TWI320056B - Virus clearance of neoplastic cells from mixed cellular compositions - Google Patents

Description

1320056 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 6. 7. V. Invention description (Related invention: The invention is applied for US Provisional Patent Application No. 60/201 990, delivered on May 3, 2000 Person; No. 6〇/2〇5 389, who will be seen at May 19, 2010, and the serial number 6〇/268 〇54, delivered on February 13, 2001. Serial No. 60/276,782, delivered on March 16, 2001, under 35 USC § 1 l9(e). The complete description of each of the above provisional applications is based on reference materials. In the specification. Field of the Invention: The present invention relates to a method for selectively removing tumor cells from a mixed cell composition of a living organism, which is carried out using a virus capable of selectively infecting and killing tumor cells. The invention also provides a composition prepared according to the method, and a kit comprising the virus combination for use in the present invention. References: 1. U.S. Patent No. 6,136,307, WO 94/18992, published September 1, 1994, EJ WO 94/25627, published on January 1, 1994, WO 99/08692, at 1 Published on February 25, 999

Bar-Eli, N., et al. 'The major cytotoxic effects of new domain viruses on lymphoma cells,' Cancer Clinical Oncology Hybrids, No. 122: 409-415 (1996).

Bensinger, WJ • “Should we clean up?” <<Bone Marrow Transplantation, No. 21: 113-115 (1998).

Bischoff JR et al., "An adenovirus mutant strain selectively replicating in human tumors deficient in p53." Science, No. 274-4- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X) 297 mm) ίκ------------------ order _| (please read the notes on the back and fill out this page) 1320056

Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumer Cooperatives, M A7 B7 V. Inventions () (5286) : 373 -6 pages (1996) ° 8 · Blagoselonny, MV et al., Γρ·53_ phenotype adenovirus In vitro evaluation of cancer drugs, &lt;&lt;International Journal of Cancer&gt;&gt;, No. 6 7 (3): 386-392 (1996). 9. Bos. J.L. 拉Rast tumor genes in human cancer: a study J ’&lt;&lt;&apos; Cancer Research&gt;&apos;, No. 49 (17): 4682-4689 (1989). 10. Brooks, ed., “Jawetz, Melrick & Adellberg’s Medical Microbiology,” (1998). 11. Chang et al. 'PNAS' No. 89: 4825-4829 (1992). 12. Chang H.W. et al. &lt;&lt; Virology &gt;&gt;, No. 194: 537-547 (1993). 13. Chang et al., &lt;&lt;Journal of Virology &gt;&gt;, No. 69: 6605-6608 (1995). 14. Coffey, M. C. et al., Reovirus treatment of tumors with activated tract pathways, &lt;&lt;Science&gt;, 282: 1332-1334 (1998). 15 · Duggan, P.R., et al., "Predictors of long-term self-sourced stem cell transplantation" &lt;&lt;Bone marrow transplantation&gt;&gt;, 26 (12): 1299-1304 (2000). 16· Fueyo, J., et al., “A mutant tumor-soluble adenovirus targeting the Rb pathway produces an anti-glioma effect in vivo”, &lt;&lt;Tumor Gene&gt;&gt;&gt;, 19 (1) : 2-12 pages (2000). -5- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) ·.---------------I---订·---- (Please read the notes on the back and fill out this page.) Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Printed 1320056 A7 _ B7 V. Invention Description () 17. Gao·, JB Tombal and JT Isaacs ., “for price measurement Rapid in situ hybridization technique for cell contamination of malignant mice in human allograft tissues of nude mice and test tube cultures of such heterologous grafts, &lt;&lt;Prostate&gt;&gt;&gt; No. 39 (1) : 67-70 (1999). 18. Haig, DM et al., &lt;&lt;Immunology&gt;&gt;, No. 17: 4146-4158 (1997), 19. He. B. et al., PNAS, No. 94: 843-848 Page (1997). 20. Kawagishi-Kobayashi, M. et al., &lt;&lt;&gt; Molecular Cell Biology&gt;&gt;, No. 7: 4146-4158 (1997). 21. Nemunaitis, &lt;<New Drug Research Miscellaneous> 〉, No. 17: 375-386 (1999). 22. Nielsen, L.L., et al., "P5 3 Tumor Suppressor Gene Therapy for Cancer," &lt; Cancer Gene Therapy, vol. 5 (1): 52-63 (1998). 23. Nieto. Y. et al. "Self-derived stem cell transplantation of solid tumors in adults", &lt;&lt;hematology oncology clinical study, North America&gt;&gt;, No. 13 (5): 939-968 (Year 1999). 24. Norman. K. et al., Reovirus as a novel tumor lysing agent &lt;&lt;Journal of Clinical Research &gt;&gt;, 105 (8): 1035-1038 (2000). - 25. Richard. KW et al., "Newcastle virus selectively kills human tumor cells" ' &lt;&lt; Journal of Surgical Research' &gt;&gt;, No. 5, pp. 448-453 (1992) . 26. Stojdl. DF . et al. 'Using previously unknown tumor lytic viruses -6 - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) is--------- ------Booking! (Please read the notes on the back and fill out this page) 132〇〇56 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Print A7 B7 V. Invention Description () Interferon pathway "Tumor-specific defects" '&lt;&lt;National Journal of Medicine&gt;&gt;&gt;, No. 6 (7): 821-825 (2000). 27. Romano et al., &lt;&lt; Molecular and Cell Biology &gt;&gt;, 18: 7304-7316 (1998). 28· Sharp et al., &lt;&lt; Virology, vol. 250: pp. 301-315 (1998). 29. Spyridonidis, Α· et al., “The smallest residual disease in autologous hematopoietic harvests from breast cancer patients”, &lt;&lt;Oncology Years&gt;&gt;, No. 9: 821-826 ( 1998). 30. Steele, T_A. "The Recent Development of Cancer Virus Therapy", &lt;&lt;Social Experiments Biomedical Progress, 223, 118-127 (2000). 31. Stewart, DA et al., "From the dose-dense cyclophosphamide, etoposide cisplatin. G-CSF self-sourced stem cell movement is better than the less intensive chemotherapy regimen," &lt;&lt Bone Marrow Transplantation&gt;&gt;, 23rd (2): 111-117 (1999). 32. Strong, JE, et al., "Molecular basis for the solubilization of viral tumors: the signal pathway for larvae by the reovirus," &lt;&lt;embod&gt;&gt;, No. 17: 3351-3362 ( 1998). 33· Strong, JE, et al., “The smallest residual virus in a self-derived hematopoietic harvest from breast cancer patients”, &lt;&lt;Oncology Yearbook&gt;, No. 9: 82 1-826 (1998) ). 34· Strong, JE, et al., “Evidence for the efficiency of epithelial growth factor receptor administration on reovirus infection in host cells”, &lt;&lt;virology&gt;&gt;, 197(1): 4〇 Page 5_411 (1993). --------Book --------- (Please read the notes on the back and fill out this page)

1320056 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Employees' Consumption Cooperatives Printed Clothes 5, Inventions (5) 35. Strong, JE, et al., "v-erb.V. Tumor genes give increased cellular susceptibility to reovirus infection ", &lt;&lt; Virology &gt;&gt;&gt;, No. 70: 612-616 (1996). 36_ Wilman. K.G., "New p53-based anti-cancer medical strategy", &lt;Medical Oncology &gt;&gt;, No. 15 (4): 222-8 (1998). 3 7. Winqer. JN·“High-dose therapy and stem cell transplantation in malignant lymphoma”, &lt;&lt;Oncology&gt;&gt;, (Huntingt), Issue 1 (1 2): 1635-1645 (1999) year). 38. Yoon, SS et al., “A first type of herpes simplex virus with tumor solubility destroys osmotic liver metastasis from colon cancer”, FASEB. J., No. 14: 301-311 (2000) . 39. Zorn. U. et al. 'Inducing intercellular and cytotoxicity against tumor cells by Newcastle virus,&&lt;&lt;&lt;&gt; Cancer Biotherapy&gt;&gt;, No. 9(3): 22-235 (1994). All of the above publications, patents, and patent applications are hereby incorporated by reference in their entirety in their entireties in the entire contents of Specifies the extent to which the reference is to be incorporated in its entirety. BACKGROUND OF THE INVENTION Cell proliferation is regulated by growth promoting signals and growth inhibition signals. In general, these two signals for each cell respond to the body. This particular cell needs a way to balance. If the cell is unable to respond to the growth inhibitory signal, or overreacts to the growth-promoting signal, the cell will be --- _ 8 _ this paper size _ + 03⁄4 standard (CNS) _A4 specification (210 X 297 public Chu) -- -- (Please read the notes on the back and fill out this page)

