Antibody or antigen-binding fragment thereof

Abstract

To provide a novel antibody or antigen-binding fragment thereof against exon 21 of human periostin.SOLUTION: Provided is an antibody or antigen-binding fragment thereof having binding properties against a steric epitope on human periostin, consisting of a specific amino acid sequence A and a specific amino acid sequence B.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an antibody or an antigen-binding fragment thereof, etc. [Background technology]

[0002] Periostin is a type of extracellular matrix protein and a polypeptide with a molecular weight of approximately 90,000. Each polypeptide chain contains a signal sequence, a cysteine-rich domain, and four It contains a repeat domain and a C-terminal domain. It has been suggested that its expression is also associated with vascular restenosis, cancer, inflammation, and angiogenesis.

[0003] Patent Document 1 reports an antibody against exon 21 of periostin, and describes that the antibody can inhibit periostin having cell adhesion activity and suppress the action of a specific periostin variant whose expression increases in diseases accompanied by inflammation including vascular intimal hyperplasia, cancer, and inflammatory colitis, and angiogenesis, thereby suppressing the progression and aggravation of the disease, thereby treating the disease. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2014 / 136910 Summary of the Invention [Problem to be solved by the invention]

[0005] In the course of their research, the present inventors have found that existing antibodies against exon 21 of periostin have extremely low binding activity to human periostin secreted from human cells, and have therefore focused on the need to develop new antibodies that overcome this problem.

[0006] An objective of the present invention is to provide a novel antibody against exon 21 of human periostin or an antigen-binding fragment thereof. [Means for solving the problem]

[0007] As a result of intensive research, the present inventors have discovered that the amino acid sequence A shown in SEQ ID NO:61 and the amino acid sequence B shown in SEQ ID NO:62 A three-dimensional structure of human periosin consisting of an amino acid sequence B represented by any one of ~64 It has been found that the above-mentioned problems can be solved by an antibody or an antigen-binding fragment thereof that has binding affinity to an epitope. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0008] Item 1. An amino acid sequence A represented by SEQ ID NO: 61 and an amino acid sequence B represented by any one of SEQ ID NOs: 62 to 64 A. Binding to a conformational epitope on human periostin, which is composed of amino acid sequence B. An antibody or an antigen-binding fragment thereof having the following structure:

[0009] Item 2. The antibody or antigen-binding fragment thereof according to Item 1, wherein the human periostin is human periostin secreted from human cells.

[0010] Item 3. The antibody or antigen-binding fragment thereof according to Item 1 or 2, wherein the human cell is a human breast cancer cell.

[0011] Item 4. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 3, which is a monoclonal antibody or an antigen-binding fragment thereof.

[0012] Item 5. The heavy chain CDR1 comprises an amino acid sequence represented by SEQ ID NO: 1 and / or 2, SEQ ID NO: 13 and / or 14, SEQ ID NO: 25 and / or 26, SEQ ID NO: 37 and / or 38, or SEQ ID NO: 49 and / or 50; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 3 and / or 4, SEQ ID NO: 15 and / or 16, SEQ ID NO: 27 and / or 28, SEQ ID NO: 39 and / or 40, or SEQ ID NO: 51 and / or 52; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:5 and / or 6, SEQ ID NO:17 and / or 18, SEQ ID NO:29 and / or 30, SEQ ID NO:41 and / or 42, or SEQ ID NO:53 and / or 54; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 7 and / or 8, SEQ ID NO: 19 and / or 20, SEQ ID NO: 31 and / or 32, SEQ ID NO: 43 and / or 44, or SEQ ID NO: 55 and / or 56; Light chain CDR2 is SEQ ID NO: 9 and / or 10, SEQ ID NO: 21 and / or 22, SEQ ID NO: 33, and / or 34, SEQ ID NO: 45 and / or 46, or SEQ ID NO: 57 and / or 58, and the light chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 11 and / or 12, SEQ ID NO: 23 and / or 24, SEQ ID NO: 35 and / or 36, SEQ ID NO: 47 and / or 48, or SEQ ID NO: 59 and / or 60; Item 5. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 4.

[0013] Section 6. (A) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 2; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 3 and / or 4; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 5 and / or 6; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 7 and / or 8; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 9 and / or 10; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11 and / or 12; or (B) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 13 and / or 14; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 15 and / or 16; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 17 and / or 18; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 19 and / or 20; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 21 and / or 22; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23 and / or 24; or (C) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 25 and / or 26; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 27 and / or 28; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 29 and / or 30; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 31 and / or 32; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 33 and / or 34; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 35 and / or 36; or (D) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 37 and / or 38; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 39 and / or 40; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 41 and / or 42; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 43 and / or 44; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 45 and / or 46; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 47 and / or 48; or (E) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 49 and / or 50; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 51 and / or 52; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 53 and / or 54; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 55 and / or 56; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 57 and / or 58; and The light chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 59 and / or 60. Item 6. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 5.

[0014] Section 7. (A) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 2; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 3 and / or 4; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 5 and / or 6; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 7 and / or 8; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 9 and / or 10; and the light chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 11 and / or 12; The antibody or antigen-binding fragment thereof according to any one of items 1 to 6.

[0015] Item 8. (e) a protein or peptide comprising the amino acid sequence represented by SEQ ID NO: 65; and (f) a protein or peptide comprising an amino acid sequence X having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 65, and comprising within said amino acid sequence X an amino acid sequence A shown in SEQ ID NO: 61 and an amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64. At least one protein or peptide selected from the group consisting of and selecting an antibody that binds to human periostin secreted from human breast cancer cells from antibodies produced by the selected hybridoma.

[0016] Item 9. (e) a protein or peptide comprising the amino acid sequence represented by SEQ ID NO: 65; and (f) a protein or peptide comprising an amino acid sequence X having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 65, and comprising within said amino acid sequence X an amino acid sequence A shown in SEQ ID NO: 61 and an amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64. At least one protein or peptide selected from the group consisting of The antibody is produced from the hybridoma and has an affinity for human periostin secreted from human breast cancer cells.

[0017] Item 10. A polynucleotide comprising a coding sequence for the antibody or antigen-binding fragment thereof according to any one of items 1 to 9.

[0018] Item 11. A cell containing the polynucleotide according to Item 10.

[0019] Item 12. A conjugate of the antibody or antigen-binding fragment thereof according to any one of items 1 to 9 and a drug.

[0020] Item 13. A pharmaceutical composition comprising at least one member selected from the group consisting of the antibody or antigen-binding fragment thereof according to any one of items 1 to 9, and the conjugate according to item 12.

[0021] Item 14. The pharmaceutical composition according to Item 13, which is for preventing or treating cancer, for inhibiting vascular mimicry of cancer, for inhibiting periostin, for preventing or treating inflammation-related diseases, for inhibiting vascular intimal hyperplasia, for inhibiting angiogenesis, or for preventing or treating aneurysm.

[0022] Item 15. A reagent comprising at least one member selected from the group consisting of the antibody or antigen-binding fragment thereof according to any one of items 1 to 9, and the complex according to item 12. Effect of the Invention

[0023] According to the present invention, a novel antibody or an antigen-binding fragment thereof against exon 21 of human periostin can be provided. [Brief description of the drawings]

[0024] [Figure 1] The results of the human periostin exon 21 recognition test for each antibody (Test Example 2) are shown. (A) shows the results of Western blotting of the sample after immunoprecipitation. (B) shows a CBB stained image of the gel after Western blotting. The antibody used is shown at the top of each photograph, and the molecular weight of each band of the molecular weight marker is shown on the left. [Diagram 2] The results of Test Example 5, which investigated the effect of each antibody on vascular mimicry, are shown below. (A) shows a fluorescent photograph of a cultured cell line vascular mimicry model. The fluorescent areas indicate 4T07 mouse breast cancer cells. (B) shows the results of measuring the number of nodes, which is an index of lumen formation. (C) shows the results of measuring the number of segments, which is an index of lumen formation. [Diagram 3] 1 shows the results of Test Example 6, in which the effect of each antibody on tumor growth was examined. The vertical axis indicates tumor volume, and the horizontal axis indicates the number of days since tumor implantation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In this specification, the expression "comprise" includes the concepts of "consist essentially of" and "consist only of".

[0026] 1. Antibody or antigen-binding fragment thereof In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof (also referred to herein as the "antibody of the present invention or a fragment thereof") which is composed of an amino acid sequence A shown in SEQ ID NO: 61 and an amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64 and has binding affinity to a conformational epitope on human periostin. This will be described below.

[0027] Human periostin is not particularly limited as long as it is human periostin having exon 21 (SEQ ID NO: 65) among the multiple existing splicing variants. Preferred examples of human periostin include human periostin having exon 21 but not exon 17, and more preferably human periostin (PN-2) consisting of the amino acid sequence shown in SEQ ID NO: 66.

