JP5748656B2 - Method for determining the sensitivity of hyperproliferating cells to oncolytic viruses based on tumor suppressors - Google Patents

Description

  The present invention relates generally to the fields of virology, molecular biology and medicine. In particular, the present invention relates to a method for assessing susceptibility to oncolytic viral therapy of hyperproliferative diseases such as cancer.

  This application claims the benefit of priority of US Provisional Patent Application Serial No. 61 / 055,360, filed May 22, 2008, the entire contents of which are incorporated herein by reference. .

  Reovirus (respiratory and intestinal orphan virus) is a ubiquitous, non-enveloped virus that contains 10 double-stranded RNAs as a genome and is generally mildly infected in humans, upper respiratory tract and gastrointestinal tract It is often asymptomatic in hosts with limited immunity and functioning (Tyler, 2001). Attempts to retrograde reovirus have largely failed due to several factors, such as the double-stranded RNA genome of reovirus. Reovirus particles lacking σ1 have been shown to be non-infectious (Larson et al., 1994).

  Importantly, reovirus has long been recognized to exhibit significant cytocidal activity when infecting certain transformed cells (Duncan et al., 1978; Hashiro et al., 1977). Replicative oncolytic viruses provide an attractive anticancer therapeutic approach. These oncolytic viruses have two major advantages. First, unlike conventional chemotherapy and radiation therapy, the virus specifically targets cancer cells because of its limited ability to replicate in normal cells. Secondly, compared to vectors that do not have replication capacity, the virus is distributed on a large scale by spreading from the cancer cells that were originally infected to the surrounding cancer cells and exerts a strong anticancer effect. can do.

  Although contact with wild-type reovirus is often asymptomatic in healthy individuals, it can still pose a serious and potentially very serious risk for immunocompromised individuals. While limiting the clinical potential of reovirus therapy in patients such as cancer patients, the patient may benefit from this therapy without limitation. Until it has been shown that transformed cells containing the tumorigenic Ras signaling pathway are preferentially susceptible to reovirus infection (Type 3 Dearing strain) in vitro and in vivo (Coffey et al., 1998); Strong et al., 1998; Norman et al., 2004), the basic principle underlying the oncolytic activity of reovirus remained unknown. As Ras gene mutations are frequently observed in various types of human cancer (Dursma et al., 2003), these findings have led to the current use of reovirus therapy in clinical trials (Norman Et al., 2005).

  Nevertheless, the presence of Ras gene mutations only partially explains the sensitivity of cancer cells to reovirus therapy. From recent studies, the Ras pathway is not the only factor determining reovirus tolerance (Song et al., 2009) and reovirus resistant cells have been established from Ras transformed cells (Kim et al.). 2007), yet it was shown that Ras mutation activation can be maintained. Accordingly, there is a need for improved methods for identifying and selecting appropriate cancer cell subpopulations for treatment with reovirus and other oncolytic virus agents.

  There is a need for improved methods of identifying and selecting appropriate cancer cell subpopulations for treatment with reovirus and other oncolytic virus agents.

  The reovirus is p53, Rb, ATM, BRCA1, BRCA2, MutS, whether it is in its wild type (Deering type 3) or a modified form (eg, a partially attenuated virus with incomplete synthesis of the σ1 protein). Hyperproliferative or transformed cancer cells that contain certain defective or inactivated tumor suppressor genes, such as MutL, MutH, APC and ATBF1, It is effective to kill when exposed to the virus in vitro or in vivo. Thus, the scope of reovirus may extend beyond cases with abnormal or excessive activity of the ras proto-oncogene signaling pathway. Other viruses previously thought to be primarily responsive to the status of ras, interferon or other cellular immune signaling pathways are also susceptible to loss of function of tumor suppressors, as well as to broaden the scope of the virus. There is a possibility.

  Thus, according to the present invention, a method for determining whether a hyperproliferative disease is susceptible to oncolysis of reovirus or mixoma virus comprising the steps of: (a) providing hyperproliferative cells; b) evaluating the structure, function, or expression of at least one gene or gene product of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, or ATBF1 derived from the hyperproliferative cell And (c) changing the structure, function, or expression of the hyperproliferative cell to at least one of the gene or gene product of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, or ATBF1. Comparing to wild-type structure, function, or expression P53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, or ATBF1, a defect in the structure, function, or expression of the hyperproliferative disease is a reovirus or a mixoma virus A method is provided that demonstrates the susceptibility to tumor oncolysis.

  The method can further include treating the patient from whom the hyperproliferative cells are obtained, for example, by at least one of reovirus therapy or mixoma virus therapy. The reovirus therapy may be wild type reovirus therapy and utilizes a reovirus that expresses a defective σ1 capsid protein, expresses an undetectable σ1 capsid protein, or does not express a σ1 capsid protein It may be an attenuated reovirus therapy. Treatment may include ex vivo treatment of bone marrow cells from the patient with reovirus and / or mixoma virus therapy, and may also include applying reovirus and / or mixoma virus therapy to the patient. Good. In the case of ex vivo therapy, the method may further comprise administering a second anti-hyperproliferative therapy to the bone marrow cells, and in the case of viral therapy applied to the subject, the method The method may further comprise applying a second anti-hyperproliferative therapy to the subject. The second anti-hyperproliferative therapy can be chemotherapy, radiation therapy, hormone therapy, gene therapy, or immunotherapy. The method further includes inhibitors of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, or ATBF1, such as proteins or peptides, antibodies, siRNA, antisense molecules, ribozymes or small molecules It may further include a treatment method using. Treatment can also include non-viral anti-hyperproliferative therapy such as using chemotherapy, radiation therapy, hormone therapy, gene therapy, or immunotherapy. A hyperproliferative cell may be a malignant cell or a benign cell. The method can further include obtaining hyperproliferative cells from the patient prior to step (a). Evaluation of expression can comprise Northern blot, Western blot, immunohistochemistry, RT-PCR, microarray analysis, transcriptome analysis, proteome analysis, or metabolome analysis. As another example, structural evaluation can comprise sequencing, in situ hybridization, immunohistochemistry, or structural proteomics. As yet another example, functional assessment can comprise evaluating the expression or activity of a target downstream of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, or ATBF1.

  In another embodiment, a method of treating a hyperproliferative disease in a patient, comprising: (a) treating hyperproliferative cells of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, or ATBF1. Contacting the function or expression with an inhibitor; and (b) subjecting the hyperproliferative cell to reovirus and / or mixoma virus therapy. The reovirus therapy may be wild type reovirus therapy, such as a reovirus that expresses a defective σ1 capsid protein, expresses an undetectable σ1 capsid protein, or does not express a σ1 capsid protein. It may also be attenuated reovirus therapy. The treatment may comprise ex vivo treatment of bone marrow cells from the patient or may comprise in vivo treatment of the patient. The method further comprises administering to the bone marrow cells a second anti-hyperproliferation therapy, eg, a second anti-hyperproliferation therapy, eg, chemotherapy, radiation therapy, hormone therapy, gene therapy, or immunotherapy. Can do. A hyperproliferative cell may be a malignant cell or a benign cell.

  The use of the term “a” or “an” when used with the term “comprising” in at least any of the claims or specification, It may mean “one”, but the words are also “one or more”, “at least one”, and “one or more”. more than one) ”.

  Throughout this application, the term “about” includes the inherent variation in error associated with the device, method or method being used to determine that value, or the variation present in the study object. Used to indicate

  The use of the term “or” in the claims supports the definition that the disclosure refers only to alternatives and “and / or at least one of and / or”. Are used to mean “and / or at least and / or” unless explicitly stated to refer only to alternatives, or alternatives are not mutually exclusive.

  As used herein and in the claims, the term “comprising” (and any form thereof, eg, “comprise” and “comprises”), “having” (And any form thereof, such as “have” and “has”), “comprising” (and any form thereof, such as “includes” and “include”) Or “containing” (and any form thereof, eg, “contains” and “contain”) is a comprehensive or open-ended, additional enumeration It does not exclude elements or method steps that are not done.

  Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the invention, are provided by way of illustration only, will occur to those skilled in the art. This is because various changes and modifications within the spirit and scope of the present invention will become apparent from this detailed description.

  The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 5 shows preferential infection of reovirus and mixoma virus to L3 defective in p53 − / − MEF and ATM. (FIG. 1A) p53 − / − MEF and p53 + / + MEF (p53 knockout mice or p53 wild type mouse fetal fibroblasts, respectively) were infected with WT / AV reovirus with MOI of 40 or MOI 5 was infected with a GFP-expressing myxoma virus (Myx-GFP (myxv MYXV type Lausanne (ATCC) strain is described in Johnston et al., 2003)). On day 3 post infection, cells were immobilized / permeabilized and analyzed by FACS using reovirus and secondary FITC antisera. The results show that both wild type and attenuated reovirus were able to preferentially infect p53 − / − MEF. For the mixoma infection experiment, cells were infected for 24 hours and visualized by phase contrast and fluorescence microscopy. Cells expressing GFP represent a mixoma virus infection. Mock: mock infection. (FIG. 1B) BT (lymphoblastoid C3ABR cells with normal ATM; Kozlov et al., 2003) and L3 (lymphoblastoid cells lacking ATM; Kozlov et al., 2003) In contrast, MOI was 40 and WT / AV reovirus was infected, or MOI was 5 and myxoma virus (Myx-GFP) was infected. On day 3 post infection, cells were immobilized / permeabilized and analyzed by FACS using reovirus and secondary FITC antisera. The results show that both wild type and attenuated reovirus were able to preferentially infect p53 − / − MEF. For the mixoma infection experiment, cells were infected for 48 hours and visualized by phase contrast and fluorescence microscopy. Cells expressing GFP represent a mixoma virus infection. The C35ABR (B3) and L3 cell lines are respectively Dr. M Lavin (Queensland Institute of Medical Research) and Dr. Y Shiloh (Dr. Y Shiloh). (Tel Aviv University) Mock: mock infection. The figure shows preferential infection of reovirus and mixoma virus to human lymphomas where p53 and ATM do not function normally. (FIG. 2A) p53 and ATM defects enhance the response to IR stimulation. Mantle cell lymphoma (Granta, HBL-2 (p53 mutant, Turker et al., 2006), Z138C, JVM-2) and Burkitt lymphoma (Raji, and Ramos) were irradiated with 2 Gy for 2 hours, and the cells were Collected. Whole cell lysates were generated by treatment with NET-N lysis buffer (1% NP-40) followed by sonication. 50 μg of protein was then run on 8% or 10% SDS-PAGE, transferred to nitrocellulose, and displayed antibodies (phosphorylated ATM (Phospho-ATM); pSer1981 ATM and phosphorylated p53 (Phospho-p53)) PSer15 and p53 were purchased from Rockland Immunochemicals for Research and Cell Signaling; ATM-specific rabbit polyclonal antibody 4BA was a gift from Dr. M. Lavin; It investigated using. BT cells and L3 cells were used as controls. The figure shows preferential infection of reovirus and mixoma virus to human lymphomas where p53 and ATM do not function normally. (FIG. 2B) Cells to which p53 and ATM normally respond (BT, Z138C, JVM-2, Ramos), and dysfunctional cells to which at least one of p53 or ATM does not respond normally (L3, HBL-2) , Grant, Raji), the virus susceptibility was plotted against both reovirus and mixoma virus. 1; Virus sensitivity. 0: Virus resistance. Cells that respond normally are resistant, but dysfunctional cells that do not respond normally are susceptible to virus infection. The figure shows preferential infection of reovirus and mixoma virus to human lymphomas where p53 and ATM do not function normally. (FIG. 2C) Cells were infected with a WT / AV reovirus with an MOI of 40 or a Myxoma virus (Myx-GFP) with an MOI of 5. Previous studies have shown that Raji cells are sensitive to Reovirus and Ramos cells are resistant (Alain et al., 2002). On day 3 post infection, cells were immobilized / permeabilized and analyzed by FACS using reovirus and secondary FITC antisera. The results showed that both reovirus and mixoma virus preferentially infected p53 and / or ATM non-responsive lymphomas. For the mixoma infection experiment, cells were infected for 48 hours and visualized by phase contrast and fluorescence microscopy. Cells expressing GFP represent a mixoma virus infection. Mock: mock infection. The figure which shows the retinoblastoma cell reovirus and the mixoma virus infection. Retinoblastoma cells (Y79 and WERI-Rb-1, purchased from ATCC) were infected with WT / AV reovirus with an MOI of 40 or Myxoma virus (Myx-GFP) with an MOI of 5. . At designated days after infection, cells were fixed / permeabilized and analyzed by FACS using reovirus and secondary FITC antisera. For the mixoma infection experiment, cells were infected for 48 hours and visualized by phase contrast and fluorescence microscopy. Cells expressing GFP represent a Mixoma infection. Mock: mock infection.

  Carcinogenesis is a multi-step process involving a combination of oncogene activation and inactivation of tumor suppressor genes by mutation events that affect normal proto-oncogenes and tumor suppressor genes. Interestingly, oncolytic viruses utilize various intracellular signaling pathways driven by oncogenes to selectively replicate and kill cells infected by the virus. In addition to oncogene-dependent oncolysis, the inventors speculated that some tumor suppressor genes may also play an important role in promoting viral oncolysis. Therefore, we tested various murine and human cancer cell lines with modulated tumor suppressor genes, and tumor suppressor gene dysfunction affects tumor destruction by reovirus and mixoma virus Investigate whether or not.

  The molecular basis of reovirus oncolysis has unexpectedly advanced through efforts to characterize cellular receptors for reovirus. In 1993, a group of the same cell line transfected with a gene encoding EGFR while two mouse cell lines that do not express epidermal growth factor receptor (EGFR) are relatively resistant to reovirus infection Reported significantly higher sensitivity (Strong et al., 1993). This result suggested that reovirus utilizes an activated intracellular signaling pathway brought about by the presence of functional EGFR on the host cell. The same investigators then demonstrated that NIH3T3 cells that are resistant to reovirus infection become susceptible when transformed with activated Sos, Ras, or verbB (downstream of EGFR). (Strong et al., 1996; Strong et al., 1998). The use of the oncogenic Ras signaling pathway is thus considered an important step in reovirus oncolysis.

  Many human cancers show activation of the Ras pathway, highlighting the strong impact of this signaling network on tumorigenesis, and reovirus exerts a wide range of potential oncolytic potential in various types of tumors. It is suggested that From initial experiments, it was found that over 80% of cell lines originating from various types of tumors are susceptible to reovirus-mediated killing (Coffey et al., 1998). More interestingly, another oncogene (Myc) also contributes to reovirus oncolysis. Overexpression of Myc increases the sensitivity of reovirus resistant cells to make them reovirus permissive (Egan et al., 2003).

  Mixoma virus is a rabbit-specific poxvirus that appears to be a promising oncolytic virus base (reviewed by Stanford and McFadden, 2007). The host orientation of the virus is highly restricted to European rabbits, and the virus is nonpathogenic to all other vertebrate species tested, including humans (McFadden, 2005). Despite this strict specificity, Mixoma virus can infect and kill a wide variety of human tumor cell lines (Sypula et al., 2004). The directionality of the mixoma virus at the cellular level is primarily regulated by intracellular events downstream of viral binding and entry, not at the level of specific host receptors (McFadden, 2005).

  The molecular basis of myxoma virus oncolysis was presented through studies on viral host range determinants that control virus tolerance in human cancer cells. Viral M-T5 host range determining protein interacts with cellular Akt, and this interaction has been shown to enhance the oncolytic ability of Mixoma in human cancer cells (Wang et al., 2006). . M-T5 KO (knockout) myxoma virus shows reduced viral oncolytic potential in certain human cancer cell groups (cancer cells called type II), but constitutively activated Akt Cancer cells are still tolerant to M-T5 KO mixoma virus, and this is why the up-regulation of Akt, which is frequently seen in many cancer cells, is important in determining the ability of oncology to disrupt tumors. It is suggested that it plays a role (Wang et al., 2006).