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1320056 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Description of invention (6) Abnormally rapidly proliferating (called tumor cells), and may eventually develop into cancer ‘a malignant tumor. Chemotherapy is a modern method of treating cancer that is generally based on the rapidly proliferating nature of cancer cells. Because cancer cells proliferate very quickly, they are more sensitive to drugs that inhibit cell proliferation. In theory, by carefully selecting the dose of chemotherapeutic drugs, we can inhibit cancer cell growth without seriously damaging normal cells. However, some normal cells such as hematopoietic stem cells also proliferate very quickly. Therefore, any dose that is harmful to cancer cells is often harmful to hematopoietic stem cells. On the other hand, if the dose is not high enough to kill cancer cells, the cancer cells will be at risk of reappearing shortly after the termination of chemotherapy. Because it is difficult to find a dose that selectively kills cancer cells, sputum dose chemotherapy followed by stem cell transplantation from the source of hematopoiesis has been widely used as a medical treatment for many cancers (see, for example, winter, 1999) ; Nieto and Shpall·, 1999). In this method, a portion of the hematopoietic stem cells are removed from the cancer patient, and then high doses are used to rapidly kill the rapidly growing cells such as cancer cells and hematopoietic stem cells. Immediately thereafter, the patient received a self-derived hematopoietic stem cell transplant, which was previously removed from the same patient to regenerate the hematopoietic system. A serious disadvantage of this type of therapy is that when the stem cells are removed from the disease, they are often contaminated by cancer cells. This is especially a problem when patients have cancers that are at the root of blood, but patients with solid tumors can also suffer from hematopoietic stems, such as cell contamination, especially if the solid tumor has migrated. Therefore, when the transplanted cells are planted back to rebuild the bloodline, "the cancer cells should be labeled with the $ standard (please read the notes on the back and fill out this page). I----book.-- ------ -9- 1320056 Ministry of Economic Affairs Intellectual Property Office Staff Consumption Cooperatives Printing Solid System A7 _____B7 V. Invention Description (8) The activated tumor cells, the virus that expresses the wild-type p 5 3 gene will have The dysfunctional p53 tumor cells are selective, and any interferon-sensitive virus is selective for tumor cells with a blocked interferon pathway. Accordingly, one aspect of the invention is directed to the selective removal of neoplastic cells in a mixed cell composition suspected of having tumor cells, wherein the composition is in vitro. The method comprises the steps of: (a) mixing The cell composition is contacted with the virus under conditions which result in substantial killing of the tumor cells; and (b) the treated cell composition is collected. In another embodiment of the invention, the invention further comprises freezing the virus-treated cell composition and presenting it in a solution containing DMS. DMSO is routinely used to freeze and store animal cells, but it denatures the virus. Thus, DMSO treatment removes the infectious virus from the cell composition while preserving the composition for an extended period of activity in the frozen state. In another embodiment of the present invention, a virus-treated cell composition is treated by combining a virus-specific antiviral antibody or an anti-viral antibody with complement to dissolve a reovirus. Remove the virus. Alternatively, or in addition to the 'antiviral antibody', the surface molecule capable of recognizing the reovirus can be used to remove the viral particle by applying the cell composition to the immobilized antibody by antibody deuteration. The fraction of the chelate that cannot bind to the antibody is collected. A specific antibody against a particular virus can be administered to a recipient of the transplant to reduce the reovirus in the body, and a conventional stimulant is used for this purpose. 'Improve I r -------- order --------- line - (please read the notes on the back and fill out this page)

This paper Ma scale suitable wealth E3 which standard (CNS) A4 specifications 297 mm) 1320056

V. INSTRUCTIONS (10) - The Ministry of Economic Affairs' Intellectual Property Office staff consumption cooperative prints viruses and parapoxviruses. Each of these natural forms of the virus develops a mechanism that inhibits the double-stranded RNA protein kinase (PKR) to aid in the protein synthesis of the virus, which would otherwise be inhibited by PKR. Therefore, these viruses can replicate in any cell not associated with PKR. When these viral PKR inhibitors are mutated or modified, the virus is sensitive to pKR inhibition and does not replicate in normal cells, since normal cells have a functional pKR pathway. These mutated or modified viruses can be used to selectively remove tumor-activated tumor cells because the RAS-activated tumor cells lack PKR function and thus cannot inhibit the replication of these viruses. In another aspect of the invention, the virus selectively kills tumor cells by virtue of which it carries a tumor suppressor gene. For example, p53 is a cellular tumor suppressor that inhibits uncontrolled proliferation of normal cells. Approximately half of all tumors have functionally impaired p 5 3 and proliferate in an uncontrolled manner. Therefore, a virus exhibiting the wild-type p53 gene selectively kills tumor cells which become a result of inactivation of the p53 gene product. A similar specific example relates to a viral inhibitor of a cell tumor suppressor gene. Some viruses encode proteins that inhibit tumor suppressors, thereby allowing the virus to replicate in cells. By mutating these viral inhibitors, a virus is produced that cannot replicate in normal cells because of the presence of tumor suppressors. However, it will replicate in tumor cells that have lost tumor suppressor and can be used to selectively kill tumor cells of the present invention. In another embodiment of the invention, an interferon sensitive virus is used to selectively kill tumor cells. Interferon-sensitive viruses are inhibited by interferon and cannot be used in normal cells with a complete interferon pathway. 13- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) I r --- -----^---------Line· (Please read the phonetic transcription on the back? Please fill out this page again) - Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, Printed 1320056

DESCRIPTION OF THE INVENTION (14 refers to a cell which proliferates without normal growth-inhibiting properties.) The new growth of tumor-containing cells I is a tumor or a tumor. A tumor is an abnormal cell growth that grows to form a unique biomass. It grows faster by cell proliferation than tissue growth of positive 4. The tumor may show partial or complete lack of structural organization and functional association with normal tissues. As used in this specification, The tumor is intended to cover hematopoietic tumors as well as solid tumors. The tumor may be benign (benign tumor) or malignant (malignant tumor or cancer). Malignant tumors can be broadly classified into three main types. Malignant caused by epithelial tissue Tumors are called cancers, and malignant tumors derived from connective tissues such as muscle, cartilage, fat, or osteophytes are called sarcomas, and they affect hematopoietic structures (structures related to blood cell formation), including those of the immune system, called blood cancer and lymph. Other tumors include, but are not limited to, fibrone oncology. "&quot;Rast-activated tumor cells&quot;J or "&quot;Rust-regulated tumor cells&quot;" means abnormal speed The rate of proliferation of cells is due to, at least in part, activation of the larva pathway. The ras route can be activated by structural mutations in the rassig gene, increased expression of the rassig gene, and stable information of the rass gene. Increased, or any mutation or any mechanism that causes activation of a factor upstream or downstream of a striatum or striate in the larvavirus pathway, thereby activity of the singapore pathway. For example, EGF receptor, PEGF receptor or SOS Activation will result in activation of the rib pathway. The rib-regulated tumor cells include, but are not limited to, sir-regulated cancer cells, which are cells that proliferate in a malignant manner due to activation of the larva pathway. "&quot;Cell composition &quot;" means a composition comprising cells. The composition may comprise a non-cellular substance. For example, whole blood is a cellular composition, and its paper size is applicable to the Chinese National Standard (CNS) A4 specification (210). X 297 mm) &quot; ' (Please read the note on the back and fill out this page)

1320056 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. Description of Invention (15) Contains plasma, platelets, hormones and other non-cellular substances other than cells such as red blood cells and white blood cells. Cellular compositions can contain various types, sources or tissues. Cell. For example, tissues and organs containing different cell forms in a defined structural arrangement are considered to be cellular compositions. A "mixed cell composition" means a cell composition containing at least two types of cells. Typically, the mixed cell composition contains both normal cells and tumor cells. Preferably, most of the cells in the cell composition are dividing cells, and the virus selectively kills the tumor cells while leaving the cells in the division intact. A &quot;suspected tumor cell&quot; cell composition is a cell composition that may contain tumor cells. For example, any resulting autograft of a tumor-bearing subject may contain tumor cells. A cell culture that has been cultured for a long time spontaneously contains tumor cells. ""Significantly killing &quot;" means reducing the survival of target tumor cells by at least about 2%. Survivability can be measured by the number of viable cells in the treated cells, and the degree of reduction can be determined by comparing the number of viable cells in the treated and untreated cells, or by comparing survival before and after virus treatment. Cell count to determine. The decrease in viability is preferably about 50%, more preferably about 7 %, and even more preferably about 8 %, and most preferably about 90%. Tumor cells can be killed in a variety of ways. For example, it can be dissolved by a virus capable of dissolving sexy tumor cells (tumor lysis). The tumor cells can be wilted by cells that are directly or indirectly caused by the virus. The cells can also be killed by the immune system that has been activated by the virus, although this is less preferred. For example, the virus (please read the note on the back and then fill out this page) -------- order --------- line

