TWI302458B - Pharmaceutical compositions and kits for preventing reovirus recognition for the treatment of cellular proliferative disorders - Google Patents

Description

1302458 A7 B7 V. INSTRUCTIONS OF THE INVENTION (Background of the Invention) The present invention relates to a method for avoiding rectal virus identification for treating a cell proliferative disorder in a mammal, in particular a ras-mediated cell proliferative disorder. The invention provides a proliferative disorder in a mammal which is immunosuppressed or immunodeficient by a reovirus. Repression, suppression or induction of immunodeficiency in a mammal may make the reovirus more effective. The method comprises selectively excluding a systemic agent that may interfere with the virus. The immune component is transmitted; the host immune system is prevented from recognizing the reovirus; and after the reovirus treatment, the virus is excluded from the host which is immunosuppressed or immunologically unsuitable. References The following publications, patent applications and applications are extracted from the application. Patent case: US Patent No. 5,023,252

Armstrong, GD et al. (1984), Virology 138:37 Aronheim, A., et aL, (1994) Cell, 75:949-961 Barbacid, M., Annu. Rev, Biochem., 56:779-827 ( 1987)

Berrozpe, G., et al (1994), Int. J. Cancer, 55: 185-191 Bischoff, J.R. and Samuel, C.E., (1989) Virology, 772:106-115

Bos, J. (1989) Cancer Res. 49:4682

Cahill, M.A., etaL, Curr Biol, 6:16-19 (1996)

Chandron and Nibert, "Protease cleavage of reovirus capsid protein mul and mu 1C is blocked by alkyl sulfate detergents, yielding a new type of infectious subvirion particleM, 7. of Virology 72(1):467-75 (1998) Applicable to China National Standard (CNS) A4 specification (210x 297 mm) 1302458 A7 B7___ V. Invention description (2)

Chaubert, P. et al (1994), Am. 7. Path, 144:161

Cuff et al., "Enteric reovirus infection as a probe to study immunotoxicity of the gastrointestinal tractM Toxicological Sciences 42(2):99-108 (1998)

Der, S.D. etal, Proc. Natl Acad. ScL USA 94:3279-3283 (1997)

Dudley, D.T. etaL, Proc. Natl Acad. ScL USA 92:7686-7689 (1995)

Duncan et al., "Conformational and functional analysis of the C-terminal globular head of the reovirus cell attachment proteinr, Virology 182(2):810-9 (1991)

Fields, B.N. et al, (1996), Fundamental Virology, 3rd Edition, Lippincott-Raven

Gentsch, J.R.K. and Pacitti, A.F. (1985), J. Virol. 56:356 E. Harlow and D. Lane, "Antibodies: A laboratory manual", Cold Spring Harbor Laboratory (1988)

Helbing, C.C. etalt Cancer Res. 57:1255-1258 (1997)

Hu, Y. and Conway, T.W. (1993), /. Interferon Res., 75:323-328 4

James, P.W., et al (1994) Oncogene 9:3601

Laemmli, U;K., (1970) Nature, 227:680-685

Lee. J.M. et al. (1993) PNAS 90: 5742-5746

Lee, P.W.K. et aL (1981) Virology, 705:134-146

Lee, P.W.K. et aL (1999) Reovirus for the Treatment of Neoplasia, PCT International Application No. PCT/CA98/00774

Levitzki, A. (1994) Eur. J. Biochem. 226:1

Lowe. S.W. etal (1994) Science, 266:807-810

Lyon, H., Cell Biology, A Laboratory Handbook, JE Celis, ed., Academic Press, 1994, p. 232 -5- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 x 297 mm) 1302458 A7 B7 V. Description of invention (3)

Mah et al., 'The N-terminal quarter of reovirus cell attachment protein sigma 1 possesses intrinsic virion-anchoring functioiT 179(1): 95-103 (1990)

McRae, M.A. and Joklik, W.K., (1978) Virology, 59:578-593

Millis, NE et al (1995) Cancer Res, 55:1444

Mundschau, L.J. and Faller, D.V., (1992) J. Biol Chem., 267:23092-23098

Nagata, L., et aL, (1984) Nucleic Acids Res., 72:8699-8710

Paul R.W. etal. (1989) Virology 772:382-385

Raybaud-Diogene. H. et al (1997)/. Clin, Oncology, 75(3): 1030-1038 1 Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia ί PA 17th ed. (1985)

Robinson, M.J. and Cobb, M.H., Curr, Opin. Cell. Biol. 9:180-186 (1997)

Rosen, L. (1960) /. Hyg, 71:242

Sabin, A.B. (1959), Science 130:966

Samuel, CE-and Brody, M., (1990) Virology, 776: 106-113 Smith, RE et al, (1969) Virology, 59:791-800 Stanley, NF (1967) Br. Med. Bull 25:150 Strong, JE et al., (1993) Virology, 797:405-411 Strong, JE and Lee, PWK, (1996) 7. Virol, 70:612-616 Trimble, WS etal (1986) Nature, 527:782-784

Turner and Duncan, "Site directed mutagenesis of the C-terminal portion of reovirus protein sigma 1:evidence for a conformation-dependent receptor binding domain" Virology 186(1):219-27 (1992)

Waters, SD etal, 7. Biol. Chem. 270:20883-20886 (1995) -6 - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1302458 A7 B7 ___ V. Description of invention ( 4)

Wiessmuller, L. and Wittinghofer, F. (1994), Cellular Signaling 6(3): 247-267 Wong, H., et aL, (1994) Anal Biochem., 225:251*25.8 Yang, YL et al EMBOJ. 74:6095-6106 (1995)

Yu, D. et al (1996) Oncogene 75:1359 All of the above publications, patent applications and patents are hereby incorporated by reference in their entirety as if individually, individually, individually The manner of citation is fully incorporated herein. Related Art Normal cell proliferation is regulated by the balance between the proto-oncogene that promotes growth and the tumor suppressor gene that limits growth. Gene changes into the genomic group lead to mutations in cell elements that control cell signal interpretation, such as strengthening the activity of the primary oncogene or inactivating the action of suppressing the tumor, which can cause tumors to occur. I understand that the most painful interpretation of these signals can affect cell growth and differentiation, and the misinterpretation of these signals causes twin cells to grow (tumor formation). The genetic alteration of the native oncogene Ras is responsible for about 30% of all human tumors (Wiessmuller, L. and Wittinghofer, F. (1994), Cellular Signaling 6(3): 247-267; Barbacid, Μ. (1987 ) A Rev. Biochem· 56, 779-827). The role of Ras in human oncology depends on the type of tumor. Activating mutations in Ras itself occur in most human malignancies, and are mostly concentrated in pancreatic cancer (80%), sporadic colorectal cancer (40-50%), human lung adenocarcinoma (15-24%), thyroid neoplasms. (50%) and spinal cord leukemia (30%) (Millis, NE et al. (1995) Cancer Res. This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458 A7 B7 V. Invention Description (5) 55:1444; Chaubert, P. et al. (1994), Am. J. Path. 144:767; Bos, J. (1989) Cancer Res. 49:4682). Ras activation can also be confirmed by upstream mitogen elements, mainly tyrosine receptor kinases (TRKs). If these upstream elements have been amplified or overexpressed, they will eventually provide Ras activity due to the signal transduction activity of Ras. Examples include PDGFR in some forms of glioblastoma and c-erbB-2/neu overexpression 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) o Current methods of treating neoplasia include surgery, chemotherapy, and radiation therapy. Surgery is the primary treatment for early cancer; however, many tumors cannot be completely excluded using surgical procedures. In addition, metastatic growth of the tumor may prevent surgery to completely treat cancer. Chemotherapy involves the administration of compounds with antitumor activity such as alkylating agents, antimetabolites, and anti-tumor antibodies. The efficacy of chemotherapy is often caused by serious side effects, including nausea and vomiting, inhibition of bone marrow, kidney damage, and inhibition of the central nervous system. Radiation therapy relies on normal cells to have greater self-repairing ability than neoplastic cells after receiving radiation treatment. However, due to the sensitivity of the tissue surrounding the tissue, radiation therapy cannot be used to treat many types of tumors. In addition, some tumors have developed resistance to radiation therapy and may depend on the oncogene or anti-oncogene status of the cell (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): 103 0-1038). Since there are still shortcomings in the current treatment of neoplasms, there is still a need to treat most cancers. -8 1 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458 A7

