WO2023106284A1 - 抗体又はその抗原結合性断片 - Google Patents
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
-
- A—HUMAN NECESSITIES
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/09—Recombinant DNA-technology
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- C12N5/10—Cells modified by introduction of foreign genetic material
Definitions
- the present invention relates to antibodies or antigen-binding fragments thereof.
- Periostin is a type of extracellular matrix protein and consists of a polypeptide with a molecular weight of about 90,000. Each polypeptide chain contains a signal sequence, a cysteine-rich domain, a 4-repeat domain, and a C-terminal domain. So far, it has been suggested that the expression of the periostin gene is also associated with the pathology of vascular restenosis, cancer, inflammation, and angiogenesis.
- Patent Document 1 reports an antibody against exon 21 of periostin, and this antibody can inhibit periostin, which has cell adhesion activity, and also induce intimal hyperplasia, cancer, and inflammatory colitis. It is described that diseases are treated by suppressing the action of a specific periostin variant whose expression is increased in diseases such as inflammation and angiogenesis, thereby suppressing exacerbation of disease conditions.
- An object of the present invention is to provide a novel antibody against exon 21 of human periostin or an antigen-binding fragment thereof.
- the present inventors have found that the three-dimensional epitope on human periostin, which consists of the amino acid sequence A shown in SEQ ID NO: 61 and the amino acid sequence B shown in any of SEQ ID NOS: 62 to 64,
- the present inventors have found that the above problems can be solved by an antibody or an antigen-binding fragment thereof that has a binding property to . Based on this finding, the inventors have further studied and completed the present invention. That is, the present invention includes the following aspects.
- Item 1 An antibody or an antigen-binding thereof, which is composed of 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, and has binding affinity to a stereoscopic epitope on human periostin sex fragment.
- Section 2 The antibody or antigen-binding fragment thereof according to Item 1, wherein the human periostin is human periostin secreted from human cells.
- Section 3 The antibody or antigen-binding fragment thereof according to Item 1 or 2, wherein the human cells are human breast cancer cells.
- 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.
- Heavy chain CDR1 comprises an amino acid sequence represented by SEQ ID NO: 1 and / or 2
- Heavy chain CDR2 comprises an amino acid sequence represented by SEQ ID NO: 3 and / or 4
- heavy chain CDR3 comprises an amino acid sequence represented by SEQ ID NO: 5 and / or 6
- light chain CDR1 comprises an amino acid sequence represented by SEQ ID NO: 7 and/or 8
- light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 9 and/or 10
- light chain CDR3 comprises the amino acid sequence represented by SEQ ID NO: 11 and/or 12
- Heavy chain CDR2 comprises an 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 an amino acid sequence represented by SEQ ID NO: 19 and/or 20
- light chain CDR2 comprises
- Heavy chain CDR1 comprises an amino acid sequence represented by SEQ ID NO: 1 and / or 2
- Heavy chain CDR2 comprises an amino acid sequence represented by SEQ ID NO: 3 and / or 4
- heavy chain CDR3 comprises an amino acid sequence represented by SEQ ID NO: 5 and / or 6
- light chain CDR1 comprises an amino acid sequence represented by SEQ ID NO: 7 and/or 8
- light chain CDR2 comprises an amino acid sequence represented by SEQ ID NO: 9 and/or 10
- light chain CDR3 comprises an amino acid sequence represented by SEQ ID NO: 11 and/or 12
- Item 7 The antibody or antigen-binding fragment thereof according to any one of items 1 to 6.
- Item 8. a protein or peptide comprising the amino acid sequence represented by SEQ ID NO: 65; At least one protein or peptide selected from the group consisting of proteins or peptides containing the amino acid sequence A shown in SEQ ID NO: 61 and the amino acid sequence B shown in any of SEQ ID NOS: 62-64 Obtained from immunized animals obtained by a method comprising the step of selecting antibodies that bind to human periostin secreted from human breast cancer cells from antibodies produced by the hybridomas obtained by the method.
- Item 9 (e) a protein or peptide comprising the amino acid sequence represented by SEQ ID NO: 65; At least one protein or peptide selected from the group consisting of proteins or peptides containing the amino acid sequence A shown in SEQ ID NO: 61 and the amino acid sequence B shown in any of SEQ ID NOS: 62-64 Obtained from immunized animals an antibody having binding to human periostin, which is produced from a hybridoma derived from a genus and is secreted from human breast cancer cells.
- 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.
- Item 11 A cell containing the polynucleotide according to Item 10.
- Item 12 A complex of the antibody or antigen-binding fragment thereof according to any one of Items 1 to 9 and a drug.
- Item 13 A pharmaceutical composition comprising at least one 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.
- Item 14 For the prevention or treatment of cancer, the inhibition of angiomimic cancer, the inhibition of periostin, the prevention or treatment of inflammation-related diseases, the inhibition of intimal thickening, the inhibition of angiogenesis, or the prevention or treatment of aneurysms. 14. The pharmaceutical composition according to item 13.
- Item 15 A reagent containing at least one 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.
- novel antibodies against exon 21 of human periostin or antigen-binding fragments thereof can be provided.
- the results of a human periostin-exon 21 recognition test (Test Example 2) for each antibody are shown.
- (A) shows the results of Western blotting of post-immunoprecipitation samples.
- (B) shows a CBB-stained image of the gel after Western blotting. The antibody used is shown above each photograph, and the molecular weight of each band of molecular weight markers is shown on the left.
- Test Example 5 shows the effects of each antibody on blood vessel mimicry.
- (A) shows a fluorescence photograph image of a vascular mimetic model of a cultured cell line. Fluorescence indicates 4T07 mouse breast cancer cells.
- (B) shows the measurement results of number of nodes, which is an index of tube formation.
- (C) shows the measurement results of number of segments, which is an index of tube formation. Shown are the results of Test Example 6 in which the influence of each antibody on tumor growth was examined. The vertical axis indicates the tumor volume, and the horizontal axis indicates the number of days elapsed after tumor implantation. The results of a human periostin-exon 21 recognition test (Test Example 7) for each antibody are shown.
- a to E indicate immunoprecipitation samples of antibodies A to E, IgG indicates immunoprecipitation samples of negative control antibody (IgG), and Beads are beads for immunoprecipitation without antibodies. A control is shown. Shown are the results of Test Example 8 in which the effects of each antibody on blood vessel mimicry were investigated.
- the vertical axis indicates total master segments length, which is an index of tube formation.
- mPN2 indicates samples added with mouse periostin
- #8, A, B, C, and D indicate samples added with antibody #8, A, B, C, or D
- (-) indicates antibody.
- Cont. indicates samples to which neither mouse periostin nor antibody was added.
- the vertical axis indicates the total mesh area (the total area of the formed lumen), which is an index of lumen formation.
- PN2 indicates samples added with mouse periostin, #8, antibody A
- antibody E indicates samples added with antibody #8, antibody A, or antibody E
- (-) indicates no antibody added. Cont. indicates samples to which neither mouse periostin nor antibody was added.
- an antibody or an antigen-binding fragment thereof on human periostin comprising 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 antibody or antigen-binding fragment thereof (herein sometimes referred to as the "antibody or fragment thereof of the present invention"), which has binding properties to a conformational epitope of This will be explained below.
- Human periostin is not particularly limited as long as it has exon 21 (SEQ ID NO: 65) among multiple splicing variants.
- Human periostin preferably includes human periostin without exon 17 but with exon 21, more preferably human periostin (PN-2) consisting of the amino acid sequence shown in SEQ ID NO: 66. .
- Human periostin also includes variants occurring within the human species as long as the structure and sequence of the conformational epitope described below does not change. From this point of view, as human periostin, for example, a protein described in (a) below and a protein described in (b) below: (a) a protein comprising the amino acid sequence shown by SEQ ID NO: 65; At least one human periostin selected from the group consisting of proteins comprising the amino acid sequence A represented by number 61 and the amino acid sequence B represented by any one of SEQ ID NOs: 62 to 64, Preferably, the protein described in (c) below and the protein described in (d) below: (c) a protein consisting of an amino acid sequence represented by SEQ ID NO: 66, and (d) an amino acid sequence represented by SEQ ID NO: 66 and 85% At least one kind of human periostin selected from the group consisting of proteins consisting of amino acid sequences having the above identity and comprising the amino acid sequence shown in SEQ ID NO:65 can be mentioned
- the identity is more preferably 90% or more, still more preferably 95% or more, and even more preferably 98% or more.
- Identity of amino acid sequences refers to the degree of matching of two or more comparable amino acid sequences to each other. Therefore, the higher the identity or similarity between two amino acid sequences, the higher the identity or similarity between those sequences.
- the level of amino acid sequence identity is determined, for example, using the sequence analysis tool FASTA, using default parameters. or Algorithm BLAST by Karlin S, Altschul SF. SF. "Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX based on such a BLAST algorithm has been developed.
- proteins described in (b) and (d) above include, for example, (b') substitution, deletion, addition, or insertion of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 65 ( (preferably substituted, more preferably conservatively substituted) amino acid sequence X, and the amino acid sequence A represented by SEQ ID NO: 61 and the amino acid sequence B represented by any one of SEQ ID NOs: 62 to 64 in the amino acid sequence X
- the plural number is, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, even more preferably 2 or There are 3.
- Constant substitution means that an amino acid residue is replaced with an amino acid residue having a similar side chain.
- substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine correspond to conservative substitutions.
- amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine, tryptophan; amino acid residues with ⁇ -branched side chains such as threonine, valine, isoleucine; aromatic side chains such as tyrosine, phenylalanine, tryptophan, histidine. Substitutions between amino acid residues are also conservative substitutions.
- 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 in this respect. . Therefore, recombinant human periostin obtained by expression and purification using non-human cells (eg, bacteria) is not included in "human periostin secreted from human cells”.
- Human cells are not particularly limited as long as they are cells capable of secreting human periostin.
- Human cells include, for example, cancer cells.
- cancers from which cancer cells are derived include brain tumor, 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, and ovarian cancer.
- 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, etc., preferably breast cancer, colon cancer,
- lung cancer and malignant melanoma particularly preferably breast cancer.
- the antibody or fragment thereof of the present invention has binding to a conformational epitope on human periostin.
