Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2002-01-28
2004-08-17
Chan, Christina (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007950, C435S326000, C435S975000, C530S388800
Reexamination Certificate
active
06777194
ABSTRACT:
DESCRIPTION
1. Technical Field
The present invention relates to monoclonal antibodies against human protein Mcm3, processes for their production and their use.
2. Prior Art
Mcm proteins were first described in the barm S. cerevisiae. It is known, that these proteins play an important role in the initiation of DNA replication, which was shown in the barm by its decisive role in the transmittance of extra chromosome DNA segments, minichromosomes (Maine et al., Genetics, 1984, 106:365-385). This feature was the basis for the naming for these proteins, minichromosome maintenance, Mcm. The proteins of the Mcm family are highly conserved with respect to evolution.
At present six proteins (Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, Mcm7) are described in the human system which with other cell cycle dependent structures form a protein complex that is necessary for DNA replication, and which were already postulated as DNA replication licence factors by J. J. Blow and R. A. Laskey in 1988 (Nature, 332:546-548). Mcm3 protein plays an important role by forming a biochemical strong bond with Mcm5 (A. Richter, R. Knippers, Eur. J. Biochem., 1997, 247:136-141). Since Mcm3 and the other members of the Mcm family have such a basic function in the cell cycle, detection systems are desired, preferably immunobiochemical and immunohistological detections. Such detections are of need because new parameters for medical diagnosis, preferably in cancer diagnosis, can be achieved therewith.
It is known that human Mcm protein is immunogenic in the rabbit (Thommes et al., Nucleic Acid Res., 1992, 20:1069-1074). But the known polyclonal antisera either do not react monospecifically in immunobiochemical analyses (Western Blot) and/or are not applicable quickly and without problems in routine immunohistology (Hu, B., et al., Nucleic Acid Res., 1993, 21: 5289-5293). Therefore, there is no tool at hand that can serve as a detection method for Mcm3 in medical diagnosis.
SUMMARY OF THE INVENTION
The object of the present invention is therefore to provide means that detect quickly and monospecifically Mcm3 protein in biochemical and also in histological systems conducted alone or together in combination. This detection can be conducted alone or in combination with other known markers.
According to the invention this is achieved by a monoclonal antibody directed against Mcm3 protein and being applicable both in immunobiochemical and in immunohistochemical detection systems, whereby these detections can be conducted alone or in combination.
Further, hybridomas producing monoclonal antibodies according to the invention are disclosed.
Another aspect of the invention is the provision of diagnostic compositions and detection kits comprising the monoclonal antibody according to the present invention. Yet another aspect is the use of the monoclonal antibody according to the present invention for the detection of Mcm3 in a sample.
Moreover, processes are disclosed relating to the production of a monoclonal antibody and hybridoma, respectively, according to the present invention.
Finally, the present invention relates to pharmaceutical preparations and medicines containing the monoclonal antibody and the use of the monoclonal antibody for the preparation of a medicament for the treatment of certain diseases.
Also, within the scope of the invention are methods for treating diseases or disorders which are associated with an aberrant Mcm3 level or activity or which can benefit from modulation of the activity or level of Mcm3. The methods comprise administering, e.g., either locally or systemically to a subject, a pharmaceutically effective amount of a composition comprising an MCm3 antibody according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
According to the invention a monoclonal antibody directed against Mcm3 protein and being applicable both in immunobiochemical and in immunohistochemical detection systems, whereby these detections can be conducted alone or in combination is provided.
The monoclonal antibody according to the present invention can be obtained from any animal or the human being, whereby the monoclonal antibodies of the mouse are preferred.
Further, the monoclonal antibody may be altered biochemically, by genetic manipulation, or it maybe synthetic, with the antibody possibly lacking portions completely or in parts, said portions being necessary for the recognition of Mcm3 and being substituted by others imparting further advantageous properties to the antibody.
A hybridoma cell line producing a preferred monoclonal antibody of the present invention, namely, a monoclonal mouse antibody with said above-mentioned detection, was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124, Braunschweig, Germany under Accession No. DSM ACC2388 on Feb. 16, 1999.
The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) Mcm3. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)
2
fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides monoclonal antibodies that bind Mcm3. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of Mcm3. A monoclonal antibody composition thus typically displays a single binding affinity for Mcm3 with which it immunoreacts.
A disease, a disorder or condition “associated with” or “charterized by” an aberrant Mcm3 activity refers to a disease, disorder or condition in a subject which is caused by or contributed to by an aberrant Mcm3 activity.
The term “treating” as used herein is intended to encompass curing as well as ameliorating at least one symptom of the condition or disease.
Monoclonal anti-Mcm3 antibodies can be prepared by immunizing a suitable subject with an Mcm3 immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed Mcm3 protein or a chemically synthesized Mcm3 polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agents. Immunization of a suitable subject with an immunogenic Mcm3 preparation induces an anti-Mcm3 antibody response.
The anti-Mcm3 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized Mcm3. If desired, the antibody molecules directed against Mcm3 can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Koehler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. US.4 76:2997-3 1; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing a monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A new Dimension in Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J: Biol. Med., 54:387402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:23136). Briefly, an immortal ce
Baron-Lühr Bettina
Endi Elmar
Gerdes Johannes
Hahn Margrit Kernbach
Knippers Rolf
Chan Christina
Dakocytomation Denmark A/S
Haddad Maher
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