MXPA99010176A - Reovirus for the treatment of neoplasia - Google Patents

Abstract

Methods for treating neoplasia, by administering reovirus to a Ras-mediated neoplasm, and use of reovirus for manufacture of a medicament for the treatment of neoplasia, are disclosed. The reovirus is administered so that it ultimately directly contacts cells of the neoplasm. Human reovirus, non-human mammalian reovirus, and/or avian reovirus can be used. If the reovirus is human reovirus, type 1 (e.g., strain Lang), type 2 (e.g., strain Jones), type 3 (e.g., strain Dearing or strain Abney), as well as other serotypes or strains of reovirus can be used. Combinations of more than one type and/or strain of reovirus can be used, as can reovirus from different species of animal. Either solid neoplasms or hematopoietic neoplasms can be treated.

Description

REOVIRUS FOR THE TREATMENT OF NEOPLASIA FIELD AND BACKGROUND OF THE INVENTION Normal cell proliferation is regulated by an equilibrium between tumor suppressor genes _ that restrict growth and proto-oncogens that promote growth. Tumorigenesis can be caused by genetic alterations to the genome that result in the mutation of those cellular elements that control the interpretation of cellular signals, such as potentiation of proto-oncogenic activity or inactivation of tumor suppression. It is believed that the interpretation of these signals lately influences the growth and differentiation of a cell, and that the misinterpretation of these signals can result in neoplastic growth (neoplasia). The genetic alteration of proto-oncogene Ras is believed to contribute to approximately 30% of all human tumors (Wiessmuller, L. and Wittinghofer, F. - (1994), Cellular Signaling 6 (3): 247-267; Barbacid, M. (1987) A Rev. Biochem. 56, 779-827). The function that exhibits' Ras in the pathogenesis of human tumors is specific to the type of tumor. Activation mutations in Ras RF ^.: 31211 by themselves are found in more types of human malignancies, and are highly represented in pancreatic cancer (80%), sporadic colorectal carcinomas (40-50%), human lung adenocarcinomas (15-24%) , thyroid tumors (50%) and myeloid leukemia (30%) (Milus, NE et al. (1995) Cancer Res. 55: 1444; Chaubert, P. et al. (1994), Am. J. Path. 144: 767; Bos, J. (1989) Cancer Res. 49: 4682). Activation of Ras is also demonstrated by countercurrent mitogenic signaling elements, notably by tyrosine receptor kinases (RTKs). These countercurrent elements, if amplified or overexpressed, ultimately result in Ras activity raised by the transduction activity of the Ras signal. Examples of this include overexpression of PDGFR in certain forms of glioblastomas, as well as in c-erbB-2 / neu in breast cancer (Levitzki, A. (1994) Eur. J. Biochem. 226: 1; James, PW , et al. (1994) Oncogene 9: 3601; Bos, J. (1989) Cancer Res. 49: 4682). Common methods of treatment for neoplasia include surgery, chemotherapy and radiation. Surgery is typically useful as the primary treatment for early stages of cancer; however, many tumors can not be removed completely by surgical means. In addition, the metastatic growth of neoplasms can prevent the complete cure of cancer by surgery. Chemotherapy involves the administration of compounds having antitumorigenic activity, such as alkylating agents, antimetabolites, and antitumorigenic antibiotics. The efficacy of chemotherapy is often limited by several side effects, including nausea and vomiting, depression of the bone marrow, kidney damage, and central nervous system depression. Radiation therapy results in the greater ability of normal cells, in contrast to neoplastic cells, to repair themselves after radiation treatment. Radiation therapy can not be used to treat many neoplasms, however, it causes the sensitivity of the tissue surrounding the tumor. In addition, certain tumors have demonstrated resistance to radiotherapy and such may be dependent on the cell's oncogenic or antineogenic state (Lee, JM et al. (1993) PNAS 90: 5742-5746; Lowe, SW et al. (1994). ) Science, 266: 807-810; Raybaud-Diogene, H. et al. (1997) J. Clin. Oncology, 15 (3): 1030-1038). In view of the disadvantages associated with common means for the treatment of neoplastic growth, there is still a need for improved methods for the treatment of more types of cancers.

DESCRIPTION OF THE INVENTION The present invention pertains to methods for the treatment of neoplasia in a mammal, using reovirus, and to the use of reovirus for the manufacture of a medicament for the treatment of neoplasia. The reovirus is administered to a neoplasm, in which an element of the Ras signaling path (either in downstream or countercurrent) is activated to an extent that results in reovirus-mediated oncolysis of neoplasm cells. The reovirus can be administered in a single dose or in multiple doses; In addition, more than one neoplasm in an individual mammal can be treated in a common way. Both solid neoplasms and hematopoietic neoplasms can be white. The reovirus is administered in such a way that it makes contact with mammalian cells (for example, by injection directly into a solid neoplasm, or intravenously in the mammal for a hematopoietic neoplasm). The methods can be used to treat neoplasia in a variety of mammals, including mice, dogs, cats, sheep, goats, cows, horses, pigs, and non-human primates. Preferably, the methods are useful for treating neoplasia in humans. The methods and uses of the invention provide an effective means to treat neoplasms, without the side effects associated with other forms of cancer therapy. In addition, because the reovirus is not known to be associated with disease, any safety concerns that are associated with deliberate administration of a virus are minimized.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a representation of the molecular basis of reovirus oncolysis, in which reovirus usurps the Ras signaling path of the host cell. Figure 2 is a graphical representation of the effects over time of serotype 3 of activated reovirus (empty circles) or inactivated (full circles) (Dearing strain) on the size of murine THC-11 tumors grown in mice with severe combined immunodeficiency (for its acronym in English, SCID). The marked values represent the means of measurement with the standard error of the medium also shown. Figure 3 is a graphical representation of the effect during the period of serotype 3 of activated reovirus (empty circles) or inactivated (full circles) (Dearing strain) on the size of human glioblastoma U-87 xenografts growing in SCID mice. The marked values represent the average of the measurements with the standard error of the average also known. Figure 4 is a graphical representation of the effects during the period of serotype 3 of the activated reovirus (empty circles, empty squares) or inactivated (full circles, filled squares) (Dearing strain) in the size of bilateral human glioblastoma xenograft U-87 treated (circles) or untreated (squares) growing in SCID mice. The marked values represent the average of the measurements with the standard error of the average also known. The foregoing and other objects, features and advantages of the invention will appear from the following more particular description of the preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same. parts in all different views. The drawings are not necessarily to scale, they emphasize more than being placed as an illustration of the principles of the invention.

DETAILED DESCRIPTION OF THE INVENTION. The invention pertains to methods of treating a neoplasm in a mammal, administering reovirus to the neoplasm. The name reovirus (orphan respiratory and enteric virus) is a descriptive acronym that suggests that these viruses, although not associated with any known disease state in humans, can be isolated from both respiratory and enteric tracts (Sabin, AB (1959 ), Science 130: 966). The mammalian reovirus consists of three serotypes: type 1 (strain Lang or TIL), type 2 (strain Jones, T2J) and type 3 (strain Dearing or strain Abney, T3D). The three serotypes are easily identifiable in the bases of neutralization and hemagglutinin inhibition assays (Sabin, A.B. (1959), Science 130: 966); Fields, B.N. et al. (1996), Fundamental Virology, 3rd Edition, Lippincott-Raven; Rosen, L. (1960) Am. J. Hyg. 71: 242; Stanley, N.F. (1967) Br. Med. Bull. 23: 150). Although reovirus is not known to be associated with any particular disease (Tyler, KL and Fields, BN, in Fields Virology (Fields, BN, Knipe, DM, and Ho law, PM eds), Lippincott-Raven, Philadelphia, 1996, p. 1597), many people have been exposed to 'reovirus' by the time they reach maturity (ie, less than 25% in children <5 years of age, greater than 50% in those of 20- 30 years of age (Jackson GG and Muldoon RL (1973) J. Infect.

