WO2018199107A1 - 組織再生を誘導するためのペプチドとその利用 - Google Patents
組織再生を誘導するためのペプチドとその利用 Download PDFInfo
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- WO2018199107A1 WO2018199107A1 PCT/JP2018/016654 JP2018016654W WO2018199107A1 WO 2018199107 A1 WO2018199107 A1 WO 2018199107A1 JP 2018016654 W JP2018016654 W JP 2018016654W WO 2018199107 A1 WO2018199107 A1 WO 2018199107A1
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- gkmssyaffv qtcreehkkk
- qtcreehkkk hpdasvnfse
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- C—CHEMISTRY; METALLURGY
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0669—Bone marrow stromal cells; Whole bone marrow
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to a peptide for inducing tissue regeneration and use thereof.
- tissue stem cells that maintain their structural and functional homeostasis exist in each organ and tissue in the living body.
- cardiac stem cells exist in the heart
- neural stem cells in the brain and epidermal stem cells and hair follicle stem cells exist in the skin.
- cardiomyocytes, nerve cells, epidermal cells, and hair follicle epithelial cells are supplied throughout the life of the heart, brain Maintains the structure and function of the skin.
- hematopoietic stem cells that differentiate into blood cells such as red blood cells, white blood cells, and platelets exist in the bone marrow.
- Blood cells derived from these hematopoietic stem cells circulate through all organs and tissues in the body through the bloodstream, and perform essential functions for life support such as oxygen supply, immune reaction, hemostasis and repair of damaged tissues. That is, it can be said that bone marrow hematopoietic stem cells contribute to the maintenance of homeostasis of all tissues in the body through peripheral circulation rather than the maintenance of homeostasis of bone marrow and bone tissues, which are localized tissues.
- bone marrow contains mesenchymal stem cells that can differentiate into mesodermal tissues such as bone, cartilage, and fat, as well as ectoderm tissues such as nerves and epidermis, in addition to hematopoietic stem cells.
- mesenchymal stem cells that can differentiate into mesodermal tissues such as bone, cartilage, and fat, as well as ectoderm tissues such as nerves and epidermis, in addition to hematopoietic stem cells.
- mesenchyme is also present in the bone marrow.
- Stem cells also contribute to maintaining the homeostasis of living tissues by providing cells that can differentiate into bone, cartilage, fat, nerves, epithelium, etc. via peripheral circulation to the tissues and organs that need them. Expected to be.
- bone marrow mesenchymal stem cells are collected by bone marrow blood collection, proliferated by cell culture, and then transplanted into refractory tissue damage sites or peripheral blood to regenerate damaged tissues. Is being developed. Bone marrow mesenchymal stem cell transplantation has already been clinically applied in regenerative medicine such as cerebral infarction, myocardial infarction, and refractory skin ulcer. In addition, transplanted bone marrow mesenchymal stem cells have been shown to exert inflammatory / immune response inhibitory effects and fibrotic scar formation inhibitory effects locally in vivo, which is a serious side effect after bone marrow transplantation and blood transfusion.
- bone marrow mesenchymal stem cell transplantation therapy has been started as a new treatment for scleroderma, which is a host response (graft versus host disease, GVHD) and autoimmune disease.
- graft versus host disease GVHD
- autoimmune disease graft versus host disease, GVHD
- bone marrow blood containing bone marrow mesenchymal stem cells is collected only by the open technique of repeatedly inserting a thick needle into the iliac bone.
- bone marrow mesenchymal stem cells gradually lose their proliferation ability and pluripotency when they are subcultured in vitro.
- bone marrow mesenchymal stem cell culture based on high quality control that guarantees the safety of in vivo transplantation requires special culture facilities such as CPC (cell processing center).
- HMGB1 High mobility group group 1 protein
- A-box The N-terminal DNA binding domain
- B-box the C-terminal DNA binding domain
- HMGB1 protein is administered from the tail vein to mobilize PDGFR ⁇ -positive cells from the bone marrow and accumulate in the skin ulcer or cerebral infarction. It has been reported that infarct regeneration is strongly induced. It has already been reported that PDGFR ⁇ -positive cells in bone marrow are mesenchymal stem cells that can differentiate into bone, cartilage, fat, and even nerves and epithelium. In other words, by mobilizing PDGFR ⁇ -positive mesenchymal stem cells in the bone marrow to the peripheral circulation by administration of HMGB1, many mesenchymal stem cells can be accumulated in damaged tissues in vivo without taking them out of the body and performing special culture. It is possible.
- HMGB1 as a drug that induces regeneration of damaged tissue by mobilization of bone marrow mesenchymal stem cells in vivo, so that all medical institutions can perform regeneration-inducing medicine using bone marrow mesenchymal stem cells.
- many of the problems faced by the above-described regenerative medicine using bone marrow mesenchymal stem cells are solved.
- the HMGB1 drug is a revolutionary therapeutic agent that promotes the recruitment of bone marrow mesenchymal stem cells into blood and accumulation in damaged tissues, and induces tissue regeneration in the body.
- HMGB1 it has already been reported that not only HMGB1 full-length protein but also fragments of HMGB1 protein have similar effects.
- the problem to be solved by the present invention is to provide a peptide having migration activity of bone marrow-derived stromal cells, that is, a peptide for inducing tissue regeneration.
- the present inventors have found that in peptides consisting of amino acids 1 to 44 of HMGB1 protein, amino acids substituted at amino acids 43 and / or 44, and peptides 2 to 44 of HMGB1 protein.
- the peptide consisting of amino acids the peptide in which the amino acid was substituted at the 43rd and / or 44th amino acid was found to have migration activity of mouse bone marrow-derived stromal cells, and the present invention was completed.
- the present invention (1) a peptide having any amino acid sequence set forth in SEQ ID NOs: 1-399 and 403, In the peptide having any one of the amino acid sequences described in SEQ ID NOs: 1 to 399 and 403, one or more (for example, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5) Or a peptide having an amino acid sequence to which four, three or two amino acids have been added and having an activity of stimulating cell migration, and any one of the amino acid sequences of SEQ ID NOS: 1 to 399, 1 to 42 amino acids of the peptide having the amino acid sequence, or 1 to 41 amino acids of the peptide having the amino acid sequence set forth in SEQ ID NO: 403, one or more (eg, 2 to 40, 2 to 30, 2 to 20) 2-10, 2-5, or 4, 3 or 2 amino acid sequences comprising or consisting of amino acid substitutions, deletions or insertions, and A peptide selected from the group consisting of peptides having an activity of stimulating ves, and
- a peptide having any one of the amino acid sequences of SEQ ID NOs: 1 to 5 and 403 In the peptide having any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 5 and 403, one or more (for example, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5) Or a peptide having an amino acid sequence to which four, three or two amino acids have been added, and having an activity of stimulating cell migration, and any one of the amino acid sequences of SEQ ID NOS: 1 to 5.
- the peptide of the present invention is used for, for example, a composition used for stimulating the migration of PDGFR ⁇ -positive cells, a composition used for mobilizing bone marrow mesenchymal stem cells from the bone marrow to peripheral blood, and a tissue regeneration.
- the composition can be used as an active ingredient of a composition used to stimulate migration of bone marrow-derived stromal cells.
- Fig. 1 shows the plasma of 6 types of peptides (2nd to 44th peptides (2-44) of human HMGB1 and 5 types of peptides ⁇ (Peptide-1 to 5) modified with 43rd and 44th amino acids))
- the result of a medium stability test is shown.
- P1 to P5 mean peptides of Peptide-1 to 5.
- the vertical axis indicates the residual ratio calculated with the peptide immediately after plasma addition as 100, and the horizontal axis indicates the time after plasma addition.
- the residual rate could not be plotted in FIG. Fig.
- FIG. 2 shows the results of measuring the cell migration activity of two types of peptides (2nd to 44th peptide (2-44) and 43th and 44th amino acid modified peptides (Peptide-6) of human HMGB1). It is. NC indicates negative control.
- Fig. 3 shows plasma of three types of peptides (2nd to 44th peptide (2-44) and 43th and 44th amino acid modified peptides (Peptide-3 and 6) of human HMGB1). The result of a medium stability test is shown. The vertical axis indicates the residual ratio calculated with the peptide immediately after plasma addition as 100, and the horizontal axis indicates the time after plasma addition.
- the present invention provides the peptide according to any of the following (a) to (c).
