KR20110029129A - Tumor suppressor-based susceptibility of hyperproliferative cells to oncolytic viral therapy - Google Patents

Abstract

The present invention relates to a method of treating cancer using oncolytic viruses such as liovirus and myxoma virus. In particular, the present invention is based on the observation that cancer cells having abnormalities in tumor suppressors are more sensitive to infection of oncolytic viruses than cells containing normal or "wild-type" tumor suppressors. In particular, defects associated with each of p53, Rb and ATM make cancer cells susceptible to oncolytic virus therapy.

Description

TUMOR SUPPRESSOR-BASED SUSCEPTIBILITY OF HYPERPROLIFERATIVE CELLS TO ONCOLYTIC VIRAL THERAPY}

This application claims priority to US Provisional Patent Application 61 / 055,360, filed May 22, 2008, the entirety of which is incorporated by reference.

1. Field of the Invention

The present invention relates generally to the fields of virology, molecular biology and medicine. More specifically, the present invention relates to a method for assessing the sensitivity to anticancer viral therapy of hyperproliferative diseases including cancer.

2. Description of related technology

Respiratory Enteric Orphan Virus is an ubiquitous, non-enveloped virus that contains 10 fragments of double-stranded RNA in its genome and is generally weak human infectious, localized to the upper respiratory and gastrointestinal tract, and is immune In functioning hosts it is often an asymptomatic virus ( Tyler , 2001 ). Attempts to reverse engineer riovirus have been largely unsuccessful due to several factors, including the lyovirus's double-stranded RNA genome. Riovirus particles lacking σ1 are recognized to be non-infectious ( Larson et al , 1994 ).

Importantly, it has been recognized for many years that rioviruses exhibit surprising cytotoxic activity when infected with certain types of transformed cells ( Duncan et al , 1978; Hashiro). et al , 1977 ). Replication-capable anticancer viruses (tumor soluble viruses) provide attractive anticancer therapies. These anticancer viruses have two major advantages. First, unlike conventional chemotherapy and radiotherapy, these viruses specifically target cancer cells because of their limited ability to replicate in normal cells. Second, when compared to replication-incapable vectors, these viruses multiply from the first infected cancer cells to surrounding cancer cells, yielding significant amounts, and strong anti-cancer effects can be obtained.

Exposure to wild-type riovirus, although often asymptomatic in healthy individuals, is nevertheless substantially and potentially very seriously dangerous for immunocompromised individuals, and in patients such as cancer patients. The clinical viability of riovirus therapy is limited, and without such restrictions, such therapy may be beneficial for cancer patients. Transformed cells comprising the oncogenic Ras-signaling pathway are in in vitro and in Evidence for riovirus anticancer activity is unknown until it appears to be preferentially sensitive to in vivo riovirus (type 3 Dearing strain) infection ( Coffey et al , 1998; Strong et al , 1998; Norman et al , 2004 ). Ras gene mutations are frequently observed in various types of cancer in humans ( Dursma et al , 2003 ), these findings have made Riovirus therapy current in clinical practice ( Norman et al , 2005 ).

Nevertheless, the presence of the Ras gene mutation only partially explains the sensitivity of cancer cells to riovirus therapy. In a recent study, the Ras pathway is not the only factor in determining recent riovirus permissiveness in a recent study.Song et al , 2009And riovirus resistant cells can be established from Ras-transformed cells while still retaining active Ras mutations (Kim et al, 2007It turned out. Thus, there remains a need for improved methods of identifying and selecting appropriate cancer cell sub-groups to be treated with riovirus and other anticancer viral agents.

Summary of the Invention

Wild type (type 3 Dearing) or variant (e.g., partially attenuated virus with defective synthesis of σ1 protein) rioviruses are composed of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, and ATBFl. such a particular defective or inactive tumor suppressor substance an organic moiety or transformed hyperproliferative containing tea transform cancer cells in vitro Or in Exposure to riovirus in vivo is effective for killing these cancer cells. Thus, the scope of application of lioviruses can be extended beyond those associated with abnormal or excessive activity of the ras proto-oncohene signaling pathway. Other viral, interferon or other cellular immune signaling pathways previously considered to be preferentially responsive to the ras state are also susceptible to loss of tumor suppressor function and may similarly extend to their application. Thus, according to the present invention, there is provided a method for determining whether a hyperproliferative disease can be affected by liovirus or myxoma virus oncolysis, wherein the method comprises (a) providing a hyperproliferative cell and ; (b) assessing the structure, function or expression of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, and / or ATBFl, genes or gene products of hyperproliferative cells; And (c) comparing the structure, function or expression of hyperproliferative cells with the wild type structure, function or expression of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, and / or ATBFl, gene or gene product. Wherein the defect in the structure, function, or expression of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, and / or ATBF1 is a lyovirus or myxoma virus It may be affected by oncolysis.