[0028] Human periostin also includes variants occurring within the human species, so long as the conformational epitope structure and sequence described below remain unchanged. From this perspective, human periostin includes: For example, the protein described in (a) below and the protein described in (b) below: (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 65, and (b) a protein comprising an amino acid sequence X having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 65, and comprising within said amino acid sequence X an amino acid sequence A shown in SEQ ID NO: 61 and an amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64. At least one human periostin selected from the group consisting of Preferably, the protein described in (c) below and the protein described in (d) below: (c) a protein consisting of the amino acid sequence shown in SEQ ID NO: 66, and (d) an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 66; and comprising the amino acid sequence set forth in SEQ ID NO:65. The present invention includes at least one type of human periostin selected from the group consisting of:

[0029] In the above (b) and (d), the identity is more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more.

[0030] "Identity" of amino acid sequences refers to the degree to which two or more comparable amino acid sequences are related to each other. It refers to the degree of identity of amino acid sequences. Therefore, the higher the identity between two amino acid sequences, The identity or similarity of their sequences is high. The level of identity of amino acid sequences is determined, for example, using the sequence analysis tool FASTA with default parameters. For example, the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes”Proc Natl Acad Sci USA.87:2264-2268(1990), Karlin S, Altschul SF. Based on this BLAST algorithm, a program called BLASTX is available. Specific methods for these analysis methods are publicly known and are being developed at the National Center of For details, please refer to the National Center for Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of base sequences is also defined in the same manner as above.

[0031] Examples of the proteins described in (b) and (d) above include (b') an amino acid sequence X in which one or more amino acids are substituted, deleted, added, or inserted (preferably substituted, more preferably conservatively substituted) with respect to the amino acid sequence shown in SEQ ID NO: 65, and the amino acid sequence X contains the amino acid sequence A shown in SEQ ID NO: 61 and the sequence A protein comprising an amino acid sequence B represented by any one of Nos. 62 to 64. (d') a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted (preferably substituted, more preferably conservatively substituted) relative to the amino acid sequence shown in SEQ ID NO: 66, and comprising the amino acid sequence shown in SEQ ID NO: 65. Examples include:

[0032] In the above (b') and (d'), "plural" means, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 or 3.

[0033] "Conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution. Other conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0034] The human periostin is preferably human periostin secreted from human cells. Human periostin secreted from human cells is human periostin produced based on mRNA transcribed from the human periostin gene in human cells and secreted outside the cells, and is not particularly limited thereto. Thus, recombinant human periostin obtained by expression and purification using non-human cells (e.g., bacteria) is not included in "human periostin secreted from human cells."

[0035] There are no particular limitations on the human cells, so long as they are capable of secreting human periostin. Examples of human cells include cancer cells. Examples of cancers from which cancer cells are derived include brain tumors. Examples of cancers that can be caused by the cancer include leukemia, osteosarcoma, breast cancer, colon cancer, malignant melanoma, bone cancer, stomach cancer, lung cancer, liver cancer, kidney cancer, pancreatic cancer, gallbladder cancer, skin cancer, uterine cancer, ovarian cancer, rectal cancer, colon cancer, fallopian tube cancer, esophageal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, prostate cancer, bladder cancer, malignant lymphoma, and the like, of which breast cancer, colon cancer, lung cancer, and malignant melanoma are preferred, and breast cancer is particularly preferred.

[0036] The antibody or fragment thereof of the present invention has binding activity to a conformational epitope on human periostin. The conformational epitope on human periostin is the amino acid sequence A shown in SEQ ID NO:61. and an amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64. As long as it is within this range, there is no particular limitation. In other words, the above-mentioned binding ability of the antibody or fragment thereof of the present invention can recognize a conformation on human periostin or bind to said conformation. The antibody or fragment thereof of the present invention preferably has specific binding ability to a conformational epitope on human periostin. The amino acid sequence B is preferably SEQ ID NO: No. 62 is an example.

[0037] In a preferred embodiment, the antibody or fragment thereof of the present invention comprises: heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 1 and / or 2, SEQ ID NO: 13 and / or 14, SEQ ID NO: 25 and / or 26, SEQ ID NO: 37 and / or 38, or SEQ ID NO: 49 and / or 50; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 3 and / or 4, SEQ ID NO: 15 and / or 16, SEQ ID NO: 27 and / or 28, SEQ ID NO: 39 and / or 40, or SEQ ID NO: 51 and / or 52; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:5 and / or 6, SEQ ID NO:17 and / or 18, SEQ ID NO:29 and / or 30, SEQ ID NO:41 and / or 42, or SEQ ID NO:53 and / or 54; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 7 and / or 8, SEQ ID NO: 19 and / or 20, SEQ ID NO: 31 and / or 32, SEQ ID NO: 43 and / or 44, or SEQ ID NO: 55 and / or 56; Light chain CDR2 is SEQ ID NO: 9 and / or 10, SEQ ID NO: 21 and / or 22, SEQ ID NO: 33, and / or 34, SEQ ID NO: 45 and / or 46, or SEQ ID NO: 57 and / or 58, and the light chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 11 and / or 12, SEQ ID NO: 23 and / or 24, SEQ ID NO: 35 and / or 36, SEQ ID NO: 47 and / or 48, or SEQ ID NO: 59 and / or 60; It may be an antibody or a fragment thereof.

[0038] In the preferred embodiment, the amino acid sequences represented by odd-numbered SEQ ID NOs are identified by Kabat The CDR sequences are defined as follows: The CDR sequences are as follows:

[0039] In a more preferred embodiment, the antibody or fragment thereof of the present invention comprises: (A) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 2; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 3 and / or 4; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 5 and / or 6; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 7 and / or 8; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 9 and / or 10; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11 and / or 12; or (B) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 13 and / or 14; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 15 and / or 16; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 17 and / or 18; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 19 and / or 20; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 21 and / or 22; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23 and / or 24; or (C) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 25 and / or 26; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 27 and / or 28; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 29 and / or 30; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 31 and / or 32; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 33 and / or 34; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 35 and / or 36; or (D) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 37 and / or 38; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 39 and / or 40; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 41 and / or 42; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 43 and / or 44; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 45 and / or 46; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 47 and / or 48; or (E) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 49 and / or 50; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 51 and / or 52; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 53 and / or 54; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 55 and / or 56; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 57 and / or 58; and The light chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 59 and / or 60. It may be an antibody or an antigen-binding fragment thereof.

[0040] Of the antibodies or antigen-binding fragments thereof of the present invention, those of (A) to (E) above are more preferably the antibody or antigen-binding fragment thereof of (A) or (B) above, and particularly preferably the antibody or antigen-binding fragment thereof of (A) above.

[0041] In a more preferred embodiment, the antibody or fragment thereof of the present invention comprises: (A1) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:1; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:3; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:5; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:7; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:9, and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:11; or (A2) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:2; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:4; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:6; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:8; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 10; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:12; or (B1) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 13; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 15; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 17; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 19; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 21, and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:23; or (B2) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 14; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 16; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 18; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 20; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 22; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:24; or (C1) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:25; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:27; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:29; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:31; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 33; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:35; or (C2) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:26; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:28; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 30; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 32; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 34, and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:36; or (D1) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 37 and / or 38; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 39 and / or 40; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 41 and / or 42; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 43 and / or 44; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 45 and / or 46; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 47 and / or 48; or (D2) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:38; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 40; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 42; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 44; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 46; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:48; or (E1) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:49; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:51; the heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:53; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:55; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:57; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:59; or (E2) heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:50; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:52; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:54; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:56; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:58; and The light chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 60. It may be an antibody or an antigen-binding fragment thereof.

[0042] The antibody or fragment thereof of the present invention is, among the antibodies or antigen-binding fragments thereof of (A1) to (E2) above, more preferably the antibody or antigen-binding fragment thereof of (A1), (A2), (B1), or (B2) above, and particularly preferably the antibody or antigen-binding fragment thereof of (A1) or (A2) above.

[0043] The antibody or antigen-binding fragment thereof of (A) has an amino acid sequence AH1 shown in SEQ ID NO:67, and has a binding affinity of 85% or more (preferably 90% or more, more preferably 95% or more, further preferably 10% or more, more preferably 15% or more, more ... a heavy chain variable region comprising an amino acid sequence AH2 having an identity (more preferably 98% or more, and even more preferably 99% or more) with said amino acid sequence AH1, or an amino acid sequence AH3 which is a humanized sequence of said amino acid sequence AH1. an amino acid sequence AL1 having an identity of 85% or more (preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more) to said amino acid sequence AL1; It is preferred that the humanized sequence of sequence AL1 is a light chain variable region comprising amino acid sequence AL3.

