JP6695795B2 - Hematopoietic cell growth peptide and uses thereof - Google Patents

Description

  The present invention relates to a novel peptide that is effectively used for proliferating hematopoietic cells (hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells) in vitro or for producing them. More specifically, it relates to a peptide that can be effectively used to grow hematopoietic cells by culturing hematopoietic stem cells or hematopoietic progenitor cells in the presence of existing cell stimulating factors.

  The present invention also relates to the use of the peptide, in particular to grow hematopoietic cells in vitro, or as a reagent (or preparation) used for producing these, specifically, existing cell stimulators. The present invention relates to the use as a growth auxiliary agent used for supporting the growth of hematopoietic cells in the presence of a factor. Furthermore, the present invention relates to a method for proliferating hematopoietic cells in vitro using the peptide, in other words, a method for producing hematopoietic cells in vitro, using the peptide.

  Furthermore, the present invention relates to an antibody against the peptide.

If hematopoietic stem cells are produced in vitro from pluripotent stem cells, or if hematopoietic stem cells are taken out in vitro and cultured, and only hematopoietic stem cells can be amplified without tumor formation, many transplant-related problems will be solved, Not only will the application of hematopoietic stem cell transplantation therapy expand, but it will also open up new regenerative medicine such as the development of other organ-specific stem cell transplantation therapy. However, the following problems have been pointed out in the methods for producing and expanding hematopoietic stem cells that have been developed to date.
1. 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 expanded but acute leukemia is induced.
3. The addition of cytokine induces differentiation of mature blood cells, and it is difficult to maintain the undifferentiated state of hematopoietic stem cells.
4. Addition of low molecular weight compounds such as demethylating agents increases the efficiency of hematopoietic stem cell expansion, but it is necessary to confirm the safety as it affects epigenetics.

  Although pluripotent stem cells can theoretically differentiate into all cell lineages, the technology to determine the fate of their differentiation in vitro is still under development, and hematopoietic stem cells that can be applied clinically are produced from pluripotent stem cells. The technology to do so is also developing. Pluripotent cells retain the property of losing the ability to differentiate into other lineages when differentiated in a certain direction, and by suppressing the differentiation into cells other than hematopoietic stem cells, pluripotent stem cells are transformed into hematopoietic stem cells. Manufacturing can be expected. Hematopoietic stem cells have the seemingly contradictory abilities of self-renewal and pluripotency, and differentiate into mature blood cells upon stimulation with cytokines. In other words, in order to expand hematopoietic stem cells, it is necessary to accelerate self-renewal and brake differentiation into various cell lineages.

  In order to develop a new method for producing and expanding hematopoietic stem cells while avoiding the above problems, a factor that suppresses cell differentiation other than hematopoietic stem cells, a factor that promotes only self-renewal of hematopoietic stem cells, or from hematopoietic stem cells The search and development of factors that brake differentiation are important. The place of expansion of hematopoietic stem cells in vivo is mainly in the embryonic liver, and for the search and development of new factors, the mechanism of expansion of hematopoietic stem cells in this liver should be clarified, and a straightforward method to reproduce the results in vitro can be taken. It's a shortcut.

  Patent Document 1 discloses a hematopoietic stem cell proliferating agent containing a pulverized product of placenta-constituting cells as an active ingredient, but such a biological material has problems such as infection and difficulty in preparation of the material.

  Patent Document 2 discloses a hematopoietic stem cell maintenance factor or amplification promoting factor consisting of the membrane protein Thsd1 / Tmtsp, but such a membrane protein is difficult to prepare in an active form.

JP, 2007-106760, A JP, 2007-238473, A WO2011 / 040500A1

  Hematopoietic stem cell transplantation therapy is widely applied not only to hematopoietic diseases but also to autoimmune diseases, solid tumors, and regenerative medicine. Currently, autologous bone marrow, donor bone marrow, and cord blood are used as sources of hematopoietic stem cells for transplantation. When using donor bone marrow or umbilical cord blood, insufficient absolute numbers of transplanted hematopoietic stem cells, insufficient donor, engraftment failure due to immune response, and graft-versus-host disease have become problems.

  The ability to produce hematopoietic stem cells in vitro from pluripotent stem cells could provide a new therapeutic tool independent of sources such as bone marrow, peripheral blood, and cord blood. Also, if hematopoietic stem cells can be cultured and expanded in vitro, it will be possible to supply a sufficient amount of hematopoietic stem cells for transplantation treatment at any time simply by collecting a small amount of hematopoietic stem cells from the bone marrow of the patient's own, etc. It can be safely ported without waiting. In other words, the number of hematopoietic stem cells required for transplantation can be secured, and autologous transplantation without rejection can be performed. Even if autologous transplantation is not possible, if amplification technology can be established, transplantation treatment will be possible by simply collecting a small amount of hematopoietic stem cells from the bone marrow of the donor, the risk of the donor will disappear, and the number of donors registered in the bone marrow bank will increase, The chances of finding a donor can be expected to increase. If cord blood can also be cultured in vitro to expand hematopoietic stem cells, it will be possible to treat adults.

  Thus, establishment of in vitro culture and expansion technology of hematopoietic stem cells makes it possible to save the lives of many patients with intractable blood diseases. In addition, hematopoietic stem cells required for gene therapy can be easily obtained, safe gene transfer can be performed without using retrovirus, and the spread of gene therapy can be expected. The development of in vitro culture and expansion of hematopoietic stem cells has become an important technical issue to be urgently solved for transplantation therapy for patients with intractable blood diseases, gene therapy for regenerative medicine, and the like.

  From such a viewpoint, the present invention has a proliferative effect on hematopoietic stem cells of vertebrates, particularly mammals, and proliferates hematopoietic progenitor cells which are hematopoietic stem cells and their differentiated cells in vitro to produce them. The purpose of the present invention is to provide a novel peptide useful in. Another object of the present invention is to provide a novel peptide that has a proliferative effect also on hematopoietic progenitor cells, proliferates mature hematopoietic cells in vitro, and is useful for producing them.

  Furthermore, the present invention uses the peptide as (i) a hematopoietic cell (hematopoietic stem cell, hematopoietic progenitor cell, and mature hematopoietic cell) as a growth aid, and a method for proliferating the hematopoietic cell in vitro using the peptide, In other words, it aims to provide a method for producing hematopoietic cells.

  The present inventor suppresses the differentiation and proliferation of hematopoietic stem cells in a plurality of peptides designed based on the transmembrane domains of various membrane surface proteins expressed in hepatoblasts of human fetal liver, which is the place of proliferation of hematopoietic cells. A peptide having the 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, myocytes, 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).