Dis. 128: 811; Stanley N.F. (1974) In: Comparative Diagnosis of Viral Diseases, edited by E. Kurstak and K. Kurstak, 385-421, Academic Press, New York). For mammalian reovirus, the cell surface recognition signal is sialic acid (Armstrong, GD et al. (1984), Virology 138: 37, Gentsch, JRK and Pacitti, AF (1985), J. Virol. 356; Paul RW et al. (1989) Virology 172: 382-385). Due to the ubiquitous nature of sialic acid, the reovirus binds efficiently to a multitude of cell lines and as such can potentially be white, many different tissues; However, there are significant differences in the susceptibility to infection by reovirus between cell lines, as described here., Applicants have discovered that cells which are resistant to reovirus infection become susceptible to reovirus infection when transformed with a gene in the Ras path. '"Resistance" of cells to reovirus infection indicates that infection of the cells with the virus does not result in significant viral yield or yield. The cells that are "susceptible" are those that demonstrate induction of cytopathic effects, synthesis of the viral protein, and / or production of the virus. Resistance to reovirus infection was found to be at the level of gene translation, rather than in the initial transcript: while viral transcripts were produced, the virus proteins were not expressed. The transcription of the viral gene in resistant cells correlated with the phosphorylation of a cellular protein of approximately 65 kDa, determined to be protein kinase activated by double-stranded RNA (PKR), which was not observed in the transformed cells. Phosphorylation of PKR leads to translation inhibition. When phosphorylation was suppressed by 2-aminopurine, a known inhibitor of PKR, dramatic improvement of reovirus protein synthesis occurred in non-transformed cells. In addition, in a mouse model with combined immunodeficiency, severe, (for its acronym in English, SCID) in which the tumors were created in both the right and left hind legs reveal that the reovirus significantly reduces the size of the tumor when is injected directly into the tumor on the right side; in addition, the significant reduction in tumor size was also shown in the tumor on the left side which was not injected directly with reovirus, indicating that the oncolytic capacity of the reovirus was systemic as well as local.

These results indicate that reoviruses use the Ras path machinery of the host cell to regulate PKR and therefore reproduce it. Figure 1 shows the usurpation of the Ras signaling path of the host cell by reovirus. As shown in Figure 1, both for non-transformed cells (resistance to reovirus) and transformed by EGFR, Sos or ras (susceptible to reovirus), virus binding, internalization, uncoated, and early transcription of viral genes all proceeded normally . In the case of untransformed cells, the secondary structures in the more recent viral transcripts inevitably activate phosphorylation of PKR, thereby activating it, leading to phosphorylation of translational initiation factor eIF-2a, and consequently, inhibition. of translation of viral genes. In the case of cells transformed by EGFR, Sos or ras, the phosphorylation step of PKR is prevented or reversed by Ras or one of its lower elements, by which it makes it possible to assure the translation of the viral gene. The action of Ras (or a lower element) can be mimicked by the use of 2-aminopurine (2-AP), which promotes translation of the viral gene (and consequently reovirus infection) into non-transformed cells by blocking the phosphorylation of PKR.

Based on these findings, the Applicants have developed methods for the treatment of neoplasms in mammals. Representative mammals include mice, dogs, cats, sheep, goats, cows, horses, pigs, non-human primates, and humans. In a preferred embodiment, the mammal is a human. In the methods of the invention, the reovirus is administered to a neoplasm in the individual mammal. Representative types of human reoviruses that may be used include type 1 (e.g., strain Lang or TIL); type 2 (for example, strain Jones or T2J); and type 3 (e.g., strain Dearing or strain Abney, T3D or T3A); other strains of reovirus can also be used. In a preferred embodiment, the reovirus is Dearing strain. Alternatively, the reovirus can be a non-human mammalian reovirus (e.g., non-human primate reovirus, such as mandrel reovirus; equine; or canine reovirus), or a non-mammalian reovirus (e.g., bird reovirus). A combination of different serotypes and / or different strains of reovirus, such as reovirus from different animal species, can be used. Reovirus is 'naturally present', that is, it can be isolated from a natural source and not intentionally modified by humans in the laboratory, for example, the reovirus can be from a * field source ": , from a human patient. If desired, the reovirus can be chemically or biochemically treated (eg, by treatment with a protease, such as chymotrypsin or trypsin) prior to administration to the neoplasm. Such pretreatment removes the external coating of the virus and therefore, can result in better virus infectivity. The neoplasm can be a solid neoplasm (for example, sarcoma or carcinoma), or a cancerous growth that affects the hematopoietic system (a 'hemopoietic neoplasm', for example, lymphoma or leukemia) A neoplasm is an abnormal tissue growth, which generally forms a different mass, which grows by cell proliferation more rapidly than the growth of normal tissue Neoplasms show partial or total lack of structural organization and functional coordination with normal tissue As used here, a 'neoplasm', also referred to as a 'tumor', it is intended to encompass hematopoietic neoplasms as well as solid neoplasms.At least one of the cells of the neoplasm has a mutation in which the Ras gene (or an element of the Ras signaling path) is activated, either directly (for example, by an activation mutation in Ras) or indirectly (for example, by activation of an upstream or countercurrent element in the Ras path). Activation of an element countercurrent in the Ras path includes, for example, transformation with epidermal growth factor receptor (EGFR) or Sos. A neoplasm that results, at least in part, from the activation of Ras, a countercurrent Ras element, or an element in the Ras signaling pathway is referred to herein as a "Ras-mediated neoplasm." A neoplasm that is particularly susceptible to treatment by the methods of the invention is pancreatic cancer, because of the prevalence of Ras-mediated neoplasms associated with pancreatic cancer Other neoplasms that are particularly susceptible to treatment by the methods of the invention, include breast cancer, cancer of brain (eg glioblastoma), lung cancer, prostate cancer, colorectal cancer, thyroid cancer, kidney cancer, adrenal cancer, liver cancer, and leukemia Reovirus' is typically administered in a physiologically acceptable vehicle or carrier , such as phosphate buffered saline., to the neoplasm. 'Administration to a neoplasm' indicates that the reovirus is administered This is such that it makes contact with the neoplasm cells (also referred to herein as "neoplastic cells"). The route by which the reovirus is administered, as well as the formulation, carrier or vehicle, will depend on the location as well as the type of the neoplasm. A wide variety of administration routes can be employed. For example, for a solid neoplasm that is accessible, the reovirus can be administered by injection directly to the neoplasm. For a hematopoietic neoplasm, for example, the reovirus can be administered intravenously or intravascularly. For neoplasms that are not readily accessible within the body, such as metastases or brain tumors, the reovirus is administered in such a way that it can be transported systemically through the body by the mammal and thereby reaches the neoplasm (e.g. intrathecally, intravenously or intramuscularly). Alternatively, the reovirus can be administered directly to a single solid neoplasm, where it is then transported systemically through the body to metastasis. The reovirus can also be administered subcutaneously, intraperitoneally, topically (for example, for melanoma), orally (for example, for oral or esophageal neoplasm), rectally (for example, for colorectal neoplasm) vaginally (for example, for cervical or vaginal neoplasm) nasally or by spray for inhalation (for example, for neoplasm of the lung).

The reovirus is administered in an amount that is sufficient to treat the neoplasm (eg, an "effective amount"). A neoplasm is 'treated' when the administration of the reovirus to cells of the neoplasm is effected by oncolysis of the neoplastic cells, resulting in in a reduction in size of the neoplasm, or in a complete elimination of the neoplasm. The reduction in size of the neoplasm, or elimination of the neoplasm, is usually caused by lysis of neoplastic cells (Oncolysis) by the reovirus.The effective amount will be determined on an individual basis and may be based, at least in part, on consideration of the type of reovirus, the size of the individual, age, gender, and the size and other characteristics of the neoplasm For example, for treatment of a human, plaque formation units of approximately 103 to 1012 can be used (for its acronym in English). , PFU) of reovirus, depending on the type, size and number of tumors present.Repovirus can be administered in a single dose, or multiple doses (ie, more than one dose) .Multiple doses can be commonly administered, or 'Consecutive form (for example, over a period of days or weeks.) Reovirus can also be administered to more than one neoplasm in the same individual.

The invention is further illustrated by the following Embodiment Example.

