Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-06-16
2004-10-05
Falk, Anne-Marie (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S325000
Reexamination Certificate
active
06800483
ABSTRACT:
INTRODUCTION
1. Field of the Invention
This invention relates to novel polynucleotides identified and sequenced which encode a carboxylesterase enzyme, polypeptides encoded by these polynucleotides and vectors and host cells comprising these vectors which express the enzyme. This enzyme is capable of metabolizing chemotherapeutic prodrugs and inactive metabolites into active drug. The instant invention thus relates to compositions comprising these polynucleotides and methods for sensitizing selected tumor cells to a chemotherapeutic prodrug by transfecting the tumor cells with a polynucleotide placed under the control of a disease-specific responsive promoter. Sensitized tumor cells can then be contacted with a chemotherapeutic prodrug to inhibit tumor cell growth. Compositions of the present invention can also be used in combination with chemotherapeutic prodrugs to purge bone marrow of tumor cells. The invention further includes novel drug screening assays for identifying chemotherapeutic prodrugs that are activated by this enzyme.
2. Background of the Invention
Cancer is a disease resulting from multiple changes at the genomic level. These changes ultimately lead to the malfunction of cell cycle machinery and finally to autonomous cell proliferation. Neoplastic transformation involves four types of genes: oncogenes, tumor-suppressor genes, mutator genes, and apoptotic genes. Different types of cancer can involve alteration of any one or any combination of these genes.
Proto-oncogenes of the myc family are overexpressed in many different types of human tumors including tumors of the breast, colon, cervix, head and neck, and brain. Many solid tumors amplify or overexpress c-myc, with up to a 50-fold increase in c-myc RNA in tumor cells relative to normal cells having been reported (Yamada, H. et al. 1986
. Jpn. J. Cancer Res
. 77:370-375). For example, three of the six most common solid tumors, including up to 100% of colon adenocarcinomas, 57% of breast cancers, and 35% of cervical cancers, demonstrate increased levels of c-myc protein. Enforced expression of c-myc in nontumorigenic cells causes immortalization but not transformation; however, elevated levels of c-myc protein are rare in benign cancers and normal differentiated tissue. While solid tumors can oftentimes be removed surgically, overexpression of c-myc has been linked with amplification of the c-myc gene and correlated with poor prognosis and an increased risk of relapse (Nagai, M. A. et al. 1992
. Dis. Colon Rectum
35:444-451; Orian, J. M. et al. 1992
. Br. J. Cancer
66:106-112; Riou, G. et al. 1987
. Lancet
2:761-763; Field, J. K. et al. 1989
. Oncogene
4:1463-1468).
Another member of the myc oncogene family, N-myc, has been linked with development of neuroblastomas in young children. Overexpression of this member of the myc family of proto-oncogenes has also been correlated with advanced stages of disease and poor prognosis (Brodeur, G. M. et al. 1997
. J. Ped. Hematol. Oncol
. 19:93-101). Primary tumors for this specific condition usually arise in the abdomen and as many as 70% of patients have bone marrow metastases at diagnosis (Matthay, K. E. 1997
. Oncology
11:1857-1875). Treatment of children with Stage 4 disease using surgery, chemotherapy, and purged autologous or allogeneic marrow transplant produces a progression-free survival rate of 25 to 49% in patients four years post transplant (Matthay, K. K. et al. 1994
. J. Clin. Oncol
. 12:2382-2389). Most relapses after autotransplant occur at sites of bulk disease and/or previously involved sites. Estimates of the rate of local recurrence vary depending upon the study. However, recurrence of tumor at an original site has been estimated to occur in approximately 25% of high risk neuroblastoma patients.
Further, definitive evidence from gene marking studies indicates that autologous marrow, free of malignant cells by standard clinical and morphologic criteria, contributes to relapse at both medullary and extramedullary sites (Rill, D. R. et al. 1994
. Blood
84:380-383). In a recent pilot clinical study, bone marrow involvement at diagnosis correlated with specific relapse at that site in children receiving autologous purged marrow (Matthay, K. K. et al. 1993
. J. Clin. Oncol
. 11:2226-2233). Accordingly, improvements in surgery, detection of tumor margins, development of new anticancer drugs or application of novel therapies are required to prevent local tumor regrowth. In particular, more effective treatment strategies are needed for elimination of “minimal residual disease” or “MRD” which results from the presence of a small number of tumor cells at the site of disease after treatments such as tumor resection or purging bone marrow of tumor cells.
CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is a prodrug currently under investigation for the treatment of cancer that is converted to the active drug known as SN-38 (7-ethyl-10-hydroxy-camptothecin) (Tsuji, T. et al. 1991
. J. Pharmacobiol. Dynamics
14:341-349; Satoh, T. et al. 1994
. Biol. Pharm. Bull
. 17:662-664). SN-38 is a potent inhibitor of topoisomerase I (Tanizawa, A. et al. 1994
. J. Natl. Cancer Inst
. 86:836-842; Kawato, Y. et al. 1991
. Cancer Res
. 51:4187-4194), an enzyme whose inhibition in cells can result in DNA damage and induction of apoptosis (Hsiang, Y. -H. et al. 1989
. Cancer Res
. 49:5077-5082). The specific enzyme responsible for activation in vivo of CPT-11 has not been identified, although serum or liver homogenates from several mammalian species have been shown to contain activities that convert CPT-11 to SN-38 (Tsuji, T. et al. 1991
. J. Pharmacobiol. Dynamics
14:341-349; Senter, P. D. et al. 1996
. Cancer Res
. 56:1471-1474; Satoh, T. et al. 1994
. Biol. Pharm. Bull
. 17:662-664). Uniformly, these activities have characteristics of carboxylesterase (CE) enzymes (Tsuji, T. et al. 1991
. J. Pharmacobiol. Dynamics
14:341-349; Senter, P. D. et al. 1996
. Cancer Res
. 56:1471-1474; Satoh, T. et al. 1994
. Biol. Pharm. Bull
. 17:662-664). In fact, SN-38 can be detected in the plasma of animals and humans minutes after the administration of CPT-11 (Stewart, C. F. et al. 1997
. Cancer Chemother. Pharmacol
. 40:259-265; Kaneda, N. et al. 1990
. Cancer Res
. 50:1715-1720; Rowinsky, E. K. et al. 1994
. Cancer Res
. 54:427-436), suggesting that a CE enzyme present in either serum or tissues can convert the camptothecin analog to its active metabolite.
CEs are ubiquitous serine esterase enzymes that are thought to be involved in the detoxification of a variety of xenobiotics. CEs are primarily present in liver and serum, however, the physiological role of this class of enzymes has yet to be identified. A recent biochemical analysis of 13 CEs compared their ability to metabolize CPT-11 to SN-38. While the efficiency of conversion varied between enzymes, those isolated from rodents were the most efficient (Satoh, T. et al. 1994
. Biol. Pharm. Bull
. 17:662-664). The amino acid sequence of a rabbit liver CE has been disclosed (Korza, G. and J. Ozols. 1988
. J. Biol. Chem
. 263:3486-3495). In addition, there are currently 13 CDNA sequences encoding CE in the GenBank and EMBL databases, including a rat serum and rat liver microsomal CE. Interestingly, CEs purified from human tissues demonstrated the least efficient conversion of CPT-11 to SN-38, with less than 5% of the prodrug being converted to active drug (Leinweber, F. J. 1987
. Drug Metab. Rev
. 18:379-439; Rivory, L. P. et al. 1997
. Clin. Cancer Res
. 3:1261-1266).
In addition to metabolism to SN-38, in humans CPT-11 is also metabolized to a compound known as APC (Haaz, M. C. et al. 1998
. Cancer Res
. 58:468-472). APC has little, if any, anti-tumor activity and is not converted to an active metabolite in humans (Rivory, L. P. et al. 1996
. Cancer Res
. 56:3689-3694).
In preclinical studies, CPT-11 administered to immune-deprived mice bearing human tumor xenografts produces complete regression of glioblastomas, rhabdomyosarcomas (RMS), neuroblastomas, and colon adenocarcinomas (
Danks Mary K.
Houghton Peter J.
Potter Philip M.
Falk Anne-Marie
Licata & Tyrrell P.C.
Qian Celine
St. Jude Children's Research Hospital
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