Improved method of disease. SUMMARY OF THE INVENTION The present invention is directed to methods for enhancing current respiratory efficacy. Viral Therapy for the Treatment of Proliferative Disorders The present invention relates to a method of preventing rectal virus recognition for the treatment of a mammalian proliferative disorder, in particular a ras-mediated cell proliferative disorder. Milk animals can be selected from the group consisting of dogs, cats, sheep, goats, cows, horses, pigs, mice, humans, and non-human spirits. The method comprises suppressing or inhibiting the immune system of the mammal, and administering the effective amount of one or more of the proliferating cells simultaneously or sequentially under the condition that the proliferating cells are dissolved in a large amount, the method provides the call Enterovirus treats a proliferative disorder in which a mammal is immunosuppressed or immunodeficient. Repressive immunization, suppression of immunity, or immune deficiency in mammals can make reovirus more effective. The method may comprise selectively removing an immune component that may interfere with the systemic transmission of the virus; avoiding recognition of the reovirus by the primary immune system; and excluding the virus from a host that is immunosuppressed or under-immunized after treatment with the reovirus . Alternatively, the resuscitation can be administered to a mammal whose immune system has decreased in response to a large amount of proliferating cells. One or more of the following methods can be used to disrupt the immune system. HI V infection; side effects of chemotherapy or radiation therapy; selective exclusion of B and/or T cell populations; exclusion of antibodies (anti-reoviral antibody all antibodies), and the like. Repression or immunosuppression can be achieved by immunosuppression, immunosuppressive agents or any other method that inhibits the immune system of a mammal or immunocompromised a mammal. When using an immunosuppressive agent, the Chinese National Standard (CNS) A4 specification (210 X 297 mm) can be applied to the -9-paper scale of the reovirus application! 302458 A7

2 2 and ° lactation (4) cause immunosuppression, immunodeficiency or immunosuppression before or at the same time as the reovirus is administered. The primary 2 virus can be a mammalian resuscitation or a bird reovirus. Respiratory disease:: "Modification to remove the outer capsid 'embed the virion in the vesicle or gel I, or to make the coat of the protein H reovirus and / or immune pressure / can be early - dose or multiple dose The administration of a disease can be like a tumor: Includes solid tumors and hematopoietic twins. Repressive immunization results in more effective reovirus treatment. Accordingly, the present invention provides a method of treating a vegetative disorder in a mammal (1) comprising the steps of: a) performing a step selected from the group consisting of: 0 administering an effective amount of immunity to proliferating cells in the mammal a repressor; ii) excluding B-fine or tau cells of the mammal; iii~ excluding the anti-reovirus antibody of the mammal; iv) displacing the antibody of the mammal; v) treating the mammal Administering an anti-anti-reovirus antibody; and vi) suppressing the immune system of the mammal; and b) administering an effective amount of one or more of the mammalian immune system under conditions effective to substantially dissolve the proliferating cell Enterovirus. The present invention also provides a method for treating neoplasm formation in human ras, which comprises suppressing or destroying the immune system of a mammal, and simultaneously or subsequently administering to the tumor a reovirus sufficient to substantially dissolve the tumor cells. . Reovirus can also be injected into or near a solid tumor.

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Red phobia also provides a method of inhibiting metastasis of a tumor in a mammal, which comprises the animal's immunization & system, and simultaneously or subsequently, the reovirus in an amount sufficient to substantially dissolve the tumor cells in the mammal. The present invention also provides a method for treating a tumor of a mammalian suspected ra S, which comprises surgically removing substantially all tumors, suppressing or inhibiting the main immune system of the mammal, and administering an effective amount of call at or near the surgical site. Intestinal disease, causing any residual tumor cells to dissolve. The present invention also provides a pharmaceutical composition comprising an immunosuppressive agent or an immunosuppressive agent such as an anti-anti-reovirus antibody, a reovirus, and an acceptable excipient. A method for excluding a reovirus and another component such as an immunosuppressive agent, a B-cell or a T-cell, or a mammalian anti-reovirus antibody, excluding a mammal Antibody methods, anti-anti-reovirus antibodies, and methods of suppressing the mammalian immune system. The invention also provides a pharmaceutical composition comprising an immunosuppressive or immunosuppressive agent, a modified reovirus, and a pharmaceutically acceptable excipient. Further, the method and pharmaceutical composition of the present invention provide a method for the effective treatment of neoplasia without side effects associated with other forms of cancer therapy. Inhibition or suppression of the immune system increases the ability of reovirus infection and lysing mediators of ras, as it is formed by anti-reovirus antibodies. Since it is not known that the virus is related to disease, the safety issues associated with the planned administration of the virus have been minimized. The above and other objects, features and advantages of the present invention will become apparent from -11-This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1302458 A7 B7 V. Description of the invention (9) Detailed description of the invention We have confirmed that the human reovirus needs to be activated by Ras signal Pathway, cells for infection culture (Strong, JE et al 1998 EMBO J 17:3351) In addition, we have confirmed that reovirus can be used as a lytic tumor cell agent in many animal models (Lee, PWK et al (1999) )). Severe combined immunodeficiency mice (SCID) with tumors formed by V-erbB-transformed murine NIH 3T3 cells or human U 8 7 glial cell tumor cells were treated with virus (Coffey, MC et al 1998 Science 282: 1332). Injecting a virus into the tumor results in tumor regression. We further confirmed that a unilateral injection of reovirus into the ipsilateral tumor of an animal bearing a xenograft U8 7 tumor on both sides resulted in a reduction in the contralateral tumor. This shrinking of tumors at distant tumor locations is the result of systemic dissemination of the virus. To examine the possible effects of a functional immune system on this therapy, an immunologically active mouse model was established. Treatment of immunocompetent C3 mice with tumors formed by ras_transformed CSH-loflQ cells also caused tumor regression, but a series of injections were required (Coffey, MC et al. 1998). Therefore, the concentration and/or frequency of reovirus treatment can be reduced when the virus is administered simultaneously with the immunosuppressant, or when the animal with a reduced immune response is administered. In addition, suppression of animal immunity can improve the rate of response away from the mediators of the synchronous r a s of the injected tumor or systemic delivery. Repressive immunity also facilitates systemic delivery by increasing the bioavailable virus. The present invention relates to a method of treating a proliferative disorder modulated by a mammalian r a s, which is to suppress immunity, suppress immunity or cause immunodeficiency in a mammal, and simultaneously or subsequently administer a reovirus to the proliferating cells. -12- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458 A7 B7 V. Description of invention (10) Reovirus (breathing and enterovirus) is a typeface language, indicating that The virus can be isolated from the intestines from the intestines, but is not associated with any known human condition (Sabin, AB (1959), Science 130: 966). The term "reovirus" refers to all viruses classified as reoviruses. Reovirus is a virus with a double-stranded RN A genome with a virion diameter of 60-80 nm and two central capsids, each in the form of a icosahedron. The genome consists of 10-12 segments of double-stranded RN A with a total genome of 16-27 kbp. Individual RNA segments vary in size. Three different but related reovirus patterns have been recovered from a variety of species. All three types have a common complement-immobilized antigen. The human reovirus includes three serotypes · type 1 (Lang or T 1 L strain), type 2 (Jones strain, T2J) and type 3 (Dearing strain or Abney strain, T3D). These three serotypes are easily identified by neutralization and hemagglutinin inhibition assays (Sabin, Α·Β. (f959), Science 130:966; Fields, BN: et al. (1996), Fundamental Virology, 3rd edition , Edition, Lippincott-Raven; Rosen, L. (1960) Am. J. Hyg. 71:242; Stanley, NF (1967) Br. Med. Bull. 23:150). Although reovirus is not known to be associated with any specific disease, many people have been exposed to reovirus when they grow up (ie, under 25% of children under 5 years old, and over 50% of children under 20-30 years old, Jackson GG And Muldoon RL (1973) J. Infect. Dis. 128:811; Stanley NF (1974) In: Comparative Diagnosis of Viral Diseases, edited by E. Kurstak and K. Kurstak, 385-421, Academic Press, New York) . -13 - This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) 1302458 A7 B7 V. Description of invention (11) The cell surface recognition signal of mammalian enterovirus is sialic acid (Armstrong, GD et al. ( 1984), Virology 138:37; Gentsch, JRK· and Pacitti, AF· (1985), J. Virol. 56:356; Paul RW et al. (1989) Virology 172:382-385). Due to the ubiquitous nature of sialic acid, reovirus can be effectively formed with many cell lines and thus can be targeted against many different tissues; however, there is a significant difference in the susceptibility of reovirus infection between cell lines. As described herein, the Applicant has found that cells that are resistant to reovirus infection are susceptible to reovirus infection when transformed by the Ras pathway gene. Cell "resistance" Reovirus infection means that cells infected with the virus do not produce or release the virus in large quantities. "Therapeutic" cells are cells that have been shown to inhibit cytopathic effects, viral protein synthesis and/or virus production. Cells have been found to resist reovirus infection during the gene translation phase, rather than at the early transcriptional stage; viral transgenes are produced at this time, but no viral protein is present. Without being bound by theory, it is believed that the viral gene transcription of resistant cells is associated with phosphorylation of approximately 65 kDa cellular protein, which was determined to be a double-stranded RN A-activated protein kinase (PKR). Now in the transformed cells. Phosphorylation of P KR inhibited translation. When 2-aminoguanidine (a known PKR inhibitor) suppresses phosphorylation, the recombination protein synthesis of untransformed cells is greatly enhanced. In addition, the severely combined immunodeficiency (SCID) mouse model in which both the left side and the right posterior abdomen form a tumor showed that when the reovirus was injected directly into the right tumor, the tumor was significantly reduced; in addition, the reovirus injection was not directly received. The tumor on the left side was also significantly reduced. This represents the lytic tumor cells of the reovirus -14- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458

