepared and the neutralization activity was evaluated. The comparison was made by comparing the antibody or inhibitor molar concentration ND50 required to neutralize recombinant human TACE activity by 50%.

Specifically, measurement was performed in the same manner as in “ Comparison of TACE inhibitory ability of affinity purified polyclonal antibody and low molecular weight inhibitor TAPI-2 ” in Example 1 except that a monoclonal antibody was used.

The results are shown in Fig. 3. Compared to TAPI-2 (385252 pM), both monoclonal antibodies C6-30 (2094 pM) and C6-62 (728 pM) exhibited ND50 values that were three orders of magnitude lower and had a very high ability to inhibit TACE activity.

Example 3
Epitope mapping TACE extracellular domain short polypeptide No. 9 (VMYPIAVSGDHENNKMFSNCSKQ), 8 peptides with 8 amino acid residues shorter than the N-terminal, shifted by 2 amino acid residues, total of 8 types were requested from the Institute of Medical Biology And synthesized. The sequence of the epitope mapping peptide requested for synthesis is shown below. The actually measured purity of each peptide by HPLC was 94.6-99.8%.

A sample was prepared by adding each peptide 9A to 9H to the purified monoclonal antibody, and these were added to each well of an ELISA plate coated with recombinant human TACE and blocked with BlockAce and incubated. Thereafter, an HRP-labeled anti-mouse IgG antibody was reacted as a detection antibody, and after further washing the well, absorbance at 650 nm was measured using TMB as a reaction substrate for HRP. By adding each peptide 9A to 9H to the purified monoclonal antibody, the purified monoclonal antibody binds to the peptide that is the epitope and cannot bind to recombinant human TACE. This shows the epitope sequence of the purified monoclonal antibody. Specifically:

Epitope mapping TACE extracellular domain short chain polypeptide No. 9 of monoclonal antibodies C6-30 and C6-62 consists of 23 amino acid residues. Among these 23 amino acid residues, 8 amino acids are used for further narrowing down the epitope region. Epitope sequence identification of the monoclonal antibody was carried out by synthesizing 8 types of peptides with a residue length shifted by 2 amino acid residues from the N-terminus, and investigating which peptide the obtained monoclonal antibody bound strongly.

First, we were concerned that the 8 amino acid residue-length peptide would not bind to the ELISA plate sufficiently, and after adding an equal amount of each 8 amino acid residue peptide to the monoclonal antibody solution and reacting with the peptide, react with the peptide. The antibody was added to an ELISA plate coated with recombinant human TACE, and the epitope was identified by using a peptide that reduces the amount of monoclonal antibody bound to recombinant human TACE as an epitope sequence.

More specifically, samples were prepared by adding each peptide 9A-9H to the purified monoclonal antibody, and they were added to each well of an ELISA plate coated with recombinant human TACE and blocked with BlockAce and incubated. Thereafter, an HRP-labeled anti-mouse IgG antibody was reacted as a detection antibody, and after further washing the well, absorbance at 650 nm was measured using TMB as a reaction substrate for HRP. The data is shown in the upper graph of FIG.
In the figure, * indicates that there is a significant difference from the sample in which PBS was added to the monoclonal antibody at p <0.05.
A significant decrease in the numerical value was observed in the monoclonal antibody sample to which peptide 9B was added, suggesting that peptide 9B is an epitope of the obtained monoclonal antibody. The data shows the result of monoclonal antibody C6-30, but the same result was obtained with C6-62 (Data not shown).

As another method for detecting a purified monoclonal antibody epitope sequence, each peptide 9A-9H is coated on an ELISA plate to directly observe the reactivity of the purified monoclonal antibody to each peptide 9A-9H, and after BlockAce blocking After adding and incubating the purified monoclonal IgG sample to each well, an HRP-labeled anti-mouse IgG antibody was reacted as a detection antibody, and absorbance at 650 nm was measured using TMB as a reaction substrate for HRP. This shows that the peptide coated with the well having a high value in this detection system is the epitope sequence of the purified monoclonal antibody.

