Method of screening apoptosis inducing substances

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism

Reexamination Certificate

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C435S004000

Reexamination Certificate

active

06579692

ABSTRACT:

This Application is 35 USC §371 of PCT/JP97/00702, filed Mar. 6, 1997.
1. Technical Field
The present invention relates to a method of screening substances having property of causing apoptosis and the like, in particular, relates to a novel screening method which makes it possible to screen readily and highly efficiently the substances, such as monoclonal antibodies and the like, that have property of causing apoptosis on myeloid cells by using cells which are expressing IAP (Integrin Associated Protein), and relates to the substances having property of causing apoptosis obtained by the above screening method, pharmaceutical compositions containing as the active ingredient the above substances, and relates to substances having property of causing apoptosis which have a specific binding activity to IAP, pharmaceutical compositions containing as the active ingredient the above substances.
2. Background Art
Granulocyte colony-stimulating factors, for example, recombinant granulocyte colony-stimulating factors (rG-CSF), have been known primarily as humoral factors to stimulate the differentiation and proliferation of granulocyte cells, and it has been reported in an experiment upon mice in vivo that the administration of rG-CSF enhances the hematopoiesis of the bone marrow and in addition causes remarkable extramedullary hematopoiesis in the spleen to proliferate hematopoietic stem cells and all hematopoietic precursor cells in the spleen. And it has been thought as extramedullary hematopoietic mechanism in the spleen that hematopoiesis occurs due to a splenic hematopoietic microenvironment modifications according to the stimulation of rG-CSF to enhance hematopoietic potential.
Hence, the present inventors have noted splenic stromal cells administered rG-CSF with a view to clarifying the hematopoietic potential in the spleen, and established a hematopoietic stromal cell line (CF-1 cells) from the spleen of a mouse administered rG-CSF with a view to attempting the analysis of the enhancement of the hematopoietic potential by stromal cells with rG-CSF, and examined the potential effect on hematopoiesis using the hematopoietic stromal cells, and as a result, colony-stimulating activities in vitro and potency supportive of hematopoietic stem cells in vivo have been recognized [Blood, 80, 1914 (1992)].
However, though some of splenic stromal cells have been established as a cell line (CF-1 cells), and cytological characteristics thereof have been examined, a specific antibody recognizing surface antigens thereof has been hardly prepared so far, and characteristics thereof have been scarcely known.
Hence, the present inventors have engaged in assiduous studies with a view to developing specific antibodies capable of recognizing splenic stromal cells on the basis of the above information upon splenic stromal cells and the results of the studies, and prepared monoclonal antibodies using the splenic stromal cell lines as antigens for immunization, and as a result, novel monoclonal antibodies unreported so far have been obtained.
And as a result of examining the properties of the obtained monoclonal antibodies, the inventors found that they had the property of causing apoptosis on myeloid cells, and had reported this result previously, and further have found that the antigens recognized by the monoclonal antibodies are identical to IAP (Integrin Associated Protein) and the IAP have functions relating to apoptosis, and that it is possible to differentiate, identify and screen the substances, such as antibodies and the like, that have property of causing apoptosis by using cells which are expressing the IAP, and further have engaged in assiduous studies, which have led to the completion of the present invention.
SUMMARY OF INVENTION
It is the objective of the present invention to provide a method of screening substances having property of causing apoptosis and the like.
This invention relates to a method of screening substances having property of causing apoptosis characterized by using cells which are expressing IAP (Integrin Associated Protein) and screening the substances. The invention relates to the screening method, wherein the cells used are myeloid cells, and relates to pharmaceutical compositions containing as the ingredient the substances obtained by the above screening method. The invention makes it possible to differentiate, identify and screen readily and highly efficiently the substances, such as antibodies and the like, that have property of causing apoptosis on myeloid cells by using cells which are expressing IAP while using specific binding reactions of the substances. The above specific substances thus obtained by the screening method of the invention can be used by virtue of their characteristics as the active ingredient of pharmaceutical compositions such as anticancer agents and medicines for myelocytic leukemia and the like which are useful in the field of remedies for myelocytic leukemia and the like.
DISCLOSURE OF INVENTION
It is the objective and purpose of the present invention to provide a method of screening substances having property of causing apoptosis by using cells which are expressing IAP, and further to provide novel substances having property of causing apoptosis on cells obtained by the above screening method and pharmaceutical compositions as the active ingredient the above substances.
The above monoclonal antibody is remarkably useful as an antibody recognizing antigens causing the apoptosis [it is also called self-destruction of cells, phenomenon that a nuclear chromatin DNA is digested at a nucleosome unit (so-called ladder formation) to result in the death of cells] of myeloid cells and having a function of identifying them or a function of causing apoptosis on myeloid cells. Incidentally, myeloid cells include cells other than lymphoid cells, such as neutrophils, megakaryocytes, myeloblasts, myelocytes, mast cells, macrophages, monocytes and erythroblasts, and the myeloid cells according to the present invention also mean the same as mentioned above. No monoclonal antibody having the property of causing apoptosis on myeloid cells has been known so far other than the above monoclonal antibody, and hence the above monoclonal antibodies are defined to include all monoclonal antibodies having the property of causing apoptosis on myeloid cells.
The monoclonal antibody may be prepared basically as stated below.
Namely, the above monoclonal antibody may be prepared, for example, by using splenic stromal cells derived from an animal administered rG-CSF as antigens, immunizing them according to an ordinary immunization method, cell-fusing the immunized cells according to an ordinary cell fusion method, and cloning the fused cells according to an ordinary cloning method.
As a method of preparing the above monoclonal antibody can be preferably exemplified a method comprising using CF-1 cells, splenic stromal cells of an animal administered rG-CSF established as culture cell line by the present inventors, as the antigen [Blood, Vol. 80, 1914 (1992)], fusing plasma cells (immunocyte) of a mammal immunized with the antigen with myeloma cells of a mammal such as a mouse, cloning the obtained fused cells (hybridomas), selecting clones producing the above antibody recognizing the above cell line among them, and culturing them to recover objective antibody. However, the method is only an example, and in this case, for example, not only the above CF-1 cells but also cell lines derived from human splenic stromal cells obtained according to the case of CF-1 cells may be used as the antigens properly to prepare antibodies binding to objective human myeloid cells in the same manner as in the case of the above CF-1 cells.
In the method of preparing such monoclonal antibodies, mammals to be immunized with the above antigen are not particularly restricted; it is preferable to make selection taking into account suitability with myeloma cells to be used in cell fusion, and preferably a mouse, a rat and a hamster and the like are used.
Immunizatio

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