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V. INSTRUCTIONS (16) (Please read the notes on the back and fill out this page) θ induces interleukin productivity, which activates natural killer cells to selectively kill tumor cells (Z〇rn et al. 1994). A "&quot;copy-capable" virus is a virus that can replicate in at least one cell type. Contrary to the replication of competent viruses, "viruses that do not replicate the viability", which are in the genome of the genome that is necessary for replication, are mutated and cannot be replicated in any cell. "&quot;Adenovirus" is a virus containing approximately 3.6 kb of double-stranded DNA. In humans adenovirus can replicate and in the eye and breath. The gastrointestinal tract and the urinary tract cause diseases. Approximately one-third of the forty-seven known human serotypes are involved in most cases of human adenoviral disease (Br〇〇ks et al., 1998). Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed clothing "mutant adenovirus" or "modified adenovirus," as used in this specification, refers to the gene product that prevents pKR activation. Lack of inhibition or mutation causes PKR activation to be blocked. Adenovirus encodes several gene products that compete with antiviral host defense mechanisms. The virus-associated RNA (VAI RNA or VA RNA) of adenovirus is small, and the RNA of the structure is accumulated in the cytoplasm at a high concentration at the end of adenovirus infection. These VAI RNAs bind to the PKR's double-stranded RNA (dsRNA) binding motif and block the ds rNA-dependent PKR activation by autophosphorylation. Therefore, pKR does not work and the virus can be replicated in the cell. Excessive growth of virions ultimately leads to cell death. In mutant or modified adenoviruses, VAI RNA is preferably not transcribed. Such a mutated or modified adenovirus cannot replicate in normal cells that do not have an activated larva pathway; however, it can infect and be used in the fine ___-19-_____ paper scale with activated ras route National Standard (CNS) A4 specification (210 X 297 mm)

Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Printing 1320056 V. Inventions (17) Intercellular replication. "Herpes simplex virus" (HSV) refers to herpes simplex virus (HSV-D or herpes simplex virus 2 (HSV-2). _ Gene ri34 5 encodes a cellular protein 34.5 infected with a gene product (ICP 34 5) , 纟 can prevent the antiviral effect produced by pKR ❶ ICP 34·5 has a unique mechanism to prevent pKR activity, by interacting with protein phosphatase and redirecting its activity to dephosphorylate eIF-2a (He et al., 1997). In genetically engineered viruses deleted by the wild-type or μ34·5 gene, elF_2a is phosphorylated and the protein is stopped in cells infected with a virus free of rl34.5. It is expected that a virus containing no rl 34.5 will be replication competent in cells having an activated ras route, and wherein the activity of Icp 34 5 is excessive. "&quot;mutated HSV"" or "&quot The term "modified HSV," as used in this specification, means that the gene product that prevents PKR activation is deficient. PKR activation cannot be inhibited by inhibition or mutation. Preferably, the HSV gene"! 34.5 Transcribed. Such mutation or modification hSV is unable to replicate in normal cells that do not have an activated ritux pathway, however it can infect and replicate in cells with an activated lacquer pathway. ""Vaccivirus orf"" is a poxvirus. Viruses that cause acute skin damage in different mammalian species, including humans. Paraneovirus orf naturally infects sheep, goats, and humans through ruptured or damaged skin, and replicates in regenerated epithelial cells. It will turn into a pustular 'damage' (Haig et al. 1998). The paralogous virus gene OV20.0L is involved in the activity of PKR (Haig et al., 1998). -20- This paper scale applies. Chinese national standard (CNS> A4 specification (210 X 297 mm) ill· -------- order--------- line (please read the notes on the back and fill out this page)

1320056 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 ---------- Β7 _ 5, invention description (19) The tumor cells defined above. To test whether a virus is sensitive to interferon, the culture of normal cells can be incubated with the virus in the presence of different concentrations of interferon, and the viability of the cells can be determined according to methods known in the art. The virus is considered to be sensitive to interferon, and if the normal cell t is less than 20%, preferably less than 10% is killed in a high concentration of interferon (e.g., 100 units per ml). "Resistance of cells" to viral infection means that infection of the cells with the virus does not result in significant virus production or yield. - "Vitro's tumor lysate" is a composition prepared by treating a tumor cell with a virus capable of dissolving a tumor in a test tube, and the composition is subsequently administered to a tumor patient having the same tumor to induce The tumor patient is immune to the tumor in vivo. Therefore, the tumor lysate of the virus is essentially a virus-modified cancer cell membrane. As used in this specification, ", the recipient of the graft" is a mammal that receives a transplant of a cell composition. Preferably, the recipient is a human, and more preferably the recipient is a human being transplanted to treat cancer. Method The present invention relates to the use of a virus to selectively remove tumor cells from a cellular composition suspected of containing a mixture of tumor cells. There are many viruses that can be used in this method. 'Each virus is selective for a tumor or a group of tumors. Although reovirus is used as an example, the general skill in the art can be used according to this In the instructions in the specification, the method is used to remove any mixed cell composition using a virus other than a holovirus. -22- This paper scale applies to China National Standard (CNS) A4 specification mo X 297 public) !!1 Order! ! - Line 1 (Please read the note on the back and fill out this page) .IIIII —1 1320056 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed A7 B7 V. Invention Description (20 ) 1 · Reovirus Recently, we found The enterovirus selectively dissolves the lassatin-activated tumor cells in vitro, in vitro and in vitro (Coffey et al., 1998; WO 99/08692). In general, cells are less susceptible to infection by reovirus. However, when the ras route is activated, the reovirus can successfully replicate in the cell and eventually cause the host cell to dissolve. For example, when a reovirus-resistant NIH 3T3 cell is transformed with activated Ras or s〇s, a protein that activates Ras, reovirus infection is enhanced (Strong et al. 1998) ). Similarly, fibroblasts from mice that are resistant to reovirus infection become susceptible to infection after infection with the EGF receptor gene or v_erb B tumor gene transfer (Strong et al., 1993; Strong et al., 1996)_. Under the condition of unrestricted cable theory, it seems that the nakedness of reovirus will be regulated at the translation level (Strong et al., 1998; Norman et al., 2000), in untransformed 3 T 3 cells. In the early stage, the viral transcript sequence activates the protein chymase (PKR) activated by double-stranded RNA, which inhibits translation and thus inhibits viral replication. Activated Ras (or an active element of the striate pathway) is hypothesized to inhibit or reverse the activation of PKR. Therefore, the synthesis of viral proteins proceeds, viral particles are produced, and the cells are finally dissolved. The RAS tumor gene can explain a large number of tumor genesis. Activation of mutations in the Ras gene itself occurs in approximately 30% of all human tumors (Bos, JL 1989), mainly in the pancreas (90%), sporadic colorectal (5〇〇/.) And lung (40%) cancer, and bone marrow blood cancer (30%). Activation of factors upstream or downstream of the ribose gene in the rib pathway is also associated with tumors. For example, at the -23- paper scale, the Chinese National Standard (CNS) A4 specification (210 X 297 mm) is applied. LI l·----------------- Order---- -----Line (please read the note on the back and then fill out this page) 1320056 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 ---------____ V. Invention description (21 ) Breast cancer Overexpression of HER2/Heu/ErbB2 or epithelial growth factor (EGF) receptors is common (25-30%), while platelet-derived growth factor (PDGF) is overexpressed in gliomas and glioblastomas. Receptors and receptors are common (4〇-50%). £0 [receptor and? The 〇0? receptor is known to activate the laccase gene when it binds to its opposite ligand, while v_erbB constitutively encodes an activating receptor lacking the extracellular domain. We first measured the ability of reovirus to kill cancer cells. Reovirus can efficiently cause tumor lysis of three breast cancer model systems MCF7, SKBR3 and HTB 132, which is achieved by inducing cell wilting in infected cells (Example 1). Therefore, 'reovirus treatment resulted in a significant decrease in the viability of MCF7, SKBR3 and HTB 132 cells, whereas the control group treated with no virus or dead virus grew normally (Fig. 1 A_1 〇). Decreased viability will be accompanied by features associated with cell wilting, such as Dna fragmentation, positive results of serotonin V or APO 2.7 staining (Fig. 2 A - 2 G) and cytopathic effects as observed under the microscope The cell membrane is blistered, the nucleus is concentrated, and the chromatin is concentrated. Because reovirus infection is usually blocked at the translational level of normal cells, but not in the tumor cells regulated by lins, we examined protein synthesis in reovirus-treated MCF7 cells and CD34+ stem cells. Degree (Example 2). In fact, viral proteins are synthesized in cancer cell lines infected with reovirus, but not in CD34+ stem cells that are also infected with reovirus (data not shown). This result suggests the fact that it is safe to treat hematopoietic stem cells with reovirus because the reovirus protein is not used in reovirus-treated stem cells-24- This paper scale applies _ i : Standard (CNS) A4 specification curry 29 ill · -------- order --------- line (please read the note on the back and fill out this page)

