Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
1999-02-19
2001-08-14
Spector, Lorraine (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C530S350000, C530S324000, C514S002600, C514S012200, C424S085200
Reexamination Certificate
active
06274709
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a protein which induces the interferon-&ggr; (hereinafter abbreviated as “IFN-&ggr;”) production by immunocompetent cells, and a monoclonal antibody specific to the protein.
2. Description of the Prior Art
It is said that IFN-&ggr; is a protein which has antiviral-, antioncotic- and immunoregulatory-activities and which is produced by immunocompetent cells stimulated with antigens and/or mitogens. Because of these biological activities, IFN-&ggr; is expected to be used as an antitumor agent since the discovery, and energetically studied on clinical trials as a therapeutic agent for malignant tumors in general including brain tumors. IFN-&ggr; preparations now commercially available are roughly classified into 2 groups, i.e. natural IFN-&ggr;s produced by immunocompetent cells and recombinant IFN-&ggr;s produced by transformants prepared by introducing DNAs which encode the natural IFN-&ggr;s into microorganisms of the species
Escherichia coli
. In such clinical trials, either of these IFN-&ggr;s is administered to patients as an “exogenous IFN-&ggr;”.
Among these IFN-&ggr;s, the natural IFN-&ggr; is usually produced by culturing established immunocompetent cells in nutrient culture media supplemented with IFN-&ggr; inducers to form IFN-&ggr;, and purifying the IFN-&ggr;. It is known that the type of IFN-&ggr; inducers greatly influence on the production yield and the facility of IFN-&ggr; purification, as well as the safeness of the final products. Generally, mitogens such as concanavalin A (Con A),
Lens culinaris, Phytolacca americana
, endotoxin and lipopolysaccharide are used. These mitogens, however, have problems of their molecular and quality varying dependently on their origins and purification methods, as well as the difficulty of obtaining a desired amount of preparations with a constant IFN-&ggr; inducibility. In addition, most of these mitogens induce unfavorable side effects when administered to living bodies, and some of them may cause toxicity, so that it is substantially difficult to induce the IFN-&ggr; production by the direct administration to living bodies.
During the study of cytokines produced by mammalian cells, the present inventors have found in mouse liver a novel substance which induces the IFN-&ggr; production. They isolated the substance using two or more conventional purification methods including column chromatography mainly, studied the property and feature and revealing that the reality is a protein having the following physicochemical properties:
(1) Molecular weight
19,000±5,000 daltons on gel filtration sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE);
(2) Isoelectric point (pI)
4.8±1.0 on chromatofocusing;
(3) Partial amino acid sequence
Possessing partial amino acid sequences corresponding to amino acid residues 26-43 and 79-103 of SEQ ID NO:2; and
(4) Biological activity
Inducing the interferon-&ggr; production by immunocompetent cells.
Such a protein with these physicochemical properties has never been reported, and the data concludes that the protein is novel. The present inventors energetically studied on mouse liver cells and have succeeded to isolate a DNA encoding the protein. The decoding of the protein revealed that the DNA consists of 471 base pairs and encodes the amino acid sequence in SEQ ID NO:3. When the DNA was introduced into microorganisms of the species
Escherichia coli
to express the production of the present protein, the protein was produced in the culture in a satisfactorily high yield. These findings are disclosed in Japanese Patent Application No.184,162/94 applied by the present applicant.
As is described above, the present protein has an activity of inducing the IFN-&ggr; production by immunocompetent cells, and is expected to be used in a variety of uses as an anti-virus agent, antioncotic agent, antiseptic, immunoregulatory agent or a platelet-increasing agent. Generally, in the case of incorporating biologically active proteins into pharmaceuticals, the developments of methods for purifying such proteins highly and effectively and those for assaying samples containing these proteins are inevitable. The material most suitable for the purification and assay is a monoclonal antibody, but such a monoclonal antibody specific to the protein is not established.
SUMMARY OF THE INVENTION
In view of the foregoing, the object of the present invention is to provide a novel protein which induces the IFN-&ggr; production by immunocompetent cells.
It is another object of the present invention to provide a DNA encoding the protein.
It is further object of the present invention to provide a replicable recombinant DNA which contains the DNA and a self-replicable vector.
It is yet another object of the present invention to provide a transformant obtainable by introducing the recombinant DNA into an appropriate host.
It is another object of the present invention to provide a process for preparing the protein by using the recombinant DNA technology.
It is another object of the present invention to provide a novel monoclonal antibody which is specific to the protein having the aforesaid physicochemical properties.
It is another object of the present invention to provide a hybridoma which produces the monoclonal antibody.
It is another object of the present invention to provide a process for preparing the monoclonal antibody.
It is another object of the present invention to provide a purification method with the monoclonal antibody.
It is another object of the present invention to provide a method for detecting the protein with the monoclonal antibody.
The first object of the present invention is attained by a protein having the following physicochemical properties:
(1) Molecular weight
19,000±5,000 daltons on gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE);
(2) Isoelectric point (pI)
4.8±1.0 on chromatofocusing;
(3) Partial amino acid sequence
Possessing partial amino acid sequences corresponding to amino acid residues 26-43 and 79-103of SEQ ID NO:2; and
(4) Biological activity
Inducing the IFN-&ggr; production by immunocompetent cells.
The second object of the present invention is attained by a DNA which encodes the protein.
The third object of the present invention is attained by a replicable recombinant DNA which contains the DNA and a self-replicable vector.
The fourth object of the present invention is attained by a transformant obtainable by introducing the replicable recombinant DNA into an appropriate host.
The fifth object of the present invention is attained by a process for preparing the protein comprising culturing the transformant in a nutrient culture medium, and collecting the formed protein from the resultant culture.
The sixth object of the present invention is attained by a monoclonal antibody which is specific to a protein having the following physicochemical properties:
(1) Molecular weight
19,000±5,000 daltons on gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE);
(2) Isoelectric point (pI)
4.8±1.0 on chromatofocusing;
(3) Partial amino acid sequence
Possessing partial amino acid sequences corresponding to amino acid residues 26-43 and 79-103 of SEQ ID NO:2; and
(4) Biological activity
Inducing the IFN-&ggr; production by immunocompetent cells.
The seventh object of the present invention is attained by a hybridoma which can produce the monoclonal antibody.
The eighth object of the present invention is attained by a process comprising culturing in vivo or in vitro hybridomas capable of producing the monoclonal antibody, and collecting the monoclonal antibody from the resultant body fluids or cultures.
The ninth object of the present invention is attained by a method for purifying the present protein comprising contacting a monoclonal antibody specific to the protein to a mixture containing the protein and impurities to adsorb the protein on the monoclonal antibody, and desorbing the adsorbe
Kohno Keizo
Kunikata Toshio
Kurimoto Masashi
Okamura Haruki
Taniguchi Mutsuko
Browdy and Neimark
Jiang Dong
Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo
Spector Lorraine
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