The method may also include treating patients with hyperproliferative cells obtained with lyovirus and / or myxoma virus therapy. Reovirus therapy is wild type reovirus therapy or attenuated reovirus therapy and utilizes a reovirus that expresses an σ1 capsid protein with binding, an detectable σ1 capsid protein or does not express an σ1 capsid protein. How is it treated the ex marrow cells of patients with Rio virus and / or myxoma virus therapy treatment in vivo or administering a lyovirus and / or myxoma virus therapy to the patient. ex In vivo therapy, the method further comprises contacting the bone marrow cells with a second anti-hyperproliferative regimen, or in a viral therapy administered to an individual, the method administers a second anti-hyperproliferative agent to the individual It may include doing. The second anti-hyperproliferative therapy may be chemotherapy, radiotherapy, hormone therapy, gene therapy, or immunotherapy. The method may be an inhibitor of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, or ATBF1 function or expression, eg, protein or peptide, antibody, siRNA, anti-antisense molecule, ribozyme or It may further comprise therapy with small molecules. Therapies may include non-viral anti-hyperproliferative therapies such as chemotherapy, radiotherapy, hormone therapy, gene therapy, or immunotherapy. Hyperproliferative cells may be malignant or benign cells. The method may further comprise obtaining a hyperproliferative cell from the patient prior to step (a). Assessment of expression can be performed using Northern blot, Western blot, immunohistochemistry, RT-PCR, microarray analysis, transcriptome analysis, proteome analysis or metabolome analysis. Include. Alternatively, evaluation of the structure may include sequencing, in situ hybridization, immunohistochemistry or structural proteomics. In addition, assessing the function may include assessing the expression or activity of a downstream target 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 is provided, the method comprising: (a) p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, or ATBF1 function in hyperproliferative cells; Or contacting an inhibitor of expression; And (b) contacting the hyperproliferative cell with a lyovirus and / or myxoma virus therapy. Reovirus therapy can be wild type reovirus therapy or attenuated reovirus therapy, which is a reovirus that expresses a binding σ 1 capsid protein, an undetectable σ 1 capsid protein, or does not express σ 1 capsid protein. Treatment ex patient's bone marrow in vivo , or in in vivo processing. The method may further comprise contacting the bone marrow cells with a second anti-proliferative therapy, wherein the second anti-proliferative therapy may be, for example, chemotherapy, radiotherapy, hormone therapy, gene therapy, or immunotherapy. Can be. Hyperproliferative cells may be malignant or benign cells.

The indefinite article used in connection with the wording “comprising” in the specification and / or claims may mean “one” but may also mean “one or more”, “at least one” and “one or more than one”. .

Throughout this application, the term “about” refers to the numerical value of the inherent variation of the device's error, the method used to determine the value, or the variables present in the research task.

In the claims, “or” is used to mean “and / or” unless the alternative expressly refers to only or the alternative is mutually exclusive, and the specification defines only the alternative or refers to “and / or”. Is backing. As used in the specification and claims, “comprising” (and any form such as “comprise and comprise”), “having” (and “have and has” ) "," Including "(and any form such as" includes and include "), or" containing "(" contains and contain ") Are inclusive or open, and do not exclude additional, unspecified elements or method steps. Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it is to be understood that the specific embodiments that are directed to the description and specific embodiments of the invention are provided for the purpose of illustration, for reasons without departing from the scope and spirit of the invention and various changes and modifications from the description. This is because it will be apparent to those skilled in the art.