The systemic effect of the ability is as good as the local effect. Although reovirus can be effectively used to treat the tumor-producing tumors of immunocompetent mice, 'we have found that to achieve the desired optimal effect, a larger amount (about " times) of reovirus and multiple treatments of beta are required. Intratumoral injection is required to achieve maximum effect on immunocompetent animals. These results indicate that the reovirus uses the Ras pathway of the host cell to turn down to regulate PKR, and then regenerate, turn (regressive virus resistance) and EG=R-, S〇S_ or ras-transformation Viral susceptibility) For both cells, the diseased gene I binds to the virus, internalizes, removes the coating, and early transcription can proceed normally. In the case of untransformed cells, the secondary structure of the early viral transcript cannot prevent the phosphorylation of PKR, thereby activating the phosphorylation of the translation initiation factor eIF-2α, thereby inhibiting viral gene transcription. In the case of EGFR-, s〇s_ or ras-transformed cells, the ρ κ R 嶙 acidification step is blocked or reversed by Ras or one of the downstream elements, thus ensuring viral gene translation. The action of (or downstream elements) can be performed using an aminopurine (2_Ap) mimetic that promotes viral gene translation of untransformed cells (and thus promotes reovirus infection) by blocking PKR phosphorylation. Methods for implanting human tumor cells in SCID mice have been recognized as a well-known model system for testing the effectiveness of various anti-tumor agents in humans. It has been found in the past that the efficacy of a human tumor against a SCID mouse can be predicted to be effective against the same tumor in the human body. Based on these findings, the Applicant has developed a method of treating a vector-borne disease in mammalian r a s. Representative mammals include dogs, green sheep, goats, cows, horses, pigs, mice, non-human primates, and human-15- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458 A7 B7 V. INSTRUCTIONS (13). In a preferred embodiment, the mammal is a human. In the method of the present invention, a reovirus is administered to a proliferating cell of R a s in an individual mammal. In a specific embodiment of the invention, the course of re-intestinal therapy is administered one or more times. After the first administration of reovirus therapy, specific immune components that may interfere with the next reovirus administration are excluded from the patient. Such immune components include B cells, T cells, antibodies, and the like. B cell or T cell populations can be removed using several methods. One of these methods filters blood and dialysates the blood matrix. Another method is to filter blood coupled to an in vitro compound of another removable cell population. For example, mixing with an immobilized antibody that recognizes a specific receptor on a cell population that is planned to be removed. Another way to remove cell populations is to suppress immunity. It can be accomplished using first line radiation therapy or a cyclic steroid such as cyclosporine. Selective removal of anti-reovirus antibodies can also eliminate the patient's immune system to remove the recurrent enterovirus. Avoiding the interaction of antibodies with the virus also contributes to systemic treatment. Antibodies can be removed by several methods, including blood matrix dialysis and passage of blood through immobilized reovirus (selective removal of antibodies); blood matrix dialysis and blood passage through immobilized protein A (commercially available PROSORBA, Cypress Bioscience) , San Diego, CA) to remove all IgG antibodies; or to anthropomorphic anti-genetic antibodies, wherein the genotype is resistant to reovirus. Another method of the present invention is to allow the reovirus to exert a systemic effect by masking or damaging the immunological susceptibility of the reovirus without damaging the normal immune function. In order to avoid the patient's immune system to recognize reovirus, another -16- paper size of this invention applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458

Specific embodiments are _ series administration of reovirus and reovirus recombination or reovirus can be coated with non-viral toxicity anthropomorphic antibodies, such as antibody coated Fab protein, or coated in micelles. . In addition, the virus can be treated with chymotrypsin to produce a salty secondary virion (1_). Lin can be used alone or in combination with a complete virus, and the resulting formulation is not easily recognized by the patient's immune system or has not been previously prevented by the patient's immune system. The present invention is a "35" specific implementation < column comprising a reovirus that removes a patient after administration. Since this method can be used in immunosuppressed or immunocompetent patients, it is necessary to remove the virus in the bloodstream after the treatment process, and the reovirus can utilize the affinity layer method, 彳吏(4) external anti-reovirus antibody, In combination with blood matrix dialysis, B-cell proliferator, or a transfection agent that stimulates an immune response against the virus, such as UV deactivated virus or Freund's adjuvant. ♦ In the present invention, the reovirus is administered to a vector of US mammalian proliferating cells of an individual mammal. Representative human reovirus types that can be used include type 1 (eg, Lang strain or T1L), type 2 (eg, J〇nes)

For species T 2 J ) and 3 (for example, Dearing or Abney species, T 3 D or T3A), other reovirus species may also be used. In a preferred embodiment, the reovirus is human reovirus serotype 3, more preferably, the reovirus is human reovirus serotype 3, Dearing species, or the reovirus can be non-human breastfeeding Animal reovirus (eg, non-human primate reovirus, such as: sputum reovirus; horse or canine reovirus), or non-mammalian reovirus (eg, avian reovirus). Different serum can be used -17-

1302458 A7 B7 V. Combination of inventions (15) and/or combinations of reoviruses of different species (eg, reovirus from different animal species). Reovirus can be naturally occurring or modified. When the reovirus is isolated from the natural source and has not been deliberately modified by humans in the laboratory, it is a "naturally occurring" For example, the reovirus can come from "natural", that is, from a human patient. Reovirus can be modified but still capable of lysogenic infection of mammalian cells with active r a s pathway. The reovirus can be pretreated by chemical or biochemical methods prior to administration of the proliferating cells (e.g., by protease treatment, such as chymotrypsin or trypsin). Protease pretreatment removes the outer sheath or capsid of the virus and increases the infectivity of the virus. Reovirus cleavage of reovirus capsid protein mul and mulC is blocked by alkyl sulfate detergents, yielding a new type of infectious subvirion particle11, J. of Virology 72(1): 467-75 (1998)) to reduce or avoid an immune response in mammals that have developed immunity to reovirus. For example, virions can be treated with chymotrypsin in the presence of an alkyl sulfate detergent which forms a micelle concentration to produce new infective secondary virions. Reovirus can be a recombinant reovirus of two or more reoviruses with different pathogenic phenotypes, and thus contains different antigenic determinants, thereby reducing or avoiding breastfeeding of past exposed reovirus subtypes. The immune response of animals. The recombinant virion (also known as recombination agent) can be produced by co-infection of different subtypes of reovirus by mammalian cells, and the results can be applied to the Chinese National Standard (CNS) A4 specification ( 210 X 297 mm) 1302458 A7 B7 V. INSTRUCTIONS (16) Selecting and introducing different subtypes of coat protein to the virion capsid produced. The virion modification method can be used to mutate proteins (eg, for example, : σΐ) Introduced virions in the outer capsid. Proteins can undergo substitution, intercalation or deletion mutations. The displacement method involves embedding a different amino acid in place of the native amino acid. Interference insertion involves the insertion of other amino acid residues into one or more positions in the protein. Deletion involves deleting one or more amino acid residues in the protein. Such mutations can be generated using methods known in the art. For example, an oligonucleotide-directed mutagenesis of a gene encoding a coat protein produces the desired coat protein. The mammalian cells infected with reovirus produce mutant proteins in vitro (eg, COS1 cells). The mutant proteins are introduced into the reovirus virions (Turner and Duncan, "Site directed mutagenesis of the C-terminal portion of reovirus Protein sigmal: evidence for a conformation-dependent receptor binding domainM Virodogy 186(1):219-27 (1992); Duncan et al., MConformational and functional analysis of the C-terminal globular head of the reovirus cell attachment protein" Virology 182 (2): 810-9 (1991); Mah et al.?, fThe N-terminal quarter of reovirus cell attachment protein sigma 1 possesses intrinsic virion-anchoring function" Virology 179(1): 95-103 (1990)) 0 Preferably, the reovirus is modified to reduce or eliminate the immune response to the reovirus. This modified reovirus is called "immunoprotective reovirus". This modification method includes packaging of reovirus in vesicles, micelles -19 · This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458 A7 __________ B7 V. Description of invention In (17) or other carriers, to mask the reovirus to avoid the immune system of the mammal. Alternatively, the capsid of the reovirus particles can be removed because the protein contained in the capsid is the major determinant of the humoral and cellular responses of the host. Anything that is faster than normal tissue growth ί 1i This "co-cultured cell" refers to a much faster than normal sputum proliferative disorder including (but not limited to): a tumor. Tumors are abnormal tissue growth 'usually forming a separate substance that grows faster than normal tissue by cell proliferation. The tumor is partially or completely lacking in structural organization and functional coordination with normal tissues. A malignant neoplasm produced by the epidermal structure is called a carcinoma. A malignant neoplasm from a connective tissue such as muscle, cartilage, fat or bone is called a sarcoma, and affects the hematopoietic structure (structure associated with blood cell formation) including the immune system. The malignant tumors of the composition are called leukemias and lymphomas. Tumors are tumor growth of cancer. As used herein, "tumor" is also referred to as "tumor" and includes hematopoietic neoplasms and solid tumors. Other proliferative disorders include (but are not limited to, X: neurofibromatosis. At least some cells of proliferative disorders) Mutations in which the Ras gene (or an element of the Ras signaling pathway) is directly activated (eg, activating mutations in Ras) or indirectly activated (eg, activation of upstream elements in the Rus pathway). Activation of upstream elements in the Ras pathway includes, for example, : Transdermal growth factor receptor (EGFR) or Sos transformation. Proliferative disorders caused at least in part by activation of elements in the upstream elements of Ras, Ras or Ras signaling pathways are referred to herein as "Ras·Media proliferative disorders B-cell " refers to B-lymphocytes. B lymphocytes have two major subgroups, B-1 and B-2 cells. B-1 cells can regenerate themselves and often secrete high levels of anti-20- The paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458 A7 B7 V. Description of the invention (18) The combination of multiple bodies with multiple prion (multiple specificity) but the binding is quite low. Most B-cells For B'2 The cell produces a precursor directly from the bone marrow and secretes a highly specific antibody. T-cell refers to: a τ-lymphocyte, which differentiates in the cell gland and is specialized to fight cells with intracellular organisms. Τ_ cells can only recognize antigens on the surface of body cells. ''Anti-reovirus antibody" refers to antibodies that bind to reovirus."Ig (j antibody, immunoglobulin G antibody, IgG is the most abundant antibody) Type, Wang wants to neutralize bacterial toxins and bind to microorganisms to enhance their phagocytic effects. "Anthropomorphic antibodies" means that the amino acid sequence in the non-antigen binding region of the antibody molecule has changed, making the antibody more human-like. Body, but still retain its original binding ability. In particular, it can be felt that one of the tumors of the present invention is pancreatic cancer, because Ras-mediated tumors are generally associated with pancreatic cancer. In particular, he can feel the heart of the present invention. Tumors include breast cancer, central nervous system cancer (eg, cerebral blastoma and glioblastoma), peripheral nervous system cancer, lung cancer, prostate cancer, and rectal cancer , thyroid cancer, kidney cancer, gluteal adenocarcinoma, liver cancer, lymphoma and leukemia. It is particularly sensitive to the treatment of the present invention. Other proliferative disorders include neurofibromatosis due to activation of the ras pathway. To proliferating cells or tumors, indicating that the reovirus is administered in contact with cells of proliferating cells or tumors (also known as "neoplastic cells,"), the route of administration of reovirus, and the formulation The carrier or carrier will depend on the location and type of the tumor. There are many routes of administration that can be used. For example, treatment can be - 21 - ^^i^(CNS) A4^(210X297^) 1302458

