Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
1999-09-29
2003-07-29
Goldberg, Jerome D (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C424S093210
Reexamination Certificate
active
06599909
ABSTRACT:
BACKGROUND OF THE INVENTION
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.
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 an 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, which was 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 b e 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.
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 radiation therapy, (e.g., external beam or brachytherapy) of the tumor. This method results in tumor regression and prolonged tumor growth inhibition compared to control treatments with molecular chemotherapy or radiation therapy alone. Also disclosed is an adenoviral-conjugate mechanism to circumvent current limitations of cancer gene therapy to solid gastrointestinal malignancies.
Specifically, the present invention utilizes an adenoviral vector under the control of a cytomegalovirus promoter (AdCMVCD) encoding cytosine deaminase in combination with 5-FC and single fraction radiotherapy to demonstrate enhanced cytotoxicity to WiDr human colon carcinoma cells in vitro. The present invention also demonstrates such gene therapy/prodrug treatment strategy employing a fractionated radiation dosing schema in animal models of WiDr human colon carcinoma and SK-ChA-1 human cholangiocarcinoma. A prolonged WiDr tumor regrowth delay was obtained with AdCMVCD infection in combination with systemic delivery of 5-FC and fractionated external beam radiation therapy compared to control animals treated without radiation, without 5-FC, or without AdCMVCD. The present invention further discloses redirection of adenovirus vector (AdCMVCD) binding via a ligand to a cellular receptor, e.g., the basic fibroblast growth factor (FGF2) receptor, to improve the effectiveness of gene therapy in combination with 5-FC treatment and radiation therapy.
Clinical applications for cancer gene therapy are limited by the inability to genetically modify a majority of tumor cells to achieve a therapeutic effect. In this regard, the enzyme/prodrug strategy consisting of cytosine deaminase/5-fluorocytosine (CD/5-FC) relies on diffusion of the cytotoxic enzymatic product 5-FU to kill non-transduced tumor cells. Methods to increase solid tumor transduction in situ may augment therapeutic gene expression and response to therapy. To this end, gene delivery was improved via vector binding to molecules expressed on the cells of tumors. Fibroblast growth factor (FGF) receptors are overexpressed in a majority of pancreatic carcinomas, but poorly characterized in cholangiocarcinoma. Targeted adenovirus via basic fibroblast growth factor (FGF2) to the fibroblast growth factor receptor was used as a vehicle for the delivery of cytosine deaminase to hepatobiliary tumor cells for combination molecular chemotherapy and radiation therapy studies.
FGF2 redirected adenoviral delivery of firefly luciferase gene (AdCMVLuc) expression was evaluated in vitro. Transduction efficiencies using adenoviral delivered
E. coli
&bgr;-galactosidase gene (AdCMVLacZ) expression also was determined. The methodology to redirect adenoviral gene delivery employed the Fab fragment of a neutralizing anti-adenoviral knob monoclonal antibody which ablates native adenoviral tropi
Buchsbaum Donald J.
Curiel David T.
Pederson Lee C.
Stackhouse Murray A.
Adler Benjamin Aaron
Goldberg Jerome D
UAB Research Foundation
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