The following drawings are part of this specification and are included to further illustrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.
Degree. 1A-B p53-/-Lyovirus and myxoma virus preferred infection of L3 with MEF and ATM binding.
Degree. 1A
p53-/-MEF and p53 + / + MEF (in turn, p53 knockout or p53 wild-type murine embryonic fibroblasts) are WT / AV reovirus at MOI 40 or GFP-expressing myxoma virus at MOI 5 (Myx-GFP is Johnston et al , 2003 , which is the GFP expression version of the Lausanne (ATCC) strain of MYXV). Three days after infection, cells were fixed / infiltrated and analyzed by FACS using riovirus antiserum and secondary FITC antiserum. In the results, both wild type and attenuated lioviruses were able to preferentially infect p53 − / − MEF. In myxoma infection studies, cells were infected for 24 hours and viewed under relative fluorescence microscopy. GFP-expressing cells indicate myxoma virus infection. Mock: False infection.
Degree. 1B
BT (ATM-lymphoblastoid C3ABR cells; Kozlov et al , 2003) and L3 (ATM-deficient lymphoblast cells; Kozlov et al , 2003 ) were infected with WT / AV reovirus at MOI 40 or myxoma virus (Myx-GFP) at MOI 5. Three days after infection, cells were fixed / infiltrated and analyzed by FACS, using riovirus antiserum and secondary FITC antiserum. In the results, both wild-type and attenuated riovirus were able to preferentially infect p53 − / − MEF. In myxoma infection studies, cells were infected for 48 hours and viewed under relative fluorescence microscopy. GFP-expressing cells indicate myxoma virus infection. The C35ABR (B3) and L3 cell lines were in turn dr. M Lavin (QueenS1and Institute of Medical Research) and Dr. Y Shiloh (Tel Aviv University). Mock: False infection.
Degree. 2A-C-p 53 and ATM dysfunctional lyovirus and myxoma virus preferred infection of human lymphoma.
Degree. 2A
p53 and ATM lack a potentiating response to IR stimulation. Mantle cell lymphoma (Granta, HBL-2 (p53 mutation, Turker et al , 2006 ), Z138C, JVM-2)) and Burkitt's lymphoma ( Raji , and Ramos ) was irradiated at 2 Gy for 2 hours, and cells were obtained. Treatment with NET-N Lysis Buffer (1% NP-40) and sonication produced total cell lysate. 50 μg of protein was placed on 8% or 10% SDS-PAGE, transferred to nitrocellulose, and the indicated antibodies (Phospho-ATM; pSerl981 ATM and Phospho-p53; pSerl5, and p53) were obtained from the Rockland Immunochemicals for Research and Cell signal. Purchased from ring; ATM specific rabbit polyclonal antibody 4BA It's a gift from M Lavin. BT and L3 cells were used as reference. (FIG. 2B) p53 and ATM-normal-reactive cells (BT, Z138C, JVM-2, Ramos), and p53 and / or ATM-dysfunctional-reactive cells (L3, HBL-2, Grant, Raji) Plot for virus susceptibility for both virus and myxoma virus. One; Virus-sensitive. 0; Virus-resistant. Normally-reactive cells are resistant to viral infection, but dysfunctional cells are sensitive to viral infection.
Figure 2C.
The cells were infected with WT / AV reovirus at MOI 40 or myxoma virus (Myx-GFP) at MOI 5. Prior work showed that Raji cells are susceptible to riovirus, and Ramos cells are resistant to riovirus ( Alain et al , 2002 ). Three days after infection, cells were fixed / infiltrated and analyzed by FACS, using riovirus antiserum and secondary FITC antiserum. In the results, both lyovirus and myxoma virus were able to preferentially infect p53 and / or ATM non-reactive lymphoma. In myxoma infection studies, cells were infected for 48 hours and viewed under relative fluorescence microscopy. GFP-expressing cells indicate myxoma virus infection. Mock: False infection.
Degree. 3-Reovirus and Myxoma Virus Infection of Retinoblastoma Cells
Retinoblastoma cells (Y79 and WERI-Rb-I, purchased from ATCC) were infected with WT / AV reovirus at MOI 40 or myxoma virus (Myx-GFP) at MOI 5. At the time point designated after infection, cells were fixed / infiltrated and analyzed by FACS, using Riovirus antiserum and secondary FITC antiserum. In myxoma infection studies, cells were infected for 48 hours and viewed under relative fluorescence microscopy. GFP-expressing cells indicate myxoma virus infection. Mock: False infection.

Carcinogenesis is a multistep process involving multiple iterations of mutual oncogene activation and inactivation of tumor suppressor genes through mutant events that affect normal primary oncogenes and tumor suppressor genes. Interestingly, oncolytic viruses use different oncogene-driven cell line signaling pathways to selectively replicate and kill the cells they infect. In addition to oncogene-dependent oncolysis, the inventors thought that some tumor suppressor genes would play an important role in carrying out tumor lysis by viruses. Therefore, these inventors regulated tumor suppressor genes of various murine and human cancer cell lines to test whether tumor suppressor gene dysfunction affects lyovirus and myxoma virus oncolysis.