1320056 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (3G) and / or cell mitigation signal. Although interferon is theoretically useful for inhibiting the growth of tumor cells, such attempts have never been very successful because of mutations in members of the tumor-specific interferon pathway. However, by destroying the interferon pathway to avoid growth inhibition by interferon, tumor cells simultaneously cope with their antiviral response. In fact, studies have shown that VS V 'a shelled, antisense RNA virus replicates in many human tumor cell lines in the presence of interferon and kills the cell line' while normal human primary cell culture Obviously it will be protected by interferon. Intratumoral </ RTI> VSV also reduced tumor burden in nude mice bearing subcutaneous human melanoma xenografts (Stojdl et al., 2000). Thus, in another embodiment of the invention, VSV is used to remove tumor cells from the mixed cell composition in the presence of interferon. However, it is intended that this aspect of the invention can be applied to any interferon-sensitive virus (W0 99/187&quot;), i.e., a virus that does not replicate in normal cells in the presence of interferon. This virus can be determined by the following method: culturing a normal cell culture&apos; The culture is contacted with an interesting virus at different concentrations of interferon, and then the percentage of killed cells measured after a period of incubation is determined. Preferably, less than 2% of normal cells are killed, and more preferably less than 10% of normal cells are killed. We may also take advantage of the fact that certain tumor cells exhibit a high concentration of an enzyme and construct a virus that depends on this enzyme. For example, 'ribose nuclear reductase is abundant in liver metastasis, but it is rare in normal liver. Therefore, a herpes simplex virus 1 (HSV-1) mutant with a defective expression of ribonucleoside reductase, hrR3 showed that it would be applicable to the Chinese National Standard (CNS) A4 specification on the scale of colon cancer. 210 X 297 mm) --------^-------- (Please read the notes on the back and fill out this page) 1320056 A7 B7 V. Invention description (32 related, but Έ : It is more infectious to cancers whose immune system is weakened by chemotherapy. Therefore, if a composition containing hematopoietic stem cells is treated with reovirus, and the composition is transplanted to a cancer patient immediately, the resuscitation The orphan virus can be removed prior to transplantation of the cell composition. Thus, in another embodiment of the invention, the virus-treated cell composition is frozen in a solution containing DMS mash and prior to transplantation. Although D is routinely used for ice silk storage of animal cells', it removes infectious viruses from dry, cell preparations. This reduces the risk of the virus causing unwanted infections. When it is introduced through a stem cell transplant In the case of the recipient, the virus-treated cell composition is treated with a specific antibody against a specific virus or a combination of a specific antibody and a complement to inactivate or dissolve the virus. In addition, antibodies that recognize specific viral molecules can be used to remove viral particles from the virus-treated cell group a. Therefore, the antibody is immobilized in other materials or devices known as tube lines. The cell composition is applied to the solid: part of the composition which is not bound to the antibody, and is collected in a special immobilization method. Another type can be used to remove the virus from the virus-treated mixture. The method of removing the mixture is to layer the mixture with cells that can open the cells and the disease material. In another specific case, the patient is given a treatment to stimulate the immune system to reduce the virus infection. Risk. This treatment can be done at the same time or after, but it is better to do it before transplanting. Do =1 35- This paper scale applies to China National Standard (CNS) A4 specifications (2) 〇χ 公 13 1320056 A7 B 7 V. INSTRUCTIONS (34) Toxic. (Please read the note on the back and then fill out this page.) The following examples are provided to illustrate the invention and should not be construed as limiting the scope of the invention in any way. In the following examples, the following abbreviations have the following meanings. Undefined abbreviations have their generally accepted meanings. °c = degrees Celsius hr. = hours min = minutes μ Μ = micro-mole concentration mM = milli Ear concentration M = molar concentration m 1 = ml μ 1 = microliter mg = mg β g = microgram PAGE = polypropylene guanamine colloidal electrophoresis rpm = number of revolutions / minute FBS - fetal bovine serum economic department intellectual property bureau employee consumption Co-operative printing DTT = dithiothreitol SDS = sodium lauryl sulfate PBS = phosphate buffer DMEM = Dulbecco's modified Eagle's medium or - MEM = or - modified Eagle's medium - 37 - paper The scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). V. Description of invention (36) 16%. In MDA MB 468 cells, the number of intact cells treated with the virus fell to 12.7% ' 8.8% and 3.6% of the original cells after 24, 48, and 72 hours after infection. Therefore, the reovirus will efficiently induce tumor lysis in all three cancer cells. The cell died by cell wilting. A typical cell hole marker such as CPE, a pool of _#DNA ladders, can be observed in a time course parallel to a decrease in survival. ^A_2G shows the percentage of DN+A fragmentation (2A_2C) combined with v-staining (2d) or (iv) 2.7+ cells (2E-2G) at different time points after reovirus infection. The reovirus-treated cells exhibited a dramatic degree of cell wilting compared to the virus-free or dead-virus control group. ^Recommended reovirus induced cell wilting in all three cell lines. The cell wilting in the control group also seems to increase slowly over time, which is because the cells begin to die when they grow too densely. Example 2 彳 Enterovirus selectively inhibits protein synthesis in cancer cells but does not inhibit synthesis in CD34+ stem cells. In order to further demonstrate that the virus selectively infects cancer cells, the viral protein is labeled/SDS/ PAGE. In the re-reovirus-stained=CF7 cells, the protein synthesis of the virus is very significant, while the cell=white matter synthesis is simultaneously reduced. #出出肠病毒 has taken over the cells, and there is no protein synthesis on it. Was detected to 'this has built up the following facts. g卩娇古's all cells have been killed. In the control group where the cells were infected with a diseased mother or not infected with the virus, no protein was synthesized, and the protein synthesis of the cells was normal. Relative 35s ^ in the presence or absence of reovirus, CD34+ stem cells paper-scale thorn (four) national standard (CNS) A4 specification (210-x 297 mm) 1320056 A7

The S mark printed by the Ministry of Economic Affairs' Intellectual Property Office employee consumption cooperative shows that after adding the virus, the virus is as long as 72,], and the virus has the protein Ρ»成 so the 'reovirus will selectively infect 7 cells' but It does not infect CD34+ stem cells. Example 3 Treatment of sputum enterovirus neither inhibited cell proliferation nor altered the differentiation potential of CD34+ cells and protein synthesis results - the cell count of the survival 'survival indicates that reovirus treatment does not cause CD34+ cells The number of viable cells was reduced (Fig. 3A), which is the result of comparison with the virus-free control group. Although the number of CD34+ cells is not affected by reovirus infection, there is still a question remaining: whether the reovirus changes the potential of CD34+ stem cells to differentiate into all hematopoietic lineages at an appropriate ratio. If this is the case, stem cells treated with reovirus will not be a good candidate for reconstitution of the entire hematopoietic system I. To investigate this possibility, we cultured CD34+ cells with reovirus for 2, 24, 48 or 72 hours, respectively. The reovirus was then removed and the cells were diluted and incubated in fresh medium for 14 days to allow colony formation. Each colony was tested to determine whether it belonged to granulocyte, erythropoietin or granulocyte-type erythrocyte macrophage megakaryocyte lineage. As shown in Figure 3B, stem cells treated with live virus (LV) produce a similar number of granulocytes (G), red blood cells (E) or granulocyte-type erythrocyte macrophage megakaryocytes (GEMM), as if there were no virus (NV). The control group is generally. Therefore, reovirus treatment does not alter the differentiation potential of CD34+ cells. Example 4 Reovirus selectively removes cancer cells from a mixture of cell compositions -40 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------- ----^----Order---------Line (please read the note on the back and fill in this page) 1320056 ,, A7 _B7 V. Description of invention (38) Separation of tumor cells The product is mixed and infected with reovirus to investigate whether the reovirus selectively removes the tumor cells from the mixture of cell compositions. The isolate product was prepared according to the procedure previously described (Stewart et al., 1999; Duggan et al., 2000). When the mixture of the separation product (90%) and MCF7 (10%) was treated with reovirus, and the daily test cell count and survival rate, the cytokeratin-positive MCF 7 was fined. CD34+ stem cells remain intact and alive. Figures 4A-4C show the cleaning effect of a mixture of reovirus isolation products with MCF7, SKBR3, or MDA MB 468 cells. These results demonstrate that the reovirus selectively kills the tumor cells in the cell mixture while leaving the stem cells intact. ---------# (Please read the notes on the back and fill out this page) Order---------Line. Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 41 paper scale Applicable to China National Standard (CNS) A4 specification (210 X 297 mm) A7 B7