- the conformational epitope on human periostin is a conformation composed of the amino acid sequence A shown in SEQ ID NO: 61 and the amino acid sequence B shown in any one of SEQ ID NOs: 62-64, and is not particularly limited in this respect.
- the antibody or fragment thereof of the present invention is capable of recognizing or binding to a conformation on human periostin.
- Antibodies or fragments thereof of the invention preferably have specific binding to a conformational epitope on human periostin.
- Amino acid sequence B preferably includes SEQ ID NO:62.
- the antibody or fragment thereof of the present invention is Heavy chain CDR1 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 including Heavy chain CDR2 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 including heavy chain CDR3 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 including Light chain CDR1 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
- amino acid sequences indicated by odd-numbered SEQ ID NOs are CDR sequences defined by Kabat, and the amino acid sequences indicated by even-numbered SEQ ID NOS are CDR sequences defined by IMGT.
- the antibody or fragment thereof of the present invention is, among the antibodies (A) to (E) or antigen-binding fragments thereof, more preferably the antibody (A) or (B) or an antigen-binding fragment thereof, Particularly preferred is the above antibody (A) or an antigen-binding fragment thereof.
- the antibody or fragment thereof of the present invention is more preferably the antibody (A) or (E) or an antigen-binding fragment thereof, and particularly preferably the antibody (E) or the fragment thereof. It is an antigen-binding fragment.
- the antibody or fragment thereof of the present invention is (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; wherein the light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO: 7; light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 9, and light chain CDR3 comprises the amino acid sequence represented by 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; wherein the light chain CDR1 comprises the amino acid sequence represented by SEQ ID NO:8; light chain CDR2 comprises the amino acid sequence represented by SEQ ID NO: 10, and light chain CDR3 comprises the amino acid sequence represented by SEQ ID NO:
- the antibody or fragment thereof of the present invention is among the above antibodies (A1) to (E2) or antigen-binding fragments thereof, more preferably the above (A1), (A2), (B1), or (B2) antibody or an antigen-binding fragment thereof, particularly preferably the antibody of (A1) or (A2) or an antigen-binding fragment thereof.
- the antibody or fragment thereof of the present invention is more preferably the above (A1), (A2), (E1), or (E2) antibody or antigen-binding fragment thereof, particularly preferably is the above (E1) or (E2) antibody or antigen-binding fragment thereof.
- the antibody or antigen-binding fragment thereof of (A) has the amino acid sequence AH1 shown in SEQ ID NO: 67, and is 85% or more (preferably 90% or more, more preferably 95% or more, more preferably 95% or more of the amino acid sequence AH1) 98% or more, more preferably 99% or more), or a heavy chain variable region comprising an amino acid sequence AH3 that is a humanized sequence of the amino acid sequence AH1, and/or represented by SEQ ID NO: 68 85% or more (preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more) with the amino acid sequence AL1 It preferably comprises a light chain variable region comprising the sequence AL2 or the amino acid sequence AL3 which is a humanized sequence of said amino acid sequence AL1.
- the antibody or antigen-binding fragment thereof of (B) has an amino acid sequence BH1 represented by SEQ ID NO: 69, and 85% or more (preferably 90% or more, more preferably 95% or more, more preferably 95% or more of the amino acid sequence BH1) 98% or more, more preferably 99% or more), or a heavy chain variable region comprising an amino acid sequence BH3 that is a humanized sequence of the amino acid sequence BH1, and / or represented by SEQ ID NO: 70 85% or more (preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more) with the amino acid sequence BL1 that is identical to the amino acid sequence BL1 It preferably comprises a light chain variable region comprising the sequence BL2 or the amino acid sequence BL3 which is a humanized sequence of said amino acid sequence BL1.
- the antibody or antigen-binding fragment thereof of (C) has the amino acid sequence CH1 shown in SEQ ID NO: 71, and is 85% or more (preferably 90% or more, more preferably 95% or more, more preferably 95% or more of the amino acid sequence CH1) 98% or more, more preferably 99% or more), or a heavy chain variable region comprising an amino acid sequence CH3 that is a humanized sequence of the amino acid sequence CH1, and / or represented by SEQ ID NO: 72 amino acid sequence CL1, an amino acid having 85% or more (preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more) identity to the amino acid sequence CL1 It preferably comprises a light chain variable region comprising the sequence CL2 or the amino acid sequence CL3 which is a humanized sequence of said amino acid sequence CL1.
- the antibody of (D) or an antigen-binding fragment thereof has the amino acid sequence DH1 shown in SEQ ID NO: 73, and is 85% or more (preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, more preferably 99% or more), or a heavy chain variable region comprising an amino acid sequence DH3 that is a humanized sequence of the amino acid sequence DH1, and / or represented by SEQ ID NO: 74 amino acid sequence DL1, an amino acid having 85% or more (preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more) identity to the amino acid sequence DL1 It preferably comprises a light chain variable region comprising the sequence DL2 or the amino acid sequence DL3 which is a humanized sequence of said amino acid sequence DL1.
- the antibody or antigen-binding fragment thereof of (E) has the amino acid sequence EH1 shown in SEQ ID NO: 75, 85% or more (preferably 90% or more, more preferably 95% or more, more preferably 95% or more of the amino acid sequence EH1) 98% or more, more preferably 99% or more), or a heavy chain variable region comprising an amino acid sequence EH3 that is a humanized sequence of the amino acid sequence EH1, and / or represented by SEQ ID NO: 76 amino acid sequence EL1, an amino acid having 85% or more (preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more) identity to the amino acid sequence EL1 It is preferred to include a light chain variable region comprising the sequence EL2 or the amino acid sequence EL3 which is a humanized sequence of said amino acid sequence EL1.
- Antibody is used in the sense of including monoclonal antibodies and polyclonal antibodies.
- Antibodies can be of any isotype, eg, IgG (eg, IgG1, IgG2, IgG3, IgG4), IgA (eg, IgA1, IgA2), IgD, IgE, IgM, and the like.
- An 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, and the like.
- the antibody may be a chimeric antibody, such as a mouse-human chimeric antibody.
- Antibodies can be partially or fully humanized antibodies.
- Antibodies of the present invention are preferably monoclonal antibodies.
- the antigen-binding fragment of an antibody is not particularly limited as long as it contains heavy chain CDRs 1-3 and light chain CDRs 1-3. -Fc, Fv, scFv, diabody, triabody, tetrabody and the like.
- Fab includes a heavy chain fragment containing CH1 in the heavy chain variable region and the heavy chain constant region, and a light chain containing the light chain variable region and the light chain constant region (CL). It has a structure in which it associates with the chain variable region through the above-described non-covalent intermolecular interaction or binds through a disulfide bond.
- CH1 and CL may form a disulfide bond between the thiol groups of the cysteine residues present in each.
- F(ab') 2 has a structure in which two pairs of the above Fab are formed by disulfide bonding between thiol groups of cysteine residues contained in CH1.
- a minibody is a structure in which two fragments in which CH3 is bound to the heavy chain variable region that constitutes the scFV below are associated through non-covalent intermolecular interactions between CH3.
- scFv-Fc means that two antibody fragments containing the following scFv, CH2, and CH3 are associated by non-covalent intermolecular interactions between CH3, similar to the above minibody, and the cysteine residue contained in each CH3 It is a structure in which the thiol groups of the groups are disulfide-bonded.
- Fv is also called the minimum structural unit of an antibody, and is a structure in which heavy chain variable regions and light chain variable regions are associated through non-covalent intermolecular interactions.
- the thiol groups of cysteine residues present in the heavy chain variable region and the light chain variable region may be disulfide bonded to each other.
- scFv is a structure in which the C-terminus of the heavy chain variable region and the N-terminus of the light chain variable region are connected by a linker, or the N-terminus of the heavy chain variable region and the C-terminus of the light chain variable region are connected by a linker.
- structure also called a single-chain antibody.
- the antibody or fragment thereof of the present invention may be conjugated or fused with other peptides, oligopeptides, or proteins.
- Other peptides, oligopeptides, or proteins include albumin (eg, serum albumin), protein tags (eg, biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tags), fluorescent proteins (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, ⁇ -galactosidase, chloramphenicol acetyltransferase, ⁇ -glucuronidase
- secretory signal sequences e.g. Ig ⁇ signal sequences
- protease recognition sequences e.g.
- TEV protease recognition sequence
- expression-enhancing sequence solubilization 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 ⁇ 1 protein domain, hepatitis delta antigen domain
- the antibody or fragment thereof of the present invention is bound or fused to a multimerization domain
- the multimerization domain is further bound by an antibody or antigen-binding fragment thereof homologous or heterologous to the antibody or fragment thereof of the present invention. Alternatively, they may be fused to form multimers (homo-multimers or hetero-multimers).
- the antibody or fragment thereof of the present invention may be chemically modified as long as the binding to human periostin is not significantly impaired.
- the antibody or fragment thereof of the present invention may have a carboxyl group (--COOH), carboxylate ( --COO- ), amide group (--CONH 2 ), or ester group (--COOQ) at the C-terminus.
- Q in the ester group includes, for example, C 1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl and n-butyl; C 3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl; C 6-12 aryl groups such as phenyl, ⁇ -naphthyl; phenyl-C 1-2 alkyl groups such as benzyl, phenethyl; C 7 such as ⁇ -naphthyl-C 1-2 alkyl groups such as ⁇ -naphthylmethyl -14 aralkyl group; pivaloyloxymethyl group and the like are used.
- carboxyl groups (or carboxylates) other than the C-terminus may be amidated or esterified.
- ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
- the amino group of the amino acid residue at the N-terminus is protected with a protecting group (for example, a formyl group, a C1-6 acyl group such as a C1-6 alkanoyl such as an acetyl group, etc.).
- a protecting group for example, a formyl group, a C1-6 acyl group such as a C1-6 alkanoyl such as an acetyl group, etc.
- Antibodies or fragments thereof of the present invention can be produced by various methods.
- Antibodies or fragments thereof of the present invention are, for example, (e) a protein or peptide comprising the amino acid sequence represented by SEQ ID NO: 65; At least one protein or peptide selected from the group consisting of proteins or peptides containing the amino acid sequence A shown in SEQ ID NO: 61 and the amino acid sequence B shown in any of SEQ ID NOS: 62-64 Obtained from immunized animals It can be produced by a method comprising a step of selecting antibodies that bind to human periostin secreted from human breast cancer cells from antibodies produced by the hybridomas thus obtained.
