Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1997-04-08
2001-07-31
Ungar, Susan (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S388100, C530S413000, C530S388230
Reexamination Certificate
active
06268486
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel monoclonal antibody, and more particularly, to a monoclonal antibody which is specific to a polypeptide capable of inducing the interferon-&ggr; (hereinafter abbreviated as “IFN-&ggr;”) production by immunocompetent cells.
2. Description of the Prior Art
It is known that IFN-&bgr; is a protein which has antiviral-, antioncotic- and immunoregulatory-activities, and is produced by immunocompetent cells stimulated with antigens or mitogens. Because of these biological activities, IFN-&ggr; is expected to be used as an antitumor agent from the beginning of the finding, and is actively studied in 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 obtained by introducing DNA molecules which encode natural INF-&ggr;s into microorganisms of the species
Escherichia coli
. In the above clinical trials, one of these 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 cells in nutrient culture media supplemented with IFN-&ggr; inducers to form IFN-&ggr;s, and purifying the formed IFN-&ggr;s. It is known that the type of IFN-&ggr; inducers greatly influence the IFN-&ggr; yield, as well as the ease of IFN-&ggr; purification and the safety of the final products. Generally, mitogens such as concanavalin A (Con A),
Lens culinaris, Phytolacca americana,
endotoxin and lipopolysaccharide are used as an IFN-&ggr; inducer. However, these mitogens have problems with their molecular varieties and quality depending 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 even cause toxicity, so that it is substantially difficult to induce the IFN-&ggr; production by direct administrations to living bodies.
The present inventors found in mouse liver a substance which induces IFN-&ggr; production during their research of cytokines produced from mammalian cells. They isolated the substance by using a variety of purification methods comprising column chromatography as a main technique, and studied the properties and features, which revealed a protein having the following physicochemical properties:
(1) Molecular weight
Exhibiting a molecular weight of 19,000±5,000 daltons on sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE);
(2) Isoelectric point (pI)
Exhibiting an isoelectric point of 4.8±1.0 on chromatofocusing;
(3) Partial amino acid sequence
Having the partial amino acid sequences in SEQ ID NOs:4 and 5; and
(4) Biological activity
Inducing the IFN-&ggr; production by immunocompetent cells.
It can be concluded that it is a novel substance because no protein with these physicochemical properties has been known. The present inventors continued studies on mouse liver cells and have found that the DNA (SEQ ID NO:6) of the substance consists of 471 base pairs and encodes the amino acid sequence in SEQ ID NO:7.
Based on these findings, the present inventors continued studies on human liver cells and obtained a DNA which encodes another novel substance that induces the IFN-&ggr; production by immunocompetent cells. They revealed that the novel substance is a polypeptide, decoded its DNA and further revealed that the polypeptide has the amino acid sequence in SEQ ID NO:1. They introduced the DNA into
Escherichia coli
to express the polypeptide and to obtain the polypeptide in the resultant culture in a considerably high yield. These findings were disclosed in Japanese Patent Application Nos.184,162/94 and 304,203/94, applied by the present inventors.
As is described above, the polypeptide has a property of inducing the IFN-&ggr; production by immunocompetent cells, and is expected to be used in a variety of fields as an IFN-&ggr; inducer, antiviral agent, antitumor agent, antibacterial agent, immunoregulatory agent, and blood platelet enhancing agent. In general, the developments of methods for efficiently purifying biologically active polypeptides to give a relatively-high purity and those for assaying many samples in parallel are inevitably required when the polypeptides should be incorporated into pharmaceuticals. Although the best material for enabling purification and assay of the polypeptide is a monoclonal antibody, no monoclonal antibody specific to the polypeptide has been established.
SUMMARY OF THE INVENTION
In view of the foregoing, the object of the present invention is to provide a monoclonal antibody which is specific to the polypeptide.
It is another object of the present invention to provide a hybridoma capable of producing the monoclonal antibody.
It is further object of the present invention to provide a method for preparing the monoclonal antibody.
It is yet another object of the present invention to provide a purification method for purifying the polypeptide using the monoclonal antibody.
It is another object of the present invention to provide a detection method for assaying the polypeptide using the monoclonal antibody.
The first object of the present invention is attained by a monoclonal antibody which is specific to a polypeptide having either the amino acid sequence in SEQ ID NO:1 or a homologous amino acid sequence thereunto, and induces the IFN-&ggr; production by immunocompetent cells.
The second object of the present invention is attained by a hybridoma capable of producing the monoclonal antibody.
The third object of the present invention is attained by a process for preparing the monoclonal antibody comprising culturing the hybridoma capable of producing the antibody in vitro, i.e. in a nutrient culture medium, or in vivo, i.e. in an animal, and collecting the antibody from the resultant culture or the body fluid.
The fourth object of the present invention is attained by a purification method for a polypeptide comprising contacting the monoclonal antibody with a mixture containing the polypeptide and impurities to adsorb the polypeptide thereunto, and desorbing the polypeptide from the antibody.
The fifth object of the present invention is attained by a method for detecting the polypeptide comprising contacting samples with the monoclonal antibody to effect immunological reaction to detect the polypeptide.
REFERENCES:
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Johnstone & Thorpe Immunochemistry in Practice, 1987.*
Blackwell Scientific Publications, Oxford, p. 30.*
Nakamura et al, “Endotoxin-induced serum factor that stimulates gamma interferon production”,Infection and Immunity, 57:590-595 (Feb. 1989).
Nakamura et al, “Purification of a factor which provides a costimulatory signal for gamma interferon production”,Infection and Immunity, 61:64-70 (Jan. 1993).
Okamura et al, “A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock”,Infection and Immunity, 63:3966-3972 (Oct. 1995).
Okamura et al, “Cloning of a new cytokine that induces IFN-gamma”,Nature, 378:88-91 (Nov. 1995).
Goding, J.W., “Antibody production by hybridomas”,Journal of Immunological Methods, 39:285-308 (1980).
Sevier et al, “Monoclonal antibodies in clinical immunology”,Clinical Chemistry, 27(11):1797-1806 (1981).
Toyama, et al,Experimental Manual for Monoclonal Antibody, pp. v-153 (1987).
Tijssen, P., “Practice and Theory of Enzyme Immunoassays”,Laboratory Techniques in Biochemistry and Molecular Biology; Burden, et al (eds), Elsevier Science Publishers B.V. (Biomedical Division), (A
Kohno Keizo
Kunikata Toshio
Kurimoto Masashi
Taniguchi Mutsuko
Browdy and Neimark
Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo
Ungar Susan
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