`
`PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 6 :
`
`(11) International Publication Number:
`
`WO 97/12623
`
`A61K 35/76, 48/00, 41/00, C12Q U70 ll
`(A61K 35/76, 31:40)
`
`(43) International Publication Date:
`
`10 April 1997 (10.04.97)
`
`(21) International Application Number:
`
`PCT/US96/ 16047
`
`(22) International Filing Date:
`
`7 October 1996 (07.10.96)
`
`(30) Priority Data:
`08/540,343
`
`6 October 1995 (06.10.95)
`
`US
`
`ARCH DEVELOPMENT CORPORATION
`(71) Applicants:
`[US/US]; 1101 East 58th Street, Chicago. IL 60637 (US).
`DANA—FARBER CANCER INSTITUTE [US/US];
`44
`Binney Street, Boston, MA 02115 (US).
`
`(72) Inventors: HALLAHAN, Dennis, E.; 231 N. Elmore, Park
`Ridge. IL 60068 (US). WEICHSELBAUM, Ralph, R.; 2031
`N. Sedgwick, Chicago,
`IL 60614 (US). KUFE, Donald;
`179 Grove Street, Wellesley, MA 02181 (US). SIBLEY,
`Gregory, S.; 102 Weymouth Place, Chapel Hill, NC (US).
`ROlZMAN, Bemard; 5555 South Everett Avenue, Chicago,
`IL 60637 (US).
`
`(74) Agent: SERTICH, Gary, 1.; Arnold, White & Durkee, P.O. Box
`4-433, Houston, TX 77210 (US).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE,
`HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS,
`LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL,
`PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, "IT, UA,
`UG, UZ, VN, ARIPO patent (KE, LS, MW, SD, 52, UG),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, CH, DE. DK, ES, FI, FR, GB,
`GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ,
`CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`radiation, the virus is an adenovirus. and the increase in cell killing is synergistic when compared to radiation alone.
`
`(54) Title: METHODS AND COMPOSITIONS FOR VIRAL ENHANCEMENT OF CELL KILLING
`
`(57) Abstract
`
`The present invention is directed to novel methods of enhancing the effectiveness of DNA damaging agents by exposing cells to
`viruses prior to or during exposure to the damaging agent. In certain embodiments of the invention, the DNA damaging agent is ionizing
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`
`Viet Nam
`
`United Kingdom
`Georgia
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Kenya
`Kyrgystan
`Democratic People‘s Republic
`of Korea
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Liberia
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`Mali
`Mongolia
`Mauritania
`
`Armenia
`Austria
`Ausu'alia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`COte d'lvoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Germany
`Denmark
`Estonia
`Spain
`Finland
`France
`Gabon
`
`

`

`
`
`W0 97/l 2623
`
`PCT/US96/l 6047
`
`DESCRIPTION
`
`METHODS AND COMPQSITIQNS FOR VIRAL
`
`ENHANCENIENT OF CELL KILLING
`
`BACKGROUND OF THE D‘IVENTION
`
`1.
`
`Field of the Invention
`
`The present invention relates generally to the fields of cell and tumor killing
`
`utilizing DNA damaging agents. More particularly, it concerns the use of selected
`
`viruses to enhance the effects of ionizing radiation and other DNA damaging agents to
`
`kill cells and potentiate the therapeutic effect of these modalities.
`
`10
`
`2.
`
`Description of the Related Art
`
`Recently, there has been a renewed interest in the potential use of cytolytic
`
`viruses in the treatment of cancer (Lorence et al., 1994; Mineta er al. , 1994; Kenney et
`
`al., 1994). The rationale for such an approach stems from case reports in the clinical
`
`literature describing tumor regression in human cancer patients during virus infection
`
`(Cassel et al., 1992). In one clinical trial, regression of tumors occurred in cancer
`
`patients treated with a wild-type mumps virus (Cassel et al., 1992). In another report,
`
`complete remission occurred in a chicken farmer with widely metastatic gastric cancer
`
`during a severe outbreak of Newcastle disease (NDV) within the chicken population
`
`(Csatary, 1992).
`
`Biomedical investigation has focused on the utilization of viruses as either
`
`direct therapeutics or for gene therapy, including the experimental therapy of brain
`
`tumors. For the experimental treatment of malignant gliomas, two approaches have
`
`predominated (Daumas-Duport et al., 1988; Kim et a1. , 1991; Culver et a1. , 1992;
`
`Ram et al., 1993(a); Ram et al., 1993(b); Ram et al., 1994) (Takamiya et al., 1992;
`
`Martuza et a1. , 1991; Martuza et a1. , 1991). The first involves deliberate in situ
`
`inoculation of cells infected with a retrovirus (producer cells) expressing the herpes
`
`15
`
`20
`
`25
`
`