- the antibody or fragment thereof of the present invention is (e) a protein or peptide comprising the amino acid sequence represented by SEQ ID NO: 65; At least one protein or peptide selected from the group consisting of proteins or peptides containing the amino acid sequence A shown in SEQ ID NO: 61 and the amino acid sequence B shown in any of SEQ ID NOS: 62-64 Obtained from immunized animals obtained by a method comprising the step of selecting antibodies that bind to human periostin secreted from human breast cancer cells from antibodies produced by the hybridomas thus obtained.
- the antibody or fragment thereof of the present invention is (e) a protein or peptide comprising the amino acid sequence represented by SEQ ID NO: 65; At least one protein or peptide selected from the group consisting of proteins or peptides containing the amino acid sequence A shown in SEQ ID NO: 61 and the amino acid sequence B shown in any of SEQ ID NOS: 62-64 Obtained from immunized animals
- the antibody can be an antibody produced from a hybridoma derived from a periostin and having binding to human periostin secreted from human breast cancer cells.
- Animals to be immunized are not particularly limited as long as they can prepare hybridomas, but preferably include autoimmune disease animals (in the case of mice, BXSB mice, etc.). 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. Section 11.4-11.11).
- the evaluation method for selecting antibodies that bind to human periostin secreted from human breast cancer cells is not particularly limited as long as it is a method that utilizes antigen-antibody reaction using human periostin secreted from human breast cancer cells. However, for example, the method described in Test Example 2 below can be adopted.
- the binding property of an antibody or a fragment thereof to a target can be measured by the surface plasmon resonance (SRP) method.
- the dissociation constant (KD value) between the antibody or fragment thereof of the present invention and a target is, for example, 9 ⁇ 10 ⁇ 6 M or less, preferably It is 9 ⁇ 10 ⁇ 8 M or less, more preferably 9 ⁇ 10 ⁇ 10 M or less.
- the lower limit of the dissociation constant is not particularly limited, but can be, for example, 1 ⁇ 10 ⁇ 14 M, 1 ⁇ 10 ⁇ 13 M, 1 ⁇ 10 ⁇ 12 M, 1 ⁇ 10 ⁇ 11 M.
- antibodies of the invention or fragments thereof can be obtained by culturing a host transformed with, for example, a polynucleotide comprising a coding sequence for an antibody of the invention or a fragment thereof (polynucleotide of the invention), It can be produced by a method comprising the step of collecting a fraction containing the antibody.
- 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 fragment thereof.
- the polynucleotide of the invention comprises the above coding sequence in a state capable of expressing the antibody of the invention or fragment thereof.
- Polynucleotides of the present invention may contain other sequences in addition to the coding sequences described above. Other sequences include secretory signal peptide coding sequences, promoter sequences, enhancer sequences, repressor sequences, insulator sequences, origins of replication, drug resistance gene coding sequences, etc., which are located adjacent to the antibody coding sequences of the invention. be done.
- the polynucleotides of the present invention may be linear polynucleotides or circular polynucleotides (such as vectors).
- polynucleotide of the present invention include (I) a nucleotide sequence encoding at least one selected from the group consisting of the heavy chain, heavy chain variable region, and heavy chain CDRs 1-3 of the antibody of the present invention.
- polynucleotide (II) Even a polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of the light chain, light chain variable region, and light chain CDRs 1-3 of the antibody of the present invention, ( III) A nucleic acid comprising a nucleotide sequence encoding at least one selected from the group consisting of the heavy chain, heavy chain variable region, and heavy chain CDRs 1-3 of the antibody of the present invention, and the light chain and light chain of the antibody of the present invention Polynucleotides containing nucleotide sequences encoding at least one selected from the group consisting of chain variable regions and light chain CDRs 1-3.
- the host is not particularly limited, and examples include insect cells, eukaryotic cells, mammalian cells, and the like. Among them, mammalian cells such as HEK cells, CHO cells, NS0 cells, and SP2/O cells are preferable from the viewpoint of more efficient antibody expression.
- the methods of transformation, culture, and recovery are not particularly limited, and known methods for antibody production can be adopted.
- the antibody of the present invention may be purified as necessary. Purification can be performed by known methods for antibody production, such as chromatography, dialysis, and the like.
- Polynucleotides such as DNA and RNA may be chemically modified as exemplified below.
- the phosphate residue of each nucleotide can be replaced with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. can.
- PS phosphorothioate
- methylphosphonate methylphosphonate
- phosphorodithioate phosphorodithioate
- the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is replaced by -OR (R is, for example, -CH 3 , -CH 2 CH 2 OCH 3 , -CH 2 CH 2 NHC (NH) NH 2 , -CH 2 CONHCH 3 , -CH 2 CH 2 CN, etc.).
- the base moiety pyrimidine, purine
- the antibody of the present invention is obtained as a humanized antibody, it can be produced based on the following information and, if necessary, according to other known information.
- Immunoglobulin G (hereinafter simply referred to as "IgG”) consists of a light polypeptide chain with a molecular weight of about 23000 (hereinafter referred to as “light chain”) and a heavy polypeptide chain with a molecular weight of about 50000 (hereinafter referred to as “heavy chain”). Consists of two pieces each. Both the heavy and light chains have a repeating structure of about 110 residues with conserved amino acid sequences, which constitute the basic unit (hereinafter referred to as "domain") of the three-dimensional structure of IgG. Heavy and light chains are composed of 4 and 2 contiguous domains, respectively.
- V domain variable domain
- the heavy chain and light chain V domains complementarily associate to form a variable region.
- the remaining domains together form the constant region.
- the constant region has sequences characteristic of each animal species. For example, the constant region of mouse IgG is different from the constant region of human IgG.
- HAMA human anti mouse antibody
- such domains generally have an elongated cylindrical structure in which antiparallel ⁇ -sheets consisting of 3 to 5 ⁇ -strands are stacked in two layers.
- variable region three loops are assembled in each of the heavy chain and light chain V domains to form an antigen-binding site.
- Each of these loops is called a complementarity determining region (hereinafter referred to as "CDR"), and has the most significant amino acid sequence variation.
- CDR complementarity determining region
- the portions other than the CDRs of the variable region generally have a role of retaining the structure of the CDRs and are called "framework". Kabatt et al.
- each framework was classified into multiple subgroups with common amino acid sequence features. Furthermore, it was found that there is a corresponding framework between human and mouse. Based on such research on the structural characteristics of IgG, the following methods for producing humanized antibodies have been devised. In the early stages of research, a chimeric antibody was proposed in which the variable region of a mouse-derived antibody was conjugated to a human-derived constant region (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 can induce HAMA responses, especially when administered for a long time (Begent et al., Br. J. Cancer, (1990 )62, 487).
- a non-human mammal-derived antibody that has a CDR to be transplanted is defined as a "donor”
- a human antibody to which the CDR is transplanted is defined as an "acceptor”.
- Queen et al. proposed a design method in which the amino acid residues of the framework of the donor are transplanted into the acceptor together with the CDR sequence when at least one of the following criteria is met (Japanese Patent Publication No. 4-502408): (a) an amino acid in the framework region of the acceptor is rare at that position and the corresponding amino acid of the donor is common at said position of the acceptor (b) the amino acid is in close proximity to one of the CDRs ( c) that the amino acid has a side chain atom within about 3 angstroms of the CDR in a three-dimensional immunoglobulin model and is expected to be able to interact with the antigen or with the CDRs of a humanized antibody.
- the antibody of the present invention is obtained as a human antibody, it can be produced based on the following information and, if necessary, according to other known information.
- Human antibody or “human immunoglobulin” in the present invention refers to an H chain variable region (VH), an H chain constant region (CH), an L chain variable region (VL) and an L chain that constitute an immunoglobulin.
- VH H chain variable region
- CH H chain constant region
- VL L chain variable region
- L chain that constitute an immunoglobulin is an immunoglobulin whose entire region, including the constant region (CL), is derived from genes encoding human immunoglobulins.
- it means an antibody whose H chain is derived from a human immunoglobulin heavy chain gene and whose light chain is derived from a human immunoglobulin light chain gene.
- Human antibodies can be obtained by immunizing a transgenic animal prepared by integrating at least a human immunoglobulin gene into a locus of a non-human mammal such as a mouse, with an antigen according to a conventional method. It can be produced in the same manner as the antibody production method. For example, transgenic mice that produce human antibodies have been reported (Mendez MJ et al. Nature Genetics (1997) 15, 146-56, Green LL et al. Nature Genetics (1994) 7, 13-21, Omotehei 4-504365). Publication: International Application Publication No. WO94/25585; Nikkei Science, June issue, pp. 40-50, 1995; Nils Lonberg et al. ) can be prepared according to the method described in .
- the present invention relates to cells containing the polynucleotides of the present invention.
- the cells include, for example, cells transduced with the polynucleotide of the present invention.
- the cells are preferably isolated cells.
- the cells may be cultured cells, primary cultured cells, or subcultured cells.
- the cells may be cell lines.
- the cells may be iPS cells.
- Examples of the cells include Escherichia coli K12 and other Escherichia coli, Bacillus subtilis MI114 and other Bacillus bacteria, Saccharomyces cerevisiae AH22 and other yeasts, Spodoptera frugiperda-derived Sf cell line or Trichoplusia ni-derived HighFive cell line, olfactory neurons, and the like.
- Animal cells are preferably cultured cells derived from mammals, specifically COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells and the like. be done.
- the present invention relates to conjugates of the antibody of the present invention or a fragment thereof and a drug.
- the drug is not particularly limited as long as it has physiological activity.
- examples of drugs include proteins, peptides, low-molecular-weight compounds, nucleic acids, sugar chains, and the like.
- the method for conjugating the antibody or fragment thereof of the present invention with a drug is not particularly limited, and known methods can be employed.
- the conjugate of the present invention is formed by binding the antibody of the present invention and the drug directly or indirectly via a linker or the like.
- the binding mode is not particularly limited, and examples thereof include covalent bonds, coordinate bonds, ionic bonds and the like.