  Further studies have shown that specific modifications of the above peptides have the effect of autonomously proliferating (amplifying) and differentiating and proliferating hematopoietic stem cells in the presence of cell stimulating factors such as cytokines in vitro, for example, in vitro. It was confirmed that the peptide was extremely effective as a "proliferation cofactor for hematopoietic stem cells and hematopoietic progenitor cells" that assists the proliferation of hematopoietic stem cells and hematopoietic progenitor cells which are differentiated cells thereof. It was also confirmed that the peptide has an action of differentiating and proliferating hematopoietic progenitor stem cells in vitro in the presence of a cell stimulating factor to generate mature hematopoietic cells. In addition, it was also confirmed that it has an action of maintaining hematopoietic stem cells in vitro, that is, maintaining a trait as a hematopoietic stem cell without differentiating the hematopoietic stem cell into any cell.

  From this, by culturing the hematopoietic stem cells or hematopoietic progenitor cells in the presence of the peptide developed by the present inventor together with the cell stimulating factor, hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells in vitro (in the present invention, , Collectively referred to as "hematopoietic cells"). That is, by culturing the hematopoietic stem cells or hematopoietic progenitor cells in the presence of the peptide developed by the present inventor, and a cell stimulating factor, the hematopoietic cells (hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic progenitor cells) can be efficiently produced in vitro. Cells) and these can be cultured. As described above, since the peptide of the present invention can proliferate hematopoietic stem cells and produce them, 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. ..

The present invention has been completed based on these findings and has the following embodiments.
(I) Novel peptide and modified product thereof (I-1) Peptide represented by the following general formula (I) (SEQ ID NO: 6) or modified product thereof:

[In the formula, 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), m and p are 1 An integer of 3, and q is 0 or 1 (provided that when y is 1, q is 1)].
(I-2) The peptide of (I-1) or a modified product thereof, wherein the peptide represented by the general formula (I) is a peptide having the amino acid sequence of the following formula (SEQ ID NO: 1):

(II) Hemopoietic cell growth auxiliary agent, peptide described in its usage (II-1) (I-1) or (I-2), modified product thereof, pharmaceutically acceptable salt thereof, and these An agent for proliferating hematopoietic cells, which comprises as an active ingredient at least one selected from the group consisting of solvates 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 for proliferating hematopoietic cells, which comprises a step of culturing hematopoietic stem cells or hematopoietic progenitor cells in a medium containing a seed and a cell stimulating factor.
(II-3) 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. The method for proliferating hematopoietic cells according to (II-2), which is at least one selected from the group consisting of:
(III) Peptide described in the method for proliferating hematopoietic cells in vitro (III-1) (I-1) or (I-2), a modified product thereof, a pharmaceutically acceptable salt thereof, and a solvate thereof. A method for proliferating hematopoietic cells in vitro, comprising the step of culturing hematopoietic stem cells or hematopoietic progenitor cells in a medium containing at least one selected from the group consisting of the following: and a cell stimulating factor.
(III-2) 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. The method for proliferating hematopoietic cells in vitro according to (III-1), which is at least one selected from the group consisting of:
(IV) 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 in vitro production of hematopoietic cells, which comprises a step of culturing hematopoietic stem cells or hematopoietic progenitor cells in a medium containing at least one selected from the group consisting of the following: and a cell stimulating factor.
(IV-2) 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. The method for producing a hematopoietic cell in vitro according to ( IV- 1), which is at least one selected from the group consisting of:
(V) A cell population containing hematopoietic cells, and a cell population containing hematopoietic cells obtained by the method described in (V-1) (III-1) or (III-2).
(V-2) A pharmaceutical composition having the cell population described in (V-1).
(V-3) The pharmaceutical composition according to (V-2), which is a hematopoietic function improving agent.

(VI) Antibody (VI-1) An antibody against at least one peptide described in (I-1) or (I-2).
(VI-2) The antibody described in (VI-1) which is a monoclonal antibody.

  For example, in vitro by culturing hematopoietic stem cells (such as autologous hematopoietic stem cells or cord blood hematopoietic stem cells with relatively few immune reactions), preferably in the presence of the peptide of the present invention, together with cell stimulating factors such as cytokines. In a culture vessel such as a tube, hematopoietic stem cells can be autonomously proliferated to increase their absolute number, or they can be differentiated and proliferated to increase the number of hematopoietic progenitor cells and mature hematopoietic cells. This makes it possible to solve the problems of the present invention and solve the problem of the supply source of hematopoietic stem cells for transplantation and hematopoietic progenitor cells in conventional hematopoietic stem cell transplantation therapy.

  Further, by culturing hematopoietic progenitor cells in the presence of the peptide of the present invention, preferably together with a cell stimulating factor, in a culture container such as a test tube in vitro, the hematopoietic stem cells are differentiated and proliferated to generate hematopoietic mature cells. It is possible to increase the number.

  Furthermore, by using the peptide of the present invention and its antibody, it becomes possible to analyze the mechanism of differentiation and proliferation of hematopoietic stem cells and autonomous proliferation.