[0044] The antibody or antigen-binding fragment thereof of (B) has an amino acid sequence BH1 shown in SEQ ID NO:69, and has a binding affinity of 85% or more (preferably 90% or more, more preferably 95% or more, more preferably 10% or more, more preferably 1 ... a heavy chain variable region comprising an amino acid sequence BH2 having an identity (more preferably 98% or more, and even more preferably 99% or more) with said amino acid sequence BH1, or an amino acid sequence BH3 which is a humanized sequence of said amino acid sequence BH1; 70, an amino acid sequence BL2 having an identity of 85% or more (preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more) to the amino acid sequence BL1, or It is preferred that the humanized sequence of sequence BL1 is a light chain variable region comprising the amino acid sequence BL3.

[0045] The antibody or antigen-binding fragment thereof of (C) has an amino acid sequence CH1 shown in SEQ ID NO: 71, and has a sequence identity of 85% or more (preferably 90% or more, more preferably 95% or more, further preferably 10% or more, more preferably 15% or more, more ... a heavy chain variable region comprising an amino acid sequence CH2 having an identity (more preferably 98% or more, and even more preferably 99% or more) with said amino acid sequence CH1, or an amino acid sequence CH3 which is a humanized sequence of said amino acid sequence CH1; 72, an amino acid sequence CL2 having an identity of 85% or more (preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more) to the amino acid sequence CL1, or It is preferred that the humanized sequence of sequence CL1 is a light chain variable region comprising an amino acid sequence CL3.

[0046] The antibody or antigen-binding fragment thereof of (D) has an amino acid sequence DH1 shown in SEQ ID NO: 73, and has a binding affinity of 85% or more (preferably 90% or more, more preferably 95% or more, further preferably 10% or more, more preferably 15% or more, more ... a heavy chain variable region comprising an amino acid sequence DH2 having an identity (more preferably 98% or more, and even more preferably 99% or more) with said amino acid sequence DH1, or an amino acid sequence DH3 which is a humanized sequence of said amino acid sequence DH1; 74, an amino acid sequence DL2 having an identity of 85% or more (preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more) to said amino acid sequence DL1, or It is preferred that the humanized sequence of sequence DL1 is a light chain variable region comprising the amino acid sequence DL3.

[0047] The antibody (E) or the antigen-binding fragment thereof has an amino acid sequence EH1 shown in SEQ ID NO: 75, and has a sequence identity that is 85% or more (preferably 90% or more, more preferably 95% or more, further preferably 10% or more) of the amino acid sequence EH1. a heavy chain variable region comprising an amino acid sequence EH2 having an identity (more preferably 98% or more, and even more preferably 99% or more) with said amino acid sequence EH1, or an amino acid sequence EH3 which is a humanized sequence of said amino acid sequence EH1. 76, an amino acid sequence EL2 having an identity of 85% or more (preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more) to said amino acid sequence EL1, or It is preferred that the antibody comprises a light chain variable region comprising the amino acid sequence EL3, which is a humanized version of sequence EL1.

[0048] The term "antibody" is used to encompass monoclonal and polyclonal antibodies. The antibody can be of any isotype, such as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, IgM, etc. The antibody may be a human antibody or a non-human antibody. Examples of non-human antibodies include, but are not limited to, mouse antibodies, rat antibodies, rabbit antibodies, monkey antibodies, chimpanzee antibodies, etc. The antibody may be a mouse-human antibody. The antibody may be a chimeric antibody, such as a humanized antibody. The antibody may be a partially or fully humanized antibody. The antibody of the present invention is preferably a monoclonal antibody.

[0049] In the present specification, the antigen-binding fragment of an antibody is not particularly limited as long as it contains heavy chain CDR1 to 3 and light chain CDR1 to 3, and may be, for example, Fab, F(ab')2, minibody, scFv-Fc, Fv , scFv, diabody, triabody, tetrabody, and the like.

[0050] Fab comprises a heavy chain fragment containing a heavy chain variable region and CH1 in the heavy chain constant region, and a light chain containing a light chain variable region and a light chain constant region (CL), and has a structure in which the heavy chain variable region and the light chain variable region are associated with each other through the above-mentioned non-covalent intermolecular interaction or are bonded by a disulfide bond. In Fab, CH1 and CL may be disulfide-bonded by the thiol groups of cysteine ​​residues present in each.

[0051] F(ab')2 is a peptide having two pairs of the above-mentioned Fab, in which the CH1s are linked by a cysteine ​​residue bond. It has a structure formed by disulfide bonds between all groups.

[0052] A minibody is a combination of two fragments of the heavy chain variable region of the scFV shown below, each of which is bound to CH3. The structure is held together by non-covalent intermolecular interactions.

[0053] scFv-Fc is a combination of two antibody fragments, including the following scFv, CH2, and CH3, and the same structure as the above minibody. Similarly, the CH3s associate with each other through non-covalent intermolecular interactions, and the thiol groups of the cysteine ​​residues contained in each CH3 are disulfide-bonded.

[0054] Fv is also called the minimum structural unit of an antibody, and is a structure in which a heavy chain variable region and a light chain variable region are associated with each other through non-covalent intermolecular interactions. In Fv, the thiol groups of cysteine ​​residues present in the heavy chain variable region and the light chain variable region may be disulfide-bonded.

[0055] An scFv has a structure in which the C-terminus of a heavy chain variable region and the N-terminus of a light chain variable region are linked by a linker, or a structure in which the N-terminus of a heavy chain variable region and the C-terminus of a light chain variable region are linked by a linker, and is also called a single-chain antibody.

[0056] Diabodies, triabodies, and tetrabodies are structures in which the above scFv forms a dimer, trimer, and tetramer, respectively, and have non-covalent intermolecular interactions between the variable regions, similar to Fv. This structure is formed by associating with other molecules in a structurally stable state.

[0057] The antibody of the present invention or a fragment thereof may be bound or fused to another peptide, oligopeptide, or protein. Examples of other peptides, oligopeptides, or proteins include albumin (e.g., serum albumin), protein tags (e.g., biotin, His tag, FLAG tag), and the like. tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag), fluorescent protein (e.g. GFP , Azami-Green, ZsGreen, GFP2, HyPer, Sirius, BFP, CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, Jred , KillerRed, Cherry, HcRed, mPlum), photoproteins (e.g. luciferase, β-galactosidase, tosidase, chloramphenicol acetyltransferase, β-glucuronidase) , secretion signal sequence (e.g., Igκ signal sequence), protease recognition sequence (e.g., TEV protease recognition sequence), expression enhancing sequence, solubility sequence, multimerization domain (e.g., cartilage oligomeric matrix protein domain, leucine zipper domain, collagen-like domain, cholera toxin B subunit domain, tetrabrachion coiled core domain, reovirus Examples of such antibodies include hepatitis C σ1 protein domain, Hepatitis C delta antigen domain, etc. When the antibody or fragment thereof of the present invention is bound to or fused with a multimerization domain, an antibody or an antigen-binding fragment thereof of the same or different species as the antibody or fragment thereof of the present invention may be further bound to or fused to the multimerization domain to form a multimer (homomultimer or heteromultimer).

[0058] The antibody or fragment thereof of the present invention may be chemically modified so long as its binding ability to human periostin is not significantly impaired.

[0059] The antibody or fragment thereof of the present invention has a carboxyl group (-COOH) at the C-terminus, a carboxylate group (-COOH) at the C-terminus, (-COO - ), an amide group (-CONH2), or an ester group (-COOQ). Here, Q in the ester group is, for example, C such as methyl, ethyl, n-propyl, isopropyl, and n-butyl. 1-6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3-8Cycloalkyl groups; for example, C phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl group; α-naphthyl α-Naphthyl-C such as methyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0060] The antibody or fragment thereof of the present invention has a carboxyl group (or carboxylate group) other than the C-terminus. ) may be amidated or esterified. In this case, the ester may be, for example, the above-mentioned C-terminal ester.

[0061] In the antibody or fragment thereof of the present invention, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., C such as ruthenium and acetyl groups 1-6 Alkanoyl C 1-6 acyl group, etc.), and the N-terminal glutamine residue that can be cleaved in vivo to produce pyroglutamine Oxidized, the substituents on the side chains of the amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by suitable protecting groups (e.g., C groups such as formyl group, acetyl group, etc.). 1-6 Alkanoyl group etc. 1-6 Also included are those protected by an acyl group, or conjugated proteins such as so-called glycoproteins to which a sugar chain is bound.

[0062] 2. Method for producing an antibody or an antigen-binding fragment thereof The antibodies or fragments thereof of the present invention can be produced in a variety of ways.

[0063] The antibody or fragment thereof of the present invention may, for example, (e) a protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 65, and (f) a protein or peptide comprising an amino acid sequence X having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 65, and comprising within said amino acid sequence X an amino acid sequence A shown in SEQ ID NO: 61 and an amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64. At least one protein or peptide selected from the group consisting of The antibody can be produced by a method comprising a step of selecting an antibody that binds to human periostin secreted from human breast cancer cells from antibodies produced by the selected hybridoma.