- the binding between the antibody of the present invention and the drug can be carried out according to or according to known methods, depending on the binding mode.
- Covalent bonding can be carried out, for example, by reacting the functional groups possessed by the antibody of the present invention and the drug, or the functional groups introduced as necessary.
- Combinations of the functional groups include, for example, an amino group and a carboxyl group, a carboxyl group and a hydroxyl group, a maleimide group and a thiol group, a thiol group and a thiol group, a hydrazide group and a ketone group, a hydrazide group and an aldehyde group, and an amino group and an aldehyde group.
- the present invention relates to pharmaceutical compositions, reagents, etc. containing at least one selected from the group consisting of the antibody of the present invention or a fragment thereof, and the complex of the present invention.
- the pharmaceutical composition of the present invention can be used for the prevention or treatment of cancer, the suppression of cancer angiomimics, the inhibition of periostin, the prevention or treatment of inflammation-related diseases, the suppression of intimal thickening, the suppression of angiogenesis, or the arterial It can be suitably used for prevention or treatment of aneurysms.
- the antibody or fragment thereof of the present invention, or the conjugate of the present invention can be used to prevent or treat inflammation-related diseases involving periostin, which has cell adhesion activity.
- Inflammation-related diseases involving periostin are diseases in which the periostin gene, which has cell adhesion activity, is highly expressed and the production of the protein encoded by the gene is increased during the disease state. It also refers to a disease whose condition is aggravated by an increase in the gene or protein.
- Inflammation-related diseases with such cell adhesion activity include, but are not limited to, diseases mainly caused by intimal thickening, cancer, and other inflammation-related diseases.
- Diseases mainly caused by intimal thickening include arteriosclerosis and restenosis mainly caused by intimal thickening observed after coronary angioplasty.
- cancers to which the antibody or fragment thereof of the present invention or the complex of the present invention can be applied are not limited to these, but examples include brain tumor, leukemia, osteosarcoma, breast cancer, colon cancer, malignant melanoma, Bone cancer, gastric 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 Examples include cancer, prostate cancer, bladder cancer, and malignant lymphoma, and particularly preferred examples include breast cancer, colon cancer, lung cancer, and malignant melanoma.
- inflammation-related diseases include autoimmune arthritis, atopic dermatitis, asthma, emphysema, Behçet's disease, multiple sclerosis, spinocerebellar degeneration, uveitis, Guillain-Barré syndrome, Fisher's syndrome, chronic inflammatory prolapse.
- Polymyelinitis Polymyositis, Scleroderma, Autoimmune hepatitis, Sarcoidosis, Chronic pancreatitis, Inflammatory enteritis, Crohn's disease, Solid tumor, Multiple myeloma, Angiofibroma, Atherosclerosis, Arteriovenous Malformation, granulation, hemangioma, hypertrophic scar, keloid, premature aging, psoriasis, febrile granuloma, wart, hemorrhagic joint, non-union fracture, rheumatoid arthritis (e.g.
- Angiogenesis is involved in many of the inflammation-related diseases exemplified above.
- Another aspect of the present invention includes a diagnostic agent for inflammation-related diseases associated with periostin having cell adhesion activity, prepared by labeling the above antibody with a marker.
- the marker is not particularly limited, and enzymes, radioactive isotopes, fluorescent dyes, and the like can be used. Enzymes used here must have a large turnover number, be stable even when bound to the enzyme, and be able to react specifically with the substrate to develop color. There is no particular limitation, and enzymes used in ordinary enzyme immunoassay (EIA) can be used.
- Examples of preferred enzymes include peroxidase, ⁇ -galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase and the like. Enzyme inhibitors, coenzymes, and the like can also be used.
- Binding of these enzymes and antibodies can be carried out by a known method using a known cross-linking agent such as a maleimide compound.
- a known cross-linking agent such as a maleimide compound.
- a known substance can be used 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, para-nitrophenol and the like can be used.
- the radioisotope used as a marker those used in ordinary radioimmunoassay (RIA) such as 125I and 3H can be used.
- the fluorescent dye those used in ordinary fluorescent antibody methods such as fluorescence isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) can be used.
- the present diagnostic agent can be used as an immunohistological stain capable of specifically staining cancer cells and surrounding fibroblasts.
- radioisotopes when radioisotopes are labeled, they can be administered into the body and used to image lesions such as cancer pathologies.
- One aspect of the present invention is the use of the antibody or fragment thereof of the present invention, or the conjugate of the present invention to enhance cell adhesion activity in a biological sample prepared by preparing serum from human or animal blood, that is, in serum.
- the present invention provides a method for detecting or quantifying periostin having periostin, and a method for diagnosing an inflammation-related disease involving periostin having cell adhesion activity, such as heart failure, by detecting or quantifying periostin.
- periostin having cell adhesion activity can be detected by a so-called sandwich ELISA method (Enzyme-linked immunosorbent assay).
- the sample is brought into contact with a plate on which an anti-periostin primary antibody has been immobilized so that the two are bound, and this conjugate is bound with a marker-labeled anti-periostin secondary antibody.
- Periostin having cell adhesion activity can be detected or quantified by measuring the signal intensity of the marker in the tripartite conjugate.
- periostin which has cell adhesion activity, is a splicing variant that is specifically expressed during pathological conditions such as cancer, and therefore pathological conditions such as cancer can be diagnosed by monitoring the production of periostin.
- one aspect of the present invention includes a pharmaceutical composition for inhibiting periostin having cell adhesion activity, which contains the antibody or fragment thereof of the present invention or the complex of the present invention as an active ingredient. Since the antibody, antibody fragment and/or derivative has inhibitory activity against periostin having cell adhesion activity, the pharmaceutical composition suppresses angiogenesis, suppresses intimal hyperplasia, treats cancer, Also, aneurysms can be prevented or treated. That is, the present invention provides a composition for suppressing intimal hyperplasia, treating cancer, suppressing angiogenesis, and preventing or treating aneurysm, comprising an anti-periostin antibody that recognizes a splicing variant of periostin that has a cell adhesion effect. be.
- composition of the present invention can be used for treatment and prevention of restenosis mainly caused by intimal thickening observed after coronary angioplasty, prevention or treatment of cancer, treatment or prevention of diseases accompanied by angiogenesis, prevention or prevention of aneurysm, and can be used to treat
- one aspect of the present invention includes a pharmaceutical composition for the prevention or treatment of inflammation-related diseases associated with periostin, which has cell adhesion activity, and which contains the antibody, antibody fragment and/or derivative of the present invention as an active ingredient.
- a pharmaceutical composition for suppressing intimal hyperplasia containing the antibody, antibody fragment and/or antibody derivative as an active ingredient, and the antibody, antibody fragment and/or antibody derivative effective
- a pharmaceutical composition for treating cancer containing as an ingredient, a pharmaceutical composition for suppressing angiogenesis containing the above antibody, antibody fragment and/or antibody derivative as an active ingredient, and the above antibody, antibody fragment and/or antibody derivative as an active ingredient and a pharmaceutical composition for preventing or treating aneurysm containing as.
- composition for cancer treatment of the present invention has the effect of suppressing the growth of cancer lesions and the effect of suppressing cancer metastasis. Therefore, it can be used in anticipation of the effect of suppressing the growth of cancer pathogenic foci, anticipation of the effect of suppressing metastasis of cancer, or in anticipation of both.
- the antibody or fragment thereof of the present invention or a pharmaceutical composition containing the conjugate of the present invention as an active ingredient is used in normal formulation, and is prepared using known pharmacologically acceptable carriers, excipients, and other additives. are prepared with the following additives:
- the active ingredient of the pharmaceutical composition according to the present invention is mixed with known pharmacologically acceptable carriers, excipients, diluents, etc., and administered by a method generally used for medicine (e.g., oral administration, Alternatively, it is preferably administered by a parenteral administration method such as intravenous administration, intramuscular administration or subcutaneous administration).
- the pharmaceutical composition of the present invention comprises, for example, an active ingredient, a pharmacologically acceptable carrier, a flavoring agent, an excipient, a stabilizer, a diluent, an emulsifying agent, a solution, a suspension, a syrup and the like. It can be produced by appropriately mixing with.
- the dosage form of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include tablets, powders, granules, solutions and the like.
- additives that can be mixed with tablets and the like include binders such as gelatin and lubricants such as cornstarch.
- Pharmaceutical compositions may also be coated with sugar coatings or films of gastric or enteric substances.
- the composition may further contain a liquid carrier.
- Sterile compositions for injection can also be prepared by applying conventional pharmaceutical formulations.
- Aqueous solutions for injection include isotonic solutions containing glucose and the like, and may be used in combination with suitable solubilizers such as polyethylene glycol.
- compositions may also be formulated with buffers, stabilizers, preservatives, antioxidants, soothing agents, and the like.
- oral administration if the active ingredient is susceptible to degradation in the gastrointestinal tract, it is also possible to administer orally as a formulation that is resistant to degradation in the gastrointestinal tract (e.g., microcapsules encapsulating the active ingredient in liposomes).
- the drug through mucous membranes other than the gastrointestinal tract, such as rectal, intranasal, sublingual, and pulmonary. In this case, it can be administered in the form of suppositories, nasal drops, sublingual agents, and pulmonary agents.
- an effective dosage for treatment is determined. determined on a case-by-case basis.
- the daily dose for adults is usually about 0.1 to 1000 mg, which may be administered in 1 to several (2, 3, etc.) divided doses.
- the dosage range can be 0.01 ⁇ g/kg/min to 1.0 ⁇ g/kg/min, and the dosage range is 0.025 ⁇ g/kg/min to 0.1 ⁇ g/kg/min. is desirable.
- a method for inhibiting periostin having cell adhesion activity characterized by using a periostin antibody
- anti-periostin recognizing a splicing variant of periostin having a therapeutically effective amount of cell adhesion activity A method for preventing or treating an inflammation-related disease involving periostin, which has cell adhesion activity, which comprises administering an antibody to a cancer patient.
- “Use of a periostin antibody for manufacturing a pharmaceutical composition for inhibiting periostin with cell adhesion activity” "Pharmaceutical for prevention or treatment of inflammation-related diseases involving periostin with cell adhesion activity”
- Use of Periostin Antibody for Manufacture of Compositions The anti-periostin antibodies include the above-described antibodies, antibody fragments and/or antibody derivatives. Inflammation-related diseases include those mentioned above.