EXAMPLIFICATION MATERIALS AND METHODS Cells and viruses The major NIH-3T3 cell lines in conjunction with NIH-3T3 cells transformed with a number of oncogens were obtained from a variety of sources. The major NIH-3T3 and NIH-3T3 cells transfected with the Harvey-ras (H-ras) and EJ-ras oncogenes were a generous graft from Dr. Douglas Faller (Boston University School of Medicine). The NIH-3T3 cells together with their counterparts transformed by Sos (designated TNIH # 5) were a generous graft from Dr. Michael Karin (University of California, San Diego). The major NIH-3T3 cells together with NIH-3T3 cells transfected with the v-erbB oncogene (designated THC-11) donated generously by Dr. H. -J. Kung (Case Western Reserve University). 2H1 cells, a derivative of the murine fibroblast line C3H 10T1 / 2, containing the Harvey-ras gene under the transcriptional control of the mouse metallothionein-I promoter were obtained from Dr. Nobumichi Hozumi (Mount Sinai Hospital Research Institute). These 2H1 cells are transformants of the conditional ras that expresses the H-ras oncogene in the presence of 50 μM ZnS04. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). NIH-3T3 tet-myc cells were obtained from Dr. R.N. Johnston (University of Calgary) and were cultured in DMEM containing 10% hot inactivated FBS and antibiotics in the presence or absence of 2 μg / ml tetracycline (Helbing, CC et al., Cancer Res. 57: 1255-1258 ( 1997)). In the presence of tetracycline, the expression of the human gene c-iayc is represented. The removal of tetracycline results in increased expression of c-myc by up to 100-fold in these cells, which also exhibits a transformed phenotype. Fibroblasts from the mouse embryo (MEFs) PKR + / + and PKR ° / ° were obtained from Dr. B.R.G. Williams (the Cleveland Clinic Foundation) and cultured in a-MEM containing fetal bovine serum and antibiotics as previously described (Yang, YL et al., EMBO J. 14: 6095-6106 (1995); Der, SD et al. al., Proc. Nati, Acad. Sci. USA 94: 3279-3283 (1997)).

The Dearing strain of serotype 3 of the reovirus used in these studies was propagated in suspension cultures of L cells and purified according to Smith (Smith, R.E. et al., (1969) Virology, 39: 791-800) with the exception that β-mercaptoethanol (β-ME) was omitted from the extraction buffer. The reovirus labeled with [35 S] methionine was cultured and purified as described by McRae and Joklik (McRae, M.A. and Joklik, W.K., (1978) Virology, 89: 578-593). The particle / PFU ratio for the purified reovirus was typically 100/1.

Immuno-fluorescent analysis of reovirus infection For immunofluorescent studies, NIH-3T3, TNIH # 5, H-ras, EJ-ras, 2H1 (+/- ZnSO.), And THC-11 cells were grown on cover strips or coverslips, and were infected with reovirus in a multiplicity of infection (MOI) of cells ~ 10 PFU or infected by simulation by the application of the carrier agent (phosphate buffered saline, PBS) to the cells in an identical manner as the administration of virus to the cells. In 48 hours postinfection, the cells were fixed in a mixture of ethanol / acetic acid (20/1) for 5 minutes, then rehydrated by sequential washes in 75%, 50% and 25% ethanol, followed by four washes with solution salt buffered with phosphate (PBS). The fixed and rehydrated cells were then exposed to the primary antibody (rabbit polyclonal antireovirus type 3 serum, diluted 1/100 in PBS) [antiserum prepared by injection of rabbits with reovirus serotype 3 in complete Freund's adjuvant, and subsequent blood flows ] for 2 hours at room temperature. Following three washes with PBS, the cells were exposed to secondary antibody [conjugated. of IgG anti-rabbit goats (complete molecule) -fluorescein isothiocyanate (FITC) [Sigma ImmunoChemicals F-0382] diluted 1/100 in PBS containing 10% goat serum and 0.005% Evan Blue] for 1 hour at room temperature . Finally, the fixed and treated cells were washed three more times with PBS and then once with distilled water twice, dried and mounted on slides in 90% glycerol containing 0.1% phenylenediamine, and observed with a Zeiss Axiophot microscope in which the Carl Zeiss camera was mounted (magnification or magnification for all images was 200X).

Detection of MAP Kinase Activity (ERK) The set of Antibodies (Thr202 / Tyr204) of MAP Kinase PhosphoPlus p44 / 42 (New England Biolabs) was used for the detection of MAP kinase in cell lysates according to the manufacturing instructions . Briefly, subconfluent monolayer cultures were treated with lysis, with the sample buffer containing SDS recommended, and subjected to SDS-PAGE, followed by electro-drying on nitrocellulose paper. The membrane was then tested with the primary antibody (anti-total MAPK or anti-phospho-MAPK), followed by the secondary antibody conjugated with horseradish peroxidase (HRP) as described in the manufacturer's instruction manual.

Radiolabelling of cells infected with reovirus and preparation of lysates The confluent monolayers of NIH-3T3, TNIH # 5, H-ras, EJ-ras, 2H1 (+/- ZnSO_), and THC-11 cells were infected with reovirus (MOI-10 PFU / cell). At 12 hours post-infection, the medium was replaced with methionine-free DMEM containing 10% dialyzed FBS and 0.1 mCi / ml [35S] methionine. After further incubation for 36 hours at 37 ° C, the cells were washed in phosphate-buffered saline (PBS) and lysed in the same buffer containing 1% Triton X-100, 0.5% deoxycholate sodium and 1 mM EDTA. The nuclei were then removed by low speed centrifugation and the supernatants were stored at -70 ° C until use.

Preparation of cytoplasmic extracts for in vitro kinase assays The confluent monolayers of the various cell lines were grown in 96-well cell culture plates. At the appropriate time post-infection the medium was removed by aspiration and the cells were lysed with a buffer containing 20 mM HEPES [pH 7.4], 120 mM KCl, 5 M MgCl2, 1mM dithiothreitol, 0.5% Nonidet P-40, 2 μg / ml leupeptin, and 50 μg / ml aprotinin. The nuclei were then removed by low speed centrifugation and the supernatants were stored at -70 ° C until use. The cytoplasmic extracts were normalized to protein concentrations before use by the micro-assay method of the Bio-Rad protein. Each in vitro kinase reaction contained 20 μl of cell extract, 7.5 μl of reaction buffer (20 mM HEPES [pH 7.4], 120 mM KCl, 5 mM MgCl 2, 1 mM dithiothreitol, and 10% glycerol) and 7.0 μl of a mixture of ATP (1.0 μg [? -32P] ATP in 7 μl of reaction buffer), and incubated for 30 minutes at 37 ° C (Mundschau, LJ, and faller, DV, J. Biol. Chem. , 267: 23092-23098 (1992)). Immediately after incubation the labeled extracts were boiled in Laemmli SDS-sample buffer or precipitated with poly (I) poly (I) agarose beads or immunoprecipitated with an anti-PKR antibody.

Precipitation of Agarose poly (I) poly (C) To each in vitro kinase reaction mixture, 30 μl of a Type 6 slurry of 50% poly (I) Poly (C) Ag (Pharmacia LKB Biotechnology) was added, and the mixture was incubated at 4 ° C for 1 h. The poly (I) Poly (C) Ag beads with the tagged proteins absorbed, were then washed four times with buffer (20 mM HEPES [7.5 pH], 90 mM KCl, 0.1 mM EDTA, 2 mM dithiothreitol, glycerol%) at room temperature and mixed with 2X Laemmli SDS sample buffer. The beads were then boiled for 5 minutes, and the released proteins were analyzed by SDS-PAGE.