`

`W0 97/ 12623
`
`PCT/U896“ 6047
`
`simplex virus 1 (HSV—l) thymidine kinase gene into the tumor mass followed by
`
`treatment with ganciclovir (GCV), an antiviral drug (Culver et al., 1992). The
`
`retrovirus is secreted from the producer cells and infects the tumor cells. GCV is
`
`selectively phosphorylated by the HSV-l thymidine kinase to its mono—phosphate
`
`derivative and by cellular enzymes to a triphosphate derivative, which kills the tumor
`
`cells. Limitations of this approach include the quantity of nondividing cells that can
`
`be inoculated directly into the brain tumor, the relatively low yield of retroviruses, and
`
`the requirement for administration of GCV, a drug that has significant hematopoietic
`
`toxicity and does not penetrate the central nervous system to a great extent.
`
`An alternative approach utilizes genetically engineered HSV. Among the
`
`mutants tested for this purpose were viruses lacking the thymidine kinase or
`
`ribonucleotide reductase gene or a genetically engineered virus lacking the 734.5 gene
`
`(Markert et al., 1993). Although some of the viruses tested to date prolonged the
`
`survival of tumor-bearing animals. none totally destroyed the tumor mass. Some of
`
`the deletion mutants tested. notably those that are thymidine kinase-negative. are
`
`potentially hazardous, since such viruses can cause encephalitis in animal models and
`
`are not treatable by drugs that depend on the viral thymidine kinase for their activity
`
`(Erlich et al., 1989). The interest in testing of 734.5- viruses stems from studies on
`
`the function of the 734.5 gene and the phenotype of these viruses carrying deletions
`
`and substitutions in that gene. The 734.5 gene maps in the sequences flanking the long
`
`unique sequence and is present in two copies in the viral genome (Chou et al., 1990;
`
`Ackermann et al., 1986; Chou er al., 1986). Mutants lacking both y34.5 genes (e.g.,
`
`recombinant R3616) are apathogenic and fail to replicate in the central nervous
`
`system of mice (30). In cell culture, particularly in human fibroblasts and in the
`
`SK-N-SH human neuroblastoma cells, R3616 fails to prevent a stress response
`
`induced by the onset of viral DNA synthesis (Chou et a1. , 1992). In consequence,
`
`protein synthesis is totally and prematurely shut off, resulting in cell death and
`
`significantly reduced viral yields. Although R3616 possesses many of the properties
`
`desired for cancer therapy, its effectiveness may be limited because its host range is
`
`10
`
`15
`
`20
`
`25
`
`30
`
`very restricted.
`
`

