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WO2015152305A1 - Peptide de croissance pour cellules hématopoïétiques et utilisation dudit peptide de croissance pour cellules hématopoïétiques - Google Patents

Peptide de croissance pour cellules hématopoïétiques et utilisation dudit peptide de croissance pour cellules hématopoïétiques Download PDF

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WO2015152305A1
WO2015152305A1 PCT/JP2015/060273 JP2015060273W WO2015152305A1 WO 2015152305 A1 WO2015152305 A1 WO 2015152305A1 JP 2015060273 W JP2015060273 W JP 2015060273W WO 2015152305 A1 WO2015152305 A1 WO 2015152305A1
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cells
hematopoietic
peptide
hematopoietic stem
cell
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杉山　大介
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Science Lustre Co Ltd
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Science Lustre Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2306—Interleukin-6 (IL-6)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to a novel peptide that is effectively used for proliferating or producing hematopoietic cells (hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells) in vitro. More specifically, the present invention relates to a peptide that can be used effectively to grow hematopoietic cells by culturing hematopoietic stem cells or hematopoietic progenitor cells in the presence of an existing cell stimulating factor.
  • the present invention also relates to the use of the peptide, in particular, as a reagent (or preparation) used for growing hematopoietic cells in vitro or for producing them, specifically, an existing cell stimulation.
  • the present invention relates to a use as a growth aid used to assist the growth of hematopoietic cells in the presence of factors.
  • the present invention relates to a method for proliferating hematopoietic cells using the peptide in vitro, in other words, a method for producing hematopoietic cells in vitro, as a use of the peptide.
  • the present invention relates to an antibody against the peptide.
  • hematopoietic stem cells can be produced in vitro from pluripotent stem cells, or if hematopoietic stem cells can be removed from the body and cultured, and only hematopoietic stem cells can be amplified without becoming tumorous, many transplant-related problems will be solved.
  • new regenerative medicine such as the development of other organ-specific stem cell transplantation therapy can be cultivated.
  • the following problems have been pointed out in the methods for producing and amplifying hematopoietic stem cells that have been developed so far. 1.
  • hematopoietic stem cells When the HoxB4 gene is introduced into pluripotent stem cells, hematopoietic stem cells can be produced, but acute leukemia is induced. 2. When the HoxB4 gene is introduced into hematopoietic stem cells, hematopoietic stem cells are amplified, but acute leukemia is induced. 3. The addition of cytokines induces differentiation of mature blood cells and it is difficult to maintain the undifferentiated nature of hematopoietic stem cells. 4). Addition of low molecular weight compounds such as demethylating agents increases the efficiency of hematopoietic stem cell amplification, but safety is necessary because it affects epigenetics.
  • pluripotent stem cells can theoretically differentiate into all cell lineages, technology to determine the fate of their differentiation in vitro is under development, and hematopoietic stem cells that can be applied clinically are produced from pluripotent stem cells Technology to develop is also in the process of development. Multipotent cells retain the property of losing the ability to differentiate into other lineages when differentiated in a certain direction. If the differentiation into cells other than hematopoietic stem cells is suppressed, Manufacturing can be expected. Hematopoietic stem cells have seemingly contradictory capabilities of self-renewal and multipotency, and differentiate into mature blood cells upon stimulation with cytokines. In other words, in order to amplify hematopoietic stem cells, it is necessary to brake differentiation into various cell lineages while accelerating self-replication.
  • Patent Document 1 discloses a hematopoietic stem cell proliferating agent containing a placental constituent cell pulverized product as an active ingredient, but such a biological material has problems such as infection and difficulty in preparing the material.
  • Patent Document 2 discloses a hematopoietic stem cell maintenance factor or amplification promoting factor comprising the membrane protein Thsd1 / Tmtsp, but such a membrane protein is difficult to prepare in an active form.
  • Hematopoietic stem cell transplantation is widely applied not only to hematopoietic diseases but also to autoimmune diseases, malignant solid tumors, and regenerative medicine.
  • autologous bone marrow, donor bone marrow, and umbilical cord blood are used as a source of hematopoietic stem cells for transplantation.
  • donor bone marrow or umbilical cord blood is used, there are problems of lack of absolute number of transplanted hematopoietic stem cells, donor shortage, failure of engraftment due to immune response, and graft-versus-host disease.
  • hematopoietic stem cells can be produced from pluripotent stem cells in vitro, it can be a new therapeutic tool that does not depend on the source of bone marrow, peripheral blood, umbilical cord blood, and the like.
  • hematopoietic stem cells can be cultured and amplified in vitro, a sufficient amount of hematopoietic stem cells can be supplied at any time by simply collecting a small amount of hematopoietic stem cells from the patient's own bone marrow, etc. It is possible to transplant safely without waiting. That is, the number of hematopoietic stem cells necessary for transplantation can be secured and autotransplantation without rejection can be performed.
  • transplantation treatment can be performed simply by collecting a small amount of hematopoietic stem cells from the donor's bone marrow, eliminating the risk of the donor and increasing the number of registered donors in the bone marrow bank. We can expect an increased chance of finding donors. If umbilical cord blood can also be cultured in vitro to amplify hematopoietic stem cells, treatment for adults becomes possible.
  • the present invention has an effect of amplifying hematopoietic stem cells of vertebrates, particularly mammals, and proliferates hematopoietic stem cells and hematopoietic progenitor cells, which are differentiated cells thereof, in vitro.
  • An object of the present invention is to provide a novel peptide useful in the above.
  • Another object of the present invention is to provide a novel peptide that has a proliferative action on hematopoietic progenitor cells, is useful for proliferating mature hematopoietic cells in vitro, and producing them.
  • the present invention relates to (i) the use of the peptide as a growth assistant for hematopoietic cells (hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells), and a method for proliferating hematopoietic cells in vitro using the peptide,
  • an object is to provide a method for producing hematopoietic cells.
  • the present inventor suppresses the differentiation and proliferation of hematopoietic stem cells among a plurality of peptides designed based on the extra-membrane domain of various membrane surface proteins expressed in the hepatoblasts of human fetal liver, which is the growth site of hematopoietic cells.