[0064] In this respect, in one embodiment of the present invention, the antibody or fragment thereof of the present invention comprises: (e) a protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 65, and (f) a protein or peptide comprising an amino acid sequence X having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 65, and comprising within said amino acid sequence X an amino acid sequence A shown in SEQ ID NO: 61 and an amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64. At least one protein or peptide selected from the group consisting of The antibody may be an antibody obtained by a method comprising a step of selecting an antibody that binds to human periostin secreted from human breast cancer cells from antibodies produced by the selected hybridoma.

[0065] In view of the above, in one aspect of the present invention, the antibody or fragment thereof of the present invention comprises: (e) a protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 65, and (f) a protein or peptide comprising an amino acid sequence X having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 65, and comprising within said amino acid sequence X an amino acid sequence A shown in SEQ ID NO: 61 and an amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64. At least one protein or peptide selected from the group consisting of The antibody may be an antibody produced from a hybridoma isolated from the human breast cancer cell line and having binding activity to human periostin secreted from human breast cancer cells.

[0066] The above proteins and peptides (e) and (f) are similar to the above proteins (a) and (b).

[0067] The animal to be immunized is not particularly limited as long as it is an animal from which a hybridoma can be prepared, but preferably includes an animal with an autoimmune disease (in the case of mice, for example, BXSB mice). Immunization and preparation of hybridomas can be performed according to known methods (for example, Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Sections 11.4 to 11.11). The method for evaluating the selection of an antibody that binds to human periostin secreted from human breast cancer cells is not particularly limited as long as it is a method that utilizes an antigen-antibody reaction using human periostin secreted from human breast cancer cells, but for example, the method described in Test Example 2 described later can be adopted.

[0068] In the present specification, the binding affinity of an antibody or a fragment thereof to a target (e.g., human periostin secreted from human breast cancer cells) is measured by a surface plasmon resonance (SRP) method. In one embodiment of the present invention, the dissociation constant (KD value) between the antibody or fragment thereof of the present invention and a target (e.g., human periostin secreted from human breast cancer cells) can be measured by, for example, 9×10 -6 M or less, preferably 9 x 10 -8 M or less, preferably 9×10 -10 The dissociation constant is less than M. The lower limit of the number is not particularly limited, but is, for example, 1×10 -14 M, 1×10 -13 M, 1×10 -12 M, 1×10 -11 It can be M.

[0069] As another example, the antibody of the present invention or a fragment thereof can be produced by a method comprising the steps of culturing a host transformed with a polynucleotide comprising the coding sequence of the antibody of the present invention or a fragment thereof (the polynucleotide of the present invention) and recovering a fraction comprising the antibody of the present invention.

[0070] The polynucleotide of the present invention is not particularly limited as long as it contains the coding sequence of the antibody of the present invention or a fragment thereof. Preferably, the polynucleotide of the present invention contains the above coding sequence in a state capable of expressing the antibody of the present invention or a fragment thereof. The polynucleotide of the present invention may contain other sequences in addition to the above coding sequence. Examples of such other sequences include a secretory signal peptide coding sequence, a promoter sequence, an enhancer sequence, and the like, which are located adjacent to the coding sequence of the antibody of the present invention. Examples of the polynucleotide include a sequence, a repressor sequence, an insulator sequence, an origin of replication, and a drug resistance gene coding sequence. The polynucleotide of the present invention may be a linear polynucleotide or a circular polynucleotide (such as a vector).

[0071] Specific examples of the polynucleotide of the present invention include: (I) the heavy chain and heavy chain variable regions of the antibody of the present invention; (II) a polynucleotide comprising a base sequence encoding at least one selected from the group consisting of the light chain, the light chain variable region, and the light chain CDRs 1-3 of the antibody of the present invention; A polynucleotide comprising a base sequence encoding at least one selected from the group consisting of (III) a nucleic acid comprising a base sequence encoding at least one selected from the group consisting of the heavy chain, the heavy chain variable region, and the heavy chain CDRs 1-3 of the antibody of the present invention, and a light chain of the antibody of the present invention, Examples of such polynucleotides include a polynucleotide comprising a base sequence encoding at least one selected from the group consisting of a light chain variable region and light chain CDRs 1-3.

[0072] The host is not particularly limited, and examples thereof include insect cells, eukaryotic cells, mammalian cells, etc. Among these, mammalian cells such as HEK cells, CHO cells, NS0 cells, and SP2 / O cells are preferred from the viewpoint of more efficient expression of the antibody.

[0073] The methods for transformation, culture, and recovery are not particularly limited, and known methods for antibody production can be used.

[0074] After recovery, the antibody of the present invention may be purified, if necessary, by known methods for antibody production, such as chromatography and dialysis.

[0075] Polynucleotides such as DNA and RNA may be chemically modified as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide may be replaced with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be replaced with -OR (R represents, for example, -CH3, -CH2CH2OCH3, -CH2CH2NHC(NH)NH2, -CH2CONHCH3, -CH2CH2CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by replacing the carbonyl group at the 2-position with a thiocarbonyl. Further examples include those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc., but are not limited to these.

[0076] 2-1. Creation of human antibodies When obtaining the antibody of the present invention as a human antibody, it can be produced based on the information below, and, if necessary, in accordance with or in accordance with other known information.

[0077] Immunoglobulin G (hereinafter referred to simply as "IgG") is a light polypeptide with a molecular weight of about 23,000. a light chain having a molecular weight of about 50,000 (hereinafter referred to as the heavy chain); Both the heavy and light chains have a repeating structure of a region with a conserved amino acid sequence consisting of approximately 110 residues, which constitute the basic unit of the three-dimensional structure of IgG (hereinafter referred to as "domain"). The heavy chain and light chain are composed of four consecutive domains and two consecutive domains, respectively. In both the heavy chain and the light chain, the amino-terminal domain has greater variation in amino acid sequence between antibody molecules than the other domains, and this domain is called the variable domain (hereinafter referred to as "V domain"). At the amino terminal of IgG, the V domains of the heavy and light chains associate in a complementary manner to form the variable region. In contrast, The remaining domains as a whole form a constant region. The constant region has a sequence characteristic of each animal species. For example, the constant region of mouse IgG is different from the constant region of human IgG. Mouse IgG is recognized as a foreign substance by the human immune system, resulting in a human anti-mouse antibody (HAMA) response (Schroff RW. et al Cancer Res. (1985) 45, 879-85). Therefore, repeated administration of mouse antibodies to humans is not effective. No. In order to administer such antibodies to humans, it is necessary to modify the antibody molecule so that it does not induce a HAMA response while retaining the antibody's specificity.

[0078] X-ray crystallography has shown that such domains generally consist of three to five β-strands. The variable region has a long cylindrical structure in which two layers of antiparallel β-sheets consisting of β-sheets are stacked. In the variable region, three loops are assembled for each of the V domains of the heavy chain and the light chain to form the antigen-binding site. Each of these loops is called a complementarity determining region (CDR). The amino acid sequence of the variable region is called the nucleotide sequence. Generally, it plays a role in maintaining the structure of CDR and is called "framework". A large number of primary sequences of heavy and light chain variable regions were collected, and a table was created in which each primary sequence was classified into CDR and framework based on sequence conservation (Kabatt et al. SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No.91-3242, EA).

[0079] In addition, each framework was classified into several subgroups with common amino acid sequence characteristics. Furthermore, it was found that there are corresponding frameworks between humans and mice. Based on such research on the structural characteristics of IgG, the following method for producing humanized antibodies was considered. In the early stages of research, chimeric antibodies were proposed in which the variable regions of a mouse-derived antibody were joined to the constant regions of human origin (Morrison SL. et al Proc Natl Acad Sci U S A. (1984)81,6851-5). However, such chimeric antibodies still contain many non-human amino acid residues and may induce HAMA responses, especially when administered for a long period of time (Begent et al., Br. J. Cancer, (1990)62, 487).

[0080] As a method for further reducing the number of amino acid residues derived from non-human mammals that may induce HAMA responses in humans, a method for incorporating only the CDR portion into an antibody derived from a human has been proposed (Peter T et al. Nature, (1986) 321, 522-5). Grafting only the CDRs was insufficient to maintain activity. Using X-ray crystal structure analysis data, (a) the amino acid sequence of the CDR contains a molecule that directly binds to the antigen. (b) There are regions that maintain the structure of the CDR itself and regions that maintain the structure of the CDR itself, and the three-dimensional structures that the CDR can take are classified into several typical patterns (canonical structures). It is determined by the types of amino acids at specific positions in the framework as well as in the CDRs. (Chothia C. et al. J. Mol. Biol. (1987)196, 901-17). Based on these findings, it has been suggested that when using CDR grafting, it is necessary to graft some framework amino acid residues as well as the CDR sequences onto a human antibody (Patent Publication No. 4-502408).