`

`
`
`W0 97/l 2623
`
`PCT/US96/l 6047
`
`While treatnent with viruses alone or with DNA damaging agents alone
`
`provide some relief measure of cell killing, the overall cell death rate is generally
`
`below that obtained utilizing other treatment modalities. One type of cancer that
`
`would benefit from an increased therapeutic potential is malignant glioma. These
`
`cancers are the most common primary intracranial malignant tumor, accounting for
`
`30% of primary brain tumors (Levin et a1. , 1989). The estimated tumor incidence in
`
`the United States is 14.7 per 100 thousand, resulting in 5000 new cases annually
`
`(Mahaley et al., 1989). In spite of aggressive surgical therapy, radiotherapy, and
`
`chemotherapy of patients with malignant gliomas, the overall 5-year survival is
`
`<5 5%, and the median survival is 52 weeks. This poor survival has remained
`
`virtually unchanged over the past 20 years (Levin er al., 1989; Mahaley et al., 1989;
`
`Salazar et al., 1979; Walker et al., 1980; Daumas-Duport et al., 1988; Kim et al.,
`
`1991). These abysmal survival rates have reinforced the need for new modalities of
`
`therapy. In view of such statistics, it would therefore be of great importance to
`
`develop methods of improving the therapeutic ability of current techniques of treating
`
`neoplastic disease.
`
`SUMMARY OF THE INVENTION
`
`The present invention, in a general and overall sense, concerns the use of
`
`viruses in combination with radiotherapy to potentiate the therapeutic effect. In
`
`particular, the inventors have discovered that certain viruses, for example adenovirus
`
`and herpes simplex virus, act in an additive manner in vitro or, surprisingly and
`
`unexpectedly, in a synergistic manner in vivo to enhance cell killing following
`
`exposure to ionizing radiation. In particular, tumor cell growth is controlled using the
`
`methods and compositions of the invention. As used herein, tumor cell formation and
`
`growth describes the formation and proliferation of cells that have lost the ability to
`
`control cellular division, thus forming cancerous cells. Using the methods of the
`
`invention, a number of different types of transformed cells are potential targets for
`
`control, such as carcinomas, sarcomas, melanomas, gliomas, lymphomas, and a wide
`
`10
`
`15
`
`20
`
`25
`
`

`

`W0 97/] 2623
`
`PCT/US96/l 6947
`
`variety of solid tumors. While any tissue having malignant cell growth may be a
`
`target, brain, lung and breast tissue are preferred targets.
`
`In certain embodiments, the invention is a method of potentiating the response
`
`of a cell to DNA damaging agents that comprises first administering at least one virus
`
`to the cell, followed by exposing the cell to a DNA damaging agent, such as, for
`
`example, ionizing radiation or DNA damaging agents. The viruses that are
`
`contemplated to be within the scope of the invention include, but are not limited to,
`
`adenovirus, Herpes Simplex Virus (HSV-l ), retrovirus, or Newcastle Disease Virus
`
`(NDV). In exemplary embodiments, the virus is an adenovirus. As used herein,
`
`"potentiate" means to increase the level of cell killing above that seen for a treatment
`
`modality alone. The potentiation may be additive. or it may be synergistic.
`
`Ionizing radiation is considered to be included in exemplary embodiments of
`
`the invention. The radiation may be delivered by external sources, such as from
`
`gamma or beta sources, or it may be supplied from linear accelerators and the like. In
`
`other embodiments, the ionizing radiation may be delivered to a cell by radioisotopes
`
`or by providing a radiolabeled antibody that immunoreacts with an antigen of the
`
`tumor, followed by delivering an effective amount of the radiolabeled antibody to the
`tumor.
`
`In addition to ionizing radiation, other DNA damaging agents are
`
`contemplated to be within the scope of the invention. DNA damaging agents or
`
`factors are defined herein as any chemical compound or treatment method that
`
`induces DNA damage when applied to a cell. Such agents and factors include
`
`ionizing radiation and waves that induce DNA damage, such as, y-irradiation, X-rays,
`
`UV-irradiation, microwaves, electronic emissions, and the like. A variety of chemical
`
`compounds, also described as "chemotherapeutic agents", function to induce DNA
`
`damage, all of which are intended to be of use in the combined treatment methods
`
`disclosed herein. Chemotherapeutic agents contemplated to be of use, include, e. g.,
`
`alkylating agents such as mitomycin C, adozelesin, cis-platinum, and nitrogen
`
`mustard. The invention also encompasses the use of a combination of one or more
`
`10
`
`15
`
`2O
`
`25
`
`