- 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 of the present invention or a fragment thereof and the complex of the present invention.
- the composition may contain other ingredients as needed.
- Other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, and perfumes. , chelating agents and the like.
- 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 may contain instruments, reagents, and the like that can be used to detect and isolate human periostin. 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), and the like.
- Test example 1 Preparation of monoclonal antibody against human periostin (1)
- Preparation of antigen Peptides in which a Cys residue was added to the N-terminus of the amino acid sequence (SEQ ID NO: 65: EVTKVTKFIEGGDGHLFEDEEIKRLLQG) constituting human periostin exon 21 were chemically synthesized by the Fmoc method. , 10 mg of antigenic peptide with a purity of 90% or more was obtained. 5 mg of this antigen peptide was combined with 5 mg of KLH (manufactured by CALBIOCHEM) as a carrier protein to obtain an antigen solution.
- KLH manufactured by CALBIOCHEM
- KLH was dissolved in PBS (0.01 M) to adjust to 3.3 mg/mL, 0.2524 mg/mL MBS solution (manufactured by GE Healthcare Bioscience) was added dropwise, and the mixture was stirred at room temperature for 60 minutes to react. . Free MBS was removed using dichloromethane to give KLH-MB. 5 mg of this KLH-MB and 5 mg of antigen peptide dissolved in 0.01 M sodium phosphate buffer (pH 7.2) were mixed and stirred at 4° C. for 12 hours to react to obtain an antigen solution.
- RPMI medium manufactured by Kohjin Bio Co., Ltd.
- the lymph nodes were crushed and passed through a mesh with a pore size of about 10 ⁇ m to obtain lymph node cells suspended in RPMI medium. This was centrifuged at 1000 rpm for 10 minutes to obtain lymph node cells as a sediment fraction. 1 ml of a solution of 0.84% ammonium chloride solution and 20 mM HEPES buffer (pH 7.4) was added to the precipitated fraction to lyse the erythrocytes, followed by centrifugation at 1,000 rpm for 5 minutes. . The resulting precipitated fraction (cell fraction) was washed several times with RPMI medium and used for cell fusion.
- HAT solution manufactured by Invitrogen
- Each well of a 96-well microtiter plate (Falcon 353912) was coated with 1 ⁇ g/ml of the above conjugate by standing overnight at 4°C. After washing the plate, 50 ⁇ l of the culture supernatant (containing monoclonal antibody) from (4) was added dropwise to each well, left in an incubator at 37°C for 2 hours, and then washed with PBS (-) (phosphate buffer). washed. Alkaline phostase-conjugated sheep anti-mouse IgG antibody (manufactured by Zymed) was added thereto, left in an incubator at 37°C for 1 hour, washed with PBS (-), and then a coloring substrate (ALP) was added to develop color for 20 minutes.
- PBS phosphate buffer
- Alkaline phostase-conjugated sheep anti-mouse IgG antibody manufactured by Zymed
- Antibody-producing cell lines were cloned by limiting dilution from the cells in the positive wells in which reactivity with the antigen peptide was confirmed by the ELISA method in (5). That is, the cells in the positive wells were seeded into each well of a 96-well culture plate and cultured in a 5% CO 2 ⁇ 37° C. incubator for 2 weeks. The culture supernatant of each well was tested 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).
- the positive wells were cloned again by the limiting dilution method, and cells with high reactivity with the antigen peptide and good cell colony growth were obtained. These cells were transferred to a 24-well culture plate and cultured in a 5% CO2 /37°C incubator for 2 weeks. From these, cells in 5 wells, ie, 5 hybridoma cell lines (A, B, C, D, E) were selected. The selection was performed by an immunoprecipitation test according to Test Example 2 described later.
- This cell suspension was injected into the peritoneal cavity of BALB/c mice pretreated with pristane, and after about 3 weeks, leaking ascites was collected from the abdomen with a syringe. After filtering the collected ascites using a filter with a pore size of 0.22 ⁇ m ⁇ , the filtrate was purified by affinity chromatography using a protein G-Sepharose column (Millipore, 11511324) according to a conventional method, and anti-human periostin-exon 21 monoclonal antibody 5 was added. Seeds (antibodies AE) were prepared.
- Test example 2 Human Periostin-Exon 21 Recognition Test 1 Monoclonal antibodies (antibodies A and B) obtained from hybridoma A and hybridoma B, respectively, and antibody #8 (antibody-producing cell line (hybridoma) No. 8 (independent administrative agency product evaluation) reported to be produced in Patent Document 1 From the international deposit at the National Institute of Technology Patent Microorganisms Depositary (NPMD) (acceptance date: February 26, 2013, accession number: NITE BP-01546, identification: KS-0259#8,080611 Kohjin Bio) Using the produced antibody), a binding test to human periostin secreted from human cells was performed, specifically as follows.
- immunoprecipitation was performed using 1.0 ml of the supernatant. After binding 5 ⁇ g each of antibody A, antibody B, antibody #8, and control mouse IgG antibody to immunoprecipitation beads (Invitrogen immunoprecipitation kit - Dynabeads Protein G cat #10007D), incubate with culture supernatant overnight at 4°C. bottom. After that, the proteins bound to each antibody were extracted and subjected to Western blotting using an antibody against periostin/exon 12 antigen (N-terminal antigen of periostin) as a primary antibody to confirm the binding of each exon 21 antibody to periostin.
- immunoprecipitation beads Invitrogen immunoprecipitation kit - Dynabeads Protein G cat #10007D
- Test example 3 Determination of Antibody CDR Sequences From the genomic information of the hybridomas (A to E) cloned in Test Example 1, the amino acid sequences of antibodies A to E were determined. The amino acid sequences of the heavy chain variable regions and light chain variable regions of antibodies A to E are as follows (SEQ ID numbers are shown in parentheses at the end of the sequences).
- the amino acid sequence was obtained from databases of amino acid sequences of known antibodies (IgBLAST website: https://www.ncbi.nlm.nih.gov/igblast/ and IMGT website: http://www.imgt. org/) to determine the amino acid sequences of the CDRs. That is, "CDR sequences defined by Kabat” were determined according to comparison with the database of the IgBLAST website, and "CDR sequences defined by IMGT” were determined according to comparison with the database of the IMGT website.
- the CDR sequences defined by Kabat (Kabat) and CDR sequences defined by IMGT (IMGT) for antibodies AE, respectively, are shown in Tables 1-5.
- Test example 4 Determination of epitopes within exon 21
- Epitopes within human periostin-exon 21 were determined for antibodies AE. Specifically, it was carried out as follows.
- antibodies A to E recognize the three-dimensional structure on human periostin-exon 21, which is composed of amino acid sequence A and amino acid sequence B shown in the table below.
- Vascular mimicry test 1 A phenomenon has been reported in which tumor mimics blood vessels in cancer cells and allows 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 such as breast cancer, and has been reported as a poor prognostic factor. In addition, it has been reported to evaluate tube formation of cancer cells in a vascular mimic model of this cultured cell system. Therefore, the antibody was compared and examined using this culture model.
- Mouse mammary carcinoma cells 4T07 were plated on u-Slide angiogenesis plates coated with Matrigel (Corning Matrigel Matrix Growth Factor Reduced 5 ml cat#356230) to induce vascular mimicry.
- RPMI-1640 was used as a culture medium in a serum-free state, and the number of seeded cells was 3 ⁇ 10 5 cells/ml.
- Mouse periostin (PN2) and periostin antibody (antibody #8, antibody A) were used as drugs to be added.
- a final concentration of 10 ⁇ g/ml PN2 and 20 ⁇ g/ml antibody was used and analyzed for vascular mimicry after overnight incubation with 4T07 cells.
- Image J Angiogenesis Analyzer was used for analysis.
- Test example 6 Cancer cell transplantation test
- Mouse breast cancer cells 4T07 were seeded on tissue culture treated 10 cm dishes (Greiner) using RPMI1640 (Gibco) containing 10% bovine serum albumin (Invitrogen) and Penicillin-Streptomycin Mixed Solution (Nacalai Tesque). was cultured in an incubator for 24 hours. Thereafter, the culture supernatant was removed, washed with PBS, and suspended with trypsin/EDTA. The cells were collected, centrifuged for 5 minutes, counted to 1 ⁇ 10 6 breast cancer cells/mouse, and suspended in 100 ⁇ l of PBS. The conditioned cells were implanted into mammary glands of 8-week-old female BALB/c nude mice using a 25G injection needle.
- tumor volume diameter x minor axis 2 x 0.5. was calculated, and when the volume reached 100 mm 3 , they were divided into 3 groups: a control group, an antibody #8-administered group, and an antibody A-administered group (6 animals each). After that, 10 mg/kg of the antibody was administered twice/week through the tail vein using a 30G injection needle, and the subsequent tumor volume was compared and examined.
- Test example 7 Human Periostin-Exon 21 Recognition Test 2 Antibodies A to E were tested for binding to human periostin secreted from human cells in the same manner as in Test Example 2 of the present application.
- Test example 8 Vascular mimicry test 2 Antibodies A, B, C, and D were evaluated in the same manner as in Test Example 5 of the present application for cancer cell tube formation in a blood vessel mimic model. However, as an index of tube formation, total master segments length was measured.
- Test example 9 Vascular mimicry test 3 Antibodies A, B, C, and D were evaluated in the same manner as in Test Example 5 of the present application for cancer cell tube formation in a blood vessel mimic model. However, as an index of tube formation, the total mesh area (total area of tube formed tube) was measured.