  • a peptide having an action of promoting the autonomous proliferation (amplification) of mesenchymal stem cells which are said to have the ability to differentiate into mesenchymal cell lineages such as osteoblasts, adipocytes, muscle cells, and chondrocytes It was found that there are peptides; and peptides having an action of specifically inducing hematopoietic stem cells from pluripotent stem cells such as ES cells (see Patent Document 3).
  • a specific modification of the above peptide has the effect of autonomously proliferating (amplifying) and differentiating and proliferating hematopoietic stem cells in vitro, for example, in vitro in the presence of a cell stimulating factor such as a cytokine. It was confirmed that the peptide is extremely effective as a “proliferative cofactor for hematopoietic stem cells and hematopoietic progenitor cells” that assists in the proliferation of hematopoietic stem cells and their differentiated cells.
  • the peptide has the effect of generating mature hematopoietic cells by differentiating and proliferating hematopoietic progenitor stem cells in vitro in the presence of cell stimulating factors. It was also confirmed that hematopoietic stem cells are maintained ex vivo, that is, has the effect of maintaining the characteristics of hematopoietic stem cells without differentiation from hematopoietic stem cells into any cells.
  • hematopoietic stem cells or hematopoietic progenitor cells together with cell stimulating factors in the presence of the peptide developed by the present inventor, hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells (in the present invention, These are collectively referred to as “hematopoietic cells”).
  • hematopoietic stem cells hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells
  • hematopoietic stem cells hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells
  • Cells can be grown and cultured.
  • hematopoietic stem cells can be proliferated and produced, and therefore, it is highly expected as a material that can solve the problem of the source of hematopoietic stem cells for transplantation in hematopoietic stem cell transplantation therapy. .
  • n is an integer of 3 to 15, x and y are 0 or 1 (provided that when one of x and y is 1, the other y and x are 0, respectively), and m and p are 1 to An integer of 3 as well as q is 0 or 1 (provided that when y is 1, q is 1)].
  • (I-2) The peptide according to (I-1) or a modified product thereof, wherein the peptide represented by the general formula (I) is a peptide consisting of the amino acid sequence of the following formula (SEQ ID NO: 1):
  • (II) Hematopoietic cell growth assistant and its usage (II-1) (I-1) or peptide described in (I-2), a modified product thereof, a pharmaceutically acceptable salt thereof, and these
  • a hematopoietic cell growth assistant comprising as an active ingredient at least one selected from the group consisting of: (II-2) at least one selected from the group consisting of the peptide described in (I-1) or (I-2), a modified product thereof, a pharmaceutically acceptable salt thereof, and a solvate thereof
  • a method of proliferating hematopoietic cells comprising culturing hematopoietic stem cells or hematopoietic progenitor cells in a medium containing a seed and a cell stimulating factor.
  • the cell stimulating factor is derived from a stem cell stimulating factor, thrombopoietin, interleukin-6, soluble interleukin-6 receptor, granulocyte colony stimulating factor, interleukin-3, interleukin-11, and Flt3 ligand.
  • III-1) Method for in vitro proliferation of hematopoietic cells
  • III-1 Peptides described in (I-1) or (I-2), modified products thereof, pharmaceutically acceptable salts thereof, and solvates thereof
  • a method for in vitro proliferation of hematopoietic cells comprising culturing hematopoietic stem cells or hematopoietic progenitor cells in a medium containing a cell stimulating factor and at least one selected from the group consisting of substances.
  • the cell stimulating factor is selected from stem cell stimulating factor, thrombopoietin, interleukin-6, soluble interleukin-6 receptor, granulocyte colony stimulating factor, interleukin-3, interleukin-11, and Flt3 ligand.
  • IV-1 In vitro production method of hematopoietic cells (IV-1) Peptides described in (I-1) or (I-2), modified products thereof, pharmaceutically acceptable salts thereof, and solvates thereof
  • a method for producing hematopoietic cells in vitro comprising a step of culturing hematopoietic stem cells or hematopoietic progenitor cells in a medium containing at least one selected from the group consisting of substances and a cell stimulating factor.
  • the cell stimulating factor is a stem cell stimulating factor, thrombopoietin, interleukin-6, soluble interleukin-6 receptor, granulocyte colony stimulating factor, interleukin-3, interleukin-11, and Flt3 ligand.
  • V-2) A pharmaceutical composition comprising the cell population described in (V-1).
  • V-3) The pharmaceutical composition described in (V-2), which is a hematopoietic function improving agent.
  • VI An antibody against at least one peptide described in the antibody (VI-1) (I-1) or (I-2).
  • VI-2) The antibody according to (VI-1), which is a monoclonal antibody.
  • hematopoietic stem cells for example, autologous hematopoietic stem cells or cord blood hematopoietic stem cells with a relatively low immune response
  • a cell stimulating factor such as a cytokine
  • the number of hematopoietic progenitor cells and mature hematopoietic cells can be increased by autonomously proliferating hematopoietic stem cells to increase the absolute number thereof or by causing differentiation to proliferate.
  • the problems of the present invention can be solved, and the problem of the source of transplanted hematopoietic stem cells and hematopoietic progenitor cells in conventional hematopoietic stem cell transplantation therapy can be solved.
  • hematopoietic stem cells are differentiated and proliferated in a culture container such as a test tube in vitro. It becomes possible to increase the number.
  • FIG. 1 (A) The total number of cells (FIG. 1 (A)) and the cell viability [FIG. 1 (B)] on day 7 and day 11 of culturing human cord blood CD34-positive hematopoietic stem cells are shown (Experimental Example 1).
  • - ⁇ -(Peptide A) is peptide A (10 ⁇ g / mL) and cell stimulating factor (hSCF 50ng / mL, hTPO 10ng / mL, hFlt3L 20ng / mL, hIL-6 20ng / mL, hsIL-6R ⁇ 20ng
  • Peptide A means peptide A (10 ⁇ g / mL) and cell stimulating factor (hSCF 50 ng / mL, hTPO 10 ng / mL, hFlt3L 20 ng / mL, hIL-6 20 ng / mL, and hsIL-6R ⁇ 20 ng / mL
  • Control indicates the result of culturing in the presence of only the cell stimulating factor without peptide A (10 ⁇ g / mL). The same applies to FIGS. 3 to 6 below.