Efforts to characterize cellular receptors for riovirus have advanced the molecular basis of riovirus oncolysis. In 1993, two mouse cell lines without the expression of epidermal growth factor receptors (EGFRs) were reported to be relatively resistant to riovirus infection, and that the same cell lines transfected with EGFR-encoding genes showed significantly higher sensitivity. Strong et al , 1993 ). As a result, it has been suggested that lioviruses utilize activated cellular signal transduction pathways imparted by the presence of EGFR functioning in host cells. After that, when these same researchers have shown that the resistance to NIH3T3 Rio virus infected cells transformed with an activated Sos, Ras or verbB in the downstream of the EGFR, were able to describe that the sensitivity (Strong et al , 1996; Strong et al , 1998 ). Thus, development of oncogenic Ras signaling pathways is considered a critical step in lyovirus oncolysis.

Many cancers in humans demonstrate enhanced Ras pathway activation, highlighting the potential effects of this signaling network on tumorigenesis, and suggesting that rioviruses will exert broad oncolytic capacity in various tumor types. Initial experiments have shown that more than 80% of cell lines from various tumor types are sensitive to riovirus-mediated killing ( Coffey et al , 1998 ). More interestingly, another oncogene, Myc, also contributes to lyovirus oncolysis. Myc overexpression permits riovirus permissiveness to riovirus resistant cells ( Egan et al, 2003 ).

Myxoma virus is a rabbit-specific poxvirus that is considered a promising oncolytic virus basis ( Stanford). and McFadden , 2007 ). Its host tropism is significantly limited to European rabbits and is non-pathogenic for all other vertebrate species, including humans ( McFadden , 2005 ). Despite this limited specificity, myxoma viruses can infect and kill a wide variety of human tumor cell lines ( Sypula et al , 2004 ). Myxoma virus affinity at the cellular level is usually regulated by downstream intracellular events of viral binding and influx, rather than at specific water receptor receptor levels ( McFadden , 2005 ).

Through the study of viral host range factors that control virus tolerance in human cancer cells, the molecular basis of myxoma virus oncolysis has been advanced. Viral M-T5 host range protein interacts with cellular Akt, and this interaction enhances myxoma oncolytic ability in human cancer cells ( Wang et al , 2006 ). M-T5 KO (knock-out) myxoma virus exhibited attenuated viral oncolytic capacity in a class of human cancer cells (type II cancer cells), while constitutive Akt activated cancer cells were M-T5 KO myxoma virus. and a still acceptable, it has been proposed up-regulation of Akt that is frequently found in many cancer cells that play an important role in determining the solubility myxoma tumors capacity (Wang et al , 2006 ).

Adenovirus studies have led to the first progress in the relationship between viral oncolysis and tumor suppressor genes. The human adenovirus ElB gene encodes a 55 kD protein that binds to and inactivates the cellular tumor suppressor protein p53. Mutated adenoviruses that do not express such viral proteins replicate in p53 dysfunctional human tumor cells and can kill them, but not in functional p53 ( Bisschoff). et al , 1996 ). Basis for the mutated adenoviral oncolytic is that due to a defect or mutant p53, at least p53 function in the cancer cells are adenovirus-induced E1A- p53- dependent oh paptop system not to function (Bischoff et al , 1966 ). However, subsequent studies have shown that p53-dependent adenovirus oncolysis is more complex and responsive to additional pathways in various models ( O'Shea et al , 2004; Abou et al , 2004; Royds et al , 2006 ). In fact, this led the inventors to think that p53 dysfunctional cancer cells could disrupt cellular anti-viral mechanisms through genomic instability. ( Carroll et al ., 1999 ). Because of the importance of genome maintenance by p53, the p53 gene is considered a guardian of the cellular genome ( Gomez - Lazaro et al , 2004; Vogelstein et al , 2000 ). Because of p53-mediated genomic maintenance, normal cells may be able to maintain their own anti-viral mechanism. However, p53 dysfunction will ultimately lead to viral susceptibility (via anti-viral preexisting loss) caused by overall genomic instability. In a recent study, it was found that p53 contributes to intrinsic immunogenicity by enhancing IFN-dependent anti-viral activity as a pro-apoptotic and tumor suppressor gene ( Takaoka). et al , 2003; Munoz - Fontela et al , 2008 ). The p53 transcription role is important for activating the IFN pathway in viral infections. p53 can activate the transcription of IFN regulatory factor 9 upon viral challenge ( Munoz- Fontella et al. al , 2008 ). Moreover, when p53 expression was reduced in cancer cells, significantly higher levels of viral replication were observed ( Dharel et al , 2008 ). These findings explain the significant antiviral activity of p53. This may be true for other tumor suppressor genes that are important for DNA repair and genomic stability. Here, the inventors have shown that cancer cell dysfunction at p53, ATM or Rb is highly sensitive to both lyovirus and myxoma virus challenge, suggesting that sensitivity may be enhanced by activation of specific tumor gene signaling pathways. In addition to this, it may help to outline how oncolytic virus distinguishes normal cells from cancer cells. The identification of a link between this genetic abnormality and oncolytic virus susceptibility greatly increases the likelihood of applying oncolytic virus therapy.