Patent application No. 132005fe〇90109803 Replacement page of Chinese manual (September 96) V. Description of invention (7) It may also be returned to cancer patients, and these cancer cells may proliferate and cause cancer to recur. Therefore, we hope to purify the autograft before transplantation. Several methods have been used to purify autografts (Spyridonidis et al. '1998; Bensinger, 1998). This autograft can be treated with chemotherapy to kill contaminating tumor cells in vitro. However, as discussed above, it is difficult to find a dose of a chemotherapeutic drug that selectively kills tumor cells or cancer cells while keeping the main hematopoietic stem cells intact. Autologous grafts can also be treated with an anti-binding toxin that recognizes tumor cell-specific antigens' but such tumor-specific antigens are not always present. It is also possible to separate stem cells from other cells by using a flow blood count&apos; affinity column or magnetic beads, depending on the stem cell specific surface marker (CD34). However, by selecting only certain hematopoietic cells, such as CD34+ cells, other hematopoietic cells such as sputum cells, sputum cells, monocytes and natural killer cells are also excluded, and immune response is delayed (Bensinger, 1998). ^ This method also causes about half of the loss of CD34+ cells and retains some contaminating cancer cells (Spyridonidis et al., 1998). Therefore, we maintain a need for highly selective methods with reasonable yields to clean up autografts that may contain tumor cells. SUMMARY OF THE INVENTION: The present invention is directed to a method of selectively removing tumor cells from a mixed cell composition, such as an autograft, using a virus that exhibits selective killing of tumor cells. There are many viruses that selectively remove tumor cells without removing normal cells. For example, reovirus will selectively kill the paper size for the Chinese national standard (C school S) A4 rule bar (210X 297 mm)

132005^〇9〇ι〇98〇3 Patent Application Chinese Manual Replacement Page (%年^月) V. Description of the Invention (Removal in the cell composition of another embodiment of the present invention is achieved by utilization The gradient of separating the virus from the cells by treatment with the virus / in a preferred embodiment of the invention, the mixed cell composition is packaged so that the hematopoietic stem cells can be transplanted or any desired use^ The tumor cells are removed from the sputum. The hematopoietic stem cells can be collected from the bone marrow. The application of the present invention is not limited to the purification of hematopoietic stem cells. In another embodiment of the invention, the invention can be applied to any tissue, organ, tissue/organ a combination, or a part of a tissue or organ, to remove tumor cells. However, the invention also has a tissue or organ required for purifying any other purpose, wherein the tumor cells present in the tissue or organ need to be removed. In another embodiment of the invention, a virus is used in a cell line that is in a resting state to remove spontaneously transformed cells. This method can also be used to treat artificial insemination or other The pre-procedural semen or the donor's egg. In another aspect of the invention, the virus is a competent competent virus. In contrast to the replication-deficient virus, the re-pulse competent virus can be susceptible to the virus. Replication in cells 'and often causes such cell lysis. There is a replication competent virus for use in the present invention which selectively selectively lyses tumor cells in a phenomenon known as "tumor lysis." Another embodiment of the present invention The virus is a mutant or modified virus selected from the group consisting of: adenovirus, herpes simplex virus, bovine cancer

70681-960914.DOC

&amp;V.月^修正- 补亦. 1320056^〇9〇ι〇98〇3 Patent application Chinese manual replacement page (96 years 9' month) V. Invention description (u) Copy. Because the interferon pathway of certain tumor cells is disrupted, it can be selectively killed by an interferon-sensitive virus. An interferon-sensitive virus is preferably a vesicular stomatitis virus (VSV). Interferons may be added as needed to remove interferon-sensitive viruses to remove tumor cells. The invention also provides a cellular composition that is virally treated to remove neoplastic cells and leave viable non-neoplastic cells. Such compositions can be used in in vitro studies, or in transplants, fertilization, or other in vivo procedures, which can be self-derived, homologous, or even heterologous. Preferably, the transplant is self-derived, and the composition preferably comprises hematopoietic stem cells. Another aspect of the invention provides a kit comprising at least two viruses of different selectivities, such as a reovirus, a diseased mother expressing a functional p53 protein (5 2 4 ' ONYX-015 'Newcastle virus or water Cancerous stomatitis virus. Brief description of the figure: Figure 1 Figure 1A-1C shows the number of cells surviving in MCF7^1A), SKBR3 (Figure 1B) or HTBi32 (Figure 1 C), as indicated by Live reovirus, dead reovirus or non-viral infection, Figure 1D shows the percentage of survival of MCF7 cells at various time points after reovirus infection. Figure 2 Figure 2 shows that cell wilting is induced by infection of reovirus in cells of MCF7 (Figure 2A), SKBR3 (Figure 2B) or HTB132 (Figure 2C). Figures 2A-2C show the percentage of DNA becoming fragments after reovirus infection. Figure 2D shows the percentage of marker Annex 5 (Annexin V) staining for cell wilting after reovirus infection. Figure 2 £_2(} is shown in each of the points indicated

70681-9609U.DOC This paper scale applies to China National Standard (Cf^s) A4 specification (210 x 297 mm) 132005^090109803 Patent Application Replacement Chinese Manual (September 96) V. Invention Description ( 12) Α7 Β7

96. 9. 14-year FJ correction 补充 Supplement a cell type (Figure 2Ε: MCF7; Figure 2F: ΗΤΒ 132; Figure 2G: SKBR3) ΑΡΟ 2.7+ cell 妁 percentage. Figure 3 Figure 3 shows the number of viable cells at different time points after CD34+ stem cells were infected with reovirus. Figure 3 Β I member shows the effect of reovirus on long-term stem cell culture. The stem cell line was infected with reovirus and cultured for 2, 24, 48 or 72 hours, respectively, and then the cells were diluted and cultured for 4 days to allow individual colonies to form. Then, the number of cells infected with virus-free (N V) or live virus (L V), respectively, was determined for each of the globular blood cells (G), erythroid cells (E), or granulocyte-type erythrocyte macrophage megakaryocytes (gemM). For example, the n V - G strain refers to a granulocyte colony obtained from cells not treated with a virus, and the L v - G strain refers to a granulocyte colony obtained from cells treated with a live virus. Figure 4 Figure 4 A-4C shows the cleaning effect of reovirus on the mixture of MCF7 (Figure 4A), MDA MB 468 (Figure 4B) or SKBR3 (Figure 4C) cells, respectively. The present invention is directed to a method of selectively removing tumor cells from a mixed cell composition, such as an autograft, using a virus that exhibits selective killing of tumor cells. There are many viruses that are useful in the present invention. For example, a mixed cell composition can be treated with a reovirus capable of selectively killing the 'synthesized tumor cells. The RAS-activated tumor cells can also be selectively removed by a virus in which the double-stranded protein kinase (PKR) inhibitor of the virus is mutated or modified. If the composition is suspected of having a tumor lacking p 5 3

70681-960914.DOC This paper music scale applies to Chinese national standards (C_ Μ regulation (21〇χ297 public) 132005 continuation 090109803 patent application Chinese manual replacement page (September 96) A7 B7 9ft 9. 14

V. INSTRUCTION DESCRIPTION (13) Cells, which can be treated with a virus capable of expressing the p 5 3 tumor suppressor gene, can induce the wilting of tumor cells that are functionally defective in the p 5 3 gene product (Wiman, 1998, Nielsen et al., 1998). The vesicular stomatitis virus (VSV) or other interferon-sensitive virus can be used to kill tumor cells in which the interferon pathway is disrupted in the presence of interferon. Other examples useful in the present invention include vaccinia virus, cold virus, varicella virus, aphrodisiac virus, 'carcinoma virus and Newcastle virus, which have been reported to be associated with tumor regression or death (Nemunaitis, 1999). However, the invention encompasses any virus that selectively kills tumor cells. Before the present invention is further explained, the nouns used in the present specification are defined as follows unless otherwise indicated. Definition "&quot;Virus&quot; means any virus, whether in its original form, attenuated or modified. Modified viruses include chemically modified viruses or viruses modified in a recombinant manner. The recombinantly modified virus may be a mutated virus, a recombinant virus, or a reclassified virus. A mutated virus is one in which the viral genome is mutated, i.e., a nucleoside insertion, deletion, and/or substitution. Recombinant viruses are viruses that have coat proteins from different subtypes, usually co-infecting cells with more than one subtype of virus, producing viruses that are enveloped by other subtype-encoded coat proteins. The reclassified virus is a multiplex fragment virus in which the fragment has been reclassified, usually by co-infecting the cells with more than one subtype of the virus, allowing fragments from different subtypes to be mixed and intermixed intracellularly. ""Tumor cells&quot;j, also known as "cells with proliferative diseases", this paper scale applies to China National Standard (CSS) A4 Regulation (210X 297 mm) Patent Application No. 132005^090109803 Chinese manual replacement page (September 96) A7 B7