- (a) Peptide having any amino acid sequence set forth in SEQ ID NOs: 1-399 and 403 (b) In the peptide having any amino acid sequence set forth in SEQ ID NOs: 1-399 and 403, one or more (for example, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5, or 4, 3, or 2) amino acid sequences added, and Peptide having activity to stimulate migration (c) 1-42 amino acid of peptide having any amino acid sequence set forth in SEQ ID NO: 1 to 399 or 1-41 of peptide having amino acid sequence set forth in SEQ ID NO: 403 In the second amino acid, one or more (eg 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5, or 4, 3 or 2) a Bruno acid substituted, or comprises a deletion or insertion in the amino acid sequence, and a peptide having an activity to stimulate cell migration
- the present invention provides a composition used for stimulating the migration of PDGFR ⁇ -positive cells or bone marrow-derived stromal cells, comprising the peptide according to any of the following (a) to (c).
- the second amino acid one or more (eg 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5, or 4, 3 or 2) a A peptide comprising or consisting of an amino acid sequence in which noic acid is substituted
- the reagent composition used for stimulating the migration of PDGFR ⁇ -positive cells or bone marrow-derived stromal cells of the present invention is used, for example, as a reagent necessary for basic research and clinical research for the development of regenerative medicine and regenerative induction medicine. be able to.
- the reagent composition it becomes possible to mobilize cells to a living tissue in a laboratory animal and examine the degree of tissue repair and tissue function reconstruction.
- tissue regeneration induction studies by cell mobilization can be performed in a test tube.
- the pharmaceutical composition used for stimulating the migration of PDGFR ⁇ -positive cells or bone marrow-derived stromal cells of the present invention can be used, for example, as a medicine in regenerative medicine or regeneration-inducing medicine.
- the tissue can be regenerated by using the pharmaceutical composition.
- the pharmaceutical composition can prevent a decrease in tissue / organ function due to a decrease in tissue stem cells as a so-called preventive drug, or can delay the progression of age-related changes as an anti-aging drug. .
- the composition used for stimulating the migration of PDGFR ⁇ -positive cells or bone marrow-derived stromal cells is the agent used for stimulating the migration of PDGFR ⁇ -positive cells or bone marrow-derived stromal cells, cell migration Or a composition used for inducing cell migration, an agent used for inducing cell migration, an agent for inducing cell migration, or an agent for inducing cells.
- the cell migration stimulating activity means an activity for stimulating cell migration.
- cell migration stimulating activity is also expressed as cell migration-inducing activity or cell-inducing activity.
- the present invention provides a composition used for mobilizing bone marrow mesenchymal stem cells from bone marrow to peripheral blood, containing the substance described in any of (a) to (c) below.
- the second amino acid one or more (eg 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5, or 4, 3 or 2) a A peptide comprising or consisting of an amino acid sequence in which noic acid is substituted
- the reagent composition used for regenerating the tissue of the present invention can be used, for example, as a reagent necessary for basic research and clinical research for the development of regenerative medicine and regenerative guided medicine.
- the pharmaceutical composition used for regenerating the tissue of the present invention can be used, for example, as a medicine in regenerative medicine or regeneration-guided medicine.
- bone marrow cells can be mobilized in the peripheral circulation and the tissue can be regenerated.
- cells that have been mobilized in peripheral blood using the pharmaceutical composition can be collected outside the body, concentrated, and then administered to a tissue for treatment. In the conventional method, cells are collected from the bone marrow deep inside the body, so that the living body is invasive.
- bone marrow cells are collected from peripheral blood in a minimally invasive manner, and bone marrow cell transplantation is performed. Can be used for etc.
- a composition used for mobilizing bone marrow mesenchymal stem cells from bone marrow to peripheral blood is also expressed as a composition used for inducing bone marrow mesenchymal stem cells from bone marrow to peripheral blood.
- the present invention provides a composition used for regenerating a tissue, which contains the substance described in any of the following (a) to (c).
- the second amino acid one or more (eg 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5, or 4, 3 or 2) a A peptide comprising or consisting of an amino acid sequence in which no acid is substituted, deleted or inserted, and having an activity of stimulating cell migration
- the reagent composition used for regenerating the tissue of the present invention can be used, for example, as a reagent necessary for basic research and clinical research for the development of regenerative medicine and regenerative guided medicine.
- the pharmaceutical composition used for regenerating the tissue of the present invention can be used, for example, as a medicine in regenerative medicine or regeneration-guided medicine.
- the composition used for regenerating a tissue includes a composition used for inducing or promoting tissue regeneration, an agent used for inducing or promoting tissue regeneration, and a tissue regeneration inducing agent. Or expressed as a tissue regeneration promoter. Tissue regeneration also includes tissue repair.
- the composition used for regenerating the tissue of the present invention does not choose the administration / addition site. That is, the composition can exert its effect when administered to any tissue, such as a tissue requiring regeneration, a tissue different from the tissue requiring regeneration, or blood.
- tissue requiring regeneration a tissue different from the tissue requiring regeneration
- blood a tissue different from the tissue requiring regeneration
- by administering / adding the composition cells are mobilized to the tissue at or near the administration / addition site, and tissue regeneration is induced or promoted.
- tissue regeneration is induced or promoted.
- peripheral circulation is also referred to as “blood circulation” and “peripheral circulation blood flow”.
- tissue that needs to be regenerated examples include damaged tissue, necrotic tissue, post-operative tissue, tissue with reduced function, fibrosis, aged tissue, diseased tissue, etc.
- tissue For example, living skin tissue, body biopsy (surgical) tissue (brain, lung, heart, liver, stomach, small intestine, large intestine, pancreas, kidney, bladder, spleen, uterus, testis, blood, etc.) can be exemplified.
- administering means administration to a site other than the site that requires regeneration (a site different from the site that requires regeneration). Therefore, “a tissue different from the tissue that needs to be regenerated” refers to a site that is different from the tissue that needs to be regenerated, a site that is different from the site that needs to be regenerated, a site away from the tissue that needs to be regenerated, or a site that needs to be regenerated. It can also be expressed as a site remote from the site, a site distal to the site that needs regeneration, a tissue distal to the tissue that needs to be regenerated, a distal portion, and a distal tissue. That is, the composition of the present invention is effectively used to regenerate a tissue (brain, heart, etc.) where it is difficult to administer a drug directly from outside the body.
- tissue that need regeneration differentiate into various cells, contributing to functional regeneration, functional maintenance, and function enhancement of tissues that need regeneration.
- the tissues that need to be regenerated include tissues damaged by various pathologies caused by ischemia, ischemia / hypoxia, trauma, burns, inflammation, autoimmunity, gene abnormalities, etc. It is not limited to the cause.
- the tissue in the present invention is not particularly limited as long as bone marrow-derived cells can be differentiated.
- skin tissue, bone tissue, cartilage tissue, muscle tissue, adipose tissue, myocardial tissue, nervous system tissue, lung tissue examples include all in vivo tissues such as gastrointestinal tissue, liver / bile / pancreatic tissue, urinary / genital organs, and the like.
- mammals include humans and non-human animals, and examples include, but are not limited to, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, and whales. It is not a thing.
- tissue different from the tissue that needs to be regenerated examples include blood tissue, muscle tissue, subcutaneous tissue, intradermal tissue, and abdominal cavity.
- neural tissue examples include, but are not limited to, central nervous tissue.
- the composition used for regenerating nerve tissue can be used for the treatment of, for example, cerebral infarction, cerebral hemorrhage, cerebral contusion, but is not limited thereto.
- the composition used for regenerating bone tissue can be used, for example, for the treatment of fractures, but is not limited thereto.
- the composition used for regenerating skin tissue can be used for the treatment of, for example, skin ulcers, suture defects of surgical wounds, burns, cuts, bruises, skin erosion, abrasions, etc., but is not limited thereto.
- cells that are stimulated to migrate or cells that are mobilized from bone marrow to peripheral blood include, but are not limited to, undifferentiated cells and cells in various stages of differentiation.
- cells that are stimulated to migrate or cells that are mobilized from bone marrow to peripheral blood include, but are not limited to, stem cells and non-stem cells.
- Stem cells include circulating stem cells or non-circulating stem cells. Examples of non-circulating stem cells include tissue stem cells that are resident in tissues. Examples of circulating stem cells include blood circulating stem cells.
- hematopoietic stem cells refers to blood cells such as neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, as well as red blood cells, platelets, mast cells, dendritic cells, etc. It is known that the stem cells can be differentiated into these cells, and the markers are CD34 positive, CD133 positive in humans, CD34 negative in mice, c-Kit positive, Sca-1 positive, and Lineage marker-negative.