  • the ratio of CD34-positive CD38-negative cells to the total number of living cells on day 11 of culturing human cord blood CD34-positive hematopoietic stem cells (FIG. 2 (A)), and the total number of CD34-positive CD38-negative cells (FIG. 2 (B)) (Experimental example 1).
  • the total number of cells expressing each cell surface marker (CD13, CD34, CD38, CD45, and GPA) among the results of the cell surface marker test on day 11 of culturing human cord blood CD34 positive hematopoietic stem cells is shown.
  • the number of living cells expressing each cell surface marker (CD13, CD34, CD38, CD45, and GPA)
  • the ratio (%) in the total number of Human umbilical cord blood CD34 positive hematopoietic stem cell population with medium containing cell stimulating factor mixture (hSCF 50ng / mL, hTPO 10ng / mL, hFlt3L 20ng / mL, hIL-6 20ng / mL, and hsIL-6R ⁇ 20ng / mL) Control), or after culturing in a medium (PeptidePA) containing the cell stimulating factor mixture and peptide A (10 ⁇ g / mL) for 11 days, a colony-forming cell assay was performed to determine the number of colony-forming cells (corresponding to hematopoietic progenitor cells).
  • FIG. 6A shows the number of counts for each type of hematopoietic progenitor cells (CFU-G, CFU-M, CFU-GM, CFU-MK, BFU-E, and CFU-GEMM), and FIG. ) Indicates the total number of hematopoietic progenitor cells.
  • Example 2 Diagram showing fluorescent micrographs of human cord blood CD34 positive hematopoietic stem cells cultured with biotinylated peptide A (10 ⁇ g / mL) (Modified KS13) or without (Control) for 6 hours or 12 hours (experiment) Example 3). Nucleus in the figure indicates only nuclear staining, and Marge indicates a combination of nuclear staining and peptide A.
  • the expression level of each gene of the SOCS family (SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7) on the first day from the start of liquid culture is shown.
  • the vertical axis of the graph in the figure represents a relative value.
  • the expression level of each gene of the SOCS family (SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7) on the second day from the start of liquid culture is shown.
  • the vertical axis of the graph in the figure represents a relative value.
  • hematopoietic stem cell means “self-renewal ability” that is the ability to proliferate while maintaining undifferentiation and “multipotency” that is the ability to differentiate into all blood cells and lymphocyte cells.
  • Human hematopoietic stem cells can be characterized as CD34 (+) because they express the stem cell marker CD34. For this reason, CD34 positive cells can be used as human hematopoietic stem cells.
  • Human hematopoietic stem cells can also be characterized by other hematopoietic stem cell markers in addition to CD34 based on cell surface antigens expressed on hematopoietic stem cells.
  • hematopoietic stem cell markers including CD34 include Lin ( ⁇ ), CD34 (+), CD38 ( ⁇ ), DR ( ⁇ ), CD45 (+), CD90 (+), CD117 (+), CD123 (+) , And CD133 (+). These can be used singly or in combination. Examples of the combination include Lin ( ⁇ ) CD34 (+) CD38 ( ⁇ ), CD45 (+) CD34 (+) CD38 ( ⁇ ), etc. Can be mentioned.
  • mouse hematopoietic stem cells are known to change the expression of cell surface antigens in the embryonic and adult period, and as markers of embryonic hematopoietic stem cells, Lin (-), CD31 (+), CD34 (+), CD41 (+), C-Kit (+), as markers for adult hematopoietic stem cells, Lin (-), CD31 (+), CD34 (-/ +), CD45 (+), c-Kit (+), Sca-1 (+), CD150 (+), EPCR (+). These can be used singly or in combination. As a mode of combination, Lin ( ⁇ ) c-Kit (+) Sca-1 (+) and CD45 (+) c-Kit (+ ) Sca-1 (+).
  • hematopoietic stem cells are contained in a very small amount in bone marrow, peripheral blood, and umbilical cord blood. From these, using conventional methods such as FACS (fluorescence activated cell sorting) using the above stem cell markers as an index Can be collected.
  • FACS fluorescence activated cell sorting
  • Hematopoietic progenitor cell refers to a cell derived from a hematopoietic stem cell and not terminally differentiated. Hematopoietic progenitor cells can be classified into pluripotent hematopoietic progenitor cells that can differentiate into two to three lineages of blood cells, or unipotent hematopoietic progenitor cells that have limited differentiation into one blood cell. Hematopoietic progenitor cells differentiate into three types of progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, or red blood cell / megakaryocytic progenitor cells.
  • Myeloid progenitor cells differentiate into progenitor cells that finally differentiate into granulocytes (neutrophils, eosinophils, basophils), monocytes and the like.
  • the hematopoietic progenitor cells may be cells that differentiate into mature hematopoietic cells regardless of the above-mentioned lineage.
  • lymphoid progenitor cells differentiate into progenitor cells that finally differentiate into T cells, B cells, and NK cells. Therefore, hematopoietic progenitor cells are myeloid cells [granulocytes (eosinophils, neutrophils, basophils), monocytes, macrophages, mast cells], erythroid cells [erythrocytes], megakaryocytes [ Megakaryocytes, platelets], and precursor cells of lymphoid cells [T cells, B cells, plasma cells].
  • myeloid cells granulocytes (eosinophils, neutrophils, basophils), monocytes, macrophages, mast cells]
  • erythroid cells erythrocytes
  • megakaryocytes Megakaryocytes, platelets
  • precursor cells of lymphoid cells T cells, B cells, plasma cells.
  • “Mature hematopoietic cells” are cells in which the above “hematopoietic stem cells” and “hematopoietic progenitor cells” are terminally differentiated. As described above, myeloid cells [granulocytes (eosinophils, neutrophils, basophils) Monocytes, macrophages, mast cells], erythroid cells [erythrocytes], megakaryocyte cells [megakaryocytes, platelets], and lymphocyte cells [T cells, B cells, plasma cells].
  • myeloid cells granulocytes (eosinophils, neutrophils, basophils) Monocytes, macrophages, mast cells]
  • erythroid cells erythrocytes
  • megakaryocyte cells megakaryocyte cells
  • platelets lymphocyte cells
  • hematopoietic cell is used as a generic term for the above-mentioned hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells without distinction.