V. INSTRUCTIONS (18) The term "• 'mutated parapoxvirus orf&quot; or "&quot; modified parapoxvirus orf" as used in this specification is intended to prevent the lack of gene products that prevent PKR activation, Inhibition or mutation, such that PKR activation cannot be prevented. Preferably, the gene OV20.0L is not transcribed. Such a mutated or modified parapoxvirus orf cannot replicate in normal cells that do not have an activated rib pathway, however, it can infect and replicate in cells having an activated rib pathway. ""Vaccinium virus" means a virus of the genus Carcinoma, which infects humans and produces localized lesions (Brooks et al., 1998). Bovine cancer viruses encode two genes that play a role in reducing PKR activity via two distinct mechanisms. The E 3 L gene encodes two proteins, 20 and 25 kDa, which are expressed in the early stage of infection and have ds RNA binding activity which inhibits PKR activity. Deletion or disruption of the E3L gene causes the virus to randomly replicate the K3L gene encoding pK3 of the vaccinia virus in cells with the activated lacquer pathway, which is a pseudo-matrix of PKR. "&quot;mutated vaccinia virus&quot; or &quot;mutated vaccinia virus&quot; as used in this specification means the absence, inhibition or mutation of a gene product that prevents PKR activation, thereby activating PKR The effect is not blocked. Preferably, the E3L gene and/or the K3L gene are not transcribed. Such a mutated or modified vaccinia virus cannot be replicated in normal cells that do not have an activated larva pathway, however, it can be infected and replicated in cells having an activated rib pathway. "A virus that is sensitive to interferon" is a virus that cannot replicate or kill normal cells in normal cells in the presence of interferon. Normal cells are not applicable to the Chinese national standard (C^S) 本4 rules (210Χ 297 mm) A7 B7

132005 You 〇 90109803 Patent Application Chinese Manual Replacement Page (September 96) V. Synthesis of the invention (22), and the synthesis of cellular proteins is carried out normally. To confirm this, we measured the viability of reovirus-treated CD34+ cells at various time points after reovirus treatment (Example 3). The number of cells treated with or without live reovirus was similar at each time point (Fig. 3A), indicating that CD34+ cells are less susceptible to reovirus infection. In order for reovirus to be used for purification, high-dose chemotherapy in the treatment of hematopoietic stem cells, reovirus treatment must not alter the ability of stem cells to differentiate into each hematopoietic lineage to reconstitute the complete hematopoietic system. Therefore, we assessed the long-term effects of reovirus treatment (Example 3). Treatment of CD34+ cells with no virus or with live virus showed no difference in their ability to differentiate into granulocyte, erythropoietin or granulocyte-like erythrocyte macrophage megakaryocytes, even after 72 hours of reovirus treatment ( Figure 3B). The ratio of these three lines to each other remains unchanged after this extended treatment. Therefore, reovirus treatment neither kills CD34+ nor alters the potential of its recombinant hematopoietic system. In addition, the reovirus can purify the mixed cell composition, as evidenced by its selective killing of MCF 7, SKBR3 or HTB 132 cells in a mixture of cancer cells, and the isolation product containing CD34+ stem cells (Example 4) ). By measuring CD34 and cytokeratin, a marker specific for epithelial cells such as MCF7, SKBR3 or HTB 132, it has been shown that reovirus essentially reduces cancer cells from mixed cell compositions (Figure 4 A - 4 C The stem cells are maintained intact. Therefore, reovirus treatment is an effective method for removing tumor cells from hematopoietic stem cell compositions. This paper scale applies to Chinese national standards (Cf^S) A4 regulations_(210X 297 mm) 132005ft 090109803 Patent Application Replacement Page (96th year of the month) V. Description of the invention (23) Therefore, in the preferred embodiment of the invention I, the self-graft containing stem cells before transplantation It is treated with reovirus to remove contaminating or spontaneous sir-regulated tumor cells. This can improve the efficacy of high-dose chemotherapy/autologous hematopoietic stem cell transplantation. Particularly interesting is the treatment of odgkin's disease. Many cases of myeloma, non-Hardkin's lymphoma, acute bone marrow-generating blood cancer, germ cell (Baiwan) cancer, brain tumor, and breast tumor, due to high-dose chemotherapy and Source stem cell transplantation has been shown to be effective in patients with these tumors. However, we have observed that the method of the present invention is also useful for other cancers to remove any fine-regulated tumors: 'Because of the Lacy pathway Activation produces blood in any cell or tissue type and stem cells can be removed from the patient's bone marrow prior to treatment. Or, in cancer patients receiving traditional, non-high dose chemotherapy, there are many stem cells. Typically, it occurs in the blood of Zhouyuan. Therefore, hematopoietic progenitor stem cells can be removed from the blood in the form of an isolated product, and can be stored for a long time before being transplanted. The present invention can be applied to an autograft containing stem cells. The graft can be collected from any tissue source, including bone marrow and blood. In addition to hematopoietic stem cells, the invention is broadly applicable to the removal of lins-regulated tumor cells from many other cellular compositions. For example, reoviruses are available. Perform routine operations to "clear". (Attach the tumor cells regulated by the ribs) Any tissue-accreditable organ transplant. The application of the present invention Limited by cell or tissue type, as the receptor for reovirus is extensive as discussed above, and is used to inhibit reovirus replication in normal cells, pKR machine 70681-960914.DOC, 26 - This paper method is suitable for the national standard (CKS) A4 regulations (210 X 297 public) 132005 you 090109803 patent application replacement instructions (September 96)

The 24 system is also widely used. Therefore, any cell may become a tumor cell that regulates one and becomes susceptible to infection by reovirus. Of particular interest is the use of the method of the present invention to cleanse whole blood or any portion thereof for subsequent blood transfusion. Similarly, tissue or organ transplantation has gradually become commonplace, and it would be advantageous if the graft could remove the rib-regulated tumor cells prior to transplantation. Liver, kidney, heart, cornea, skin grafts, sputum cells, bone marrow or any part thereof are merely a few examples of tissues or organs to which the present invention can be applied. The tissue or organ can be self-derived, homologous or heterologous. The tissue or organ can also be obtained from a gene transfer animal, which is a tissue/organ that develops from a stem cell or expands in vitro in a test tube. The tissue or organ treated with the reovirus can be obtained from an embryonic or adult source, for example, the embryonic nerve cells can be treated prior to transplantation into the Alzheimer's disease. Similarly, the invention can be used to treat semen or donated eggs in vitro. The application of the invention is not limited to grafts. Moreover, any cell composition can be "cleared," with a reovirus for any purpose. Therefore, the examples described above can be applied even if the tissue or organ is not intended. The figure is also used for transplantation. The cell line can also be routinely treated to protect it against spontaneous or contaminated sir-regulated tumor cells. Further, any cell strain can be a good candidate for the present invention, of course, except for the transformation by the method of activating the lins route. The cell strain is outside. Recently, many experiments have attempted to establish a series of transplantable allografts of human prostate cancer tissue and inoculate them into immune-preserving mice. 70681-960914.DOC .27. This paper scale is applicable to the China National Standard (0) A4 (21〇Χ297 茇) 1320056^090109803 Patent Application Replacement Page (September 96) A7 B7 9· 14 /jt. X Year of the month β' νί1 Supplement V. Description of the invention (25) However, contamination of cancer cells by mice often occurs during continuous transmission of allografts, and these cells eventually grow longer than human prostate cancer cells. Deda (Gao. et al., 1999). The present invention may be a simple solution to this problem&apos; if the contaminated cancer is regulated by a lacquer and the allograft is not. The invention differs from the method of preparing viral tumor lysates. Tumor cells are often poor immune response elicitors and therefore escape the immune system. The tumor lysate of the virus is essentially a virus-modified tumor cell membrane that is used to enhance the immune response of the tumor cells. In order to prepare the tumor lysate of the virus, the tumor cells are removed from the subject with the tumor. 'And infection with a virus that dissolves the tumor cells. The resulting material is then administered to a subject with the tumor, and immunity is often induced to combat uninfected tumor cells. The mechanism by which a virus infects a tumor cell to induce immunity to an uninfected tumor cell is not known, but may involve a virus that causes tumor cells to be xenogeneized. Influenza virus infection of melanoma gland cancer, tumor lysates of ovarian tumors, and tumor lysates of colon tumors infected with Newcastle virus and tumor lysates of bovine cancer virus have been used to combat various tumors, for example, mesothelioma Suffering from tumor lysate after surgical removal of the tumor. The tumor lysate of the virus is administered weekly to week 4, or once every 2 weeks to week 52 'every 3 weeks to 1 2.0 weeks, and every 6 weeks to 160 weeks. In another clinical case, the schedule of administration of self-derived NDV tumor lysates against colorectal cancer begins two weeks after surgery and is repeated five times at two-week intervals, followed by three months. Strengthening 70681-960914.DOC - 28 - This paper scale is applicable to Chinese national standard (C investment S) A4 specification (210X 297 mm) 132005fe〇90109803 Patent application A July 14 revision Chinese manual replacement page (96 years 9 Month) B7 I Supplementary 丨 _ V. Description of invention (26 ) (Nemunaitis, 1999). Studies have shown clinically-response or the ability to produce active immunity against tumor antigens in some patients (Steele, 2000). The present invention differs from tumor lysates of viruses in that they are not associated with virally modified tumor cells. The tumor cells which are solubilized relative to the tumor lysate of the virus can be, and preferably are, removed from the virus-treated cell composition without affecting the efficacy of the present invention. Moreover, the tumor lysate of the virus is mainly prepared by using tumor cells, and the mixed cell composition of the present invention preferably contains less than 60% of tumor cells, more preferably less than 40%, and more preferably Less than 20%, preferably less than 10% of the tumor cells. 2 other viruses that selectively kill the tumor cells activated by ras

