Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Hormone or other secreted growth regulatory factor,...
Reexamination Certificate
1993-05-21
2001-04-24
Cochrane Carlson, Karen (Department: 1653)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Hormone or other secreted growth regulatory factor,...
C424S198100
Reexamination Certificate
active
06221359
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a hemopoietic stem cell augmenting agent. In particular, the present invention relates to a hemopoietic stem cell augmenting agent which has an activity to augment undifferentiated pluripotent hemopoietic stem cells. According to the present invention, therapeutic agents to restore bone marrow growth after the use of anti-cancer agents or after bone marrow transplantation or therapeutic agents for bone marrow malfunctions such as hypoplastic anemia and osteomyelodysplasia syndrome can be provided. Furthermore, this hemopoietic factor having the hemopoietic stem cell augmenting activity can be used as a useful reagent or the like for in vitro proliferation of peripheral stem cells and bone marrow stem cells and furthermore as an analytical reagent or as an antigen for preparation of antibodies.
Furthermore, the present invention also relates to a novel protein which belongs to hepatocyte growth factors and has a hemopoietic stem cell augmenting activity.
BACKGROUND OF THE INVENTION
Recently, it has been found that in the differentiation of undifferentiated pluripotent hemopoietic stem cells to mature blood cells, a number of hemopoietic factors are mutually involved In various levels and thus a complicated hemopoietic network system is formed. Furthermore, most of the hemopoietic factors are being genetically cloned and several hemopoietic factors are being mass produced by genetic recombination techniques, and their clinical applications are in progress. On the other hand, although pluripotent hemopoietic stem cells are characterized by their self-replicating ability (growth), sufficient studies have not been done on the growth factors which affect the undifferentiated pluripotent hemopoietic stem cells in the bone marrow.
It is known that bone marrow stromal cells play a major role in the growth of the pluripotent hemopoietic stem cells and their differentiation into mature cells; some kind of fluid factors secreted by the stromal cells, or intercellular reactions or the like are thought to be involved in hemopoieses in the bone marrow.
For example, it is known that bone marrow stromal cell line MC3T3-G2/PA-6(PA-6) which is established from the calvaria of C57B1/6 newborn mouse supports the growth of mouse pluripotent hemopoietic stem cells (Kodama, H. et al., J. Cell. Physiol., 112, 89, 1982).
In recent years, a ligand for a c-kit protein, which is a tyrosine kinase receptor being expressed in pluripotent stem cells, has attracted attention as a factor involved in the growth of undifferentiated stem cells and great efforts have been made to substantiate the ligand; in 1990, three research groups succeeded in genetically cloning the ligand. They were reported as SCF (stem cell factor; K. M. Zsebo et al., Cell, 63, 195-201, 1990), MGF (mast cell growth factor; D. E. Williams et al., Cell, 63, 167-174, 1990) and KL (c-kit ligand; Huang et al., Cell, 63, 225-233, 1990).
At present, the mechanism of the action of the ligand is being studied using the c-kit ligand which is mass produced by genetic recombination techniques and studies to date are clarifying that this factor acts on the stem cells which are differentiated to a certain extent (Hayashi et al., Int. J. Hematology, Suppl. No. 1, p198, 1991).
Accordingly, it is now considered that aside from this protein, other factors exist which act on pluripotent hemopoietic stem cells which are differentiated to a lesser extent in the bone marrow.
The hemopoietic factors having the above-mentioned activities can be made into useful pharmaceuticals such as therapeutic agents to restore bone marrow growth after the use of anti-cancer agents or after bone marrow transplantation and therapeutic agents for bone marrow malfunctions such as hypoplastic anemia and osteomyelodysplasia syndrome.
Furthermore, the hemopoietic factors having the above-mentioned activities can be used as a useful reagent or the like for in vitro proliferation of peripheral stem cells and bone marrow stem cells and furthermore as an analytical reagent or as an antigen for preparation of antibodies.
DISCLOSURE OF THE INVENTION
It is an object of the present invention to provide a hemopoietic stem cell augmenting agent which contains at least one hepatocyte growth factor as an active component. In particular, an object of the present invention is to provide a hemopoietic stem cell augmenting agent which has an activity for proliferating undifferentiated pluripotent hemopoietic stem cells. This hemopoietic stem cell augmenting agent eventually promotes proliferation of osteoclasts, the progeny of hemopoietic stem cells, as well as various blood cells. This hemopoietic stem cell augmenting agent may contain interleukin 3 and/or interleukin 7 as an active component(s) in addition to at least one of the hepatocyte growth factors.
A further object of the present invention is to provide a novel protein which is a type of hepatocyte growth factor and has a hemopoietic stem cell augmenting activity. In particular, such protein can be obtained from a culture fluid of human normal fibroblasts or by genetic recombination techniques using human normal fibroblasts as a gene supply source. It is a particular object of the present invention to provide a recombinant human hepatocyte growth factor protein, which is one type of hepatocyte growth factor and has the amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing or a substance which has equivalent effectiveness. The protein is derived from human normal fibroblasts and preferably from fibroblasts of the highest normality. The present invention also provides a DNA which is characterized in that it contains a nucleotide sequence encoding an amino acid sequence of a protein, which is a type of hepatocyte growth factor and has a hemopoietic stem cell augmenting activity, or a substance equivalent in terms of effectiveness; a recombinant expression vector in which said coding nucleotide sequence is so incorporated as to be expressed; a transformant which is obtained by the transformation of a host cell using the expression vector; and a process for the preparation of a recombinant human hepatocyte growth factor protein, which is one of hepatocyte growth factors and has a hemopoietic stem cell augmenting activity, which is characterized in that the transformant is cultured under the conditions in which said protein can be expressed in a culture medium and the recombinant human hepatocyte growth factor protein, which is one type of hepatocyte growth factor and has a hemopoietic stem cell augmenting activity, is prepared from the culture. In particular, the present invention relates to a recombinant which is derived from human normal fibroblasts and considered preferably from fibroblasts of the highest normality, and a process for the preparation of the same and use of the same.
An agent which contains at least one type of hepatocyte growth factor disclosed in the present invention as an active component is effective for use as a therapeutic agent for bone marrow suppression or as a therapeutic agent for bone marrow malfunctions.
An agent which contains at least one type of hepatocyte growth factor disclosed in the present invention as an active component is effective for in vitro growth of peripheral blood stem cells and bone marrow stem cells, and thus the present invention also provides a process for in vitro proliferation or the cultivation of stem cells using said agent.
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Ikebuchi et al. (1987) “Interleukin 6 Enhancement of Interleukin 3-Dependent Proliferation of Multipotential Hemopoietic Progenitors”,Proc. Natl. Acad. Sci. USA 84, 9035-90
Kawano Genji
Kojima Katsuaki
Komiyama Atsushi
Kubo Tetsuo
Nakahata Tatsutoshi
Carlson Karen Cochrane
Scully Scott Murphy & Presser
Toray Industries Inc.
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