I. Oncolytic Viruses

A. Rio virus ( Resoviridae )

Reoviruses comprise a naturally occurring, non-enveloped viral family that is divided into 10 fragments and has a double-stranded RNA (dsRNA) genome surrounded by two concentric icosahedral protein capsids. These viruses can affect the gastrointestinal system and respiratory tract. The leoviride name comes from “respiratory enteric orphan virus”. An “orphan” virus refers to a virus that is not associated with any known disease. Although the virus in the Reoviridae family has recently been identified as having various diseases, the original name is still used.

Riovirus infections are common in humans, but most are mild or subclinical. The virus can be easily detected in feces but can also be recovered from throat or nasal secretions, urine, cerebrospinal fluid, and blood. Although lyoviruses can easily be found in clinical specimens, their role in human disease or therapy is still unclear. Reovirus has a icosahedral capsid (T-13) consisting of a non-enveloped and outer and inner protein shell. Rioviride's viral genome contains 10-12 fragments, which are divided into three classes according to size: L (large), M (medium), and S (small). Fragments range from 3.9-1 kB and each fragment encodes 1-3 proteins. The rioviride protein is represented by the Greek letter corresponding to the fragment to which it is translated (L fragment encodes λ protein, M fragment encodes μ protein, and S fragment encodes σ protein ).

Because these viruses have a dsRNA genome, replication occurs exclusively in the cytoplasm, and the virus encodes several proteins that are required for replication and the dsRNA genome to be converted into (+)-RNAs. The virus can enter the host cell through receptors on the cell surface. The receptor is unknown, but is considered to include sialic acid and conjugated adsorption molecules (JAM). The virus is partially stripped by proteases in the endolisosome, in which the capsid is partially digested for further entry into the cell. The core particle then enters the cytoplasm by a process that has not yet been identified, in which the genome is conservatively transcribed, resulting in an excess of the positive sense strand, which becomes an mRNA template and the negative sense strand is synthesized. do. Viral particles begin assembly in the cytoplasm within 6-7 hours after infection.

Infectious mammalian riovirus virions are produced as particles about 85 nm in diameter. The virion outer capsid contains several distinct protein species, including sigma-1 (σ1, 50 kDa) through individual carbohydrate bonds ( Chappell et al , 1997; Chappell et al, 2000; Connolly et al , 2001 ), and the virion-anchoring domain ( Mah et al , 1990; Fernandes et al , 1994; Lee et al , 1994 ) mediating the attachment of viruses to the surface of host cells ( Lee et al , 1981; Duncan et al , 1991; Nagata et al , 1987; Turner et al , 1992 ). σ1 is a product of the bicistronic riovirus S1 gene, which also encodes a non-structural protein named σ1s using a separate but overlapping reading frame ( Ernst et al , 1985; Jacobs et al , 1985; Sarkar et al , 1985 ). Riovirus particles without σ1 have been reported as non-infectious ( Larson et al , 1994 ). The riovirus S1 gene appears to play a significant role in determining riovirus pathogenicity ( Haller et al , 1995; Wilson et al, 1994; Kaye et al , 1986; Werner et al , 1980 ).

i. Wild type in cancer therapy Rio virus

Specific methods for the production and use of wild type lioviruses in cancer treatment have been described (e.g., US Pat. 6,136,307, and 6,110,461, and US Patent Publications 2006/0165724, 2006/0073166, 2005/0123513, 2004/0265271, 2004/0126869, 2004/0109878, 2002/0037543, 2006/0029598, 2005/0026289, 2002/0006398, and 2001/0048919, each of which is incorporated by reference in its entirety without abstention).

ii . In cancer therapy Attenuated Rio virus

In hosts that fail the immune system, such as new born animals and SCID (severe combined immunodeficiency) animals, wild-type riovirus exerts significant viral etiology , particularly in neural and cardiac muscle tissues ( Sabin , 1959; Weiner). et al , 1977; Baty et al , 1993; Loken et al , 2004 ). In some cases, immune-functioning hosts, including humans, have been associated with viral pathogenesis ( Terheggen et al , 2003, Hirasawa et al , 2003 ). Thus, in nonimmune or very young hosts, wild-type riovirus does not always work in a good manner. For example, in nonimmune hosts such as very young or immunodeficient mature animals, the virus has also been shown to infect some healthy tissues such as the heart, liver, pancreas, and nervous structures. Such issues may be further applied to cancer patients treated with strong radiotherapy / chemotherapy, which should be immunosuppressed. Thus, there is clearly a need to develop malignant attenuated lioviruses for viral oncolytic therapy.