Polymerase chain reaction Cells in various postinfection periods were harvested and resuspended in ice-cooled TNE (10 mM Tris [pH 7.8], 150 mM NaCl, 1 mM EDTA) to which NP-40 was then added to a final concentration of 1%. After 5 minutes, the nuclei became pellets and the RNA was extracted from the supernatant using the phenol: chloroform procedure. Equal amounts of total cellular RNA from each sample were subjected to RT-PCR (Wong, H., et al., (1994) Anal. Biochem., 223: 251-258) using random hexanucleotide primers (Pharmacia) and RTase (GIBCO-BRL) in accordance with the manufacturer's protocol. The cDNAs of the RT-PCR step were subjected to selective reovirus amplification if cDNA using the primer 5'-AATTCGATTTAGGTGACACTATAGCTATTGGTCGGATG-3 '(SEQ ID NO: 1) and 5'-CCCTTTTGACAGTGATGCTCCGTTATCACTCG-3' (SEQ ID NO: 2) which amplifies a fragment of 116 bp predicted. These primer or activator sequences were derived from the previously determined SI sequence (Nagata, L., et al., (1984) Nucleic Acids Res., 12: 8699-8710). The GAPDH activators (Wong, H., et al., (1994) Anal.Biochem., 223: 251-258), 5'-CGGAGTCAACGGATTTGGTCGTAT-3 '(SEQ ID NO: 3) and 5'-AGCCTTCTCCATGGTGGTGAAGAC-3' (SEQ ID NO: 4) were used to amplify a predicted 306 bp GAPDH fragment, which serve as a gel loading control and PCR. Selective amplification of si and GAPDH cDNA was performed using Taq DNA polymerase (GIBCO-BRL) according to the manufacturer's protocol using a 9600 Perkin Elmer Gene Amp PCR system. PCR was performed for 28 cycles with each consisting of a denaturation step for 30 seconds at 97 ° C, annealing step for 45 seconds at 55 ° C, and the polymerization step for 60 seconds at 72 ° C. The PCR products were analyzed by electrophoresis through a 2% agarose gel of TAE impregnated with ethidium bromide and photohed under ultraviolet illumination with Polaroid 57 film.

Immunoprecipitation and analysis of SDS-PAGE Immunoprecipitation of lysates from 35S-labeled reovirus-infected cells with serum serotype 3 anti-reovirus was performed as previously described (Lee, P.W.K. et al (1981) Virology, 108: 134-146). Immunoprecipitation of lysates from 32 P-labeled cells with an anti-PKR antibody (from Dr. Michael Mathews, Cold Spring Harbor) was performed in a similar manner. The immunoprecipitates were analyzed by batch SDS-PAGE according to the protocol of Laemmli, (Laemmli, U.K., (1970) Nature, 227: 680-685).

EXAMPLE 1. Intermediaries activated in the Efficiency of Reovirus Infection in Increasing the Signaling Path or Ras Mark It has previously been shown that 3T3 cells and their derivatives lacking epidermal growth factor receptors (EGFR) are barely infectious by reovirus, whereas the same cells transformed with EGFR or v-erb B are highly infectious. susceptible as determined by cytopathic effects, viral protein synthesis, and virus outflow (Strong, JE et al., (1993) Virology, 197: 405-411; Strong, JE and Lee, PWK, (1996) J. Virol ., 70: 612-616). To determine if the descending mediators of the transduction path. of the EGFR signal may be involved, a number of different NIH 3T3-derivatives, transformed with constitutively activated oncogens downstream of the EGFR, were tested for relative susceptibility to reovirus infection. Of particular interest were the intermediaries in the ras signaling pathway (analyzed by Barbacid, M., Annu, Rev. Biochem., 56: 779-827 (1987); Cahill, M.A., et al., Curr, Biol., 6: 16-19 (1996)). To investigate the signaling path of Ras, NIH 3T3 major cell lines and NIH 3T3 lines transfected with activated versions of Sos oncogens (Aronheim, A., et al., (1994) Cell, 78: 949-961) or ras ( Mundschau, LJ and Faller, DV, (1992) J. Biol. Chem., 267: 23092-23098) were exposed to reovirus, and their ability to promote viral protein synthesis was compared. Detection of viral proteins was initially performed using the indirect immunofluorescence microscope as described above. The results indicate that while the NIH 3T3 cells adopt a dissemination morphology, typically crushed with marked contact inhibition, the transformed cells all grew as needle cells with much less contact inhibition. In comparison of the main cell lines not infected with the various transformed cell lines, it was evident that the morphology of the cells was very different in the transformation. Due to the requirement with reovirus, it becomes clear that the main NIH 3T3 line was poorly infected (<5%), despite the source of the main NIH 3T3 line. In contrast, the transfected cell lines each demonstrate relatively pronounced immunofluorescence for 48 hours post-infection (data not shown). To demonstrate that viral protein synthesis was more efficient in cell lines transformed with Sos or Ras, the cells were continuously labeled with [35S] -methionine for 12 to 48 hrs post-infection and the proteins were analyzed by polyacrylamide gel electrophoresis. sodium dodecyl sulfate (SDS-PAGE), as described above. The results clearly show that viral protein synthesis levels were significantly higher in cells transformed with Sos or Ras than in NIH 3T3 major cells. The identities of the viral bands were confirmed by immunoprecipitation of the proteins labeled with polyclonal anti-reovirus antibodies. Since uninfected NIH 3T3 cells and their transformed counterparts exhibit comparable levels of cellular protein synthesis and bending periods (data not shown), the difference observed in the level of viral protein synthesis could not be due to the intrinsic differences in protein synthesis. growth intervals or translation efficiencies for these cell lines. Prolonged period death of infected NIH 3T3 cells was continued by passing these cells for at least 4 weeks. They grew normally and seemed healthy, with no sign of lytic or persistent infection; no virus could be detected in the medium after this period (data not shown).

EXAMPLE 2. Improved Infectivity Conferred by Activated Oncogens Is Not Due to Transformation in Prolonged Periods or the Generalized Transformed State of the Cell To determine whether the differences in susceptibility may be the result of effects in prolonged periods of transformation, or the result of the activated oncogene itself, a cell line expressing a cellular Harvey-ras (cH-ras) gel that can be induced by Zinc was tested for susceptibility to reovirus infectivity, as described above. These cells, called 2H1, are derived from the C3H 10T1 / 2 cell line which is scarcely infected by reovirus (data not shown), and carry the cH-ras gene under the control of the mouse metallothionin-I promoter (Trimble, WS et al. (1986) Nature, 321: 782-784). The cells were falsely treated or pretreated with 50 μM ZnSO4 18 hours prior to infection or sham infection (administration of the carrier agent), followed by indirect immunofluorescent analysis of these cells to 48 hours postinfection or simulated infection. The results (not shown) demonstrate that the non-induced cells were poorly infected (<5%) while those induced by only 18 hours were much more susceptible (> 40%). Improved viral protein synthesis in 2H1 cells induced by Zn ~ was further confirmed by metabolic labeling of the cells with [35S] methionine, followed by SDS-PAGE analysis of virus-specific proteins (not shown). Based on these observations, the increased efficiency of reovirus infection in the transformed cells is a direct result of the activated product (s) of oncogenes, and not due to other factors such as aneuploid frequently associated with the transformation of period. prolonged, or other accumulated mutations that can be acquired under a chronically transformed state (eg, myc or p53 activation). To further show that the susceptibility to reovirus infection is not a result of transformation per se (ie, a result of the transformed state of the host cell), NIH-3T3 cells containing a human c-myc gene controlled by tetracycline (tet-myc cells) were examined (Helbing, CC et al., Cancer Res. 57: 1255-1258 (1997)). These cells are normally maintained in tetracycline (2 μg / ml) which represents the expression of c-myc. The removal of tetracycline under normal growth conditions (10% fetal bovine serum) leads to the accumulation of the c-Myc protein and the cells exhibit a transformed phenotype. It is found that these cells were unable to support the growth of the virus either in the presence or in the absence of tetracycline (data not shown), which suggest that the susceptibility to reovirus infection is not due to the general transformed state of the cell host, but requires specific transformation by elements of the Ras signaling path. A good indicator of the activation of the Ras signaling pathway is the activation of the MAP kinases ERK1 and ERK2 (for an analysis, see Robinson, MJ and Cobb, MH, Curr. Opin. Cell. Biol. 9: 180-186 (1997)). In this regard, it is found that, compared to untransformed cells, cells transformed with Ras have a significantly elevated ERK1 / 2 activity (data not shown). In addition, an analysis of a number of human cancer cell lines have revealed an excellent correlation between the level of ERK1 / 2 activity and susceptibility to reovirus infection (data not shown), although ERK1 / 2 itself does not appear to exhibit any function in this one. The mouse L cells and human HeLa cells, in which the reoviruses grow very well, both show elevated activity of ERK1 / 2 (data not shown).

