TWI248973B - Methods for the treatment of cellular proliferative disorders - Google Patents

Abstract

The present invention relates to methods of identifying the susceptibility of cells to reovirus infection by measuring constitutive ras-MAP signaling. The invention also pertains to methods using reovirus for the treatment of cellular proliferative disorders, and particularly cellular proliferative disorders wherein the proliferating cells exhibit constitutive MAPK phosphorylation, in mammals. In particular, the methods provide for reovirus treatment of mammals to treat proliferative disorders which include breast tumors, a subset of tumors in which mutation of the ras gene is not believed to play a significant role.

Description

1248973 A7 B7 V. INSTRUCTIONS OF THE INVENTION (1) Scope of the Invention The present invention relates to a method for confirming the sensitivity of a cell to a virus infection in Central Europe by measuring a structural ras-MAP. The invention also relates to a method of treating a disorder of cell proliferation using a Leovirus, and in particular a mammalian cell proliferation disorder, wherein the proliferating cell exhibits structural MAPK phosphorylation. In particular, the present method provides a mammalian solution for the treatment of proliferative disorders in mammals, including breast tumors, and tumor subpopulations in which ras gene mutations are not the primary cause. REFERENCES The following documents, patent applications, and patents are hereby incorporated by reference in its entirety in its entirety in its entirety in its entirety in W0 99/08692, published on February 5, 1999. Archer (1995), Br. J. Cancer 72: 1259-1266. Armstrong, G.D. et al. (1984), Virology 138:37. Barbacid, M., Annu Rev. Biochem, 56: 779-827 (1987) Baselga et al. (1996), J_Clin·One 14: 737-744. Bos, J. (1989) Cancer Res. 49:4682 Carter et al. (1992), PNAS 89: 4285-4289. "Chandron" and "Nibert", ''Protease cleavage of reovirus capsid protein mul and mulC is blocked by alkyl sulfate detergents, yielding a new type of infectious subvirion particle'', J. of Virology 72(1) :467-75 (1998). -4- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 public)

1248973 A7 B7 V. INSTRUCTIONS (2) Chaubert, P. et al. (1994), Am. J. Path. 144:767. Clark et al. (1995), Science 268: 233-239. Clark et al. (1996), Inti. J. Cancer 65: 186-191, Cuff et al., 'Enteric reovirus infection as a probe to study immunotoxicity of the gastrointestinal tract.' Toxicological Sciences 42(2):99 -108 (1998) DiDomenico et al. (1996), Cancer Res. 56:4516-4521. Duncan et al., "Conformational and functional analysis of the C-terminal globular head of the Reovirus cell attachment protein" Virology 182(2): 810-9 (1991). Dvorak et al. (1988), Am J Path 133: 95-109. Fields, Β·Ν· et al. (1996), Fundamental Virology, 3rd Edition, Lippincott-Raven o Gentsch, JRK and Pacitti, AF (1985), J. Virol 56:356 o E. Harlow and D. Lian, '丨Antibodies: A laboratory manualn, Cold Spring Harbor Laboratory (1988) o Harweth et al. (1992), J Biol Chem 267: 15160-15167. Hudziak et al. (1989), Mol Cell Biol 9: 1165-1172. Hung et al. (1995), Gene 159: 65-71, Jacobs et al. (1983), Cancer Res 43: 1696-1702 o Janes, PW, et al. (1994) Oncogene 9:3601. -5- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1248973 A7 B7 V. Description of invention (3) Jardines et al. (1993) Pathobiology 61:268-282. Koenders et al. (1991), Cancer Res 51:4544-4548. Lee (Lee). J. M. et al. (1993) PNAS 90: 5742-5746. Lee, P. W. K. et al. (1981) Virology, 108: 134. Levitzki, Α (1994) Eur J. Biochem. 226:1. Lowe. S. W. et al. (1994) Science, 807-8 10. M The N-terminal quarter of reovirus cell attachment protein sigma 1 possesses intrinsic virion-anchoring function丨 Virology 179(1): 95-103 (1990) o Migliaccio et al. (1996), EMBO J 15: 1292-1300 〇米格里希欧 et al. (1998), EMBO J 17:2008-2018. Millis, NE et al. (1995) Cancer Res. 55:1444. Nagy et al. (1989) Biochim Biophys Acta 948: 305-326. Paul R. W. et al. (1989) Virology 172: 3 82-3 85. Pietras et al. (1994), Oncogene 9: 1829-1838. Raybaud-Diogene H_ et al. (1997) J. Clin Oncology, 15(3): 1030-1038.

Remington’s Pharmaceutical Sciences, Mace Publishing Company, Philadelphia PA 17th ed. (1985) o Rosen, L. (1960) Am. J. Hyg. 71:242. Sabin, A.B. (1959), Science 130:966. Shackney et al. (1998), Clin Cancer Res 4: 913-928 〇 -6- This paper scale applies to China National Standard (CNS) A4 specification (210X 297 mm) 螫

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1248973 A7 B7 V. INSTRUCTIONS (4) Slamon et al. (1989), Science 244: 707-712. Smith, R.E., et al. (1969) Virology, 39:791-800. Spadiodos (1987), Anticancer Res 7:991-996 S Stanley, N.F. (1967) Br. Med. Bull 23:150. Strong J.E. et al. (1993) Virology, 197:405-41 1. Shi Zhuang, J.E. and Li, P.W.K., (1996) J. Virol., 70:612-616. Shi Zhuang, J.E., et al. (1998) EMBO J, 17: 3351-3362. Turner and Duncan, "Site directed mutageneis of the C-terminal portion of reovirus protein sigmal :evidence for a conformation-dependent receptor binding domain 11 Virology 186(1): 219_27 (1992). Verbeek et al. (1996) J Path 180: 383-388. Wiessmuller, L. and Wei; Ding Huofo (\\^1:111§]1〇€61·),? (1994), Cellular signaling 6(3): 247-267. Zhou et al. (1989), Oncogene 4: 105-108. All of the above-mentioned documents, patent applications, and patents are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the Technical Description Normal cell proliferation is regulated by promoting the balance between growth of the proto-oncogene and inhibition of growth tumor-suppressor genes. Tumor development can result from genetic variation in certain genomes, which can lead to mutations in cellular components that control cellular signal interpretation. For example, the enhancement of the activity of the original oncogene or the inhibition of tumors -7 - the paper scale is applicable to the Chinese national standard ( CNS) A4 size (210X297 mm)

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Line 1248973 A7 _B7 _ V. Description of invention (5) Activation. The signal interpretation of these signals can ultimately affect cell growth and differentiation, and misinterpretation of signals can lead to tumor growth (tumor formation). The genetic variation of the original oncogene Ras has caused about 30% of all human tumors (Vistula, L. and Wittenhofer, F. (1994), Cellular Signaling 6(3): 247-267; Barr Bassett,]^.(1987) 八1^¥· Biochem· 56, 779-827). The role of Ras in the pathogenesis of human tumors is determined by the type of tumor. The activating mutation of Ras itself is found in most human malignant tumor types, and is common in pancreatic cancer (80%), occasional in colorectal cancer (40-50%), human lung adenocarcinoma (15-24%), thyroid tumor. (50%) and myeloid leukemia (30%) (Millies, NE et al., (1995) Cancer Res. 55:1444; Joebert, Ρ· et al. (1994), Am. J. Path· 144 :767 ; Boss, J. (1989) Cancer Res. 49:4682). Ras activation can also be manifested by upstream mitotic signaling elements, particularly via tyrosine acceptor kinases (RTKs). These upstream elements (if amplified or overexpressed) eventually lead to an increase in Ras activity by the signal transduction activity of Ras. Examples include certain glioblastoma types, and PDGFR overexpression of c-erbB-2/neu in breast cancer (Revizki, A. (1994) Eur. J. Biochem 226:1; Janes, PW·, Striving (1994) Oncogene 9:3601; Boss, 1. (1989) Cancer