When the solid tumor is reached, the reovirus can be injected directly into the tumor. Where = hematopoietic tumor, for example: intravenous or intravascular injection of two. When dealing with tumors that are not easily accessible in the body, such as metastasis or brain tumors, the method and means of resuscitation of the venom are transmitted to the body through the body of the mammal (for example, intraspinal, intravenous or intramuscular) ). Alternatively, resuscitation can be directly administered to a single solid tumor and then transmitted to the body through the body. 、·········································································································· For example, when treating a colorectal neoplasm, when administered intravaginally (for example, when treating a cervical or vaginal tumor), intranasally or by inhalation (for example, when a pulmonary tumor is present). , Enterovirus can be systemically administered to a mammal that is immunocompromised or has not developed immunity to the reovirus antigen. In this case, the reovirus can be administered systemically, ie, by intravenous injection, it can be combined with proliferating cells: the cells are dissolved. Immunologically active mammals that have been exposed to reovirus subtypes in the past can develop humoral and/or cellular immunity to reovirus subtypes. Despite this, it has been found that when direct injection of enteropathy 4 to free active mammalian tumors, it causes twin cells to dissolve. On the other hand, when the reovirus is administered systemically to an immunocompetent mammal, the mammal will immunoreact with the reovirus. If the reovirus is a subtype in which the mammal has not developed immunity, or if the reovirus has been modified to be immunoprotective as described herein, for example, when the outer capsid is decomposed by a protease or packaged in a micelle This immune response can be avoided. -22- 1302458 A7 B7 V. INSTRUCTIONS (20) Alternatively, by prior or concurrently administering medicines known to suppress the general immune system (Cuff et al., "Enteric reovirus infection as a probe to study Immunotoxicity of the gastrointestinal tract "Toxicological Sciences 42(2):99-108 (1998)) or administration of such an immunosuppressant as an anti-anti-reovirus antibody can suppress the immune activity of a mammal against reovirus. Humoral immunity of mammals against reovirus can also be temporarily reduced or suppressed by removal of anti-reovirus antibodies by plasmapheresis of mammalian blood. The non-specific immunoglobulin can also be administered intravenously to a mammal to temporarily reduce or suppress the humoral immunity of the mammal against reovirus. The reovirus can be administered to an immunocompetent mammal that has been immunized against reovirus by administering an immunosuppressive agent and/or an immunosuppressant. Such immunosuppressive agents and immunosuppressive agents are known to those skilled in the art and include, for example, cyclosporine, rapamycin, tacrolimus, mycophenolic acid, and sulfonium. Azathioprine and its analogues, and the like. Other formulations having immunosuppressive properties are also known (see, for example, Goodman and Gilma, 7th Edition, page 1242, the contents of which are incorporated herein by reference). Such immunosuppressive agents also include "anti-anti-reovirus antibodies" which are antibodies against anti-reovirus antibodies. Such antibodies can be made according to methods known in the art, see, for example, "Antibodies: A laboratory manual(R) E. Harlow and D. Lane, Cold Spring Harbor LabQratory (1988). This anti-anti-reovirus antibody can be administered between, simultaneously with or after administration of the reovirus. It is best to administer an effective amount of anti-reovirus antibody for a sufficient period of time to reduce or eliminate lactation. -23- This paper size applies to Chinese National Standard (CNS) A4 size (210 X 297 mm) 1302458

The immune response of the substance to the reovirus. "Immune repressor, or repressive immunologic preparations", including known immunosuppressive agents, immunosuppressive agents, antibodies, and, for example, radiation therapy or HI V infection, which causes damage to the immune system. Dissolve at least 10% of proliferating cells to at least 5 0 /. More preferably, it is best to dissolve at least 7 5 ❶ / ❶ cells. The percentage of tumor cell lysis is determined by measuring the degree of tumor shrinkage or the degree of in vitro tumor cell lysis in a mammal. A mammal suspected of having a proliferative disorder means that the mammal may have a proliferative disorder or tumor or is diagnosed with a proliferative disorder or tumor or has been diagnosed with a proliferative disorder or tumor in the past, and the tumor or substantially all of the tumor has been surgically removed but The mammal is still suspected of remaining some tumor cells. The present invention also encompasses a pharmaceutical composition comprising as an active ingredient one or more immunosuppressive or immunosuppressive agents and one or more reoviruses and π pharmaceutically acceptable carriers or agents The preparation of the composition of the present invention, the active ingredient/immunosuppressor or immunosuppressant and the reovirus are usually mixed with an excipient, and are excipiently selected or embedded in the carrier. It may be in the form of a capsule, a pack, a paper pack or other container. When a pharmaceutically acceptable excipient serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a carrier, carrier or Thus, the composition may be in the form of tablets, pills, powders, buccal tablets, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, An ointment (either as a solid or in a liquid medium), containing an ointment of up to 1% by weight of active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaging powders. Examples include: lactose, dextrose, sucrose, Yamanashi-24 -

1302458 A7 B7 V. INSTRUCTIONS (22) Sugar alcohol, mannitol, starch, acacia gum, calcium, alginate, tragacanth H, calcium citrate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose , sterile water, sugar, and methyl cellulose. The formulation may further comprise: a lubricant such as talc, magnesium stearate, and mineral oil; a wetting agent; an emulsifier and a suspending agent; a preservative such as methyl and propyl hydroxybenzoate; a sweetener; Withered sword. The compositions of the present invention can be formulated, and the active ingredients can be quickly, continuously or continuously released using the relevant art. When a solid composition such as a tablet is prepared, the main active ingredient/immunosuppressive or immunosuppressive agent and the reovirus are mixed with a pharmaceutical excipient to form a solid pre-formulation composition containing a homogeneous mixture of the present invention. When such pre-35ge compositions are said to be homogeneous, it means that the active ingredient is dispersed in the composition so that the composition is readily reconstituted into equivalent effective unit dosage forms such as tablets, pills and capsules. The present invention <Pills or pills may be coated or formulated to form a dosage form which provides long-lasting advantages. For example, tablets or pills may contain inner and outer doses, while the latter may be coated in the form of a jacket. These two ingredients can be separated by an enteric layer to resist disintegration in the stomach and allow the ingredients to pass intact into the duodenum or to be delayed. A wide variety of materials can be used for such enteric layers or coatings, including mixtures of polymeric acids and polymeric acids with materials such as: shellac, fresh ground alcohol, and cellulose acetate. Liquid forms which may comprise the compositions of the present invention for oral or injectable administration include water-soluble, suitably flavored syrups, aqueous or oily suspensions, and flavoring emulsions containing edible oils such as corn oil, Cottonseed oil, sesame oil, coconut oil, or peanut oil, and tinctures and similar pharmaceutical carriers. -25-