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Abstract
Description
重鎖CDR2が配列番号3及び/若しくは4、配列番号15及び/若しくは16、配列番号27及び/若しくは28、配列番号39及び/若しくは40、又は配列番号51及び/若しくは52で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号5及び/若しくは6、配列番号17及び/若しくは18、配列番号29及び/若しくは30、配列番号41及び/若しくは42、又は配列番号53及び/若しくは54で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号7及び/若しくは8、配列番号19及び/若しくは20、配列番号31及び/若しくは32、配列番号43及び/若しくは44、又は配列番号55及び/若しくは56で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号9及び/若しくは10、配列番号21及び/若しくは22、配列番号33及び/若しくは34、配列番号45及び/若しくは46、又は配列番号57及び/若しくは58で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号11及び/若しくは12、配列番号23及び/若しくは24、配列番号35及び/若しくは36、配列番号47及び/若しくは48、又は配列番号59及び/若しくは60で表されるアミノ酸配列を含む、
項1~4のいずれかに記載の抗体又はその抗原結合性断片。
重鎖CDR1が配列番号1及び/若しくは2で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号3及び/若しくは4で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号5及び/若しくは6で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号7及び/若しくは8で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号9及び/若しくは10で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号11及び/若しくは12で表されるアミノ酸配列を含む;或いは(B)
重鎖CDR1が配列番号13及び/若しくは14で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号15及び/若しくは16で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号17及び/若しくは18で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号19及び/若しくは20で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号21及び/若しくは22で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号23及び/若しくは24で表されるアミノ酸配列を含む;或いは(C)
重鎖CDR1が配列番号25及び/若しくは26で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号27及び/若しくは28で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号29及び/若しくは30で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号31及び/若しくは32で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号33及び/若しくは34で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号35及び/若しくは36で表されるアミノ酸配列を含む;或いは(D)
重鎖CDR1が配列番号37及び/若しくは38で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号39及び/若しくは40で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号41及び/若しくは42で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号43及び/若しくは44で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号45及び/若しくは46で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号47及び/若しくは48で表されるアミノ酸配列を含む;或いは(E)
重鎖CDR1が配列番号49及び/若しくは50で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号51及び/若しくは52で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号53及び/若しくは54で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号55及び/若しくは56で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号57及び/若しくは58で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号59及び/若しくは60で表されるアミノ酸配列を含む、
項1~5のいずれかに記載の抗体又はその抗原結合性断片。
重鎖CDR1が配列番号1及び/若しくは2で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号3及び/若しくは4で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号5及び/若しくは6で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号7及び/若しくは8で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号9及び/若しくは10で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号11及び/若しくは12で表されるアミノ酸配列を含む、
項1~6のいずれかに記載の抗体又はその抗原結合性断片。
(f)配列番号65で示されるアミノ酸配列と85%以上の同一性を有するアミノ酸配列Xを含み、且つ前記アミノ酸配列X内に配列番号61で示されるアミノ酸配列A及び配列番号62~64のいずれかで示されるアミノ酸配列Bを含む、タンパク質又はペプチド
からなる群より選択される少なくとも1種のタンパク質又はペプチド免疫した動物から得られたハイブリドーマが産生する抗体から、ヒト乳がん細胞から分泌されたヒトペリオスチンに結合する抗体を選別する工程を含む方法により得られた、抗体。
(f)配列番号65で示されるアミノ酸配列と85%以上の同一性を有するアミノ酸配列Xを含み、且つ前記アミノ酸配列X内に配列番号61で示されるアミノ酸配列A及び配列番号62~64のいずれかで示されるアミノ酸配列Bを含む、タンパク質又はペプチド
からなる群より選択される少なくとも1種のタンパク質又はペプチド免疫した動物から得られたハイブリドーマから産生され、且つヒト乳がん細胞から分泌されたヒトペリオスチンに対する結合性を有する、抗体。
本発明は、その一態様において、配列番号61で示されるアミノ酸配列Aと配列番号62~64のいずれかで示されるアミノ酸配列Bとから構成される、ヒトぺリオスチン上の立体エピトープに対して結合性を有する、抗体又はその抗原結合性断片(本明細書において、「本発明の抗体又はその断片」と示すこともある。)、に関する。以下に、これについて説明する。
例えば下記(a)に記載するタンパク質及び下記(b)に記載するタンパク質:
(a)配列番号65で示されるアミノ酸配列を含むタンパク質、及び
(b)配列番号65で示されるアミノ酸配列と85%以上の同一性を有するアミノ酸配列Xを含み、且つ前記アミノ酸配列X内に配列番号61で示されるアミノ酸配列A及び配列番号62~64のいずれかで示されるアミノ酸配列Bを含む、タンパク質
からなる群より選択される少なくとも1種のヒトぺリオスチンが挙げられ、
好ましくは下記(c)に記載するタンパク質及び下記(d)に記載するタンパク質: (c)配列番号66で示されるアミノ酸配列からなるタンパク質、及び
(d)配列番号66で示されるアミノ酸配列と85%以上の同一性を有するアミノ酸配列からなり、且つ配列番号65で示されるアミノ酸配列を含むタンパク質
からなる群より選択される少なくとも1種のヒトぺリオスチンが挙げられる。
(b’)配列番号65で示されるアミノ酸配列に対して1若しくは複数個のアミノ酸が置換、欠失、付加、又は挿入(好ましくは置換、より好ましくは保存的置換)されたアミノ酸配列Xを含み、且つ前記アミノ酸配列X内に配列番号61で示されるアミノ酸配列A及び配列番号62~64のいずれかで示されるアミノ酸配列Bを含む、タンパク質
(d’)配列番号66で示されるアミノ酸配列に対して1若しくは複数個のアミノ酸が置換、欠失、付加、又は挿入(好ましくは置換、より好ましくは保存的置換)されたアミノ酸配列からなり、且つ配列番号65で示されるアミノ酸配列を含むタンパク質
が挙げられる。
重鎖CDR1が配列番号1及び/若しくは2、配列番号13及び/若しくは14、配列番号25及び/若しくは26、配列番号37及び/若しくは38、又は配列番号49及び/若しくは50で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号3及び/若しくは4、配列番号15及び/若しくは16、配列番号27及び/若しくは28、配列番号39及び/若しくは40、又は配列番号51及び/若しくは52で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号5及び/若しくは6、配列番号17及び/若しくは18、配列番号29及び/若しくは30、配列番号41及び/若しくは42、又は配列番号53及び/若しくは54で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号7及び/若しくは8、配列番号19及び/若しくは20、配列番号31及び/若しくは32、配列番号43及び/若しくは44、又は配列番号55及び/若しくは56で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号9及び/若しくは10、配列番号21及び/若しくは22、配列番号33及び/若しくは34、配列番号45及び/若しくは46、又は配列番号57及び/若しくは58で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号11及び/若しくは12、配列番号23及び/若しくは24、配列番号35及び/若しくは36、配列番号47及び/若しくは48、又は配列番号59及び/若しくは60で表されるアミノ酸配列を含む、
抗体又はその断片であることができる。
(A)
重鎖CDR1が配列番号1及び/若しくは2で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号3及び/若しくは4で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号5及び/若しくは6で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号7及び/若しくは8で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号9及び/若しくは10で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号11及び/若しくは12で表されるアミノ酸配列を含む;或いは(B)
重鎖CDR1が配列番号13及び/若しくは14で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号15及び/若しくは16で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号17及び/若しくは18で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号19及び/若しくは20で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号21及び/若しくは22で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号23及び/若しくは24で表されるアミノ酸配列を含む;或いは(C)
重鎖CDR1が配列番号25及び/若しくは26で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号27及び/若しくは28で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号29及び/若しくは30で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号31及び/若しくは32で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号33及び/若しくは34で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号35及び/若しくは36で表されるアミノ酸配列を含む;或いは(D)
重鎖CDR1が配列番号37及び/若しくは38で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号39及び/若しくは40で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号41及び/若しくは42で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号43及び/若しくは44で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号45及び/若しくは46で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号47及び/若しくは48で表されるアミノ酸配列を含む;或いは(E)
重鎖CDR1が配列番号49及び/若しくは50で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号51及び/若しくは52で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号53及び/若しくは54で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号55及び/若しくは56で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号57及び/若しくは58で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号59及び/若しくは60で表されるアミノ酸配列を含む、
抗体又はその抗原結合性断片であることができる。
(A1)
重鎖CDR1が配列番号1で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号3で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号5で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号7で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号9で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号11で表されるアミノ酸配列を含む;或いは