Moreover, in many cases, wild-type virus in Rio It possesses undesirable virulence and infectivity that limits their use in vitro . In particular, exposure of cell cultures (eg, bone marrow implants taken from cancer patients) to wild-type riovirus may cause undesirable consequences for the cancer system's immune system to kill cells such as bone marrow cells again. Thus, the enhanced pathogenesis of riovirus is associated with exposing cells to riovirus in Significantly limits clinical applicability in vitro .

The attenuated riovirus described herein lacks a detectable full-length σ1 capsid protein, but is still unexpectedly infectious, and σ1 binds and attaches riovirus to cells via cell surface sialic acid residues at an early stage of viral replication infection. Involved in Lee et al ., 1981; Duncan et al ., 1991; Nagata et al , 1987; Turner et al , 1992; Chappell et al , 1997; Chappell et al , 2000; Connolly et al , 2001 ). Despite the lack of σ 1 of full length, the attenuated lyoviruses described herein are capable of host cell entry and cytolytic virus replication. Additionally, the attenuated riovirus is one or more on non-malignant cells as compared to the level of cytopathic effect seen on non-malignant cells by naturally occurring, non-damped rioviruses. Showing surprising properties that lead to reduced levels of cytopathic effect (eg, statistically significant reduction). Thus, as described in more detail below, the attenuated lioviruses provided herein provide improvements over existing rioviruses, which improve the tropism and cellularity of normal cells (eg, non-malignant cells). It is suitable for use as an oncolytic substance without undesirable side effects such as dissolution. In particular, these attenuated viruses are in Rio for the selective killing of cancer cells or renal cell formation It can also be used in vitro .

In certain embodiments, U.S. Patent application dated August 5, 2006. The attenuated lyovirus described in Application 60 / 704,604 and filed July 31, 2006, described in International Patent Application PCT / US2006 / 029881 (these documents are incorporated by reference in their entirety), can be used in the present invention. In certain embodiments, the attenuated reovirus lacks the wild type Reovirus S1 gene. The attenuated riovirus may carry a S1 gene that produces a reduced and / or undetectable amount of S1 gene product (σ1) or truncated, mutated, and / or dysfunctional σ1. The attenuated riovirus may carry a S1 gene comprising one or more mutations when compared to the wild type S1 gene, where the mutation is a nucleotide substitution, nucleotide deletion or nucleotide insertion.

Wherein the “damped” riovirus is compared to the level of one or more infectious, replicable and / or hemolytic properties against or within the host cell exhibited by known, naturally occurring or wild type rioviruses, These properties include lyoviruses that have been modified (eg, reduced or increased in a statistically significant manner). In certain embodiments, the attenuated riovirus will exhibit reduced infectivity, replication capacity, and / or hemolytic potential as compared to wild type riovirus. Examples of such modified properties that can identify attenuated lyoviruses presented herein include various manifestations of viral cytopathic effects, such as infections required for productive infection of a given host cell. Ratio of multiplicity of infection (MOI, average number of virions infecting each cell), host cell cytolysis induced by viral infection (including apoptosis and / or necrosis), and proliferation after cytolytic virus replication Viral titers released from sexually infected host cells and other variables familiar to the art that can measure viral activity on host cells. Other manifestations of the cytopathic effect include altered host cell morphology, modified ability to induce attack by the host cell-mediated immune system, or to substances such as extracellular matrix proteins or semi-solid growth media or to other cells. Other modifications in cell adsorption, modified expression levels of one or more cellular genes, modified ability of the host cell to replicate, and / or cellular metabolic activity.