`

`W0 97/ 12623
`
`PCT/US96/l 6047
`
`DNA damaging agents, whether ionizing radiation-based or actual compounds, with
`
`one or more viruses.
`
`The invention also contemplates methods of controlling cell growth by
`
`administering to a cell a virus that contains foreign DNA. The DNA may be in the
`
`form of a heterologous promoter sequence or it may be a heterologous gene encoding
`
`a structural protein. Also contemplated is a heterologous promoter sequence that is
`
`operatively linked to a structural gene coding for a tumoricidal gene, such as TNF-a.
`
`In certain methods, the tumor is first treated with a therapeutically efl‘ective amount of
`
`a virus that contains a DNA molecule comprising a radiation responsive enhancer-
`
`promoter operatively linked to an encoding region that encodes a polypeptide having
`
`the ability to inhibit growth of a tumor cell. Following uptake by the tumor cells, the
`
`tumor area is exposed to an effective expression-inducing dose of ionizing radiation
`
`that results in production of the protein .
`
`To kill a cell in accordance with the present invention, one would generally
`
`contact the cell with a DNA damaging agent, such as ionizing radiation, and a virus,
`
`such as an adenovirus or HSV-l in a combined amount effective to kill the cell. The
`
`term " in a combined amount effective to kill the cell" means that the amount of the
`
`DNA damaging agent and virus that are sufficient so that, when combined within the
`
`cell, cell death is induced. Although not required in all embodiments, the combined
`
`effective amount of the two agents will preferably be an amount that induces more
`
`cell death than the use of either element alone, and even one that induces synergistic
`
`cell death in comparison to the effects observed using either agent alone. A number
`
`of in vitro parameters may be used to determine the effect produced by the
`
`compositions and methods of the present invention. These parameters include, for
`
`example, the observation of net cell numbers before and after exposure to the
`
`compositions described herein.
`
`Similarly, a "therapeutically effective amount" is an amount of a DNA
`
`damaging agent and a virus that, when administered to an animal in combination, is
`
`effective to kill cells within the animal. This is particularly evidenced by the killing
`
`of cancer cells within an animal or human subject that has a tumor. The methods of
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`W0 97/ l 2623
`
`PCT/US96/16047
`
`the instant invention are thus applicable to treating a wide variety of animals,
`
`including mice and humans. "Therapeutically effective combinations" are thus
`
`generally combined amounts of DNA damaging agents and viruses or viral agents that
`
`function to kill more cells than either element alone and that reduce the tumor burden.
`
`In certain embodiments, a process of enhancing cell death is provided, which
`
`comprises the steps of first treating cells or tumor tissue with a DNA damaging agent,
`
`such as ionizing radiation or an alkylating agent, followed by contacting the cells or
`
`tumors with a virus, such as an adenovirus, a herpesvirus, NDV, or a retrovirus.
`
`DNA damaging agents or factors are defined herein as any chemical
`
`compound or treatment method that induces DNA damage when applied to a cell.
`
`Such agents and factors include radiation and waves that induce DNA damage, such
`
`as, y-irradiation, X-rays. UV-irradiation, microwaves. electronic emissions. and the
`
`like. A variety of chemical compounds, which may be described as
`
`"chemotherapeutic agents", also function to induce DNA damage, all of which are
`
`intended to be of use in the combined treatment methods disclosed herein.
`
`Chemotherapeutic agents contemplated to be of use, include, e. g., mitomycin C
`
`(MMC), adozelesin, cis-platinum, nitrogen mustard, S-fluorouracil (SFU), etoposide
`
`(VP-l6), camptothecin, actinomycin-D, cisplatin (CDDP).
`
`The invention provides, in certain embodiments, methods and compositions
`
`for killing a cell or cells, such as a malignant cell or cells, by contacting or exposing a
`
`cell or population of cells to one or more DNA damaging agents and one or more
`
`viruses inhibitors in a combined amount effective to kill the cell(s). Cells that may be
`
`killed using the invention include, e.g. , undesirable but benign cells, such as benign
`
`prostate hyperplasia cells or over-active thyroid cells; cells relating to autoimmune
`
`diseases, such as B cells that produce antibodies involved in arthritis, lupus,
`
`myasthenia gravis, squamous metaplasia, dysplasia and the like. Although generally
`
`applicable to killing all undesirable cells, the invention has a particular utility in
`
`killing malignant cells. "Malignant cells" are defined as cells that have lost the ability
`
`to control the cell division cycle, and exhibit uncontrolled growth and a "transformed"
`
`10
`
`15
`
`20
`
`25
`
`30
`
`or "cancerous" phenotype.
`
`