  The relationship between viral oncolysis and tumor suppressor genes was initially presented through studies on adenovirus. The human adenovirus E1B gene encodes a 55 kD protein that binds to and inactivates the cellular tumor suppressor protein p53. It has been shown that mutant adenoviruses that do not express this viral protein are capable of replicating in and killing p53 dysfunctional human tumor cells, but not in cells with functional p53. (Bischoff et al., 1996). The underlying basis for this oncolysis of mutant adenoviruses is that p53-dependent apoptosis caused by adenovirus E1A is impaired in p53 dysfunctional cancer cells due to p53 deletion or mutation. (Bischoff et al., 1966). However, later studies have shown that p53-dependent adenoviral oncolysis is more complex and responds to additional pathways in various models (O'Shea et al., 2004; Abou ) Et al., 2004; Royds et al., 2006). In fact, from this, we speculated that cancer cells with p53 dysfunction may have disrupted the antiviral mechanism of the cell due to genomic instability (Carroll et al., 1999). ).

  Due to the importance of genome maintenance by p53, the p53 gene is considered to be a protector of the cell genome (Gomez-Lazaro et al., 2004; Vogelstein et al., 2000). Because the genome is maintained through p53, normal cells can retain a complete antiviral mechanism. However, p53 dysfunction may ultimately result in viral susceptibility (due to loss of antiviral mechanisms) caused by global genomic instability. Recent studies have shown that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of p53 function as a pro-apoptotic gene and tumor suppressor gene (Takaoka ( Takaoka) et al., 2003; Munoz-Fontera et al., 2008). The transcriptional role of p53 is important in the activation of the IFN pathway during viral infection. p53 can activate transcription of IFN regulatory factor 9 upon virus inoculation (Munoz-Fontera et al., 2008). Furthermore, significantly higher levels of viral replication were observed when p53 expression was reduced in cancer cells (Dhalel et al., 2008). These findings demonstrate the important antiviral activity of p53. This may also be the case for other tumor suppressor genes that are important in DNA repair and genome stability. The inventors here are that cancer cells in which either p53, ATM or Rb do not function normally are highly susceptible to inoculation with either reovirus or mixoma virus. In addition to the susceptibility that can be enhanced by activation of the oncogene signaling pathway, it may help clarify how various oncolytic viruses can distinguish normal cells from cancer cells It shows that. Verification of the relationship between these genetic abnormalities and oncolytic virus susceptibility greatly increases the potential for application of oncolytic virus therapy.

I. Oncolytic viruses A. Reovirus Reovirus (Reoviridae) is a naturally occurring non-enveloped virus that has a double-stranded RNA (dsRNA) genome divided into ten and surrounded by two concentric icosahedral protein capsids. Comprising a family. These viruses can affect the gastrointestinal system and respiratory organs. The name Reoviridae is derived from “respiratory and intestinal orphan virus”. The term “orphan” virus refers to a virus that is not associated with a known disease. Although the reoviridae family of viruses has been identified with various diseases in recent years, the original name is still used.

  Reovirus infections often occur in humans, but in most cases are mild or subclinical. The virus can be easily detected in feces and can also be detected from pharyngeal or nasal secretions, urine, cerebrospinal fluid, and blood. Despite the ease of finding reovirus in clinical laboratory materials, the role of the virus in human disease or therapy remains uncertain.

  Reoviruses are non-enveloped and have an icosahedral capsid (T-13) composed of outer and inner protein shells. The genome of the reoviridae virus contains 10-12 segments that fall into three categories corresponding to its size: L (large), M (medium) and S (small). Segments range from approximately 3.9 to 1 kB and each segment encodes 1-3 proteins. Reoviridae proteins are indicated by Greek letters corresponding to the segment into which they are translated (L segment encodes λ protein, M segment encodes μ protein, and S segment encodes σ protein ).

  Since these viruses have a dsRNA genome, replication occurs only in the cytoplasm and the virus encodes several proteins required for replication of the dsRNA genome and conversion to (+)-RNA. The virus can enter the host cell via a receptor on the cell surface. The receptor is not known, but is believed to have sialic acid and junctional adhesion molecules (JAM). The virus is partially unshelled by the protease in the endolysosome, but the capsid is partially digested in the endolysosome to allow further cell entry. The core particle then enters the cytoplasm by an unknown process, where the genome is conservatively transcribed, resulting in an excess (+) sense strand, which is used as an mRNA template to synthesize the (-) sense strand. Is done. Viral particles begin to assemble in the cytoplasm 6-7 hours after infection.

  Infectious mammalian reovirus virions occur as particles approximately 85 nm in diameter. The outer capsid of the virion comprises several distinct protein species, among which the sigma-1 (σ1, 50 kDa) is a discontinuous carbohydrate binding domain (Chappel et al., 1997; Chappell et al., 2000; Mediates viral attachment to the host cell surface via Connolly et al., 2001) and virion fixation domains (Mah et al., 1990; Fernandes et al., 1994; Lee et al., 1994) (Lee et al., 1981; Duncan et al., 1991; Nagata et al., 1987; Turner et al., 1992). σ1 is the product of the bicistronic reovirus S1 gene, which also encodes a nonstructural protein called σ1s using separate but overlapping reading frames (Ernst et al., 1985; Jacobs et al., 1985; Sarkar et al., 1985). Reovirus particles lacking σ1 have been reported to be non-infectious (Larson et al., 1994). The reovirus S1 gene has been thought to play an important role in determining reovirus virulence (Haller et al., 1995; Wilson et al., 1994; Kaye et al., 1986; Weiner (Weiner et al., 1980).

i. Wild-type reovirus in cancer therapy Certain methods have been described for the production and use of wild-type reovirus in cancer therapy (eg, US Pat. Nos. 7,300,650, 7,163,678). No. 7,049,127, No. 7,014,847, No. 6,994,858, No. 6,811,775, No. 6,703,232, No. 6, 576,234, 6,565,831, 6,528,305, 6,455,038, 6,344,195, 6,261,555, 6,136,307 and 6,110,461, and US Patent Application Publication Nos. 2006/0165724, 2006/0073166, 2005/0123513, 2004/04. No. 265271, No. 2004/0126869, No. 2004/0109878, No. 2002/0037543, No. 2006/0029598, No. 2005/0026289, No. 2002/0006398 and No. 2001/0048919. Specification, these patent documents are each incorporated by reference in their entirety without waiver).

ii. Attenuated reovirus in cancer therapy However, in immunocompromised hosts, such as newborn animals and SCID (severe combined immunodeficiency) animals, wild-type reovirus is a significant virus, especially in neural and myocardial tissues. It exerts pathogenicity (Sabin, 1959; Weiner et al., 1977; Batty et al., 1993; Loken et al., 2004). In some cases, wild-type reovirus was associated with virulence, even in hosts with immunity, including humans (Terhegen et al., 2003, Hirasawa et al., 2003). Thus, wild-type reoviruses do not always act in a harmless manner, especially in immunocompromised hosts or very young hosts. For example, in an immunocompromised host such as a very young animal or an immunodeficient adult, the virus has also been shown to infect some healthy tissue, such as the heart, liver, pancreas and nerve structures. This problem further applies to cancer patients who have received large-scale radiation / chemotherapy treatment because they are prone to immunosuppression. Accordingly, there is a clear need for the development of less virulent reovirus for viral oncolytic therapy.

  Furthermore, wild-type reoviruses often possess undesirable virulence and infectivity that limit their potential for in vitro use. Specifically, when cell cultures (eg, bone marrow transplants collected from cancer patients) are exposed to wild-type reovirus, such as stem cells required for the regeneration of the immune system of cancer patients, etc. Undesirable side effects of killing cells can result. Thus, the high virulence of wild type reovirus presents a significant limitation on the clinical potential of in vitro application involving exposure of cells to reovirus.

  Although the attenuated reovirus described herein lacks a detectable full-length σ1 capsid protein, it is still unexpectedly infectious. σ1 was associated with reovirus binding and attachment to cells via cell surface sialic acid residues in the first step of viral infectious replication (Lee et al., 1981; Duncan et al., 1991; Nagata et al., 1987; Turner et al., 1992; Chappel et al., 1997; Chappel et al., 2000; Connolly et al., 2001). Despite lacking full length σ1, the attenuated reovirus described herein has the ability to enter host cells and to disrupt cytolytic viruses. In addition, the attenuated reovirus has a level that is low (ie, statistically significantly lower) compared to the level of cytopathic effect exhibited by non-attenuated cells by naturally occurring non-attenuated reovirus. It exhibits the surprising property of causing one or more cytopathic effects on non-malignant cells. Thus, and as described in more detail below, the attenuated reovirus provided herein exhibits an improvement over prior art reoviruses and is directed to normal (eg, non-malignant) cells. It is suitable for use as an oncolytic agent without undesirable side effects such as lysis of sex and normal cells. Specifically, the attenuated reovirus can be used in vitro for selective killing of cancer cells or neoplastic cells.

  In certain embodiments, U.S. Patent Application No. 60 / 704,604, filed August 5, 2006, and International Patent Application No. PCT / US2006 / 029881, filed Jul. 31, 2006 (the patents). Attenuated reoviruses as described in the literature are incorporated herein by reference in their entirety without waiver may be used with the present invention. In certain embodiments, the attenuated reovirus may lack the wild type reovirus S1 gene. Attenuated reovirus is an amount of S1 gene product (σ1) that is either reduced or undetectable, or at least one of a truncated, mutated, or dysfunctional form You may have S1 gene which produces a certain (sigma) 1. The attenuated reovirus may have a S1 gene that contains one or more mutations compared to the wild-type S1 gene, the mutation being a nucleotide substitution, a nucleotide deletion or a nucleotide insertion .

  The “attenuated” reovirus described herein has at least one of infectivity to host cells, replication properties in host cells, or host cell lysis properties known or naturally occurring, ie wild type. Reoviruses that are altered (ie, statistically significantly increased or decreased) relative to the level of one or more such properties exhibited by that reovirus. In certain embodiments, the attenuated reovirus will exhibit reduced infectivity, replication ability and / or lysis ability compared to wild-type reovirus. Examples of such altered properties that can distinguish attenuated reoviruses as disclosed herein include various indications of viral cytopathic effects, such as growth of a given host cell Multiplicity of infection required for infection (MOI, average number of virions infecting each cell), degree of host cell lysis reaction (including apoptosis and / or necrosis) caused by viral infection, cytolytic virus replication Subsequently, the titer of virus released from the proliferatively infected host cell and other parameters by which one skilled in the art can measure viral activity against the host cell. Other signs of cytopathic effects include host cell morphological changes, changes in the ability to induce attack by the immune system via the host cell, (supports such as extracellular matrix proteins or semi-solid growth media, or Changes in cell adhesion (to other cells), changes in the expression level of one or more cellular genes, changes in the replication capacity of the host cell, and / or other changes in the metabolic activity of the cell.

  Considering that the S1 gene segment of reovirus is greatly related to reovirus virulence (Weiner et al., 1977; Weiner et al., 1980; Mann et al., 2002) The inventors surprisingly separated S1 attenuated reovirus (“AV reovirus”) from persistent reovirus infections of cultured cells (Kim et al., 2007). This attenuated reovirus strain showed a significant reduction in viral pathogenicity in an immunodeficient animal model without compromising viral oncolytic activity in vivo. Wild-type reoviruses are known to adversely affect the development of rat and mouse embryos by slowing development and inhibiting blastocyst formation (Priscott, 1983; Heggie et al. 1979), the present inventors further evaluated the pathogenicity of AV reovirus against stem cells. Therefore, we compared the pathogenicity of wild type and AV reoviruses against embryonic stem cells (ESC). As shown in the examples below, wild-type reovirus readily infects ESCs in vitro and significantly suppresses stem cell growth in a teratoma model, while AV reovirus minimizes ESCs in vitro. And does not affect stem cell growth in a teratoma model.

  In various embodiments, the attenuated reovirus can comprise one or more additional mutations. For example, in certain other embodiments, the attenuated reovirus may lack the wild type reovirus S4 gene. The reovirus wild type S4 gene encodes a reovirus capsid σ3 polypeptide involved in virion processing during reovirus replication infection of host cells (eg, Ahmed et al., 1982; Giantini et al. 1984).

  An attenuated reovirus may comprise a reovirus virion that has the ability to replicate. Further, the attenuated reovirus may have a genetic mutation (eg, substitution, insertion, deletion) in at least one of the S1 gene and the S4 gene. The S1 gene of wild type reovirus is known to those skilled in the art. For example, the wild-type S1 gene sequence is the S1 gene sequence identified in a major type of naturally occurring reovirus isolated from the respiratory or intestinal tissue of an infected person, or a common derived from such a sequence. It has an array. Several reovirus S1 gene sequences, including human reovirus, have been determined (eg, Genbank accession number of human reovirus S1 gene sequence: human type 3 reovirus S1: X01161; human type 2 Reovirus S1: M35964; human type 1 reovirus S1: M35963), the polynucleotide sequence encoding the σ1 protein as well as the amino acid sequence of the encoded σ1 protein itself (eg main human reovirus serum) Genbank accession number of type S1 gene sequence: human type 1 reovirus Lang strain (T1L) accession number M35963; human type 2 reovirus Jones strain (T2J) accession number M35964; human type 3 reovirus Dealing strain (T3D) ) Registration number X01161; Human type 3 reovirus Abney strain (T3A) accession number L37677).

  According to certain embodiments, an attenuated comprising a reovirus genome that contains a mutant reovirus S1 gene that lacks the wild-type reovirus S1 gene or cannot encode the complete reovirus σ1 capsid protein. Reoviruses are provided (Genbank accession number of human reovirus S1 gene sequence: human type 3 reovirus S1: X01161; human type 2 reovirus S1: M35964; human type 1 reovirus S1: M35963). Methods for determining whether a mutant S1 gene is present by sequencing the reovirus S1 gene will be apparent from the present disclosure and are known in the art, for example, Ausubel et al. (1989); (Ausubel et al. (1993); Sambrook et al. (1989); Maniatis et al. (1982); Glover (1985); Hames and Higgins (1985); Follow techniques described elsewhere. Thus, a mutated S1 gene is one from the corresponding S1 wild-type or consensus nucleotide sequence by one or more nucleotide substitutions, nucleotide insertions, and nucleotide deletions, so that it can be readily determined. Alternatively, it refers to an S1 gene having a polynucleotide sequence that differs at a plurality of nucleotide sequence positions. Several mutations in the murine reovirus S1 gene sequence encoding the σ1 protein have been disclosed in Hoyt et al. (2005), but there are certain of the invention described herein. According to an embodiment, the Huite et al. Mutation is clearly excluded.

  As described herein and known in the art, the outer capsid σ1 protein of a reovirus is based on its biochemical and / or immunochemical properties (eg, Mah et al., 1990; Leone et al., 1991; Chappel et al., 1997), typically one or more techniques such as immunodetection methods (eg sigma 1 specific immunoprecipitation, Western immunoblot analysis, immunoaffinity chromatography, Immunofluorescence staining, immunocytofluorimetry, electrophoresis of radioisotope-labeled reovirus polypeptides, etc.), hemagglutination, and / or the use of related methods. Accordingly, the above and other means for detecting reovirus σ1 protein have been established and, in view of the teachings herein, one of ordinary skill in the art will recognize standards accepted in the art for the detection of σ1 protein. And what is the sensitivity of the state of the art, so reovirus virions that are capable of replication and lack the “detectable” reovirus σ1 capsid protein are currently standard It will be understood that reoviruses are included where the full σ1 protein cannot be detected even if the σ1 protein detection task (if present) is used.

US patent application Ser. No. 11 / 997,537, which describes such attenuated reovirus, is incorporated herein by reference.
iii. Control of Reovirus In some embodiments, peptidyl fluoromethyl ketone (PFMK) may be added to the cell composition to repress or eliminate the reovirus. Co-pending US Provisional Patent Application Serial No. 60 / 906,706, which is hereby incorporated by reference in its entirety, describes peptidyl fluoromethyl ketone (PFMK) for inhibiting viral replication. , Incorporated herein by reference in its entirety without waiver.

iv. Reovirus production According to certain embodiments disclosed herein, a reovirus may be derived from any reovirus, which refers to those belonging to the family Reoviridae, It contains reoviruses with different orientations and can be obtained from different sources (Tyler and Fields, 1996). In certain embodiments, mammalian reoviruses and human reoviruses are envisioned, but the invention is not intended to be so limited and, based on this disclosure, for such purposes Those skilled in the art will recognize the situation where any particular reovirus may be desirable. In certain embodiments, the reovirus may be a human type 3 (Dealing), type 1 (Lang), type 2 (Jones), or type 3 (Abney) reovirus, but in certain other embodiments, the reovirus Viruses may include other mammalian species, such as non-human primates (eg, chimpanzees, gorillas, macaques, monkeys, etc.), rodents (eg, mice, rats, gerbils, hamsters, rabbits, guinea pigs, etc.), dogs, cats, It may be derived from one or more reoviruses that are directed towards cells of common livestock (eg, cattle, horses, pigs, goats, etc.), or alternatively, leos with distinct directional properties. Viruses (eg, avian reovirus) may be used.