Considering that the S1 gene fragment of riovirus is strongly associated with the virulence (virulence) of riovirus ( Weiner et al , 1977; Weiner et al , 1980; Mann et al , 2002 ) The inventors surprisingly isolated S1 attenuated riovirus (“AV riovirus”) from sustained riovirus infection of cultured cells ( Kim. et al , 2007 ). Attenuated riovirus strains in It exhibits significantly reduced viral pathogenesis in immune-deficient animal models without compromising its viral oncolytic activity in vivo . Since wild-type riovirus is known to adversely affect the development of murine and murine embryos by retarding and inhibiting blastocytst development in rats and murine embryos ( Prisscott , 1983; Heggie et al , 1979 ), the inventors further assessed the pathogenicity of AV riovirus on stem cells. Thus, the inventors compared the pathogenicity of wild type riovirus and AV riovirus on embryonic stem cells (ESCs). As shown in the examples below, the wild type riovirus is in Although ESCs are easily infected in vitro and significantly inhibited stem cell development in teratoma models, AV riovirus infects ESCs minimally in vitro and does not affect stem cell development in teratoma models.

In various embodiments, the attenuated reovirus may comprise one or more additional mutations. For example, in further specific embodiments, the attenuated riovirus may lack the wild type riovirus S4 gene. Riovirus wild type S4 gene encodes a Riovirus capsid σ3 polypeptide involved in virion processing during Riovirus proliferative infection of a host cell (eg, Ahmed et al , 1982; Giantini et al , 1984 ).

Attenuated reovirus may comprise a replication-competent reovirus virion. Moreover, attenuated lioviruses may have genetic mutations (eg, substitutions, insertions, deletions) in the S1 gene and / or the S4 gene. Wild-type riovirus S1 genes are known to those skilled in the art. For example, the wild type S1 gene sequence may comprise a S1 gene sequence identified in predominant forms of naturally-occurring riovirus isolated from respiratory or intestinal tissue of an infected individual, or a consensus sequence derived from such a sequence. (consensus sequences). S1 gene sequences for many rioviruses, including human rioviruses, including polynucleotide sequences encoding σ1 protein have been identified (eg, Genbank Accession numbers for human riovirus S1 gene sequences; human type 3 riovirus S1 : Genbank Accession numbers for the amino acid sequence of the encoded σ1 protein itself (eg, the major human Riovirus serotype S1 gene sequence) as well as the XOl 161; human type 2 liovirus S1: M35964; human type 1 riovirus S1: M35963) : Human type 1 riovirus strain Lang (TlL) Ace.No.M35963; human type 2 riovirus strain Jones (T2J) Ace.No.M35964; human type 3 riovirus strain Dealing (T3D) Ace.No.XOl 161; Human type 3 riovirus strain Abney (T3A) Ace.No. L37677).

According to certain embodiments, an attenuated riovirus comprising a riovirus genome lacking a wild type riovirus S1 gene is provided, or comprises a riovirus S1 gene that is mutated such that it cannot encode a native riovirus σ1 capsid protein Attenuated reovirus is provided (human reovirus S1 gene sequence Genbank Accession numbers: human type 3 reovirus S1: X01161; human type 2 reovirus S1: M35964; human type 1 reovirus S1: M35963). By sequencing of the lyovirus S1 gene, a method of determining whether a mutated S1 gene is present is evident from the present disclosure, Ausubel et al . (1989); Ausubel et al (1993); Sambrook et al (1989); Maniatis et al . (1982); Glover (1985); Hames and Higgins (1985) ; According to the techniques described in other literature, they are known in the art. Thus, a mutated S1 gene differs in one or more nucleotide sequences by one or more nucleotide substitutions, nucleotide insertions and nucleotide deletions (as can be readily determined) from the nucleotide sequences of the corresponding S1 wild type or consensus sequence. Refers to the S1 gene having a polynucleotide sequence. Several mutations in the murine riovirus S1 gene encoding σ1 protein are described by Hoyt et al. (2005), in accordance with certain embodiments of the invention described herein, the mutation of Hoyt et al is expressly excluded.

As described herein and known in the art, lyovirus outer capsid σ1 protein is based on its biochemical and / or immunochemical properties (eg, Mah et al, 1990; Leone et al, 1991; Chappell). et al, 1997), immunodetection (eg, σ1-specific immunoprecipitation, Western immunoblot analysis, immunoaffinity chromatography, immunofluorescence staining, immunocytofluorimetry, electrophoresis of radiolabeled riovirus polypeptides) Etc.), hemagglutination, and / or related methods, can be detected immediately using one or more techniques. Such and other means for detecting lyovirus σ1 protein have been established and, by the techniques described herein, those skilled in the art will recognize what is acceptable in the art and related technical sensitivity for detecting σ1 protein. For example, a replication-competent Riovirus virion lacking a “detectable” Riovirus σ1 capsid protein would not detect a native σ1 protein when current conventional procedures are used to determine the σ1 protein (if present). It will be understood that the Rio virus does not include.