- hematopoietic stem cells are characterized by difficulty in culturing alone when cultured in a culture dish, and requiring co-culture with stromal cells.
- bone marrow cell means a cell existing in the bone marrow
- bone marrow-derived cell means “bone marrow cell” mobilized or removed from the bone marrow to the outside of the bone marrow.
- bone marrow cells include cells that contain a population of tissue precursor cells present in the bone marrow.
- the “bone marrow-derived cell” may be a cell containing mesoangioblast or a cell excluding mesoangioblast.
- Tissue progenitor cells are defined as undifferentiated cells that have the ability to unidirectionally differentiate into specific tissue cells other than the blood system, and differentiate into mesenchymal tissue, epithelial tissue, neural tissue, parenchymal organ, and vascular endothelium as described above. Including undifferentiated cells having the ability.
- “Bone marrow cells” and “bone marrow-derived cells” are hematopoietic stem cells and differentiated cells such as leukocytes, erythrocytes, platelets, osteoblasts and fibroblasts, or bone marrow mesenchymal stem cells and bone marrow mesenchymal so far Stem cells represented by cells called stromal cells, bone marrow pluripotent stromal cells, or bone marrow pluripotent stem cells.
- “bone marrow stem cell” means a stem cell present in the bone marrow
- “bone marrow-derived stem cell” means a “bone marrow stem cell” mobilized or removed from the bone marrow outside the bone marrow. .
- cells that are stimulated to migrate or cells that are mobilized from bone marrow to peripheral blood include, but are not limited to, “bone marrow-derived stem cells”.
- “Bone marrow cells” and “bone marrow-derived cells” can be isolated by bone marrow collection (bone marrow cell collection) or peripheral blood collection.
- Hematopoietic stem cells are non-adherent cells, but some of the “bone marrow cells” and “bone marrow-derived cells” are mononuclear cell fractionated cells obtained by bone marrow collection (bone marrow cell collection) and peripheral blood collection. It is obtained as an adherent cell by culture.
- “Bone marrow cells” and “bone marrow-derived cells” include mesenchymal stem cells, and can be identified by osteoblasts (identified by the presence of calcium deposition when differentiation is induced), chondrocytes (positive for Alcian blue staining, safranin) -O staining positive, etc.), adipocytes (identified by Zudan III staining positive), mesenchymal cells such as fibroblasts, smooth muscle cells, stromal cells, tendon cells, nerve cells, It preferably has an ability to differentiate into epithelial cells (for example, epidermal keratinocytes, intestinal epithelial cells express cytokeratin family) and vascular endothelial cells.
- the cells after differentiation are not limited to the above cells, but also include the ability to differentiate into parenchymal organ cells such as liver, kidney, pancreas and the like.
- bone marrow stromal cells are cells other than hematopoietic stem cells and hematopoietic cells derived from the cells existing in the bone marrow, and are collected from the bone marrow and attached to a culture dish. A cell that can be cultured and proliferated as a cell. Bone marrow stromal cells or bone marrow stromal cells taken out of the bone marrow include pluripotent cells (bone marrow pluripotent stromal cells or bone marrow derived pluripotent stromal cells). Moreover, the bone marrow origin stromal cell may be established as a cell line.
- Examples of established bone marrow-derived stromal cells include ST2 cells (Ogawa et al., EMBO J 1988; 7: 1337-43). ST2 cells are a bone marrow derived pluripotent stromal cell line that has the ability to differentiate into osteoblasts and adipocytes.
- bone marrow mesenchymal stem cell is used interchangeably with “bone marrow mesenchymal stromal cell”, “bone marrow pluripotent stromal cell”, and “bone marrow pluripotent stem cell”.
- the “bone marrow mesenchymal stem cell” is a cell existing in the bone marrow, which is directly collected from the bone marrow or indirectly from other tissues (blood, skin, fat, other tissues), It can be cultured and proliferated as adherent cells on a culture dish (plastic or glass), and mesenchymal tissue (mesenchymal stem cells) such as bone, cartilage, and fat, or skeletal muscle, myocardium, nerve tissue, epithelium It is a cell characterized by having the ability to differentiate into a tissue (pluripotent stem cell) and can be obtained by collecting bone marrow cells.
- bone marrow-derived mesenchymal stem cells “bone marrow-derived mesenchymal stromal cells”, “bone marrow-derived pluripotent stromal cells” or “bone marrow-derived pluripotency” mobilized from the bone marrow to the outside of the bone marrow
- a “stem cell” is a cell that can be obtained by collecting peripheral blood and collecting it from mesenchymal tissue such as fat, epithelial tissue such as skin, and nerve tissue such as brain.
- these cells can be obtained directly after collection or by administering cells once attached to a culture dish to a damaged part of a living body, for example, epithelial tissues such as keratinocytes constituting the skin, nervous tissues constituting the brain. It also has the feature of having differentiation ability.
- Bone marrow mesenchymal stem cells, bone marrow mesenchymal stromal cells, bone marrow pluripotent stromal cells, bone marrow pluripotent stem cells, or these cells mobilized from the bone marrow to the bone marrow are osteoblasts (calcium when differentiation is induced
- osteoblasts calcium when differentiation is induced
- chondrocytes identifiable by positive Alcian blue staining, positive Safranin-O staining, etc.
- adipocytes identifiable by positive Sudan III staining, etc.
- fibroblasts Cells smooth muscle cells, skeletal muscle cells, stromal cells, tendon cells and other mesenchymal cells, nerve cells, pigment cells, epidermal cells, hair follicle cells (expressing cytokeratin family, hair keratin family, etc.), epithelial system
- epithelial system It is preferable to have the ability to differentiate into cells (for example, epidermal keratinocytes
- human bone marrow cells and human bone marrow derived cells include cells that can be obtained as adherent cells by obtaining bone marrow collection (bone marrow cell collection), peripheral blood collection, and fat collection, and culturing directly or after separating the mononuclear cell fraction. Yes, but you are not limited to this.
- PDGFR ⁇ positive, PDGFR ⁇ positive, Lin negative, CD45 negative, CD44 positive, CD90 positive, CD29 positive, Flk-1 negative, CD105 positive, CD73 positive, CD90 positive, CD71 positive, Stro-1 positive, CD106 positive, CD166 positive, CD31 negative, CD271 positive, and CD11b negative can be exemplified in whole or in part, but are not limited thereto.
- mouse bone marrow cells and mouse bone marrow-derived cells can be obtained as adherent cells by obtaining bone marrow collection (bone marrow cell collection), peripheral blood collection, or fat collection, and culturing directly or after separation of the mononuclear cell fraction.
- bone marrow collection bone marrow cell collection
- peripheral blood collection peripheral blood collection
- fat collection culturing directly or after separation of the mononuclear cell fraction.
- markers for mouse bone marrow cells or mouse bone marrow derived cells eg, bone marrow mesenchymal stem cells
- Flk-1 negative, CD271 positive, CD11b negative can be illustrated, it is not limited to these.
- cells that are stimulated to migrate or cells that are mobilized from bone marrow to peripheral blood include PDGFR ⁇ -positive cells, but are not limited thereto.
- markers other than PDGFR ⁇ include all or part of CD29 positive, CD44 positive, CD90 positive, CD271 positive, CD11b negative and Flk-1 negative, but are not limited thereto.
- PDGFR ⁇ -positive cells include PDGFR ⁇ -positive bone marrow-derived cells, PDGFR ⁇ -positive bone marrow-derived mesenchymal stem cells, PDGFR ⁇ -positive tissue cells (eg, fibroblasts), PDGFR ⁇ -positive cells
- PDGFR ⁇ -positive tissue cells eg, fibroblasts
- PDGFR ⁇ -positive cells examples include bone marrow collection (bone marrow cell collection) and cells obtained as adherent cells by mononuclear cell fractionation in blood obtained by peripheral blood collection, but are not limited thereto. It is not something.
- the composition of the present invention may contain another substance other than at least one of the peptides described in any of the above (a) to (c).
- the cell migration stimulating activity, the cell mobilization activity, or the tissue regeneration promoting activity is not inhibited.
- a related molecule that enhances the function of the peptide described in (a) to (c) above in addition to at least one of the peptides described in (a) to (c) above, a related molecule that enhances the function of the peptide described in (a) to (c) above. (Group), molecules (group) that suppress actions other than the expected effects of the peptides described in (a) to (c) above, factors that control cell growth and differentiation, enhancement of these factors or cell functions It can include other factors to maintain.
- the human HMGB1 protein in the present invention means a protein having the amino acid sequence set forth in SEQ ID NO: 401 as a human-derived HMGB1 protein.