EXAMPLE 3. Viral Transcripts are Generated but Not Transformed in NIH 3T3 Cells Resistant to Reovirus The passage to which the reovirus infection is blocked in non-susceptible NIH 3T3 cells was also identified. Because the virus binding and virus incorporation for non-susceptible cells was comparable to that observed by susceptible cells (Strong, JE et al., (1993) Virology, 197: 405-411), the transcription of viral genes was investigated. Relative amounts of reovirus SI transcripts generated in NIH 3T3 cells and Ras transformed cells during the first 12 hours of infection were compared after amplification of these transcripts by polymerase chain reaction (PCR), as described above. The results showed that the accumulation ratios of Si transcripts in the two cell lines were similar, at least up to 12 hours post-infection. The similar data were obtained when accumulation intervals of other reovirus transcripts were compared _ (data not shown). These results demonstrate that the block of infection in non-susceptible cells is not at the level of transcription of viral genes, but preferably, at the translational level of the transcripts. In later periods, the level of viral transcripts present in non-transformed NIH-3T3 cells decreases significantly while the transcripts in the transformed cells continue to accumulate (data not shown). The inability of these transcripts is transferred into NIH-3T3 cells that probably contribute to their degradation. As expected, the level of viral transcripts in infected L cells was at least comparable with that in cells transformed with infected Ras (data not shown).

EXAMPLE 4. A 65 kDa Protein is Phosphorylated in NIH 3T3 Cells Treated with Reovirus but Not in Transformed Cells Infected by Reovirus Because the viral transcripts were generated, but not transferred, in NIH 3T3 cells, it was investigated whether the kinase activated with double-stranded RNA or strand (dsRNA), PKR, is activated (phosphorylates) in these cells (e.g. , by SI siRNA transcripts which have been shown to be potent activators of PKR ((Bischoff, JR and Samuel, CE., (1989) Virology, 172: 106-115), which in turn leads to the inhibition of translation or transference of viral genes The corollary of a scenario could be that in the case of transformed cells, this activation is prevented, allowing to ensure the synthesis of the viral protein NIH 3T3 cells and cells transformed by v-erbB or Ras (designated THC-11 and H-Ras, respectively) were treated with reovirus (ie, were infected) or infected by simulation (as above), and 48 hours post-treatment, were subjected to in vitro kinase reactions, followed by autoradiographic analysis as described above. The results clearly demonstrated that there was a different phosphoprotein migration at approximately 65 kDa, the expected size of PKR, only in NIH 3T3 cells and only after exposure to the reovirus. This protein was not labeled in the lysates of either the uninfected transformed cell lines or the infected transformed cell lines. Instead, a protein that migrates at approximately 100 kDa was found to be labeled in transformed cell lines after viral infection. This protein was absent in the preinfection or post-infection lysates of the NIH 3T3 cell line, and was not a reovirus protein because it does not react with an anti-reovirus serum that precipitates all the reovirus proteins (data not shown). A similar 100 kDa protein was also found to be 32 P-labeled in in vitro kinase reactions of lysates post-infection of cell lines transformed by Sos (data not shown). These intermediaries in the Ras signaling pathway were responsible for the lack of phosphorylation of the 65 kDa protein, was further confirmed by the use of 2H1 cells which contain a Ras oncogene induced by Zn. The non-induced 2H1 cells (relatively resistant to reovirus infection, as shown above), were able to produce the 65 kDa phosphoprotein only after exposure to the virus. However, 2H1 cells subjected to Zn induction of the H-Ras oncogene show significant deterioration in the production of this phosphoprotein. This deterioration coincides with the improvement of viral synthesis. Therefore, these results eliminate the possibility that the induction of the 65 kDa phosphoprotein was a specific event of NIH 3T3, and clearly establishes the function of Ras in the prevention (or reversal) of the induction of the production of this phosphoprotein . Zn-induced 2H1 cells do not produce the 100 kDa phosphoprotein observed in chronically transformed, infected H-Ras cells.

EXAMPLE 5. Induction of 65 kDa Protein Phosphorylation Required in Active Viral Transcription Since production of the 65 kDa phosphoprotein occurs only in cells that were resistant to reovirus infection, and only after the cells were exposed to the reovirus, it was investigated whether the active viral transcription was required for the production of the phosphoprotein at 68 kDa. The reovirus was treated with UV to inactivate its genome prior to administration of the reovirus to NIH 3T3 cells. For UV treatment, the reovirus was suspended in DMEM at a concentration of approximately 4 x 10 8 PFU / ml and exposed to short-wave UV light (254 nm) for 20 minutes. The UV-inactivated viruses were not infectious as determined by the lack of cytopathic effects in mouse L-929 fibroblasts and lacked viral protein synthesis by [35 S] -methionine labeling methods as previously described. Such transcription of the viral gene abolished by UV treatment, when analyzed by PCR, and therefore viral infectivity (data not shown). The cells were then incubated for 48 hours, and the lysates were prepared and subjected to in vitro 32 P labeling as above. The results show that NIH 3T3 cells infected with untreated reovirus produced a band labeled with 32P of prominent 65 kDa was not found in disinfected cells. Cells exposed to UV-treated reoviruses behave similarly to uninfected control cells, manifesting minimal phosphorylation of the 65 kDa protein. Thus, the induction of phosphorylation of the 65 kDa phosphoprotein is not due to the dsRNA present in the entry reovirus; preferably, it requires de novo transcription of the viral genes, consistent with the identification of the 65 kDa phosphoprotein as PKR.

EXAMPLE 6. Identification of the 65 kDa Phosphoprotein as PKR To determine whether the 65 kDa phosphoprotein was PKR, an experiment that links dsRNA was performed in which the agarose beads (poly (I) -poly (C) were added to lysates labeled with 32P, as described above. incubation for 30 minutes at 4 ° C, the beads were washed, and the bound proteins were released and analyzed by SDS-PAGE. The results show that the 65 kDa phosphoprotein produced in the NIH 3T3 cell lysates of the postinfection, were able to bind to dsRNA; such a link is a well-recognized feature of PKR. In contrast, the 100 kDa phosphoprotein detected in the cell line transformed by infected H-ras not bound to the Poly (I) -poly (c) agarose. The 65 kDa phosphoprotein was also immunoprecipitable with a PKR-specific antibody (provided by Dr. Mike Mathews, Cold Spring Harbor Laboratory), which confirms that it was actually PKR.

EXAMPLE 7. Inactivation of PKR or Deletion Results in Improved Infectibility of Untransformed Cells If the phosphorylation of PKR is responsible for the closure of the viral gene transfer in 'NIH-3T3 cells, and one of the functions of the oncogene product (s) activated in the transformed cells is the prevention of this case of phosphorylation, then the inhibition of PKR phosphorylation in NIH-3T3 cells by other means (eg, drugs) should result in the improvement of viral protein synthesis, and therefore infection, in these cells. To test this idea, 2-aminopurine was used. This drug has been shown to possess relatively specific inhibitory activity towards PKR autophosphorylation (Samuel, CE and Brody, M., (1990) Virology, 176: 106-113; Hu, Y. and Conway, TW (1993), J. Inferieron Res., 13: 323-328). Accordingly, NIH 3T3 cells were exposed to 5 mM 2-aminopurine commonly with exposure to reovirus. The cells were labeled with [35 S] methionine for 12 to 48 h post-infection, and the lysates were collected and analyzed by SDS-PAGE. The results demonstrate that exposure to 2-aminopurine resulted in a significantly higher level of viral protein synthesis in NIH 3T3 cells (not shown). The improvement was pronounced particularly after the immunoprecipitation of lysates with an anti-reovirus serum. These results demonstrate that phosphorylation of PKR leads to inhibition of translation of the viral or viral gene, and that the inhibition of this phosphorylation event releases the translation or transfer block. Therefore, the intermediates in the Ras signaling pathway negatively regulate PKR, leading to the improvement of the infectious quality of the cells transformed by Ras. Interferon-ß, known to induce expression of PKR, was found to significantly reduce the replication of the reovirus in cells transformed by Ras (data not shown). A more direct approach to defining the role of PKR in reovirus infection is through the use of cells that are free of PKR. Accordingly, the primary embryo fibroblast from mouse PKR V + and PKR ° / wild-type mouse (Yang, Y.L. et al. EMBO J. 14: 6095-6106 (1995)) were compared in terms of susceptibility to reovirus infection. The results clearly show that the reovirus proteins were synthesized at a significantly high level in the PKR ° / ° cells than in the PKR + / + cells. These experiments demonstrate that inactivation of PKR or deletion improves the susceptibility of the host cell to reovirus infection in the same way as transformation by Ras or elements of the Ras signaling path, thereby providing strong function support. of elements of the signaling path of Ras in the negative regulation of PKR.

