Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-10-30
2002-03-26
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007230, C435S007900, C424S139100, C424S152100, C424S172100, C424S178100, C424S001490, C436S504000, C436S536000, C530S387900, C530S391300
Reexamination Certificate
active
06361954
ABSTRACT:
BACKGROUND OF THE INVENTION
Proliferative growth of normal cells requires an orderly progression through a series of distinct steps, a process known as the cell cycle (Alberts et al.,
Cell Growth and Division
, Garland Publishing, Inc., New York). Progression through the cell cycle is modulated by nutrient availability, cell size, and growth factors through complex signaling pathways involving phosphorylation cascades and the strictly regulated expression and stability of specific proteins required at each phase of the cell cycle. In addition, the sequence of cell cycle events is rigorously controlled at specific checkpoints to ensure that each discrete stage in the cell cycle has been completed before the next is initiated. Human diseases associated with abnormal cell proliferation result when these rigorous controls on cell cycle progression are perturbed.
SUMMARY OF THE INVENTION
The invention relates to novel genes which function in cell cycle regulation. In a particular embodiment, the genes are derived from vertebrates, including mammalian cells, particularly those derived from Xenopus or human cells, and function in the regulation of DNA replication and/or entry of a cell into mitosis. In one embodiment, the gene is a human gene called Hscdc6 and in another embodiment the gene is a Xenopus gene called Xcdc6. In one embodiment, the genes have a DNA sequence comprising at least one DNA sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and combinations thereof; the invention also pertains to the complementary DNA sequences thereof. The present invention also relates to genes which function in the regulation of DNA replication or the entry of a cell into mitosis and which have a nucleotide sequence which hybridizes under conditions of medium stringency to at least one DNA sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26.
In particular embodiments, the isolated nucleic acid molecule encodes a protein or polypeptide with the same amino acid sequence as the endogenous protein or polypeptide. In another embodiment, the isolated nucleic acid molecule has the same nucleotide sequence as the endogenous gene encoding the protein or polypeptide.
Accordingly, this invention pertains to an isolated Hscdc6 gene or an Xcdc6 gene, or an active derivative or fragment thereof. The isolated gene is characterized by its ability to regulate the cell cycle as described herein. In particular embodiments, the expressed protein or polypeptide is purified to homogeneity or is substantially free of other proteins (i.e., isolated).
The invention also pertains to novel gene products, e.g., polypeptides or proteins, encoded by the vertebrate genics described herein, or an active derivative or fragment thereof. In a particular embodiment, the polypeptide or protein has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the gene product is a recombinant human or Xenopus polypeptide or protein which regulates DNA replication and/or the entry of a cell into mitosis. In one embodiment, the encoded protein or polypeptide is a fragment having DNA replication regulation and/or mitosis regulation activity. In another embodiment, the encoded protein or polypeptide is a derivative possessing substantial sequence identity with the endogenous protein or polypeptide.
The invention also relates to DNA constructs comprising the nucleic acid molecules described above operatively linked to a regulatory sequence, and to recombinant host cells, such as bacterial cells, fungal cells, plant cells, insect cells and mammalian cells, comprising the nucleic acid molecules described above operatively linked to a regulatory sequence.
The invention also pertains to an antibody, or an antigen-binding fragment thereof, which selectively binds to the described protein or polypeptide, or an active derivative or fragment thereof. One embodiment of the invention relates to monoclonal antibodies having specificity for Hscdc6. In particular, the invention encompasses the hCdc6-26, hCdc6-37, hCdc6-34, hCdc6-39, and hCdc6-41 monoclonal antibodies or an immunoglobulin antigen binding region or fragment derived from the hCdc6-26, hCdc6-37, hCdc6-34, hCdc6-39, and hCdc6-41 monoclonal antibodies.
Another embodiment of the claimed invention relates to the monoclonal antibodies derived from the hybridoma deposited with the American Type Culture Collection (ATCC), Accession Numbers: HB-12590 and HB-12591 Oct. 30, 1998, as well as to the deposited hybridomas themselves. Additionally, the invention relates to a humanized or chimeric immunoglobulin having specificity for Hscdc6 comprising an antigen binding region of non-human origin (e.g., the complementarity determining region (CDR) that is derived from the hCdc6-26, hCdc6-37, hCdc6-34, hCdc6-39, or hCdc6-41 monoclonal antibody). The humanized or chimeric immunoglobulin can further comprise at least a portion of human origin (e.g. a human constant region and/or a human framework region (FR)).
The invention also relates to a method for assaying the presence of the described protein or polypeptide in a cell, e.g., in a sample from an individual, comprising contacting said cell with an antibody which specifically binds to the protein or polypeptide. Embodiments of the claimed invention include Enzyme-Linked Immnosorbent Assay (ELISA), competition ELISA assays, RadioImmuno-Assays (RIA), immunofluorescence and immunohistochemical assays which involve assaying Hscdc6 in a sample using the monoclonal antibodies having specificity for Hscdc6, as described herein. The invention involves determining the presence or absence of Hscdc6 comprising combining the sample to be tested with an antibody (e.g., hCdc6-26, hCdc6-37, hCdc6-34, hCdc6-39, or hCdc6-4 1) having specificity for Hscdc6, and then detecting or measuring the formation of the complex between the antibody and the antigen. The antibodies are detectably labeled (e.g., radioactive, fluorescently, biotinylated or HRP-conjugated) to facilitate detection of the complex.
The claimed invention also pertains to methods for determining the presence or absence of a proliferative disorder (e.g., cancer) comprising determining the presence, absence, or the level of hscdc6, wherein the presence of hscdc6 or an elevated level of hscdc6, as compared to a control, standard, or baseline, indicates the presence of a proliferative disorder. An embodiment of the claimed invention includes determining the presence or absence of a proliferative disorder comprising determining the levels (e.g., presence or absence) of two or more markers for proliferative disease, wherein one of the markers is hscdc6. The additional marker can be a protein from the Mcm (mini-chromosome maintenance) family (e.g., Mcm-2, Mcm-3, Mcm-4, Mcm-5, Mcm-6, and Mcm-7). The invention also embodies methods for diagnosing or aiding in the diagnosis of a proliferative disease comprising determining the presence, absence or level of hscdc6, wherein the presence of hscdc6 or ail elevated level of hscdc6, as compared to a control, standard, or baseline, indicates a positive diagnosis for a proliferative disorder. These methods utilize the hscdc6 assays and/or monoclonal antibodies having specificity for hscdc6, as described herein.
Furthermore, the invention encompasses pharmaceutical compositions comprising the genes and proteins or polypeptides described herein, as well as methods of treating disease utilizing the compositions described herein. For example, the invention relates to a method of treating a tumor in an individual. In the method, an antagonist of Hscdc6 is administered to the individual, causing at least one of two possible results: inhibition of Hscdc6 function and inhibition of tumor cell DNA replication, with concomitant inhibition of tumor growth, or mitotic division of tumor cells with failure of DNA replication and tumor cell death. Such compositions comp
Mendez Juan R.
Stillman Bruce
Williams R. Sanders
Cold Spring Harbor Laboratory
Hamilton Brook Smith & Reynolds P.C.
Saunders David
Tung Mary Beth
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