- the peptide consisting of the amino acid sequence from the 1st to the 44th amino acid sequence of HMGB1 protein means the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 402.
- the cell migration stimulating activity of the peptide can be confirmed by, for example, the method disclosed in WO2012 / 147470 in addition to the method described in Examples, but is not limited thereto.
- the plasma stability of the peptide can be confirmed by, for example, the method described in the Examples, but is not limited thereto.
- peptide of the present invention examples include, but are not limited to, peptides consisting of the following amino acid sequences.
- Examples of the peptide of the present invention further include a peptide in which the 43rd and / or 44th amino acid of the peptide described in SEQ ID NOs: 1 to 399 is replaced with an unnatural amino acid, or the peptide described in SEQ ID NO: 403 above.
- a peptide in which the amino acid at position 42 and / or 43 is replaced with an unnatural amino acid is also included.
- the peptide of the present invention may have a conservative substitution of amino acids in the peptide having any amino acid sequence described in SEQ ID NOs: 1 to 5.
- a conservative substitution refers to a substitution with another amino acid having a structure or property chemically similar to that of the original amino acid, which does not substantially change the function of the peptide.
- conservative substitutions include mutual substitution between the acidic amino acids aspartic acid and glutamic acid, mutual substitution between the basic amino acids lysine, arginine and histidine, and the hydrophilic amino acids serine, threonine, asparagine and glutamine. And the like.
- the peptide of SEQ ID NO: 167 is obtained by substituting the 44th glutamic acid (E) of the peptide of SEQ ID NO: 5 with aspartic acid (D), and is considered to have the same function as the peptide of SEQ ID NO: 5.
- the peptides described in SEQ ID NOs: 126, 128, 129, 138, 248, 254, 280, 282, 283 and 292 are also conservative of amino acids in the peptides having any of the amino acid sequences described in SEQ ID NOs: 1 to 5. It has a substitution and is considered to have a function equivalent to any of the peptides described in SEQ ID NOs: 1 to 5.
- the peptide of the present invention has any amino acid sequence set forth in SEQ ID NOs: 1-5, 126, 128, 129, 138, 167, 248, 254, 280, 282, 283, 292 and 403. Is. In one more preferred embodiment, the peptide of the present invention has any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 5, 167, and 403 from the viewpoint that it can be expected to have the above functions more reliably. In one more preferred embodiment, the peptide of the present invention has any amino acid sequence set forth in SEQ ID NOs: 1 to 5 and 403.
- one preferred embodiment of the peptide defined in (a) above is described in SEQ ID NOs: 1 to 5, 126, 128, 129, 138, 167, 248, 254, 280, 282, 283, 292 and 403.
- a peptide having any amino acid sequence and one more preferable embodiment is a peptide having any amino acid sequence described in SEQ ID NOs: 1 to 5, 167, and 403, and one more preferable embodiment is SEQ ID NO:
- a preferred embodiment of the peptide defined in (b) above is described in SEQ ID NOs: 1 to 5, 126, 128, 129, 138, 167, 248, 254, 280, 282, 283, 292 and 403.
- a peptide having any amino acid sequence one or more (for example, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5, or 4, 3, or 2)
- a peptide having an activity of stimulating cell migration and one more preferred embodiment is any one of the amino acids described in SEQ ID NOs: 1 to 5, 167 and 403
- a peptide having an amino acid sequence to which is added, and having an activity of stimulating cell migration and one more preferred embodiment has any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 5 and 403
- one preferred embodiment of the peptide defined in (c) above is any one of SEQ ID NOS: 1 to 5, 126, 128, 129, 138, and 167, 248, 254, 280, 282, 283, and 292.
- One of the more preferred embodiments is a peptide having any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 5 and 167.
- a “derivative” of a peptide of the present invention means a peptide having substantially the same biological function or activity as the peptide of the present invention, ie, an activity that stimulates cell migration.
- the C-terminus of the derivative of the peptide may be a carboxyl group, a carboxylate, an amide or an ester. Examples thereof include a modified peptide obtained by adding a modifying group, a mutant peptide obtained by altering an amino acid residue, and the like.
- the modifying group include a functional group that exhibits fluorescence and a functional group that does not participate in the formation of the three-dimensional structure of the peptide.
- amino acid residue other than the amino acid residue constituting the naturally derived peptide of the present invention for example, ⁇ -alanine.
- the functional group exhibiting fluorescence include eosin and fluorescein isothiocyanate (FITC).
- FITC fluorescein isothiocyanate
- the functional group that does not participate in the formation of the three-dimensional structure of the peptide include a spacer group typified by a ⁇ -alanine residue. Such a functional group is preferably present at the terminal.
- PEG polyethylene glycol
- Examples of the “pharmaceutically acceptable salt” of the peptide of the present invention include physiologically acceptable salts with acids or bases, and acid addition salts are particularly preferable.
- Examples of such salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, or acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, Salts with organic acids such as malic acid, succinic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid are used.
- the present invention provides the peptides described above.
- the peptide can be chemically synthesized by any of the peptide liquid phase synthesis method and the peptide solid phase synthesis method.
- a peptide synthesized by a peptide solid phase synthesis method is preferred.
- the peptide solid phase synthesis method is one of the methods generally used for chemically synthesizing peptides, and can be carried out, for example, as follows: The surface is modified with an amino group and has a diameter of about 0.1 mm. Polystyrene polymer gel beads are used as the solid phase, and amino acid chains are extended one by one through a dehydration reaction.
- the sequence of the target peptide is completed, it is cut out from the solid surface to obtain the target substance.
- solid phase synthesis it is possible to synthesize ribosomal peptides that are difficult to synthesize in bacteria, introduce non-natural amino acids such as D-forms and heavy atom substitutions, and modify peptides and protein backbones.
- synthesizing 70 to more than 100 long peptide chains in the solid phase method it is possible to synthesize by combining two peptide chains using the native chemical ligation method.
- the administration method of the composition of the present invention includes oral administration or parenteral administration, and specific examples of the parenteral administration method include injection administration, nasal administration, pulmonary administration, and transdermal administration. However, it is not limited to these.
- parenteral administration method include injection administration, nasal administration, pulmonary administration, and transdermal administration.
- injection administration include, for example, intravenous, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like, and the composition of the present invention is systemically or locally (for example, subcutaneous, intradermal, skin surface, eyeball or eyelid conjunctiva).
- Examples of the method for administering the composition of the present invention include intravascular administration (intraarterial administration, intravenous administration, etc.), intravascular administration, intramuscular administration, subcutaneous administration, intradermal administration, and intraperitoneal administration. However, it is not limited to these.
- the administration site includes a tissue site that requires regeneration or the vicinity thereof, a site different from the tissue that requires regeneration, or a site that is distal and different from the tissue that requires regeneration.
- examples include blood (intraarterial, intravenous, etc.), muscle, subcutaneous, intradermal, and intraperitoneal, but are not limited thereto.
- the administration method can be appropriately selected depending on the age and symptoms of the patient.
- the dose can be selected in the range of 0.0000001 mg to 1000 mg per kg of body weight per administration.
- the dose can be selected within the range of 0.00001 to 100,000 mg / body per patient.
- the pharmaceutical composition of the present invention is not limited to these doses.
- the pharmaceutical composition of the present invention can be formulated in accordance with a conventional method (for example, Remington's Pharmaceutical, Science, Latest Edition, Mark Publishing, Company, Easton, USA) together with pharmaceutically acceptable carriers and additives. It may be.
- a conventional method for example, Remington's Pharmaceutical, Science, Latest Edition, Mark Publishing, Company, Easton, USA
- pharmaceutically acceptable carriers and additives for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, flavoring agents
- surfactants for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, flavoring agents
- it is not limited to these, and other commonly used carriers can be used as appropriate.
- Example 1 Synthesis of various HMGB1 peptides The 43rd and / or 44th amino acid sequence was substituted in the peptide consisting of the 2nd to 44th amino acids of human HMGB1 (2-44) and the 1st to 44th peptides of human HMGB1 The following five peptides (Peptide-1 to 5) were synthesized by the solid phase method.
- Example 2 Measurement of cell migration activity ST2 cells (RCB0224, RIKEN BioResource Center), a mouse bone marrow-derived stromal cell line, were detached from the dish using TrypLE Express (Thermo Fisher Scientific) and then incubated at 25 ° C, 1500 rpm for 5 minutes. It was collected by centrifugation. Cell pellet was prepared with RPMI1640 / 10% FBS / 1% PenStrep (Gibco) to 7 x 10 5 cells / mL, and seeded at 70 ⁇ L / insert well in a 24-well plate with culture insert (Nippon Genetics) . After 24 hours, the culture insert was removed and washed once with RPMI1640.