[0081] In general, an antibody derived from a non-human mammal having a CDR to be grafted is defined as a "donor," and a human antibody to which the CDR is grafted is defined as an "acceptor." The aim is to preserve the CDR structure as much as possible and to maintain the activity of the immunoglobulin molecule. To this end, (a) the acceptor must be a member of any of the subgroups. and (b) which amino acid residues should be removed from the donor framework. There are two points to keep in mind:

[0082] Queen et al. proposed a design method in which amino acid residues in the framework of a donor are grafted onto an acceptor together with CDR sequences if they meet at least one of the following criteria: (Special Patent Publication No. 4-502408): (a) the amino acid in the framework region of the acceptor is rare at that position, the corresponding amino acid of the donor is normal at said position of the acceptor (b) the amino acid is in close proximity to one of the CDRs; (c) the amino acid is located within about 3 Å (angstroms) of the CDR in a three-dimensional immunoglobulin model; ) and capable of interacting with the antigen or with the CDR of the humanized antibody. It is expected that.

[0083] 2-2. Generation of human antibodies When obtaining the antibody of the present invention as a human antibody, it can be produced based on the information below, and, if necessary, in accordance with or in accordance with other known information.

[0084] In the present invention, a "human antibody" or "human immunoglobulin" refers to an immunoglobulin in which all regions constituting the immunoglobulin, including the variable region of the H chain (VH) and the constant region of the H chain (CH) and the variable region of the L chain (VL) and the constant region of the L chain (CL), are derived from genes encoding human immunoglobulins. In other words, the H chain is derived from a human immunoglobulin heavy chain gene, It refers to an antibody whose light chain is derived from a human immunoglobulin light chain gene.

[0085] Human antibodies can be produced in the same manner as monoclonal antibodies, for example by immunizing a transgenic animal, which is produced by incorporating at least a human immunoglobulin gene into the gene locus of a non-human mammal such as a mouse, with an antigen. For example, a transgenic mouse producing human antibodies can be produced according to the methods previously reported (Mendez MJ et al. Nature Genetics (1997) 15, 146-56, Green LL et al. Nature Genetics (1994) 7, 13-21, Table 4-504365; International Patent Publication WO94 / 25585; Nikkei Science, June issue, pp. 40-50, 1995; Nils Lonberg et al. Nature (1994) 368, 856-9, and JP-T-6-500233).

[0086] 3.Cells In one aspect, the present invention relates to a cell containing the polynucleotide of the present invention.

[0087] Such cells include, for example, cells transfected with a polynucleotide of the invention.

[0088] The cells are preferably isolated cells. The cells may be cultured cells, and may be primary cultured cells or subcultured cells. The cells may be established cell lines. The cells may be iPS cells.

[0089] Examples of such cells include Escherichia coli K12 and other Escherichia coli, Bacillus subtilis MI114, Bacillus bacteria such as Saccharomyces cerevisiae AH22 and yeast such as Spodoptera frugiperda Examples of the animal cells include the Sf cell line derived from Trichoplusia ni or the HighFive cell line derived from Trichoplusia ni, insect cells such as olfactory nerve cells, and animal cells. The animal cells are preferably cultured cells derived from mammals, specifically, COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, etc. Examples of cells include N1E-115 cells and SH-SY5Y cells.

[0090] 4. Complex In one aspect, the present invention relates to a conjugate of the antibody of the present invention or a fragment thereof and a drug.

[0091] There are no particular limitations on the drug, so long as it has physiological activity, and examples of the drug include proteins, peptides, low molecular weight compounds, nucleic acids, and sugar chains.

[0092] The method for conjugating the antibody of the present invention or a fragment thereof with a drug is not particularly limited, and any known method can be used.

[0093] The complex of the present invention is formed by binding the antibody of the present invention to a drug directly or indirectly via a linker or the like. The mode of binding is not particularly limited, and examples include covalent bonds, coordinate bonds, ionic bonds, etc. The antibody of the present invention and a drug can be bound according to or in accordance with a known method depending on the mode of binding.

[0094] Covalent binding can be achieved, for example, by reacting functional groups possessed by the antibody of the present invention and the drug, or functional groups introduced as necessary. Examples of combinations of functional groups include amino and carboxyl groups, carboxyl and hydroxyl groups, maleimide and thiol groups, thiol and thiol groups, hydrazide and ketone groups, hydrazide and aldehyde groups, amino and aldehyde groups, thiol and carboxyl groups, amino and squaric acid derivatives, dienylaldehyde and amino groups, haloester and thiol groups, azide and alkyne, etc.

[0095] 5.Applications In one aspect, the present invention relates to a pharmaceutical composition, a reagent, etc., comprising at least one selected from the group consisting of the antibody of the present invention or a fragment thereof, and the conjugate of the present invention.

[0096] The pharmaceutical composition of the present invention can be suitably used for preventing or treating cancer, inhibiting vascular mimicry of cancer, inhibiting periostin, preventing or treating inflammation-related diseases, inhibiting vascular intimal hyperplasia, inhibiting angiogenesis, or preventing or treating aneurysm.

[0097] In one embodiment of the present invention, the antibody or fragment thereof of the present invention, or the complex of the present invention is The periostin-binding protein can be used to prevent or treat an inflammatory-related disease in which periostin having binding activity is involved.

[0098] An inflammatory-related disease associated with periostin having cell adhesion activity is a disease in which expression of the gene encoding periostin having cell adhesion activity is high during the pathological condition of the disease and production of the protein encoded by the gene increases, or the pathological condition of the disease worsens due to an increase in the gene or protein.

[0099] Inflammation-associated diseases having such cell adhesion activity include, but are not limited to, diseases caused mainly by vascular intimal thickening, cancer, and other inflammation-associated diseases. Diseases caused mainly by vascular intimal thickening include arteriosclerosis and restenosis caused mainly by vascular intimal thickening after coronary angioplasty.

[0100] Furthermore, examples of cancers to which the antibody or fragment thereof of the present invention, and the complex of the present invention can be applied include, but are not limited to, brain cancer, leukemia, osteosarcoma, breast cancer, colon cancer, malignant melanoma, bone cancer, stomach cancer, lung cancer, liver cancer, kidney cancer, pancreatic cancer, gallbladder cancer, skin cancer, uterine cancer, ovarian cancer, rectal cancer, colon cancer, fallopian tube cancer, esophageal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, prostate cancer, bladder cancer, malignant lymphoma, and the like, with breast cancer, colon cancer, lung cancer, and malignant melanoma being particularly preferred.

[0101] Other inflammation-related diseases include autoimmune arthritis, atopic dermatitis, asthma, emphysema, Behcet's disease, multiple sclerosis, spinocerebellar degeneration, uveitis, Guillain-Barre syndrome, Fisher syndrome, chronic inflammatory demyelinating polyneuropathy, polymyositis, scleroderma, autoimmune hepatitis, sarcoidosis, chronic pancreatitis, inflammatory bowel disease, Crohn's disease, solid tumors, multiple myeloma, angiofibroma, atherosclerosis, arteriovenous malformation, granulation, hemangioma, hypertrophic scar, keloid, premature aging, psoriasis, febrile granuloma, warts, hemorrhagic joints, non-union fractures, rheumatoid arthritis (e.g., malignant rheumatoid arthritis), osteoarthritis, follicular cysts, ovarian hypertrophy syndrome, polycystic ovary, age-related macular degeneration, diabetic retinopathy, neovascular glaucoma, trachoma, emphysema, chronic bronchitis, obesity, dental These include periodontal disease, corneal graft angiogenesis, aneurysms, etc. Angiogenesis is involved in many of the inflammation-related diseases listed above.

[0102] Another aspect of the present invention includes a diagnostic agent for an inflammatory-related disease in which periostin having cell adhesion activity is involved, prepared by labeling the above antibody with a marker. The marker is not particularly limited, and an enzyme, a radioisotope, a fluorescent dye, or the like can be used. The enzyme used here is not particularly limited as long as it has a large turnover number, is stable even when bound to the enzyme, and can react specifically with a substrate to develop color, and the like, and enzymes used in ordinary enzyme immunoassays (EIA) can be used. Preferred examples of the enzyme include periostin. oxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase Enzymes, etc. Enzyme inhibitors, coenzymes, etc. can also be used.

[0103] The binding of these enzymes to antibodies can be carried out by known methods using known crosslinking agents such as maleimide compounds. Known substances can be used as substrates depending on the enzyme used. For example, when peroxidase is used as the enzyme, 3,3',5,5'-tetramethylbenzidine can be used, and when alkaline phosphatase is used as the enzyme, paranitrophenol or the like can be used. Radioisotopes used as markers include those used in ordinary radioimmunoassays (RIA), such as 125I and 3H. As the fluorescent dye, conventional fluorescent antibodies such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) can be used. The diagnostic agent can be used as an immunohistochemical stain that can specifically stain cancer cells and surrounding fibroblasts. When labeled with a radioisotope, it can be administered into the body to image lesions such as cancer.