This paper size applies to the Chinese National Standard (cns) Α4 specifications ((10) 297 297 gongs) 1302458 A7 B7 V. Description of the invention (23) Compositions for inhalation or insufflation include pharmaceutically acceptable aqueous or organic solvents or Solutions and suspensions in the mixture, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein. Preferably, the composition is administered orally or systemically via the oral or nasal respiratory route. The compositions contained in the preferred pharmaceutically acceptable solvent can be aerosolized using an inert gas. The aerosolized solution can be directly inhaled from the aerosolizing device or the aerosolizing device can be attached to a mask or intermittent positive pressure breathing apparatus. Preferably, the solution, suspension or powder composition can be administered orally or nasally from a device which delivers the formulation in a suitable manner. Another preferred formulation for use in the method of the present invention employs a transdermal delivery device ("stick π") which can be used for continuous or intermittent input control of a reovirus of the invention. The construction and use of the wearable patch is known in the art. See, for example, U.S. Patent No. 5,023,252, which is incorporated herein by reference. Delivery of the agent. Other formulations suitable for use in the present invention can be found in Remington's Pharmaceutical Sciences, the contents of which are incorporated herein by reference. The composition of the reovirus can be packaged into a convenient kit, and the necessary materials are packaged in a suitable container. The kit can also include a chemotherapeutic agent. The immunosuppressive or immunosuppressive agent is administered in an appropriate amount, and an appropriate administration plan is adopted. The foot causes the mammal's immune system to produce immunosuppression or immunosuppression-26- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 1302458 A7 _____B7 V. INSTRUCTIONS (24) Sex, such dosages and schedules are well known to those skilled in the art. The amount of reovirus administered is sufficient to treat proliferative disorders (eg, "effective amount"). When reovirus is administered When proliferating cells are used to dissolve proliferating cells, it is possible to treat & proliferate the disease. The result is to reduce the tumor or completely eliminate the tumor. The tumor shrinks or eliminates the normalization of the neoplastic cells by the reovirus ("lysis of cancer Preferably, the effective amount is an amount which inhibits the growth of tumor cells. Preferably, the effective amount is from about 1.0 fufu/kg body weight to about 10 i5 pfu/kg body weight, from about pfu/kg body weight to about 1013 pfu/ The weight of kilograms is better. For example, when treating human rituals, about 102 to 1 溶 plaque forming units (PFU) of reovirus can be used, depending on the type, size and quantity of the tumor present. It will depend on individual basis, and at least in part on the type of reovirus; the route of administration chosen; the size, age, sex of the individual; the severity of the patient's symptoms; the size and other characteristics of the tumor; The drug process can last from a few days to several months or until the disease is alleviated. Immunoassay or immunosuppressive agents and reovirus can be administered in single or multiple doses (ie more than one dose). Multiple doses can be administered simultaneously. Or sequential administration (eg, over several days or weeks). Reovirus can also administer more than one type of tumor to the same individual. The composition is preferably formulated into unit dosage forms, each dose containing an appropriate amount of immunosuppressive or immunosuppressive And a dose of about 1 〇 2 pfu to about 1013 pfu of reovirus. The term "unit dosage form" refers to a physical component that is suitable for administration to humans and other mammals in unit doses, each unit containing a calculated production unit. A predetermined amount of reovirus that is therapeutically effective, in combination with a suitable pharmaceutical excipient. As mentioned above, 'reovirus has been found to be effective in the treatment of immune-active mammals _ _ - 27 - Ϊ paper scale applicable to China National Standard (CNS) A4 specification (210 X 297 mm) """"" 1302458 A7 B7 V. INSTRUCTIONS (25) Solid tumor of the object. Direct administration of unreacted reovirus to tumors of immunocompetent animals results in the dissolution of neoplastic cells and shrinks the tumor. When an animal causes immunosuppression or immunodeficiency in some ways, systemic administration of reovirus will be more effective in causing tumor cell lysis. Reovirus can be administered in conjunction with surgery or removal of tumors. Accordingly, a method of treating a solid tumor is provided at this time, which includes surgical removal of the tumor and administration of a reovirus near or at the site of the tumor. Reovirus can be combined with or increased radiation therapy, and radiation therapy can suppress the immunity of mammals. Furthermore, the reovirus of the present invention may be administered in combination with or increased by known anticancer compounds or chemotherapeutic agents. Chemotherapeutic agents are compounds that inhibit tumor growth. Such preparations include, but are not limited to, 5-fluorouracil, mitomycin C, amine methotrexate, transurea urea, cyclophosphamide, dacarbazine, mitoxantrone, Anthracyclins (Epirubicinf and Doxumbicin), receptor antibodies such as: herceptin, etopside, pregnasome ), Ming compounds such as: carboplatin (carboplatin) and cisplatin, taxans such as: taxol and taxotere, hormone therapy such as: tamoxifen And anti-estrogen, interferon, aromatase inhibitors, pre-pregnancy agents and LHRH analogues. It has been found that the present invention reovirus and immunosuppressive agents can reduce the growth of metastatic tumors. In a specific embodiment of the invention, there is provided a method of reducing metastatic tumor growth in a mammal comprising administering an effective amount of Reovirus-28- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297) PCT) 1302458 A7

Give immunity to suppressed mammals. Uses Toxic and immunosuppressive agents can be used in a variety of applications. It can be used to eliminate the proliferation of the ::: two media. It can be used to reduce sputum, /, and can be used to treat metastases, which can be used in conjunction with known cancer treatments including surgery, chemotherapy, and radiation. The following clear examples are presented to illustrate the invention and its advantages, but not to limit the invention in any way. EXAMPLES In the following examples, all temperatures are expressed in degrees Celsius (unless otherwise stated) and all weights are by weight (unless otherwise stated). In the following examples, the abbreviations are defined as follows. If the abbreviation is not defined, it is generally acceptable: μΜ = micromolar concentration mM —= millimolar concentration Μ = molar concentration ml = ml β 1 =: microliter mg = mg "g = microgram PAGE Polyacrylamide gel electrophoresis rpm = FBS per minute = fetal bovine serum DTT = dithiothreitol -29 - This paper scale applies to Chinese National Standard (CNS) Α 4 specifications (210X 297 DON)

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1302458 A7 B7 V. INSTRUCTIONS (27) SDS = Teflon-based sulfuric acid steel PBS = phosphate buffered saline DMEM = Dulbecco's modified Eagle's medium a. -MEM = heart-modified Ig's Medium y5-ME = /?-Hydroxythioethanol MOI infection fold PFU plaque forming unit MAPK = MAP kinase phosph-MAPK = phosphorylation - MAP kinase HRP = horseradish peroxidase PKR = double-stranded RNA-activated protein kinase RT -PCR = reverse transcriptase-polymerase chain reaction GAPDH = glyceraldehyde-3-phosphate vinegar 4 dehydrogenase EGFR ~ = epidermal growth factor receptor MEK kinase = mitogen-activated extracellular signal-regulated kinase DMSO = dimethyl Stone Wind SCID = Severe Combined Immunodeficiency General Method Cell and Virus Parental NIH-_3T3 and NIH-3T3 Cell Line Transfected with Harvey_ras (H-ras) and EJ-ras Oncogenes from Dr*· Douglas Faller (Boston University A generous gift from the medical school. NIH-3T3 cells and their Sos-transformed equivalents -30- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 x 297 mm)