- Each peptide sample prepared in Assay medium (RPMI1640 / 0.1% FBS) to 10 ⁇ M (2nd to 44th peptides of human HMGB1 (2-44), 5 kinds of modified peptides Peptide-1 prepared in Example 1 To 5) was added and further cultured overnight.
- Example 3 Whole blood collected from Crl: CD (SD) rats was centrifuged using a stable anticoagulant EDTA ⁇ 2K in peptide plasma to obtain blank plasma. The obtained plasma was stored on ice and used only on the day of the stability test. A solution prepared to 375 ⁇ g / mL with distilled water was prepared from a stock solution obtained by dissolving 6 types of peptides used in Example 2 in 0.1% TFA aqueous solution at 1 mg / mL. 16 ⁇ L of the solution was added to 1184 ⁇ L of blank plasma to prepare a sample for stability test (assay concentration: 5 ⁇ g / mL).
- a sample for stability test was allowed to stand on ice, and 50 ⁇ L (each time point, 3 cases) was collected immediately after addition, 1, 3, 5, 15, 30, 60 minutes later.
- 50 ⁇ L sample 270 mmol / L citric acid solution / 130 mmol / L EDTA ⁇ 2K aqueous solution (4: 1, v / v) 250 ⁇ L, 0.1% TFA aqueous solution 25 ⁇ L, IS solution (human HMGB1 (4 ⁇ g / mL 0.1% TFA aqueous solution of the compound in which 9 carbon atoms and 1 nitrogen atom of the 18th, 38th, and 41st phenylalanines are substituted with stable isotopes, respectively) 25 ⁇ L was added and mixed.
- the measurement sample was introduced into a high performance liquid chromatograph-tandem quadrupole mass spectrometer, and monitor ions specific to each peptide were detected.
- a calibration curve was created from a separately prepared sample of known concentration in the concentration range of 0.1-10 ⁇ g / mL, and the peptide concentration in the sample was obtained by reverse regression from the peak area of the chromatogram obtained by measuring each sample. .
- the peptide concentration at each time point was averaged, and the residual rate (%) calculated as 100 immediately after the addition was plotted against the time after the addition (FIG. 1).
- HMGB1 peptide A peptide (Peptide-6) having the following sequence in which the 2nd to 44th peptides (2-44) of human HMGB1 were substituted with two amino acids at the C-terminus was synthesized by a solid phase method.
- Peptide-6 GKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPP (SEQ ID NO: 403)
- Example 5 Measurement of cell migration activity ST2 cells (RCB0224, RIKEN BioResource Center), a mouse bone marrow-derived stromal cell line, are removed from dishes using 2.5g / L-Trypsin / 1mmol / L-EDTA Solution (nacalai tesque) The solution was collected by centrifugation at 25 ° C. and 200 g for 3 minutes. Prepare cell pellets with RPMI1640 / 10% FBS / 1% PenStrep (nacalai tesque) to 8.6 x 10 5 cells / mL and seed them in a 24-well plate with culture inserts (Nippon Genetics) at 70 ⁇ L / insert well. did.
- Respective peptide samples (2nd to 44th peptides (2-44) of human HMGB1 and the modified peptide Peptide-6 prepared in Example 4) prepared to 20 ⁇ M with Assay medium (RPMI1640 / 0.1% FBS) 1000 ⁇ L was added and further cultured for 20 hours.
- Assay medium RPMI1640 / 0.1% FBS
- NC negative control
- the cells were imaged immediately after addition of Assay medium and after 20 hours of culture. Each group as a percentage of the area occupied by cells that migrated to the area in 20 hours to the area of the blank area created by the culture insert (the area where no cells existed when the assay medium was added) from the captured image The cell migration was evaluated.
- Example 6 Peptide stability in plasma 3 mL of whole blood collected from Crl: CD (SD) rats was placed in a 5 mL tube containing 60 ⁇ L of 10% EDTA-2K in advance, and allowed to stand at room temperature for 30 minutes, then 3000 rpm (1600 ⁇ g ), And centrifuged at 4 ° C. for 10 minutes, and the supernatant was collected to obtain blank plasma. The obtained plasma was stored on ice and used only on the day of the stability test.
- CD (SD) rats was placed in a 5 mL tube containing 60 ⁇ L of 10% EDTA-2K in advance, and allowed to stand at room temperature for 30 minutes, then 3000 rpm (1600 ⁇ g ), And centrifuged at 4 ° C. for 10 minutes, and the supernatant was collected to obtain blank plasma. The obtained plasma was stored on ice and used only on the day of the stability test.
- the peptide of the present invention is used in the field of regenerative medicine.
Abstract
Description
これら造血幹細胞に由来する血液細胞は、血流を介して体内のあらゆる臓器や組織を循環し、酸素供給、免疫反応、止血・損傷組織修復など生命維持に不可欠な機能を果たしている。即ち、骨髄造血幹細胞はその局在する組織である骨髄や骨組織の恒常性維持というよりもむしろ、末梢循環を介して生体内すべての組織の恒常性維持に寄与していると言える。
(1) 配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチド、
配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド、および
配列番号1~399に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
からなる群より選ばれるペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物;
(2) 配列番号1~5、126、128、129、138、167、248、254、280、282、283、292および403に記載のいずれかのアミノ酸配列を有するペプチド、
配列番号1~5、126、128、129、138、167、248、254、280、282、283、292および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド、および
配列番号1~5、126、128、129、138、167、248、254、280、282、283および292に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
からなる群より選ばれるペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物;
(3) 配列番号1~5、167および403に記載のいずれかのアミノ酸配列を有するペプチド、
配列番号1~5、167および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド、および
配列番号1~5および167に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
からなる群より選ばれるペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物。
(4) 配列番号1~5および403に記載のいずれかのアミノ酸配列を有するペプチド、
配列番号1~5および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド、および
配列番号1~5に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
からなる群より選ばれるペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物;
(5) 前記(1)~(4)のいずれかに記載のペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物を含有する、PDGFRα陽性細胞の遊走を刺激するために用いられる組成物;
(6) 前記(1)~(4)のいずれかに記載のペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物を含有する、骨髄間葉系幹細胞を骨髄から末梢血に動員するために用いられる組成物;
(7) 前記(1)~(4)のいずれかに記載のペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物を含有する、組織を再生するために用いられる組成物;
(8) 前記(1)~(4)のいずれかに記載のペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物を含有する、骨髄由来間質細胞の遊走を刺激するために用いられる組成物;に関する。
(a)配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチド
(b)配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド
(c)配列番号1~399に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
(a)配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチド
(b)配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド
(c)配列番号1~399に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
本発明のPDGFRα陽性細胞又は骨髄由来間質細胞の遊走を刺激するために用いられる組成物には、試薬組成物および医薬組成物が含まれる。本明細書において、試薬組成物は試薬とも表現され、医薬組成物は医薬、薬剤または薬学的組成物とも表現される。
本発明において、細胞遊走刺激活性とは、細胞遊走を刺激する活性を意味する。本明細書において、細胞遊走刺激活性は、細胞遊走誘導活性または細胞誘導活性とも表現される。
(a)配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチド
(b)配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド
(c)配列番号1~399に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
本発明の骨髄間葉系幹細胞を骨髄から末梢血に動員するために用いられる組成物には、試薬組成物および医薬組成物が含まれる。
(a)配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチド
(b)配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド
(c)配列番号1~399に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個(例えば2~40個、2~30個、2~20個、2~10個、2~5個、または4個、3個もしくは2個)のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
本発明の組織を再生するために用いられる組成物には、試薬組成物および医薬組成物が含まれる。
(1) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKP(配列番号1)
(2) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPK(配列番号2)
(3) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPP(配列番号3)
(4) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKA(配列番号4)
(5) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKE(配列番号5)
(6) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAA(配列番号6)
(7) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAC(配列番号7)
(8) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAD(配列番号8)
(9) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAE(配列番号9)
(10) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAF(配列番号10)
(11) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAG(配列番号11)
(12) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAH(配列番号12)
(13) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAI(配列番号13)
(14) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAK(配列番号14)
(15) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAL(配列番号15)
(16) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAM(配列番号16)
(17) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAN(配列番号17)
(18) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAP(配列番号18)
(19) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAQ(配列番号19)
(20) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAR(配列番号20)
(21) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAS(配列番号21)
(22) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAT(配列番号22)
(23) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAV(配列番号23)
(24) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAW(配列番号24)
(25) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSAY(配列番号25)
(26) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCA(配列番号26)
(27) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCC(配列番号27)
(28) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCD(配列番号28)
(29) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCE(配列番号29)
(30) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCF(配列番号30)
(31) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCG(配列番号31)
(32) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCH(配列番号32)
(33) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCI(配列番号33)
(34) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCK(配列番号34)
(35) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCL(配列番号35)
(36) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCM(配列番号36)
(37) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCN(配列番号37)
(38) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCP(配列番号38)
(39) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCQ(配列番号39)
(40) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCR(配列番号40)
(41) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCS(配列番号41)
(42) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCT(配列番号42)
(43) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCV(配列番号43)
(44) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCW(配列番号44)
(45) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSCY(配列番号45)
(46) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDA(配列番号46)