`

`
`
`W0 97/l 2623
`
`PCT/U596“ 6047
`
`It is envisioned that the cell that one desires to kill may be first exposed to a
`
`virus, and then contacted with the DNA damaging agent(s), or vice versa. In such
`
`embodiments, one would generally ensure that sufiicient time elapses, so that the two
`
`agents would still be able to exert an advantageously combined effect on the cell. In
`
`such instances, it is contemplated that one would contact the cell with both agents
`
`within about 12 hours of each other, and more preferably within about 6 hours of each
`
`other, with a delay time of only about 4 hours being most preferred. These times are
`
`readily ascertained by the skilled artisan.
`
`The terms "contacted" and "exposed“, when applied to a cell, are used herein
`
`to describe the process by which a virus, such as an adenovirus or a herpesvirus, and a
`
`DNA damaging agent or factor are delivered to a target cell or are placed in direct
`
`juxtaposition with the target cell. To achieve cell killing, both agents are delivered to
`
`a cell in a combined amount effective to kill the cell, i.e., to induce programmed cell
`
`death or apoptosis. The terms, "killing", "programmed cell death" and "apoptosis" are
`
`used interchangeably in the present text to describe a series of intracellular events that
`
`lead to target cell death.
`
`The present invention also provides advantageous methods for treating cancer
`
`that, generally, comprise administering to an animal or human patient with cancer a
`
`therapeutically effective combination of a DNA damaging agent and a virus.
`
`Chemical DNA damaging agents and/or viruses may be administered to the animal,
`
`often in close contact to the tumor, in the form of a pharmaceutically acceptable
`
`composition. Direct intralesional injection is contemplated, as are other parenteral
`
`routes of administration, such as intravenous, percutaneous, endoscopic, or
`
`subcutaneous injection. In certain embodiments, the route of administration may be
`
`10
`
`15
`
`20
`
`25
`
`oral.
`
`In terms of contact with a DNA damaging agent, this may be achieved by
`
`irradiating the localized tumor site with ionizing radiation such as X-rays, UV-light,
`
`y-rays or even microwaves. Alternatively, the tumor cells may be contacted with the
`
`DNA damaging agent or a virus by administering to the animal a therapeutically
`
`30
`
`effective amount of a pharmaceutical composition comprising a DNA damaging
`
`

