Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-06-18
2001-12-11
Campbell, Eggerton A. (Department: 1653)
Chemistry: molecular biology and microbiology
Vector, per se
C530S300000
Reexamination Certificate
active
06329198
ABSTRACT:
The present invention relates to human nm23 protein, DNA encoding human nm23 proteins, (or fragments or analogues of such DNA), antibodies which recognize human nm23 protein and processes and products for producing and using such materials.
Steeg et al., Journal of the National Cancer Institute 80:200-204, 1988 discloses a murine, nm23 gene, and corresponding protein which is associated with murine tumor metastatic potential.
Applicant has provided a human gene(s) encoding for a human nm23 protein(s), as well as the protein(s) and antibodies which car be used as an aid in predicting the aggressiveness of human tumors.
More specifically, the present invention relates to genetic testing for cancer susceptibility, diagnosis and prognosis. The present invention makes use of the marker of the nm23 genes, for which the human pnm23-H1 and pnm23-H2S and murine 23 and pnm23-1 recombinant cDNA clones have been described (3, 21-22). The genetic marker itself can be a whole gene, a fragment thereof, a genomic or cDNA clone, an adjacent region, or a regulatory region thereof. The purpose of this invention is to provide novel genetic methods for the detection of (a) susceptibility to cancer and (b) cancer tumorgenic and metastatic potential.
These methods are based on (a) structural and sequential evaluation of nm23 DNA and; (b) evaluation of novel nm23 expression patterns, either at the RNA, mRNA and/or protein levels. Such information is critical to the physician's selection of diagnostic and therapeutic regimens for the patient, both prior to the development of cancer and during cancer detection and treatment.
Therefore, in accordance with one aspect of the present invention, there is provided DNA, or a fragment, analogue or derivative of such DNA, which encodes a human nm23 protein.
In accordance with another aspect of the present invention, there is provided a cloning or expression vehicle which includes DNA, or a fragment, analogue or derivative of such DNA, which encodes a human nm23 protein.
In accordance with a further aspect of the present invention, there is provided a host; in particular cells, genetically engineered to include DNA, or a fragment or analogue or derivative of such DNA, which encodes a human nm23 protein or a fragment or analogue or derivative of of such DNA; i.e., such cells are modified to include human nm23 DNA.
In accordance with yet another aspect of the present invention, there is provided a human nm23 protein.
In accordance with yet a further aspect of the present invention, there is provided antibodies which recognize human nm23 protein.
In accordance with still a further aspect of the present invention, there is provided procedures for using the aforementioned DNA and antibodies for predicting the metastasic potential of tumors.
The term “human nm23 gene or DNA” as used herein means a gene or DNA which encodes a human nm23 protein, or an analogue or derivative or fragment of such DNA or gene which encompasses or includes a DNA sequence unique to DNA which encodes a human nm23 protein. Thus, the term human nm23 gene encompasses the genes or DNAs of Table 1 or fragments or derivatives or analogues of such genes.
“Human nm23 protein” means nm23 protein found in humans, or a fragment, analogue or derivative thereof which encompasses or includes an amino acid sequence which is unique to human nm23 protein and which preferably elicits an antibody which is recognized by human nm23 protein. The term human nm23 protein encompasses the proteins encoded by the genes of Table 1.
The term “antibody” as used herein encompasses polyclonal and monoclonal antibodies.
The term “nm23 antibody” means an antibody which is elicited in response to human nm23 protein or which recognizes human nm23 protein. An antibody which recognizes human nm23 protein may or may not be elicited in response to human nm23 protein.
Applicant has presently found two different and distinct human genes (DNA) which encode for two different and distinct nm23 proteins. The first gene is referred to herein as nm23-H1. The second gene is referred to herein as nm23-H2S. The gene sequences for both are shown in Table 1.
Although Applicant has presently identified two distinct human genes encoding for two different nm23 proteins, the scope of the present invention is not limited to such specific genes.
Human nm23 DNA (RNA) can be used as a diagnostic tool for detecting and/or determining RNA or DNA. For example, such DNA or RNA may be employed to detect mRNA expression in cancer cells to thereby aid in predicting the malignant potential of a human tumor. The methods which may be used include:
(1) RNA (“Northern”) blotting. RNA can be isolated from tumor samples by any of a number of standard procedures. For example, refinements of the method of Lehrach (1) can be used. RNA is subjected to denaturing gel electrophoresis and transferred to nitrocellulose or other support matrix. The nm23 mRNA can be detected by hybridization of radioactively or non-radioactively labelled nm23-H1 or nm23-H2S. mRNA in the tumor will be reflected by the intensity of hybridization. For comparison, hybridization with control probes for mRNA whose level is constant (e.g. B-actin) allows normalization of results. Detection of low levels of nm23-H1 or nm23-H2S indicates a tumor of high malignant potential.
(2) Nuclease Protection assays. RNA isolated from tumor samples can be analyzed for the content of nm23-H1 or nm23-H2s by its ability for duplexes with a labelled complementary DNA or RNA. Using the whole or part of the nm23-H1 or nm23-H2S nucleotide sequence, plasmids can be generated for the production of nucleic acid probes complementary to the corresponding mRNA. Examples of such vectors are those based on the T7 or SP6 promoter for RNA probes or m13 phage for preparation of DNA probes using oligonucleotide priming. Probes prepared from such vectors will be allowed to hybridize to completion to RNA from tumor samples under conditions of excess of probe. Either RNase can be used to remove molar unhybridized RNA probes or S1 nuclease, or other single-stranded specific DNase, can be used to remove unhybridized DNA probe. These are then subject to denaturing gel electrophoresis and autoradiography. The intensity of bands corresponding to protected probe is a measure of the amount of either nm23-H1 or nm23-H2S from the sample. Inclusion of nuclease protection experiments for mRNAs whose levels do not change will allow normalization of results. Detection of tumors with relatively low levels of nm23-H1 or nm23-H2S indicates tumors of high malignant potential.
(2) In situ hybridization of nm23-H1 or nm23-H2S in tumor sections allow analysis of the quantity of nm23-H1 or H2S mRNA in individual cells of a tumor. Probe complementary to the nm23-H1 or H2S sequence can be prepared as described above and allowed to hybridize to mRNA within thin section of tumor sample (either embedded by standard techniques such as by the use of paraffin, or otherwise preserved). Unhybridized probe can be removed by nuclease. Hybridization can be detected by autoradiography or other methods. The intensity of hybridization reflects the amount of nm23-H1 or H2S mRNA within the cells of the tumor. When tumor cells contain low levels of nm23 they are likely to be highly malignant.
Susceptibility to early onset, familial breast cancer or other cancers could be detected by several methods, including analysis of the inheritance pattern of allelic fragments of a nm23 gene. Inheritance of an allele associated with the development of breast cancer in a patient's family would signify high risk for eventual cancer development. A second method to determine cancer susceptibility is to determine the DNA sequence of an nm23 gene, or its regulatory sequences, in DNA extracted from the patient's normal tissue. The presence of a mutation in the nm23 gene which would alter its amino acid sequence from the normal sequence would signify high risk of cancer development. Other mutations occuring in the regulatory DNA regions for nm23, or in intron regions responsibl
King Charles Richter
Liotta Lance A.
Steeg Patricia Schriver
Campbell Eggerton A.
Needle & Rosenberg P.C.
The United States of America as represented by the Department of
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