(47) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDC(配列番号47)
(48) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDD(配列番号48)
(49) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDE(配列番号49)
(50) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDF(配列番号50)
(52) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDH(配列番号52)
(53) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDI(配列番号53)
(54) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDK(配列番号54)
(55) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDL(配列番号55)
(56) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDM(配列番号56)
(57) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDN(配列番号57)
(58) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDP(配列番号58)
(59) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDQ(配列番号59)
(60) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDR(配列番号60)
(61) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDS(配列番号61)
(62) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDT(配列番号62)
(63) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDV(配列番号63)
(64) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDW(配列番号64)
(65) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSDY(配列番号65)
(66) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEA(配列番号66)
(67) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEC(配列番号67)
(68) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSED(配列番号68)
(69) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEE(配列番号69)
(70) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEF(配列番号70)
(71) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEG(配列番号71)
(72) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEH(配列番号72)
(73) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEI(配列番号73)
(74) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEK(配列番号74)
(75) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEL(配列番号75)
(76) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEM(配列番号76)
(77) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEN(配列番号77)
(78) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEP(配列番号78)
(79) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEQ(配列番号79)
(80) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSER(配列番号80)
(81) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSES(配列番号81)
(82) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSET(配列番号82)
(83) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEV(配列番号83)
(84) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEW(配列番号84)
(85) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSEY(配列番号85)
(86) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFA(配列番号86)
(87) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFC(配列番号87)
(88) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFD(配列番号88)
(89) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFE(配列番号89)
(90) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFF(配列番号90)
(91) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFG(配列番号91)
(92) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFH(配列番号92)
(93) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFI(配列番号93)
(94) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFK(配列番号94)
(95) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFL(配列番号95)
(96) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFM(配列番号96)
(97) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFN(配列番号97)
(98) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFP(配列番号98)
(99) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFQ(配列番号99)
(100)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFR(配列番号100)
(102)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFT(配列番号102)
(103)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFV(配列番号103)
(104)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFW(配列番号104)
(105)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSFY(配列番号105)
(106)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGA(配列番号106)
(107)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGC(配列番号107)
(108)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGD(配列番号108)
(109)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGE(配列番号109)
(110)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGF(配列番号110)
(111)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGG(配列番号111)
(112)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGH(配列番号112)
(113)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGI(配列番号113)
(114)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGK(配列番号114)
(115)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGL(配列番号115)
(116)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGM(配列番号116)
(117)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGN(配列番号117)
(118)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGP(配列番号118)
(119)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGQ(配列番号119)
(120)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGR(配列番号120)
(121)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGS(配列番号121)
(122)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGT(配列番号122)
(123)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGV(配列番号123)
(124)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGW(配列番号124)
(125)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSGY(配列番号125)
(126)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHA(配列番号126)
(127)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHC(配列番号127)
(128)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHD(配列番号128)
(129)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHE(配列番号129)
(130)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHF(配列番号130)
(131)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHG(配列番号131)
(132)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHH(配列番号132)
(133)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHI(配列番号133)
(134)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHK(配列番号134)
(135)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHL(配列番号135)
(136)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHM(配列番号136)
(137)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHN(配列番号137)
(138)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHP(配列番号138)
(139)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHQ(配列番号139)
(140)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHR(配列番号140)
(141)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHS(配列番号141)
(142)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHT(配列番号142)
(143)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHV(配列番号143)
(144)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHW(配列番号144)
(145)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSHY(配列番号145)
(146)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIA(配列番号146)
(147)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIC(配列番号147)
(148)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSID(配列番号148)
(149)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIE(配列番号149)
(150)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIF(配列番号150)
(152)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIH(配列番号152)
(153)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSII(配列番号153)
(154)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIK(配列番号154)
(155)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIL(配列番号155)
(156)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIM(配列番号156)
(157)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIN(配列番号157)
(158)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIP(配列番号158)
(159)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIQ(配列番号159)
(160)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIR(配列番号160)
(161)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIS(配列番号161)
(162)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIT(配列番号162)
(163)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIV(配列番号163)
(164)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIW(配列番号164)
(165)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSIY(配列番号165)
(166)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKC(配列番号166)
(167)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKD(配列番号167)
(168)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKF(配列番号168)
(169)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKG(配列番号169)
(170)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKH(配列番号170)
(171)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKI(配列番号171)
(172)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKL(配列番号172)
(173)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKM(配列番号173)
(174)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKN(配列番号174)
(175)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKQ(配列番号175)
(176)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKR(配列番号176)
(177)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKS(配列番号177)
(178)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKT(配列番号178)
(179)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKV(配列番号179)
(180)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKW(配列番号180)
(181)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKY(配列番号181)
(182)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLA(配列番号182)
(183)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLC(配列番号183)
(184)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLD(配列番号184)
(185)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLE(配列番号185)
(186)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLF(配列番号186)
(187)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLG(配列番号187)
(188)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLH(配列番号188)
(189)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLI(配列番号189)
(190)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLK(配列番号190)
(191)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLL(配列番号191)
(192)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLM(配列番号192)
(193)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLN(配列番号193)
(194)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLP(配列番号194)
(195)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLQ(配列番号195)
(196)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLR(配列番号196)
(197)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLS(配列番号197)
(198)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLT(配列番号198)
(199)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLV(配列番号199)