[0104] One embodiment of the present invention is a method for the preparation of a medicament for the treatment of a cancer by using the antibody or fragment thereof of the present invention or the complex of the present invention. The present invention provides a method for detecting or quantifying periostin having cell adhesive activity in a biological sample obtained by preparing serum from human or animal blood, i.e., in serum, and further provides a method for diagnosing an inflammatory-related disease, such as heart failure, in which periostin having cell adhesive activity is involved, by detecting or quantifying periostin having cell adhesive activity. In this method, periostin having cell adhesive activity is detected by a so-called sandwich ELISA (Enzyme-linked immunosorbent assay). When the diagnostic kit of the present invention is used, a sample is first brought into contact with a plate on which an anti-periostin primary antibody is immobilized to bind the two, a marker-labeled anti-periostin secondary antibody is bound to the bound antibody, and the signal intensity of the marker in the bound antibody of the three is measured to detect or quantitate periostin having cell adhesive activity. In particular, periostin having cell adhesive activity is a splicing variant that is specifically expressed in pathological conditions such as cancer, and therefore pathological conditions such as cancer can be diagnosed by monitoring periostin production.

[0105] In this way, the antibody of the present invention can be labeled and used as a secondary antibody.

[0106] In one embodiment of the present invention, there is provided a method for producing a medicament comprising the antibody or fragment thereof of the present invention, or the complex of the present invention. An example of such a pharmaceutical composition is a pharmaceutical composition for inhibiting periostin having cell adhesion activity, which contains as an active ingredient an anti-periostin antibody that recognizes a splicing variant of periostin having cell adhesion activity. The antibody, antibody fragment, and / or derivative has inhibitory activity against periostin having cell adhesion activity, and as a result, the pharmaceutical composition can suppress angiogenesis, suppress vascular intimal thickening, treat cancer, and prevent or treat aneurysm. That is, the present invention relates to a pharmaceutical composition for inhibiting vascular intimal thickening, treating cancer, suppressing angiogenesis, preventing aneurysm, and treating aneurysm, which comprises an anti-periostin antibody that recognizes a splicing variant of periostin having cell adhesion activity. The composition of the present invention can be used for treating and preventing restenosis caused primarily by vascular intimal thickening following coronary angioplasty, preventing or treating cancer, treating or preventing diseases involving angiogenesis, and preventing or treating aneurysms.

[0107] In one embodiment of the present invention, the antibody, antibody fragment and / or derivative of the present invention is used as an effective For example, in one embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory-related disease in which periostin having cell adhesion activity is involved, the pharmaceutical composition comprising the antibody, the anti- Examples of such compositions include a pharmaceutical composition for inhibiting vascular intimal thickening, which contains the antibody, antibody fragment and / or antibody derivative as an active ingredient; a pharmaceutical composition for treating cancer, which contains the antibody, antibody fragment and / or antibody derivative as an active ingredient; a pharmaceutical composition for inhibiting angiogenesis, which contains the antibody, antibody fragment and / or antibody derivative as an active ingredient; and a pharmaceutical composition for preventing or treating aneurysms, which contains the antibody, antibody fragment and / or antibody derivative as an active ingredient.

[0108] The composition for cancer treatment of the present invention has an effect of suppressing the growth of cancer pathogenic foci and an effect of suppressing the metastasis of cancer, and therefore can be used in the hope of suppressing the growth of cancer pathogenic foci, or in the hope of suppressing the metastasis of cancer, or even in the hope of both.

[0109] Pharmaceutical compositions containing the antibody or fragment thereof of the present invention, or the conjugate of the present invention as an active ingredient are prepared using known pharmacologically acceptable carriers, excipients, and other additives that are used in conventional formulations.

[0110] The active ingredient of the pharmaceutical composition according to the present invention is preferably mixed with a known pharmacologically acceptable carrier, excipient, diluent, etc. and administered by an administration method generally used for medicines (for example, oral administration method, or parenteral administration method such as intravenous administration, intramuscular administration or subcutaneous administration). The pharmaceutical composition of the present invention can be produced, for example, by appropriately mixing the active ingredient with a pharmacologically acceptable carrier, flavoring agent, excipient, stabilizer, diluent, emulsion, solution, suspension, syrup, etc. The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include tablets, powders, granules, solutions, etc. Examples of additives that can be mixed with tablets, etc. include binders such as gelatin, and lubricants such as cornstarch. The pharmaceutical composition may also be coated with a sugar coating or a film of a gastrosoluble or enteric substance. In the case of a capsule dosage form, the composition may further contain a liquid carrier. Sterile compositions for injection can also be produced by applying a normal pharmaceutical formulation. Examples of aqueous solutions for injection include isotonic solutions containing glucose, etc., which may be used in combination with appropriate solubilizing agents such as polyethylene glycol. The pharmaceutical composition may also be mixed with buffers, stabilizers, preservatives, antioxidants, soothing agents, etc. In oral administration, when the active ingredient is easily decomposed in the digestive tract, it is also possible to orally administer the active ingredient in the form of a preparation that is not easily decomposed in the digestive tract (for example, a microcapsule in which the active ingredient is encapsulated in liposomes). In addition, administration methods that allow absorption through mucous membranes other than the digestive tract, such as rectum, nasal cavity, sublingual, and lungs, are also possible. In this case, the active ingredient can be administered in the form of a suppository, nasal drops, sublingual, or pulmonary preparation.

[0111] When the pharmaceutical composition of the present invention is used for treatment, the dosage is determined to be an effective dosage for treatment, but the dosage varies depending on the age, weight, severity of symptoms, and route of administration of the subject, and is determined for each individual case. In general, when administered orally, the daily dosage for an adult is about 0.1 to 1000 mg, which may be administered once or in divided doses (2 or 3 times, etc.). For subsequent intravenous administration, the dose should be in the range of 0.01μg / kg / min to 1.0μg / kg / min. It is possible to administer this drug at a rate of 0.025μg / kg / min to 0.1μg / kg / min.

[0112] As one embodiment of the present invention, there is provided a method for detecting cell adhesion using a periostin antibody. "Method of inhibiting periostin having a therapeutically effective amount of cell adhesion activity" One embodiment of the present invention includes a "method for preventing or treating an inflammatory-associated disease in which periostin having cell adhesion activity is involved, comprising administering to a cancer patient an anti-periostin antibody that recognizes a splicing variant of periostin." "Use of a periostin antibody for producing a pharmaceutical composition for inhibiting periostin" and "Use of a periostin antibody for producing a pharmaceutical composition for preventing or treating an inflammatory-associated disease in which periostin having cell adhesion activity is involved." Examples of the anti-periostin antibody include the above-mentioned antibodies, antibody fragments, and / or antibody derivatives. Examples of the inflammatory-associated disease include the above-mentioned inflammatory-associated diseases.

[0113] The reagent of the present invention may be in the form of a composition containing at least one selected from the group consisting of the antibody or fragment thereof of the present invention and the complex of the present invention. Other ingredients may be included as necessary, such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, and chelating agents.

[0114] The reagent of the present invention may be in the form of a kit containing at least one selected from the group consisting of the antibody of the present invention and the complex of the present invention. The kit includes: It may also include instruments, reagents, etc. that can be used to carry out separation, etc. Examples of such instruments and reagents include test tubes, microtiter plates, agarose particles, latex particles, purification columns, labeled antibodies, standard samples (positive controls, negative controls), etc. EXAMPLES

[0115] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0116] Test Example 1. Preparation of monoclonal antibodies against human periostin (1) Preparation of antigen A peptide with a Cys residue added to the N-terminus of the amino acid sequence constituting human periostin exon 21 (SEQ ID NO: 65: EVTKVTKFIEGGDGHLFEDEEIKRLLQG) was chemically synthesized by the Fmoc method to obtain 10 mg of an antigen peptide with a purity of 90% or more. 5 mg of this antigen peptide was bound to 5 mg of KLH (CALBIOCHEM) as a carrier protein to obtain an antigen solution. That is, KLH was dissolved in PBS (0.01 M) to adjust the concentration to 3.3 mg / mL, and a 0.2524 mg / mL MBS solution (GE Healthcare Biosciences) was added dropwise and stirred at room temperature for 60 minutes to react. Free MBS was removed with dichloromethane. KLH-MB was obtained by dissolving 5 mg of this KLH-MB in 0.01 M sodium phosphate buffer (pH 7.2). The mixture was mixed with 5 mg of the antigen peptide and reacted at 4° C. for 12 hours with stirring to obtain an antigen solution.

[0117] (2) Immunity Three 6-week-old BALB / c female mice were inoculated with 100 μg of the KLH-bound antigen peptide obtained in (1) into both legs. The total volume of the mixed emulsion of 50 μl of antigen solution containing and 50 μl of FCA (Freund's complete adjuvant) The antigen solution and FIA (Freund's solution) were then subcutaneously injected twice at 2-week intervals. The mice were then sacrificed by cervical dislocation, and the lymph nodes were aseptically collected.