  As described herein, certain embodiments relate to attenuated reoviruses that can be recovered after in vitro persistent infection methods, although attenuated reoviruses that can be obtained according to other methods are also contemplated. The methods include, for example, in vivo persistent infection methods, generation and identification of σ1 deficient and / or σ1 deficient mutants by molecular biology techniques (in some embodiments, additionally or alternatively, σ3 deficient and And / or the generation and identification of σ3 deficient mutants), and further isolation of naturally occurring σ1 deficient and / or σ1 deficient mutants and / or σ3 mutants, or chemical, physical, Or genetic techniques (eg, selective recombination of reovirus genes in productively infected host cells). Such .sigma.1 (and / or .sigma.3) mutation artificially induced by at least one of), at least one can be cited among.

  In addition, the inventor has determined that any other gene that affects the infectivity, replication ability or packaging ability of the virus, such as any gene encoded on the virus, such as the 10 gene segments of reovirus or its We anticipate that mutations may be observed that result in the desired attenuated phenotype in the combination. For example, a reovirus S1 gene or S4 gene mutation is one, two, three, four, five or more on ten gene segments of a reovirus compared to a reovirus wild-type gene. May be accompanied by mutations. A variety of methods can be used to generate attenuated reovirus, including, for example, those described in US Patent Application No. 60 / 704,604.

B. Myxoma virus Myxoma virus is a poxvirus and has a large double-stranded DNA genome that allows the potential insertion of eukaryotic genes associated with large (25 kb) therapy. Mixoma virus is a virus unique to rabbits and causes a fatal disease called myxoma in European rabbits (Oryctalagas cuniculus). Since its species specificity is so stringent, the virus was used in Australia in the 1950s to control the number of wild-raised rabbit populations. Importantly, the virus is non-pathogenic to all other vertebrate species tested, including humans, but certain non-rabbit cells, such as immortalized baby monkey kidney fibroblasts (BGMK) Primary mouse cells genetically deficient in IFN responses, and several different human tumor cells in vitro can be replicated in vitro. It is well known that cancer cells lack their IFN response. Recently, Mixoma virus has been shown to be an oncolytic agent in vitro, in vivo for experimental gliomas and ex vivo for human malignant glioma surgical specimens (Lun et al., 2005; 2007; Stanford et al., 2008). The virus can infect and kill human glioma cells, is safe when administered intracerebrally, and “heals” mice when administered intratumorally in an orthotopic human malignant glioma model. . Furthermore, the virus infects and kills all tested primary glioma cells obtained directly from glioma surgical specimens, and its oncolytic activity is enhanced by rapamycin.

  Mixoma viruses, like other oncolytic viruses such as reovirus, need to circumvent the antiviral defenses present in normal healthy cells so that they can replicate intracellularly. Mixoma virus and other oncolytic viruses cause interferon production and are generally sensitive to the antiviral effects of the IFN pathway. Related proteins that are induced by the antiviral response of IFN and that primarily affect viral growth include PKR, OAS synthase, and Rnase L nuclease. PKR activates eIF2α resulting in translational repression and induction of apoptosis. In normal cells, myxoma virus is directly affected by PKR and eIF2α.

  Antiviral response pathways are often confused in cancer cells. For example, a reduced response or defect to IFN is a genetic defect that often occurs during transformation and tumor development. More than 80% of tumor cell lines do not respond to interferon or show poor response to interferon. (Stojdl et al., 2003 and references cited therein; Wong et al., 1997; Sun et al., 1998; Matin et al., 2001; Balachandran et al. 2004). US Patent Application Publication No. 2006/0263333 (incorporated by reference) describes the use of a mixoma virus to infect and kill cancer cells, including human tumor cells. Is incorporated herein in its entirety.

II. Tumor suppressor Tumor suppressor genes are those that protect cells from one step on the pathway leading to cancer. When this gene is damaged, cells can progress to cancer, usually after being affected by other factors. Unlike oncogenes, tumor suppressor genes generally follow a “two-hit hypothesis” that suggests that both alleles encoding a particular gene must be affected before results appear. . This is due to the fact that if only one allele of the gene is damaged, the other can still produce the appropriate protein. In other words, tumor suppressor genes are usually haploinsufficient, in contrast to oncogenes that are generally haploinsufficient. Of course, there are obvious exceptions such as the p53 gene product that can exist as a mutated form of dominant inhibition relative to the normal p53 protein, and in such cases is therefore also haploinsufficient.

  Tumor suppressor genes, or more precisely the proteins encoded by the genes, have a diminishing or inhibitory effect on cell cycle regulation, or promote apoptosis and sometimes both. Tumor suppressor proteins function in several categories, including: (a) Repression of genes essential for cell cycle continuation (If these genes are not expressed, the cell cycle does not continue and cell division is effectively suppressed) (B) Coupling of the cell cycle to DNA damage. As long as there is damaged DNA in the cell, the cell should not divide (if the damage can be repaired, the cell cycle can continue); (c) induction of apoptosis or programmed cell death, repairing the damage If this is not possible, it removes the threat posed by the damage for greater benefit to the organism; and (d) some proteins involved in cell adhesion prevent tumor cell dispersal and contact inhibition It is classified as a protein that is known to be a metastasis inhibitor.

  In contrast, oncogenes are genes that cause cancer. Many cells are usually destined to die. In cancer, these cells survive and grow because of the presence of mutated carcinogenic DNA sequences. Most oncogenes require additional steps, such as mutation of another gene, or environmental factors, such as viral infection, to cause cancer. Since the 1980s, a number of oncogenes have been identified in human cancer. A proto-oncogene is a normal gene that can become an oncogene by mutation or increased expression. The proto-oncogene encodes a protein that helps regulate cell growth and differentiation. Oncogenes are often involved in the execution of signal transduction and mitogenic signals, usually through their protein products. When activated, the proto-oncogene (or its product) becomes a tumor-inducing agent (oncogene). Examples of proto-oncogenes include RAS, WNT, MYC, ERK and TRK.

A. p53
p53 (registration number NM_000546) is a transcription factor that regulates the cell cycle and functions as a tumor suppressor. p53 is important in multicellular organisms because it supports cancer suppression, in part through its role in regulating cellular responses that damage DNA. p53 refers to its role in maintaining stability by preventing genomic mutations, such as “the guardian of the genome”, “the guardian angel gene”, or It has been described as a “master watchman”.

  The name p53 is related to the apparent molecular weight on SDS-PAGE, but in fact it is only 43.7 kD. This difference is due to the large number of amino acid proline residues in the p53 protein that make p53 appear larger by slowing the movement of p53 in SDS-PAGE. This effect is seen in p53 from various species including humans, rodents, frogs and fish. The gene is located on human chromosome 17 (17p13.1) and has five domains: (a) an N-terminal transcription activation domain (TAD) that activates transcription factors (residues 1-42); (b) p53 Proline-rich domain important for apoptotic activity (residues 80-94); central DNA binding core domain (DBD) containing one zinc atom and several arginine amino acids (residues 100-300); homo-oligomerization domain Encodes a 393 amino acid protein with (OD) (residues 307-355); as well as the C-terminus (residues 356-393) involved in the down-regulation of DNA binding in the central domain.

  Mutations that inactivate p53 in cancer usually occur in DBD. Most of these mutations disrupt the ability of the protein to bind to its target DNA sequence and thus prevent transcriptional activation of these genes. Therefore, mutations in DBD are recessive loss-of-function mutations. A molecule of p53 with a mutation in OD dimerizes with wild-type p53, preventing p53 from activating transcription. Therefore, the OD mutation has a dominant inhibitory effect on the function of p53.

p53 has many anticancer mechanisms. For example, p53 can activate DNA repair proteins when DNA is damaged. p53 can also hold the cell cycle at the G ₁ / S regulatory point with DNA damage recognized (if p53 holds the cell long enough here, the DNA repair protein will have time to verify the damage. And the cells will be able to continue the cell cycle). p53 can also initiate apoptosis (programmed cell death) when DNA damage is found to be irreversible.

  p53 is activated in response to a myriad of types of stress, including but not limited to DNA damage (either caused by chemicals such as UV, IR or hydrogen peroxide). Oxidative stress, osmotic shock, ribonucleotide depletion and disordered oncogene expression. This activation is characterized by two major events. First, the half-life of the p53 protein is dramatically increased, resulting in rapid accumulation of p53 in stressed cells. Secondly, structural changes force p53 to assume an active role as a transcriptional regulator in these cells. A critical event leading to p53 activation is phosphorylation of its N-terminal domain. The N-terminal transcriptional activation domain contains multiple phosphorylation sites and can be considered the primary target of protein kinases that transmit stress signals.

  Protein kinases known to target this transcriptional activation domain of p53 can be roughly divided into two groups. Group 1 protein kinases belong to the MAPK family (JNK1-3, ERK1-2, p38 MAPK) and respond to several types of stress such as membrane damage, oxidative stress, osmotic shock, heat shock, etc. It has been known. A second group of protein kinases (ATR, ATM, Chk1, Chk2, DNA-PK, CAK) are molecules that detect and respond to genomic integrity checkpoints, ie some forms of DNA damage caused by genotoxic stress Related to the cascade.

  In unstressed cells, p53 levels are kept low by constant degradation of p53. A protein called Mdm2 binds to p53 and transports p53 from the nucleus to the cytosol where it is degraded by the proteasome. Phosphorylation of the N-terminal end of p53 by the protein kinase described above prevents Mdm2 binding. Subsequently, other proteins such as Pin1 are recruited to p53, causing a structural change in p53 that further prevents Mdm2 binding. A transcriptional coactivator such as p300 or PCAF then acetylates the carboxy terminal end of p53, exposing the DNA binding domain of p53, allowing p53 to activate or repress specific genes.

B. Rb
Retinoblastoma protein (Rb; NM — 00321) is a tumor suppressor protein that has been found to be dysfunctional in several types of cancer. pRb was so named because retinoblastoma cancer occurs when the protein is inactivated by mutations in both alleles of the RB1 gene encoding the protein. Rb normally exists as a phosphoprotein inside the cell and is a target for phosphorylation by several kinases as described below. One well-studied function of Rb is to prevent cell division and cell progression through the cell cycle. Thus, if Rb is ineffective in this role, the mutant cell can continue to divide and can become cancerous.

  Rb is a member of the “pocket protein family” because it has a pocket to which proteins can bind. Oncogenic proteins, such as those produced by cells infected with high-risk human papillomavirus, can bind to Rb and inactivate Rb, which can lead to cancer. . Rb prevents the cell from replicating damaged DNA by preventing the cell from going through the cell cycle and entering the S or synthesis phase, or going through the G1 or first gap phase. Rb binds to and inhibits E2F family transcription factors. The E2F transcription factor is a dimer of E2F protein and DP protein. The transcriptional activation complex of the E2 promoter-binding protein dimerization partner (E2F-DP) can push cells into S phase. As long as E2F-DP is inactivated, the cells remain stagnant in G1 phase. When Rb binds to E2F, the complex acts as a growth inhibitor and prevents progression through the cell cycle. The Rb-E2F / DP complex also attracts histone deacetylase (HDAC) protein to chromatin and further suppresses DNA synthesis.

  In a low phosphorylated state, Rb is active and performs its role as a tumor suppressor by inhibiting cell cycle progression. Rb is inactivated by phosphorylation. Rb is activated near the end of the G1 phase, when one of its residues is dephosphorylated by phosphatase, allowing binding to E2F. During the time the cell enters S phase, a cyclin-dependent kinase (CDK) and cyclin complex phosphorylates pRb and suppresses its activity. Initial phosphorylation is performed by cyclin D / CDK4,6, followed by further phosphorylation by cyclin E / CDK2. pRb maintains a phosphorylated state throughout S phase, G2 phase and M phase. The phosphorylation of pRb allows E2F-DP to dissociate from pRb and have activity. When E2F is released, E2F activates cyclin-dependent kinases to force cells to move through the cell cycle (eg, cyclins E and A), and by helping polymerase bind to DNA. It activates factors such as molecules called proliferating cell nuclear antigen (ie PCNA) that promote DNA replication and repair.

C. ATM
Capillary diastolic ataxia (AT; NM_00051) is an autosomal recessive disease caused by mutations in the ATM gene located on chromosome 11q22-23. The gene was characterized in June 1995 and consists of 66 exons dispersed in genomic DNA over 150 kb. The gene encodes a 13 kb mature transcript with an open reading frame of 9168 nucleotides. The ATM protein is approximately 370 kDa, is ubiquitously expressed, and is localized in the cell nucleus. The ATM protein is a large serine-threonine kinase that is thought to play a role in regulating cell cycle checkpoints, double-stranded DNA repair, and meiosis (similar to the BRCA gene). ATM is also known to play a role in regulating p53, BRCA1 and CHEK2. Some of ATM's role in DNA repair is known to be a role in telomere repair, since telomeres degrade more rapidly in people with AT.

  There are two types of mutations in the ATM gene: (a) a null mutation is a mutation that causes a complete loss of protein function and thus is recessive and causes AT; and (b) function It is believed that there are “missense” mutations that produce stable, sufficiently large proteins with reduced, eg, substitutions, short in-frame insertions and deletions. These mutants act by dominantly interfering with the normal copy of the protein. The vast majority of AT patients (65-70%) have a truncated mutation with a particularly common exon skipping mutation. This results in very low or undetectable levels of ATM protein. Missense mutations are the most common type of mutation found in breast cancer patients. Patients with two missense mutations are thought to have milder AT, which may be the main cause of cases of mild AT.

D. Other Tumor Suppressors A variety of other tumor suppressors can be utilized in the present invention, such as p53, ATM and Rb. For example, BRCA1 and BRCA2 play a critical role in breast cancer progression. BRCA1 is a human gene that maintains the integrity of the genome to prevent random growth. Multifactorial BRCA1 protein products are involved in DNA damage repair, ubiquitination, transcriptional regulation and other functions. Mutations in the gene have been associated with several hereditary cancers, namely breast, ovarian and prostate cancer. The BRCA1 gene is located at base pair 38,449,843 to base pair 38,530,933 in band 21 of the long arm (q) of chromosome 17 (map). BRCA1 protein is directly involved in the repair of damaged DNA. Although its exact role is unknown, it is believed that the BRCA1 protein interacts with RAD51 during repair of DNA double-strand breaks. These cuts can be caused not only by natural radiation or other exposures, but can also occur when chromosomes exchange genetic material during special types of cell division (meiosis) that produce sperm and eggs . By affecting the repair of DNA damage, this protein plays a role in maintaining the stability of the human genome.

  BRCA2 is another human gene involved in the repair of chromosomal damage. Although the structures of the BRCA1 and BRCA2 genes are very different, their functions appear to be similar. Proteins made by both genes are essential for the repair of damaged DNA. The BRCA2 protein binds to and regulates the protein produced by the RAD51 gene to ascertain the break in the DNA. Not only can these cuts be caused by natural and medical radiation or other environmental exposures, but the chromosomes exchange genetic material during a special type of cell division (meiosis) that produces sperm and eggs. Sometimes it can happen. BRCA1 protein also interacts with RAD51 protein. By repairing DNA, these three proteins play a role in maintaining the stability of the human genome. Like BRCA1, BRCA2 probably plays a critical role in embryonic development, possibly regulating the activity of other genes. The BRCA2 gene is located at base pair 31,787,616 to base pair 31,871,804 at site 12.3 (13q12.3) of the long arm (q) of chromosome 13.

  MutS is a major member of a family or protein that supports double-stranded DNA repair mismatches. The first step in this process is the recognition of mismatched DNA. In Escherichia coli, MutS binds to the mismatch site of double-stranded DNA and coordinates with the MutL and MutH proteins to target the portion of the DNA strand to be removed. Other proteins complete the repair process, i.e .: the targeted DNA portion is removed and degraded, a patch is synthesized using the complementary strand as a template, the patch is ligated in place, and there is no mismatch A repair part of double-stranded DNA occurs. Human MutS homologs have received much interest because defects in some of the human MutS homologues are responsible for certain hereditary non-polyposis colorectal cancers (HNPCC) and possibly other colorectal cancers.

