Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1999-01-07
2002-05-07
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Lymphokine
C424S085100, C424S085200, C514S885000, C514S002600, C530S300000, C530S350000, C530S399000
Reexamination Certificate
active
06383480
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Phase Application filed under 35 U.S.C §371 of International Application No. PCT/JP97/02401, filed Jul. 10, 1997.
TECHNICAL FIELD
This invention relates to a novel use of MK to promote proliferation and differentiation of hematopoietic stem cells and hematopoietic precursor cells in hematopoietic tissues, peripheral blood, or umbilical cord blood synergistically with other hematopoietic factors.
BACKGROUND ART
In blood, there exist various hemocytes having different shapes and functions, including erythrocytes, leukocytes, and platelets, which play important roles in maintaining homeostasis of the living body. These mature hemocytes have their own life-spans. For maintaining the hemocyte count at a constant level, hemocytes must be incessantly produced to make up for the number of hemocytes that is lost due to the expiration of their life-spans.
In the normal healthy individual, it is presumed that daily production of hemocytes reaches as much as 2×10
11
erythrocytes, 10
11
leukocytes, and 1 to 2×10
11
platelets. Hematopoietic stem cells play central roles in the system to produce such an enormous number of hemocytes over a long period without being exhausted. The cells have not only self-renewal capability but also multipotentiality to differentiate to various mature hemocytes including erythrocytes, granulocytes, platelets, and lymphocytes. Hematopoietic stem cells (multipotential stem cells) lose their self-renewal capability as they proliferate to become hematopoietic precursor cells (committed stem cells) destined to differentiate to the specific hemocytes. Hematopoietic precursor cells then differentiate to mature peripheral hemocytes.
It has been known that a number of cytokines regulate each step of the hematopoietic system to proliferate and differentiate hematopoietic stem cells to various mature hemocytes via hematopoietic precursor cells. At least twenty kinds of these cytokines participating in the hematopoietic system have been found at present (Masami Bessho: Igaku no Ayumi 180(13): 802-806, 1997). The genes for all have been cloned, allowing their production on a large scale by genetic engineering techniques. Stem cell factor (SCF) and flk-2 ligand are the most remarkable cytokines as factors acting on mainly hematopoietic stem cells at the early stage of hematopoiesis. SCF acts on the most undifferentiated hematopoietic stem cells. In either mice or humans, it remarkably promotes the formation of colonies of blast colony-forming unit (CFU-BL), colony-forming unit-mixed (CFU-Mix), burst forming unit-erythrocyte (BFU-e), colony-forming unit-granulocyte/macrophage (CFU-GM), eosinophil colony-forming unit (CFU-Eo), and colony-forming unit-megakaryocyte (CFU-Meg), showing a synergistic effect with various cytokines such as IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-11, G-CSF, GM-CSF, and EPO. It has been reported that SCF alone has weak colony-stimulating activity (Tsuji, K. et al., Blood 78: 1223, 1991; Shioharu, M. et al., Blood 81: 1453, 1993; Kubo, T. and Nakahata, T., Int. Hematol. 58: 153, 1993). Nevertheless, SCF is thought to be the most important cytokine for in vitro amplification of hematopoietic stem cells at present.
The gene for flk-2 ligand has been just recently cloned and its biological activity has not been fully clarified. Since it exhibits synergistic actions with many cytokines as SCF does, it is expected to be an important factor for in vitro amplification of human hematopoietic stem cells.
Some of these hematopoietic factors have been clinically applied. For example, erythropoietin (EPO), which promotes the production of erythrocytes, is used for treating renal anemia, and granulocyte colony-stimulating factor (G-CSF), which promotes the production of neutrophils is used for treating neutropenia caused by cancer chemotherapy. These contribute to improved quality of life of patients. Recently, the clinical application of thrombopoietin (TPO) for treating thrombocytopenia has been studied because it promotes the production of platelets.
On the one hand, since hematopoietic stem cells are capable of reconstituting all kinds of cells in the hematopoietic system, the transplantation of hematopoietic stem cells has been widely performed for hematopoietic tumors. Recently, the transplantation of peripheral blood stem cells has rapidly become prevalent, and gathered attention as the powerful fundamental therapy for the chemotherapy-sensitive malignant tumors including the hematopoietic organ tumors. Furthermore, as a future prospect, the transplantation of hematopoietic stem cells is expected to be introduced to many cell therapy and gene therapy protocols. For that purpose, it is necessary to establish a method for amplifying hematopoietic stem cells in vitro. However, even now, human hematopoietic stem cells have been neither isolated nor clarified as to what extent they can repeat self-renewal.
DISCLOSURE OF THE INVENTION
An objective of the present invention is to provide a novel cytokine capable of synergistically promoting the proliferation and differentiation of hematopoietic stem cells or hematopoietic precursor cells in combination with known cytokines.
Another objective of the present invention is to provide a novel cytokine capable of promoting the proliferation and differentiation of granulocyte/monocyte precursor cells.
Still another objective of the present invention is to provide a novel cytokine capable of promoting the proliferation and differentiation of erythroblast precursor cells in combination with known cytokines.
The present inventors have found that single use of a novel growth factor called midkine (MK) or pleiotrophin (PTN) promotes the proliferation and differentiation of hematopoietic stem cells and hematopoietic precursor cells (also called hematopoietic cells) of mammals such as mice and humans in vitro. MK also exerts an extremely remarkable synergistic effect for proliferating and differentiating hematopoietic cells when used together with SCF, M-CSF, G-CSF, GM-CSF, IL-3, and IL-6. Furthermore, the inventors have found that MK or PTN promotes rapid recovery of neutrophils in neutropenia of mammals.
The present invention will be described in detail below.
MK was isolated as the product of a gene that is expressed at the early stage of the differentiation of mouse embryonic tumor cells by the induction of retinoic acid (Kadomatsu, K., et al., Biochem. Biophy. Res. Commun. 115: 1312-1318, 1988). PTN was found as a heparin-binding protein with neurite outgrowth capability in the newborn rat brain (Rauvaa, H., EMBO J. 8: 2933-2941, 1989). MK and PTN belong to a new class of heparin-binding growth factors, sharing a 45% homology (in amino acid sequence) to each other, and called the MK family. MK and PTN respectively exhibit characteristic expression patterns in the developmental process, indicating that they have important physiological activities for the implementation of differentiation.
Paying attention to such biological activities of the MK family, the present inventors studied their hematopoietic factor activities to proliferate and differentiate myeloid cells and peripheral blood stem cells of mammals. In general, at what stage of the proliferation/differentiation process of hematopoietic stem cells and hematopoietic precursor cells in myeloid hematopoietic factors participate and function can be studied by culturing a certain number of myelocytes in a semi-solid medium in the presence of these hematopoietic factors, selecting cells constituting colonies formed, and counting the number of colonies. In such colony formation methods, it has been proved by a number of direct or indirect methods that, a single hematopoietic precursor cell proliferates, divides, and matures, forming a single colony comprising many matured hemocytes. There are colony formation assay methods specific for cells of each hematopoietic system including granulocyte/macrophage, erythroblasts, and megakaryocytes, and stimulators specific for each hematopoietic syst
Ikematsu Shinya
Kikuchi Makoto
Muramatsu Takashi
Oda Munehiro
Sakuma Sadatoshi
Bunner Bridget E.
Fish & Richardson P.C.
Kunz Gary L.
Meiji Milk Products Co. Ltd.
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