Drug – bio-affecting and body treating compositions – Lymphokine – Interferon
Reexamination Certificate
2001-03-29
2002-06-11
Stucker, Jeffrey (Department: 1647)
Drug, bio-affecting and body treating compositions
Lymphokine
Interferon
C514S002600, C514S012200, C514S021800, C530S350000, C530S351000, C530S324000, C435S325000, C435S366000
Reexamination Certificate
active
06403079
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel protein which induces the interferon-&ggr; (hereinafter abbreviated as “IFN-&ggr;”) production by immunocompetent cells.
2. Description of the Prior Art
It is known that IFN-&ggr; is a protein which has antiviral-, antioncotic- and immunoregulatory-activities and is produced by immunocompetent cells that are stimulated with antigens or mitogens. Because of these biological activities, IFN-&ggr; has been expected for use as an antitumor agent since it was discovered, and studied energetically on clinical trials as a therapeutic agent for malignant tumors in general including brain tumors. IFN-&ggr; preparations commercially available now are roughly classified into two groups, i.e. one group of natural IFN-&ggr;s produced by immunocompetent cells and another group of recombinant IFN-&ggr;s produced by transformants obtained by introducing DNAs which encode natural IFN-&ggr;s into microorganisms of the species
Escherichia coli
. In the above clinical trials, one of these two groups of IFN-&ggr;s is administered to patients as an “exogenous IFN-&ggr;”.
Among these IFN-&ggr;s, natural IFN-&ggr;s are usually produced by culturing established immunocompetent cell lines in nutrient culture media admixed with IFN-&ggr; inducers to produce IFN-&ggr;s, and purifying the produced IFN-&ggr;s from the resulting cultures. It is known that IFN-&ggr; inducers greatly influences on the IFN-&ggr; yield, the facility of IFN-&ggr; purification, and the safety of final IFN-&ggr; preparations. Generally, mitogens such as concanavalin A (Con A), lentil lectin, pokeweed lectin, endotoxin and lipopolysaccharides can be used as IFN-&ggr; inducers. However, these mitogens have the following problems: (i) their molecules and qualities vary and change depending on their origins and purification methods, and (ii) preparations with a constant IFN-&ggr; inducibility are not readily prepared in a satisfactory yield. In addition, most of these mitogens might induce unfavorable side effects when administered to living bodies, and some of them might cause toxicity, so that it is substantially difficult to induce IFN-&ggr; production by directly administering IFN-&ggr; inducers to the living bodies.
SUMMARY OF THE INVENTION
The present invention was made based on a novel protein which induces the interferon-&ggr; production by immunocompetent cells. During the study of cytokines produced by mammalian cells, the present inventors noticed that the existence of a substance which induces IFN-&ggr; production in mouse liver cells which had been treated with a lipopolysaccharide and inactivated whole cells of Corynebacterium. They isolated the substance by many purification methods using column chromatography as a main technique and studied the properties and features, and have found that the reality is a protein having the following physicochemical properties:
(1) Molecular weight 19,000±5,000 daltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);
(2) Isoelectric point (pI) pI of 4.8±1.0 on chromatofocusing;
(3) Partial amino acid sequence Having the partial amino acid sequences of SEQ ID NOs:8 and 9; and
(4) Biological activity Inducing the IFN-&ggr; production by immunocompetent cells.
The data concluded that the substance is novel because no protein with these physicochemical properties is known. The present inventors continued studying on mouse liver cells and have succeeded to isolate a DNA which encodes the protein. The inventors decoded the DNA and have found that it consists of 471 base pairs and encodes the amino acid sequence of SEQ ID NO:10 (where the symbol “Xaa” means “methionine” or “threonine”).
Based on these findings, the present inventors further studied on human liver cells to obtain a DNA which encodes another novel substance that induces the IFN-&ggr; production by immunocompetent cells. They revealed that the reality is a polypeptide, then decoded the DNA and found that it has the amino acid sequence of SEQ ID NO:6 (where the symbol “Xaa” is “isoleucine” or “threonine”). They introduced the DNA into
Escherichia coli
to express the polypeptide and to produce it in the resulting culture in a satisfactorily high yield. These findings were disclosed in Japanese Patent Laid-Open Nos.27,189/96 and 193,098/96, applied by the present applicant. In Japanese Patent Application No.78,357/95 applied by the applicant, the polypeptide is disclosed as an agent for susceptive diseases. Although biologically active proteins which are administered to humans after mixed with pharmaceuticals should be generally human cell origin, no human cell which produces such a polypeptide is reported.
In view of the foregoing, the object of the present invention is to provide a protein of human cell origin, which induces the IFN-&ggr; production by immunocompetent cells.
The another object of the present invention is to provide a process for producing the protein.
The further object of the present invention is to provide the use of the protein as an agent for susceptive diseases.
The first object of the present invention is attained by a protein of human cell origin which induces the IFN-&ggr; production by immunocompetent cells and has the amino acid sequence of SEQ ID NO:1.
The second object of the present invention is attained by a process for producing the protein by propagating human cells which produce the protein, and collecting the protein from the propagated cells.
The third object of the present invention is attained by an agent for susceptive diseases, which contains the protein as an effective ingredient.
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Genebank/dbEST: “Public availability dates for SEQ ID NOS: 2-7 of the instant application and the database alignments”.
Schoenhaut et al., J. Immunol. 148:3433-40.
Okamura et al., “Cloning of a new cytokine that induces IFN-&ggr;”, Nature, 378:88-91 (1995).
Nakamura et al., “Purification of a factor which provides a costimulatory signal for gamma interferon production”, Infection and Immunity, 61:64-70 (1993).
Hatt et al., “American type culture collection catalogue of cell lines and hybridomas”, 5thed., (1985).
Kostura et al., “Identifiecation of a monocyte specific pre-interleukin 18 convertase activity”, Proc. Natl. Acad. Sci., vol. 86:5227-5231 (Jul. 1989).
Fujioka et al., “Combination of lymphokine-activated killer cells and interleukin-2 in treating metastatic renal cell carcinoma”, British Journal of Urology, 73:23-31 (1994).
Laemmli et al., “Cleavage of structural proteins during the assembly of the head of bacteriophage T4”, Nature, 227:680-685 (Aug. 15, 1970).
Balkwill, “Cytokines in cancer therapy”, Oxford University Press, New York, New York (1989).
Ushio et al., “Cloning of the cDNA for human IFN-gamma0inducing Factor”, Journal of Immunology, 156(11)4274-4279 (Jun. 1, 1996).
Akita Kenji
Fujii Mitsukiyo
Kurimoto Masashi
Nukada Yoshiyuki
Tanimoto Tadao
Browdy and Neimark
Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo
Seharaseyon Jegatheesan
Stucker Jeffrey
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