  APC (Adenomatosis polyposis coli) is another tumor suppressor associated with colorectal cancer. The factor helps control the frequency of cell division, controls cell adhesion to other cells in the tissue, or controls the movement of cells into or out of the tissue, and in cells that result from cell division It also helps to ensure that the number of chromosomes is appropriate. APC proteins perform the above roles primarily through association with other proteins, particularly those involved in cell adhesion and signal transduction. Specifically, the activity of a protein, β-catenin, is controlled by an APC protein that is part of the Wnt signaling pathway. Regulation of β-catenin prevents genes that stimulate cell division from working too often and prevents cell overgrowth. The APC gene is located between base pairs 112, 118, 468 to base pairs 112, 209, 532 between sites 21 and 22 of the long arm (q) of chromosome 5.

  AT-binding transcription factor 1 (ie, ATBF1) is a tumor suppressor, and its loss is associated with the progression of gastric cancer. The factor is located at 16q22.3-q23.1. The total length of DNA is 261.32 kB. Due to the use of alternative promoters in conjunction with alternative splicing, there are two isoforms ATBF1-A and ATBF1-B. The size of the protein is 3703 amino acids and 404 kDa. The protein contains 4 homeodomains and 23 zinc fingers, 1 pseudo zinc finger motif, 1 DEAD and 1 DEAH box, RNA and ATP binding sites, 2 large molecules It has an RS domain as well as multiple phosphorylation sites. The protein is localized in the nucleus and binds to the AT-rich core sequence of the AFP gene enhancer and acts as a transcription factor that down-regulates AFP gene expression, possibly involved in neuronal differentiation. Amplification in the form of non-syntenic co-amplification with extrachromosomal double microchromosome, myc, in a cell line SJNB-12 derived from the early neural crest. Lack of ATBF1 expression is observed in gastric cancer cell lines that express α-fetoprotein. This lack of ATBF1 expression is not due to mutations, deletions or translocations, but is due to strong suppression of transcriptional levels.

III. Assessment of the structure, expression or function of tumor suppressors A. Nucleic acid based diagnostics One embodiment of the present invention includes a method of detecting variations in the expression of tumor suppressors. The method can include measuring tumor suppressor levels or measuring specific alterations in the expression product. The nucleic acid used is isolated from cancer cells according to standard methods (Sambrook et al., 1989). The nucleic acid may be genomic DNA, fractionated cellular RNA, or whole cell RNA. If RNA is used, it may be desirable to convert the RNA to complementary DNA. In one embodiment, the RNA is whole cell RNA; in another embodiment, it is poly A RNA. Usually, the nucleic acid is amplified.

  Depending on its type, the particular nucleic acid of interest is identified in the sample directly using amplification or using a second known nucleic acid after amplification. The identified product is then detected. In some applications, detection may be performed by visual means (eg, ethidium bromide staining of gels). As another example, detection is by chemiluminescence, radiolabeled radioactive scintigraphy or fluorescent labeling, as well as systems using electrical or thermal shock signals (Affymax Technology; Bellus, 1994). Indirect identification of the product can be included.

  In one embodiment, the amount of tumor suppressor expressed is measured by assessing the amount of mRNA. However, further alterations in activity can be identified by examining various types of structural abnormalities. These abnormalities include deletions, insertions, point mutations and duplications. Point mutations result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are mutations that occur in non-germline tissues and are not heritable, but germline tissue mutations can be inherited. Mutations within and outside the coding region are both produced by altered transcription of the gene or by destabilizing either transcript (mRNA) or protein or otherwise altered processing. May affect the amount of tumor suppressor.

  When one allele of a tumor suppressor gene is inactivated by inheritance of germline lesions or acquisition of somatic mutations, the cells take a genetic step towards oncogenic transformation. Inactivation of the other allele of the gene is usually accompanied by small somatic mutations or chromosomal allelic deletions that result in loss of heterozygosity (LOH). As another example, both copies of the tumor suppressor gene may be lost due to homozygous deletions.

In this regard, a variety of different assays such as, but not limited to, fluorescence in situ hybridization (FISH), DNA direct sequencing, PFGE analysis, Southern or Northern blotting, single strand conformation Analysis (SSCA), RNAse protection assay, allele specific oligonucleotide (ASO), dot blot analysis, denaturing gradient gel electrophoresis, RFLP and PCR ^™ -SSCP are contemplated.

i. Primer and Probe The term primer is intended to encompass any nucleic acid capable of preparing (priming) the synthesis of nascent nucleic acids in a template dependent process, as defined herein. Typically, primers are oligonucleotides 10-20 base pairs in length, although longer sequences can be used. Primers may be provided in double-stranded or single-stranded form, but single-stranded form is preferred. Probes are defined differently but can also serve as primers. The probe is probably priming but is designed to bind to the target DNA or RNA and need not be used in the amplification process. In certain embodiments, the probe or primer uses a radioactive chemical species ( ³² P, ¹⁴ C, ³⁵ S, ³ H or other label) to produce a fluorophore (rhodamine, fluorescein) or chemiluminescence (luciferase). To be labeled.

ii. Template-dependent amplification methods Several template-dependent processes are available to amplify marker sequences present in a given template sample. One of the best known amplification methods is described in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and Innis et al. The polymerase chain reaction (referred to as ^PCRTM ) described in detail in 1990 (each of which is incorporated herein by reference in its entirety).

Briefly, PCR ^™ provides two primer sequences that are complementary to regions on opposite complementary strands of a marker sequence. To the reaction mixture, an excess amount of deoxynucleoside triphosphate is added along with a DNA polymerase (eg, Taq polymerase). If the marker sequence is present in the sample, the primer will bind to the marker and the polymerase will extend the primer along the marker sequence by adding nucleotides. By raising or lowering the temperature of the reaction mixture, the extended primer dissociates from the marker to form a reaction product, excess primer will bind to the marker and reaction product, and the process is repeated.

The reverse transcriptase PCR ^TM amplification procedure can be performed to quantify the amount of amplified mRNA. Methods for reverse transcription of RNA into cDNA are well known and are described in Sambrook et al., 1989. An alternative method of reverse transcription utilizes a thermostable RNA-dependent DNA polymerase. These methods are described in WO90 / 07641 filed on Dec. 21, 1990. Polymerase chain reaction methodologies are well known in the art.

Another amplification method is the ligase chain reaction (“LCR”), which is disclosed in European Patent Office (EPO) 320308, which is hereby incorporated by reference in its entirety. In LCR, two sets of complementary probe pairs are prepared, and in the presence of the target sequence, each pair binds to the opposite complementary strand of the target so as to contact each other. In the presence of ligase, the two probe pairs will be linked to form a single unit. Through temperature cycling, like the PCR ^™ , the bound ligation unit dissociates from the target and then serves as a “target sequence” for the ligation reaction of excess probe pairs. US Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

  Qβ replicase described in International Application No. PCT / US87 / 00880 based on the Patent Cooperation Treaty may be used as still another amplification method in the present invention. In this method, a replicating sequence of RNA having a region complementary to the target sequence is added to the sample in the presence of RNA polymerase. The polymerase will copy this replicating sequence, which can then be detected.

  An isothermal amplification method in which amplification of a target molecule containing a nucleotide 5 ′-[α-thio] -triphosphate in one strand of a restriction enzyme cleavage site using restriction endonuclease and ligase is also performed in the present invention. It may be useful for nucleic acid amplification (Walker et al., 1992).

  Strand displacement amplification (SDA) is another method for performing isothermal amplification of nucleic acids with multiple rounds of strand displacement and synthesis (ie, nick translation). A similar method, called Repair Chain Reaction (RCR), anneals several probes across the targeted region of amplification and there are only two of the four bases thereafter With a repair reaction. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3 'and 5' sequences of non-specific DNA and a central sequence of specific RNA is hybridized to DNA present in the sample. Upon hybridization, the reaction is treated with RNase H, and the product of the probe is identified as a unique product that is released after digestion. The original template is annealed to another cycling probe and the reaction is repeated.

Further amplification methods described in United Kingdom Patent Application No. 2202328 and International Application No. PCT / US89 / 01025 under the Patent Cooperation Treaty, each of which is incorporated herein by reference in its entirety, are disclosed herein. May be used according to In the former application, “modified” primers are used in template-dependent and enzyme-dependent synthesis such as PCR ^™ . The primer can be modified by labeling with at least one of a capture moiety (eg biotin) or a detection moiety (eg enzyme). In the latter application, an excess amount of labeled probe is added to the sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released in its original state and bound by excess probe. Cleavage of the labeled probe indicates the presence of the target sequence.

  Other nucleic acid amplification techniques include transcription-based amplification systems (TAS), such as nucleic acid sequence-based amplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., International Publications). Publication No. 88/10315 pamphlet, which is incorporated herein by reference in its entirety. In NASBA, nucleic acids are prepared for amplification by standard phenol / chloroform extraction, heat denaturation of clinical samples, treatment with lysis buffers and minispin columns for DNA and RNA isolation, or RNA extraction with guanidine chloride. be able to. These amplification techniques involve annealing a primer having a target specific sequence. Following polymerization, DNA / RNA hybrids are digested with RNase H, while double-stranded DNA molecules are heat denatured. In either case, the single stranded DNA becomes completely double stranded by the addition of a second target specific primer, followed by polymerization. The double stranded DNA molecule is then transcribed in a complex manner with an RNA polymerase such as T7 or SP6. In an isothermal cycle reaction, RNA is reverse transcribed into single stranded DNA, which is then converted to double stranded DNA and then transcribed again with an RNA polymerase such as T7 or SP6. The resulting product, whether truncated or full length, exhibits a target specific sequence.

  Davey et al., European Patent Office (EPO) No. 329822 (incorporated herein by reference in its entirety) cycles synthesize single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA). A nucleic acid amplification method involving the same, which may be used in accordance with the present invention. ssRNA is a template for the first primer oligonucleotide that is extended by reverse transcriptase (RNA-dependent DNA polymerase). RNA is then removed from the DNA: RNA duplex produced by the action of ribonuclease H (RNase H, RNase specific for RNA in the duplex with DNA or RNA). The resulting ssDNA is a template for the second primer, which further comprises an RNA polymerase promoter (eg, T7 RNA polymerase) sequence 5 'to the portion homologous to the template. This primer is then extended by a DNA polymerase (eg, a large “Klenow” fragment of E. coli DNA polymerase I) so that it has a sequence identical to that of the original RNA between the primer and even one end Yields a double-stranded DNA (“dsDNA”) molecule having a promoter sequence. This promoter sequence can be used by an appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle resulting in very rapid amplification. If the enzyme is selected appropriately, this amplification can be performed isothermally without adding the enzyme in each cycle. Because of the cyclic nature of this process, the initial sequence can be selected as either DNA or RNA form.

Miller et al., International Publication No. WO 89/06700 (incorporated herein by reference in its entirety) is a nucleic acid sequence amplification scheme in which promoter / primer sequences to target single-stranded DNA (“ssDNA”) Discloses a scheme based on the subsequent hybridization and subsequent transcription of multiple RNA copies of the sequence. This scheme is not cyclic, ie no new template is produced from the resulting RNA transcript. Other amplification methods include “RACE” and “one-sided PCR ^™ ”. (Frohman, 1990; Ohara et al., 1989; each incorporated herein by reference in its entirety).

  A method based on amplifying the di-oligonucleotide by ligating two (or more) oligonucleotides in the presence of the nucleic acid having the sequence of the “di-oligonucleotide” produced is the amplification of the present invention. It can also be used for steps. Wu et al., 1989, the entirety of which is incorporated herein by reference.

iii. Southern / Northern blotting Blotting techniques are well known to those skilled in the art. Southern blotting involves the use of DNA as a target, while northern blotting involves the use of RNA as a target. Although each provides a different class of information, cDNA blotting is similar in many ways to blotting RNA species.

  Briefly, probes are used to target DNA or RNA molecular species immobilized on a suitable matrix, often a nitrocellulose filter. Various molecular species must be spatially separated to facilitate analysis. This is often accomplished by gel electrophoresis of nucleic acid species and subsequent “blotting” to filters.

  The blotted target is then incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Since the probe is designed to base pair with the target, the probe will bind to a portion of the target sequence under reconstitution conditions. The unbound probe is then removed and detection is performed as described above.

iv. Separation Methods It is usually desirable to separate the amplification product from the template and excess primer at one or another stage to determine if specific amplification has occurred. In one embodiment, amplification products are separated by agarose gel electrophoresis, agarose-acrylamide gel electrophoresis or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al., 1989.

  As another example, chromatographic techniques may be used to perform the separation. There are many types of chromatographic methods that can be used in the present invention: adsorption chromatography, partition chromatography, ion exchange chromatography and molecular sieve chromatography, as well as many specialized techniques for using them, For example, column chromatography, paper chromatography, thin layer chromatography and gas chromatography (Freifelder, 1982).

v. Detection method The product may be visualized to confirm amplification of the marker sequence. One typical visualization method involves staining of a gel with ethidium bromide and visualization under ultraviolet light. As another example, if the amplification product is totally labeled with radiolabeled or fluorescently labeled nucleotides, the amplification product may be exposed to X-ray film after separation or under an appropriate stimulation spectrum. It may be visualized.

  In one embodiment, visualization is achieved indirectly. After separation of the amplification products, the labeled nucleic acid probe is brought into contact with the amplified marker sequence. The probe is preferably conjugated to a chromophore, but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.

  In one embodiment, detection is performed by a labeled probe. Related techniques are well known to those skilled in the art and can be found in many standard books on molecular protocols. See Sambrook et al., 1989. For example, targets are identified during or after amplification by chromophores or radiolabeled probes or primers.

  An example of the above is described in US Pat. No. 5,279,721, incorporated herein by reference, which discloses an apparatus and method for automated nucleic acid electrophoresis and transcription. ing. This device allows electrophoresis and blotting without external manipulation of the gel and is ideally suited for carrying out the method according to the invention.

  In addition, the amplification products described above can be subjected to sequence analysis to identify specific types of mutations using standard sequence analysis techniques. In some methods, exhaustive analysis of genes is performed by sequence analysis using primer sets designed for optimal base sequencing (Pignon et al., 1994). The present invention provides a method that can use some or all of this type of analysis. Using the sequences disclosed herein, oligonucleotide primers can be designed to allow amplification of the sequences, which can then be analyzed directly by sequencing.

vi. Kit Components All essential materials and reagents necessary for detection and sequencing of the gene of interest can be collected together in a kit. This will generally include preselected primers and probes. Enzymes suitable for nucleic acid amplification such as various polymerases (RT, Taq, Sequenase ^™, etc.), deoxyribonucleotides and buffers may also be included to provide the reaction mixture necessary for amplification. Such kits will generally also include separate containers for each individual reagent and enzyme, and for each primer or probe, by any suitable means.

vii. Relative Quantitative RT-PCR ^TM Design and Theoretical Considerations Using reverse transcription (RT) of RNA to cDNA and subsequent relative quantitative PCR ^TM (RT-PCR ^TM ) to identify specific mRNA molecular species isolated from patients. Relative concentration can be measured. By measuring that the concentration of a specific mRNA molecular species changes, it is shown that the gene encoding the specific mRNA molecular species is differentially expressed.

In PCR ^™ , the number of target DNA molecules amplified is approximately doubled every reaction cycle until some reagent reaches its limit. Thereafter, the amplification rate gradually decreases until there is no increase in the target amplified between cycles. When a graph is drawn with the number of cycles on the X-axis and the logarithm of the concentration of the amplified target DNA on the Y-axis, a characteristic curve is formed by connecting the plotted points. Starting from the first cycle, the slope of this line is positive and constant. This is said to be the straight part of the curve. After a reagent reaches its limit, the slope of the line begins to decrease and eventually becomes zero. At this point, the concentration of the amplified target DNA is asymptotic to a fixed value. This is said to be the plateau part of the curve.