Res. 49:4682) ° Current methods of treating tumor formation include surgery, chemotherapy, and radiation therapy. Surgery is typically used to treat early cancer; however, many tumors cannot be completely removed by surgery. In addition, the metastatic growth of the tumor prevents the surgery from completely curing the cancer. Chemotherapy comprises administering a compound having antitumor activity such as an alkylating agent, an antimetabolite, and an antitumor antibiotic. Chemotherapy -8- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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1248973 A7 ___B7 V. INSTRUCTIONS (6) The utility of the method is usually limited by serious side effects including nausea and vomiting, osteomyelin, renal damage, and central nervous system depression. After radiation therapy is used in radiation therapy, normal cells have greater self-healing ability than tumor cells. However, many tumors cannot be treated with radiation therapy due to tissue susceptibility around the tumor. In addition, some tumors have been shown to be resistant to radiation therapy, which can be treated with oncogene or cell anti-oncogene status (Li JM et al., (1993) PNAS 90:5742- 5746 ; Rowe SW et al. (1994) Science, 266: 807-810; Lei Bode. Dai Oujian H. et al. (1997) J. Clin. Oncology, 15(3): 1030-1038) Women who take $ see and fear cancer are estimated to have one out of every eight American women (NCI-SEER, 1998). Although breast cancer treatment has achieved great success, its mortality has not risen in the face of increasing prevalence. Most of these advances can be attributed to advances in early detection methods and non-new treatment strategies. However, there has been little progress in the actual treatment of this pain. Therefore, it is necessary to use the traditional treatment strategy of eucalyptus. In cancer biology, the development of breast cancer treatment has never been as promising. Many of these therapies have been identified for certain clinical uses of specific receptors that are over-expressed in this cancer subtype. The most promising of these new therapies are antibodies that calibrate HER2 receptors that are often overexpressed in breast cancer. The results obtained by directly using these monoclonal antibodies in the extracellular portion of the growth factor receptor are very promising, and some clinical achievements have been achieved (Ba Xiejia, 1996; see Nass, 1998). ; Hang, 1995). However, there are still many technical barriers to deal with. First of all, these antibodies against the HER2 foot alone can not kill the cells themselves, but the cells make the cells quiescent-9 - This paper ruler ® ® 国 (21〇^iy 1248973 A7 B7 V, invention description (7 (Pitris '1994, Harwerth, 1992; Hugh Tecker, 1989; Carter, 1992). The point is that in SCID or nude mouse models, anti-HER2 antibodies only inhibit tumor growth rather than In fact, the tumor is degraded. Furthermore, once the antibody administration is stopped, the inhibition of tumor growth disappears rapidly. What is more unfavorable is that there is evidence that treatment of tumors with anti-HER2 antibodies to immunoreactive potential animals can actually lead to antibody-determination. Cellular cytotoxic effect (ADCC) mediates the killing effect (Carter, ι992). Second, the importance of calibrating growth factor receptors (EGFR) may be limited, as not all breast cancers will highly express these receptors. This strategy approach only calibrates the receptor tyrosine kinase and has a minor effect on breast cancer that overexpresses non-receptor tyrosine kinases (such as Src), while Src has been identified as certain milk Related to growth (Clark, 1996). Finally, the biology of tumor blood vessels is also needed to consider the ability to deliver these giant molecules to their target cells. It has been shown that the distribution of tumor blood vessels is heterogeneous and that their blood vessels ooze out of giant molecules (eg The ability of antibodies) is determined by its spatial orientation to tumor mass (De Flack, 1988; Naji and Defrak, 1989). It has been shown that the vessels with the greatest permeability to these molecules are mainly distributed in tumor-hosts. The contact surface, and the least permeable blood vessels are those who actually penetrate the tumor mass (Develak, 1988). Different permeability makes these tumor-specific antibodies and their cell-killing cells may not penetrate the tumor mass. It can only act around the mass. In addition to the fact that ras activating mutations are rare in breast cancer, 'increasing evidence suggests that the activation of the Ras/MAPK pathway is important for the activation and progression of the disease. The upstream components of the Ras pathway In particular, the receptor tyrosine kinase (RTK) is often overexpressed in breast cancer. About -10- of this paper scale is suitable for all breast cancers. National Standards (CNS) A4 size (210 X 297 mm)

1248973 A7 B7 V. Description of invention (8)

30% have HER-2/neu (erbB-2) overexpressed (Sbandodiods, 1987; Zhou, 1989; Aya, 1995) and have a prognosis for patients with adverse conditions (Slammon, 1989) . When overexpressed in NIH-3T3 cells, HER-2 can mediate transformation; however, it shows that overexpression must reach a baseline concentration to promote transformation (Jardins, 1993; Clark, 1995). The ability to transform HER_2 appears to be dependent on Ras activity, as cell lines overexpressing HER-2 exhibit dramatic increases in MAP kinase activity, which is a reflection of Ras activity (Janes, 1994). The growth factor receptor EGFR, which is closely related to HER-2 but is different from each other, has also been observed to be overexpressed in breast tumors and is also associated with the prognosis of adverse patients (Charkny, 1998, Yan Deides, 1991).

Additional components upstream of Ras are also implicated in breast cancer etiology. Non-receptor The tyrosine kinase c-Src is an important candidate for the development of breast cancer. Some studies have shown that c-Src activity in primary breast cancer tumors is increased by -4 to _30 times compared to normal breast tissue (Verbeeks, 1996; Jakob and Rubsamen) ), 1983). C-Src should play an important role in signal transmission from both estrogen and progesterone receptors via the Ras/MAPK pathway. Some groups have observed that treatment of breast cancer-derived MCF-7 cells with estradiol can lead to activation of c-Src kinase activity, ultimately resulting in activation of MAPK (Didomenico, 1996; Migliacio) , 1996). Recently, Migliacio (1998) has demonstrated that cell proliferation stimulated by progesterone is dependent on Src/Ras/MAPK in T47D breast cancer cells. The fact that steroid receptors can use Ras as their signaling shows that Ras may play a more important role in promoting the growth of ER and PR-positive breast tumors. -11 - The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1248973 A7 B7 V. Description of invention (9)

Ras itself may play a more critical role in the development of breast cancer than it was originally imagined. Although activating mutations in Ras are rare in the development of breast cancer, excessive expression of normal H-Ras in breast tumors has been observed (Charkny, 1998; Spenced Otis, 1987). This overexpression of Ras provides an additional mechanism for uncoupled normal signal transduction and can promote tumorigenesis. Combining these data demonstrates the feasibility of calibrating the activation of the Ras pathway in the development of novel breast cancer therapies. It has been previously stated that the Leo virus can only replicate in cells containing the activated Ras signaling pathway, which can be directly mutated by Ras itself or via upstream elements that can lead to activation (Shi Zhuang, 1993; Shi Zhuang., 1998) . In addition, evidence suggests that Central European viruses can be used in organisms to combat tumors that contain Ras activation (Coffey, 1998). The SCID mouse model of tumor xenograft was established using the human glioblastoma cell line U87. U87 cells can be selected as a suitable model because they overexpress RTKPDGF and cause activation of Ras, and can show acute susceptibility to influenza virus infection in vitro. The U87 tumor xenograft strain was implanted into the SCID mouse, and the tumor was treated with a single intratumoral injection of the Ley virus after the tactile tumor was determined. This single treatment can lead to dramatic tumor regression. While considering the shortcomings of current methods for treating neoplasm growth, there remains a need for improved methods of treating most cancer types, specifically breast cancer. It is useful if there is a method for determining the sensitivity of the Central European virus to predict the effectiveness of the treatment of Central European virus. DESCRIPTION OF THE INVENTION The present invention relates to the identification of fine -12- by means of measuring structural ras-MAP. The paper is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm).