`

`WO 97/12623
`
`PCT/US96/16047
`
`compound, such as mitomycin C, adozelesin, air-platinum, and nitrogen mustard
`
`and/or a virus. A chemical DNA damaging agent may be prepared and used as a
`
`combined therapeutic composition, or kit, by combining it with a virus, as described
`above.
`
`The methods of enhancing the effectiveness of radiotherapy in a mammals
`
`comprises administering to that mammal an effective amount of a pharmaceutical
`
`composition that contains a virus. As used herein, a "pharmaceutical composition"
`
`means compositions that may be formulated for in vivo administration by dispersion
`
`in a pharmacologically acceptable solution or buffer. Suitable pharmacologically
`
`acceptable solutions include neutral saline solutions buffered with phosphate, lactate,
`Tris, and the like.
`
`In certain embodiments of the invention, the number of virus particles that are
`contacted to a host are about 103 to about 10” virus particles. In other embodiments,
`the number of virus particles is about 105 to about 1012 virus particles, and in
`
`exemplary embodiments, the number of virus particles is between about 108 to about
`
`10ll virus particles.
`
`The invention further contemplates methods of assessing the cellular response
`
`to the effect of viral therapy in conjunction with exposure of cells to ionizing radiation
`
`that comprises first, growing cells in culture, which is followed by exposing the cells
`
`with a selected virus and to an effective dose of ionizing radiation. The response of
`
`the cells to this treatment modality may be assessed by techniques known in the art,
`
`such as cell survival assays or enzymatic assays of selected biomarker proteins.
`
`Suitable viruses include, for example, adenovirus, HSV-l, retrovirus, or NDV. The
`
`specificity of viral vectors may be selected to be preferentially directed to a particular
`
`target cell, such as by using viruses that are able to infect particular cell types.
`
`Naturally, different viral host ranges will dictate the virus chosen for gene transfer,
`
`and, if applicable, the likely foreign DNA that may be incorporated into the viral
`
`genome and expressed to aid in killing a particular malignant cell type.
`
`In using viruses within the scope of the present invention, one will desire to
`
`purify the virus sufficiently to render it essentially free of undesirable contaminants,
`
`10
`
`15
`
`2O
`
`25
`
`30
`
`

`

`
`
`W0 97/l 2623
`
`PCT/US96/16047
`
`such as defective interfering viral particles or endotoxins and other pyrogens, so that it
`
`will not cause any undesired reactions in the cell, animal, or individual receiving the
`
`virus. A preferred means of purifying the vector involves the use of buoyant density
`
`gradients, such as cesium chloride gradient centrifugation.
`
`Preferred viruses will be replication defective viruses in which a viral gene
`
`essential for replication and/or packaging has been deleted from the virus. In
`
`embodiments where an adenovirus is used, any gene, whether essential (e. g. ElA,
`
`ElB, E2 and E4) or non-essential (e.g. E3) for replication, may be deleted and
`
`replaced with foreign DNA, or not replaced. Techniques for preparing replication
`
`defective adenoviruses are well known in the art, as exemplified by Ghosh—
`
`Choudhury, et al., 1987. It is also well known that various cell lines may be used to
`
`propagate recombinant adenoviruses, so long as they complement any replication
`
`defect that may be present. A preferred cell line is the human 293 cell line, but any
`
`other cell line that is permissive for replication, e. g. that expresses EIA and ElB, may
`
`be employed. Further, the cells may be propagated either on plastic dishes or in
`
`suspension culture in order to obtain virus stocks.
`
`The invention is not limited to El—lacking virus and E1 expressing cells.
`
`Other complementary combinations of viruses and host cells may be employed in
`
`connection with the present invention. Where a gene that is not essential for
`
`replication is deleted and replaced, such as, for example, the E3 gene, this defect will
`
`not need to be specifically complemented by the host cell. The adenovirus may be of
`
`any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of
`
`subgroup C is the preferred starting material in order to obtain the conditional
`
`replication-defective adenovirus vector for use on the method of the present invention.
`
`The methods and compositions of the present invention are suitable for killing
`
`a cell or cells both in vitro and in viva. When the cells are to be killed are located
`
`within an animal, for example in an organ, the virus and the DNA damaging agent
`
`will be administered to the animal in a pharmacologically acceptable form. Direct
`
`intralesional injection of a therapeutically effective amount of a virus and/or a DNA
`
`damaging agent into a tumor site is one preferred method. Other parenteral routes of
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 97/12623
`
`PCT/US96/16047
`
`administration, such as intravenous, percutaneous. endoscopic, or subcutaneous
`
`injection are also contemplated.
`
`As set forth above, any number of in vitro parameters may be used to
`
`determine the effect produced by the compositions and methods of the present
`
`invention. These parameters include, for example, the observation of net cell numbers
`
`before and after exposure to the disclosed treatment methods. Also, one may be able
`
`to assess the size of cells grown in culture, such as those colonies formed in tissue
`
`culture. Alternatively, one may measure parameters that are indicative of a cell that is
`
`undergoing programmed cell death. such as. for example, the fragmentation of cellular
`
`genomic DNA into nucleoside size fragments, generally identified by separating the
`
`fragments by agarose gel electrophoresis. Staining the DNA, and comparing the DNA
`to a DNA size ladder.
`
`One may also use other means to assess cell killing. As set forth in the instant
`
`examples. one may measure the size of the tumor. either by the use of calipers. or by
`
`the use of radiologic imaging techniques, such as computerized axial tomography
`
`(CAT) or nuclear magnetic resonance (NMR) imaging.
`
`In other embodiments of the invention, kits for use in killing cells, such as
`
`malignant cells, are contemplated. These kits will generally include, in a suitable
`
`container means, a pharmaceutical formulation of a virus for contacting the host cells.
`
`In certain kit embodiments, the DNA damaging agent, such as a DNA alkylating
`
`agent or a radiopharmaceutical may be included in the kit. The kit components may
`
`be provided as a liquid solution. or a dried powder. A preferred approach is to provide
`
`a sterile liquid solution.
`
`The combination of viral infection with radiation treatment produces tumor
`
`cures which are greater than those produced by treatment with radiation alone. Viral
`
`infection alone actually had no effect on cell killing, whether the virus contained an
`
`foreign gene insert or a either modality alone. Cells that contain genetic constructs
`
`constitutively producing toxins and are targeted with ionizing radiation provides a
`
`new conceptual basis for increasing the therapeutic ratio in cancer treatment.
`
`10
`
`15
`
`20
`
`25
`
`-10-
`
`