In general, when a virus enters a cell, double-stranded RNA kinase (PKR) is activated and limits protein synthesis, and the virus cannot be replicated intracellularly. Certain viruses have developed a system that inhibits PKR and assists in viral protein synthesis and viral replication. For example, adenoviruses produce large amounts of small RNA, VA1 RNA. VA1 RNA has a large number of secondary structures and binds to PKR, but competes with double-stranded RNA (dsRNA) that normally activates PKR. Since the shortest length of dsRNA is required to activate PKR, VA1 RNA cannot activate PKR. On the contrary, PKR is hidden due to its large amount. Therefore, protein synthesis cannot be blocked, and the adenovirus can be replicated in the cell. The vaccinia virus encodes two gene products, K3L and E3L, which reduce PKR by a different mechanism. The K3L gene product has limited homology with the N-terminal region of eIF-2a, the natural receptor of PKR, and can be used as a pseudo-suffering of PKR. The E3L gene product is a double-stranded RN A binding protein, 70681-960914.DOC -29- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

Patent Application No. 090109803 to Chinese Patent Application No. 090109803 (September 1996) V. Inventive Note (27) and it is apparent that its function is to conceal the activator dsRNA. Similarly, the herpes simplex virus (HSV) gene r! 34.5 encodes a gene product that is an infected cellular protein 34.5 (ICP 34.5), which prevents the antiviral effects produced by PKR. The parapoxvirus orf virus encodes the OV20.0L gene, which is involved in the activity of blocking PKR. Therefore, these viruses can successfully infect cells without being inhibited by PKR. As discussed above, the rib-activated tumor cells are not inhibited by PKR-induced protein synthesis because the rasps inactivate PKR. Therefore, these cells are also infected with the virus, and even the virus does not have a PKR. inhibition system. Therefore, if the PKR inhibitor of adenovirus, vaccinia virus, simple cancer virus or paraneovirus orf virus is mutated and cannot inhibit PKR, the virus produced cannot infect the cell because PKR inhibits its protein. Synthesis. However, they replicate in tumor-activated tumor cells lacking PKR activity. Accordingly, the present invention provides a method for removing RAS-activated tumor cells from a mixed cell composition, which is an adenovirus, vaccinia virus, herpes simplex virus, parapoxvirus orf which is unable to inhibit PKR function by modification or mutation. virus. The modified or mutated virus is selectively replicable in sir-activated tumor cells, whereas normal cells are resistant. Preferably, the adenovirus in this particular example has a mutation in the VA1 region, the vaccinia virus is mutated in the K3L and/or E3L region, and the herpes simplex virus has a mutation in the r i34.5 gene, and the parapoxvirus orf virus is in the OV20. There is a mutation in the 0L gene.

The virus can be modified or mutated according to the known structure-function relationship of the viral PKR inhibitor. For example, because the amine end region of the E3 protein will interact with PKR • 30·

70681-960914.DOC This paper scale applies to Chinese National Standard (CiJS) A4 specification (210 X 297 mm) 96. 9. 14 /,% rp Year no replacement A7 B7 132005&amp;090109803 Patent application Chinese manual replacement Page (September 96) V. Description of the invention (^ · The interaction of the carboxy-terminal regions, so deletion or point mutations to this location would impede the function of anti-PKR (Chang et al., 1992, 1993, 1995, Sharp) Et al., 1998; Romano et al., 1998). The K3L gene encoding bovine disease is a pseudo-receptor of PKR. There is a loss-of-function mutation in K3L. By cutting off the K3L protein. Mutation of the terminal portion or placement point abolishes the inhibitory activity of PKR, wherein the c-terminal portion shares homology with residues 79 to 83 of eIF-2α (Kawagishi_Kobayashi, et al., 1997). A virus carrying a tumor suppressor gene or a gene associated with a tumor suppressor. In another aspect of the invention, the virus selectively kills a tumor cell by carrying a tumor suppressor gene. For example, p53 is a cell capable of inhibiting normal cells. Uncontrolled proliferating cell tumor suppressor However, approximately half of all tumors have functionally impaired p 5 3 and proliferate in an uncontrolled manner. Thus, a virus capable of expressing the wild-type p53 gene can selectively kill due to inactivation of the ρ53 gene product. Tumor cell. This virus has been constructed and shown to induce cell wilting of cancer cells that exhibit mutations p 5 3 (Blagosklonny et al., 1996). A similar approach involves viral inhibitors of tumor suppressors For example, 'some adenoviruses, SV40 and human papilloma viruses contain proteins that cause p53 to lose/tongue' and thus replicate itself (Nemunaitis, 1999). For serotype 5 adenoviruses, this The protein is a 5 5 kd protein encoded by the E16 region. If the E 1 b region encoding this 5 5 kd protein is deleted 'as in the ONYX-015 virus (Bischoff et al., 1996; Heise et al., 2000; W0 94/18992), then the 55 kd p53 inhibitor is not

70681-960914.DOC This paper scale is the common Chinese national standard (CSS) A4 regulation (210 X 297 gong) I32005fe〇90109803 patent application 9%9·14 amendment Chinese manual replacement page (September 96) _ Supplement V. Invention Description (29) Re-exist. Therefore, when ONYX-015 enters normal cells, p 5 3 has the function of inhibiting cell proliferation and virus replication, and virus replication depends on the cell proliferation. Therefore, ONYX-015 is not replicated in normal cells. On the other hand, in tumor cells with disrupted p53 function, ONYX-015 can replicate and eventually cause cell death. Therefore, this virus can be used to selectively infect and remove p 5 3 -deficient tumor cells from mixed cell compositions. Those of ordinary skill in the art can also mutate or disrupt the p53 inhibitor gene in adenovirus 5 according to established techniques, and the resulting virus is useful for mixing cellular compositions in the present method. Removed from tumor cells. Another example is the Delta 24 virus, which is a mutant adenovirus with 24 base pair deletions in the E1A region (Fueyo et al., 2000). This region is responsible for binding to tumor suppressors of cells and inhibiting R b function, thereby enabling cell proliferation and viral replication to proceed in an uncontrolled manner. Delta 24 is deleted in the Rb junction and cannot be combined with Rb. Therefore, replication of a mutant virus is inhibited by Rb in positive cells. However, if Rb is inactivated and the cells become tumors, then Delta 24 is no longer inhibited. Instead, the mutant virus is efficient - replication and lysis of cells lacking R b . Again, the virus is selective for tumor cells and can be used to purify mixed cell compositions and remove cells lacking Rb. 4. Other viruses The vesicular stomatitis virus (VSV) selectively kills tumor cells in the presence of interferon. Interferon is a circulating factor that binds to cell surface receptors, which ultimately leads to an antiviral response and induces growth inhibition in target cells. 70681-960914.DOC -32- This paper scale applies to Chinese National Standard (CfiS) A4 Rule &quot;(210 X 297 mm) 132005 Grab 090109803 Patent Responsibility 丨B Amendment Chinese Manual Replacement Page (September 96) g Year; Day Supplement V, Invention Description (31) Copy, but not Replication in normal liver cells (Yoon et al., 2000).

