Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-02
2001-11-27
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100
Reexamination Certificate
active
06322982
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of the invention is modulation of drug resistance.
The concentrations of proteins in biological cells are regulated by elegant biochemical mechanisms. By way of these mechanisms, cells eliminate damaged proteins and can, by altering the concentrations of biologically active proteins, such as enzymes, alter cellular processes that are important for the overall well being of the organism.
In eukaryotic cells, proteins can be selectively degraded via the ubiquitin pathway. Ubiquitin is a highly conserved protein that is covalently ligated to proteins in a process referred to as ubiquitination. Proteins that have been ubiquitinated are committed to degradation by a 26S protease complex. The ubiquitin pathway is thought to play an important role in regulating cellular processes by regulating protein levels. Numerous review articles have described various aspects of the ubiquitin pathway. For example, the molecular genetics of the ubiquitin system have been reviewed by Finley et al. (
Ann. Rev. Cell Biol.
7:25-69, 1991) and Jentsch et al. (
Biochim. Biophys. Acta
1089:127-139, 1991); the involvement of the system in pathological states has been reviewed by Mayer et al. (
Biochim. Biophys. Acta
1089:141-157, 1991); and the biochemistry and enzymology of various stages of the ubiquitin pathway have been reviewed by Hershko and Ciechanover (
Ann. Rev. Biochem.
61:761-807, 1992). Interest in the ubiquitin pathway is due in part to the wide variety of physiological processes that are affected by ubiquitination of proteins. These processes include the heat shock response, DNA repair, cell cycle progression, the modification of histones and of receptors, and the possible pathogenesis of selected neurodegenerative diseases.
Only the protein conjugated to ubiquitin is degraded via the proteasome; ubiquitin itself is recycled by ubiquitin carboxy-terminal hydrolases, which cleave the bond between ubiquitin and the protein targeted for degradation. These enzymes constitute a family of thiol proteases, and homologues have been found in, for example, yeast (Miller et al.,
BioTechnology
7:698-704, 1989; Tobias and Varshavsky,
J. Biol. Chem.
266:12021-12028, 1991; Baker et al.,
J. Biol. Chem.
267:23364-23375, 1992), bovine (Papa and Hochstrasser,
Nature
366:313-319, 1993), avian (Woo et al.,
J. Biol. Chem.
270:18766-18773, 1995), Drosophila (Zhang et al.,
Dev. Biol.
17:214, 1993) and human (Wilkinson et al.,
Science
246:670, 1989) cells.
SUMMARY OF THE INVENTION
As described herein for the first time, the expression of ubiquitin carboxy-terminal hydrolase (UCH; sometimes abbreviated UCTH) is aberrent in cells that are resistant to treatment with chemical agents, for example, pharmaceutical agents. Accordingly, the invention features methods for diagnosing and treating drug resistant cells (e.g., tumor cells) by examining and modulating the expression or activity of UCH. The cells can be resistant to one or more pharmaceutical agents, and so may, as appropriate, be referred to as drug resistant or multidrug resistant cells.
The present invention provides a method of assessing expression, especially aberrant expression, of a cellular UCH gene, which may indicate the presence, persistence, or reappearance of tumor cells in an individual's tissue. UCH gene expression is assessed by obtaining a sample of cellular tissue from a mammal (e.g., a human), preferably from a site in the body suspected of containing malignant tissue, and obtaining RNA from the tissue. The RNA is combined with a UCH oligonucleotide under standard conditions for hybridization, and the resulting mixture is assayed for the presence of hybrids consisting of the UCH oligonucleotide and a cellular UCH gene transcript. As further described below, the oligonucleotide can be detectably labeled or otherwise modified. For example, the oligonucleotide can have a peptide-nucleic acid backbone. The presence and/or relative abundance of hybrids indicates aberrant expression of a cellular UCH gene, and correlates with the occurrence of transformed cells in situ, especially transformed cells having a drug-resistant phenotype.
Information obtained by practicing the diagnostic methods described herein will be useful in determining the prognosis for patients with malignancies (tumors) that are characterized by expression of UCH and thus by a drug-resistance phenotype. The information will also assist the clinician in designing chemotherapeutic or other treatment regimes to eradicate such malignancies from the body. The methods described herein can be practiced with a sample of any body tissue type, such as that from the mammary, respiratory, urogenital, endocrine, or immune systems. The present methods are particularly useful in assessing biopsy tissue obtained from the breast, respiratory system (e.g., by bronchoalveolar lavage), ovary, uterus, cervix, prostate, testes, pancreas, spleen, bone marrow, or lymphatic system.
In addition, the invention features therapeutic compositions, such as those containing a compound that modulates expression of UCH protein, and therapeutic methods in which these compounds are administered to a patient. Accordingly, the invention provides means for mitigating (detectably decreasing or otherwise affecting) aberrant expression of a UCH gene or protein, and for attenuating an undesirable drug-resistance phenotype, particularly a phenotype contributed by UCH. More particularly, the invention features methods for mitigating aberrant expression of a UCH gene, and/or aberrant activation or alteration of a UCH polypeptide. One embodiment involves the administration of a pharmaceutical composition containing an antisense UCH oligonucleotide to a mammal suffering from the effects of altered expression and/or function of UCH. Another embodiment involves the administration of an antibody or fusion polypeptide that specifically binds UCH. In either embodiment, the therapeutic agent is administered systemically or locally under conditions sufficient to mitigate or attenuate the phenotype associated with aberrant expression or activity of UCH (including undesirably high or undesirably low levels of UCH expression or activity). Preferably, the therapeutic agent is administered under conditions sufficient to destroy cells producing aberrant levels or UCH or cells in which the activity of UCH is higher than that in a comparably cell. For example, either of the foregoing therapeutic agents can be administered as an adjunct to conventional chemotherapy. That is, either of the foregoing therapeutic agents can be coadministered together with one or more chemotherapeutic drugs. In this manner, the invention provides for destruction of drug-resistant tumor cells in situ. The present antisense or fusion polypeptide therapeutic agent can be administered prior to, concomitant with, or following administration of one or more chemotherapeutic drugs. In such embodiments, the antisense pharmaceutical composition mitigates resistance of UCH-expressing cells to the cytotoxic effects of the chemotherapeutic drug. That is, the antisense composition attenuates the phenotype associated with an aberrant level of UCH expression or activity, which is characterized by the property of drug resistance. This is accomplished by disrupting activation or transcription of the UCH gene, or by destabilizing RNA transcripts thereof. Diminished or discontinued expression of UCH renders cells more susceptible to the cytotoxic effects of a chemotherapeutic drug. Similarly, a therapeutically administered cytotoxic fusion polypeptide localized in the vicinity of cells aberrantly expressing UCH can produce cytolysis thereof. Alternatively (or in addition), a chemoattractant fusion polypeptide localized to UCH-expressing cells can stimulate their destruction by macrophages, killer T cells, or cytotoxic T cells.
Another aspect of the invention features methods for identifying a modulator of UCH. In general, this aspect of the invention relies on the use of a UCH-expressing host cell. Prokaryotic or eukaryotic
MacBeth Kyle J.
Shyjan Andrew W.
Fish & Richardson P.C.
McKelvey Terry
Millennium Pharmaceuticals Inc.
Sandals William
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