Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-11-03
2003-04-22
Reamer, James H. (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S093200, C435S069100, C435S455000, C435S320100
Reexamination Certificate
active
06552005
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular biology, radiation oncology and cancer therapy. More specifically, the present invention relates to the finding that a combination of molecular chemotherapy and radiation therapy enhances therapeutic effects against cancer.
2. Description of the Related Art
Clinical applications of cancer gene therapy have had limited success due to a variety of factors, including ineffective therapeutic gene delivery in situ. The physiologic milieu of the target tumor may have deleterious effects on the delivery of therapeutic genes. This limitation may be disease specific, and variable depending on the specific tumor type and tumor location. Most clinical gene therapy trials thus far have utilized compartmental models of malignant disease (1, 2). In this regard, thoracic malignancies and intra-abdominal carcinomatosis represent common body compartmentalized diseases that have been explored in a n experimental therapeutic context. Attempts to address the issue of achieving viral vector delivery to cancer cells in the face of a physiologic infection medium of pleural fluid or abdominal ascites have been examined (3, 4). Yang et al. demonstrated retroviral transduction of pancreatic cancer cells in the presence of human ascites, similar to the results obtained in culture medium (3). Batra et al. reported significant inhibition of retroviral transduction of mesothelioma cells in the presence of malignant pleural fluid, specifically the chondroitin sulfate proteoglycan fraction (4).
Radiotherapy combined with the radiosensitizing chemotherapeutic drug 5-fluorouracil (5-FU) has been studied as a therapeutic modality in many human tumor types (5). Systemic toxicity limits the amount of 5-FU that can be administered for many clinical anti-cancer applications (6, 7). Radiation therapy and gene therapy have the potential to be combined to enhance effectiveness of cancer therapy without enhancing dose limiting toxicity. To this end, reports have investigated this interaction (8). These include: TNF&agr; under the control of a radiation inducible promoter (9, 10), conversion of prodrugs to toxic metabolites that are also radiosensitizers (11-15), p53 mediated radiosensitization (16, 17) and the genetic induction of membrane receptors that can be targeted with radiolabeled peptides (18-21).
With respect to enzymatic conversion of nontoxic prodrugs into radiation sensitizing agents, the genes for bacterial and yeast cytosine deaminase (CD) have been cloned and studied (22, 23, 40). Cytosine deaminase converts a nontoxic prodrug 5-fluorocytosine (5-FC) into 5-FU. The cytosine deaminase gene has been used in gene therapy strategies to mediate intracellular conversion of 5-FC to 5-FU, and has been shown to be effective in animal tumor models of human colon carcinoma (24). Human colon cancer cells that have been stably transduced to express the cytosine deaminase gene have been shown to be radiosensitized by the addition of 5-FC in vitro and in vivo (13). Adenoviral vectors have been used to achieve efficient gene delivery in a variety of tissues in vitro and in vivo. Adenoviral vectors encoding the cytosine deaminase gene have been described (25, 26).
Presently available assays for determining intratumoral 5-FU concentration are problematic. They require the removal of a tumor, the homogenization of that tumor and the collection of the cellular lysate in order to directly measure 5-FU concentration, usually by high-pressure liquid chromatography. No noninvasive method of detection existed, which could allow for continuous in vivo monitoring of 5-FU production.
In the context of multiple administrations of adenoviral vectors, the host immunologic response, with generation of neutralizing anti-adenovirus antibodies and cytotoxic T cells, is thought to limit the potential effectiveness of secondary administration of adenoviral vectors. A means to overcome this problem may be to improve the effectiveness of infection of the initial viral challenge, i.e., to enhance the transduction efficiency of the adenoviral vector for the target cells at the initial adenoviral administration. This goal may be achieved by utilizing a ligand to a cellular receptor overexpressed in the target carcinoma cells to redirect adenovirus vector binding.
Primary central nervous system (CNS) tumors, arising in both the brain and spinal cord, are the leading cause of cancer-related deaths in children less than 15 years of age (42-44). They are the most common solid neoplasia in children with an estimated incidence of 3.77 newly diagnosed pediatric patients per 100,000 children at risk each year in the US (45). Despite aggressive treatment with radiation and/or chemotherapy, children with intrinsic brainstem gliomas and high-grade astrocytomas rarely survive more than a few years from diagnosis (46-49). The long-term sequelae of radiation are significant, especially in very young children, militating against its use as standard therapy in children less than 36 months old (50-52). In this context, gene therapy offers a promising approach for pediatric brain tumors.
The main factor currently limiting the clinical potential of gene therapy is the poor level of in situ tumor cell transduction achievable by existing gene transfer vectors (53). Of these, adenovirus (Ad) is particularly attractive due to its well-characterized mechanism of cellular entry, and its propensity to efficiently infect a wide variety of cell types within the CNS (54-56). This is presumably due to their expression of the cellular receptors necessary for efficient Ad entry, the coxsackie-adenovirus receptor (CAR) and av integrins (57, 58). Ad vectors have shown utility in several animal models of glioma (59, 60) and are currently being investigated in at least three separate clinical trials in the US in adult patients with malignant glioblastomas. Yet as all three employ direct intratumoral or intracavitary injection, expression of Ad receptors on these tumors will likely determine the overall success of these and future Ad cancer gene therapy trials. Two of these trials are investigating replication-defective Ad as a vector for enzyme/prodrug therapy using herpes simplex virus thymidine kinase/gancyclovir (HSV-tk/GCV, 61). While HSV-tk/GCV enzyme/prodrug therapy is promising for malignant gliomas, several alternatives have been described (reviewed in 62).
The third trial involves a replication-competent Ad lacking an exogenous transgene (ONYX-015) (76) and is being conducted in adult patients. ONYX-015 harbors an E1B-55K gene deletion that permits the selective replication in and lysis of cells with mutations in the gene encoding p
53
(77). Replication-competent viruses such as Ad have distinct advantages over non-replicative viruses in cancer gene therapy (reviewed in 78, 79). First, Ad replication in tumor cells results in cell lysis (lytic infection) and hence tumor destruction (viral oncolysis). Second, lateral spread of progeny Ad virions within the productively infected tumor mass dramatically increases exogenous transgene expression compared with replication-defective vectors (80).
The prior art is deficient in the lack of effective means of treating of human cancers by chemotherapy combined with radiation therapy to produce enhanced therapeutic effects against cancer and reduced normal tissue toxicity. In addition, the prior art is deficient in the lack of effective means of redirecting adenovirus vector binding via a cellular receptor to improve the effectiveness of gene therapy. Furthermore, the prior art is deficient in the lack of a noninvasive method for continuously monitoring therapeutic transgene expression in tumors therefore improving the gene therapy. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention is directed to a method of transfecting established tumors in vivo with an adenovirus encoding the cytosine deaminase gene, administration of systemic 5-FC, and
Buchsbaum Donald J.
Garver, Jr. Robert J.
Gillespie G. Yancey
Miller C. Ryan
Adler Benjamin Aaron
Reamer James H.
UAB Research Foundation
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