EXAMPLE Inactivation of PKR in Transformed Cells Does Not Involve MEK Tyrosine receptor kinases such as EGFRs are known to stimulate kinases regulated by extracellular signal or activated by mitogens (ERK1 / 2) via Ras (see Robinson, M.J. and Cobb, M.H., Curr. Opin. Cell.

Biol. 9: 180-186 (1997)). This stimulation requires the phosphorylation of ERK1 / 2 by the kinase regulated by mitogen-activated extracellular signal, MEK kinase, which itself is activated (phosphorylated) by Raf, a serine-threonine kinase from below Ras. To determine whether the MEK activity was required for the inactivation of PKR in transformed cells, the effect of the recently identified EK inhibitor PD98059 (Dudley, DT et al., Proc. Nati. Acad. Sci. USA 92: 7686- 7689 (1995); Waters, SD et al., J. Biol. Chem. 270: 20883-20886 (1995)) in cells transformed by infected Ras. Cells transformed by H-Ras were cultured at 80% confluence and were infected with reovirus at one m.o.i. of about 10 p.f .u. /cell. PD98059 (Calbioche), dissolved in dimethylsulfoxide (DMSO), was applied to the cells at the same time as the virus (final concentration of PD98059 was 50 μM). The control cells receive an equivalent volume of DMSO. Cells were labeled with 35S-methionine for 12 to 48 hours post-infection. The lysates were then prepared, immunoprecipitated with the serotype 3 anti-reovirus polyclonal serum and analyzed by SDS-PAGE. The results (data not shown) show that PD98059, in a concentration that effectively inhibits the phosphorylation of ERK1 / 2, does not inhibit the synthesis of reovirus protein in the transformed cells. In contrast, treatment of PD98059 consistently causes a slight improvement in viral protein synthesis in these cells; The reason for this is under investigation. Consistent with the lack of inhibition of viral protein synthesis in the presence of PD98059, the PKR in these cells remains unphosphorylated (data not shown). As expected, PD98059 has not been effected on phosphorylation of PKR induced by reovirus in non-transformed NIH 3T3 cells (data not shown). These results indicate that MEK and ERK1 / 2 are not involved in the activation of PKR.

EXAMPLE 9. In Vivo Oncolytic Capacity of Reovirus A "severe combined immunodeficiency host tumor (SCID) model was used to test the efficacy of reovirus utilization for tumor reduction." Female and Male Mice with SCID (Charles River, Canada) were injected with NIH 3T3 mouse fibroblasts transformed by v-erbB (designated THC-11 cells) in two subcutaneous sites that suffer from hind legs. In a first assay, an injection bolus of 2.3 X 105 cells in 100 μl of sterile PBS was used. In a second trial, an injection bolus of 4.8 X 10 6 cells in 100 μl of PBS was used. The palpable tumors were evident approximately two to three weeks post-injection. Three serotypes of reovirus (Dearing strain) were injected into the right-sided tumor mass (the 'mass of the treated tumor') in a volume of 20 μl at a concentration of 1.0 × 10 9 units (PFU) / ml forming plaques The tumor mass on the left side (the 'mass of the untreated tumor') was not treated. Mice were observed for a period of seven days followed by injection with reovirus, measurements of tumor size were taken every two days using calibrators, and the weight of the tumors was measured after slaughter of the animals. All mice were sacrificed on the seventh day. The results were shown in Table 1.

Table 1 Tumor Mass after Reovirus Treatment The mass of the treated tumor was 47% of that of the untreated tumor mass in trial 1, and 31.6% of the mass of the untreated tumor in trial 2. These results indicate that tumors treated with virus are substantially smaller. that untreated tumors, and that they may have an additional systemic effect of the virus on the mass of the untreated tumor.

Similar experiments were also conducted using unilateral introduction of the tumor cells. The SCID mice were injected subcutaneously and unilaterally into the hind paw with NIH 3T3 mouse fibroblasts transformed with v-erbB (THC-11 cells). The palpable tumors (average area 0.31 cm2) were established after two weeks. Then, eight animals were given a single intratumoral injection of 1.0 x 107 PFUs of reovirus serotype 3 (Dearing strain) in phosphate buffered saline.

(PBD). Control tumors (n = 10) were injected with equivalent amounts of UV inactivated virus. The development of the tumor was followed for 12 days, during which time no additional reovirus treatment was administered. The results, shown in Figure 2, demonstrate that treatment of these tumors with a single dose of active reovirus (empty circulations) results in dramatic suppression of tumor growth by 13 days (end point), when tumors in animals of control injected with a single dose of inactivated reovirus (filled circles) exceeds the acceptable tumor weight. This experiment was repeated several times and found to be highly reproducible, thus demonstrating in addition the efficacy of reovirus in suppressing tumor growth.

EXAMPLE 10. In Vivo Oncolytic Capacity of Reovirus Against Cell Lines Derived from Human Breast Cancer In vivo studies were also performed using carcinoma cells from the human breast in a SCID mouse model. The female SCID mice were injected with 1 x 109 MDAMB468 cells in two subcutaneous sites, suffering both hind legs. The palpable tumors were evident approximately two to four weeks post injection. Serotype three of undiluted reovirus (Dearing strain) was injected into the tumor mass of the right side in a volume of 20 μl at a concentration of 1.0 x 1010 PFU / ml. Results are shown in table 2.

Table 2 Tumor Mass After Treatment with Reovirus * Note: One of the control mice died early during the treatment phase. None of the mice treated with reovirus died.

Although these studies were preliminary, it was clear that the size of the tumors in animals treated with reovirus was substantially lower than that in the untreated animals. However, the size of the tumors on the right (treated) side of the animals treated with reovirus was slightly higher on average than the left side (untreated). This was unexpected but can be explained by the composition of the mass that is taken up by the inflammatory cells with subsequent fibrosis, as well as by the fact that these tumors were originally larger on the right side than on average. the left one The histological composition of the tumor masses is being investigated. These results also support the systemic effect that the reovirus has on the size of the untreated tumor on the contralateral reovirus injection slide.

EXAMPLE 11. Susceptibility of Human Tumors Additional to Reovirus Oncolysis In view of the in vivo results present above, the oncolytic capacity observed in murine cells was investigated in cell lines derived from additional human tumors.

A. Materials and Methods Cells and Viruses All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). The Dearing serotype 3 strain of reovirus used in these studies was propagated in suspension cultures of L cells and purified according to Smith (Smith, RE et al., (1969) Virology, 39: 791-800) with the except that ß-mercaptoethanol (ß-ME) was omitted from the extraction buffer. The reovirus labeled with [35S] methionine was cultured and purified as described by McRae and Joklik (McRae, M.A. and Joklik, W.K., (1978) Virology, 89: 578-593). The particle / PFU ratio for purified reovirus was typically 100/1.

Cytotoxic effects of reovirus in cells Confluent monolayers of cells were infected with reovirus serotype 3 (Dearing strain) at a multiplicity of infection (MOI) of approximately 40 plaque-forming units (PFU) per cell. The images were taken at 36 hours postinfection for both cells infected by reovirus as infected in a simulated manner.