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1248973 A7 B7 V. INSTRUCTIONS (1〇) Methods of susceptibility of cells to Central European virus infection. The present invention also provides a method of using a Leo virus to treat a disorder of cell proliferation, and in particular, a cell proliferation disorder in a mammal in which the proliferating cells exhibit structural MAPK phosphorylation. Specifically, the method provides for the treatment of mammalian dysregulation with the Leo virus, which comprises a breast tumor, i.e., a tumor subpopulation in which the iras gene mutation is believed to not play an important role. The present invention also relates to a method for preventing recognition of a virus in the body to treat a disorder of cell proliferation, and in particular a mammalian cell proliferation disorder exhibiting structural MAPK phosphorylation. Mammals may be selected from the group consisting of dogs, cats, sheep, goats, cows, horses, pigs, rats, humans, and non-human primates. The method comprises administering to the proliferating cells an effective amount of one or more Central European viruses to cause substantial expansion of the proliferating cells. The Leo virus can be a mammalian or avian Central European virus. The Leo virus can be engineered to remove the capsid, fill the virions in liposomes or colloidal particles, or mutate the capsid protein. The Leo virus agent can be administered in a single dose or in multiple doses. The disorder of proliferation may be a tumor. Both solid and hematopoietic tumors are acceptable for calibration. Pre-, simultaneous or subsequent use of immunosuppression can lead to more effective treatment with Central European virus. Thus, in one aspect of the present invention, a method for measuring the susceptibility of a cell to a Leu virus infection by measuring structural ras-MAP signaling is provided, wherein the appearance of the message exhibits sensitivity to a virus infection in Rio. Regardless of the presence of mitogens, structural ras-MAP signaling can lead to MAP kinase activation, whereas MAP kinase activation leads to phosphorylation of MAP kinase. Therefore, the phosphorylation status of MAP kinase can be determined as the equivalent of the Chinese National Standard (CNS) A4 specification (210 X 297 mm) for the knot-13- paper scale.

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1248973 A7

Any of the techniques: Sending a measurement. The phosphorylation state of MAP kinase can be determined by a method known in <=Technology#, and specifically by using an antibody specific for the MAP MAP MAP kinase. Because: 匕, the present invention can be used to diagnose the proliferative loss that can be treated by the Leo virus, and the biological sample can be collected from a mammal suspected of having a proliferative disorder: the method of the present invention is used to test the structural traces of the cells in the sample. . A dysplastic disorder can be any condition associated with abnormally active cells such as neurofibromatosis. Proliferative disorders are preferably 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 m Tiaozhong special is for breast cancer. The invention is applicable to any animal suffering from a disorder of proliferation. Preferably, the animal is a mammal; the animal is preferably selected from the group consisting of: dogs, miscellaneous, sheep, goats, cows, horses, pigs, r, non-human primates, and humans. The animal is best for humans. In a further aspect, the present invention provides a method of treating proliferative loss in a mammal characterized by a proliferating cell exhibiting structural MAPK phosphorylation, the method comprising the condition of causing substantial dissolution of the proliferating cell An effective amount of one or more Central European viruses is administered to the proliferating cells of the mammal. The method further comprises a group selected from the group consisting of administering to the proliferating cells of the mammal an effective amount of an immunosuppressant 'removing B cells or T-cells from the mammal; removing the anti-antigen from the mammal - Leo virus antibody; remove antibodies from the mammal; apply to the Chinese National Standard (CNS) A4 specification (210X 297 mm) for the mammal-14- paper scale

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5. Description of the invention (administering an anti-anti-European virus antibody; and simultaneously inhibiting the mammalian immune system' and further comprising a method of administering a chemotherapeutic agent. Also providing a method for treating a human tumor, the tumor A sputum cell characterized by a structural MAPK phosphorylation, the method comprising administering to the tumor a Leo virus sufficient to cause a substantial lytic dose of the tumor cell. Preferably, the Leo virus is administered systemically or by injection. In or near the parenchyma. The method further comprises the steps of pre-, simultaneously or subsequently inhibiting the immune system of the mammal or including other methods of detracting from suppressing the immune system of the mammal. In the method of metastasis, the tumor is characterized by the fact that the proliferating cells exhibit structural MAPK phosphorylation, and the method comprises administering to the mammal a Leo disease mother sufficient to effectively cause a dose of sputum lysis of the tumor cells. - the step of pre-, simultaneously or subsequently inhibiting the mammalian immune system or including other methods of compromising the mammal Immune system. Also provided is a method for treating a suspected tumor in a mammal characterized by a proliferating cell exhibiting structural MAPK phosphorylation, including surgical removal of all tumors and a dose of the Levi virus. The surgical site or adjacent area to dissolve any residual tumor cells. The Central European virus can also be administered systemically. The method includes the steps of inhibiting mammalian immunity at the same time, or including other methods. Repressing the immune system of a mammal.: A pharmaceutical-medicinal composition comprising an effective amount of a Leo virus and a pharmaceutically acceptable agent. Also providing an immunosuppressive -15- National Standard (CNS) A4 Specification (2ΐ〇χ 297 公爱) 1248973 A7 B7_ V. Inventive Note (13) Agent (immunosuppressant) or immunoinhibitant, Liouvirus and pharmaceutically acceptable excipients Pharmaceutical composition. The kit includes the Leo virus and, if necessary, an immunosuppressant or an immunological preparation. The method and pharmaceutical composition of the invention provide a An effective method for the treatment of neoplasms, which excludes the side effects of other types of cancer therapy. When used, suppression or inhibition of the immune system can help the Leo virus to infect and dissolve proliferating cells that exhibit structural MAPK phosphorylation. The formation of anti-Leovirus antibodies. Since it is known that the Leo virus does not cause disease, any safety considerations in its use can be minimized. The above and other objects, features and advantages of the present invention are more specific to the present invention The description of the specific embodiments and the accompanying drawings will be more apparent. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the transformation effect of v-src on a Leu virus-infected host cell. Transformation of non-infectious NIH-3T3 parental cells and v-src NIH-3T3 cells were cultured in a 24-well assay plate to 80% cell confluency and subsequently exposed to the Leo virus, with an estimated MOI of 80 PFU per cell. After infection, cells and medium were collected at the indicated times, and the resulting lysate was subjected to plaque valence analysis. V-src can transform NIH-3T3 cells (closed loop), NIH-3T3 parental cells (open loop). (average soil standard error). Figures 2A through 2C illustrate in vitro Euravirus replication in human breast tumor cell lines. (A) Simulated-infection and Leo virus protein synthesis in breast cancer cell lines after infection with Leo virus. Cells were labeled with [35S]-methionine 46 to 48 hours after infection. The lysate was prepared and subsequently immunoprecipitated with a plurality of anti-Liouvirus type 3 sera and analyzed by SDS-PAGE. Leo virus -16- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm)

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V1248973 A7 B7 V. INSTRUCTIONS (14) The protein is shown on the right. (B) The Leu virus infectivity is related to structural MAPK phosphorylation. The breast tissue cell line HBL-100 and the breast tumor cell lines MDA-MB-468, MCF7, MDA-MB-435, T47D and SK-BR-3 were placed in a six-well test plate. The cells were cultured in 10% FCS or low serum concentration (0.5% FCS) for 48 hours. The monolayer was washed with PBS and cell lysate was prepared for SDS-PAGE analysis. After the samples were coated onto nitrocellulose paper, they were probed with a specific anti-phosphorus-MAPK antibody. All phosphorus-containing MAPK concentrations were normalized to the total MAPK concentration. (C) Addition of MAPK concentrations in human breast tissue and breast cancer cell lines. Figure 3 illustrates the lysis of Leovirus-vector tumor cells in vivo against human breast tumor xenografts. MDA-MB-468 human breast tumor xenografts were implanted subcutaneously into SCID mice. After confirming the palpable tumor mass, a single intratumoral injection was given with a viable virus (open loop) or a UV-inactivated virus (closed loop) and the tumor growth was followed for four weeks. (Average standard error). DETAILED DESCRIPTION OF THE INVENTION It has previously been shown that in the SCID murine model (WO 99/08692), human Leuvirus can be used as an effective oncolytic agent against human glioblastoma xenografts. Here, the inventors have found that the sensitivity of a cell to a virus infection in Central Europe can be determined by measuring the structural ras-MAP of the cell, and the appearance of the signal can show the sensitivity to the infection of the Leu virus. The present invention also suggests that the Leo virus can be used as an oncolytic agent against breast tumors. Although ras mutations are rare in breast cancer etiology, it is common to send abnormal Ras/θΑΡΚ via upstream signaling elements such as receptor tyrosine kinase and non-receptor tyrosine kinase (eg c-Src). . -17- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm)