The total number of cells on day 7 and day 11 of culturing human cord blood CD34-positive hematopoietic stem cells [Fig. 1 (A)] and the cell viability [Fig. 1 (B)] are shown (Experimental Example 1). In the figure,-●-(Peptide A) is peptide A (10 μg / mL) and cell stimulating factor (hSCF 50 ng / mL, hTPO 10 ng / mL, hFlt3L 20 ng / mL, hIL-6 20 ng / mL, hsIL-6Rα 20 ng). / mL), the results of culture in the presence of-◆-(Control) show the results of culture in the presence of only the above-mentioned cell stimulating factor without containing peptide A (10 μg / mL). Ratio of CD34-positive CD38-negative cells to the total number of viable cells [Figure 2 (A)] and total number of CD34-positive CD38-negative cells [Figure 2 (B)] on day 7 of culturing human cord blood CD34-positive hematopoietic stem cells ] Is shown (Experimental example 1). In the figure, “Peptide A” means peptide A (10 μg / mL) and cell stimulating factor (hSCF 50 ng / mL, hTPO 10 ng / mL, hFlt3L 20 ng / mL, hIL-6 20 ng / mL, and hsIL-6Rα 20 ng / mL). In the presence of)), "Control" shows the result of culturing in the presence of only a cell stimulating factor without containing peptide A (10 µg / mL). The same applies to FIGS. 3 to 6 below. The ratio of CD34-positive CD38-negative cells to the total number of viable 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)] ] Is shown (Experimental example 1). The total number of cells expressing each cell surface marker (CD13, CD34, CD38, CD45, and GPA) in the results of the cell surface marker test on day 11 of culturing human cord blood CD34-positive hematopoietic stem cells is shown. Of the results of the cell surface marker test on day 11 of culturing human cord blood CD34-positive hematopoietic stem cells, the number of viable cells expressing each cell surface marker (CD13, CD34, CD38, CD45, and GPA) Shows the ratio (%) to the total number. Human umbilical cord blood CD34-positive hematopoietic stem cell population was supplemented with a cell stimulating factor mixture (hSCF 50ng / mL, hTPO 10ng / mL, hFlt3L 20ng / mL, hIL-6 20ng / mL, and hsIL-6Rα 20ng / mL) ( Control), or after culturing for 11 days in a medium (Peptide A) containing the above-mentioned cell stimulating factor mixture and peptide A (10 μg / mL), a colony forming cell assay was performed to determine the number of colony forming cells (corresponding to hematopoietic progenitor cells). The result of having measured is shown (Experimental example 2). FIG. 6 (A) shows the number of measurements 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. Human umbilical cord blood CD34-positive hematopoietic stem cell population was supplemented with a cell stimulating factor mixture (hSCF 50ng / mL, hTPO 10ng / mL, hFlt3L 20ng / mL, hIL-6 20ng / mL, and hsIL-6Rα 20ng / mL) ( Control), or after culturing for 7 days in a medium (Peptide A) containing the above-mentioned cell stimulating factor mixture and peptide A (10 μg / mL), a colony forming cell assay was performed to determine the number of colony forming cells (corresponding to hematopoietic progenitor cells). The result of measuring is shown (Experimental example 2). Diagram showing fluorescence micrograph images of human cord blood CD34-positive hematopoietic stem cells cultured for 6 hours or 12 hours with or without (Modified KS13) biotinylated peptide A (10 μg / mL) (Control). Example 3). Nucleus in the figure shows only nuclear staining, and Marge shows the combination of nuclear staining and peptide A. The graph which shows the result of the expression gene analysis shown in Experimental example 4. The expression level of each gene of MYC, CCND1, and CCND2 on the 1st and 2nd day from the start of liquid culture is shown. The vertical axis of the graph in the figure represents a relative value. The graph which shows the result of the expression gene analysis shown in Experimental example 4. 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 graph which shows the result of the expression gene analysis shown in Experimental example 4. 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.

Definitions of terms used in the present invention The abbreviations of amino acid sequences and the like in the present specification refer to IUPAC-IUB rules [IUPAc-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)], Guidelines for preparation of specifications including base sequences or amino acid sequences "(edited by the Patent Office) and symbols conventionally used in the art.

  In the present invention, "hematopoietic stem cells" means "self-renewal ability", which is the ability to proliferate while maintaining undifferentiated state, and "multipotency", which is the ability to differentiate into all hematopoietic cells and lymphoid cells. In addition, it refers to cells that have the ability to reconstitute hematopoiesis over a long period of several months or more.

  Human hematopoietic stem cells express CD34, which is a stem cell marker, and thus can be characterized as CD34 (+). Therefore, 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. For example, hematopoietic stem cell markers including CD34, Lin (-), CD34 (+), CD38 (-), DR (-), CD45 (+), CD90 (+), CD117 (+), CD123 (+) , And CD133 (+). These can be used singly or in combination of two or more, and as a combination mode, Lin (-) CD34 (+) CD38 (-), CD45 (+) CD34 (+) CD38 (-), etc. Can be mentioned.

On the other hand, mouse hematopoietic stem cells are known to have altered expression of cell surface antigens during the fetal and adult stages, and as markers for fetal hematopoietic stem cells, Lin (-), CD31 (+), CD34 (+), CD41 (+), C-Kit (+), as a marker of adult hematopoietic stem cells, Lin (-), CD31 (+), CD34 (-/ +), CD45 (+), c-Kit (+), Sca-1 (+), CD150 (+), EPCR (+) can be mentioned. These can be used alone or in combination of two or more, and as an embodiment of the combination, Lin (-) c-Kit (+) Sca-1 (+) and CD45 (+) c-Kit (+ ) Sca-1 (+) and the like.

  It has been clarified that hematopoietic stem cells are contained in a very small amount in bone marrow, peripheral blood, and umbilical cord blood. From these, using the above-mentioned stem cell marker as an index, a conventional method such as FACS (fluorescence activated cell sorting) is used. Can be collected.

  “Hematopoietic progenitor cells” are cells that are derived from hematopoietic stem cells and are not terminally differentiated. Hematopoietic progenitor cells can be classified into pluripotent hematopoietic progenitor cells that can differentiate into 2-3 blood cells, or unipotent hematopoietic progenitor cells that have limited differentiation into one blood cell. Hematopoietic progenitor cells are differentiated into three types of progenitor cells: myeloid progenitor cells, lymphoid progenitor cells, or erythroid / megakaryocytic progenitor cells. The myeloid progenitor cells differentiate into progenitor cells that undergo terminal differentiation 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 lineage.

  In addition, the lymphoid progenitor cells differentiate into progenitor cells that undergo terminal differentiation into T cells, B cells, and NK cells. Therefore, hematopoietic progenitor cells include myeloid cells [granulocytes (eosinophils, neutrophils, basophils), monocytes, macrophages, mast cells], erythroid cells [erythrocytes], megakaryocyte cells [ Megakaryocytes, platelets] as well as progenitor cells of lymphoid cells [T cells, B cells, plasma cells].

  “Mature hematopoietic cells” are cells obtained by terminally differentiating the above “hematopoietic stem cells” and “hematopoietic progenitor cells”, and as described above, myelocytic cells [granulocytes (eosinophils, neutrophils, basophils) , Monocytes, macrophages, mast cells], erythroid cells [erythrocytes], megakaryocytes (megakaryocytes, platelets), and lymphoid cells [T cells, B cells, plasma cells].

  In the present invention, the “hematopoietic cell” is used as a generic term for the above-mentioned hematopoietic stem cells, hematopoietic progenitor cells, and mature hematopoietic cells, without any distinction.

  The hematopoietic cells targeted by the present invention are preferably derived from vertebrates, more preferably birds (such as chicken) or mammals (human, mouse, rat, rabbit, monkey, chimpanzee, pig, horse, goat). , Sheep, cattle, dogs, cats, wallabies, kangaroos, etc.). Preferred are cells derived from mammals. Among mammals, humans or rodents (mouse, rat, rabbit, etc.) commonly used as experimental animals are particularly preferable.