`

`W0 97/l 2623
`
`PCT/US96/16047
`
`BRIEF DESCRIPTION OF THE DRAW1N§§
`
`The following drawings form part of the present specification and are included
`
`to further demonstrate certain aspects of the present invention. The invention may be
`
`better understood by reference to one or more of these drawings in combination with
`
`the detailed description of specific embodiments presented herein.
`
`FIG. 1 Shows U-87MG glioblastoma cell growth in hindlimbs of mice
`
`following exposure to HSV-l, radiation, and radiation plus HSV-l. Also shown is the
`
`effect ganciclovir on tumor volume, when given in combination with virus or virus
`
`plus radiation.
`
`10
`
`FIG. 2 Shows the regression rate of large tumors compared to small
`
`xenografts following treatment with radiation alone or adenovirus construct
`
`Ad.Egr-TNF plus radiation.
`
`FIG. 3 Shows the regression rate of large tumors compared to small
`
`xenografts following treatment with radiation alone or adenovirus construct Ad.LacZ
`
`15
`
`plus radiation.
`
`DETAILED DESCRIPTION OF THE PREFERRED EMBODINIENTS
`
`The present invention presents methods that are a novel combination of viral
`
`infection and radiotherapy that act together to enhance cell killing in vitro and in viva.
`
`VIR ES
`
`20
`
`Adenovirus
`
`Adenoviruses have been widely studied and well-characterized as a model
`
`system for eukaryotic gene expression. Adenoviruses are easy to grow and
`
`manipulate, and they exhibit broad host range in vitro and in vivo. This group of
`
`viruses may be obtained in a highly infective state and at high titers, e. g., 109-10ll
`
`25
`
`plaque-fanning unit (PFU)/ml. The Adenovirus life cycle does not require integration
`
`into the host cell genome, and foreign genes delivered by these vectors are expressed
`
`episomally, and therefore, generally have low genotoxicity to host cells.
`
`-11-
`
`