k 1248973 A7 B7 V. INSTRUCTIONS (15) Based on the same reason that the efficacy of monoclonal antibodies for breast cancer may be diminished (see above), the Leo virus can be a compelling cancer therapy. First, the Leo virus itself is a cell killer and does not have to rely on immune-activated cells to cause tumor regression. In fact, the original machine that kills infected cells is directly lysed by viral replication (Tyler and Fieldsfields, 1996). Second, it can calibrate breast cancer with Ras activation. This activation is not limited to triggering Ras mutations (a well-recognized breast tumor subtype) and also includes Ras activation by the upstream elements of Ras itself. These elements include not only receptor tyrosine kinases (such as EGFR and HER2), but also non-receptor tyrosine kinases (such as members of the Src population). Taken together, this type of therapy can be used to combat heterogeneous tumor types (such as breast cancer) with high efficiency and is not as restrictive as the treatment of only the receptor. Finally, the fact that antibodies cannot penetrate into solid tumor masses suggests that replication should be easily carried out if the Central European virus is delivered intratumorally. Therefore, it is useful to understand whether proliferative cells are susceptible to infection with the Leo virus, which can be used to predict the efficacy of the treatment. To assess whether the Src family of kinases can mediate the infection with the Leu virus, the uninfected NIH-3T3 cells were transformed with v-src and challenged with the Leo virus. The determination of v-src signaling can be referred to as the susceptibility to the Leo virus. Next, examine five breast cancer cell lines: MDA-MB-468, MCF7, MDA-MB-435, T47D, and SK-BR-3, and cell lines derived from normal breast tissue (HBL-100), and examine them in vitro. European virus replication capability. All five tumor-derived tumors can be infected with the virus, while HBL-100 does not replicate the virus. -18- This paper scale applies to Chinese National Standard (CNS) A4 specification (210X 297 mm) 1248973 A7 B7 V. Description of invention (16) To determine whether the Ras pathway in these cell lines is indeed activated, in serum presence and deficiency MAPK phosphorylation concentration was assessed under serum conditions. Infected cell lines exhibit structural MAPK phosphorylation even in the absence of mitogens, whereas cell lines that are not susceptible to infection by the Leo virus exhibit MAPK phosphorylation only in the presence of serum. Thus, structural MAPK phosphorylation is a sensory marker for infection with Leu. MDA-MB-468 human tumor xenografts were implanted into SCID mice to determine whether the Leo virus could be used as an oncolytic agent in vivo to combat breast tumors. After confirming the palpable tumor, the mice were treated with a single injection of Central European virus and the tumor size was monitored over a four-week period. A single injection can result in dramatic degradation of tumor size. Finally, the ability of the Central European virus to develop antigenic breast cancer tumors can be determined, and the results show that the Leo virus can be replicated in the biopsy samples collected by various patients. Therefore, many breast tumors are susceptible to infection by the Leo virus. The reovirus is an abbreviation for Respiratory and Enteric orphan virus. The full name indicates that the virus is isolated from the respiratory tract and the intestine, although it is not related to any known human disease state. Sabin, AB. (1959), Science 130: 966). ‘’Leo virus π — The word means all viruses classified under the genus Leo. The Leo virus is a double-stranded RNA genomic virus. The virions are 60-80 nm in diameter and have two concentric shells that are all icosahedral. The genome consists of 10-12 unpaired fragments of double-stranded RNA with a total genome size of 16-27仟 bases. The size of individual RNA fragments is diverse. There are three different but related types of Leoviruses that have been separated from many biological species. -19- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X297 mm).

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1248973 A7 B7 V. Description of invention (17). All three types share the same complement-binding antigen. The human Central European virus system consists of three serotypes: type 1 (Lang virus strain or TIL), type 2 (Jones virus strain, T2J) and type 3 (Dearing) Viral strain or Abney virus strain, T3D). These three serotypes can be easily distinguished by neutralization and hemagglutinin-inhibition analysis (Sabin, AB (1959), Science 130: 966; Fields, Debate (1996), Fundamental Virology. 3rd Edition. Lippincott- Raven; Rosen, L. (1960) Am. J. Hyg. 71: 242; Steiner, NF (1967) Br. Med. Bull· 23: 150). Although the Leo virus is not associated with any specific disease known, many people have been exposed to the Leo virus before adulthood (ie, less than 25% of young children under 5 years old, and more than 50% of adults between 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) 细胞 For mammalian Central European viruses, the cell surface recognition signal is sialic acid (Amsz, GD et al. (1984) , Virology 138:37; Jane, JRK and Pasiti, A_F. (1985), J. Virol 56:356; Paul (RW) RW et al. (1989) Virology 172:382-385). The ubiquitous nature of the acid, the Leo virus can effectively bind to a variety of cell lines and thus can effectively calibrate many different tissues; however, the sensitivity of each cell line to the infection of the Leo virus is clearly different. As described herein, the applicant It has been found that the presence of structural MAPK phosphorylation -20- this paper scale is applicable National Su quasi (CNS) A4 size (210X297 mm)

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1248973 A7 B7 V. INSTRUCTIONS (18) Cells are susceptible to infection with Central European viruses. The π-resistance π of the cell against the infection of the Leu virus means that no virus is produced or produced after the virus infects the cell. A cell line that is susceptible to π infection can show cytopathic, viral protein synthesis, and/or virus production. Resistance to Leo virus infection is seen in the gene translation phase, but not in the early transcription: the virus transcript does not appear after production. Viral proteins. The implantation of human tumor cells into SCID mice has been recognized as a well-known model system for testing the effectiveness of various anti-tumor agents on humans. It has been shown to be effective against SCID mice implanted with human tumors. The pharmaceuticals are also expected to be effective against humans, tumors in humans. Based on these findings, the Applicant has developed a method for measuring the sensitivity to infection of the Leu virus by measuring structural ras-MAP signals and treating mammals. A method of proliferative disorders, wherein the proliferating cell line can exhibit a structural MAPK phosphorylation. Representative mammals include dogs, cats, sheep, goats, cows, horses, pigs, mice, non-human primates, and humans. In a preferred embodiment, the mammal is a human. In the diagnostic method of the invention, the proliferating cell MAPK phosphorylation process The degree of mitogen is determined by the presence or absence of the mitogen. The presence of the structural ras-MAP signal in the cell indicates the susceptibility to the infection of the Leo virus. In the treatment method of the present invention, the drug of the Leo virus is presented to the proliferating cell. In a specific embodiment of the present invention, the course of the Leo virus can be administered once or several times. In the treatment method of the present invention, the drug of the Leo virus is finely propagated - 21 - This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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1248973 A7 B7 V. INSTRUCTIONS (19) Cells are mammalian individuals that exhibit structural MAPK phosphorylation. Typical human Leuviruses that can be used include type 1 (eg, beam strain *T1L), type 2 (eg, Jones strain or T2J), and type 3 (eg, Deerin strain or Aberni strain) , T3D or T3A), other types of Leo virus can also be used. In a preferred embodiment, the Leo virus is a human Leovirus serotype 3, and the Leo virus is preferably a human Leovirus serotype 3, a Deerlin virus strain. Or the 'Lio virus can be a non-human mammalian virus (such as a non-human-primate Leo virus, such as a leo virus, a horse, or a canine leu virus), or a non-mammalian Central European virus ( Such as the bird Central European virus). Combinations of different serotypes and/or different strains of Central European viruses (such as the Central European virus from different species of animals) can be used. The Leo virus can be naturally occurring or modified. "Naturally produced" are Central European viruses that are isolated from natural sources and have not been specially modified by humans in the laboratory. Such as the Leo virus can come from, the source of the field:: from the patient. The Leo virus can be modified but still can cause soluble infection of mammalian cells exhibiting structural MAPK citrate. Can be chemistry before administration of proliferating cells Or biochemical pre-treatment of the Central European virus (such as the use of protease treatment, such as chymotrypsin or trypsin). Pretreatment with protease can remove the outer layer or capsid of the virus and may increase the virus infectivity. For liposome or colloidal particles (Pr〇teaSe cleavage of reovirus capsid protein mul and mulC is blocked by alkyl sulfate detergents, yielding a new type of infectious subvirion particle1丨, J. of Virology 72(1) ): 467-75 (1998)) To reduce or prevent immunity to mammals that have developed immunity to the Leo virus-22- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X297 mm)