The concentration of target DNA in the linear part of PCR ^TM amplification is directly proportional to the starting concentration of the target before the reaction begins. Measure the relative concentration of a specific target sequence in the original DNA mixture by measuring the concentration of the target DNA amplification product in the PCR ^TM reaction that has completed the same number of cycles and is in its linear range It is. If the DNA mixture is cDNA synthesized from RNA isolated from various tissues or cells, the relative abundance of the specific mRNA from which the target sequence was obtained can be determined for each tissue or cell. This direct proportional relationship between the PCR ^TM product concentration and relative mRNA abundance is only true in the linear region of the PCR ^TM reaction.

The final concentration of target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mixture and is independent of the original concentration of target DNA. Thus, the first condition that must be met before the relative abundance of mRNA species can be measured by RT-PCR ^TM on a collection of RNA populations is when the PCR ^TM reaction is in the linear part of the curve This means that the concentration of amplified PCR ^TM product must be sampled.

A second condition that must be met for RT-PCR ^™ experiments in order to successfully measure the relative abundance of a particular mRNA molecular species is that the relative concentration of amplifiable cDNA is normalized to some independent standard It must be done. The purpose of the RT-PCR ^TM experiment is to measure the abundance of a particular mRNA molecular species relative to the average abundance of all mRNA molecular species in the sample.

Most protocols for competitive PCR ^™ utilize an internal PCR ^™ standard that is present in approximately the same amount as the target. This strategy is effective when the product of PCR ^TM amplification is sampled during its linear step. If the product is sampled as the reaction is approaching the plateau stage, less product will be shown in relative excess. The relative abundance comparisons made for many different RNA samples are distorted in such a way that the difference in relative abundance of RNA appears to be less than it actually is, as is the case when examining RNA samples for differential expression. I will become This is not a serious problem if the internal standard is much larger than the target. If the internal standard is larger than the target, a direct linear comparison between RNA samples can be performed.

The above discussion describes the theoretical considerations of the RT-PCR ^TM assay for clinically derived materials. The problems inherent in clinical samples are that their quantity is variable (makes normalization difficult) and their quality is variable (preferably simultaneous amplification of a reliable internal control of a size larger than the target) Is required). RT-PCR ^TM is a relative quantitative RT-PCR ^TM that uses an internal standard, wherein the internal standard is an amplifiable cDNA fragment larger than the target cDNA fragment, and the abundance of mRNA encoding the internal standard All of the above problems are overcome when is performed as RT-PCR ^TM approximately 5-100 times more than the mRNA encoding the target. This assay measures the relative abundance, not the absolute abundance of each mRNA molecular species.

Other studies can be performed using a more conventional relative quantitative RT-PCR ^TM assay using an external standard protocol. In this assay, the PCR ^TM product is sampled in the linear portion of its amplification curve. The optimal number of PCR ^TM cycles for sampling must be determined experimentally for each target cDNA fragment. In addition, the reverse transcriptase product of each RNA population isolated from various tissue samples must be carefully normalized to an equal concentration of amplifiable cDNA. The above considerations are very important since the absolute abundance of mRNA is measured in this assay. The absolute abundance of mRNA can be used as a measure of differential gene expression only in normalized samples. Although experimental determination of the linear range of the amplification curve and normalization of cDNA preparations is a tedious and time-consuming sequence, the RT-PCR ^TM assay results obtained are relative quantitative RT- It can be superior to results derived from the PCR ^TM assay.

One reason for this advantage is that without an internal standard / competitor, all of the reagents are converted to a single PCR ^TM product in the linear region of the amplification curve, thus increasing the sensitivity of the assay. . Another reason is that if there is only one PCR ^TM product, the display of the product in an electrophoresis gel or another display method is simpler, the background is lower, and it is easier to interpret. .

viii. Chip Technology Particularly contemplated by the present inventors are chip-based DNA technologies such as those described in Hasia et al. (1996) and Shoemaker et al. (1996). . Briefly, these techniques involve quantitative methods for the rapid and accurate analysis of a large number of genes. Using chip technology to separate the target molecules as a high-density array by tagging genes with oligonucleotides or using an array of fixed probes, and screening these molecules based on hybridization It is possible. See also Pease et al. (1994); Fodor et al. (1991).

B. Immunodiagnosis The antibodies of the present invention can be used in characterizing tumor suppressor content of healthy and diseased tissues through techniques such as ELISA and Western blotting. This is a screening for the presence of malignant lesions or as a predictor of future cancer or as a predictor in current cases as a strategy to predict possible responses to oncolytic virus exposure. Can be provided.

  The use of the antibodies of the present invention in an ELISA assay is contemplated. For example, an anti-tumor suppressor antibody is immobilized on a selected surface, preferably a surface exhibiting protein affinity, such as a well of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, the wells of the assay plate are antigenically neutral with respect to the test antiserum, such as a solution of bovine serum albumin (BSA), casein, or milk powder. It is desirable to bind to or coat with a non-specific protein known to be. This allows blocking of non-specific adsorption sites on the immobilized surface, thus reducing the background caused by non-specific binding of antigen to the surface.

  After binding of the antibody to the well, coating with non-reactive material to reduce background, and washing to remove unbound material, the immobilization surface becomes immune complex (antigen / antibody ) It comes into contact with the test sample in a way that leads to formation.

  After formation of a specific immune complex between the test sample and the immobilized antibody, and subsequent washing, the second antibody has specificity for a tumor suppressor different from the first antibody. It is possible to measure even the occurrence and amount of immune complex formation. Suitable conditions preferably include diluting the sample with diluents such as BSA, bovine gamma globulin (BGG), and phosphate buffered saline (PBS) / Tween®. These additives tend to help reduce non-specific background. The laminated antiserum is then incubated for about 2 to about 4 hours, preferably at a temperature on the order of about 25 ° C to about 27 ° C. Following incubation, the surface that has been contacted with the antiserum is washed to remove material that has not formed immune complexes. Preferred washing procedures include washing with a solution such as PBS / Tween® or borate buffer.

  In order to provide a detection means, the second antibody will preferably be bound to an enzyme that develops color when incubated with a suitable chromogenic substrate. Thus, for example, incubating the surface to which the second antibody is bound in contact with anti-human IgG conjugated with urease or peroxidase for a time and under conditions that aid in the formation of immune complexes (eg, PBS / Incubation for 2 hours at room temperature in a PBS-containing solution such as Tween® would be desirable.

After incubation with a second enzyme-labeled antibody and following washing to remove unbound material, with chromogenic substrates, for example with urea and bromocresol purple, or when using peroxidase as an enzyme label Is quantified by incubating with 2,2′-azino-di- (3-ethyl-benzthiazoline) -6-sulfonic acid (ABTS) and H ₂ O ₂ . Quantification is then achieved, for example, by measuring the degree of color development using a visible spectrum spectrophotometer.

  The foregoing scheme may be modified by first binding the sample to the assay plate. The primary antibody is then incubated with the assay plate, followed by detection of bound primary antibody using a labeled secondary antibody having specificity for the primary antibody.

  The antibody composition of the present invention is considered to be very useful in immunoblot analysis or Western blot analysis. The antibody can be used as a high affinity primary reagent to identify proteins immobilized on a solid support matrix such as nitrocellulose, nylon, or combinations thereof. Use these as a single-step reagent for use in the detection of antigens, in conjunction with immunoprecipitation followed by gel electrophoresis, where secondary reagents used for the detection of antigens cause adverse background. Can do. Immunologically based detection methods for use with Western blotting include enzyme-labeled, radiolabeled, or fluorescently labeled secondary antibodies against tumor suppressors and are considered particularly useful in this regard.

IV. Anti-hyperproliferative treatment Hyperproliferative diseases are a group of diseases characterized by abnormal or otherwise unlimited cell proliferation. These diseases generally fall into two categories: more typical cancers, ie “malignant” diseases, and less common benign hyperproliferative diseases such as benign prostatic hyperplasia, benign epithelial hyperplasia of the breast. It is classified into formation, endometrial hyperplasia, thyroid hyperplasia and epithelial hyperplasia of the skin or dermis. Many aspects of the discussion below focus on cancer treatment, but it should be understood that the same approach is equally applicable to benign illnesses where appropriate.

  In contrast to benign diseases, which are typically abnormal only in cell numbers, cancer is cancerous, dysplastic, neoplastic, or otherwise inappropriately growing. Refers to the presence of a cell and / or transformed cell in a subject, such as a neoplastic cell, a tumor cell, a cell that does not exhibit contact inhibition or a tumorigenically transformed cell (eg, melanoma) , Carcinoma such as adenocarcinoma, squamous cell carcinoma, small cell carcinoma, oat cell carcinoma, sarcoma such as fibrosarcoma, chondrosarcoma, osteosarcoma, liver cancer, neuroblastoma, melanoma Hematopoietic malignancies such as lymphoma, leukemia, myeloma, etc.), which are well known in the art and have established criteria for their diagnosis and classification. Without being bound by any theory, the oncolytic properties of reovirus are as defined herein for viral tropism and sensitive intracellular environment for malignant transformed cells. In addition, this may result from a response to impaired PKR phosphorylation in Ras activated cells, as described in the art, or to a defective function of tumor suppressors.

A. Oncolytic viral therapy Certain methods regarding the production and use of wild-type reovirus in the treatment of cancer have been described (eg, US Pat. Nos. 7,300,650, 7,163,678, ibid). No. 7,049,127, No. 7,014,847, No. 6,994,858, No. 6,811,775, No. 6,703,232, No. 6,576, No. 234, No. 6,565,831, No. 6,528,305, No. 6,455,038, No. 6,344,195, No. 6,261,555, No. 6,136,307 and 6,110,461, as well as U.S. Patent Application Publication Nos. 2006/0165724, 2006/0073166, 2005/0123513, 2004/0265. No. 271, No. 2004/0126869, No. 2004/0109878, No. 2002/0037543, No. 2006/0029598, No. 2005/0026289, No. 2002/0006398 and No. 2001/0048919. Specification, each of which is incorporated herein by reference in its entirety without waiver). Mixoma virus therapy is described in US Patent Application Publication No. 2006/0263333, also incorporated by reference.

B. Purge Therapy The present invention, in one embodiment, provides a method for removing (purging) cancer cells from a cell population. For example, patients with hematopoietic or solid cancer treated with myeloablative therapy will then receive hematopoietic stem cell relief using purged bone marrow tissue collected from the patient prior to chemotherapy There is a case. In these embodiments, the bone marrow can be treated with oncolytic viruses to remove or kill neoplastic cells while reducing or eliminating hematopoietic stem cell damage.

  By using the patient's own adult stem cells for cell therapy, problems such as host immunity, graft-versus-host disease and ethical issues may be avoided. Therefore, human adult stem cells have been widely evaluated for a variety of tissue regeneration therapies. However, it has been reported that spontaneous transformation may occur during in vitro culture growth of adult stem cells (Tolar et al., 2007; Romano, 2005). Therefore, there is a need for an appropriate purge strategy to eliminate naturally transformed cells present in cultured grown adult stem cells for safer use in transplantation and tissue regeneration. Wild-type reoviruses have shown utility in the removal of cancer cells present in in vitro autologous hematopoietic stem cell populations (Tirukukumaran et al., 2003; Thirukukumaran et al., 2005). It is also conceivable to use attenuated reovirus and mixoma virus in a similar manner. We anticipate that the present invention can be used with virtually any adult or embryonic stem cell line that is currently known or discovered in the future.

  In certain embodiments, it may be desirable to remove the reovirus from the cell composition, for example after a time sufficient to allow killing of the neoplastic cells (if present). For example, bone marrow may be harvested from a patient and treated with reovirus to destroy neoplastic cells; from the composition before reinjecting the bone marrow into a patient (eg, an immunocompromised cancer patient) It may be desirable to destroy, eliminate and / or remove viruses. In these embodiments, an antiviral agent may be added to the cell composition. These antiviral drugs are successfully released from anti-reovirus antibodies and complements, inhibitors of important viral enzymes, such as RNA-dependent RNA polymerase inhibitors, or viral packaging, assembly, or infected cells The agent which interferes with this is mentioned.

  Additional purification steps, such as cell washing and / or centrifugation, may be used to further remove virus and / or dead cells from the cell composition. These methods are well known in the art and will enrich for the desired type of cells, eg, fluorescence activated cell sorting or adhesion that allows positive selection of desired cells such as stem cell populations. There is a method based on sex.

C. Combination therapy To increase the effectiveness of selective killing of cancer cells by oncolytic viruses, in certain embodiments, the cells are treated with additional anticancer agents, such as chemotherapeutic agents, radiation therapy, immunotherapy, hormones. It may be desirable to administer therapy, toxin treatment, treatment with another natural or recombinant virus, or gene therapy.

  Chemotherapeutic agents include, but are not limited to, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding action Drug, etoposide (VP16), farnesyl-protein transferase inhibitor, gemcitabine, ifosfamide, mechloretamine, melphalan, mitomycin, navelbine, nitrosourea, pricomycin, procarbazine, raloxifene, tamoxifen, taxolite, taxol temazolomide (aqueous form of DTIC), transplatinu (transplatinu) ), Vinblastine and methotrexate, vincristine, or any analog or derivative thereof, and the like. These agents or drugs are classified according to the mode of action in the cell, for example, whether the agent or drug affects the cell cycle and at what stage it affects the cell cycle. As another example, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate with DNA, or to cause chromosomal and mitotic abnormalities by affecting nucleic acid synthesis. Good. Most chemotherapeutic agents have the following categories: alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormonal agents, mixed agonists, and these Classified as any analog or derivative.

  Oncolytic therapy may be concurrent with the application of other agents to the cancer cells, or may follow or precede the application of other agents with an interval of minutes to weeks. For example, in such cases, it is contemplated that cancer cells, tissues, organs or organisms are given multiple treatments at about the same time as the attenuated reovirus (ie, within less than about 1 minute). In other embodiments, the one or more agents are about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about at least either before or after administration of the attenuated or wild type reovirus. 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours About 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours About 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 4 hours About 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, About 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 Within about a day, about 20 days, about 21 days, about 1, about 2, about 3, about 4, about 5, about 6, about 7 or about 8 weeks or more, and any range of time derivable therein, or It may be administered at about the same time.

Various combination regimens of attenuated or wild-type reovirus and one or more agents can be used. A non-limiting example of such a combination is shown below, in which the oncolytic virus is “A” and the anticancer agent is “B”:
A / B / A B / A / B B / B / A A / A / B A / B / B
B / A / A A / B / B / B B / A / B / B B / B / B / A
B / B / A / B A / A / B / B A / B / A / B A / B / B / A
B / B / A / A B / A / B / A B / A / A / B A / A / A / B
B / A / A / A A / B / A / A A / A / B / A
i. Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with the present invention. The term “chemotherapy” refers to the use of drugs to treat cancer. A “chemotherapeutic agent” is used to encapsulate a compound or composition that is administered in the treatment of cancer. These agents or drugs are classified according to the mode of action in the cell, for example, whether the agent or drug affects the cell cycle and at what stage it affects the cell cycle. As another example, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate with DNA, or to cause chromosomal and mitotic abnormalities by affecting nucleic acid synthesis. Good. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosourea.

  [Alkylating agent] An alkylating agent is a drug that directly interacts with genomic DNA to prevent cancer cells from proliferating. This category of chemotherapeutic agents represents agents that affect all phases of the cell cycle, ie not phase specific. Alkylating agents can be applied to treat chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, and certain breast, lung, and ovarian cancers. Alkylating agents include: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan (R)), dacarbazine, ifosfamide, mechlorethamine (musstargen (R)) and melphalan. Troglitazaone can be used to treat cancer in combination with any one or more of these alkylating agents, some of which are discussed below. The

  Busulfan (also known as myleran®) is a bifunctional alkylating agent. Busulfan is chemically known as 1,4-butanediol dimethanesulfonate.

  Busulfan is not a structural analog of nitrogen mustard. Busulfan is available in tablet form for oral administration. Each scored tablet contains 2 mg busulfan and the inactive ingredients magnesium stearate and sodium chloride.

  Busulfan is indicated for palliative treatment of chronic myeloid (myeloid, myelocytic, granulocytic) leukemia. Although not curative, busulfan reduces total granulocyte mass, reduces the symptoms of the disease, and improves the patient's clinical status. Approximately 90% of adults with chronic myelogenous leukemia who have not been previously treated will obtain hematological remission with regression or stabilization of organomegaly after use of busulfan. Busulfan has been shown to be superior to spleen radiotherapy in maintaining survival and hemoglobin levels, and equivalent to radiotherapy in controlling splenomegaly.