  “Proliferation” refers to increasing the total number of cells that have not been terminally differentiated and cells that have been terminally differentiated. Here, cells that have not been terminally differentiated include hematopoietic stem cells and hematopoietic progenitor cells, and cells that have been terminally differentiated include hematopoietic mature cells. Thus, "hematopoietic cell proliferation" includes increasing the total number of hematopoietic stem cells, hematopoietic progenitor cells, and hematopoietic mature cells without distinguishing them.

  The type of cells that grow depends on the type of cells used. For example, when hematopoietic stem cells are used as cells, hematopoietic progenitor cells differentiated from hematopoietic stem cells are subjected to terminal differentiation in addition to expansion of hematopoietic stem cells (autonomous proliferation of hematopoietic stem cells while maintaining undifferentiated state of hematopoietic stem cells). The total number of hematopoietic stem cells and hematopoietic progenitor cells increases because they grow in the absence of hematopoietic stem cells. When hematopoietic progenitor cells are used as cells, the hematopoietic progenitor cells differentiate and proliferate, and thus the total number of cells terminally differentiated from the hematopoietic progenitor cells (hematopoietic mature cells) increases.

  There are two phenomena (proliferation) in which the number of cells increases (differentiation / proliferation) that involves differentiation or maturation, and “autonomous growth” that does not involve differentiation or maturation. May be referred to as "autonomous growth" (corresponding to "amplification" in the present invention). Note that maturation refers to a state of differentiation regardless of the final differentiation.

  In the present invention, the "proliferation aid for hematopoietic cells" means a reagent (formulation) effective for increasing the total number of these hematopoietic cells without distinguishing between hematopoietic stem cells, hematopoietic progenitor cells, and hematopoietic mature cells. To do. When this is applied to, for example, hematopoietic stem cells, the total number of hematopoietic stem cells and hematopoietic progenitor cells can be increased as described above, and when applied to hematopoietic progenitor cells, the total number of hematopoietic mature progenitor cells is increased. Can be increased.

  In order to individually evaluate the proliferation of hematopoietic stem cells, hematopoietic progenitor cells, and hematopoietic mature cells, analysis of hematopoietic cell markers (for example, counting of cells corresponding to CD34 (+) by FACS) and colony assay method are used. A quantitative analysis or the like may be adopted.

  "Umbilical cord blood" refers to blood that can be obtained from umbilical cords of mammals, preferably humans. "Bone marrow-derived blood" refers to blood contained in cerebrospinal fluid present in the bone marrow of mammals, preferably humans. Cord blood and bone marrow can be obtained from a cord blood bank and a bone marrow bank, respectively. It can also be obtained from volunteers.

(I) Novel peptide (peptide of the present invention)
The peptide targeted by the present invention (hereinafter referred to as "the peptide of the present invention") has an action of acting on hematopoietic stem cells and hematopoietic progenitor cells to proliferate hematopoietic stem cells, hematopoietic progenitor cells and mature hematopoietic cells. And

  Specifically, the 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 to differentiate and proliferate mature hematopoietic cells. be able to.

  One aspect of such a peptide is a peptide consisting of the following amino acid sequence (SEQ ID NO: 1):

  In the present invention, the above-mentioned peptide is the most preferable peptide, but as shown in the following general formula (I), the number (n) of arginine residues in the Arg-rich region on the N-terminal side of the amino acid sequence is The number is not limited to eight and can be selected from the range of 3 to 15, for example. It is preferably 7 to 15, more preferably 7 to 11, and particularly preferably 7 to 8.

[In the formula, 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), m and p are 1 to An integer of 3, and q is 0 or 1 (provided that when y is 1, q is 1)].

Further, as shown in the above formula (1), the peptide of the present invention has an Arg-rich region consisting of 3 to 15 arginine residues and an amino acid sequence [Cys Gln Lys Lys Asp Gly Pro Cys] which is a core of the peptide of the present invention. Val Ile Asn Gly Ser] (SEQ ID NO: 2; hereinafter referred to as “core sequence”) (hereinafter, this site is referred to as “core sequence N side”) via a polyethylene glycol chain. (In the above general formula (I), represented by “(NH— [CH ₂ —CH ₂ —O] m—CO) x”, hereinafter also referred to as “PEG chain”). Instead of the site, a polyethylene glycol chain (in the general formula (I) above, “(NH- [ CH ₂ —CH ₂ —O] p-CO) y ”, which may hereinafter be referred to as“ PEG chain ”).

In addition, when having a PEG chain 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 Or when p is 1), the unit may be repeated two or more (polymerization). The polymerization degree (m, p) of the PEG chain is preferably 2 to 3, and more preferably 3.

When the PEG chain is present on the core sequence N side (x = 1), the PEG chain is not present on the core sequence C side (y = 0), and conversely, when the PEG chain is present on the core sequence C side (y = 1). ), The core sequence N side does not have a PEG chain (x = 0). Further, a polyethylene glycol chain _{(-NH- [CH 2 -CH 2 -O} ] m-CO-, or _{-NH- [CH 2 -CH 2 -O]} p-CO-) in place, aminocaproic acid residue ( -NH- [CH _{2] 5} -CO-) may have a.

  The peptide of the present invention may have an amino group at its C terminus (q = 1) or may have no amino group (q = 0). However, when the peptide of the present invention has a PEG chain on the C side of the core sequence (x = 0 and y = 1), its C-terminal is an amino group (q = 1). When the peptide of the present invention does not have a PEG chain on the C side of the core sequence and has an amino group at the C terminus (y = 0 and q = 1), the carboxyl group of serine at the C terminus is amidated. Has been done.

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 achieved by using a peptide synthesizer. This can be easily performed by changing the type of protected amino acid. In addition, inserting a PEG chain into a peptide can also be performed according to known techniques. For the insertion of the PEG chain, for example, “mini-PEG (registered trademark)” (Fmoc-mini-PEG ^™ , Fmoc-mini-PEG-3 ^™ , Boc-mini-PEG ^™ , commercially available from Peptides International, Boc-mini-PEG-3 ^™ ) can be used.

  The peptide of the present invention may be in a free state, a salt form, or a solvate form including a hydrate. Examples of the salt include physiologically acceptable acid addition salts and basic salts that are cell biologically or pharmaceutically acceptable. Such 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. Examples thereof include organic acid salts. Examples of the base salt include alkali metal salts such as sodium, potassium and lithium; or alkaline earth metal salts such as calcium and magnesium. The peptide of the present invention may be composed of amino acid residues of L-form, D-form, or both.

  The peptide of the present invention represented by the above formula (I) (including the peptide shown in SEQ ID NO: 1, hereinafter the same) may have a modifying group commonly used in the field of peptides. This is referred to as a peptide modified product in the present invention.