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1248973 A7 B7 V. INSTRUCTIONS (2〇) Reaction. For example, virions can be treated with chymotrypsin in the presence of a colloidal molecule forming concentration of an alkyl sulphate cleaner to produce new infectious subviral particles (ISVP). ISVP can be used alone or mixed with a complete virus to provide an agent that is not readily identifiable or that is not pre-prepared by the patient's immune system. The Leo virus can be a recombinant Leo virus, which can be composed of two or more Central European viruses with different pathogenic phenotypes, each containing the same epitope to thereby reduce or prevent the mammal from being exposed to some The immune response produced by the European virus subtype. The recombinant virion (also referred to as a recombinant) can be produced by co-infecting mammalian cells with different subtypes of the Central European virus, which can comprise the resulting recombinant and different sub-assemblies in the resulting virion capsid. Type of shell protein mixture. The Leo virus can be modified by introducing a mutant coat protein (such as σΐ) to the virion outer capsid. Proteins can be mutated by substitution, insertion or deletion. Substitution involves the insertion of a different amino acid to replace the original amino acid. Insertion involves the insertion of an additional amino acid residue into one or more protein sites. The deletion contains one or more amino acid residues in the deleted protein. This mutation can be produced by methods known in the art. A single nucleotide position-specific mutation that encodes a gene for a coat protein can result in a mutation in the desired coat protein. In vitro, mammalian cells infected with Leovirus (such as COS 1 cells) exhibit mutant proteins that allow the mutated protein to enter the Leo virus virion particles (Turner and Duncan, ''Site directed mutagenesis of the Cterminal Portions of reovirus protein sigmal: evidence for a conformation-dependent receptor binding domain11 Virology 186(1):219-27 (1992); Duncan et al., "Conformational and functional analysis of -23-

1248973 A7 B7 5. The C-terminal globular head of the reovirus cell attachment protein" Virology 182(2): 810-9 (1991); Ma et al., "N-terminal quarter of reovirus cell attachment protein sigma 1 possesses intrinsic virion-anchoring function” Virology 179(1): 95-103 (1990)). The Leo virus is preferably a Central European virus modified to reduce or remove the immune response to the Leo virus. The modified Central European virus is referred to as π-immunoprotective Central European virus. The modification may include loading the Leo virus into liposomes, colloidal particles or other excipients to protect the Leo virus against mammalian immunity. System. Alternatively, the Leo virus virion capsid can be removed due to the protein body host fluid present in the outer capsid and the main determinant of cellular response. ''Proliferation disorder π means that its cell proliferation is much faster than normal tissue growth. Any cell is dysregulated. Thus, "proliferating cell" refers to a cell that proliferates much faster than normal cells. Proliferation disorders include, but are not limited to, neoplasms. A tumor is an abnormal tissue that grows, usually forming a distinct mass. It grows far faster than normal tissues due to cell proliferation. The tumors show partial or total lack of structural organization and cannot coordinate with normal tissues. These tumors can be broadly classified into three main types. The malignant tumors originating from epithelial tissues are called For cancer, a malignant tumor derived from connective tissue (such as muscle, cartilage, fat or bone) is called a sarcoma and affects the hematopoietic group. Malignant tumors (including components of the immune system) (including components of the immune system) are called blood cancers and lymphomas. Tumor-based growth of disease-causing cancers. As used herein, tumors (also known as " Tumor π) is a hematopoietic tumor and a solid tumor. Other proliferative disorders include, but are not limited to, neuroblastoma. -24- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm)

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k 1248973 A7 B7 V. INSTRUCTIONS (22) At least some of the cells in the proliferative disorder treated by the method of the present invention exhibit structural MAPK phosphorylation. Similarly, at least some of the cells identified to be susceptible to infection with the Leu virus using the methods of the present invention exhibit structural ΜΑρκ phosphorylation. ΠΒ-cells refer to B-lymphocytes. B_lymphocytes have two major subgroups, B-1 and B-2 cells. B-1 cells can be autologously renewed and often secrete high concentrations of antibodies. Binding to multiple antigens (multiple specificity) but low affinity. Most B cells are Β2 cells, which are directly produced by the precursors in the bone marrow and secrete highly specific antibodies. '' Τ-cells' Finger lick - lymphocytes. The cell line differentiates within the thymus and is specialized to combat cells that produce intracellular organisms. Cells only recognize antigens that are located on the surface of body cells. "Anti-Liouvirus antibody" refers to an antibody that binds to the Leo virus. "IgG antibody" refers to an immunoglobulin G antibody. Ig(} (the most abundant antibody type) is the main work of neutralizing bacterial toxins and It can bind to microorganisms to enhance its phlegm. ''Humanized antibody' means that the amino acid sequence of the non-antigen-binding region has been changed to an antibody molecule that is closer to a human-like antibody, and retains its original binding ability.' 'Administration to proliferating cells or neoplasms' refers to the administration of the Leo virus in a manner that allows it to contact proliferating cells or tumor cells (also referred to herein as tumor cells). The route of administration, as well as the formulation, carrier or excipient, depends on the site and the type of tumor. There are a variety of routes of administration available. For a parenchymal tumor that can be accessed, the Leo virus can be directly injected by injection. In the case of hematopoietic tumors, the Leo virus can be administered by intravenous injection. 25- This paper size is applicable to the towel@S standard (CNS) A4 size (210 X 297 mm) ----^

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1248973 A7 B7 V. Description of invention (

Or intravascular administration. For tumors that are not easily accessible in the body (such as metastatic or brain tumors), the Leo virus can be administered to the tumor through a mammalian body (such as intramedullary, intravenous or intramuscular). Alternatively, the Leo virus can be administered directly to a single parenchymal tumor and then delivered to the metastatic site via a systemic system. The Leo virus can also be administered subcutaneously, intraperitoneally, topically (eg for melanoma), orally (eg for oral or esophageal tumors), rectal (eg for colorectal tumors), intravaginal administration (eg For example, for cervical or vaginal tumors, intranasal administration or by inhalation spray (such as for pulmonary tumors). The Leo virus can be administered systemically to a mammal that is immunocompromised or has not developed immunity to the Leo virus antigenic epitope. In this case, the systemically administered Central European virus (i.e., by intravenous injection) can be exposed to proliferating cells to cause its dissolution. When the mammal being treated has a higher anti-Liouvirus antibody price, more Leo virus needs to be administered in order to function. Mammals that are first exposed to the immunological activity of the Leo virus subtype may have developed humoral and/or cellular immunity to the Viol virus subtype. However, it has been found that direct injection of the Central European virus into a solid tumor of an immunocompetent mammal can result in lysis of the tumor cells. On the other hand, when the Leo virus is administered systemically to an immunocompetent mammal, the mammal will develop an immune response against the Leu virus. If the Leo virus is a subtype that does not develop immunity in mammals' or the Leo virus has been previously modified to be immunoprotective as described above (eg, by enzymatic digestion of the outer capsid or in a colloidal particle), this can be avoided. An immune response. Or 'may consider prior or simultaneous administration of a known drug of the art to carry out -26 -

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k 1248973 A7 B7 V. INSTRUCTIONS (24) Integral suppression of the immune system to achieve the purpose of inhibiting the immune activity of mammals against the Leo virus (Kraft 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 an immunosuppressive agent such as an anti-European virus antibody. Mammalian immunity against the Leo virus can also be used by mammalian blood. Slurry-pulling removes anti-Liouvirus antibodies for temporary reduction or suppression. Administration of non-specific immunoglobulins to mammals by intravenous injection can also temporarily reduce or suppress humoral immunity of the mammal against the Leo virus. The Leo virus is co-administered with a immunosuppressant and/or an immunosuppressant to a mammal that has been immunologically active against the Leu virus. The immunosuppressant and immunosuppressant are well known to those skilled in the art and include cyclosporine. (cyclosporin), rapamycin, tacrolimus, mycophenolic acid, achaisep Azathioprine and its analogues, and similar substances. Other agents known to have immunosuppressive properties are known (see, for example, Goodman & Gilman, 7th ed., page 1242, which discloses The disclosure is incorporated herein by reference.) The immunosuppressive agent also comprises a π anti-anti-European antibody π, which is an antibody specific against an anti-Liovirus antibody. Prepared by known methods. See, for example, ''Antibodies: A laboratory manual' Ε· Harlow and D. Lane, Cold Spring Harbor Laboratory (1988) 〇The anti-Erivirus antibody can be administered before the Leo virus, with Rio The virus is administered at the same time or after administration of the Leo virus. An effective amount of anti-European virus antibody is preferably administered for a sufficient period of time to reduce or eliminate mammalian administration. -27- This paper scale applies to Chinese National Standard (CNS) Α4 specification (210X297 mm)