  Chlorambucil (also known as leukeran®) is a nitrogen mustard type bifunctional alkylating agent that has been found active against certain human neoplastic diseases. Chlorambucil is chemically known as 4- [bis (2-chloroethyl) amino] benzenebutanoic acid.

Chlorambucil is available in tablet form for oral administration. Chlorambucil is rapidly and completely absorbed from the gastrointestinal tract. After a single oral dose of 0.6-1.2 mg / kg, maximum plasma chlorambucil levels are reached within 1 hour, and the terminal half-life of the original drug is estimated at 1.5 hours. 0.1-0.2 mg / kg / day or 3-6 mg / m < ² > / day or alternatively 0.4 mg / kg can be used for antineoplastic therapy. Treatment plans are well known to those skilled in the art and can be found in “Physicians Desk Reference” and “Remington's Pharmaceutical Sciences” referenced herein.

  Chlorambucil is indicated in the treatment of chronic lymphocytic (lymphocyte) leukemia, malignant lymphoma such as lymphosarcoma, giant follicular lymphoma and Hodgkin's disease. Chlorambucil is not curative in any of the above diseases, but may provide clinically useful relief. Therefore, it can be used in combination with troglitazone in the treatment of cancer.

Cisplatin is widely used to treat cancers such as metastatic testicular or ovarian cancer, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Have been used. Cisplatin can be used with effective doses used in clinical applications of 15-20 mg / m ² for 5 days every 3 weeks for a total of 3 courses, either alone or in combination with other agonists. Typical ^{dose, 0.50mg / m 2, 1.0mg /} m 2, 1.50mg / m 2, 1.75mg / m 2, 2.0mg / m 2, 3.0mg / m 2, 4. It may be 0 mg / m ² , 5.0 mg / m ² , 10 mg // m ² . Of course, all of the above doses are exemplary, and any dose between the above values is expected to be useful in the present invention.

Cisplatin is not absorbed orally and must therefore be delivered by intravenous, subcutaneous, intratumoral or intraperitoneal injection.
Cyclophosphamide is 2H-1,3,2-oxazaphosphorin-2-amine, N, N-bis (2-chloroethyl) tetrahydro-, 2-oxide, monohydrate; Cytoxan (R ) (Available from Mead Johnson); and Neosar® (available from Adria). Cyclophosphamide can be obtained by reacting 3-amino-1-propanol with N, N-bis (2-chloroethyl) phosphoramidic acid dichloride [(ClCH ₂ CH ₂ ) ₂ N-POCl ₂ under the action of triethylamine in a dioxane solution. ] And condensing together. This condensation is a double condensation and involves both hydroxyl and amino groups and thus cyclizes.

  Cyclophosphamide, unlike other β-chloroethylaminoalkylating agents, does not readily cyclize to the active ethyleneimonium form until activated by liver enzymes. Therefore, this substance is stable in the gastrointestinal tract, well tolerated and effective by oral and parenteral routes and does not cause local rash, necrosis, phlebitis or even pain.

Suitable doses for adults are orally 1-5 mg / kg / day (usually in combination); or 1-2 mg / kg / day depending on gastrointestinal tolerability; 50 mg / kg in divided doses for 2-5 days, 10-15 mg / kg every 7-10 days, or 3-5 mg / kg twice a week, or 1.5-3 mg / kg / day . A dose of 250 mg / kg / day may be administered as an antineoplastic agent. The intravenous route is preferred for administration due to gastrointestinal side effects. During maintenance, a total white blood cell count of 3000-4000 / mm ³ is usually desirable. The drug may optionally be administered intramuscularly, infiltrated, or administered into a body cavity. The drug is available in 100, 200, and 500 mg injectable dosage forms, as well as 25 mg and 50 mg tablets, and details of dosages for administration can be found by those skilled in the art as “Remington” incorporated herein by reference. See 's Pharmaceutical Sciences' 15th edition, chapter 61.

  Melphalan, also known as alkeran (R), L-phenylalanine mustard, phenylalanine mustard, L-PAM, or L-sarcolicine, is a phenylalanine derivative of nitrogen mustard. Melphalan is a bifunctional alkylating agent that has activity against certain human neoplastic diseases. Melphalan is chemically known as 4- [bis (2-chloroethyl) amino] -L-phenylalanine.

Melphalan is the L-isomer with the activity of the above compound, first synthesized in 1953 by Bergel and Stock; the D-isomer known as medphalan is an animal tumor The dose required to produce a chromosomal effect is greater than that required for the L-isomer. The racemic (DL-) form is known as melphalan or sarcolicin. Melphalan is insoluble in water and has a pKa ₁ of ~ 2.1. Melphalan is available in tablet form for oral administration and has been used to treat multiple myeloma.

Effective evidence suggests that about one-third to one-half of multiple myeloma patients show a favorable response to oral administration of the drug.
Melphalan has been used to treat epithelial ovarian cancer. In one regimen commonly used for the treatment of ovarian cancer, melphalan has been administered as a course at 0.2 mg / kg daily for 5 days. Depending on the hematological tolerance, the course repeats every 4-5 weeks (Smith and Rutledge, 1975; Young et al., 1978). As another example, the dose of melphalan used can be as low as 0.05 mg / kg / day or as high as 3 mg / kg / day, and any dose between these doses can be used at these doses. Dosages above may be used. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will in any case determine the appropriate dose for the individual patient.

  Antimetabolites Antimetabolites interfere with DNA and RNA synthesis. Unlike alkylating agents, antimetabolites specifically affect the cell cycle during the S phase. The agent has been used to treat chronic leukemia in addition to breast, ovarian and gastrointestinal tumors. Antimetabolites include 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.

  5-Fluorouracil (5-FU) has the chemical name 5-fluoro-2,4 (1H, 3H) -pyrimidinedione. The mechanism of action is thought to be by blocking the methylation reaction of deoxyuridylic acid to thymidylic acid. Thus, 5-FU prevents deoxyribonucleic acid (DNA) synthesis and, to a lesser extent, ribonucleic acid (RNA) formation. Since DNA and RNA are essential for cell division and proliferation, it is thought that the action of 5-FU is to cause thymidine deficiency that leads to cell death. Thus, the action of 5-FU is found in rapidly dividing cells that are characteristic of metastatic cancer.

Antitumor antibiotics Antitumor antibiotics have both antimicrobial activity and cytotoxicity. The drug further interferes with DNA by chemically inhibiting enzymes and mitosis and altering cell membranes. Since the agonist is not cell cycle specific, it acts in all phases of the cell cycle. Therefore, the agonist is widely used for various cancers. Examples of antitumor antibiotics include bleomycin, dactinomycin, daunorubicin, doxorubicin (adriamycin), and idarubicin, some of which are discussed in detail below. Is widely used in a clinical setting for the treatment of neoplasms, administration of these compounds, the intravenous bolus injection of 21 day intervals at doses ranging 25 to 75 mg / ^{m 2} for adriamycin, the etoposide 35 to 100 mg / ^{m 2} Extends to intravenous or oral administration.

  Doxorubicin hydrochloride, ie, 5,12-naphthacenedione, (8s-cis) -10-[(3-amino-2,3,6-trideoxy-aL-lyxo-hexopyranosyl) oxy] -7,8,9 , 10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-hydrochloride (hydroxydaunorubicin hydrochloride, adriamycin) is used in a broad anti-neoplastic spectrum. The drug binds to DNA and inhibits nucleic acid synthesis, inhibits mitosis and promotes chromosomal abnormalities.

  When administered alone, the drug is the first-line drug used for the treatment of goiter and primary hepatocellular carcinoma. The drugs include ovarian tumors, endometrial and breast tumors, bronchogenic oat cell carcinoma, non-small cell lung cancer, gastric adenocarcinoma, retinoblastoma, neuroblastoma, mycosis fungoides, Pancreatic cancer, prostate cancer, bladder cancer, myeloma, diffuse histiocytic lymphoma, Wilms tumor, Hodgkin's disease, adrenal tumor, osteogenic sarcoma soft tissue sarcoma, Ewing sarcoma, rhabdomyosarcoma and acute lymphoid It is a component of 31 first-line combination therapies used to treat leukemia. The drug is an alternative drug used for the treatment of islet cell cancer, cervical cancer, testicular cancer and adrenocortical cancer. The drug is also an immunosuppressant.

  Doxorubicin is difficult to absorb and must be administered intravenously. Pharmacokinetics are multi-compartmental. The distributed phase has a half-life of 12 minutes and 3.3 hours. The elimination half-life is about 30 hours. 40-50% is secreted into the bile. Most of the rest is metabolized in the liver and partly becomes the active metabolite (doxorubicinol), but a few percent is excreted in the urine. If liver dysfunction is present, the dose should be reduced.

Appropriate doses are intravenous administration, adults, 60-75 mg / m ² as 21-day intervals, or 25-30 mg / m ² or once a week for 2 consecutive days or 3 days, repeating every 3 or 4 weeks. 20 mg / m ² . Lower doses should be used in older patients if there is already myelosuppression caused by prior chemotherapy or neoplastic bone marrow infiltration, or if the drug is used in combination with other myelopoietic agents. is there. If serum bilirubin is 1.2-3 mg / dL, the dose should be reduced by 50% and if it exceeds 3 mg / dL, it should be reduced by 75%. The total lifetime dose should not exceed 550 mg / m ^{2 for} patients with normal cardiac function and 400 mg / m ² for those who received mediastinal radiation. Another example is 30 mg / m ² for 3 consecutive days, repeated every 4 weeks. Typical ^{dose, 10mg / m 2, 20mg /} m 2, 30mg / m 2, 50mg / m 2, 100mg / m 2, 150mg / m 2, 175mg / m 2, 200mg / m 2, 225mg / m 2 ^{, 250mg / m 2, 275mg /} m 2, 300mg / m 2, 350mg / m 2, 400mg / m 2, 425mg / m 2, 450mg / m 2, 475mg / m 2, may be 500 mg / ^{m 2.} Of course, these doses are all exemplary and any dose between the above values is expected to be useful in the present invention.

  Daunorubicin hydrochloride, ie 5,12-naphthacenedione, (8S-cis) -8-acetyl-10-[(3-amino-2,3,6-trideoxy-aL-lyxo-hexanopyranosyl) Oxy] -7,8,9,10-tetrahydro-6,8,11-trihydroxy-10-methoxy-hydrochloride; also named cerbidine (R) and available from Wyeth. Daunorubicin intercalates with DNA, inhibits DNA-dependent RNA polymerase, and suppresses DNA synthesis. Daunorubicin can suppress cell division at doses that do not interfere with nucleic acid synthesis.

  In combination with other drugs, daunorubicin is included in first-line chemotherapy for adult acute myeloid leukemia (to induce remission), acute lymphoblastic leukemia, and acute chronic myeloid leukemia. Poor oral absorption and must be administered intravenously. The distribution half-life is 45 minutes and the elimination half-life is about 19 hours. Its active metabolite, daunorubicinol, has a half-life of about 27 hours. Daunorubicin is mostly metabolized in the liver and is also secreted into the bile (approximately 40%). The dose must be reduced for liver or kidney failure.

The appropriate dose (base equivalent) is intravenously administered, 45 mg / m ² / day for adults under 60 years (30 mg / m ² for patients older than 60 years) 1 every 3-4 weeks Daily, 2 or 3 days, or 0.8 mg / kg / day every 3-4 weeks for 3-6 days; should not be administered more than 550 mg / m ² in lifetime, except for breast If irradiated, only 450 mg / m ² should be administered; for children, if an adult-based plan based on weight is used, the age is less than 2 years or the body surface is 0 Once a week at 25 mg / m ² unless less than 5 m. Available as an injectable dosage form (base equivalent) 20 mg (as a base equivalent to 21.4 mg hydrochloride). Typical ^{dose, 10mg / m 2, 20mg /} m 2, 30mg / m 2, 50mg / m 2, 100mg / m 2, 150mg / m 2, 175mg / m 2, 200mg / m 2, 225mg / m 2 ^{, 250mg / m 2, 275mg /} m 2, 300mg / m 2, 350mg / m 2, 400mg / m 2, 425mg / m 2, 450mg / m 2, 475mg / m 2, may be 500 mg / ^{m 2.} Of course, all of these doses are exemplary, and any dose between the above values is expected to be useful in the present invention.

  Mitomycin (also known as mutamicin® and / or mitomycin C) is an antibiotic isolated from a culture of Streptomyces caespitosus that has been shown to have antitumor activity It is. The compound is resistant to heat, has a high melting point, and is easily soluble in an organic solvent.

  Mitomycin selectively inhibits the synthesis of deoxyribonucleic acid (DNA). Guanine and cytosine content is related to the degree of mitomycin-induced crosslinking. High concentrations of the drug also inhibit cellular RNA and protein synthesis.

  In humans, mitomycin is rapidly cleared from serum after intravenous administration. The time required for a 50% reduction in serum concentration after 30 mg bolus injection is 17 minutes. 30 mg, 20 mg, or 10 mg of I.D. V. After injection, the maximum serum concentrations were 2.4 mg / ml, 1.7 mg / ml, and 0.52 mg / ml, respectively. Clearance is mainly done by metabolism in the liver, but metabolism is done in other tissues as well. The clearance rate is inversely proportional to the maximum serum concentration, which may be due to degradation of the degradation pathway. Approximately 10% of the mitomycin dose is excreted intact in the urine. As the metabolic pathway saturates at relatively low doses, the proportion of dose excreted in urine increases with increasing dose. In children, excretion of intravenous mitomycin is similar.

Actinomycin D _{(dactinomycin) [50-76-0]; C 62 H} 86 N 12 O 16 (1255.43) is an antineoplastic drug that inhibits DNA-dependent RNA polymerase. The drug is a component of a first-line combination therapy used for the treatment of choriocarcinoma, fetal rhabdomyosarcoma, testicular tumor and Wilms tumor. Tumors that do not respond to systemic therapy may respond to local perfusion therapy. Dactinomycin enhances radiation therapy. The drug is a secondary (efferent) immunosuppressive drug.

  Actinomycin D is used in combination with first-line surgery, radiation therapy and other drugs, particularly vincristine and cyclophosphamide. Anti-neoplastic activity has also been shown in Ewing tumors, Kaposi sarcomas and soft tissue sarcomas. Dactinomycin may be effective in women with advanced cases of choriocarcinoma. The drug also produces a consistent response when combined with chlorambucil and methotrexate in patients with metastatic testicular cancer. Sometimes a response is observed in patients with Hodgkin's disease and non-Hodgkin's lymphoma. Dactinomycin has also been used to inhibit immunological responses, particularly rejection of kidney transplants.

Half of the dose is excreted as it is in bile and 10% in urine; the half-life is about 36 hours. The drug does not cross the blood brain barrier. Actinomycin D is supplied as a lyophilized powder (0/5 mg in each vial). The usual daily dose is 10-15 mg / kg; it is administered intravenously for 5 days; however, additional courses may be taken at 3-4 week intervals if no toxicity is encountered. Daily injections of 100-400 mg have been given to children for 10-14 days; other regimens have used 3-6 mg / kg, a total of 125 mg / kg, and a weekly maintenance dose of 7.5 mg / kg . It is safer to administer the drug into an intravenous infusion tube, but a direct intravenous injection is made, along with precautions to discard the needle used to withdraw the drug from the vial to avoid a subcutaneous reaction. I came. Typical ^{dose, 100mg / m 2, 150mg /} m 2, 175mg / m 2, 200mg / m 2, 225mg / m 2, 250mg / m 2, 275mg / m 2, 300mg / m 2, 350mg / m 2 ^{, 400mg / m 2, 425mg /} m 2, 450mg / m 2, 475mg / m 2, may be 500 mg / ^{m 2.} Of course, all of these doses are exemplary, and any dose between the above values is expected to be useful in the present invention.

  Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus. The exact mechanism of action of bleomycin is unknown, but from the available evidence, the main mode of action is inhibition of DNA synthesis, with some evidence that inhibition of RNA and protein synthesis is milder Seems to suggest.