  • the hematopoietic cells targeted by the present invention are preferably derived from vertebrates, more preferably birds (such as chickens) or mammals (human, mouse, rat, rabbit, monkey, chimpanzee, pig, horse, goat) , Sheep, cattle, dogs, cats, wallabies, kangaroos, etc.).
  • Cells derived from mammals are preferred.
  • rodents mouse, rat, rabbit, etc.
  • widely used as humans or experimental animals are particularly preferable.
  • “Proliferation” refers to increasing the total number of undifferentiated and terminally differentiated cells.
  • the cells that are not terminally differentiated include hematopoietic stem cells and hematopoietic progenitor cells, and the cells that are terminally differentiated include hematopoietic mature cells.
  • “hematopoietic cell proliferation” includes increasing the total number of these cells without distinguishing hematopoietic stem cells, hematopoietic progenitor cells, and hematopoietic mature cells.
  • hematopoietic stem cells when hematopoietic stem cells are used as cells, hematopoietic progenitor cells differentiated from hematopoietic stem cells in addition to amplification of hematopoietic stem cells (the hematopoietic stem cells autonomously proliferate while maintaining the undifferentiated nature of hematopoietic stem cells). The total number of hematopoietic stem cells and hematopoietic progenitor cells increases. When hematopoietic progenitor cells are used as cells, hematopoietic progenitor cells are differentiated and proliferated, and the total number of terminally differentiated cells (hematopoietic mature cells) is increased.
  • hematopoietic cell growth assistant means a reagent (formulation) effective to increase the total number of hematopoietic cells without distinguishing hematopoietic stem cells, hematopoietic progenitor cells, and hematopoietic mature cells. To do. When this is applied to, for example, hematopoietic stem cells, as described above, the total number of hematopoietic stem cells and hematopoietic progenitor cells can be increased. Can be increased.
  • Ultrabilical cord blood refers to blood that can be obtained from the umbilical cord of mammals, preferably humans.
  • “Bone marrow-derived blood” refers to blood contained in spinal fluid present in the bone marrow of mammals, preferably humans.
  • Umbilical cord blood and bone marrow can be obtained from umbilical cord blood bank and bone marrow bank, respectively. It is also possible to obtain from volunteers.
  • Novel peptide peptide of the present invention
  • the peptide targeted by the present invention acts on hematopoietic stem cells and hematopoietic progenitor cells to proliferate hematopoietic stem cells, hematopoietic progenitor cells and mature hematopoietic cells.
  • this action is classified into an action that acts on hematopoietic stem cells to amplify hematopoietic stem cells, an action that differentiates and proliferates hematopoietic progenitor cells, and an action that acts on hematopoietic progenitor cells and differentiates and proliferates mature hematopoietic cells. be able to.
  • a peptide consisting of the following amino acid sequence (SEQ ID NO: 1) can be mentioned:
  • the above peptide is the most preferred peptide.
  • the number (n) of arginine residues in the Arg-rich region on the N-terminal side of the amino acid sequence is as described above.
  • the number is not limited to 8, and can be selected from a range of 3 to 15, for example.
  • the number is preferably 7 to 15, more preferably 7 to 11, and particularly preferably 7 to 8.
  • n is an integer of 3 to 15
  • x and y are 0 or 1 (provided that when one of x and y is 1, the other y and x are each 0)
  • m and p are 1 to An integer of 3 as well as q is 0 or 1 (provided that when y is 1, q is 1)].
  • the peptide of the present invention comprises an Arg-rich region composed of 3 to 15 arginine residues and an amino acid sequence [Cys Gln Lys Lys Asp Gly Pro Cys that forms the core of the peptide of the present invention.
  • Val Ile Asn Gly Ser] SEQ ID NO: 2; hereinafter referred to as “core sequence”
  • core sequence N side a polyethylene glycol chain via a peptide bond.
  • the PEG chain When the PEG chain is present on either the core sequence N side or the core sequence C side, the PEG chain may have one unit of [CH 2 —CH 2 —O] (m Alternatively, p may be 1), or the unit may be repeated two or more (polymerization).
  • the degree of polymerization (m, p) of the PEG chain is preferably 2 to 3, more preferably 3.
  • an aminocaproic acid residue ( -NH- [CH 2 ] 5 -CO-) may be included.
  • the peptide of the present invention can be synthesized by a solid phase synthesis method or a liquid synthesis method using a peptide synthesizer based on the above formula, and amino acid substitution, addition or deletion can be performed when a peptide synthesizer is used. This can be done easily by changing the type of protected amino acid.
  • insertion of a PEG chain into a peptide can also be performed according to a known technique.
  • mini-PEG® Fmoc-mini-PEG TM , Fmoc-mini-PEG-3 TM , Boc-mini-PEG TM , commercially available from Peptides International, Boc-mini-PEG-3 TM
  • mini-PEG® Fmoc-mini-PEG TM , Fmoc-mini-PEG-3 TM , Boc-mini-PEG TM , commercially available from Peptides International, Boc-mini-PEG-3 TM
  • the peptide of the present invention may be in a free state, in the form of a salt, or in the form of a solvate containing a hydrate.
  • the salt include physiologically acceptable acid addition salts and base salts, for example, cell biologically or pharmaceutically acceptable.
  • acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, nitrate and sulfate; or sulfonates such as methanesulfonic acid and toluenesulfonic acid, trifluoroacetic acid, succinic acid and acetic acid.
  • Organic acid salt etc. are mentioned.
  • the base salt include alkali metal salts such as sodium, potassium and lithium; or alkaline earth metal salts such as calcium and magnesium.
  • this invention peptide may be comprised from the amino acid residue of L body, D body, or both.
  • the peptide of the present invention represented by the above formula (I) may have a modifying group commonly used in the peptide field. This is referred to as a modified peptide in the present invention.
  • the modified product includes a carboxyl group (—COOH), a carboxylate (—COO—), an amide (—CONH 2 ), or an ester (—COOR) at the C-terminus of the peptide of the present invention represented by the general formula (I). Certain peptides are included.