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1248973 A7 B7 When the pharmaceutically acceptable excipient is a diluent, it may be a solid, semi-solid, or liquid substance which acts as an excipient, carrier or active ingredient, pill, powder, lozenge , small drug suspension, emulsion, solution, sugar, containing up to 1% by weight of live, suppository, sterile injection solution, and 5, invention instructions (25) European virus immune response. The terms "π immunosuppressant" or "immunosuppressive agent" include traditional immunosuppressive agents, immunosuppressive agents, antibodies, and conditions that can cause damage to the immune system, such as radiation therapy or HIV infection. The term "substantially dissolved" means at least 10% of proliferating cells are lysed, more preferably at least 50% 'and optimally at least 75% of cells are lysed. The percentage of tumor cells dissolved can be determined by measuring the tumor size reduction in mammals or the dissolution status of in vitro tumor cells. "A mammal suspected of having a proliferative disorder" means that the mammal may have a proliferative disorder or tumor or has been diagnosed with a proliferative disorder or tumor or has been previously diagnosed with a proliferative disorder or tumor, the tumor or substantially all of the tumor has Surgical removal and suspected that the mammal may still have some residual tumor cells. The present invention also encompasses a pharmaceutical composition comprising as an active ingredient one or more immunosuppressants or immunosuppressive agents and one or more European virus and "pharmaceutically acceptable carrier or excipient". In the preparation of the compositions of the invention, the active ingredient is exempted The inhibitor or immunosuppressive agent and the Central European virus are usually mixed with an excipient, diluted with an excipient or packaged in a container, such as a capsule, sachet, paper or other container type. Therefore, the composition may be a tablet capsule, a capsule, an elixirs slurry, an aerosol (solid or liquid medium) compound ointment, soft and hard capsules -28- The paper size is applicable to the Chinese country. Standard (CNS) A4 size (210X 297 mm)

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A7 ______B7 V. Description of invention (%) 1248973 Aseptic packaging powder type. Examples of certain suitable excipients include lactose, glucose, sucrose, sorbitol, telitol, starch, gum arabic, calcium phosphate, alginate, yellow gum, gelatin, calcium citrate, microcrystalline cellulose, Polyethylene port than pyrrolidine, cellulose, soda water, syrup, and methyl cellulose. The formulation may additionally comprise a lubricant (such as talc, magnesium stearate, and mineral oil), a wetting agent, an emulsifying pair, a suspending agent, a preservative (such as methyl benzoate and propyl benzoate), Aromatizers, and flavoring agents. The compositions of the present invention can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a patient using procedures known in the art. When preparing a solid composition such as a tablet, the main active ingredient/immunosuppressive or immunosuppressive agent and the Central European virus can be mixed with a pharmaceutical excipient to form a solid premixed composition of the compound of the present invention. . The reference to k that the pre-compound composition is homogeneous means that the active ingredient has been dispersed throughout the composition, so that the composition can be easily dispensed into equivalent unit dosage forms such as tablets, pills, and capsule. The tablets or pills of the present invention may be coated or otherwise combined to provide a dosage form having the advantage of prolonged action. For example, tablets or pills may include internal and external ingredients, the latter being used as the former. These two components are separated by an intestinal layer, which acts to resist the decomposition of gastric acid and to allow the inner layer components to pass intact to reach the duodenum or to delay release. A variety of materials can be used for the enteric layer or coating, which is a polymeric acid mixture comprising certain polymeric acids and materials including, for example, shellac, cetyl alcohol and cellulose acetate. Liquid type containing the novel composition of the present invention for oral administration or borrowing -29- This paper scale applies to Chinese National Standard (CNS) A4 specification (210X297 mm)

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1248973 5 invention description A7 B7 oily suspension for injection, coconut oil, pharmaceutical excipients can be 3 aqueous drops, suitable flavored syrup, water or 2 edible oils (such as corn oil, cottonseed oil, sesame oil Or probiotic oil) flavored emulsions, as well as aromatic liquors and similar medical waters:: 2 or:: The in-situ composition comprises solutions and suspensions present in pharmaceutically acceptable, / /, or a mixture thereof And the powdered dimer or solid composition may be suitably pharmaceutically acceptable as described herein. Preferably, the administration of the composition can be administered systemically by oral or intranasal inhalation. The group stored in the preferred pharmaceutically acceptable solvent can be atomized by using the raw gas. The atomized solution can be inhaled via the atomization device or can be attached to the mask or periodic positive pressure. a ^All/liquid, suspension, or powder compositions may be administered in a suitable regimen, preferably via oral or nasal administration. The preferred formulation is a transdermal delivery device (., patch). The Tibetan transdermal patch can be used to provide continuous or intermittent injection control of the present invention. The structure and usage of the transdermal patch for delivery of the drug is It is well known in the art. See, for example, U.S. Patent No. 5, Q23, 252, which is incorporated herein by reference. Suitable formulations for the present invention can be found in Remmgton's Pharmaceutical Sciences, the disclosure of which is incorporated herein by reference. Immunosuppressants or immunosuppressants and Leovirus or immunosuppression -30- This paper scale is applicable to China National Standard (CNS) A4 specification (21〇x 297 mm) 1248973 A7 _B7 V. Invention description (28) or pharmaceutical composition of immunosuppressive agent and Leo virus can be packaged in convenience The kit contains the necessary substances for packaging in a suitable container. The kit may also be considered to contain a chemotherapeutic agent. The immunosuppressive or immunosuppressive agent is administered at an appropriate dose and is immunosuppressing the immune system of the mammal. Or an appropriate dosing schedule for immunosuppression. The dosage and schedule are well known to those skilled in the art. The parental disease is administered in an amount sufficient to treat the proliferative disorder (i.e., an effective amount). "Treatment" of a disorder of proliferation refers to the administration of a Leo virus to a proliferating cell to cause proliferating cell lysis. It can result in a reduction in the size of the tumor or a complete removal of the tumor. A reduction in the size of the tumor or eradication of the tumor is usually caused by the dissolution of the tumor cells by the Central European virus ("tumor cell lysis"). The effective amount is preferably a dose which inhibits the growth of tumor cells. Preferably, the effective amount is from about 1 pfu/kg body weight to about 1015 pfu/kg body weight, more preferably from about 1〇2 pfu/kg body weight to about 1〇3 pfu/kg body weight. In the treatment of humans, about 102 to 1017 plaque forming units (PFU) of the Levi virus can be used depending on the type, size, and amount of tumor appearance. The effective amount should depend on each individual and can be based, at least in part, on the type of Leo virus, the route of administration chosen, the size of the individual, the age, sex, the severity of the symptoms of the patient, the size of the tumor and other characteristics, and the like. Etc. The course of treatment can last from a few days to a few months or until the disease is relieved. The immunosuppressive or immunosuppressive agent and the Central European virus can be administered in a single dose or in multiple doses (i.e., more than one dose). Multiple doses can be administered simultaneously, or consecutively (eg, over a period of days or weeks). The Leo virus can also be administered to more than one tumor of the same body. The composition is preferably formulated as a unit dosage form, each dose containing an appropriate amount of immunization -31 -

This paper scale applies to the national A4 specification (2lQX297^iT 1248973 A7 B7 5. Inventive Note (29) Inhibitor or immunosuppressive agent and the Central European virus from about 102 pfu to about l〇u pfu. π unit dose type" Words refer to discrete units that are practically suitable for single doses in human patients and other mammals, each unit containing a predetermined dose of Leovirus (which is calculated to produce the desired therapeutic effect), and appropriate medical morphing As mentioned above, it has been found that Leovirus can effectively treat parenchymal tumors in immunocompetent mammals. Direct administration of unmodified Lipovirus to tumors can lead to tumor cell lysis and reduce the tumor size of animals with immunological activity. When some methods are used to make the animal immunosuppressive or immunodeficiency, the systemic administration of the Leo virus is more effective in producing tumor cells. After consideration, the administration of the Leo virus can be combined with surgery or tumor removal. Therefore, a method for treating a parenchymal tumor can be provided, which includes surgical removal of the tumor and administration of a Leo virus to the tumor or a nearby part thereof. The toxic drug is combined with radiation therapy which can immunosuppress mammals. The present invention further contemplates co-administration of the Leo virus of the present invention with a known anticancer compound or chemotherapeutic agent. The chemotherapeutic agent is a compound which inhibits tumor growth. The agent includes, but is not limited to, 5-fluorouracil, mitomycin C, chlorpyrifos, sputum H urine, ring-amplifier, dacarbazine, mitoxantrone, Anthracyclins (Epirubicin and D〇xUrubicin, receptor antibody) such as rabbit herceptin, 依普普德德(etopside), pregn a some, platinum compounds (such as carboplatin and cisplatin), taxanes -32- This paper size applies to China National Standard (CNS) A4 Specification (210 X 297 mm)