(A2)
重鎖CDR1が配列番号2で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号4で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号6で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号8で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号10で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号12で表されるアミノ酸配列を含む;或いは
(B1)
重鎖CDR1が配列番号13で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号15で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号17で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号19で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号21で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号23で表されるアミノ酸配列を含む;或いは
(B2)
重鎖CDR1が配列番号14で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号16で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号18で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号20で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号22で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号24で表されるアミノ酸配列を含む;或いは
(C1)
重鎖CDR1が配列番号25で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号27で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号29で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号31で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号33で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号35で表されるアミノ酸配列を含む;或いは
(C2)
重鎖CDR1が配列番号26で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号28で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号30で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号32で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号34で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号36で表されるアミノ酸配列を含む;或いは
(D1)
重鎖CDR1が配列番号37及び/若しくは38で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号39及び/若しくは40で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号41及び/若しくは42で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号43及び/若しくは44で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号45及び/若しくは46で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号47及び/若しくは48で表されるアミノ酸配列を含む;或いは(D2)
重鎖CDR1が配列番号38で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号40で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号42で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号44で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号46で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号48で表されるアミノ酸配列を含む;或いは
(E1)
重鎖CDR1が配列番号49で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号51で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号53で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号55で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号57で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号59で表されるアミノ酸配列を含む;或いは
(E2)
重鎖CDR1が配列番号50で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号52で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号54で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号56で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号58で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号60で表されるアミノ酸配列を含む、
抗体又はその抗原結合性断片であることができる。
本発明の抗体又はその断片は、様々な方法で製造することができる。
(e)配列番号65で示されるアミノ酸配列を含むタンパク質又はペプチド、及び
(f)配列番号65で示されるアミノ酸配列と85%以上の同一性を有するアミノ酸配列Xを含み、且つ前記アミノ酸配列X内に配列番号61で示されるアミノ酸配列A及び配列番号62~64のいずれかで示されるアミノ酸配列Bを含む、タンパク質又はペプチド
からなる群より選択される少なくとも1種のタンパク質又はペプチド免疫した動物から得られたハイブリドーマが産生する抗体から、ヒト乳がん細胞から分泌されたヒトペリオスチンに結合する抗体を選別する工程を含む方法によって、製造することができる。
(e)配列番号65で示されるアミノ酸配列を含むタンパク質又はペプチド、及び
(f)配列番号65で示されるアミノ酸配列と85%以上の同一性を有するアミノ酸配列Xを含み、且つ前記アミノ酸配列X内に配列番号61で示されるアミノ酸配列A及び配列番号62~64のいずれかで示されるアミノ酸配列Bを含む、タンパク質又はペプチド
からなる群より選択される少なくとも1種のタンパク質又はペプチド免疫した動物から得られたハイブリドーマが産生する抗体から、ヒト乳がん細胞から分泌されたヒトペリオスチンに結合する抗体を選別する工程を含む方法により得られた、抗体、であることができる。
(e)配列番号65で示されるアミノ酸配列を含むタンパク質又はペプチド、及び
(f)配列番号65で示されるアミノ酸配列と85%以上の同一性を有するアミノ酸配列Xを含み、且つ前記アミノ酸配列X内に配列番号61で示されるアミノ酸配列A及び配列番号62~64のいずれかで示されるアミノ酸配列Bを含む、タンパク質又はペプチド
からなる群より選択される少なくとも1種のタンパク質又はペプチド免疫した動物から得られたハイブリドーマから産生され、且つヒト乳がん細胞から分泌されたヒトペリオスチンに対する結合性を有する、抗体、であることができる。
本発明の抗体をヒト型抗体として得る場合は、以下の情報に基づいて、必要に応じてその他の公知の情報に従って又は準じて、作製することができる。
(a)アクセプターのフレームワーク領域中のアミノ酸がその位置において稀であり、ドナーの対応するアミノ酸がアクセプターの前記位置において普通であること
(b)該アミノ酸がCDRのひとつのすぐ近くであること
(c)該アミノ酸が三次元免疫グロブリンモデルにおいてCDRの約3Å(オングストローム)以内に側鎖原子を有し、そして抗原と又はヒト化抗体のCDRと相互作用することができると予想されること。
本発明の抗体をヒト抗体として得る場合は、以下の情報に基づいて、必要に応じてその他の公知の情報に従って又は準じて、作製することができる。
本発明は、その一態様において、本発明のポリヌクレオチドを含有する、細胞、に関する。
本発明は、その一態様において、本発明の抗体又はその断片と薬剤との複合体、に関する。
本発明は、その一態様において、本発明の抗体又はその断片、及び本発明の複合体からなる群より選択される少なくとも1種を含有する、医薬組成物、試薬等、に関する。
いるものを使用することができる。蛍光色素としては、フルオレッセンスイソチオシアネート(FITC)やテトラメチルローダミンイソチオシアネート(TRITC)等の通常の蛍光抗体法に用いられるものを使用することができる。また、本診断薬は、癌細胞と周辺の線維芽細胞を特異的に染色することが可能な、免疫組織学的染色として使用することができる。また、放射性同位体を標識した場合には、体内に投与することによって、癌病態時等の病変部を画像化するために使用することもできる。
(1)抗原の作製
ヒトペリオスチンエクソン21を構成するアミノ酸配列(配列番65:EVTKVTKFIEGGDGHLFEDEEIKRLLQG)のN末端にCys残基を付加したペプチドを、Fmoc法にて化学合成し、純度90%以上の抗原ペプチド10mgを得た。この抗原ペプチド5mgにキャリアータンパク質としてKLH(CALBIOCHEM社製)5mgを結合させ、抗原溶液を得た。すなわち、KLHをPBS(0.01M)に溶解して3.3mg/mLに調整し、0.2524mg/mLのMBS溶液(GEヘルスケアバイオサイエンス社製)を滴下して室温で60分間攪拌し反応させた。ジクロロメタンを用いてフリーのMBSを除き、KLH-MBを得た。このKLH-MB5mgと、0.01Mリン酸ナトリウム緩衝液(pH7.2)に溶解した抗原ペプチド5mgとを混合し、4℃で12時間攪拌して反応させ、抗原溶液を得た。
6週齢のBALB/c雌性マウス3匹の両足に、(1)で得られたKLH結合抗原ペプチド100μgを含む抗原溶液50μlと、FCA(フロイント完全アジュバント)50μlとの混合乳濁液全量を皮下注射した。その後2週間間隔で2回、用時調製した上記抗原溶液とFIA(フロイント不完全アジュバント)との混合乳濁液を両足に投与した。その後、そのマウスを頸椎脱臼により致死させ、無菌的に足部リンパ節を採取した。
8-アザグアニン耐性でかつイムノグロブリン非分泌型のマウスミエローマ細胞株P3X63Ag8U.1(P3U1株)を、20%のウシ胎児血清(FCS)を含むRPMI培地で、10%CO2・37℃インキュベーター内で培養し、対数増殖期にある細胞を集め、1,000rpm、5分間の遠心分離にかけて沈殿画分として細胞のみを取得し、RPMI培地に懸濁させた。
(2)で得た免疫化リンパ節細胞1×108~3×108個を含むRPMI培地と、(3)で得たミエローマ細胞108個を含むRPMI培地とを混合した後、1,000rpm、10分間の遠心分離にかけた。上清を静かに除いて沈殿画分として細胞を取得し、これに25%(w/v)のポリエチレングリコール1500(PEG 1500、ベーリンガー社製)1mlを加えた後、更にRPMI培地をゆっくりと加えて総量を10mlとした。これに20% FCSを含むRPMI培地10mlを加えて、少し静置させた後、1,000rpm、5分間の遠心分離にかけ、得られた沈殿画分(細胞画分)に20% FCSを含むRPMI培地を加えて細胞濃度が106個/mlになるように調整した細胞懸濁液を、コーニング社製の96穴培養プレートに200μL/ウェルずつ分注した。5%CO2・37℃インキュベーター中で24時間培養後、HAT溶液(インビトロジェン社製)を添加した後、更に2週間培養した。
培養上清が抗原ペプチドと反応する陽性ウェルのスクリーニングを行った。アッセイ用の抗原溶液には、(1)で得られた抗原ペプチド、ヒトぺリオスチンPN1(全長)、又はマウスぺリオスチンPN2(エクソン17欠損)2mgに、キャリアータンパク質として卵白アルブミン(OVA)を結合させたコンジュゲートを用いた。
(5)のELISA法で抗原ペプチドとの反応性が確認された陽性ウェル内の細胞から、限界希釈法により、抗体産生細胞株のクローニングを行った。すなわち、陽性ウェル内の細胞を96穴培養プレートの各ウェルに撒き込み、5%CO2・37℃インキュベーター内で2週間培養した。各ウェルの培養上清について、(5)の方法と同様に、ELISA法にて、抗原ペプチド、ヒトぺリオスチンPN1(全長)、又はマウスぺリオスチンPN2(エクソン17欠損)との反応性を確認し、陽性ウェルについて、再度、限界希釈法によるクローニングを行い、抗原ペプチドとの反応性が高く、細胞コロニーの発育が良好な細胞を得た。これら細胞を24穴培養プレートに移し、5%CO2・37℃インキュベーター内で2週間培養した。これらの中から、5ウェル内の細胞、すなわち5個のハイブリドーマ細胞株(A、B、C、D、E)を、選別した。なお、選別は、後述の試験例2に従った免疫沈降試験により行った。
BALB/cマウスの腹腔内にプリスタン[2,6,10,14-テトラメチルペンタデカン(和光純薬製)]0.5mlを投与し、2~3週間飼育した。予め、対数増殖期に維持しておいたモノクローナル抗体産生ハイブリドーマA~Eを回収し、培養上清を除いた沈殿画分の細胞にFCS不含のRPMI培地を加え、細胞数が1×107個/mlになるように細胞液を調製した。この細胞液を、プリスタン前投与したBALB/cマウスの腹腔中に注入し、3週間後頃から漏出した腹水を腹部より注射器で回収した。採取した腹水を、孔径0.22μmφのフィルターを用いて濾過した後、濾液をプロテインG-セファロースカラム(Millipore、11511324)によるアフィニティークロマトグラフィーによって常法に従い精製し、抗ヒトぺリオスチン-エクソン21モノクローナル抗体5種(抗体A~E)を調製した。
ハイブリドーマA及びハイブリドーマBそれぞれから得られたモノクローナル抗体(抗体A及びB)と、特許文献1で作製が報告されている抗体#8(抗体産生細胞株(ハイブリドーマ)No.8(独立行政法人製品評価技術基盤機構特許微生物寄託センター(NPMD)に国際寄託されている(受託日:2013年2月26日、受託番号:NITE BP-01546、識別の表示:KS-0259#8,080611 Kohjin Bio)から産生される抗体)を使用して、ヒト細胞から分泌されるヒトぺリオスチンに対する結合試験を行った。具体的には以下のようにして行った。
試験例1でクローニングしたハイブリドーマ(A~E)のゲノム情報より、抗体A~Eのアミノ酸配列を決定した。抗体A~Eの重鎖可変領域(Heavy chain variable region)及び軽鎖可変領域(Light chain variable region)のアミノ酸配列は以下のとおりである(配列番号を、配列末尾のカッコ内に示す。)。