[0118] The lymph nodes were crushed while supplying RPMI medium (Kohjin Bio Co., Ltd.), and passed through a mesh with a pore size of approximately 10 μm to obtain lymph node cells suspended in RPMI medium. The lymph node cells were centrifuged at 1000 rpm for 10 minutes to obtain a precipitated fraction of lymph node cells. The precipitated fraction was dissolved by adding 1 ml of a solution of 0.84% ​​ammonium chloride solution and 20 mM HEPES buffer (pH 7.4). After removing red blood cells, the mixture was centrifuged at 1,000 rpm for 5 minutes. The cells (cell fraction) were washed several times with RPMI medium and used for cell fusion.

[0119] (3) Preparation of myeloma cells 8-Azaguanine-resistant and non-immunoglobulin-secreting mouse myeloma cell line P3X63Ag8U.1 (P3U1 line) was cultured in RPMI medium containing 20% ​​fetal calf serum (FCS) in a 10% CO2 / 37°C incubator, and cells in the logarithmic growth phase were collected and centrifuged at 1,000 rpm for 5 minutes. The cells alone were collected as a precipitate fraction by subjecting them to a culture medium, and suspended in RPMI medium.

[0120] (4) Cell fusion 1 × 10 immunized lymph node cells obtained in (2) 8 ~3×10 8 RPMI medium containing 10 cells of the myeloma cells obtained in (3) 8 After mixing with RPMI medium containing 1,000 rpm, the mixture was centrifuged for 10 minutes. The supernatant was gently removed to obtain the cells as a precipitate fraction, to which 1 ml of 25% (w / v) polyethylene glycol 1500 (PEG 1500, Boehringer) was added, and then RPMI medium was gently added. To this was added 10 ml of RPMI medium containing 20% ​​FCS, and after leaving it to stand for a while, it was centrifuged at 1,000 rpm for 5 minutes, and the resulting precipitated fraction (cell fraction) was diluted with 20% FCS. Add RPMI medium containing 10 mL of 10% ethanol to the cell 6The cell suspension was adjusted to 100 cells / ml and then After culturing for 24 hours in a 5% CO2 / 37°C incubator, HAT solution (Invitrogen) was added and the cells were further cultured for 2 weeks.

[0121] (5) Screening by ELISA Screening of positive wells in which the culture supernatant reacted with the antigen peptide was performed. The antigen solution used for the assay was a conjugate of 2 mg of the antigen peptide obtained in (1), human periostin PN1 (full length) or mouse periostin PN2 (exon 17 deleted), bound to ovalbumin (OVA) as a carrier protein.

[0122] Each well of a 96-well microtiter plate (Falcon 353912) was coated with 1 μg / ml of the above conjugate by leaving it to stand overnight at 4° C. After washing the plate, 50 μl of the culture supernatant (containing the monoclonal antibody) from (4) was dropped into each well and incubated in an incubator at 37° C. After leaving the plate in a 37°C incubator for 2 hours, it was washed with PBS(-) (phosphate buffer solution). Alkaline phosphatase-conjugated sheep anti-mouse IgG antibody (Zymed) was added to the plate, and the plate was left in the incubator at 37°C for 1 hour. After washing with PBS(-), a color-developing substrate (ALP) was added to develop color for 20 minutes, and the absorbance (antibody titer) at OD490 nm of each well was measured using a plate reader (BIO-RAD, Model 680 MICRO PLATE READER) to confirm reactivity with the antigen peptide, and positive wells in which the culture supernatant reacted with the antigen peptide were determined.

[0123] (6) Cloning of antibody-producing cells From the cells in the positive wells in which reactivity with the antigen peptide was confirmed by the ELISA method in (5), antibody-producing cell lines were cloned by the limiting dilution method. That is, the cells in the positive wells were seeded into each well of a 96-well culture plate and cultured for 2 weeks in a 5% CO2 / 37°C incubator. The culture supernatant from each well was examined for reactivity with the antigen peptide, human periostin PN1 (full length) or mouse periostin PN2 (exon 17 deleted) by ELISA in the same manner as in (5), and positive wells were cloned again by limiting dilution to obtain cells with high reactivity with the antigen peptide and good cell colony growth. These cells were transferred to a 24-well culture plate and cultured for 2 weeks in a 5% CO2 / 37°C incubator. From the above, cells in 5 wells, i.e., 5 hybridoma cell lines (A, B, C, D, E), were selected. The selection was carried out by an immunoprecipitation test according to Test Example 2 described below.

[0124] (7) Large-scale preparation and purification of monoclonal antibodies Pristane [2,6,10,14-tetramethylpentadecane ( The mice were then fed 0.5 ml of 100 ml of 100% ethanol (Hikari Junyaku Co., Ltd.) and kept for 2 to 3 weeks. The monoclonal antibody-producing hybridomas A to E were collected, and the cells in the precipitate fraction after removing the culture supernatant were Add RPMI medium without FCS and allow the cells to reach a density of 1 × 10 7 The cell suspension was prepared so that the number of cells was 1 / ml. The cell suspension was injected into the abdominal cavity of BALB / c mice that had been pre-administered with pristane, and the ascites that leaked out after about 3 weeks was collected from the abdomen with a syringe. The collected ascites was filtered through a filter with a pore size of 0.22 μm. After filtering, the filtrate was purified by affinity chromatography using a protein G-Sepharose column (Millipore, 11511324) according to a conventional method to prepare five types of anti-human periostin exon 21 monoclonal antibodies (antibodies A to E).

[0125] Test Example 2. Human periostin-exon 21 recognition test A binding test for human periostin secreted from human cells was carried out using monoclonal antibodies (antibodies A and B) obtained from hybridoma A and hybridoma B, respectively, and antibody #8, the production of which has been reported in Patent Document 1 (an antibody produced from antibody-producing cell line (hybridoma) No. 8 (internationally deposited with the National Institute of Technology and Evaluation Patent Microorganisms Depositary (NPMD) (deposit date: February 26, 2013, deposit number: NITE BP-01546, identification: KS-0259#8,080611 Kohjin Bio)). Specifically, the test was carried out as follows.

[0126] Human breast cancer cells Hs578t were cultured for 3 days in 0% FBS, and immunoprecipitation was performed using 1.0 ml of the supernatant. 5 μg each of antibody A, antibody B, antibody #8, and a control mouse IgG antibody were used as immunoprecipitation beads. After binding to the antibody (Invitrogen immunoprecipitation kit - Dynabeads Protein G cat #10007D), the antibody was incubated with the culture supernatant overnight at 4°C. Protein was extracted and periostin exon 12 antigen (periostin N-terminal antibody) was used as the primary antibody. Western blotting was performed using an antibody against exon 21 (H1N1), and binding of each exon 21 antibody to periostin was confirmed.

[0127] The results are shown in Figure 1. Antibody #8 was completely unable to recognize periostin exon 21 secreted from the supernatant of human breast cancer cells, whereas antibodies A and B were shown to strongly recognize the periostin exon 21 (Figure 1(A)). In addition, CBB staining was performed after Western blotting to confirm that there was no bias in the antibody dose in each lane (Figure 1(B)). Antibodies C to E We also performed a similar test on antibodies C to E, and found that all of them could recognize periostin exon 21 secreted from the supernatant of human breast cancer cells.

[0128] Test Example 3. Determination of CDR sequence of antibody From the genomic information of the hybridomas (A to E) cloned in Test Example 1, the nucleotide sequences of the antibodies A to E were determined. The amino acid sequences of the heavy chain variable regions of antibodies A to E ( Heavy chain variable region ) and Light chain variable region ( Light chain variable region The amino acid sequence of The sequence number is shown in parentheses at the end of the sequence.

[0129] < Antibody A> Heavy chain variable region KVQLVQSGAEVVKPGASVKLSCKASGYTFTEYIIHWVKQASGQGLEWIGWFYPGRGIIKYNEKFKDKATLTADKSISTVYMDLSRLRSEDTAVYFCARHGITTATGAMDYWGQGTLVTVSS (SEQ ID NO: 67) Light chain variable region DVVMTQTPLSLSVTLGQPASISCRSSQSLLHSNGNTYLHWYLQKPGQAPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPFTFGQGTKLEIK (sequence number 68).

[0130] < Antibody B> Heavy chain variable region EVQLVESGGGLVKPGGSLRLSCAASGITFSSYAMSWVRQAPGKGLEWVASISSGGSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVAIWDGSSYGYWGQGTTVTVSS (SEQ ID NO: 69) Light chain variable region DIVMTQTPLSLSVTPGQPASISCKSGQSLVHSNGKTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCSQSTHVPFTFGQGTKLEIK (sequence number 70).

[0131] < Antibody C> Heavy chain variable region EVQLVESGGGLVKPGGSLRLSCAASGFIFSSYAMSWVRQPPGKRLEWVASIGSGGTTYYPDSVRGRFTISRDNARNILYLQMRSLRAEDTAVYYCAAIWDGSSHGYWGQGTLLTVSS (SEQ ID NO: 71) Light chain variable region DVVMTQSPLSLPVTLGQPASISCRSSQNLVHSNGNTYLHWYLQRPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCSQSTHVPYTFGPGTKLEIK (sequence number 72).