The said modifications, in the C-terminal peptide of the invention represented by the general formula (I) is a carboxyl group (-COOH), a carboxylate (-COO-), an amide (-CONH _2), or an ester (-COOR) Certain peptides are included. Here, the group R in the ester includes, for example, an alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl and n-butyl; a cycloalkyl group having 3 to 8 carbon atoms such as cyclopentyl and cyclohexyl; C _7-14 Aralkyl group (for example, aryl group having 6 to 12 carbon atoms such as phenyl and α-naphthyl; phenyl-C _1-2 alkyl group such as benzyl and phenethyl; α-naphthyl-C ₁ such as α-naphthylmethyl) _-2 alkyl group, etc.); pivaloyloxymethyl group and the like. When the peptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the peptide modified product of the present invention.

In the modified product of the peptide of the present invention, 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.). ), A fatty acid (C _8-18 saturated fatty acid) -modified, a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, an amino group, an imidazole group, Indole group, guanidino group, etc.) protected by 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 Also included are complex peptides such as so-called glycopeptides in which chains are linked.

  Further, the modified products of the peptide of the present invention include those in which an imidazolyl group or an SH group is alkylated (eg methylated), aralkylated (eg benzylated) or acylated (eg acetylated, benzoylated). The modified fatty acid peptide includes a myristoylated peptide in which the amino group of the peptide main chain of the N-terminal arginine residue is modified with myristic acid; or the side chain guanidyl group of the N-terminal arginine residue. A myristoylated peptide in which an amino group constituting (also referred to as a guanidine group or a guanidino group) is modified with myristic acid (the amino group and a carboxyl group of myristic acid form an amide bond) is included.

  In addition, the above-mentioned N-terminal acetylated peptide means that 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 guanidyl group of the side chain is acetylated.

(II) hematopoietic cell growth auxiliary agent peptide of the present invention, as it is, or after physiologically acceptable if necessary, for example, after mixing with a physiologically or pharmaceutically acceptable carrier to form a composition, It can be used as a growth aid for hematopoietic cells. The peptides of the present invention may be composed of one kind alone, or may be used in any combination of two or more kinds.

  Here, the growth auxiliary agent is a growth auxiliary agent used together with a cell stimulating factor in the growth of hematopoietic stem cells or hematopoietic progenitor cells, and has a role of promoting the growth of hematopoietic stem cells or hematopoietic progenitor cells. Specifically, when proliferating hematopoietic stem cells, by using such a growth auxiliary together with a cell stimulating factor, hematopoietic stem cells can be amplified or hematopoietic progenitor cells can be differentiated and proliferated to produce them. Further, when the hematopoietic progenitor cells are proliferated, by using such a growth auxiliary together with the cell stimulating factor, it becomes possible to differentiate and proliferate mature hematopoietic cells and to produce them.

  The growth auxiliary agent is prepared, for example, by dissolving the peptide of the present invention in water or an appropriate buffer solution (eg, phosphate buffer solution, PBS, Tris-HCl buffer solution, etc.) so as to have an appropriate concentration. be able to. Further, if necessary, a preservative, a stabilizer, a reducing agent, an isotonicity agent and the like which are commonly used may be added.

  The growth-promoting agent of the present invention is obtained by, for example, adding an effective amount of the peptide of the present invention to a medium and culturing the hematopoietic stem cells or hematopoietic cells in the presence of a cell stimulating factor to give hematopoietic cells (hematopoietic stem cells, hematopoietic precursors Cells and / or mature hematopoietic cells) can be cultured and expanded and used to produce them. Therefore, the present invention also provides a method for expanding hematopoietic cells, which comprises culturing hematopoietic stem cells or hematopoietic progenitor cells in the presence of the peptide of the present invention (or the growth auxiliary agent of the present invention). This will be described later.

  Thus, by culturing hematopoietic stem cells or hematopoietic progenitor cells in the presence of the polypeptide of the present invention (or the growth auxiliary agent of the present invention) and a cell stimulating factor, the proliferation of hematopoietic cells can be promoted. Therefore, the peptide of the present invention can 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.

(III) In vitro proliferation method of hematopoietic cells (hematopoietic cell in vitro proliferation method)
(IV) In vitro production method of hematopoietic cells (in vitro production method of hematopoietic cells)
The method for proliferating hematopoietic cells of the present invention includes a step of culturing hematopoietic stem cells or / and hematopoietic progenitor cells in the presence of the peptide of the present invention. It can be said that the proliferation method is a method for producing hematopoietic cells by culturing hematopoietic stem cells or / and hematopoietic progenitor cells. Therefore, the following “method for proliferating hematopoietic cells” can be restated as “method for producing hematopoietic cells”.

  Examples of the hematopoietic stem cells used in the method of the present invention include cell groups containing at least hematopoietic stem cells, and hematopoietic stem cells may be isolated. The cell group may be a hematopoietic stem cell-containing fraction that is fractionated from a hematopoietic stem cell-containing cell group. The hematopoietic progenitor cells used in the method of the present invention also include a cell group containing at least hematopoietic progenitor cells, and may be those in which hematopoietic progenitor cells have been isolated. The cell group may be a fraction containing hematopoietic progenitor cells, which is fractionated from a cell group containing hematopoietic progenitor cells.

  The source of hematopoietic stem cells or hematopoietic progenitor cells may be any tissue containing vertebrates such as birds and mammals, preferably mammalian stem cells such as mice and rats belonging to humans and rodents. For example, hematopoietic stem cells can be collected from fetal liver, fetal bone marrow, bone marrow, peripheral blood, umbilical cord blood containing hematopoietic stem cells, or peripheral blood to which stem cells have been recruited by administration of cytokines and / or anticancer agents.

  In culturing hematopoietic stem cells or hematopoietic progenitor cells in the presence of the peptide of the present invention, a so-called culturing plate, a culturing method using a petri dish or a flask is possible, but the medium composition, pH, etc. are mechanically controlled. , The culture system can be improved by a bioreactor capable of culturing at 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 culturing is not particularly limited as long as the growth and survival of hematopoietic stem cells or hematopoietic progenitor cells are not impaired. For example, minimal essential medium (MEM) containing about 5 to about 20% fetal bovine serum, Dulbecco's modified Eagle medium (DMEM), IMDM medium, RPMI1640 medium, 199 medium, SF-02 medium (Sanko Junyaku), Opti- MEM medium (GIBCO BRL) and hematopoietic stem cell culture medium X-VIVO 10 (Lonza), StemSpan SFEM (serum-free growth medium) and StemSpan H3000 (animal component-free medium) (above, Veritas) are preferable. As. The pH of the medium is preferably about 6-8.