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t 1302458 A7 B7 V. Inventive Note (28) (referred to as TNIH#5) is a generous gift from Dr. Michael Karin (University of California, San Diego) 6 Dr. H.-J. Kung (Cas Western Reserve University) Parental NIH-3T3 cells and NIH-3T3 cells transfected with v-erbB oncogene (referred to as THC-11) were given. 2H1 cells are derivatives of the C3H 10T 1/2 murine fibroblast strain containing the Harvey-ras gene regulated by the metal thione-1 promoter of the mouse, which was obtained from Dr. Nobumichi Hozumi (Sinai Hill Hospital Research Institute). ). These 2H1 cells are conditional ras-transformers that express H-ras oncogenes in the presence of 50 vMZnS04. All cell lines were grown in Duchenne modified Ig's medium (DMEM) containing 10% fetal calf serum (F B S ). NIH-3T3 tet-myc cells were obtained from Dr. RN·Johnston (Calgary University) and grown in DMEM containing 10% heat-inactivated FBS and antibiotics with or without 2 μg/ml tetracycline (Helbing CC et al) ·, Cancer Res. 57: 1255-1258 (1997)). When all tetracycline is present, the human c-myc table f is suppressed. When tetracycline is excluded, the performance of c-myC in these cells is increased by a factor of 100, which also exhibits a phenotype of transformation. PKR + wide and PKR. /. Mouse embryonic fibroblasts (MEFs) were obtained from Dr. RG Williams (Cleveland Clinic) and grown from -MEM medium containing fetal calf serum and antibiotics as described above (Yang, YL et al, EMBO) J. 14: 6095-6106 (1995); Der, SD et al., Proc. Natl. Acad. Sci. USA 94: 3279-3283 (1997)). These studies - Deering strains of Reovirus serotype 3 used were propagated in suspension cultures of L cells and purified according to Smith (Smith, Smith et al., (1969) Virology 39:791-800), but Where the extraction buffer is omitted -31 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458 A7 B7 V. Description of invention (29) Different /? - thiol ethanol (/5-ME ). The recombinant virus McRae labeled with [35S]methionine was grown and purified as described by Joklik (McRae, M. A. and Joklik, W. K., (1978) Virology, 89: 578-593). The particle/PFU ratio of the reovirus after purification is typically 100/1. *NIH-3T3, TNIH#5, H-ras, EJ·ras, 2H1 (+/- ZnS04) and THC-11 cells on coverslips when immunofluorescence assay for reovirus infection For growth, about 10 PFU of cells are infected with reovirus according to the multiplicity of infection (MOI), or a vector preparation (phosphate buffered saline, PBS) is applied, and the cells are pseudo-infected in the same manner as the virus is administered to the cells. 48 hours after infection, the cells were fixed in an ethanol/acetic acid (20/1) mixture for 5 minutes, then washed in 75%, 50% and 25% ethanol, then hydrated, then buffered with saline. Wash 4 times with PBS). After fixation and rehydration, the cells were exposed to the primary antibody (multiple rabbit anti-resect scorpion venom type 3 serum, diluted 1/10 PBS in PBS) [antiserum was prepared by injecting rabbits into Freud's Complete adjuvant adjuvant reovirus serotype 3, then blood draw), at room temperature for 2 hours. After washing 3 times with PBS, the cells were exposed to a secondary antibody [goat anti-rabbit IgG (whole molecule)-luciferin isothiocyanate conjugate (FITC) [Sigma Immuno Chemicals F-0382], containing 10 % goat serum was diluted 1/100 with 0.005% Evan's Blue in PBS for 1 hour at room temperature. Finally, the fixed treated cells were washed 3 times with PBS, washed once with double distilled water, dried, placed on a slide containing 90% glycerol of 0.1% phenylenediamine, and observed with a Zeiss Axiophot microscope using a Carl Zeiss camera (all The magnification of the photos is 200 times). -32- This paper size applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458 A7 B7 V. Description of invention (3〇) Detection of MAP kinase (ERK) activity According to the manufacturer's instructions, PhosphoPlus is used. The p44/42 MAP kinase (Thr 202/Tyr 204) antibody kit (New England Biolabs) was assayed for MAP kinase in lysates. Briefly, SDS-PAGE was performed by dissolving the proposed monolayer culture with the SDS-sample buffer containing the solution, and then spotting the ink on nitrocellulose paper. The membrane was tested with a primary antibody (anti-total MAPK or anti-phosphorus-MAPK) according to the manufacturer's instructions for use, followed by horseradish peroxidase (HRP). Conjugated secondary antibody detection. Radiolabeling of cells infected with reovirus and preparation of lysate by fusion of relapsing virus NIH-3T3, TNIH#5, H-ras, EJ-ras, 2H1 (+/ ZnS04) and THC-11 cells Monolayer (M0I about 10 PFU/cell). Twelve hours after infection, the medium was changed to methionine-free, but contained 10% dialyzed FBS and 0.1 mCi/ml [35S]methionine DMEM. After further incubation at 37 ° C for 3 6 hours: the cells were washed with phosphate buffered saline (PBS) in the same buffer containing 1% Triton X-100, 0.5% sodium deoxycholate and 1 mM EDTA. Dissolve the cells in the solution. The nucleus was removed by low-speed centrifugation, and the supernatant was stored at -70 ° C until use. The cytoplasmic extract for the in vitro kinase assay was used. The fused monolayer of each cell strain was grown on a 96-well cell culture plate. At the appropriate time after infection, the medium was aspirated to contain 20 mM HEPES (pH 7.4), 120 mM KC1, 5 mM MgCl2, 1 mM dithiothreitol, 0.5% Nonidet P-40, 2 μg/ml antifibrinolytic solution. The cells were lysed in a buffer of the enzyme peptide and 50 μg/ml of anti-protease peptide. Using low-speed centrifugation -33 - This paper scale is applicable to China National Standard (CNS) A4 specification (210X 297 mm) 1302458 A7 B7 V. Description of invention (31) Removal of nuclei, supernatant is stored at -70 °C until use until. The cytoplasmic extract was quantified using Bio-Rad protein microassay prior to use. Each in vitro kinase reaction contains 20 microliters of cell extract, 7.5 microliters of reaction buffer (20 mM HEPES [pH 7.4], 120 nM KC1, 5 mM MgCl2, 1 mM dithiothreitol, and 10% glycerol) And 7·0 μl of ATP mixture (1.0 "Ci[r-32P]ATP, contained in 7 μl of reaction buffer), incubated at 37 ° C for 30 minutes (Mundschau, LJ, and Faller, DV· J. Biol Chem., 267: 23092-23098 (1992)). Immediately after the culture, the labeled extract was boiled in Laemmli SDS-sample buffer or precipitated using agarose-poly(I) poly(C) beads or immunoprecipitated with an anti-PKR antibody. Agarose poly(I) poly(C) precipitation method In each of the in vitro kinase reaction mixtures, 30 μl of 50% Ag poly(I) poly(C) 6 type slurry (Pharmacia LKB Biotechnology) was added, and the mixture was 4 Incubate for 1 hour at °C_. The Ag containing the absorbed and labeled protein was washed at room temperature with washing buffer (20 mM HEPES [7.5 pH], 90 mM KC1, 0.1 mM EDTA, 2 mM dithiothreitol, 10% glycerol). Poly(I) poly(C) beads were 4 times and mixed with 2X Laemmli SDS sample buffer. The beads were boiled for 5 minutes and the released proteins were analyzed by SDS-PAGE. Polymerase chain reaction The cells were harvested at different time points of infection and resuspended in ice-cold TNE (10 mM Tris (pH 7.8), 150 mM NaCl, 1 mM EDTA), then NP-40 was added to a final concentration of 1%. After 5 minutes, the nuclei were centrifuged to agglomerate, so that the ?34-^ paper scale common Chinese national standard (CNS) A4 specification (210X 297 mm) 1302458 A7 B7 V. Description of invention (32) Extraction of the supernatant with phenol: chloroform method RNA. Take equal amounts of RN A from each sample. RT-PCR was performed using random six-nucleic acid primers (Pharmacia) and RTase (GIBCO-BRL) according to the manufacturer's instructions (Wong, H., et al., (1994). Anal. Biochem·, 223:251-258). The cDNA of the RT-PCR step was used to selectively amplify the reovirus SI cDNA using a suitable primer sequence derived from the S 1 sequence determined in the past (Nagata, L., et al., (1984) Nucleic Acids Res., 12:8699-8710). A predetermined 306 bp GAPDH fragment was amplified using the GAPDH primer (Wong, H., et al., (1994) Anal. Biochem, 223:251-258) as a P C R and a control added to the gel. The selective amplification of S1 and GAPDH cDNA was performed using Takin DNA Polymerase (GIBCO-BRL) using Perkin Elmer Gene Amp PCR System 9600 according to the manufacturer's instructions. The P C R was subjected to a cycle of 28 cycles each including a denaturation step of 30 ° C for 30 ° C, a bonding step of 55 ° C for 45 seconds, and a polymerization step of 72 ° C for 60 seconds. It was analyzed by agarose gel electrophoresis of 'impregnated 3-,8-diamino-5-ethyl-chunphenylphenanthridine TAE-2%, and photographed with Polaroid 57 under ultraviolet light. . Immunoprecipitation and SDS-PAGE analysis were performed by immunoprecipitation of Reovirus cell lysate infected with 35S and anti-reovirus serotype 3 serum according to the method described previously (Lee, PWK et al. (1981) Virology, 108: 134-146). In a similar manner, immunoprecipitation was performed by cell lysate labeled 32P with anti-PKR antibody (from Dr. Michael Mathews, Cold Spring Harbor). Immunoprecipitates were analyzed by discontinuous SDS-PAGE according to the method of Laemmi (Laemmli, U, K., (1970) Nature, 227: 680-685). -35- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458 A7 B7 V. Description of invention (33) Example 1. Effect of exposure of reovirus on survival rate according to the above injection Enterovirus attacks C 3 Η mice. After 2 weeks, antibodies to the reovirus of these animals were tested. Animals that had been exposed to reovirus and animals that had not been exposed to reovirus received C3 tumor allografts. After the tumor is formed, all animals are administered live or UV deactivated reovirus. The proportion of tumors in animals that had been exposed to reovirus completely disappeared was lower than that of mice that had not been exposed to reovirus. Three of the 9 animals (33%) who had been attacked had a complete regression of the tumor, while 9 of the 9 mice that had not been exposed to the reovirus completely resolved (66%). Example 2. Co-administration of reovirus and immunosuppressive agents to reduce effective doses Reovirus has been shown to act as a lytic cancer agent for immunodeficient animals and immunocompetent animals, but immunocompetent animals need to increase viral dose and increase treatment frequency. . In order to demonstrate that the co-administration of reovirus in the presence of an immunosuppressive agent can be used to lower the dose and frequency of immunosuppressive animals, a tumor derived from ras-transformed C3H 10Τ 1/2 cells is implanted in C3 sputum mice. The animals are then treated with an immunosuppressant prior to treatment or at the beginning of treatment, or sham treatment. The two groups of animals will receive a single intratumoral injection of reovirus or a pseudo-virus with U V deactivated reovirus. Animals in the second and third trials received injections at intervals of 3 or 5 or false treatments. Animal tumors were allowed to resolve for 4 weeks and killed at the end of week 4. The results showed that, in addition to the respiratory virus, when the immunosuppressive agent was used, the tumor was more effectively regressed than when the reovirus was used alone. Animals treated with immunosuppressive agents need to have a small amount of reovirus required for cell lysis and apply to Chinese National Standard (CNS) A4 size (210 X 297 mm) 1302458 A7 B7. DESCRIPTION OF THE INVENTION (34) The number of times of the drug is small. Example 3: The 1^· 蜃"^ agent is jointly administered to the spleen to treat the tumor. In the second and third tests, animals were intravenously administered a total of 3 or 5 reoviruses or uv deactivated reovirus every other day.