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1248973 A7 B7 V. Description of invention (3〇) (such as taxol and taxotere), hormone therapeutics (such as tamoxifen and antiestrogens), interferon, Aromatase inhibitors, prostaglandin agents and LHRH analogues. The Leu virus and immunosuppressive agents of the present invention have been found to reduce the growth of metastatic tumors. In a specific embodiment of the invention, there is provided a method of reducing the growth of metastatic tumors in a mammal comprising administering an effective amount of a Leo virus to a mammal that suppresses immunization. Utility The diagnostic method of the present invention can be used to determine the susceptibility of a cell to a virus infection in Central Europe by measuring structural ras-MAp signaling. It can be used to determine patients who may have achieved success in treating cell proliferation disorders with Leydig virus. The Leo virus and immunosuppressant of the present invention can be used for various purposes. It can be used in an oral therapy method that exhibits structural dysregulation of mammalian κρκ phosphorylation. It can be used to reduce or eliminate tumors. It can be used to treat metastases&lt;methods. It can be used in combination with known methods of treating cancer, including surgery, chemotherapy, and radiation therapy. The invention and its advantages are set forth in the <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; 1~ Examples are ϋ]; ^All temperatures are in Celsius ((10) (four) degrees (unless otherwise stated), all ratios are weight ratios (unless otherwise specified), A sound, write and meaning as follows. Undefined, I am generally accepted. I am 297 mm. 1248973 A7 B7 V. Description of invention (31) μΜ = microequivalent mM = milliequivalent Μ = equivalent ml = ml μΐ = microliter mg = milligram Kg = microgram PAGE = polyacrylamide gel electrophoresis rpm = revolutions per minute FBS = fetal bovine serum DTT = dithioxitol SDS = twelve alkyl sulfate steel PBS = phosphate buffer solution DMEM = Dubecco ' s) Modified Eagli s medium a -MEM = α-modified yege medium β -ME = β-mercaptoethanol MOI = infection rate PFU = plaque forming unit MAPK = MAP kinase phosph-MAPK = phosphorylated MAP kinase HRP = horseradish peroxidase PKR = double-stranded DNA activated protein kinase RT-PCR = reverse transcription kinase - polymerase chain reaction - 34 - This paper scale applies to Chinese National Standard (CNS) A4 specification (210X 297 MM)

1248973 A7 B7 V. INSTRUCTIONS (32) GAPDH = oleylaldehyde-3-phosphate dehydrogenase EGFR = epithelial growth factor receptor MEK kinase = mitogen-activated extracellular signal-regulated kinase DMSO = dimethyl hydrazine SCID = . General Methods for Severe Complex Immunodeficiency Cell and viral parental NIH-3T3 cells and the NIH-3T3 cell line transformed with v-Src were generously presented by Dr. Jove (University of Florida). Breast tumor cell lines MDA-MB-468, MCF7, MDA-MB-435, T_47D, SK-BR-3 and control group HBL100 cells were generously presented by Dr. Karl Riabowol (University of Calgary) . All cell lines were grown in 10% fetal bovine serum (FBS) (Dubecide-modified yege medium) (DMEM). The Deerlin virus strains used in these studies were tested in L cell suspension medium and purified by Smith et al. (1969) except for beta-mercaptoethanol (β-ΜΕ). ) is deleted from the extraction buffer solution. Cellular infection and virus quantification The clustered monolayer cells were cultured on a 24-well plate and infected with a Levi virus estimated to have an infection rate of 80 PFU/cell. After incubation at 37 ° C for 1 hour, the monolayer cells were washed with warm DMEM-10% FBS and incubated in the same medium. Mix NP-40 and sodium deoxycholate at different times after infection -35- This paper size applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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1248973 A7 B7 V. INSTRUCTIONS (S3) The substances were directly added to the infected monolayer cells of the medium to a final concentration of 1% and 0.5%, respectively. The lysate was collected and the L-929 cells were tested for plaque strength to determine virus production. Preparation of radiolabeled Central European virus-infected cells and lysates Secondary cluster monolayer (80% cluster) cells were infected with Leovirus (MOI ～10 PFU/cell). At 46 hours post infection, the medium was replaced with methionine-free DMEM containing 10% FBS and 0.1 microcurie/ml [35S]methionine. After further incubation for 2 hours at 37t, the cells were washed with phosphate-buffered solution (PBS) and dissolved in the same buffer solution containing 1% (Triton) X·100, and 0.5% sodium deoxycholate. The lysate was boiled again and stored at -70 °C until use. Immunoprecipitation and SDS-PAGE analysis were performed by 35S-calibrated Leovirus using the anti-Liovirus serum type 3 serum as described above (Li, PWK et al. (1981) Virology, 108: 134-146). Infected with cell lysates.

Detection of Phosphorus-MAPK and Total MAPK Using the PhosphoPlus p44/42 MAP Kinase (Thr202/Tyr204) Antibody Kit (New England Biolabs), MAP Kinase for Detection of Cell Lysates by Original Factory Instructions . Briefly, the secondary cluster monolayer culture was dissolved using the recommended SDS-containing sample buffer solution and subjected to SDS-PAGE and electro-dyed onto nitrocellulose paper. The primary antibody (anti-total MAPK or anti-phosphorus-MAPK) was first probed according to the original manual and probed with a secondary antibody that binds horseradish peroxidase (HRP). Severe Mixed Immunodeficiency (SCID) Rats Purchased by Charles River Canada, Canada -36- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm)

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1248973 A7 _B7__ V. INSTRUCTIONS (34) Male SCID rats up to eight weeks of age were treated according to the program approved by the University of Calgary Animal Care Committee. Implantation of allografts and xenografts Actively growing MDA-MB-468 human breast cancer cells were harvested, washed, and resuspended in sterile PBS at a density of 2 x 107 cells/ml. One hundred microliters of the finished product containing 2.0 X 106 cells was injected subcutaneously into the extreme posterior lumbar region. The implanted tumor is allowed to grow for 2-3 weeks to reach a tumor of 0. 5 X 0.5 cm. Intratumoral injection of the Leu virus Once the tumor has reached the size of the treatment, a single intratumoral injection of 10 μl of sterile PBS containing 1.0 X 107 PFU of live or UV-inactive Liouvirus (serum type 3, Dilling virus strain) is performed. . Tumor size was measured twice a week for 2 to 4 weeks. Animals are sacrificed when they show severe conditions due to excessive tumor burden or any significant pain. Tissue immunology analysis of the Leu virus infection will be performed using a formalin-fixed, paraffin-embedded tumor section, which is covered with a coverslip and subjected to immunofluorescence analysis. After the paraffin was removed with xylene, the sections were rehydrated and exposed to primary antibodies (rabbit multiple anti-leovirus type 3 serum, diluted 100-fold with PBS) for 2 hours at room temperature. After washing three times with PBS, the sections were exposed to a secondary antibody [goat anti-rabbit IgG (intact molecule)-luciferin isothionate complex (FITC) at room temperature or according to the test. Cy3 was diluted 100-fold with 10% goat serum and 0.005% Evan's Blue in contrast-stained PBS] for 1 hour. For additional contrast staining, nuclear staining DAPI was also used. Finally, rinse again with PBS three times. -37- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm)