(200)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSLW(配列番号200)
(202)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMA(配列番号202)
(203)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMC(配列番号203)
(204)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMD(配列番号204)
(205)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSME(配列番号205)
(206)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMF(配列番号206)
(207)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMG(配列番号207)
(208)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMH(配列番号208)
(209)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMI(配列番号209)
(210)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMK(配列番号210)
(211)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSML(配列番号211)
(212)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMM(配列番号212)
(213)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMN(配列番号213)
(214)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMP(配列番号214)
(215)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMQ(配列番号215)
(216)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMR(配列番号216)
(217)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMS(配列番号217)
(218)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMT(配列番号218)
(219)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMV(配列番号219)
(220)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMW(配列番号220)
(221)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSMY(配列番号221)
(222)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNA(配列番号222)
(223)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNC(配列番号223)
(224)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSND(配列番号224)
(225)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNE(配列番号225)
(226)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNF(配列番号226)
(227)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNG(配列番号227)
(228)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNH(配列番号228)
(229)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNI(配列番号229)
(230)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNK(配列番号230)
(231)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNL(配列番号231)
(232)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNM(配列番号232)
(233)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNN(配列番号233)
(234)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNP(配列番号234)
(235)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNQ(配列番号235)
(236)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNR(配列番号236)
(237)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNS(配列番号237)
(238)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNT(配列番号238)
(239)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNV(配列番号239)
(240)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNW(配列番号240)
(241)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSNY(配列番号241)
(242)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPA(配列番号242)
(243)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPC(配列番号243)
(244)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPD(配列番号244)
(245)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPE(配列番号245)
(246)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPF(配列番号246)
(247)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPG(配列番号247)
(248)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPH(配列番号248)
(249)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPI(配列番号249)
(250)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPL(配列番号250)
(252)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPN(配列番号252)
(253)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPQ(配列番号253)
(254)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPR(配列番号254)
(255)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPS(配列番号255)
(256)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPT(配列番号256)
(257)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPV(配列番号257)
(258)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPW(配列番号258)
(259)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPY(配列番号259)
(260)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQA(配列番号260)
(261)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQC(配列番号261)
(262)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQD(配列番号262)
(263)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQE(配列番号263)
(264)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQF(配列番号264)
(265)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQG(配列番号265)
(266)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQH(配列番号266)
(267)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQI(配列番号267)
(268)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQK(配列番号268)
(269)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQL(配列番号269)
(270)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQM(配列番号270)
(271)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQN(配列番号271)
(272)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQP(配列番号272)
(273)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQQ(配列番号273)
(274)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQR(配列番号274)
(275)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQS(配列番号275)
(276)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQT(配列番号276)
(277)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQV(配列番号277)
(278)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQW(配列番号278)
(279)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSQY(配列番号279)
(280)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRA(配列番号280)
(281)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRC(配列番号281)
(282)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRD(配列番号282)
(283)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRE(配列番号283)
(284)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRF(配列番号284)
(285)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRG(配列番号285)
(286)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRH(配列番号286)
(287)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRI(配列番号287)
(288)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRK(配列番号288)
(289)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRL(配列番号289)
(290)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRM(配列番号290)
(291)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRN(配列番号291)
(292)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRP(配列番号292)
(293)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRQ(配列番号293)
(294)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRR(配列番号294)
(295)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRS(配列番号295)
(296)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRT(配列番号296)
(297)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRV(配列番号297)
(298)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRW(配列番号298)
(299)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSRY(配列番号299)
(300)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSA(配列番号300)
(302)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSD(配列番号302)
(303)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSE(配列番号303)
(304)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSF(配列番号304)
(305)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSG(配列番号305)
(306)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSH(配列番号306)
(307)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSI(配列番号307)
(308)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSK(配列番号308)
(309)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSL(配列番号309)
(310)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSM(配列番号310)
(311)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSN(配列番号311)
(312)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSP(配列番号312)
(313)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSQ(配列番号313)
(314)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSR(配列番号314)
(315)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSS(配列番号315)
(316)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSST(配列番号316)
(317)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSV(配列番号317)
(318)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSW(配列番号318)
(319)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSSY(配列番号319)
(320)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTA(配列番号320)
(321)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTC(配列番号321)
(322)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTD(配列番号322)
(323)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTE(配列番号323)
(324)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTF(配列番号324)
(325)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTG(配列番号325)
(326)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTH(配列番号326)
(327)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTI(配列番号327)
(328)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTK(配列番号328)
(329)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTL(配列番号329)
(330)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTM(配列番号330)
(331)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTN(配列番号331)
(332)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTP(配列番号332)
(333)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTQ(配列番号333)
(334)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTR(配列番号334)
(335)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTS(配列番号335)
(336)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTT(配列番号336)
(337)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTV(配列番号337)
(338)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTW(配列番号338)
(339)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSTY(配列番号339)
(340)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVA(配列番号340)
(341)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVC(配列番号341)
(342)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVD(配列番号342)
(343)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVE(配列番号343)
(344)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVF(配列番号344)
(345)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVG(配列番号345)
(346)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVH(配列番号346)
(347)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVI(配列番号347)
(348)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVK(配列番号348)
(349)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVL(配列番号349)
(350)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVM(配列番号350)