[0132] < Antibody D> Heavy chain variable region EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQTPEKRLEWVASINSGGVTTYYPDSVKGRFTISRDNAINILYLQMRSLRSEDTALYYCAAIWDGSSHGYWGQGTLVTVSS (SEQ ID NO: 73) Light chain variable region DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYLQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDFGVYFCSQSTHVPYTFGPGTKLEIK (sequence number 74).

[0133] < Antibody E> Heavy chain variable region EVQLVESGGGLVQPGGSLKLSCAASGFTFSDAWMDWVRQAPGKGLEWVAEIRNKANNHATYFTESVKGRFTISRDDSKNTVYLQMNSLRAEDTAVYYCTTATFVYWGQGTLVTVSS (SEQ ID NO: 75) Light chain variable region DVVMTQSPLSLPVSLGDRASISCRSSQSLVRNGITYLHWYQQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISSVEAEDFGVYYCSQTPHVPYTFGQGTKLEIK (sequence number 76). The amino acid sequence was compared with the known antibody amino acid sequence database (IgBLAST website The amino acid sequences of the CDRs were determined by comparing the sequences with the databases on the IgBLAST website (https: / / www.ncbi.nlm.nih.gov / igblast / and the IMGT website (http: / / www.imgt.org / ) to examine the homology. That is, the "CDR sequences defined by Kabat" were determined according to the comparison with the database on the IgBLAST website, and the "CDR sequences defined by IMGT" were determined according to the comparison with the database on the IMGT website. The CDR sequences defined by Kabat for each of antibodies A to E were determined. The sequences (Kabat) and the CDR sequences as defined by IMGT (IMGT) are shown in Tables 1-5.

[0134] [Table 1]

[0135] [Table 2]

[0136] [Table 3]

[0137] [Table 4]

[0138] [Table 5]

[0139] Test Example 4. Determination of epitopes within exon 21 The epitopes within human periostin exon 21 were determined for antibodies A to E. Specifically, this was done as follows.

[0140] Polypeptides consisting of 10 amino acids each from the N-terminus of human periostin exon 21 amino acid sequence This polypeptide was arrayed on a glass slide. After blocking the array, the peptide array was incubated with the AE primary antibody and the AlexaFluor 647-labeled secondary antibody. After washing, the antibodies bound to the epitopes were detected using an Axon Genepix Scanner 4300 SL50. The fluorescence of each peptide spot was captured by a laser scanner, and positive peptides with strong signals were analyzed using the Spot-recognition software GenePix.

[0141] As a result, it was found that antibodies A to E recognize the three-dimensional structure in human periostin exon 21, which is composed of amino acid sequence A and amino acid sequence B shown in the table below.

[0142] [Table 6]

[0143] Test Example 5. Vascular Mimetic Test It has been reported that tumors mimic blood vessels within cancer cells, allowing blood flow inside and outside the tumor. This phenomenon is called vasculogenic mimicry (Hendrix et al. Nature. 520, pages 300-302 (2015)). This phenomenon has also been observed in clinical cases of breast cancer, and is associated with poor prognosis. It has been reported that this cultured cell line mimics the vascular system and is a good factor for evaluating the lumen formation of cancer cells. Therefore, we used this cultured cell line to compare the antibodies.

[0144] Mouse breast cancer cells 4T07 were seeded on a u-Slide angiogenesis plate coated with Matrigel (Corning Matrigel Matrix Growth Factor Reduced 5ml cat#356230) to induce vascular mimicry. The culture medium used was serum-free RPMI-1640, and the number of cells seeded was 3x10 5 The concentration of periostin was 10 μg / ml. The antibodies were added at a final concentration of 20 μg / ml. After incubation with 4T07 cells overnight, vascular mimicry was analyzed. Analysis was performed using an Image J Angiogenesis Analyzer.

[0145] The results are shown in Figure 2. PN2 administration significantly induced vascular mimicry in breast cancer cells (P<0.05 When antibodies were administered in addition to PN2, the use of antibody #8 did not suppress indices of lumen formation, such as the number of nodes and the number of segments, but the use of antibody A suppressed these indices. The index was significantly suppressed (P<0.01).

[0146] Test Example 6. Cancer cell transplantation test Mouse breast cancer cells 4T07 were seeded on 10 cm tissue culture treated dishes (Greiner) using RPMI1640 (Gibco) containing 10% bovine serum albumin (Invitrogen) and Penicillin-Streptomycin Mixed Solution (Nacalai Tesque) and cultured for 24 hours in an incubator at 37°C. The culture supernatant was then removed, washed with PBS, and suspended with trypsin / EDTA. The cells were collected and centrifuged for 5 minutes, after which 1x10 breast cancer cells were added. 6 The cells were counted so that each mouse had 100 cells and suspended in 100 μl of PBS. The cells were then injected into the mammary glands of 8-week-old female BALB / c nude mice using a 25G needle. One week after transplantation, the mammary gland area where the cells had taken up was measured with a caliper to determine the diameter (mm) and length (mm). The diameter (mm) was measured and calculated using the formula: tumor volume = diameter x minor axis 2 Tumor volume was calculated using a ratio of 100 × 0.5. 3 When the mice reached this age, they were divided into three groups (6 mice each): a control group, an antibody #8 administration group, and an antibody A administration group. Thereafter, the antibody was administered at 10 mg / kg twice a week via the tail vein using a 30G injection needle. The subsequent tumor volumes were compared.

[0147] The results are shown in Figure 3. No tumor shrinkage was observed in the antibody #8 administration group compared to the control group, but a significant tumor suppression effect was observed in the antibody A administration group (P<0.01).

Claims

1. An antibody or an antigen-binding fragment thereof, which comprises amino acid sequence A shown in SEQ ID NO: 61 and amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64 and has binding affinity to a conformational epitope on human periostin secreted from human cells.

2. The antibody or antigen-binding fragment thereof of claim 1 , wherein the human cell is a human breast cancer cell.

3. 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a monoclonal antibody or an antigen-binding fragment thereof.

4. （B1） heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 13; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 15; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 17; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 19; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 21, and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:23; or （B2） heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 14; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 16; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 18; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 20; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 22; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:24; or （C1） heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:25; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:27; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:29; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:31; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 33; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:35; or （C2） heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:26; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:28; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 30; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 32; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 34, and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:36; or （D1） heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:37; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:39; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 41; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 43; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 45; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:47; or （D2） heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:38; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 40; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 42; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 44; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 46; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:48; or （E1） heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:49; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:51; the heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:53; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:55; The light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:57; and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:59; or （E2） heavy chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:50; heavy chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:52; heavy chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:54; light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:56; the light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO:58; and The light chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:

60. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. (e) a protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 65; and (f) A protein or peptide comprising an amino acid sequence X having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 65, and comprising within said amino acid sequence X an amino acid sequence A shown in SEQ ID NO: 61 and an amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64. An antibody or an antigen-binding fragment thereof obtained by a method comprising a step of selecting an antibody that binds to human periostin secreted from human breast cancer cells from antibodies produced by hybridomas obtained from an animal immunized with at least one protein or peptide selected from the group consisting of:

6. (e) a protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 65; and (f) A protein or peptide comprising an amino acid sequence X having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 65, and comprising within said amino acid sequence X an amino acid sequence A shown in SEQ ID NO: 61 and an amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64. An antibody or an antigen-binding fragment thereof which is produced from a hybridoma obtained from an animal immunized with at least one protein or peptide selected from the group consisting of: and has binding affinity to human periostin secreted from human breast cancer cells.

7. (e) a protein or peptide comprising the amino acid sequence set forth in SEQ ID NO: 65; and (f) A protein or peptide comprising an amino acid sequence X having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 65, and comprising within said amino acid sequence X an amino acid sequence A shown in SEQ ID NO: 61 and an amino acid sequence B shown in any one of SEQ ID NOs: 62 to 64. and selecting an antibody that binds to human periostin secreted from human breast cancer cells from antibodies produced by hybridomas obtained from an animal immunized with at least one protein or peptide selected from the group consisting of:

8. A polynucleotide comprising a coding sequence for the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

9. A cell containing the polynucleotide of claim 8.

10. A conjugate of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a drug.

11. A pharmaceutical composition comprising at least one member selected from the group consisting of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and the complex according to claim 10.

12. The pharmaceutical composition according to claim 11, which is for preventing or treating cancer, inhibiting vascular mimicry of cancer, inhibiting periostin, preventing or treating inflammation-related diseases, inhibiting vascular intimal hyperplasia, inhibiting angiogenesis, or preventing or treating aneurysm.

13. The pharmaceutical composition described in claim 12, wherein the inflammation-related disease is diabetic retinopathy.

14. A reagent comprising at least one member selected from the group consisting of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and the complex according to claim 10.

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