  • examples of the group R in the ester include alkyl groups having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl and n-butyl; cycloalkyl groups having 3 to 8 carbon atoms such as cyclopentyl and cyclohexyl; C 7-14 Aralkyl groups (for example, aryl groups having 6 to 12 carbon atoms such as phenyl and ⁇ -naphthyl; phenyl-C 1-2 alkyl groups such as benzyl and phenethyl; ⁇ -naphthyl-C 1 such as ⁇ -naphthylmethyl) -2 alkyl groups); pivaloyloxymethyl groups and the like.
  • the peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the modified
  • the amino group of the N-terminal amino acid residue (Arg) is a protecting group such as C 1-6 acyl group (for example, C 1-6 alkanoyl such as formyl group, acetyl group, etc.).
  • fatty acids saturated fatty acids of C 8-18
  • substituents on the side chains of amino acids in the molecule eg —OH, —SH, amino groups, imidazole groups, Indole group, guanidino group, etc.
  • a suitable protecting group such as C 1-6 acyl group (eg, C 1-6 alkanoyl group such as formyl group, acetyl group, etc.), or sugar
  • C 1-6 acyl group eg, C 1-6 alkanoyl group such as formyl group, acetyl group, etc.
  • complex peptides such as so-called glycopeptides with chains attached.
  • modified products of the peptide of the present invention include those in which an imidazolyl group or SH group is alkylated (for example, methylated), aralkylated (for example, benzylated), or acylated (for example, acetylated or benzoylated).
  • the peptide modified with the above fatty acid includes a myristoylated peptide in which the amino group of the peptide main chain is modified with myristic acid of the N-terminal arginine residue; or the side-chain guanidyl group of the N-terminal arginine residue.
  • Examples include myristoylated peptides in which the amino group constituting (also referred to as guanidine group or guanidino group) is modified with myristic acid (the amino group and the carboxyl group of myristic acid are amide-bonded).
  • the peptide in which the N-terminal is acetylated as described above refers to a peptide in which the amino group of the peptide main chain of the N-terminal arginine residue is acetylated (also referred to as ethanoylation); or the N-terminal arginine residue.
  • the amino group constituting the side chain guanidyl group is acetylated.
  • peptide of the present invention is used as it is or after mixing with a physiologically acceptable carrier as necessary, for example, a cell physiologically or pharmaceutically acceptable carrier. It can be used as a hematopoietic cell growth aid.
  • a physiologically acceptable carrier for example, a cell physiologically or pharmaceutically acceptable carrier. It can be used as a hematopoietic cell growth aid.
  • peptides consist of 1 type individually, 2 or more types can also be used in arbitrary combinations.
  • the proliferation assistant is a proliferation assistant used together with a cell stimulating factor when hematopoietic stem cells or hematopoietic progenitor cells proliferate, and plays a role of promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells.
  • a proliferation assistant used together with a cell stimulating factor when hematopoietic stem cells or hematopoietic progenitor cells proliferate, and plays a role of promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells.
  • the proliferation assistant is a proliferation assistant used together with a cell stimulating factor when hematopoietic stem cells or hematopoietic progenitor cells proliferate, and plays a role of promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells.
  • the proliferation assistant is a proliferation assistant used together with a cell stimulating factor when hematop
  • the growth aid is prepared, for example, by dissolving the peptide of the present invention in water or an appropriate buffer (eg, phosphate buffer, PBS, Tris-HCl buffer, etc.) so as to have an appropriate concentration. be able to. Moreover, you may mix
  • an appropriate buffer eg, phosphate buffer, PBS, Tris-HCl buffer, etc.
  • the growth assistant of the present invention is prepared by, for example, adding hematopoietic stem cells or hematopoietic progenitors by adding an effective amount of the peptide of the present invention to the medium and culturing hematopoietic stem cells or hematopoietic cells in the presence of a cell stimulating factor.
  • Cells and / or mature hematopoietic cells can be grown in culture and used to produce them. Therefore, the present invention also provides a method for proliferating hematopoietic cells, comprising culturing hematopoietic stem cells or hematopoietic progenitor cells in the presence of the peptide of the present invention (or the growth assistant of the present invention). This will be described later.
  • the growth of hematopoietic cells can be promoted by culturing hematopoietic stem cells or hematopoietic progenitor cells in the presence of the polypeptide of the present invention (or the growth assistant of the present invention) and a cell stimulating factor. Therefore, the peptide of the present invention can also be used as a component of a reagent kit for in vitro proliferation of hematopoietic cells. Such a kit may be used for the purpose of maintaining hematopoietic stem cells in vitro.
  • the method for proliferating hematopoietic cells of the present invention includes a step of culturing hematopoietic stem cells and / or hematopoietic progenitor cells in the presence of the peptide of the present invention.
  • the proliferation method can be said to be a method for producing hematopoietic cells by culturing hematopoietic stem cells and / or hematopoietic progenitor cells. Therefore, the following “hematopoietic cell growth method” can be restated as “hematopoietic cell production method”.
  • the hematopoietic stem cells used in the method of the present invention include a group of cells containing at least hematopoietic stem cells, and the hematopoietic stem cells may be isolated.
  • the cell group may be a fraction containing hematopoietic stem cells fractionated from a cell group containing hematopoietic stem cells.
  • examples of the hematopoietic progenitor cells used in the method of the present invention include a group of cells containing at least hematopoietic progenitor cells, and hematopoietic progenitor cells may be isolated.
  • the cell group may be a fraction containing hematopoietic progenitor cells fractionated from a cell group containing hematopoietic progenitor cells.
  • the collection source of hematopoietic stem cells and hematopoietic progenitor cells may be any tissue as long as it contains vertebrates such as birds and mammals, and preferably mammalian stem cells such as mice and rats belonging to humans and rodents.
  • hematopoietic stem cells can be collected from fetal liver, fetal bone marrow, bone marrow, peripheral blood, umbilical cord blood, or peripheral blood to which stem cells are recruited by administration of cytokines and / or anticancer agents.
  • a culture method using a so-called culture plate, petri dish or flask is possible, but the medium composition, pH, etc. are controlled mechanically.