(352)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVP(配列番号352)
(353)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVQ(配列番号353)
(354)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVR(配列番号354)
(355)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVS(配列番号355)
(356)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVT(配列番号356)
(357)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVV(配列番号357)
(358)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVW(配列番号358)
(359)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSVY(配列番号359)
(360)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWA(配列番号360)
(361)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWC(配列番号361)
(362)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWD(配列番号362)
(363)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWE(配列番号363)
(364)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWF(配列番号364)
(365)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWG(配列番号365)
(366)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWH(配列番号366)
(367)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWI(配列番号367)
(368)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWK(配列番号368)
(369)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWL(配列番号369)
(370)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWM(配列番号370)
(371)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWN(配列番号371)
(372)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWP(配列番号372)
(373)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWQ(配列番号373)
(374)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWR(配列番号374)
(375)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWS(配列番号375)
(376)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWT(配列番号376)
(377)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWV(配列番号377)
(378)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWW(配列番号378)
(379)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSWY(配列番号379)
(380)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYA(配列番号380)
(381)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYC(配列番号381)
(382)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYD(配列番号382)
(383)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYE(配列番号383)
(384)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYF(配列番号384)
(385)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYG(配列番号385)
(386)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYH(配列番号386)
(387)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYI(配列番号387)
(388)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYK(配列番号388)
(389)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYL(配列番号389)
(390)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYM(配列番号390)
(391)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYN(配列番号391)
(392)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYP(配列番号392)
(393)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYQ(配列番号393)
(394)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYR(配列番号394)
(395)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYS(配列番号395)
(396)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYT(配列番号396)
(397)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYV(配列番号397)
(398)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYW(配列番号398)
(399)MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSYY(配列番号399)
(400) GKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPP(配列番号403)
修飾基としては、蛍光性を呈する官能基、ペプチドの立体構造の形成には関与しない官能基などが挙げられる。また、天然由来の本発明のペプチドを構成するアミノ酸残基以外のアミノ酸残基(例えば、β-アラニンなど)であってもよい。蛍光性を呈する官能基としては、エオシンやフルオレセインイソチオシアネート(FITC)などが挙げられる。ペプチドの立体構造の形成には関与しない官能基としては、β-アラニン残基などに代表されるスペーサー基などが挙げられる。かかる官能基は末端に存在することが好ましい。さらには、ポリエチレングリコール(PEG)修飾したペプチドも「誘導体」に包含される。
固相合成法により、バクテリア中で合成させることの難しいリボソームペプチドの合成や、D体や重原子置換体などの非天然アミノ酸の導入、ペプチド及びタンパク質主鎖の修飾なども可能である。固相法において70から100個を超える長いペプチド鎖を合成する場合、ネイティブケミカルライゲーション法を用いて、2つのペプチド鎖を結合させる事により合成することが可能である。
各種HMGB1ペプチドの合成
ヒトHMGB1の2番目から44番目までのアミノ酸からなるペプチド (2-44)およびヒトHMGB1の1番目から44番目までのペプチドにおいて43番目および/または44番目のアミノ酸配列を置換した以下の5種のペプチド(Peptide-1 から5)を固相法で合成した。
2-44 GKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKK(配列番号400)
Peptide-1 MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKP(配列番号1)
Peptide-2 MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPK(配列番号2)
Peptide-3 MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPP(配列番号3)
Peptide-4 MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKA(配列番号4)
Peptide-5 MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKE(配列番号5)
細胞遊走活性測定
マウス骨髄由来間質細胞株であるST2細胞 (RCB0224,理化学研究所バイオリソースセンター) をTrypLE Express (Thermo Fisher Scientific) を用いてディッシュから剥がしたあと、25℃、1500 rpm、5分間の遠心により回収した。細胞ペレットをRPMI1640/10% FBS/1% PenStrep (Gibco) で7 x 105cells/mLに調製し、カルチャーインサート (日本ジェネティクス) をセットした24 well plateに、70 μL/insert wellで播種した。24時間後、カルチャーインサートを抜き取り、RPMI1640で1回洗浄した。Assay medium (RPMI1640/0.1% FBS) で10 μMに調製した各ペプチドサンプル(ヒトHMGB1の2番目から44番目までのペプチド (2-44)、実施例1で作成した5種の改変ペプチドPeptide-1から5)を550 μL添加し、さらに一晩培養した。陰性コントロール (NC) には、Assay mediumのみを添加した。培養後、細胞を固定化・DiffQuik染色し、撮像した。撮像した画像から各群の細胞遊走度を5段階のスコア (-,±,+,++,+++) で評価した。また、同一の条件で、各ペプチドサンプルとも3回試験を行った。
ヒトHMGB1の2番目から44番目までのペプチド(2-44)、実施例1で作成した5種類改変ペプチドPeptide-1 から5 およびNCの遊走活性の有無を確認したところ、5種の改変ペプチドのいずれにも遊走活性が認められた (表1)。
ペプチドの血漿中の安定性
抗凝固剤EDTA・2Kを用いてCrl:CD(SD)ラットから採取した全血を遠心し、ブランク血漿を得た。得られた血漿は氷上保管し、安定性試験当日のみに使用した。
実施例2で用いた6種のペプチドをそれぞれ0.1% TFA水溶液に1 mg/mLで溶解した原液から、蒸留水で375 μg/mLに調製した溶液を調製した。その溶液16 μLをブランク血漿1184 μLに対して添加し安定性試験用サンプル (アッセイ濃度:5 μg/mL) を調製した。安定性試験用サンプルを氷上に静置し、添加直後、1、3、5、15、30、60分後に50 μL (各時点、3例)を採取した。採取した50 μLのサンプルに対して、270 mmol/L クエン酸溶液/130 mmol/L EDTA・2K水溶液 (4:1, v/v) 250 μL、0.1% TFA水溶液 25 μL、IS溶液(ヒトHMGB1の1番目から44番目のペプチドのうち、18番目、38番目、41番目のフェニルアラニンの炭素原子9個、窒素原子1個をそれぞれ安定同位体に置換した化合物の4 μg/mL 0.1% TFA水溶液)25 μLを加え混合した。混合物に対してメタノール350 μLを加えて混合した後、さらにアセトニトリル100 μLを加えて混合し、ドライアイスでいったん凍結した。室温で融解し、遠心して得られた上清を別の容器に移し、撹拌したものを測定用試料とした。
結果
ヒトHMGB1の2番目から44番目までのペプチド(2-44)と43番目、44番目のアミノ酸を改変した5種のペプチド (Peptide-1から5) のラット血漿中 (氷上) における安定性試験の結果、2-44は添加後15分以降のサンプルは定量下限 (0.5 μg/mL) 以下であった。一方、Peptide-1から5の5種の改変ペプチドはいずれも2-44と比較して安定であることが確認できた(図1)。
HMGB1ペプチドの合成
ヒトHMGB1の2番目から44番目までのペプチド(2-44)においてC末端の2つのアミノ酸を置換した以下の配列からなるペプチド(Peptide-6)を固相法で合成した。
Peptide-6 GKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSPP(配列番号403)
細胞遊走活性測定
マウス骨髄由来間質細胞株であるST2細胞 (RCB0224,理化学研究所バイオリソースセンター) を2.5g/L‐Trypsin/1mmol/L‐EDTA Solution (nacalai tesque)を用いてディッシュから剥がしたあと、25℃、200 g、3分間の遠心により回収した。細胞ペレットをRPMI1640/10% FBS/1% PenStrep (nacalai tesque) で8.6 x 105 cells/mLに調製し、カルチャーインサート(日本ジェネティクス) をセットした24 well plateに、70 μL/insert wellで播種した。18時間後、カルチャーインサートを抜き取り、RPMI1640で1回洗浄した。Assay medium (RPMI1640/0.1% FBS) で20 μMに調製した各ペプチドサンプル(ヒトHMGB1の2番目から44番目までのペプチド(2-44)、および実施例4で作成した改変ペプチドPeptide-6)を1000 μL添加し、さらに20時間培養した。陰性コントロール (NC) には、Assay mediumのみを添加した。Assay medium添加直後および20時間培養後に、細胞を撮像した。撮像した画像から、カルチャーインサートにより作成した空白領域(Assay medium添加時点で細胞が存在していない領域)の面積に対する、20時間で当該領域へ遊走した細胞が占める面積の割合(%)として各群の細胞遊走度を評価した。
結果
ヒトHMGB1の2番目から44番目までのペプチド(2-44)、実施例4で作成した改変ペプチド(Peptide-6)およびNCの遊走活性の有無を確認したところ、改変ペプチド(Peptide-6)にはヒトHMGB1の2番目から44番目までのペプチド(2-44)と同等の遊走活性が認められた(図2)。
ペプチドの血漿中の安定性
あらかじめ10%EDTA・2Kを60μL入れた5mLチューブにCrl:CD(SD)ラットから採取した全血を3mL入れ、室温で30分間静置した後、3000rpm(1600×g)、4℃で10分間遠心し、上清を回収してブランク血漿とした。得られた血漿は氷上保管し、安定性試験当日のみに使用した。
ヒトHMGB1の2番目から44番目までのペプチド(以下、2-44)、実施例1で作成した改変ペプチド(Peptide-3)および実施例4で作成した改変ペプチド(Peptide-6)をそれぞれ0.1% TFA水溶液に1 mg/mLで溶解した原液から、蒸留水で375 μg/mLに調製した溶液を調製した。その溶液8 μLをブランク血漿592 μLに対して添加し安定性試験用サンプル (アッセイ濃度:5 μg/mL) を調製した。安定性試験用サンプルを氷上に静置し、添加直後、30分後および60分後に50 μL を採取した。採取した50 μLのサンプルに対して、270 mmol/L クエン酸溶液/130 mmol/L EDTA・2K水溶液 (4:1, v/v) 250 μL、0.1% TFA水溶液 50 μLを加え混合した。混合物に対してメタノール350 μLを加えて混合した後、さらにアセトニトリル100 μLを加えて混合し、ドライアイスでいったん凍結した。5~60分後、当該サンプルを室温で融解し、14000×g、4℃で3分間遠心して得られた上清を400μL採取し、遠心式濃縮機(スピンドライヤーライト VC-36R、タイテック株式会社製)で乾固した後、1% TFA/ 2% ACN Buffer 200 μLに溶解し、フィルターろ過(0.22μm)したものを測定用試料とした。
測定用試料を高速液体クロマトグラフ-シングル四重極型質量分析計に導入し、各ペプチドに特有のモニターイオンを検出した。別途調製した濃度既知試料から0.1 - 10 μg/mLの濃度範囲で検量線を作成し、各サンプルを測定して得られたクロマトグラムのピーク面積から逆回帰してサンプル中のペプチド濃度を得た。各時点におけるペプチド濃度について、添加直後を100として算出した残存率 (%) を添加後時間に対してプロットした(図3)。
結果
ラット血漿中 (氷上) における安定性試験の結果、2-44は添加後30分の時点で定量下限 (0.5 μg/mL) 以下であった。一方、Peptide-3およびPeptide-6はいずれも2-44と比較して安定であることが確認できた。また、Peptide-3とPeptide-6の安定性は同等であると認められた(図3)。
Claims (8)
- 配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチド、
配列番号1~399および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド、および
配列番号1~399に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
からなる群より選ばれるペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物。 - 配列番号1~5、126、128、129、138、167、248、254、280、282、283、292および403に記載のいずれかのアミノ酸配列を有するペプチド、
配列番号1~5、126、128、129、138、167、248、254、280、282、283、292および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド、および
配列番号1~5、126、128、129、138、167、248、254、280、282、283および292に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
からなる群より選ばれるペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物。 - 配列番号1~5、167および403に記載のいずれかのアミノ酸配列を有するペプチド、
配列番号1~5、167および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド、および
配列番号1~5および167に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
からなる群より選ばれるペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物。 - 配列番号1~5および403に記載のいずれかのアミノ酸配列を有するペプチド、
配列番号1~5および403に記載のいずれかのアミノ酸配列を有するペプチドにおいて、1若しくは複数個のアミノ酸が付加されたアミノ酸配列からなり、且つ、細胞の遊走を刺激する活性を有するペプチド、および
配列番号1~5に記載のいずれかのアミノ酸配列を有するペプチドの1-42番目のアミノ酸または配列番号403に記載のアミノ酸配列を有するペプチドの1-41番目のアミノ酸において、1若しくは複数個のアミノ酸が置換、欠失若しくは挿入されたアミノ酸配列を含み又はからなり、且つ、細胞の遊走を刺激する活性を有するペプチド
からなる群より選ばれるペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物。 - 請求項1~4のいずれかに記載のペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物を含有する、PDGFRα陽性細胞の遊走を刺激するために用いられる組成物。
- 請求項1~4のいずれかに記載のペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物を含有する、骨髄間葉系幹細胞を骨髄から末梢血に動員するために用いられる組成物。
- 請求項1~4のいずれかに記載のペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物を含有する、組織を再生するために用いられる組成物。
- 請求項1~4のいずれかに記載のペプチド、その誘導体、それらの製薬上許容される塩又はそれらの溶媒和物を含有する、骨髄由来間質細胞の遊走を刺激するために用いられる組成物。
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RU2019137603A (ru) | 2021-05-25 |
SG11201909851SA (en) | 2019-11-28 |
BR112019021230A2 (pt) | 2020-06-02 |
JP7113007B2 (ja) | 2022-08-04 |
EP3617222A4 (en) | 2021-01-20 |
CN110612307B (zh) | 2023-02-14 |
RU2019137603A3 (ja) | 2021-07-05 |
KR20190141179A (ko) | 2019-12-23 |
AU2018257071B2 (en) | 2021-11-11 |
CN110612307A (zh) | 2019-12-24 |
EP3617222A1 (en) | 2020-03-04 |
CA3059544A1 (en) | 2018-11-01 |
US20210163552A1 (en) | 2021-06-03 |
JPWO2018199107A1 (ja) | 2020-02-27 |
AU2018257071A1 (en) | 2019-10-31 |
MX2019012738A (es) | 2020-02-07 |
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