Specifically, in order to directly observe the reactivity of the purified monoclonal antibody to each peptide 9A-9H, coat each peptide 9A-9H on an ELISA plate and add the purified monoclonal IgG sample to each well after BlockAce blocking After incubation, an HRP-labeled anti-mouse IgG antibody was reacted as a detection antibody, and absorbance at 650 nm was measured using TMB as a reaction substrate for HRP. This shows that the peptide coated with the well having a high value in this detection system is the epitope sequence of the purified monoclonal antibody. The results are shown in the second graph from the top of FIG.
In the figure, * indicates that there is a significant difference from non-coated wells at p <0.05.

High absorbance was observed only in wells coated with peptide 9B compared to wells not coated with peptide, indicating that the obtained monoclonal antibody was bound only to peptide 9B. The data show the result of monoclonal antibody C6-30, but the same result was obtained with C6-62 (Data not shown).

From the above, it was shown that the epitope sequence of the obtained monoclonal antibody was “YPIAVSGD”.

Example 4
Test of TACE neutralizing ability of purified monoclonal antibody in cell culture system TACE from activated mouse T cells was used to determine whether the monoclonal antibody C6-62 obtained in the cell culture system exhibited a predetermined TACE inhibitory effect in the cell culture system. This was confirmed by measuring the release of bone cell differentiation promoting cytokines.

Specifically, a mouse T cell cell line EL4-TK cell suspension (8 × 10 ⁴ cells / ml, 1 ml) was seeded in a 24-well plate. Purified monoclonal antibody or normal mouse IgG was added to a final concentration of 0.32 μg / ml and reacted for 30 minutes. In the stimulation group, Phorbol-12-Myristate-13-Acetate (PMA) (final concentration 25 ng / ml; LC Laboratories, Woburn, MA) and Ionomycin (final concentration 500 ng / ml; Enzo Life Sciences, Inc., Farmingdale, NY) were added to the culture system to stimulate the cells. After 2 hours, the culture solution was collected for measuring TNF-α in the culture solution. For measurement of sRANKL in the culture solution, the culture solution was collected 3 days after the start of stimulation. TNF-α and sRANKL concentrations were measured and evaluated by the following method.

Measurement of mouse osteoclast differentiation promoting factor release from stimulated cells The concentrations of mouse TNF-α and sRANKL in the culture medium were determined using anti-mouse TNF-alpha ELISA kit (BioLegend, Inc., San Diego, Calif.) Rodent sRANKL ELISA kit (PeproTech, Inc., Rocky Hill, NJ) was used as recommended protocol.

The statistical significance test was repeated three times for each sample, confirming that similar results were obtained. The statistical significance test of these experimental data was performed using Tukey's multiple comparison method, and a significance level of 5% or less was considered significant.

The results are shown in FIG. In the figure, * indicates that p <コ ン ト ロ ー ル 0.05 and there is a significant difference between the control and stimulation groups. Mouse T cell cell line EL4TK released TNF-α and free RANKL (soluble RANKL: sRANKL) into the culture supernatant upon stimulation with a combination of PMA and ionomycin. When cells were stimulated with monoclonal antibody C6-62 added to the culture system, both TNF-α and sRANKL in the culture supernatant decreased markedly, and monoclonal antibody C6-62 was also active in TACE in the cell culture system. It was shown that the release of cytokines promoting osteoclast differentiation from activated T cells can be prevented.

Example 5
Animal Experiments 8 week-old female C57BL6 mice were divided into 6 control groups, 6 experimental periodontal disease groups, and 6 experimental periodontal disease + antibody administration groups.
For experimental periodontal disease group and experimental periodontal disease + antibody administration group, according to the method of Kimura et al. (J Periodontol. 2000 Jul; 71 (7): 1167-73.) Bone destruction was induced by inserting 6-0 silk thread between the first and second molars. In order to investigate how much the anti-TACE monoclonal antibody established against bone destruction shows the inhibitory effect, PBS in the palatal interdental papillary gingiva between the maxillary first and second molars in the control group and experimental periodontal disease group In the experimental periodontal disease + antibody administration group, 2 microL each of the anti-TACE monoclonal antibody (C6-62) was given on the day of the experiment (in the experimental periodontal disease group and experimental periodontal disease + antibody administration group) (Within 5 minutes after inserting the silk thread). The mice in each group were sacrificed on the 5th day from the start of the experiment, and micro CT was taken to evaluate the degree of bone destruction.