Immuno-luorescence analysis of reovirus infection For immunofluorescent studies the cells were grown on coverslips, and were infected with reovirus at a multiplicity of infection (MOI) of ~ 10 PFU / cell or infected by simulation as described above. In several postinfection periods, the cells were fixed in a mixture of ethanol / acetic acid (20/1) for 5 minutes, then rehydrated by subsequent washes in 75%, 50% and 25% ethanol, followed by 4 washes with solution salt buffered with phosphate (PBS). The fixed and rehydrated cells were then exposed to the primary antibody (rabbit polyclonal anti-reovirus type 3 serum diluted 1/100 in PBS) for 2 hours at room temperature. Followed by 3 washes with PBS, the cells were exposed to the secondary antibody [fluorescein isothiocyanate conjugate (FITC) of IgG (complete molecule) goat anti-rabbit diluted 1/100 in PBS containing 10% goat serum and 0.005% of contrasted Evan's Blue] for 1 hour at room temperature. Finally, the fixed and treated cells were washed 3 more times with PBS, followed by 1 wash with distilled water twice, dried and mounted on slides in 90% glycerol containing 0.1% phenylenediamine, and observed with a Zeiss microscope Axiophot mounted with a Carl Zeiss camera (the magnification for all images was 200 x).

Cell infection and virus quantification Confluent monolayers of cells cultured in 24-well plates were infected with reovirus at an estimated multiplicity of 10 PFU / cell. After one hour of incubation at 37 ° C, the monolayers were washed with DMEM-10% hot FBS, and then incubated in the same medium. In several post-infection periods, a mixture of NP-40 and sodium deoxycholate was added directly to the medium in the infected monolayers at final concentrations of 1% and 0.5%, respectively. The lysates were harvested and the viruses produced were determined by plaque titration in L-929 cells.

Radiolabelling of cells Infected by reovirus and preparation of lysates The confluent monolayers of cells were infected with reovirus (MOI ~ 10 PFU / cell). In several postinfection periods, the average was placed with methionine-free DMEM containing 10% dialyzed PBS and 0.1 mCi / ml [35 S] methionine. After further incubation for 1 hour at 37 ° C, the cells were washed in phosphate-buffered saline (PBS) and treated with lysis in the same buffer containing 1% Triton X-100, 0.5% deoxycholate sodium and 1 mM EDTA. The nuclei were then removed by low speed centrifugation and the supernatants were stored at 70 ° C until use.

Immunoprecipitation and analysis of SDS-PAGE Immunoprecipitation of lysates from reovirus-infected cells labeled with [35S] serotype 3 reovirus was performed, as previously described (Lee, P.W.K., et al (1981) Virology, 108: 134-146). The immunoprecipitates were analyzed by discontinuous SDS-PAGE according to the protocol of Laemmli (Laemmli, U.K., (1970) Nature, 227: 680-685).

B. Breast Cancer The c-erbB-2 / neu gene encodes a transmembrane protein with extensive homology to EGFR that is overexpressed in 20-30% of patients with breast cancer (Yu, D. et al. (1996) Oncogene 13: 1359) . Since it has been established here that the activation of Ras, either through the point of mutations or through cascade elements of increased signaling upstream of Ras (including the c-erbB-2 / neu EGFR homolog) finally creates a Hospital environment for reovirus replication, an array of cell lines derived from human breast cancers were tested for susceptibility to reovirus. The cell lines include MDA-MD-435SD (deposit of ATCC HTB-129), MCF-7 (deposit of ATCC HTB-22), T-27-D (deposit ATCC HTB-133), BT-20 (deposit of ATCC HTB-19), HBL-100 (deposit of ATCC HTB-124), MDA-MB-468 (deposit of ATCC, HTB-132), and SKBR-3 (deposit of ATCC HTB-30). Based on the induction of cytopathic effects, and viral protein synthesis as measured by radioactive metabolic labeling and immunofluorescence as described above, it is found that five of seven of the breast cancers tested were susceptible to infection by -reovirus: MDA- MB-435S, MCF-7, T-27-D, MDA MB-468, and SKBR-3 were exquisitely sensitive to infection, whereas BT-20 and HBL-100 did not demonstrate infectivity.

C Glioblastoma of the Brain Next, a variety of cell lines derived from glioblastomas of the human brain were investigated. The cell lines include A-172, U-118, U-178, U-563, U-251, U-87 and U-373 (the cells were a generous graft from Dr. Wee Yong, University of Calgary). Six of seven glioblastoma cell lines demonstrated susceptibility to reovirus infection, including U-118, U-178, U-563, U-251, U-87 or U-373, while A-172 does not demonstrate any infectibility , as measured by cytopathic effects, immunofluorescence and labeled with [-35S_] -methionine of reovirus proteins. The glioblastoma cell line U-87 was further investigated. To test the sensitivity of U-87 cells to reovirus, U-87 cells (obtained from Dr. Wee Yong, University of Calgary) were cultured at 80% confluence and tested with reovirus at a multiplicity of infection (MOI ) of 10. Within a period of _48 hours there was a broad, dramatic cytopathic effect (data not shown). To further demonstrate that the lysis of these cells was due to the replication of resvirus, then the cells were pulse labeled with [35 S] methionine for periods of three hours at various post-infection periods and the proteins were analyzed by polyacrylamide gel electrophoresis. sodium disodium sulfate (SDS-PAGE) as described above. The results (not shown) clearly demonstrate the replication of the effective reovirus within these cells with penetration resulting from the synthesis of host protein for 24 hours post-infection. U-87 cells were also introduced as xenografts of human tumors in the hind legs of 10 SCID mice. U-87 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, as described above. The cells were harvested, washed, and resuspended in sterile PBS; 2.0 x 10 6 cells in 100 μl were injected subcutaneously at a site below the hind paw in male SCID mice five to eight weeks of age (Charles River, Canada). Tumor growth was measured twice weekly for a period of four weeks. The results, shown in Figure 3, demonstrated that the treatment of U-87 tumors with a single intratumoral injection of 1.0 x 107 PFUs of live reovirus (empty circles, n = 5) resulted in drastic suppression of tumor growth, including regression of the tumor by the fourth week post-treatment (P = 0.008), compared to treatment of tumors with a single intratumoral injection of the same amount of UV-inactivated reovirus (full circles, n = 5). Staining by Hematoxylin / eosin (HE) of the remaining microfoci of tumors treated with active virus, performed as described (H. Lyon, Cell Biology, A Laboratory Handbook, JE Celis, ed., Academic Press, 1994, p.232 ) reveals that the remaining tumor mass consists largely of normal stroma without appreciable numbers of viable tumorigenic cells, nor was any evidence of infiltration of tumorigenic cells in fundamental skeletal muscle (data not shown). The necrosis of tumorigenic cells was due to direct lysis by the virus, the same mechanism of death in the cells as by reovirus in vitro. To determine if there was further viral expansion of the tumor mass, immunofluorescent microscopy using antibodies directed against total reovirus proteins was conducted as described above, in sections of the tumor and tissue attachment. It was found that reovirus-specific proteins are confined to the tumorigenous mass; no substance of viral coloration was detected in the skeletal muscle dependent (data not shown). As expected, the viral proteins were not present in tumors injected with the virus inactivated by UV (data not shown). These results demonstrate that the replication of reovirus in these animals was highly specific tumor with viral amplification only in white U-87 cells. Since more tumors are highly vasculized, it was likely that some viruses could enter the bloodstream followed by lysis of the cells of infected tumors. To determine if there was systemic expansion of the virus, blood was collected from the treated and control animals, and severely diluted by subsequent plaque titration. It was found that infectious viruses are present in the blood at a concentration of 1 x 105 PFUs / ml (data not shown). The high degree of specificity of the virus tumor, combined with systemic expansion, suggests that the reovirus might be able to replicate in glioblastoma tumors remote from the initially infected tumor, as demonstrated above with respect to breast cancer cells. To verify this hypothesis, the SCID mice were implanted bilaterally with xenografts of U-87 human tumors in sites under each hind foot of the animals. These tumors were allowed to grow until they measured 0.5 x 0.5 cm. The tumors on the left side were then given a unique reovirus infection in treated animals (n = 5); Control animals (n = 7) were treated by simulation with UV inactivated virus. The tumors were again measured twice a week for a period of four weeks. The results, shown in Figure 4, demonstrate that inhibition and eventual regression of both untreated (squared) and treated (circles) tumorigenic masses occur only in animals treated with live reovirus (squares and empty circles), in contrast to animals treated with inactivated reovirus (squares and filled circles). Subsequent immunofluorescent analysis reveals that the reovirus proteins were present in both the ipsilateral (treated) tumor and the contralateral (untreated) tumor, indicating that the regression on the untreated side was a result of oncolysis by reovirüs (data not shown ).