  • the culture system can also be improved by a bioreactor capable of culturing at a high density (Schwartz, Proc. Natl. Acad. Sci. USA, 88: 6760, 1991; Koller, MR, Bio / Technology, 11). : 358, 1993; Koller, MR, Blood, 82: 378, 1993; Palsson, BO, Bio / Technology, 11: 368,1993).
  • the medium used for the culture is not particularly limited as long as the proliferation and survival of hematopoietic stem cells or hematopoietic progenitor cells are not impaired.
  • minimum essential medium MEM
  • DMEM Dulbecco's modified Eagle medium
  • IMDM IMDM medium
  • RPMI1640 medium
  • 199 medium SF-02 medium
  • Opti- MEM medium GIC BRL
  • hematopoietic stem cell culture medium X-VIVO 10 Longza
  • StemSpan SFEM serum-free growth medium
  • StemSpan H3000 animal component-free medium
  • Veritas animal component-free medium
  • the pH of the medium is preferably about 6 to 8.
  • the medium contains cell stimulating factors (cytokines and hematopoietic hormones such as EPO (erythropoietin)), hormones such as insulin, Wnt (Thimoth, A. W., Blood, 89: 3624-3635, 1997) Differentiation and growth regulators such as gene products, transport proteins such as transferrin, demethylating agents such as 5azaD and TSA (Exp. Hematol. 34: 140, 2006), extracellular matrix proteins such as Fibronectin and Collagen (Curr) Opin Biotechnol. 2008 October; 19 (5): 534-540 .; Cell 2007 June; 129 (7): 1377-1388.) And the like.
  • cytokines and hematopoietic hormones such as EPO (erythropoietin)
  • hormones such as insulin
  • Wnt Thimoth, A. W., Blood, 89: 3624-3635, 1997)
  • Differentiation and growth regulators such as gene products, transport proteins such
  • the hematopoietic cells can be proliferated and amplified more efficiently. Can do.
  • the cell stimulating factor is a factor that gives stimulation such as proliferation, differentiation, survival, and migration to tissue-specific cells such as hematopoietic stem cells and hematopoietic progenitor cells.
  • Such cell stimulating factors are not particularly limited as long as they do not interfere with the proliferation of hematopoietic stem cells and hematopoietic progenitor cells.
  • SCF stem cell growth factor
  • IL-3 interleukin
  • GM-CSF granulocyte / macrophage colony-stimulating factor
  • IL-6 interleukin-6
  • sIL-6R soluble IL-6 receptor
  • IL- 11 interleukin-11
  • Flt-3L Flt-3L (fms-like tyrosine kinase-3 (Flt-3) ligand)
  • EPO erythropoietin
  • TPO thrombopoietin
  • G-CSF granulocyte colony stimulating factor
  • TGF- ⁇ Transforming growth factor- ⁇
  • MIP-1 ⁇ George, D., J. Exp. Med. 167: 1939-1944, 1988
  • Flt3 / Flk2-ligand FGF (fibroblast growth factor), etc. It is done.
  • These stimulating factors are detailed in Gallard, R.E., The cytokine facts book, AcademicPress, 1994.
  • the cell stimulating factor to be mixed in the medium may be one type or two or more types.
  • SCF, G-CSF, IL-3, IL-6, IL-11, Flt-3L, sIL-6R, and TPO can be mentioned, and in particular, SCF is essential.
  • the concentration of the cell stimulating factor added to the medium is 1 to 500 ng / mL, preferably 5 to 300 ng / mL, more preferably 10 to 100 ng / mL.
  • the peptide of the present invention can be added to the medium so that the final concentration in the medium is 1 to 500 ⁇ g / mL, preferably 5 to 300 ⁇ g / mL, more preferably 10 to 100 ⁇ g / mL.
  • hematopoietic stem cells or hematopoietic progenitor cells can be added to the medium so that the cell density is normally used in the art. Cultivation is usually carried out in an atmosphere of about 30 to 40 ° C. and about 5 to 10% CO 2 for a time to achieve the desired growth. You may perform ventilation
  • (V) Cell population containing hematopoietic cells obtained by the proliferation method (manufacturing method) of the present invention and use thereof “hematopoietic cells obtained by the above-described proliferation method (III) or production method (IV) of the present invention” Can be used as a composition (graft) for blood cell transplantation or regenerative medicine in place of conventional bone marrow transplantation or umbilical cord blood transplantation.
  • the “cell population containing hematopoietic cells” refers to hematopoietic cells (hematopoietic stem cells) obtained by the above-described proliferation method (III) or production method (IV) of the present invention. , Hematopoietic progenitor cells and / or mature hematopoietic cells).
  • the cell population obtained by the proliferation method (III) or the production method (IV) of the present invention is further subjected to flow cytometry and the like, and a hematopoietic stem cell group, a hematopoietic progenitor cell group, or a mature hematopoietic cell group is obtained using a conventional technique. It can also be purified and collected.
  • the cell population produced and expanded by the method of the present invention can be used for various diseases in addition to these treatments when performing systemic X-ray therapy or advanced chemotherapy for leukemia.
  • treatment in which suppression of hematopoietic function in the bone marrow as a side effect such as chemotherapy and radiation therapy for solid cancer patients
  • bone marrow is collected before the operation, hematopoietic stem cells are amplified in vitro,
  • hematopoietic system failure due to side effects can be recovered early, more powerful chemotherapy can be performed, and the therapeutic effect of chemotherapy can be improved.
  • the cell population of the present invention includes diseases associated with impaired hematopoietic function, such as aplastic anemia, congenital immunodeficiency, congenital metabolic disorders, myelodysplastic syndrome, leukemia, malignant lymphoma, multiple myeloma, Myelofibrosis, chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, immune deficiency syndrome such as acquired immune deficiency syndrome (AIDS), thalassemia, hemolytic anemia due to enzyme deficiency, sickle-like It can be used as a preventive or therapeutic agent for congenital anemia such as erythrocytosis, lysosomal storage diseases such as Gaucher disease and mucopolysaccharidosis, and adrenoleukodysplasia.
  • diseases associated with impaired hematopoietic function such as aplastic anemia, congenital immunodeficiency, congenital
  • Such a cell population can be used in the form of a pharmaceutical composition by mixing with a pharmacologically acceptable carrier or buffer, if necessary, in addition to the hematopoietic cells grown or produced by the method of the present invention. .