  In mice, high concentrations of bleomycin are found in the skin, lungs, kidneys, peritoneum and lymphatic vessels. Skin and lung tumor cells were found to have high concentrations of bleomycin as opposed to the low concentrations found in hematopoietic tissue. The low concentration of bleomycin found in the bone marrow may be related to the high level of bleomycin-degrading enzyme found in this tissue.

  In patients with creatinine clearance> 35 ml per minute, the terminal elimination half-life of bleomycin serum or plasma is approximately 115 minutes. In patients with creatinine clearance <35 ml / min, the terminal elimination half-life of plasma or serum increases exponentially as creatinine clearance decreases. In humans, 60% to 70% of the dosage is recovered in the urine as active bleomycin. Bleomycin can be administered by intramuscular route, intravenous route, or subcutaneous route. Bleomycin is soluble in water.

  Bleomycin should be considered palliative therapy. The drug may be used as a single agent or in proven combination with other approved chemotherapeutic agents in squamous cell carcinomas such as the following neoplasms: head and neck (mouth, tongue, tonsils, nasopharynx , Including oropharynx, sinus, palate, lip, buccal mucosa, gingiva, epiglottis, larynx), skin, penis, cervix and vulva. The drug has also been used to treat lymphoma and testicular cancer.

  Because of the possibility of an anaphylactoid reaction, lymphoma patients should be treated with no more than 2 units for the first 2 doses. If an acute response does not occur, then the usual dosing schedule can be followed.

  The improvement in Hodgkin's disease and testicular tumors is rapid and is shown within 2 weeks. If there is no improvement by this time, there is no prospect of improvement. The response of squamous cell carcinoma is slower and sometimes takes up to 3 weeks before any improvement is shown.

  Mitotic inhibitors Mitotic inhibitors include plant alkaloids and other natural substances that can inhibit either protein synthesis or mitosis required for cell division. These act during specific phases of the cell cycle. Mitotic inhibitors comprise docetaxel, etoposide (VP16), paclitaxel, taxol, taxotere (R), vinblastine, vincristine, and vinorelbine.

  VP16, also known as etoposide, is used primarily in the treatment of testicular tumors in combination with bleomycin and cisplatin and for small cell lung cancer in combination with cisplatin. VP16 is also active against Kaposi's sarcoma associated with non-Hodgkin lymphoma, acute non-lymphocytic leukemia, breast cancer, and acquired immune deficiency syndrome (AIDS).

VP16 can be used as a solution for intravenous administration (20 mg / ml) and as a capsule containing 50 mg liquid for oral use. For small cell carcinomas of the lung, the intravenous dose (in combination therapy) may be as high as 100 mg / m ² or as low as 2 mg / m ² , customarily at 35 mg / m ² for 4 days daily Also used at ˜50 mg / m ² for 5 consecutive days. When administered orally, the dose should be doubled. Therefore, the dose may be a high dose of about 200 to 250 mg / ^{m 2} for small cell lung cancer. The intravenous dose for testicular cancer (in the case of combination therapy) is 50-100 mg / m ² for 5 consecutive days, or 100 mg / m ² administered 3 times every other day. The cycle of treatment is usually repeated every 3-4 weeks. The drug should be administered gradually with an infusion over 30-60 minutes to avoid hypotension and bronchospasm possibly due to the solvent used in the formulation.

Taxol is an experimentally obtained antimitotic agent isolated from the bark of the ash tree Taxus brevifolia. Taxol binds to tubulin (at a site different from that used by vinca alkaloids) and promotes microtubule assembly. Taxol is currently undergoing clinical evaluation; it has activity against malignant melanoma and ovarian carcinoma. Maximum dose is administered once daily 30 mg / ^{m 2} for 5 days, or 210~250mg / ^{m 2} of every 3 weeks. Of course, all of these doses are exemplary, and any dose between the above values is expected to be useful in the present invention.

  Vinblastine is another example of a plant acryloid that can be used in combination with troglitazone for the treatment of cancer and precancerous conditions. When cells are incubated with vinblastine, microtubule degradation occurs.

  Unexpected absorption after oral administration of vinblastine or vincristine has been reported. At normal clinical dose, the peak concentration of each drug in plasma is approximately 0.4 mM. Vinblastine and vincristine bind to plasma proteins. The drug concentrates strongly on platelets and to a lesser extent on leukocytes and erythrocytes.

  After intravenous injection, vinblastine has a multifaceted clearance mode from plasma; after distribution, the drug disappears from plasma with a half-life of approximately 1 and 20 hours. Vinblastine is metabolized in the liver to biologically activate the derivative desacetylvinblastine. Approximately 15% of the dose is detected as is in the urine and about 10% is recovered in the stool after biliary excretion. In patients with liver dysfunction, the dose should be reduced. A plasma bilirubin concentration of 3 mg / dl (about 50 mM) or higher suggests a dose reduction of at least 50%.

Vinblastine sulfate is available as an injectable preparation. The drug is administered intravenously; special precautions must be taken against subcutaneous extravasation because extravasation can cause painful irritation and ulceration It is. The drug should not be injected into the limb with circulatory failure. After a single dose of 0.3 mg / kg (body weight), myelosuppression reaches its maximum in 7-10 days. If a reasonable level of leukopenia (approximately 3000 cells / mm ³ ) is not reached, the weekly dose may be gradually increased in increments of 0.05 mg / kg (body weight). In regimens aimed at treating testicular cancer, vinblastine is used at a dose of 0.3 mg / kg every 3 weeks, regardless of blood count or toxicity.

  The most important clinical use of vinblastine is in combination with bleomycin and cisplatin in the curative treatment of metastatic testicular tumors. Beneficial responses have been reported in various lymphomas (especially Hodgkin's disease), which can show significant improvement in 50-90% of cases. The effectiveness of vinblastine in most lymphomas is not diminished even when the disease is resistant to alkylating agents. The drug is also active in Kaposi's sarcoma, neuroblastoma, and Letterer-Sive disease (histiocytosis X), and breast and female choriocarcinoma.

The dose of vinblastine will be determined by the clinician according to the needs of the individual patient. 0.1 to 0.3 mg / kg may be administered, or 1.5 to ² mg / m ² may be administered. As another ^{example, 0.1mg / m 2, 0.12mg /} m 2, 0.14mg / m 2, 0.15mg / m 2, 0.2mg / m 2, 0.25mg / m 2, 0.5mg / ^{m 2, 1.0mg / m 2,} 1.2mg / m 2, 1.4mg / m 2, 1.5mg / m 2, 2.0mg / m 2, 2.5mg / m 2, 5.0mg / m ² , 6 mg / m ² , 8 mg / m ² , 9 mg / m ² , 10 mg / m ² , 20 mg / m ² may be administered. Of course, all of these doses are exemplary, and any dose between the above values is expected to be useful in the present invention.

  Vincristine prevents mitosis and causes metastasis. Most of the biological activity of this drug seems to be explained by its ability to specifically bind to tubulin and block the ability of proteins to polymerize into microtubules. . The disruption of the microtubules of the mitotic apparatus stops cell division in the middle phase. Failure to accurately segregate chromosomes during mitosis probably results in cell death.

  The relatively low toxicity of vincristine on normal bone marrow and epithelial cells makes this agent an exceptional anti-neoplastic agent that can be included in combination with other myelosuppressive agents There are many cases.

Unexpected absorption has been reported after oral administration of vinblastine or vincristine. At normal clinical dose, the peak concentration of each drug in plasma is approximately 0.4 mM.
Vinblastine and vincristine bind to plasma proteins. The drug concentrates strongly on platelets and, to a lesser extent, collects on white blood cells and red blood cells.

  Vincristine has a pleiotropic clearance mode from plasma; the terminal half-life is about 24 hours. The drug is metabolized in the liver, but no derivative with biological activity has been identified. In patients with liver dysfunction, the dose should be reduced. A plasma bilirubin concentration of 3 mg / dl (about 50 mM) or higher suggests a dose reduction of at least 50%.

Vincristine sulfate is available as a solution for intravenous injection (1 mg / ml). Vincristine used with corticosteroids is currently the best treatment to provide remission of childhood leukemia; the optimal dose of these drugs is 2 mg / m ² body surface area given vincristine intravenously per week. Once daily, and prednisone orally at 40 mg / m ² daily. Adult patients with Hodgkin's disease or non-Hodgkin's lymphoma are usually given vincristine as part of a complex protocol. When used in MOPP regimen, the recommended dose of vincristine is 1.4 mg / ^{m 2.} High doses of vincristine appear to be better tolerated in children with leukemia than adults who may experience severe neurotoxicity. Administration of the drug more frequently or at higher doses every 7 days appears to increase the onset of toxic symptoms without a commensurate improvement in response rate. Precautions should also be used to avoid extravasation during intravenous administration of vincristine. Vincristine (and vinblastine) can also be injected into the arterial blood supply of tumors at doses several times higher than can be administered intravenously with comparable toxicity.

  Vincristine was effective in Hodgkin's disease and other lymphomas. The drug is slightly less effective than vinblastine when used alone in Hodgkin's disease, but when used with mechloretamine, prednisone and procarbazine (so-called MOPP regimen), this disease in advanced stage (III and IV) Is a preferred treatment. In non-Hodgkin lymphoma, vincristine is a potent agent, especially when used with cyclophosphamide, bleomycin, doxorubicin and prednisone. Vincristine is more useful than vinblastine in lymphocytic leukemia. Beneficial responses have been reported in a variety of other neoplasms, particularly those with Wilms tumor, neuroblastoma, brain tumor, rhabdomyosarcoma, and breast, bladder and gender reproductive cancer.

The dose of vincristine at the time of use will be determined by the clinician according to the needs of the individual patient. 0.01-0.03 mg / kg or 0.4-1.4 mg / m ² may be administered, and 1.5-2 mg / m ² may be administered. As another example, 0.02 mg / m ² , 0.05 mg / m ² , 0.06 mg / m ² , 0.07 mg / m ² , 0.08 mg / m ² , 0.1 mg / m ² , 0.12 mg / m ² , 0.14 mg / m ² , 0.15 mg / m ² , 0.2 mg / m ² , 0.25 mg / m ² may be administered as a continuous intravenous infusion. Of course, all of these doses are exemplary, and any dose between the above values is expected to be useful in the present invention.

Camptothecin is an alkaloid derived from the Chinese tree, Camptotheca accumata Decne. Camptothecin and its derivatives are unique in their ability to inhibit DNA topoisomerase by stabilizing a covalent reaction intermediate termed “cleavage complex” and ultimately cause tumor cell death. It is widely believed that camptothecin analogs have shown significant anti-tumor and anti-leukemia activity. Clinical application of camptothecin is limited due to serious side effects and insufficient water solubility. Currently, several synthetic or semi-synthetic camptothecin analogs (topotecan; irinotecan) have been applied to cancer treatment and have shown satisfactory clinical effects. Molecular formula of camptothecin is _{C 20 H 16 N 2 O 4} , a molecular weight 348.36. Camptothecin is provided as a yellow powder, but may be solubilized into a clear yellow solution of 50 mg / ml in DMSO 1N sodium hydroxide. It is stable for at least 2 years when stored at 2-80 ° X in a dry, airtight, light-resistant environment.

  [Nitrosourea] Nitrosourea, like alkylating agents, inhibits DNA repair proteins. Nitrosourea is used to treat non-Hodgkin lymphoma, multiple myeloma, and malignant melanoma in addition to brain tumors. Examples include carmustine and lomustine.

  Carmastine (sterile carmastine) is one of the nitrosoureas used in the treatment of certain neoplastic diseases. Carmastine is 1,3 bis (2-chloroethyl) -1-nitrosourea. This is a lyophilized light yellow flake or clot with a molecular weight of 214.06. It is highly soluble in alcohol and lipids and poorly water soluble. Carmastine is administered by intravenous infusion after reconstitution as recommended. Sterile carmastin is generally available as a 100 mg single dose vial of lyophilizate.

  Although it is generally agreed that karmastin alkylates DNA and RNA, karmastin is not cross-resistant with other alkylating agents. Like other nitrosoureas, calmastin can also inhibit several important enzymatic processes by carbamoylation of amino acids in the protein.

  Carmastine is a palliative treatment as a single agent in brain tumors such as glioblastoma, brainstem glioma, medullobladioma, astrocytoma, ventricular ependymoma, and metastatic brain tumor, Or in established combination therapy with other approved chemotherapeutic agents. In addition, it has been used in combination with prednisone to treat multiple myeloma. Carmastine is useful as a second-line therapy in combination with other approved drugs in patients with Hodgkin's disease and non-Hodgkin's lymphoma who relapse or are unresponsive to first-line therapy while being treated with first-line therapy I know that there is.

The recommended dose of carmastine as a single agent in patients who have not received prior therapy is 150-200 mg / m ² intravenously every 6 weeks. This may be administered as a single dose or divided into daily injections such as 75-100 mg / m ² for 2 consecutive days. If carmastine is used in combination with other myelosuppressive drugs or in patients with depleted bone marrow reserves, the dose should be adjusted accordingly. The dose following the initial dose should be adjusted according to the patient's hematological response to the previous dose. It should be understood that other doses such as 10 mg / m ² , 20 mg / m ² , 30 mg / m ² , 40 mg / m ² , 50 mg / m ² , 60 mg / m ² , 70 mg / m m ² , 80 mg / m ² , 90 mg / m ² or 100 mg / m ² may be used. Those skilled in the art should refer to “Remington's Pharmaceutical Sciences” 15th edition, Chapter 61. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will in any case determine the appropriate dose for the individual patient.

Lomustine is one of the nitrosoureas used for the treatment of certain neoplastic diseases. Lomustine is 1- (2-chloro-ethyl) -3-cyclohexyl-1nitrosourea. This is a yellow powder with a composition formula C ₉ H ₁₆ ClN ₃ O ₂ and a molecular weight of 233.71. Lomustine is soluble in 10% ethanol (0.05 mg / mL) and absolute alcohol (70 mg / mL). Lomustine is relatively insoluble in water (<0.05 mg per ml). Lomustine is not very ionized at physiological pH. Inactive ingredients in lomustine capsules are: magnesium stearate and mannitol.

  Although it is generally agreed that lomustine alkylates DNA and RNA, lomustine is not cross-resistant with other alkylating agents. Like other nitrosoureas, lomustine can also inhibit several important enzymatic processes by carbamoylation of amino acids in the protein.

Lomustine may be administered orally. After oral administration at a dose ranging and lomustine having radioactivity of ^{30mg / m 2 ~100mg / m 2} , about half of the administered radioactivity was excreted in the form of degradation products within 24 hours. The serum half-life of metabolites ranges from 16 hours to 2 days. The tissue concentration is comparable to the plasma concentration 15 minutes after intravenous administration.

  Lomustine is a single agent in addition to other therapies, or other approved chemotherapeutic agents in both primary and metastatic brain tumors, in patients already undergoing appropriate surgical and / or radiation therapy It has been shown to be useful in combination therapies established together with it. Furthermore, lomustine has also been shown to be effective in second-line therapy for Hodgkin's disease in combination with other approved drugs in patients who relapse while receiving treatment in first-line therapy or do not respond to first-line therapy.

The recommended dose of lomustine in adults and children as a single agent in patients who have not received prior treatment is 130 mg / m ² as a single oral dose every 6 weeks. In patients with impaired bone marrow function, the dose should be reduced to 100 mg / m ² every 6 weeks. If lomustine is used in combination with other myelosuppressive drugs, the dose should be adjusted accordingly. Of course, other doses such as 20 mg / m ² 30 mg / m ² , 40 mg / m ² , 50 mg / m ² , 60 mg / m ² , 70 mg / m ² , 80 mg / m ² , 90 mg / m ² , 100 mg / m ^{2 2} , 120 mg / m ² or any dose between the above values determined by the clinician to be necessary for the patient to be treated may be used.