In addition to the viruses discussed above, there are many other viruses that are associated with killing tumors, although the underlying mechanisms are not always clear. Newcastle virus (NDV). Replicates preferentially in malignant cells, and the most commonly used strain is 7 3 - T (Reichard et al., 1992; Zorn et al. 1994; Bar-Eli et al., 1996). Clinical antitumor activity is found in many tumors, including: neck, colorectal, pancreas, stomach, melanoma, and renal pelvis, in tumor burdens with reduced NDV after tumor inoculation (WO 94/25627; Nemunaitis, 1999) year). Thus, NDV can be used to remove tumor cells from a mixed cell composition. Moreover, vaccinia virus multiplies in several malignant cell lines. The encephalitis virus is shown to have a tumor lytic effect in the sarcoma of mice, but may need to be adjusted to reduce its infectivity in normal cells. Tumor regression has been described in cancer patients infected with cancer, hepatitis virus, influenza, chickenpox and apothecosis (for review, see Nemunaitis, -f 1999). In accordance with the methods disclosed herein and techniques well known in the art, one skilled in the art can test these or their ability to selectively kill tumor cells in a virus to determine which virus can be used to treat tumor cells. Remove from the mixed cell composition of interest. 4. Removal of the virus after virus treatment Although the virus used in the present invention is not replicated in normal cells, we may wish to remove the virus prior to use of the virus-treated cell composition. For example, reovirus is not associated with any known disease 70681-960914.DOC -34- This paper scale applies Chinese National Standard (CSS) A4 specification (210X 297 mm) A7 B7

132005You 090109803 Patent Application Replacement Page (September 96) V. Invention Description (33) A treatment method or combination with an immune system stimulator can give the recipient a specific antibody against a specific virus to reduce the virus infection. danger. Compositions The present invention provides a composition prepared by virus treatment of a mixed cell composition, wherein the virus substantially kills tumor cells contained in the cell composition. Such a composition is not a tumor lysate of the virus. A tumor lysate of a virus is a composition produced by tumor lysis of tumor cells by a virus, which comprises a virus-modified tumor cell membrane as an active ingredient. In contrast, the active ingredient in the virus-treated cell combination in the present invention is a viable non-neoplastic cell. Kits All of the viruses discussed above can be used to purify a cellular composition that may contain a mixture of tumor cells. If desired, it can be determined which virus or agents can be used to purify a particular cell composition. For example, when the mixed cell composition contains hematopoietic stem cells derived from a cancer patient, a biopsy of the cancer can be harvested in advance, and different viruses can be tested to determine which virus can effectively kill the cancer cells. The virus can then be used to cleanse the hematopoietic stem cells. Alternatively, the mixed cell composition can be treated with the viral cocktail method without first determining the efficacy of each virus. Accordingly, the present invention provides a kit comprising a population of viruses having different or overlapping specificities. For example, the kit may comprise a reovirus for sputum activation, a reovirus for tumor cells, a p53 expression virus for p53-deficient tumor cells, and a Delta 24 for Rb-deficient tumor cells, for Onyx-015 of p 5 3-deficient tumor cells, vesicular stomatitis virus for anti-interferon tumor cells, or its secondary disease paper size is applicable to Chinese National Standard (CiiS) A4 Regulation (210X 297) 1320056 No. 090109803 Patent Application Replacement Page (September 96) 96. 9.14 A7 Year Month B7 Revision 丨•Li .5-Supplement V, Invention Description (35) β -ΜΕ= yS-巯基Ethanol ΜΟΙ = multiplicity of staining PFU - leukoplakia unit PKR = double-stranded RNA-activated protein kinase EGF = epithelial growth factor PDGF = platelet-derived growth factor DMSO = dimethyl sulfoxide CPE = cytopathic effect GCSF = granulocyte Community Stimulating Factor Example 1 Reovirus-induced Breast Cancer Cell Tumor Lysis and Cell Wilt To determine the effect of reovirus on tumor cell viability, we first used three breast cancer model systems, MCF7 (ATCC No. HTB-22). , SKB R3 (ATCC number HTB-30) and MDA MB 468 (ATCC number HTB 132). The cells of each cell line were grown to a concentration of 50-60% and infected with a serotype 3 reovirus (ie, Dearling strain>, with a multiplicity of infection of 40, we obtained a reciprocal The virus is maintained as described in U.S. Patent No. 6,136,307. Reovirus-infected and uninfected cells are harvested at 0, 24, 48 and 72 hours after infection, and their viability is determined. 1A-1D, after infection, MCF7 (Fig. 1A), SKBR3 (Fig. 1B) or MDA MB 468 cells (Fig. 1 C), which were infected with reovirus, showed a significant drop in viable cell counts and was infected by dead virus. Or uninfected cells proliferated as we expected. Reovirus caused MCF 7 (Fig. 1D) and SKBR3 viability to fall from 93% to 70681-960914 at 72 hours post infection. DOC -38- This paper scale Applicable to Chinese National Standard (C5iS) A4 Rule &quot;klOX 297 mm)

Claims (1)

13:200^911^8^3 Patent Application A8 Chinese Shenxiang Patent Scope Replacement (October 98) bs Year Month b Amendment ^---:__^__ Supplement _ VI. Patent Application Scope WHAT IS CLAIMED IS: 1. A method of selectively removing tumor cells from a mixed cell composition for transplantation, wherein the composition is in vitro of a living organism, and the method comprises the steps of: (a) selecting a substrate comprising 3 s) a mixed cell composition that activates the tumor cell and a second cell having a functional double-stranded RN A protein kinase (p KR ) pathway; and (b) contacts the mixed cell composition with the virus, resulting in a lats-activated tumor The cell parenchyma is killed' to selectively remove the squamous activating tumor cells from the mixed cell composition; wherein the virus is selected from a bovine cancer virus having a mutation at the 艮31 or £3]1 gene, a herpes simplex virus having a mutation at the 〇34,5 gene, a parapoxvirus having a mutation at the OV20.0L gene, and an adenovirus having a mutation at the VA1 gene; wherein the K3l, E3L, γ134.5, OV20.0 Or the LVA1 gene is mutated to activate pKR2 in the second cell. The system will be blocked. The method of claim 1, wherein the virus has vaccinia # mutated at the K3L gene such that activation of PKR is not inhibited. 3. The method of claim 3, wherein the virus has Bovine Disease 4 mutated at the E3L gene such that activation of the sputum is not inhibited. 4. According to the method of claim i, the virus has a simple sore virus with a large change in the γ1.34_5 gene, so that the activation of pKR is not hindered. 70681-981021.DOC This paper scale applies to China National Standard (CNS) Α4^(2ΐ〇χ 297/^7 1320056 5. The method of claim 3, wherein the virus has a parapoxvirus mutated at the OV20.0L gene such that activation of pKR is not blocked. 6. The method of claim 3, wherein the virus has an adenovirus mutated at the VA1 gene such that activation of pKR is not inhibited. 7. The method according to any one of the preceding claims, wherein the mixed cell composition comprises hematopoietic stem cells. 8. According to the method of claim 7, the gold-forming stem cell line is collected from the bone marrow. 9. The method of claim 7, wherein the ostomy cell line is collected from blood. The method according to any one of claims 1 to 6, wherein the cell composition comprises any part of a tissue, an organ, or a tissue or an organ. , wherein the tissue or organ is a heart, a horn, a skin, and an islet 11. The method according to claim 10 is selected from the group consisting of liver, kidney, cells, and whole blood β, wherein the disease is still The method cell composition according to any one of the preceding claims, wherein the cell composition comprises cultured cells, semen or eggs. 13. According to the scope of the application for patents! The method of any of the six items is to copy the competent virus, and the interferon is added to the mixed cell composition according to the method of any one of claims 1 to 6. 70681-981021.DOC 1320056 VI. Applying for a patent garden In addition, a method according to item 14 of the patent scope is added, before adding the virus or adding it at the same time as the virus.疋16_ According to any one of the scope of the patent application, items ^ to 6, the virus is removed from the virus-treated cell composition, and the method further comprises: 17. According to the patent, the patent is _ The method ^ includes the step of storing the virus-treated cell composition. 'to 0' 18. According to the scope of the patent application, ton π 叼 ,, left' where the cell composition is stored in a solution containing DMSO. 19. The method according to any one of the preceding claims, wherein the K3L, E3L, γΐ34.5, 〇V2〇 〇^vai gene lines are not transcribed in the virus. The method according to any one of claims 1 to 6, wherein the K3L, E3L, γ134.5, OV20.0 or LVA1 gene is deleted. 21. A kit for removing tumor cells from a mixed cell composition selected to comprise a sloving activating tumor cell, wherein the kit comprises at least two viruses selected from the group consisting of: having K3L Or a vaccinia virus having a mutation at the E3L gene, a herpes simplex virus having a mutation at the Η34.5 gene, a parapoxvirus having a mutation at the OV20.0L gene, and an adenovirus having a mutation at the VA1 gene, wherein the K3L, E3L, The γι34.5, OV20.0 or LVA1 gene was mutated so that the activation of PKR was not blocked. 70681-981021.DOC The paper size is based on the Chinese National Standard (CNS) Α4 specification &lt;210X297 mm)