Evaluation of bone destruction by micro CT Each mouse skull sample after sacrifice was fixed with formaldehyde soaking overnight, and then micro CT images were taken with ScanXmate-E090 (Comscan Techno). The captured micro CT image is reconstructed with ConeCTexpress (com scan techno) and converted into a DICOM file, then 3D rendering processing is performed using Pluto (http://pluto.newves.org/trac). Above, we compared the degree of alveolar bone destruction of the first molar distal palate by calculating the distance from the cement enamel junction to the alveolar bone surface.

Statistical processing <br/> Comparison between other groups is based on Tukey test (MEPHAS; http://www.gen-info.osaka-u.ac.jp/testdocs/tomocom/tukey-e.html) Was tested. p <0.05 was determined to be significant. The results are shown in FIG. Specifically, a representative three-dimensional rendering image of each group is shown on the left of FIG. The white line indicates the distance from the cement enamel junction to the alveolar bone surface. As an inter-group comparison of the distance from cement enamel junction to the alveolar bone surface, experimental periodontal disease + antibody administration when the average distance in the control group is 0% and the average distance in the experimental periodontal disease group is 100% The average distance of the group is converted to% and shown on the right side of FIG. The closer to 100%, the greater the amount of bone destruction, and the closer to 0%, the more bone destruction is prevented. As is apparent from FIG. 16, it can be seen that the monoclonal antibody used in this example prevented bone destruction.

The antibody according to the present invention has a very high ability to inhibit TACE function and inhibits RANKL-RANK signaling. The diseases resulting from RANKL-RANK signaling are very useful because they are diverse.

Claims (14)

1. An anti-human TACE protein antibody that specifically binds to a region of the amino acid sequence represented by VMYPIAVSGDHENNKMFSNCSKQ or MAKSYPNEEKDAW.
2. The antibody according to claim 1, which specifically binds to a region of the amino acid sequence represented by YPIAVSGD.
3. The antibody according to claim 1 or 2, which is a monoclonal antibody.
4. TACE peptide epitope which is a peptide consisting of at least 7 consecutive amino acids in the amino acid sequence represented by VMYPIAVSGDHENNKMFSNCSKQ or a peptide consisting of an amino acid sequence represented by MAKSYPNEEKDAW.
5. The TACE peptide epitope according to claim 4, comprising the amino acid sequence YPIAVSG or PIAVSGD.
6. A hybridoma producing the antibody according to any one of claims 1 to 3.
7. A therapeutic or prophylactic agent for a disease caused by RANKL-RANK signaling, comprising an effective amount of the antibody according to any one of claims 1 to 3.
8. The therapeutic or prophylactic agent according to claim 7, wherein the disease caused by RANKL-RANK signaling is at least one selected from the group consisting of periodontal disease, rheumatoid arthritis and metastatic bone tumor.
9. A method for treating or preventing a disease caused by RANKL-RANK signaling, comprising a step of administering an effective amount of the antibody according to any one of claims 1 to 3 to a subject.
10. The method according to claim 9, wherein the disease caused by RANKL-RANK signaling is at least one selected from the group consisting of periodontal disease, rheumatoid arthritis, and metastatic bone tumor.
11. The antibody according to any one of claims 1 to 3, for treating or preventing a disease caused by RANKL-RANK signaling.
12. The antibody according to claim 11, wherein the disease caused by RANKL-RANK signaling is at least one selected from the group consisting of periodontal disease, rheumatoid arthritis, and metastatic bone tumor.
13. Use of the antibody according to any one of claims 1 to 3 for producing a therapeutic or prophylactic agent for a disease caused by RANKL-RANK signaling.
14. The use according to claim 13, wherein the disease caused by RANKL-RANK signaling is at least one selected from the group consisting of periodontal disease, rheumatoid arthritis and metastatic bone tumor.
    

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