  • the pharmacologically acceptable carrier various organic or inorganic carrier substances commonly used as pharmaceutical materials are used.
  • suspending agents isotonic agents, buffers, and soothing agents in suspensions.
  • formulation additives such as preservatives, antioxidants, thickeners and stabilizers can be used.
  • Transplantation of a cell population or a pharmaceutical composition prepared therefrom may be performed in the same manner as conventional bone marrow transplantation or umbilical cord blood transplantation.
  • parenteral administration eg, intravenous injection, local infusion, etc.
  • Suitable formulations of the pharmaceutical composition include aqueous and non-aqueous isotonic sterile injection solutions.
  • the dosage of the cell population of the present invention or the pharmaceutical composition prepared therefrom depends on the activity of the peptide of the present invention, the severity of the disease, the species of animal to be administered, the drug acceptability, sex, weight, age, etc. of the administered subject.
  • the amount of hematopoietic stem cells per adult is 1 ⁇ 10 6 cells / kg or more, preferably 1 ⁇ 10 6 to 1 ⁇ 10 10 cells / kg, more preferably 2 ⁇ 10 6 ⁇ 1 ⁇ 10 9 cells / kg.
  • the antibodies of the present invention include polyclonal antibodies and monoclonal antibodies.
  • a monoclonal antibody is preferable.
  • Preferred antibodies include an antibody against the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 among the peptides of the present invention.
  • the monoclonal antibodies of the present invention are immunoglobulins derived from vertebrates such as mammals including birds and rodents, preferably mammals such as humans, mice or rats, and the class and subclass thereof are not particularly limited.
  • a preferred class and subclass is immunoglobulin M (IgM), more preferably IgM ( ⁇ chain).
  • Monoclonal antibodies can be prepared according to known methods such as those described in Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989) and the like.
  • the monoclonal antibody of the present invention is preferably a humanized antibody from the viewpoint of reducing antigenicity against humans.
  • a humanized antibody is a chimeric antibody in which a portion other than the variable region (or hypervariable region) of a non-human animal antibody is replaced with an amino acid sequence of human immunoglobulin, and the peptide of the present invention, particularly the amino acid sequence shown in SEQ ID NO: 1
  • Humanized monoclonal antibodies can be prepared according to known techniques.
  • Example 1 Preparation of Peptides As peptides of the present invention, the following four types of peptides were designed (hereinafter referred to as peptides A to D), and synthesis was requested from Scrum Co., Ltd.
  • peptides A to D the following four types of peptides were designed (hereinafter referred to as peptides A to D), and synthesis was requested from Scrum Co., Ltd.
  • PEG means an ethylene glycol chain (—CH 2 —CH 2 —O—).
  • Peptide A [Arg] 8 - [ Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] -NH 2 ( SEQ ID NO: 1)
  • Peptide B [Arg] 8 - [ Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] -NH- (PEG) 3 -CO-NH 2 ( SEQ ID NO: 3)
  • Peptide C [Arg] 8- NH- (PEG) 3- CO- [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] (SEQ ID NO: 4)
  • Peptide D Ac- [Arg] 8 -NH- (PEG) 3 -CO- [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] -NH 2 ( SEQ ID NO: 5).
  • [Arg] 8 means polyarginine in which eight arginines are peptide-bonded in a straight chain without passing through their side chains.
  • Experimental Example 1 Proliferation of human cord blood hematopoietic stem cells using peptide A
  • Experimental method (1-1) Human cord blood CD34-positive hematopoietic stem cell population fractionated by FACS from human cord blood cells (hematopoietic stem cells and differentiation slightly advanced) Cell population mixed with hematopoietic progenitor cells), peptide A (10 ⁇ g / mL) and various cell stimulating factors (Full: hSCF 50ng / ml, hTPO 10ng / mL, hFIt3L 20ng / mL, hIL-6 20ng / mL) And cultured in a serum-free liquid medium (StemSpan) supplemented with hsIL-6R ⁇ (20 ng / mL) (1.5 ⁇ 10 4 cells / well, 100 ⁇ L media / well) and attempted in vitro growth (invention test).
  • StemSpan serum-free liquid medium
  • peptide A was not added, and a mixture of various cell stimulating factors (Full: hSCF 50 ng / ml, hTPO 10 ng / mL, hFIt3L 20 ng / mL, hIL-6 20 ng / mL, hsIL-6R ⁇ 20 ng /
  • a human cord blood CD34 positive hematopoietic stem cell population was cultured using a serum-free liquid medium (StemSpan) supplemented with mL).
  • the viable cell ratio is Trypan Blue staining for cells cultured in liquid.
  • the ratio of live cells ( ⁇ (number of live cells / (number of live cells + number of dead cells))
  • the cell stimulating factors used above are factors used in the existing amplification method of human CD34-positive hematopoietic stem cells, and it has been reported that these act on the amplification of human hematopoietic stem cells. (Sui X, et al., Proc Natl Acad Sci USA, 1995,92,2859-2863; Ebihara Y, et al., Blood 1997, 90, 4363-4368).
  • CD13 is a glycoprotein expressed in myeloid cells (neutrophils, eosinophils, basophils, monocytes) and progenitor cells destined for myeloid differentiation;
  • CD34 Is a phosphorylated glycoprotein expressed in undifferentiated pluripotent stem cells and hematopoietic progenitor cells of all strains;
  • CD38 is a glycoprotein expressed in activated T cells, B cells, NK cells, monocytes, plasma cells, etc .;
  • CD45 is a glycoprotein expressed in cells of all leukocyte lineages (lymphocytes, neutrophils, eosinophils, basophils, monocytes);
  • GPA CD235a
  • FIGS. 1A and 1B show the total cell number and viable cell rate, respectively.
  • FIG. 1 (A) it was confirmed that the total number of cells was increased by culturing human cord blood CD34-positive hematopoietic stem cells in the presence of peptide A (10 ⁇ g / mL) and a cell stimulating factor. That is, it has been found that peptide A improves the proliferation of human umbilical cord blood CD34-positive hematopoietic stem cells.