Packing A single injection of reovirus has been shown to cause tumor regression when it is delivered to the body in an immunodeficient mouse. (4) The systemic delivery of reovirus can abolish the tumor of an immunocompetent animal, but it is necessary to increase the dose and frequency of treatment in order to achieve the same result as an immunodeficient animal. It has been demonstrated that the use of immunosuppressive agents reduces the higher doses of such immunocompetent animals, and the tumors derived from ras-transformed C3H 1〇τ 1/2 cells are implanted ventrally in the posterior aspect of the animal. The animals are then treated with an immunosuppressive agent or treated sham. The two groups of animals received the virus once, or υ ν to deactivate the disease.

All animals were killed at the end of the fourth week. ^^ The results showed that in addition to the reovirus plus the treatment of the fog-inhibitor, the results were more effective than the use of the reovirus alone. Animals treated with immunosuppressive agents require a lower number of reoviruses and fewer doses to achieve lysis. Example 4: In Vitro Immunoadsorption In order to prevent the host immune system from excluding reovirus administered for medical treatment, immunological components such as B cells: T cells and possibly interfering with systemic transmission are selectively excluded prior to medical delivery of reovirus. antibody. The elimination of B cells and T cells, or both, can be combined with the in vitro compounds that can remove the cell population by blood percolation (eg, identifiable to be removed -37-

1302458 A7 B7 V. Inventive Note (35) The immobilized antibody of the specific receptor on the cell population is carried out. Selective exclusion of antibodies also prevents the host's immune system from excluding medical viruses, preventing antibody-to-virus interactions and contributing to systemic medical treatment of reoviruses. It can be accomplished by several methods: 1) using blood matrix dialysis and passing blood through immobilized reovirus, excluding anti-reovirus antibodies (selective exclusion of antibodies); 2) using blood matrix dialysis and passing blood Immobilized protein A, such as PROSORBATM (Cypress Bioscience, San Diego, CA), excludes all IgG antibodies; 3) Administration of anthropomorphic anti-genetic antibodies, wherein the hereditary type is resistant to reovirus. The process of in vitro immunosorbent assays is described in U.S. Patent Nos. 4,711,839, issued toSinghal, and 4, 681, 870 (Balint et al), the entire disclosure of which is incorporated herein by reference. < In the salty understanding method, the reovirus is administered, the immune component is excluded, and the reovirus can be administered to the patient again. Repeat these steps as necessary. The number of repetitions of the present invention can be determined by a professional medical professional based on the condition of the patient. While the invention has been described with respect to the preferred embodiments and the embodiments of the invention, -38- This paper size applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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1302458 Application for the period 90.4.25 Case No. 090109903 Category (The above are filled by this Council) Chinese Manual Replacement Page (January 97) y? A4 C4 11 Patent Specification Chinese Name Pod Avoid Reovirus Identification to Treat Cells PHARMACEUTICAL COMPOSITIONS AND KITS FOR PREVENTING REOVIRUS RECOGNITION FOR THE TREATMENT OF CELLULAR PROLIFERATIVE DISORDERS'1 Name Nationality Invention Creation> [• Matthew C. Coffe 2 Brandy G. Thompson

MATTHEW C. COFFEY BRADLEY G. THOMPSON Canada Residences, Residences 1 · 2231, Pauli Road, North West District, Aberdeen, Canada 2. No. 1775, 7th Street, North West District, Kegari City, Alberta, Canada Name) ONCOLYTICS BIOTECH, INC. Canada Citizenship Canada 3, Applicant Residence, Residence (Office) 3〇1, 1211, Cansington Road, Kerry, Alberta, Canada Name of Representative

Matthew C. Coffey MATTHEW C. COFFEY This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

Claims (1)