`

`WO 97/12623
`
`PCT/US96/16047
`
`Adenoviruses appear to be linked only to relatively mild diseases, since there is no
`
`known association of human malignancies with Adenovirus infection. Moreover, no
`
`side effects have been reported in studies of vaccination with wild-type Adenovirus
`
`(Couch e! a]. , 1963; Top er al. , 1971), demonstrating their safety and therapeutic
`potential as in viva gene transfer vectors.
`
`Adenovirus vectors have been successfully used in eukaryotic gene expression
`(Levrero et a1. , 1991; Gomez—Foix et al., 1992) and vaccine development (Grunhaus
`
`and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies
`
`demonstrated that recombinant Adenoviruses could be used for gene therapy
`
`10
`
`(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich
`
`er al., 1993). Successful studies in administering recombinant Adenovirus to different
`
`tissues include trachea instillation (Rosenfeld er al.. 1991; Rosenfeld et al., 1992),
`
`muscle injection (Ragot et a1., 1993), peripheral intravenous injection (Her: and
`
`Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et a1.,
`1993).
`
`15
`
`Generation and propagation of the current Adenovirus vectors depend on a
`unique helper cell line, 293, which was transformed from human embryonic kidney
`cells by ADS DNA fragments and constitutively expresses E1 proteins (Graham,
`
`et al., 1977). Since the E3 region is dispensable from the Adenovirus genome (Jones
`
`and Shenk, 1978), the current Adenovirus vectors, with the help of 293 cells, carry
`foreign DNA in either the E1, the E3 or both regions (Graham and Prevec, 1991). In
`
`nature, Adenovirus can package approximately 105% of the wild-type genome
`
`(Ghosh-Choudhury, er 0]., 1987), providing capacity for about 2 extra kb of DNA.
`
`Combined with the approximately 5.5 kb of DNA that is replaceable in the E1 and E3
`
`regions, the maximum capacity of the current Adenovirus vector is under 7.5 kb, or
`
`about 15% of the total length of the vector. More than 80% of the Adenovirus viral
`
`genome remains in the vector backbone and is the source of vector-bome cytotoxicity.
`
`As used herein, the term "recombinant" cell is intended to refer to a cell into
`
`which a recombinant gene, such as a gene from the adenoviral genome has been
`
`introduced. Therefore, recombinant cells are distinguishable from naturally occurring
`
`20
`
`25
`
`3O
`
`-12-
`
`

`

`
`
`WO 97/12623
`
`PCT/US96/16047
`
`cells that do not contain a recombinantly introduced gene. Recombinant cells are thus
`
`cells having a gene or genes introduced through the hand of man. Within the present
`
`disclosure, the recombinantly introduced genes encode radiation sensitizing or
`
`radiation protecting factors and are inserted in the E1 or E3 region of the adenovirus
`
`genome. It is recognized that the present invention also encompasses genes that are
`
`inserted into other regions of the adenovirus genome, for example the E2 region.
`
`It is understood that the adenovirus vector construct may therefore, comprise
`
`at least 10 kb or at least 20 kb or even about 30 kb of heterologous DNA and still
`
`replicate in a helper cell. By "replicate in a helper cell," it is meant that the vector
`
`encodes all the necessary cis elements for replication of the vector DNA, expression
`
`of the viral coat structural proteins, packaging of the replicated DNA into the viral
`
`capsid and cell lysis, and further that the trans elements are provided by the helper
`
`cell DNA. Replication is determined by contacting a layer of uninfected cells with
`
`virus particles and incubating said cells. The formation of viral plaques, or cell free
`
`areas in the cell layers is indicative of viral replication. These techniques are well
`
`known and routinely practiced in the art. It is understood that the adenoviral DNA
`
`that stably resides in the helper cell may comprise a viral vector such as an Herpes
`
`Simplex virus vector, or it may comprise a plasmid or any other form of episomal
`
`DNA that is stable, non—cytotoxic and replicates in the helper cell.
`
`In certain embodiments. heterologous DNA is introduced into the viral
`
`genome. By heterologous DNA is meant DNA derived from a source other than the
`
`adenovirus genome, which provides the backbone for the vector. This heterologous
`
`DNA may be derived from a p