  If necessary, cell stimulating factors (cytokines and hematopoietic hormones such as EPO (erythropoietin)), hormones such as insulin, Wnt (Thimoth, AW, Blood, 89: 3624-3635, 1997) gene may be added to the medium. Differentiation and growth regulators such as 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 can be further contained. In particular, by adding a cell stimulating factor to a medium together with the peptides of the present invention and culturing the hematopoietic stem cells or hematopoietic progenitor cells in the presence of the peptides of the present invention and the cell stimulating factors, the hematopoietic cells can be more efficiently proliferated and amplified. You can

  The cell stimulating factor is a factor that gives stimulation to tissue-specific cells such as hematopoietic stem cells and hematopoietic progenitor cells such as proliferation, differentiation, survival and migration. Such a cell stimulating factor is not particularly limited as long as it does not prevent proliferation of hematopoietic stem cells or hematopoietic progenitor cells, and specifically, SCF (stem cell factor), IL-3 (interleukin). -3), GM-CSF (granulocyte / macrophage colony-stimulating factor), IL-6 (interleukin-6), soluble IL-6 receptor (sIL-6R), IL- 11 (interleukin-11), Flt-3L (fms-like tyrosine kinase-3 (Flt-3) ligand), EPO (erythropoietin), TPO (thrombopoietin), G-CSF (granulocyte colony stimulating factor), TGF-β. (Transforming growth factor-β), MIP-1α (George, D., J. Exp. Med. 167: 1939-1944, 1988), Flt3 / Flk2-ligand, FGF (fibroblast growth factor) and the like. Be done. Details of these stimulating factors are described in Gallard, R.E., The cytokine facts book, Academic Press, 1994, etc.

  The cell stimulating factor to be mixed in the medium may be one type or two or more types. Preferable examples include SCF, G-CSF, IL-3, IL-6, IL-11, Flt-3L, sIL-6R, and TPO, and 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, and more preferably 10 to 100 ng / mL.

Upon culturing, the peptide of the present invention can be added to the above medium so that the final concentration in the medium is 1 to 500 µg / mL, preferably 5 to 300 µg / mL, and more preferably 10 to 100 µg / mL. In addition, hematopoietic stem cells or hematopoietic progenitor cells can be added to the above-mentioned medium so that the cell density is the one usually used in the art. Culturing is usually performed under an atmosphere of about 30 to 40 ° C. and about 5 to 10% CO ₂ for a time at which desired growth is achieved. Aeration and agitation may be performed as necessary.

(V) Cell population containing hematopoietic cells obtained by the proliferation method (production method) of the present invention , and use thereof "Hematopoietic cells obtained by the proliferation method (III) or production method (IV) of the present invention described above Can be used as a composition (graft) for blood cell transplantation or regenerative medicine in place of conventional bone marrow transplantation or cord blood transplantation.

  Here, the “cell population containing hematopoietic cells” (hereinafter, also simply referred to as “cell population”) means 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 is subjected to a conventional technique. It can also be purified and harvested.

  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. For example, when performing a treatment such as chemotherapy of solid cancer patients, suppression of hematopoietic function in bone marrow such as radiation therapy, the bone marrow is collected before the treatment, and hematopoietic stem cells are amplified in vitro, By returning to the patient after the treatment, the hematopoietic system damage due to side effects can be recovered early, more powerful chemotherapy can be performed, and the therapeutic effect of chemotherapy can be improved.

  Further, the cell population of the present invention is a disease associated with impairment of hematopoietic function, for example, aplastic anemia, congenital immunodeficiency, congenital metabolic disorder, myelodysplastic syndrome, leukemia, malignant lymphoma, multiple myeloma, Myelofibrosis, chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, immunodeficiency syndromes such as acquired immunodeficiency syndrome (AIDS), thalassemia, hemolytic anemia due to enzyme deficiency, sickle It can be used as a prophylactic or therapeutic agent for congenital anemia such as erythrocytosis, Gaucher disease, lysosomal storage diseases such as mucopolysaccharidosis, and adrenoleukodystrophy.

  Such a cell population can be used in a state of being mixed with a pharmacologically acceptable carrier or a buffer, if necessary, in addition to the hematopoietic cells grown or produced by the method of the present invention to prepare a pharmaceutical composition. ..

  Here, as the pharmacologically acceptable carrier, various organic or inorganic carrier substances that are commonly used as pharmaceutical materials are used, and examples thereof include suspending agents in suspensions, isotonic agents, buffers, and soothing agents. It is mixed as an agent. If necessary, formulation additives such as antiseptics, antioxidants, thickeners and stabilizers can also be used.

  Transplantation (administration) of the cell population or a pharmaceutical composition prepared therefrom may be performed in the same manner as conventional bone marrow transplantation or cord blood transplantation, for example, parenteral administration (eg, intravenous injection, local infusion, etc.). ) Can be mentioned. Suitable formulations of the pharmaceutical composition include aqueous and non-aqueous isotonic sterile injection solutions.

The dose of the cell population of the present invention or a pharmaceutical composition prepared from the cell population depends on the activity of the peptide of the present invention, the severity of disease, the animal species to be administered, the drug acceptability of the administration target, sex, body weight, age, etc. Although it cannot be said unequivocally, the amount of hematopoietic stem cells per adult is usually 1 × 10 ⁶ cells / kg or more, preferably 1 × 10 ⁶ to 1 × 10 ¹⁰ cells / kg, more preferably 2 × 10 ⁶ ~ 1 x 10 ⁹ cells / kg.

(VI) Antibodies Against Peptides of the Present Invention The present invention also provides antibodies to the peptides of the present invention described above.

  The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies. It is preferably a monoclonal antibody.

  Examples of preferred antibodies include antibodies against the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 among the peptides of the present invention. The monoclonal antibody of the present invention is an immunoglobulin derived from vertebrates such as mammals including birds and rodents, preferably mammals such as humans, mice or rats, and its class and subclass are not particularly limited. The preferred class and subclass is immunoglobulin M (IgM), more preferably IgM (kappa chain).

  The monoclonal antibody can be produced according to a known method as 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 to humans. The humanized antibody is a chimeric antibody in which a portion other than the variable region (or hypervariable region) of an animal antibody other than human is replaced with the amino acid sequence of human immunoglobulin, and the peptide of the present invention, particularly the amino acid sequence represented by SEQ ID NO: 1. It is an antibody with reduced antigenicity for humans, while retaining the affinity for the peptide consisting of. The humanized monoclonal antibody can be produced according to a known method.

  Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the present invention is not limited to these experimental examples.

Example 1 Preparation of Peptide The following four types of peptides were designed as the peptides of the present invention (hereinafter referred to as peptides A to D), and synthesis was requested to Scrum Co., Ltd. In the following formulas, the following as "PEG" refers to 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 that eight arginines are linear arginines that are linearly peptide-bonded without interposing the side chains.

"-" At the right end of [Arg] ₈ and "-" at both ends of [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] are groups or amino acids adjacent to each other via "-". It means that it has an amide bond (or peptide bond) with a residue (peptide residue).

Here, shown in peptide A and peptide B [Arg] ₈ - "-" in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] For this arginine residue at the C-terminus of the [Arg] ₈ And the amino group of the cysteine residue existing at the N-terminal in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] that is adjacent via “-” is peptide-bonded.

  Regarding "-" in CO- [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] shown in peptide C and peptide D, this means N in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]. It means that the amide group of the cysteine residue located at the terminal and the “CO” (ketone group) adjacent via the “−” form an amide bond.

For the "-" in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] -NH ₂ shown in Peptide A and Peptide D, this is within [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] the carboxyl group of a serine residue located at the C-terminus, "-" and "NH _2" adjacent through means that are to form an amide bond. That is, it means that such a C-terminal serine residue has an amide group.

  Regarding "-" in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser] -NH shown in peptide B, this is located at the C-terminal in [Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly Ser]. It means that the carboxyl group of the serine residue and the “NH” that is adjacent via the “-” form an amide bond.

Regarding "-" in [Arg] ₈ -NH shown in Peptide C and Peptide D, this means that the carboxy group of the C-terminal arginine residue in [Arg] ₈ and the adjacent "NH" via "-". Form an amide bond.

Regarding "-" in Ac- [Arg] ₈ shown in Peptide D, this is an amide group constituting the amide group of the peptide main chain of the N-terminal arginine residue in [Arg] ₈ and / or the guanidyl group of the side chain. Is acetylated (Ac).

  The following experiment was performed using peptide A among these peptides.

Experimental Example 1 Proliferation of Human Cord Blood Hematopoietic Stem Cells Using Peptide A (1) Experimental Method (1-1) Human Cord Blood CD34-Positive Hematopoietic Stem Cell Population Fractionated from Human Cord Blood Cells by FACS Cell population in which hematopoietic progenitor cells are mixed), peptide A (10 μg / mL) 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-6Rα 20 ng / mL) was added to the cells and cultured in a serum-free liquid medium (StemSpan) (1.5 × 10 ⁴ cells / well, 100 μL media / well) to try in vitro growth (test of the present invention). As a control test, a mixture of various cell stimulating factors without adding peptide A (Full: hSCF 50ng / ml, hTPO 10ng / mL, hFIt3L 20ng / mL, hIL-6 20ng / mL, hsIL-6Rα 20ng / mL) A human cord blood CD34-positive hematopoietic stem cell population was similarly cultured using a serum-free liquid medium (StemSpan) supplemented with (mL).

  Then, regarding the cells obtained by liquid culture on the 7th and 11th days from the start of the culture, the total number of cells, the ratio of viable cells (viable cell rate), and the number of CD34 positive CD38 negative cells (hematopoietic stem cells and hematopoietic progenitor cells Was measured. The viable cell rate was determined by performing Trypan Blue staining on liquid-cultured cells and calculating the ratio of viable cells from the measured viable and dead cell numbers ({(viable cell number / (viable cell number + dead cell number))). The cell stimulating factors used above were those used in the existing method for expanding human CD34-positive hematopoietic stem cells, and it was reported that these act on the expansion 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).

  (1-2) Expression of lineage-specific antigen markers (CD13, CD34, CD38, CD45, and GPA) on the cell surface of cells obtained by liquid culture on the 11th day from the start of the culture using the flow cytometory method Cells were fractionated, and the total number and the ratio (%) to the total number of living cells were calculated (cell surface marker test). Among lineage-specific antigen markers, CD13 is a glycoprotein expressed on myeloid cells (neutrophils, eosinophils, basophils, monocytes) and progenitor cells destined for myeloid differentiation; CD34 Is a phosphorylated glycoprotein expressed on undifferentiated pluripotent stem cells and hematopoietic progenitor cells of all lineages; CD38 is a glycoprotein expressed on activated T cells, B cells, NK cells, monocytes, plasma cells, etc .; CD45 is a glycoprotein expressed on cells of all leukocyte lineages (lymphocytes, neutrophils, eosinophils, basophils, monocytes); GPA (CD235a) is expressed on human erythrocytes and erythroid cells It is a glycoprotein.

(2) Experimental results (2-1) Time-dependent changes in total cell number and viable cell rate FIGS. 1 (A) and 1 (B) show the total cell number and viable cell rate, respectively. As shown in 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 μg / mL) and a cell stimulating factor. That is, it was revealed that peptide A improves the proliferative property of human cord blood CD34-positive hematopoietic stem cells. On the other hand, regarding the cell viability, as shown in FIG. 1 (B), no difference was observed between the results of the test of the present invention 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 even if it is extremely low.

(2-2) Time-dependent change in the number of CD34-positive CD38-negative cells From each of the cultures on the 7th day and the 11th day after the start of culture, the flow cytometory method was used to analyze the CD34-positive CD38-negative cells which are hematopoietic stem cell populations. The total number and the ratio of the total number (the total number of living cells obtained by culture) were calculated. The results on the 7th day are shown in FIG. 2 and the results on the 11th day are shown in FIG. 3, respectively. In each figure, (A) shows the ratio (%) of CD34-positive CD38-negative cells to the total number of viable cells obtained by culture, and (B) shows the total number of CD34-positive CD38-negative cells. From these figures, it can be seen that the number of CD34-positive CD38-negative cells is significantly increased by the culture for 7 to 11 days as compared with the ratio (%) of the CD34-positive and CD38-negative cells to the total number of viable cells. .. From this, by culturing the human cord blood 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, resulting in a highly pure hematopoietic cell population. It was suggested to be obtained. It is also suggested that the hematopoietic stem cells contained in cord blood maintain their trait for a long period of time in vitro.