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1248973 A7

The treated sections were then rinsed once with double distilled water. The sections were dried and sealed with 90% eucalyptus oil containing 0.1% phenylenediamine, and a Zeiss Axiophot microscope observation with a Carl Zeiss camera (magnification of all photos) The multiple is 2〇〇χ). Leo virus infection of primary breast tumor samples Biopsy samples of breast tumors were immersed in 950/0 ethanol and sterilized several times with sterile PBS. The sample was then cut into small pieces and placed in a 24-well test disk containing DMEM (containing i0% FCS). Join the Leo virus (1X108PFU). Samples were washed with sterile PBS and fixed with formalin at various times after infection. The samples were then embedded in paraffin and sectioned for immunohistochemical analysis using antibodies specific against the total Leovirus protein. i Example 1 · Fish I-six non-receptor tyrosine kinase family member Hanization than 舫 舫 舫 田 田 田 田 田 田 田 田 田 田 田 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然 虽然-3T3 cells are sufficient for replication of the Leo virus (Shi Zhuang, 1993), but whether the transfer of non-receptor tyrosine kinase can cause the sensitivity of the Leo virus remains unknown. To determine whether Src group kinase activation, which often causes uncontrolled proliferation of many breast cancers, can result in a dose of Ras activity in the full-fledged Violvirus, transforming non-infectious NIH-3T3 cells with v-src and assessing the response to Rio Viral susceptibility. The v-src-transformed NIH-3T3 agglutinated monolayer cells or their parental cells were exposed to a Levi virus with a infection magnification (MOI) of ~50 lytic-like units (PFUs). Cells and medium were harvested at different times after infection and the resulting samples were subjected to plaque assay to determine the replication status of the virus. The results showed that after 48 hours of infection, v-src transformed cells-38- This paper scale is applicable to China National Standard (CNS) A4 specification (210X297 mm) 1248973 A7 B7 V. Invention description (36) Produces dramatic cytopathic effect (data Not shown), as well as an increase in the production of Central European virus measured by plaque strength analysis. These combinations (Figure 1) show that even non-receptor tyrosine kinases may mediate a subtype of breast tumor that is infected with the Central European virus and is therefore identifiable. Example 2. The Leo virus can infect a group of human breast cancer-derived cell lines. This type of cell line can detect the structural Ras/MAPK signal to evaluate the possibility of using the Leo virus against tumors derived from the breast. One group of breast cancer cells (including MDA-MB-468, MCF7, MDA-MB-435, T-47D, and SK-BR-3) was assayed for its sensitivity to Inovirus infection in vitro. HBL-100 cells derived from normal breast tissue were used as a control group.俟 These six cell lines grew to 80% of the cluster and then attacked with a violent virus of 10 Μ〇. Cells were labeled with [35S]methionine for 2 hours after 48 hours of infection. Rinse and dissolve the cells with a phosphate-buffer solution. The resulting lysate is then immunoprecipitated with antibodies that specifically target the total virion protein. The immunoprecipitated protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results (Fig. 2A) clearly show that the Leo virus can be efficiently replicated in a cell line derived from a breast tumor, whereas the replication of the Leu virus in the HBL-100 cell line is limited. It shows the result of Ras activation of the infectious line of these breast tumor cells, which can be via direct mutation or via upstream signaling elements. It is clearly demonstrated that a high rate of breast cancer can be treated by calibrating Ras therapy, although this particular activation is rare in this cancer type. Due to the inevitable result of MAPK phosphorylation Ras signaling, the protein phosphorylation status should only be found in cell lines with normal Ras signaling (if mitosis occurs -39 - this paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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k 1248973 A7 B7 V. Stimulation of invention (37)). In cell lines with normal Ras signaling, removal of these mitotic stimuli should result in signal disruption via this pathway, resulting in MAPK dephosphorylation. In a cell with abnormal Ras activity, whether directly activated by mutation of Ras or via upstream elements, with or without serum added, the MAPK phosphorylation pattern should always be present. In order to determine the activation of the Ras/MAPK signaling pathway of the infective line of the breast tumor cell line to be observed, Western blot analysis was performed on these cell lines, in which a specific antibody against the antibody containing Lin·ΜΑΡΚ was used. . 1^0 octa-ΜΒ-468, MCF7, MDA-MB-435, T-47D, SK-BR-3 and HBL-100 cells were plated into a six-well assay plate. The cells were then incubated in 10% fetal bovine serum (FCS) or low serum (0.5% FCS) for 48 hours. The cells were washed with phosphate buffered saline (PBS) and a protein sample buffer solution was collected. The cell lysate was subjected to SDS-PAGE and then stained onto nitrocellulose paper and detected by an anti-phosphorus-MAPK antibody according to the manufacturer's recommended method. The results (Fig. 2B) clearly show that the only non-infectious cell line (HBL-100) possesses phosphorus-MAPK in the presence of serum, but not in serum, indicating that the Ras signaling of this pathway is normal. The remaining infective cell lines possess a phosphorus-MAPK with or without mitotic signals, as would be expected to have a structural activation. Protein concentration was normalized using a specific anti-total MAPK (Fig. 2C) antibody. Example 3. Leo disease can be used as an anti-tumor agent to combat breast tumors in vivo Human breast cancer cell line MDA-MB-468 is used as a tumor xenograft and introduced into the posterior flank of SCID mice by subcutaneous injection. After confirming the palpable tumor, a single intratumoral injection of MDA-MB_468 tumor containing 1.0 X 1〇7 lysate -40- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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1248973 A7 B7 V. INSTRUCTIONS (38) PBS of Leovirus serotype 3 (Dirrin virus strain) of plaque forming unit (PFU). Animals in the control group were injected intratumorally with UV-inactivated Leovirus. Pay close attention to tumor growth within four weeks. As shown in Figure 3, treatment with Leo disease can cause dramatic deterioration in tumor size. As described above, the hematoxylin/eosin (HE) staining of the residual mass showed that a single intra-Lion virus injection resulted in complete destruction of the tumor cells when compared with the control group tumors (data not shown). Immunological light microscopy was performed to determine whether the observed tumor cell lysis was due to viral replication and to determine whether the Leo virus protein was interspersed with tumor mass. Using a monoclonal antibody against the virus of the Central European virus and a thin section of the residual tumor mass embedded in paraffin, it was examined that the replication of the Leo virus was restricted to MDA-MB-468 tumor cells and did not extend to the surrounding skeletal muscle. 4- J: European virus can be used in primary breast samples to determine the intrinsic properties of the Leo virus, not due to the inherent characteristics of the secondary cell line, to collect primary human breast cancer samples to assess the influenza virus infection. The biopsied breast tumor samples were immersed in 95% ethanol for disinfection and then washed several times with sterile PBS. The sample was cut into small pieces and placed in a 24-well test plate containing dmem (containing 10% FCS). Join the Leo virus (iX1〇8pFUs). At different times after infection, the samples were washed with sterile PBS and then fixed in formalin. The sample was then embedded in paraffin and sectioned for immunohistochemical analysis using a specific anti-European virus protein antibody. The results (not shown) clearly show the staining of the Central European virus in the challenged tumors, which shows that the virus can replicate in these primary samples. The present invention has been specifically shown and described with reference to the preferred embodiments, but it should be understood by those skilled in the art that various forms and details are still present therein, and the paper size standard (CNS) A4 Specifications (plus X 297 mm)

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线 1248973 A7 B7 V. The invention (39) does not depart from the spirit and scope of the invention as defined by the appended claims.

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-42 - This paper size applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm)

Claims (1)

I material 8 to 3 〇 126, 592 patent application AS ^ one - one Chinese application patent scope replacement (94 October) d D8^-&quot; VI, the scope of application for patents 1 · a diagnosis of available Leovirus (reovirus A method of treating a proliferative disease, comprising: U) providing a biological sample from a mammal suspected of having a proliferative disorder; (b) measuring a phosphorylation state of a map kinase in the biological sample, wherein the structural MAP kinase phosphate is absent in the absence of serum The Department of Health is an indicator of the treatment of proliferative diseases with the Central European virus. 2. The method of claim 1, wherein the MAP kinase phosphorylation state is determined using an antibody specific for phosphorylated MAP kinase. 3. The method of claim 1 or 2, wherein the proliferative disorder is fibrosarcoma. 4. The method of claim 1 or 2 wherein the proliferative disorder is a parenchymal tumor. 5. The method of claim 1 or 2 wherein the proliferative disorder is selected from the group consisting of lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer , pancreatic cancer, breast cancer and central and peripheral nerve fine cancer. 6. The method of claim 1 or 2, wherein the proliferative disorder is breast cancer. 7. The method of claim 1 or 2, wherein the mammal is selected from the group consisting of a dog, a cat, a sheep, a goat, a cow, a horse, a pig, a mouse, a non-human-primate, And humans. 8. The method of claim 1 or 2 wherein the mammal is a human. This paper scale applies to Chinese national standards ((:^8) A4 specification (21〇x297 mm)