  Other agonists Other agonists that can be used include Avastin (R), Iressa (R), Erbitux (R), Velcade (R) and Gleevec (R). Furthermore, growth factor inhibitors and small molecule kinase inhibitors have utility in the present invention as well. All therapies described in “Cancer: Principles and Practice of Oncology” (2001) are incorporated herein by reference. The following additional therapies are encompassed as well.

ii. Immunotherapy Immunotherapy is generally based on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be an antibody specific for some marker on the surface of tumor cells, for example. The antibody alone may serve as a therapeutic effector, or the antibody may recruit other cells that actually kill the cell. The antibody may further be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) to simply serve as a targeting agent. As another example, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

  Thus, immunotherapy may be used as part of a combination therapy in conjunction with reovirus therapy or other oncolytic virus therapy. The general method of combination therapy is discussed below. In general, tumor cells must have some marker that is applicable for targeting, i.e. not present on the majority of other cells. There are many tumor markers, any of which may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen Receptors, laminin receptors, erbB and p155. In addition, tumor cells or other hyperproliferative cells infected with reovirus or another oncolytic virus express viral antigens on the cell surface so that the cells are susceptible to attack by the immune system. It can also be.

  Tumor necrosis factor kills several types of cancer cells, activates cytokine production, activates macrophages and endothelial cells, promotes collagen and collagenase production, is an inflammatory mediator and septic shock A glycoprotein that is also a mediator and promotes catabolism, fever and sleep. Some infectious agents cause tumor regression by stimulation of TNF production. When used alone at an effective dose, TNF can be quite toxic, so the optimal regimen would probably be to use TNF at a lower dose in combination with other drugs. Its immunosuppressive effect is enhanced by gamma interferon, so this combination is potentially dangerous. A hybrid of TNF and interferon-α has also been found to have anticancer activity.

iii. Hormone therapy Use of sex hormones by the methods described herein in the treatment of cancer. Although the methods described herein are not limited to the treatment of certain cancers, this use of hormones is effective for breast cancer, prostate cancer, and endometrial (uterine lining) cancer. is there. Examples of these hormones are estrogens, antiestrogens, progesterone, and androgens.

  Corticosteroid hormones are useful for treating several types of cancer (lymphoma, leukemia and multiple myeloma). Corticosteroid hormones can increase the effectiveness of other chemotherapeutic drugs and are therefore frequently used in combination therapies. Prednisone and dexamethasone are examples of corticosteroid hormones.

iv. Radiation therapy Radiation therapy, also called radiation therapy, is the treatment of cancer and other diseases using ionizing radiation. Ionizing radiation provides energy that damages or destroys the cell's genetic material by damaging the cell's genetic material in the area being irradiated, making it impossible for these cells to continue to grow. Radiation damages both cancer cells and normal cells, but the latter can repair itself and function properly. Radiation therapy can be used to treat localized solid tumors such as skin, tongue, larynx, brain, breast, or cervical cancer. Radiation therapy can also be used to treat leukemias and lymphomas (hematopoietic cells and lymphoid cancers, respectively).

  Radiation therapy used by the present invention includes, but is not limited to, the use of gamma rays, X-rays, or at least one of the delivery of targeted radioisotopes to tumor cells. Other forms of DNA damage factors such as microwave and UV irradiation are also contemplated. All of these factors are very likely to cause various damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The X-ray dose range ranges from long periods (3-4 weeks) with daily doses of 50-200 X-rays to single doses of 2000-6000 X-rays. Radioisotope dose ranges vary widely, depending on the half-life of the isotope, the intensity and type of radiation emitted, and absorption by neoplastic cells.

  Radiation therapy can also include the use of radiolabeled antibodies to deliver a dose of radiation directly to the cancer site (radioimmunotherapy). Antibodies are highly specific proteins made by the body in response to the presence of antigens (substances recognized as different from self by the immune system). Some tumor cells contain specific antigens that cause the production of tumor-specific antibodies. Large quantities of these antibodies can be made in the laboratory and attached to radioactive material (a method known as radiolabeling). When introduced into the body, antibodies actively search for cancer cells, which are destroyed by the killing (cytotoxic) action of radiation. This approach can minimize the risk of radiation damage to normal cells.

  Conformal radiation therapy uses the same radiation therapy equipment (linear accelerator) as normal radiation therapy, but a metal block is placed in the X-ray beam passage so that the shape of the X-ray beam matches the shape of the cancer. To change. This ensures that a higher radiation dose is given to the tumor. Healthy surrounding cells and nearby structures receive lower radiation doses, thus reducing the possibility of side effects. Devices called multileaf collimators have been developed and can be used as an alternative to metal blocks. The multileaf collimator is composed of several metal plates fixed to a linear accelerator. Each layer can be adjusted so that the radiation therapy beam can be shaped into the treatment area without the need for a metal block. Accurate positioning of the radiotherapy device is very important for treatment with conformal radiation therapy, even if a special scanning device is used to examine the position of your internal organs at the beginning of each treatment procedure Good.

  High-resolution intensity modulated radiation therapy also uses a multileaf collimator. During this treatment, the multi-leaf collimator layer is moved while the treatment is in progress. This method appears to achieve an even more accurate shaping of the treatment beam and allows the radiation therapy dose to be constant throughout the treatment area.

  Research studies have shown that conformal radiation therapy and intensity-modulated radiation therapy can reduce the side effects of radiation therapy treatments, but by shaping the treatment area very accurately, It is possible that very small cancer cells have remained outside. This means that the risk of cancer recurrence in the future can be increased using the specialized radiotherapy techniques described above. Stereotaxic radiation therapy is used to treat brain tumors. This technique provides radiation treatment from many different angles so that the dose reaching the tumor is very high and the dose affecting the surrounding healthy tissue is very low. Prior to treatment, to ensure that radiotherapy targeting is accurately performed, several scans are analyzed by the computer and the patient's head is a specially made frame while receiving radiation therapy. Always kept inside. Several doses are given.

  Stereotactic radiosurgery for brain tumors (gamma knives) uses very precisely targeted beams of gamma radiation therapy from hundreds of different angles rather than knives. Only one radiation therapy takes about 4-5 hours. For this treatment, a specially made metal frame will be attached to the head. Thereafter, several scans and x-rays are performed to find the exact area that needs treatment. During radiation therapy, the patient lies with his head in a large protective cap, which has hundreds of holes to allow the radiation treatment beam to pass through.

  Scientists are also looking for ways to increase the effectiveness of radiation therapy. Two investigational drugs are being investigated for their effects on irradiated cells. Radiosensitizers are likely to damage tumor cells, and radioprotectors protect normal tissues from the effects of radiation. Hyperthermia (use of heat) has also been studied for its effectiveness in making tissues sensitive to radiation.

v. Subsequent surgery Although approximately 60% of cancer patients will undergo some type of surgery, surgery includes preventive surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery is a cancer treatment that can be combined with other therapies such as therapies, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy or alternative therapies of the present invention.

  Curative surgery includes resection in which the entire cancer tissue or part thereof is physically removed, removed, and / or destroyed. Tumor resection refers to physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and misoperatively managed surgery (Morse surgery). It is further contemplated that the present invention can be used with removal of superficial cancers, precancers, or associated amounts of normal tissue.

  When a cancer cell, tissue, or part of an entire tumor is removed, a cavity may form in the body. Treatment may be completed by perfusion, direct injection or topical application of the area with additional anticancer therapy. Such treatment is, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4, and 5 weeks, or 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11 or every 12 months. These treatments may also be performed by changing the dose.

V. Examples The following examples are included to demonstrate preferred embodiments of the invention. As will be appreciated by those skilled in the art, the techniques disclosed in the following examples represent techniques that have been found by the inventors to work well in the practice of the present invention, and thus are It can be considered to constitute a preferred method for implementation. However, it will be apparent to those skilled in the art in light of the present disclosure that numerous modifications may be made to the specific embodiments disclosed and still remain similar or similar without departing from the spirit and scope of the invention. It is possible to obtain the result.

Example 1 Materials and Methods [Cell Lines] L3 cells lacking p53 ^{− / −} MEF, BT and ATM, and human lymphomas HBL2, Granta, Z138C, Raji, Ramos and JVM2 cells, and retinoblastoma Cell lines (Y-79 and WERI-Rb-1) were purchased from the American Type Culture Collection. Cells were maintained in RPMI 1640 in 10% FBS.

Preparation of wild-type and attenuated reovirus The wild-type reovirus T3D strain used in this study was grown in L929 cells and purified as described above. Attenuated reovirus derived from HTR1 cultures was purified by the same method used in the preparation of wild type reovirus except that AV reovirus was propagated in HT1080 and L929 cells. Reovirus was added to the cells with a MOI of 5-10 and these were maintained at 37 ° C. for 48-72 hours. After the cytopathic effect of the virus was observed (typically 20-30% cell lysis), the virus was purified from the pelleted cells. Viral CsCl centrifugation was performed at 35,000 rpm for 7-8 hours using a SW41 rotor. Banded virus was collected and dialyzed extensively against 150 mM NaCl, 10 mM MgCl ₂ and 10 mM Tris (pH 7.5). For titration of wild type reovirus, HEK293 cells were seeded in 6-well plates as 2 × 10 ⁵ cells per well. After 2 hours of adsorption at 37 ° C., the inoculum was removed. The cell monolayer was then covered with 1% agar and fresh medium. Plaques were counted 5-7 days after infection.

  The same procedure was followed for titration of attenuated reovirus except that the agar was removed 5-7 days after infection and the cell monolayer was immunostained with reovirus antiserum and secondary FITC antibody. Immobilization / membrane permeabilization using cytofix / cytoperm (TM) (BD Bioscience). AV reovirus plaques were identified by immunofluorescence detection. As another example, for attenuated reovirus, a standard plaque assay was also performed on L929 cells. Three days after infection, the agar was overlaid with neutral red and the cell monolayer was examined for plaque formation 24 hours later. For attenuated reovirus collections from HT1080 and HTR1 infected supernatants, the virus was pelleted using ultracentrifugation at 35,000 rpm.

  [Preparation of Myxoma virus] Myx-GFP of Myxoma virus (Lausanne strain) prepared by intergenic insertion of a green fluorescent protein (GFP) cassette driven by a synthetic vaccinia virus early / late promoter was used for infection experiments. It was. Myx-GFP was grown in BGMK cells and titrated by focus formation as previously described (Opgenorth et al., 1992).

  FACS analysis For flow cytometric analysis, cells were trypsinized and fixed using cytofix / cytoperm solution (PharMingen, San Diego, CA, USA). Fixed and membrane permeabilized cells were incubated with primary reovirus antiserum and secondary FITC conjugated anti-rabbit IgG (Cedarlane, Ontario, Canada) and then analyzed by flow cytometry.

Example 2: Results [Reovirus and Mixoma virus preferentially infect cells deficient in p53 or ATM] To investigate whether modulation of tumor suppressor genes can affect susceptibility to oncolytic viruses. They selected p53 and ATM tumor suppressor genes that are highly mutated in various cancers. p53 is a prototypical tumor suppressor gene that is most commonly mutated in various cancer cells, and that more than 50% of cancers have p53 mutations. (White, 1994; Morris, 2002). ATM (Ataxia telangiectasia mutated) is a serine-threonine protein kinase that is activated in response to DNA damage. Any copy of the ATM gene is dysfunctional in telangiectasia (Takigawa et al.), A rare syndrome characterized by cancer constitution, radiosensitivity and neurodegeneration (Kitagawa et al., 2005). Therefore, we used p53 − / − MEF (mouse fetal fibroblasts) and ATM-deficient L3 lymphoblastoid cells (Kozlov et al., 2003) to investigate oncolytic virus susceptibility. . As shown in FIGS. 1A-B, reovirus (WT and AV reovirus; Kim et al., 2007) and myxoma virus, respectively, fluoresce positive cells (FITC + cells or GFP + cells) into MEF (p53 normal). And preferentially infected with p53 − / − MEF or L3 (ATM deficient) cells, as shown by FACS or microscopic detection compared to BT (normal ATM) control cells.

  [Reovirus and mixoma virus preferentially infect human lymphomas with p53 or ATM dysfunction] Reovirus and mixoma virus preferentially infects p53 or ATM deficient cells, so we Next, it was investigated whether reovirus and mixoma virus could preferentially infect human lymphoma with p53 or ATM deficiency. We tested six different lymphomas (HBL-2, Granta, Z138C, JVM2, Raji and Ramos) that show different cellular responses arising from p53 and ATM-dependent pathways upon administration of genotoxic agents ( FIG. 2A). The functional status of p53 and ATM was assessed by IR irradiation and detection of p53 and ATM phosphorylation by Western blot. BT (ATM normal) and L3 (ATM deficient) were used as controls for ATM responsiveness by IR. HBL-2 and Raji showed a p53 response that did not function normally upon genotoxic stress as evidenced by constitutive phosphorylation of serine 15 of p53 (Li et al., 2006) (FIG. 2A). Granta cells show an ATM dysfunctional response upon genotoxic stress, and ATM is not activated upon ionizing radiation (IR) stimulation (FIG. 2A). In the case of Z138C, JVM2 and Ramos cells, p53 and ATM responses were normal after genotoxic stress as shown by IR-stimulated p53 and ATM hyperphosphorylation (FIG. 2A). Interestingly, at least one of p53 or ATM is dysfunctional lymphoma (HBL2, Granta and Raji) (HBL-2 and Raji show constitutive p53 activation, Granta shows ATM deficiency upon IR stimulation ) Reovirus and mixomas as shown by FACS or microscopic detection comparing fluorescence positive cells (FITC + or GFP + cells) with ATM responsive and p53 responsive lymphomas (Z183C, JVM-2, Ramos). Both viruses are preferentially susceptible to infection (FIG. 2C). As shown in FIG. 2B, the cellular ATM or p53 dysfunction response is highly associated with both reovirus and mixoma virus susceptibility. This strongly suggests that both ATM and p53 are involved in establishing cellular resistance to viral infection, while abnormalities in either the gene or its response pathway may confer viral susceptibility. This is shown (FIG. 2A).

  [Retinoblastoma cells are susceptible to reovirus and mixoma virus] Since reovirus and mixoma virus preferentially infects p53 and ATM dysfunctional cancer cells, the inventor further infects RB. We investigated whether defective cancer cells were susceptible to infection with both reovirus and mixoma virus. As shown in FIG. 3, two different human retinoblastoma cells are susceptible to reovirus and mixoma virus, as shown by FACS or microscopic detection of fluorescence positive cells (FITC + or GFP + cells). All cells contain a deletion of the Rb gene (Reid et al., 1974).

  All methods disclosed herein and set forth in the claims can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, those skilled in the art will appreciate that the concept of the present invention, relative to the methods and sequence of methods described herein, Changes can be made without departing from the spirit and scope. More specifically, it is clear that certain or chemically related agents may be used in place of the agents described herein while achieving the same or similar results. Let's go. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

References The following references are specifically incorporated by reference herein as long as they provide exemplary procedures or other detailed explanations that supplement the content described herein. .

Claims (5)

Both oncolytic reovirus and oncolytic mixoma virus in hyperproliferative cells due to mutations in one or more tumor suppressor genes selected from the group consisting of p53 gene, Rb gene and ATM gene A method of determining susceptibility to infection of
(A) providing hyperproliferative cells;
(B) a step of infecting another group of oncolytic of myxoma virus in hyperproliferative cells with to infect one group of hyperproliferative cells with oncolytic wild-type reovirus or attenuated reovirus When,
(C) an oncolytic reovirus in a hyperproliferative cell having a mutation in one or more tumor suppressor genes selected from the group consisting of p53 gene, Rb gene and ATM gene compared to its normal control cell and whether information on both oncolytic the myxoma virus has infected preferentially, in a step of analyzing by FACS or microscopic detection, to evaluate the infection for both reovirus and myxoma virus hyperproliferative cell And wherein said assessment comprises assessing the structure, function, or expression of the p53, Rb or ATM gene or gene product from said hyperproliferative cell;
Including
The preferential infection of the hyperproliferative cell against both reovirus and mixoma virus compared to its normal control cell is selected from the group consisting of the p53 gene, Rb gene and ATM gene. and have one structure in more tumor suppressor genes, function, or a defect in the expression, mutation of the tumor suppressor gene is an indication and that may affect the oncolytic virus susceptibility, the method . The method of claim 1, wherein the hyperproliferative cell is a tumor cell.   The method according to claim 1, wherein the evaluation of expression comprises Northern blotting, Western blotting, immunohistochemistry, RT-PCR, microarray analysis, transcriptome analysis, proteome analysis, or metabolome analysis.   The method of claim 1, wherein the structural evaluation comprises sequencing, in situ hybridization, immunohistochemistry, or structural proteomics.   The method of claim 1, wherein the assessment of function comprises assessing expression or activity of a target downstream of p53, Rb, or ATM.