  In the control using the cell stimulating factor used in the existing human CD34-positive hematopoietic stem cell expansion method, although CD34-positive cells, that is, hematopoietic stem cells and hematopoietic progenitor cells also proliferate, at the same time, hematopoietic cells that are CD34-negative cells also It has been confirmed that the amount is remarkably increased, and it has been confirmed that the differentiation and proliferation of hematopoietic stem cells and hematopoietic progenitor cells are predominantly promoted. That is, in the above test, the cell stimulating factor mixed with the peptide A in the medium has the action of promoting the differentiation and proliferation of human hematopoietic stem cells, and thus the peptide A has the action of further promoting the differentiation and proliferation of hematopoietic stem cells as compared with the control. In addition, it is considered to have an action of promoting (potentially) autonomous proliferation (amplification). It is also suggested that the hematopoietic stem cells contained in cord blood maintain their trait for a long period of time in vitro.

(2-3) Cell surface marker test Among the results of the cell surface marker test, the total number of cells expressing each cell surface marker is shown in FIG. 4, and the ratio (%) to the total number of cells is shown in FIG. Show.

  From these results, by culturing a human cord blood CD34-positive hematopoietic stem cell population in the presence of peptide A, CD13-positive (myelocytic) cells, CD34-positive (hematopoietic stem / progenitor) cells, CD45-positive (white blood cell) cells , The total number of each GPA-positive (erythroid) cell group is remarkably increased, and it can be seen that the addition of peptide A promotes the proliferation of cells of each line. Therefore, peptide A was used for the human cord blood CD34-positive hematopoietic stem cell population, which was fractionated by FACS using human umbilical cord blood cells as an index to be CD34-positive, to amplify or differentiate and proliferate hematopoietic stem cells, and to proliferate and differentiate hematopoietic progenitor cells. , And mature cells (also collectively referred to as hematopoietic cell proliferative effect), as well as proliferating myeloid, leukocyte, and erythroid cells.

Experimental Example 2 Colony forming cell assay (counting the number of hematopoietic progenitor cells)
A colony-forming cell assay was performed using cells cultured in liquid for 11 days in the experimental system of Experimental Example 1 (control, addition of peptide A [10 μg / mL]), 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. Specifically, cells cultured in liquid for 11 days are mixed with a medium for measuring human hematopoietic progenitor cell colonies (MethoCult H4435 Veritas) in a Petri dish having a diameter of 35 mm at a rate of 1,000 cells / dish, and then mixed according to the manual. Cultured for a day. The medium contains SCF, IL-3, IL-6, G-CSF, GM-CSF, and Epo as cell stimulating factors. Results are shown in FIG.

  As shown in FIG. 6, CFU-G, which is a myeloid progenitor cell in which the peptide A [10 μg / mL] was added and which was liquid-cultured for 11 days, was relatively differentiated or matured compared with the control. And CFU-GM proliferation was observed, indicating that human cord blood CD34-positive hematopoietic stem cells were differentiated and proliferated. From these results, it was revealed that peptide A promotes the process of differentiation or maturation of human umbilical cord blood CD34-positive hematopoietic stem cells into myeloid cells, thereby proliferating CFU-G and CFU-GM.

  The total number of hematopoietic progenitor cells (CFU-G, CFU-M, CFU-GM, CFU-MK, BFU-E, and CFU-GEMM) is shown in FIG. 6 (B). From this, by culturing human umbilical cord blood CD34-positive hematopoietic stem cells in the presence of not only the cell stimulating factor but also peptide A, compared with 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.

  Similarly, cells cultured in liquid for 7 days instead of cells cultured in liquid for 11 days were subjected to a colony forming cell assay. The results are shown in Fig. 7.

  As shown in FIG. 7, CFU-GM, which is a myeloid progenitor cell in which the peptide A [10 μg / mL] was added and the liquid culture was carried out for 7 days, was relatively differentiated or matured as compared with the control. , BFU-E, and CFU-GEMM were observed to be proliferated, indicating that human cord blood CD34-positive hematopoietic stem cells were differentiated and proliferated. Further, from these facts, it is clear that peptide A promotes the process in which human cord blood CD34-positive hematopoietic stem cells are differentiated or proliferated into myeloid lineage cells to differentiate and proliferate CFU-G and CFU-GM. became.

  As described above, it was revealed that human cord blood CD34-positive hematopoietic stem cells promote the process of differentiation or maturation into myeloid cells in the presence of peptide A not only on the 11th day but also on the 7th day.

Experimental Example 3 Intracellular uptake of peptide A Human cord blood CD34-positive hematopoietic stem cells were cultured for 6 hours or 12 hours in the same medium as used in Experimental Example 1 containing 10 μg / ML of biotinylated peptide A and cultured. The cells were collected and used as a specimen. The cells were then treated with (TOTO-3iodide) for staining nuclei and with AlexaFluor488-labeled streptavidin and subjected to confocal fluorescence microscopy. The results are shown in Fig. 8. As a negative control, a sample cultured in a medium containing no biotinylated peptide A was prepared.

  In the case of the negative target, both Nucleus and Merge had no change in the brightness of the fluorescence micrograph image, whereas in the biotinylated peptide A, the merger with the nuclear staining and the peptide A had higher brightness, and the intracellular peptide A Has been revealed to have been incorporated. Therefore, peptide A is considered to exert its function intracellularly.

Experimental Example 4 Analysis of Expressed Gene In the experimental system of Experimental Example 1 (control, addition of peptide A [10 μg / mL]), cord blood CD34-positive hematopoiesis using FACS from cell groups 1 day and 2 days after the start of liquid culture. Stem cells were collected and the genes expressed in the cells were examined. 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 of SOCS family (SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7) on the first day from the start of culture, and FIG. 11 shows the gene expression on the second day from the start of culture of the SOCS family. The expression level is shown.

  Here, it should be noted that 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. The SOCS family is considered to be an inhibitor of the JAK-STAT signal cascade, but it should be noted that peptide A affects SOCS5 among them.

  SEQ ID NOs: 1 to 5 show specific examples of the peptide represented by the general formula (I) or a modified product thereof which is the object of the present invention.

Claims (3)

A peptide comprising the amino acid sequence of the following formula (SEQ ID NO: 1) or a modified product thereof:

The modified product is a peptide in which the main chain and / or side chain of the N-terminal arginine residue of the peptide represented by the above formula is modified with a C _1-6 acyl group, a fatty acid, or myristic acid.   An agent for proliferating hematopoietic cells, which comprises as an active ingredient at least one selected from the group consisting of the peptide according to claim 1, a modified product thereof, a pharmaceutically acceptable salt thereof, and a solvate thereof. ..   In a medium containing the peptide according to claim 1, a modified product thereof, at least one selected from the group consisting of pharmaceutically acceptable salts thereof, and solvates thereof, and a cell stimulating factor, A method for producing hematopoietic cells, comprising a step of culturing hematopoietic stem cells or hematopoietic progenitor cells.

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