Heavy chain variable region
KVQLVQSGAEVVKPGASVKLSCKASGYTFTEYIIHWVKQASGQGLEWIGWFYPGRGIIKYNEKFKDKATLTADKSISTVYMDLSRLRSEDTAVYFCARHGITTATGAMDYWGQGTLVTVSS(配列番号67) Light chain variable region
DVVMTQTPLSLSVTLGQPASISCRSSQSLLHSNGNTYLHWYLQKPGQAPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPFTFGQGTKLEIK(配列番号68)。
Heavy chain variable region
EVQLVESGGGLVKPGGSLRLSCAASGITFSSYAMSWVRQAPGKGLEWVASISSGGSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVAIWDGSSYGYWGQGTTVTVSS(配列番号69) Light chain variable region
DIVMTQTPLSLSVTPGQPASISCKSGQSLVHSNGKTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPFTFGQGTKLEIK(配列番号70)。
Heavy chain variable region
EVQLVESGGGLVKPGGSLRLSCAASGFIFSSYAMSWVRQPPGKRLEWVASIGSGGTTYYPDSVRGRFTISRDNARNILYLQMRSLRAEDTAVYYCAAIWDGSSHGYWGQGTLLTVSS(配列番号71) Light chain variable region
DVVMTQSPLSLPVTLGQPASISCRSSQNLVHSNGNTYLHWYLQRPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCSQSTHVPYTFGPGTKLEIK(配列番号72)。
Heavy chain variable region
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQTPEKRLEWVASINSGGVTTYYPDSVKGRFTISRDNAINILYLQMRSLRSEDTALYYCAAIWDGSSHGYWGQGTLVTVSS(配列番号73) Light chain variable region
DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYLQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDFGVYFCSQSTHVPYTFGPGTKLEIK(配列番号74)。
Heavy chain variable region
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDAWMDWVRQAPGKGLEWVAEIRNKANNHATYFTESVKGRFTISRDDSKNTVYLQMNSLRAEDTAVYYCTTATFVYWGQGTLVTVSS(配列番号75) Light chain variable region
DVVMTQSPLSLPVSLGDRASISCRSSQSLVRNGITYLHWYQQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISSVEAEDFGVYYCSQTPHVPYTFGQGTKLEIK(配列番号76)。そのアミノ酸配列を、既知の抗体のアミノ酸配列のデータベース(IgBLASTのウェブサイト:https://www.ncbi.nlm.nih.gov/igblast/、及びIMGTのウェブサイト:http://www.imgt.org/)と比較して相同性を調べることにより、CDRのアミノ酸配列を決定した。すなわち、IgBLASTのウェブサイトのデータベースとの比較に従って「Kabatによって定義されるCDR配列」を決定し、IMGTウェブサイトのデータベースとの比較に従って「IMGTによって定義されるCDR配列」を決定した。抗体A~Eそれぞれの、Kabatによって定義されるCDR配列(Kabat)と、IMGTによって定義されるCDR配列(IMGT)を、表1~5に示す。
抗体A~Eについて、ヒトぺリオスチン-エクソン21内のエピトープを決定した。具体的には以下のようにして行った。
癌細胞内で腫瘍が血管に擬態して、血流を腫瘍内外に通す現象が報告されている。この現象は、血管擬態(Vasculogenic Mimicry)と呼ばれている(Hendrix et al. Nature. 520, pages300-302 (2015))。この現象は乳癌などの臨床症例にも観察されており、予後不良因子として報告されている。また、この培養細胞系の血管擬態モデルにおいて、癌細胞の管腔形成を評価することが報告されている。そこで、この培養モデルを用いて抗体を比較検討した。
マウス乳癌細胞4T07を、10%牛血清アルブミン(Invitrogen)及びPenicillin-Streptomycin Mixed Solution(ナカライテスク)含有RPMI1640(Gibco)を用いてtissue culture treated 10cm dishes(グライナー)に播種し、37度にてインキュベーターにて24時間培養した。その後、培養上清を除去しPBSにて洗浄した後にトリプシン/EDTAにより浮遊させた。細胞を回収し5分間遠心した後、乳癌細胞1x106個/匹になるようにカウントし100μlのPBSに懸濁した。調整した細胞を25G注射針を用いてBALB/cヌードマウス8週齢の雌の乳腺に移植した。移植を行った1週間後から細胞が生着した乳腺部位をノギスで直径(mm)と短径(mm)を測定し、計算式:腫瘍体積=直径×短径2×0.5 を用いて腫瘍体積を算出し、体積が100mm3となった時点でコントール群、抗体#8投与群、及び抗体A投与群の3群に振り分けた(各6匹)。その後、抗体10mg/kg×2回/週で30G注射針を用いて尾静脈より投与し、その後の腫瘍体積を比較検討した。
抗体A~Eについて、ヒト細胞から分泌されるヒトぺリオスチンに対する結合試験を本願試験例2と同様にして行った。
抗体A、B、C、及びDについて、血管擬態モデルにおける癌細胞の管腔形成を本願試験例5と同様にして評価した。但し、管腔形成の指標としては、Total master segments lengthを測定した。
抗体A、B、C、及びDについて、血管擬態モデルにおける癌細胞の管腔形成を本願試験例5と同様にして評価した。但し、管腔形成の指標としては、Total mesh area(管腔形成された管腔の総面積)を測定した。
Claims (15)
- 配列番号61で示されるアミノ酸配列Aと配列番号62~64のいずれかで示されるアミノ酸配列Bとから構成される、ヒトぺリオスチン上の立体エピトープに対して結合性を有する、抗体又はその抗原結合性断片。
- 前記ヒトぺリオスチンがヒト細胞から分泌されたヒトぺリオスチンである、請求項1に記載の抗体又はその抗原結合性断片。
- 前記ヒト細胞がヒト乳癌細胞である、請求項1又は2に記載の抗体又はその抗原結合性断片。
- モノクローナル抗体又はその抗原結合性断片である、請求項1~3のいずれかに記載の抗体又はその抗原結合性断片。
- 重鎖CDR1が配列番号1及び/若しくは2、配列番号13及び/若しくは14、配列番号25及び/若しくは26、配列番号37及び/若しくは38、又は配列番号49及び/若しくは50で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号3及び/若しくは4、配列番号15及び/若しくは16、配列番号27及び/若しくは28、配列番号39及び/若しくは40、又は配列番号51及び/若しくは52で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号5及び/若しくは6、配列番号17及び/若しくは18、配列番号29及び/若しくは30、配列番号41及び/若しくは42、又は配列番号53及び/若しくは54で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号7及び/若しくは8、配列番号19及び/若しくは20、配列番号31及び/若しくは32、配列番号43及び/若しくは44、又は配列番号55及び/若しくは56で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号9及び/若しくは10、配列番号21及び/若しくは22、配列番号33及び/若しくは34、配列番号45及び/若しくは46、又は配列番号57及び/若しくは58で表されるアミノ酸配列を含み、且つ
軽鎖CDR3が配列番号11及び/若しくは12、配列番号23及び/若しくは24、配列番号35及び/若しくは36、配列番号47及び/若しくは48、又は配列番号59及び/若しくは60で表されるアミノ酸配列を含む、
請求項1~4のいずれかに記載の抗体又はその抗原結合性断片。 - (A)
重鎖CDR1が配列番号1及び/若しくは2で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号3及び/若しくは4で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号5及び/若しくは6で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号7及び/若しくは8で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号9及び/若しくは10で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号11及び/若しくは12で表されるアミノ酸配列を含む;或いは(B)
重鎖CDR1が配列番号13及び/若しくは14で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号15及び/若しくは16で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号17及び/若しくは18で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号19及び/若しくは20で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号21及び/若しくは22で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号23及び/若しくは24で表されるアミノ酸配列を含む;或いは(C)
重鎖CDR1が配列番号25及び/若しくは26で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号27及び/若しくは28で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号29及び/若しくは30で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号31及び/若しくは32で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号33及び/若しくは34で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号35及び/若しくは36で表されるアミノ酸配列を含む;或いは(D)
重鎖CDR1が配列番号37及び/若しくは38で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号39及び/若しくは40で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号41及び/若しくは42で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号43及び/若しくは44で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号45及び/若しくは46で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号47及び/若しくは48で表されるアミノ酸配列を含む;或いは(E)
重鎖CDR1が配列番号49及び/若しくは50で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号51及び/若しくは52で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号53及び/若しくは54で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号55及び/若しくは56で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号57及び/若しくは58で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号59及び/若しくは60で表されるアミノ酸配列を含む、
請求項1~5のいずれかに記載の抗体又はその抗原結合性断片。 - (A)
重鎖CDR1が配列番号1及び/若しくは2で表されるアミノ酸配列を含み、
重鎖CDR2が配列番号3及び/若しくは4で表されるアミノ酸配列を含み、
重鎖CDR3が配列番号5及び/若しくは6で表されるアミノ酸配列を含み、
軽鎖CDR1が配列番号7及び/若しくは8で表されるアミノ酸配列を含み、
軽鎖CDR2が配列番号9及び/若しくは10で表されるアミノ酸配列を含み、且つ 軽鎖CDR3が配列番号11及び/若しくは12で表されるアミノ酸配列を含む、
請求項1~6のいずれかに記載の抗体又はその抗原結合性断片。 - (e)配列番号65で示されるアミノ酸配列を含むタンパク質又はペプチド、及び
(f)配列番号65で示されるアミノ酸配列と85%以上の同一性を有するアミノ酸配列Xを含み、且つ前記アミノ酸配列X内に配列番号61で示されるアミノ酸配列A及び配列番号62~64のいずれかで示されるアミノ酸配列Bを含む、タンパク質又はペプチド
からなる群より選択される少なくとも1種のタンパク質又はペプチド免疫した動物から得られたハイブリドーマが産生する抗体から、ヒト乳がん細胞から分泌されたヒトペリオスチンに結合する抗体を選別する工程を含む方法により得られた、抗体。 - (e)配列番号65で示されるアミノ酸配列を含むタンパク質又はペプチド、及び
(f)配列番号65で示されるアミノ酸配列と85%以上の同一性を有するアミノ酸配列Xを含み、且つ前記アミノ酸配列X内に配列番号61で示されるアミノ酸配列A及び配列番号62~64のいずれかで示されるアミノ酸配列Bを含む、タンパク質又はペプチド
からなる群より選択される少なくとも1種のタンパク質又はペプチド免疫した動物から得られたハイブリドーマから産生され、且つヒト乳がん細胞から分泌されたヒトペリオスチンに対する結合性を有する、抗体。 - 請求項1~9のいずれかに記載の抗体又はその抗原結合性断片のコード配列を含む、ポリヌクレオチド。
- 請求項10に記載のポリヌクレオチドを含有する、細胞。
- 請求項1~9のいずれかに記載の抗体又はその抗原結合性断片と薬剤との複合体。
- 請求項1~9のいずれかに記載の抗体又はその抗原結合性断片、及び請求項12に記載の複合体からなる群より選択される少なくとも1種を含有する、医薬組成物。
- 癌の予防若しくは治療用、癌の血管擬態抑制用、ぺリオスチン阻害用、炎症関連疾患の予防若しくは治療用、血管内膜肥厚抑制用、血管新生抑制用、又は動脈瘤の予防若しくは治療用である、請求項13に記載の医薬組成物。
- 請求項1~9のいずれかに記載の抗体又はその抗原結合性断片、及び請求項12に記載の複合体からなる群より選択される少なくとも1種を含有する、試薬。
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