  • FIG. 1B the cell viability showed no difference between the results of the present invention test using peptide A and the control test (control). From this, it is considered that peptide A has no cytotoxicity (does not induce cell death) or is extremely low.
  • CD34-positive CD38-negative cells are markedly increased by culturing for 7 to 11 days, compared to the percentage (%) of CD34-positive CD38-negative cells in the total number of viable cells. .
  • CD34 positive hematopoietic stem cell population in the presence of peptide A, CD34 positive CD38 negative cells, that is, hematopoietic stem cells are amplified and hematopoietic progenitor cells are proliferated. It was suggested that It is also suggested that hematopoietic stem cells contained in umbilical cord blood maintain their traits for a long time in vitro.
  • CD34-positive cells that is, hematopoietic stem cells and hematopoietic progenitor cells proliferate, but at the same time, blood cells that are CD34-negative cells also It has been confirmed that it increases markedly and promotes the differentiation and proliferation of hematopoietic stem cells and hematopoietic progenitor cells.
  • peptide A since the cell stimulating factor formulated in the medium together with peptide A in the above test has an action of promoting the differentiation and proliferation of human hematopoietic stem cells, peptide A has the action of further promoting the differentiation and proliferation of hematopoietic stem cells than the control. It is also considered that it has an action of promoting (possibly) autonomous proliferation (amplification). It is also suggested that hematopoietic stem cells contained in umbilical cord blood maintain their traits for a long time in vitro.
  • peptide A is amplified or differentiated and proliferated in hematopoietic stem cells, and differentiated and proliferated in hematopoietic progenitor cells with respect to human cord blood CD34-positive hematopoietic stem cell populations fractionated by FACS using human umbilical cord blood cells as an indicator of CD34 positivity.
  • hematopoietic stem cells also referred to as the proliferation effect of hematopoietic cells
  • mice Colony forming cell assay (counting the number of hematopoietic progenitor cells) A colony-forming cell assay was performed using cells cultured for 11 days in the experimental system of Experimental Example 1 (control, peptide A [10 ⁇ g / mL] added), and colonies of hematopoietic progenitor cells [CFU-G (colony forming unit- granulocyte), CFU-M (colony forming unit- macrophage), CFU-GM (colony forming unit-granulocyte / macrophage), CFU-MK (colony forming unit-megakaryocyte), BFU-E (burst forming unit-erythrocyte), CFU -GEMM (colony forming unit-granulocyte / erythrocyte / macrophage / megakaryocyte)) was counted.
  • CFU-G colony forming unit- granulocyte
  • CFU-M colony forming unit- macrophage
  • cells cultured for 11 days were mixed with human hematopoietic progenitor cell colony measuring medium (MethoCult H4435 Veritas) in a 35 mm diameter Petri dish at a rate of 1,000 cells / dish, and 14 according to the manual. Cultured for days.
  • the medium contains SCF, IL-3, IL-6, G-CSF, GM-CSF, and Epo as cell stimulating factors. The results are shown in FIG.
  • CFU-G which is a myeloid progenitor cell in which differentiation or maturation has progressed relatively in comparison with the control in the cells cultured for 11 days with the addition of peptide A [10 ⁇ g / mL].
  • CFU-GM proliferated, indicating that human umbilical cord blood CD34-positive hematopoietic stem cells were differentiated and proliferated.
  • FIG. 6 (B) shows the total number of hematopoietic progenitor cells (CFU-G, CFU-M, CFU-GM, CFU-MK, BFU-E, and CFU-GEMM). From this, human umbilical cord blood CD34 positive hematopoietic stem cells were cultured in the presence of not only the cell stimulating factor but also peptide A, and compared to the case of culturing in the presence of only the cell stimulating factor (control), It can be seen that the number increases by about 1.4 times.
  • CFU-GM which is a myeloid progenitor cell that is relatively differentiated or matured compared to the control in cells cultured for 7 days with peptide A [10 ⁇ g / mL] added.
  • BFU-E, and CFU-GEMM were observed, indicating that human cord blood CD34-positive hematopoietic stem cells were differentiated and proliferated.
  • peptide A causes CFU-G and CFU-GM to differentiate and proliferate by promoting the process of human cord blood CD34-positive hematopoietic stem cells differentiating or proliferating into myeloid cells. became.
  • human umbilical cord blood CD34-positive hematopoietic stem cells promote the process of differentiation or maturation into myeloid cells not only on the 11th day but also on the 7th day in the presence of peptide A.
  • FIG. 9 shows the gene expression levels of MYC, CCND1, and CCND2 which are cell growth-related genes.
  • FIG. 10 shows the gene expression level on the first day from the start of culture of the SOCS family (SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7), and
  • FIG. 11 shows the gene on the second day from the start of culture of the SOCS family. The expression level is shown.
  • CCND1 the expression level of CCND1 is decreased by the addition of peptide A, and it is considered that peptide A particularly affects the cell cycle.
  • SOCS family is considered to be an inhibitor of the JAK-STAT signal cascade, and it should be noted that peptide A affects SOCS5.
  • SEQ ID NOs: 1 to 5 show specific examples of the peptide represented by the general formula (I) targeted by the present invention or a modified product thereof.

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Abstract

La présente invention concerne un nouveau peptide qui peut être utilisé efficacement pour produire ou pour faire croître des cellules souches hématopoïétiques et des cellules précurseurs hématopoïétiques à l'extérieur d'un corps vivant. Le peptide de la présente invention est un peptide représenté par la formule générale (I) ou est un de ses produits modifiés, et est caractérisé par le fait qu'il présente une activité qui favorise la différenciation/croissance autonome de cellules souches hématopoïétiques. [Dans la formule, n représente un nombre entier compris entre 3 et 15, x et y valent 0 ou 1 (à condition que lorsqu'un élément parmi x et y vaut 1, l'autre élément parmi y et x vaut 0, respectivement), m et p représentent un entier compris entre 1 et 3, et q vaut 0 ou 1 (à condition que lorsque y vaut 1, q vaut 1)].
PCT/JP2015/060273 2014-03-31 2015-03-31 Peptide de croissance pour cellules hématopoïétiques et utilisation dudit peptide de croissance pour cellules hématopoïétiques Ceased WO2015152305A1 (fr)

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