D. Pancreatic carcinoma The cell lines derived from pancreatic cancer were investigated for their susceptibility to reovirus infection. The cell lines include Capan-1 (deposit of ATCC HTB-79), BxPC3 (deposit ATCC CRL-1687), MIAPACA-2 (deposit of ATCC CRL-1420), PANC-1 (deposit of ATCC CRL-1469), AsPC -1 (deposit of ATCC CRL-1682) and Hs766T (deposit of ATCC HTB-134). Five of these six cell lines demonstrate susceptibility to reovirus infection including Capan-1, MIAPACA-2, PANC-1, AsPC-1 and Hs766T, while BxPC3 shows little infectivity when verified by cytopathological effects induced by virus, immunofluorescence and [35S] -labelled. Interestingly, four of the five cell lines demonstrating susceptibility to reovirus oncolysis have been shown to possess transformation mutations at codon 12 of the K-ras gene (Capan-1, MIAPACA-2, PANC-1 and AsPC-1). ) while one that lacks susceptibility (BxPC3) has been shown to lack mutation (Berrozpe, G., et al. (1994), Int. J. Cancer, 58: 185-191). The status of the other K-ras codons is commonly unknown by the Hs766T cell line.

EXAMPLE 12, Use of Reovirus as an Oncolytic Agent in Animals Competent to Immunity A syngeneic mouse model was developed to investigate the use of reovirus in animals competent to immunity more than in SCID mice as described above. C3H mice (Charles River) were implanted subcutaneously with 1.0 x 107 PFUs of ras-transformed C3H cells (a graft from D. Edwards, University of Calgary). Followed by the establishment of the tumor, the mice were treated with a series of injections of already. either live reovirus (1.0 x 108 PFUs) or reovirus inactivated by UV. Following an initial series (six injections during a nine-day course), the test animals received a diluted reovirus treatment (1.0 x 107 PFUs) every second day. The animals treated by simulation received an equivalent amount of virus inactivated with UV. The results demonstrate that reovirus was an effective oncolytic agent in these immune competent animals. All test animals showed regression of tumors; 5 of the 9 test animals exhibited complete regression of the tumor after 22 days, a point at which the control animals exceeded the acceptable tumor weight. In addition, lateral effects were not identifiable in the animals treated with reovirus. Verifications of the effects of previous reovirus exposure on tumor suppression and regression, one half of a test group was tested with reovirus (intramuscular injection of 1.0 x 108 PFUs, type 3 Dearing) prior to tumor establishment. Two weeks after challenge, neutralizing antibodies could be detected in all exposed animals. Following the establishment of the tumor, the animals were treated with a series of either UV or live inactivated reovirus, as described above. The results (data not shown) demonstrated that animals with circulating neutralizing antibodies to reovirus (i.e., those tested with reovirus prior to tumor establishment) exhibit tumor suppression and regression similar to those animals in which. there was no previous exposure to reovirus. Thus, reovirus can serve as an effective oncolytic agent even in animals competent in immunity with prior exposure to reovirus. While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail can be made here without departing from the spirit and scope of the invention. invention as defined by the appended claims.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property

Claims (43)

REI INDICATIONS

1. Use of a reovirus for the manufacture of a medicament for the treatment of a Ras-mediated neoplasm in a mammal.

2. The use according to claim 1, wherein the reovirus is a human reovirus.

3. The use according to claim 2, wherein the human reovirus is selected from the group consisting of: reovirus type 1, reovirus type 2, and reovirus type 34.

The use according to claim 1, wherein the reovirus is a non-human reovirus.

5. The use according to claim 4, wherein the non-human reovirus is selected from the group consisting of: mammalian reovirus and bird reovirus.

6. The use according to claim 1, wherein more than one type of reovirus is administered.

7. The use in accordance with the claim 1, wherein more than one strain of reovirus is administered.

8. The use according to claim 1, wherein the reovirus is an isolated field

9. The use according to claim 1, wherein the reovirus is treated with a protease prior to administration.

10. The use according to claim 1, wherein the neoplasm is a solid neoplasm.

11. The use according to claim 1, wherein the neoplasm in a hematopoietic neoplasm.

12. The use, according to claim 1, wherein the mammal is selected from the group consisting of: mice, dogs, cats, sheep, rams, cows, horses, pigs, and non-human primates.

13. The use according to claim 1, wherein the mammal is a human.

14. The use according to claim 1, wherein the neoplasm is selected from the group consisting of: pancreatic cancer, breast cancer and brain cancer.

15. The use according to claim 1, wherein the neoplasm is selected from the group consisting of: lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, and leukemia.

16. The use according to claim 1, wherein the ras-mediated neoplasm is metastatic.

17. A method for the treatment of a Ras-mediated neoplasm in a mammal, characterized in that it comprises administering to the neoplasm a reovirus in an amount sufficient to result in reovirus-mediated oncolysis of neoplasm cells.

18. The method according to claim 17, characterized in that the reovirus is a human reovirus.

19. The method according to claim 18, characterized in that the human reovirus is selected from the group consisting of: reovirus type 1, reovirus type 2, and reovirus type 3.

20. The method according to claim 16, characterized in that the reovirus is a non-human reovirus.

21. The method according to claim 20, characterized in that the reovirus is selected from the group consisting of: mammalian reovirus and bird reovirus.

22. The method according to claim 17, characterized in that more than one type of reovirus is administered.

23. The method according to claim 17, characterized in that more than one strain of reovirus is administered.

24. The method according to claim 17, characterized in that the reovirus is an isolated field.

25. The method according to claim 17, characterized in that the reovirus is treated with a protease prior to administration.

26. The method according to claim 17, characterized in that the neoplasm is a solid neoplasm.

27. The method according to claim 26, characterized in that the reovirus is administered by injection into the solid neoplasm.

28. The method according to claim 26, characterized in that the reovirus is administered intravenously in the mammal.

29. The method according to claim 17, characterized in that the neoplasm is a hematopoietic neoplasm.

30. The method according to claim 29, characterized in that the reovirus is administered intravenously in the mammal.

31. The method according to claim 29, characterized in that the reovirus is administered intraperitoneally in the mammal.

32. The method according to claim 17, characterized in that the mammal is selected from the group consisting of mice, dog, cats, sheep, goats, cows, horses, pigs, and non-human primates.

33. The method according to claim 17, characterized in that the mammal is a human.

34. The method according to claim 17, characterized in that the neoplasm is selected from the group consisting of: pancreatic cancer, breast cancer and brain cancer.

35. The method according to claim 17, characterized in that the neoplasm is selected from the group consisting of: lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, and leukemia.

36. The method according to claim 17, characterized in that approximately 103 to 1012 units forming plaques of reovirus. they are administered

37. The method according to claim 17, characterized in that the reovirus is administered in a single dose.

38. The method according to claim 17, characterized in that the reovirus is administered in more than one dose.

39. The method according to claim 17, characterized in that the reovirus is administered to more than one neoplasm in the mammal. 0

40. The method according to claim 17, characterized in that the ras-mediated neoplasm is metastatic.

41. - The method according to claim 40, characterized in that the reovirus is administered to a single solid neoplasm.

42. The method according to claim 40, characterized in that the reovirus is administered intravenously. ,

43. A method for treating a Ras-mediated neoplasm in a human, characterized in that it comprises administering to the neoplasm a reovirus in a sufficient amount that results in reovirus-mediated oncolysis of neoplasm cells.