I30》l (S8〇99〇3 Patent Application Chinese Application Patent Fanyuan Replacement (August 96) A8 B8 C8 D8 曰修, _) Original Patent Application 1. 2. 3. 4. 5. 7 8. A pharmaceutical composition for treating a proliferative disorder in a mammal by ras-mediated comprising an effective amount of one or more reoviruses and an effective amount of an anti-anti-reovirus antibody; The pharmaceutical composition may be used in combination with an anti-reovirus antibody excluding the mammal. The pharmaceutical composition according to claim 1, wherein the reovirus is selected from the group consisting of a mammalian reovirus and avian reovirus. The pharmaceutical composition according to claim 2, wherein the reovirus is a mammalian reovirus. The pharmaceutical composition according to claim 3, wherein the reovirus is a human reovirus 6 according to the patent composition of claim 4, wherein the reovirus is selected from the group consisting of serotype 1 reovirus, serotype 2 Reovirus and serotype 3 reovirus. A pharmaceutical composition according to claim 5, wherein the reovirus is a serotype 3 reovirus. The pharmaceutical composition according to claim 2, wherein the reovirus is a bird reovirus. The pharmaceutical composition according to claim 1 of the patent application, which comprises a reovirus of a type or higher. The pharmaceutical composition according to claim 1 of the patent application, which comprises a reovirus of a higher quality or higher. According to the pharmaceutical composition of claim 1, the reovirus system is isolated from nature. Root rubber applies for the pharmaceutical composition of the first scope of the patent, in which ras-medium 70548-960801.DOC is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) AB c D 1302458, the patent application The proliferative disorder of the park is a tumor. 12. The pharmaceutical composition according to claim 1, wherein the ras-mediated proliferative disorder is neurofibromatosis. 13. The pharmaceutical composition according to claim 11 wherein the tumor is a solid tumor. 14. The pharmaceutical composition according to claim 11 wherein the tumor is selected from the group consisting of lung cancer, prostate cancer, colorectal cancer, thyroid cancer, kidney cancer, adrenal cancer, liver cancer, pancreatic cancer, breast cancer and Central and peripheral nervous system cancer. 15. The pharmaceutical composition according to claim 14, wherein the tumor is a central nervous system cancer. 16. The pharmaceutical composition according to claim 15 wherein the tumor is breast cancer. 17. The pharmaceutical composition according to claim 11 wherein the tumor is a hematopoietic tumor. 18. A pharmaceutical composition according to claim 13 of the patent application, which is suitable for administration by injection into or near a solid tumor. 19. The pharmaceutical composition according to claim 1 of the patent application, which is suitable for intravenous administration to a mammal. 20. The pharmaceutical composition according to claim 1 of the patent application, which is suitable for intraperitoneal administration to a mammal. 21. The pharmaceutical composition according to claim 1, wherein the mammal is immunologically active. 22. The pharmaceutical composition according to Article 21 of the patent application, wherein the reovirus 70548-960801.DOC is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1302458 It is immune to protection. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. A composition, wherein a reovirus composition, wherein the mammal is embedded in a micelle according to the pharmaceutical product of claim 1 Inside. The medicine according to item 1 of the scope of patent application is human. The pharmaceutical composition of claim 3, which contains about [1015 plaque forming units of reovirus/kg body weight. According to the scope of the patent application! A pharmaceutical composition suitable for administration in a single dose. The pharmaceutical composition according to item i of the patent application is suitable for administration in one or more doses. A pharmaceutical composition according to the scope of the patent application, wherein the tumor is transferable. According to the pharmaceutical composition of claim 1, the pharmaceutical composition is used in combination with an effective amount of a chemotherapeutic agent. The pharmaceutical composition according to claim 1, wherein the reovirus is treated with a protease prior to administration. The pharmaceutical composition according to claim 11 wherein the tumor is pancreatic cancer. The pharmaceutical composition according to claim 11 of the patent application is used in combination with the step of surgically removing substantially all the tumors, and the reovirus is administered at the surgical site in an amount sufficient to cause any residual tumor cells to be large. Dissolved. The pharmaceutical composition according to the scope of patent application No. 1 is applicable to the radiation of 70548-960801.DOC. The paper size is applicable to China National Standard (CNS) A4 specification (210X297 public sauce) 1302458 _ _____ D8 VI. Patent scope therapy And use it. 34. The pharmaceutical composition according to claim 1 of the patent application, which is for use in combination with removing an anti-reovirus antibody from the mammal. 35. The pharmaceutical composition according to claim 34, wherein the anti-reovirus antibody is used for blood matrix dialysis and the blood is passed through the eye. 36. According to the pharmaceutical combination of claim 35 The object is immobilized on a solid support. Among them, reovirus 37. According to the scope of patent application! The pharmaceutical composition of the item further comprises a chemotherapeutic agent. Mouth." 38. According to the pharmaceutical composition of claim 1, the pharmaceutical composition is at least one selected from the group consisting of: a political reovirus and an immunoprotective respiratory disease. y 39. A kit for treating a proliferative disorder in a mammal by ras. The method comprising: a) a method of removing an anti-vesicovirus antibody of the mammal, and b) a pharmaceutical composition comprising a reovirus . 40. According to the kit of claim 39, it also contains a chemotherapeutic agent. 41. The kit of any one of claims 39 and 40, wherein the enterovirus is one or more recombinant reoviruses. a drug for the treatment of a mammalian proliferative disorder by a coffee medium comprising an effective amount of one or more reoviruses; wherein the pharmaceutical composition is associated with excluding the mammal The step of virus antibody or the step of administering an anti-anti-reovirus antibody to the mammal is used in combination. 43. A pharmaceutical composition according to claim 42 wherein reovirus 70548-960801.DOC 1302458 Selected from mammalian reovirus and avian reovirus. 44. The pharmaceutical composition according to claim 43, wherein the reovirus is a mammalian reovirus. The pharmaceutical composition according to claim 44, wherein the reovirus is a human reovirus. 46. The pharmaceutical composition according to claim 45, wherein the reovirus is selected from the group consisting of serotype 1 reovirus, serotype 2 reovirus and serotype 3 reovirus. 47. The pharmaceutical composition according to claim 46, wherein the reovirus is a serotype 3 reovirus v 48. The pharmaceutical composition according to claim 4, wherein the reovirus is a bird resuscitation virus. 49. The pharmaceutical composition according to item 4 of the patent application, which comprises a reovirus of a type or higher. 50. The pharmaceutical composition according to item 4 of the patent application, which comprises a reovirus of a higher quality or higher. 51. The pharmaceutical composition according to claim 4, wherein the reovirus is isolated from nature. 52. The pharmaceutical composition according to claim 42, wherein the proliferative disorder of ras-medium is a tumor. 53. The pharmaceutical composition according to claim 4, wherein the ras-mediated proliferative disorder is neurofibromatosis. 54. The pharmaceutical composition according to claim 52, wherein the tumor is a solid tumor. 70548-960801.DOC This paper scale is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 1302458 C8 D8 VI. Patent application scope 55. According to the patent application range 52 item, the tumor composition It is selected from the group consisting of lung cancer, prostate cancer, colorectal cancer, thyroid cancer, kidney cancer, adrenal cancer, liver cancer, and cancer of the peripheral nervous system of pancreatic cancer. , breast cancer and the center and 56. The pharmaceutical composition according to the scope of patent application No. 5 5, a central nervous system cancer. , wherein the tumor is medium 57. The pharmaceutical composition according to the patent application scope 56 is cancer. , wherein the tumor is milk. 58. The pharmaceutical composition according to the scope of claim 5, bloody tumor. , wherein the tumor is made. 59. The pharmaceutical composition according to claim 54 of the patent application is in or near the solid tumor. It is suitable for injection. 60. The pharmaceutical composition according to claim 4 of the patent application is administered to a mammal. It is suitable for intravenous administration. 61. The pharmaceutical composition according to claim 4 of the patent application is administered to a mammal. It is suitable for transperitoneal 62. The pharmaceutical composition according to claim 4 of the patent application is immunologically active. , wherein the mammal 63. The pharmaceutical composition according to claim 62 of the patent application is immunoprotective. , wherein the reovirus 64. The pharmaceutical composition according to claim 4 of the patent application is embedded in the micelle. , wherein the reovirus 65. The pharmaceutical composition according to claim 4 of the patent application is human. , mammals 70548-960801.DOC ·6· ^ This paper scale applies to Chinese National Standard (CNS) Α4 specifications (210 X 297 mm) 1302458 66. 67. According to the patent application, item 42 of the patent, the unreacted composition contains about 1 to the reovirus/kg body weight of the lozena forming unit. According to Article 4 of the scope of the patent application, there is a lack of a seven-spotted spear Z male and soy sauce composition which is suitable for administration in a single dose. The pharmaceutical composition according to claim 42 of the patent application is suitable for administration in more than one dose. 69. The pharmaceutical composition according to claim 42 of the patent application, wherein the cancer is a pharmaceutical composition according to claim 42 wherein the reovirus is treated with a protease prior to administration. 7L according to the pharmaceutical composition of claim 52, wherein the tumor is a cancer. According to the application of the patent scope, the pharmaceutical composition is used in combination with the surgical removal of substantially all tumors. And the reovirus is administered at the surgical site in an amount sufficient to dissolve a large amount of any remaining tumor cells. The pharmaceutical composition according to claim 42 is used in combination with radiation therapy. 74. The pharmaceutical composition according to claim 42 of the patent application, which is for use in combination with the removal of an anti-reovirus antibody in the mammal. 75. The pharmaceutical composition according to claim 74, wherein the anti-resume drug resistance system is removed by blood matrix dialysis and by passage of blood through an immobilized reovirus. 76. A pharmaceutical composition according to claim 75, wherein reovirus 70548-960801.DOC -7- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1302458 Benefit C8 ------------ __ VI. Patent application scope---- Fixed on solid support on. 77. The pharmaceutical composition according to any one of the claims U38 & 42 to % wherein at least one of the rectal diseases is a recombinant reovirus. 78. The pharmaceutical composition according to claim 77, wherein the recombinant reovirus is derived from two or more reovirus strains. The pharmaceutical composition according to claim 78, wherein the two or more reovirus strains are selected from the group consisting of Dearing strain, Abney strain, J〇nes strain and Lang strain. The pharmaceutical composition according to claim 77, wherein the reovirus is produced by co-infection of mammalian cells with reovirus of different subtypes. 81. The pharmaceutical composition according to claim 77, wherein the recombinant reovirus is naturally occurring. 82. The pharmaceutical composition according to claim 77, wherein the recombinant reovirus is non-naturally occurring. 83. The pharmaceutical composition according to claim 77, wherein the recombinant reovirus comprises various coat protein coding sequences naturally occurring. 84. The pharmaceutical composition according to claim 77, wherein the recombinant reovirus comprises a mutated coat protein coding sequence. 85. The pharmaceutical composition according to claim 77, wherein the recombinant reovirus is selected from the group consisting of reoviruses selected from the group consisting of serotype serotypes, serotype 2 and serotype 3 Got it. 86. The pharmaceutical composition according to claim 77, wherein more than one recombinant reovirus strain is administered. 70548-960801.DOC 0 This paper size applies to the Η 家 家 家 家 家 家 家 家 家 家 家 家 家 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 87 The pharmaceutical composition according to any one of items 42 to 58 and 62 to 76, which may be selected from the group consisting of intravascular, intraspinal, intravenous, intramuscular, subcutaneous, intraperitoneal, topical, oral, rectal, vaginal, intranasal, and neoplastic. The way of the group consisting of is committed. -9- 70548-960801.DOC This paper size applies to China National Standard (CNS) A4 specification (210X297 mm)