US Serial No. 11 / 997,537 describes such attenuated lyoviruses, which are incorporated by reference.

iii . Rio virus  Control of infection

In certain embodiments, peptidyl phlolomethylketones (PFMKs) can be used in cell compositions that inhibit or eliminate lyovirus from the composition. Co-Mooring US Provisional Application No. 60 / 906,706 (incorporated by reference in its entirety) describes peptidyl fluoromethylketones (PFMKs) for inhibiting viral replication, which are incorporated by reference in their entirety without abandonment.

iv . Rio virus  production

According to certain embodiments described herein, lioviruses refer to members of the Riobiride family (family), including rioviruses that can be derived from any riovirus and can be obtained from various sources ( Tyler and Fields , 1996 ). In certain embodiments, mammalian rioviruses and human rioviruses are contemplated, but the present invention is not so limited, and, based on the present disclosure, one of ordinary skill in the art would recognize that any particular riovirus would have the desired purpose. In certain embodiments, the riovirus can be human type 3 (Dealing), type 1 (Lang), type 2 (Jones), or type 3 (Abney) riovirus, and in certain other embodiments, the riovirus is human Primates (e.g., chimpanzees, gorillas, macaques, monkeys, etc.), rodents (e.g. mice, rats, gerbils, hemsters, rabbits, guinea pigs, etc.), dogs, cats, general livestock (e.g. Rioviruses derived from one or more rioviruses that exhibit tropism for cells of other mammalian species, including cows, horses, pigs, goats, and the like, or alternatively, have a unique flavor Avian lyovirus) can be used.

As described herein, certain embodiments relate to attenuated rioviruses, which can be recovered after a sustained infection regime in vitro , while attenuated rioviruses are in Generation and identification of σ1-defective and / or σ1-defective mutants by in vivo persistent infection regimen, molecular biological methods (and, in certain embodiments, additionally or alternatively, σ3-defective and / or σ3-defective) Naturally occurring σ1-defective and / or σ1-defective mutant and / or σ3 mutant, and / or such σ1 (and / or σ3) It is contemplated that mutants may be derived according to other methods, including artificial induction by chemical, physical and / or genetic techniques (eg, assortative recombination of riovirus genes in proliferative infected host cells).

Moreover, the inventors have shown that the preferred attenuation phenotypes caused by mutations affect viral infectivity, replication or packaging capacity, including any gene or combination of genes encoded on the virus, such as the ten gene fragments of riovirus. It is expected to be observed in other genes. For example, when compared to riovirus wild-type genes, mutations of the riovirus S1 or S4 gene may be combined with one, two, three, four, five or more mutations on ten gene fragments of the riovirus. Can be associated. To make attenuated reovirus, various methods can be used, including those described in US application 60 / 704,604.

B. Myxoma virus ( Myxoma Virus )

Myxoma virus is a poxvirus and has a large (25 kb) large therapeutically related, large double-stranded DNA genome that allows for potential insertion of eukaryotic genes. Myxoma virus is a rabbit-specific virus and causes a fatal disease called myxoma in European rabbits ( Oryctalagus cuniculus ). This species specificity was so limited that it was used in 1950 to control the devastating population of rabbits that caused catastrophe in Australia. Immortalized baby monkey kidney fibroblasts (BGMK), genetically deficient primary murine cells in the IFN response, and in in comprising a plurality of different human tumor cells in vitro It is possible to proliferately infect cells except certain rabbits in vitro , but importantly, they are non-pathogenic to all other vertebrates tested, including humans. Cancer cells are well known to lack an IFN response. Recently, the myxoma virus in human malignant glioma in surgical specimens in vitro , in vivo and ex It is an oncolytic substance for experimental glioma in vivo ( Lun et al , 2005; 2007; Stanford et al , 2008 ). Myxoma virus can infect human glioma cells, kill them, be safe when administered into the brain, and “treat” mice by administering into tumors in an orthotopic human malignant glioma model. Moreover, all primary glioma cells tested, directly obtained from glioma surgical specimens, are infected and killed, and their oncolytic activity is enhanced by rapamycin.

Like other lyoviruses, along with other oncolytic viruses, myxoma viruses need to bypass the anti-viral defenses present in normal healthy cells so that they can replicate intracellularly. Myxoma viruses and other oncolytic viruses induce interferon production and are generally sensitive to antiviral effects of the IFN pathway. Relevant proteins induced by the IFN anti-viral response and primarily affect viral growth include PKR, OAS synthase and Rnase L nucleases. PKR activates eIF2α to inhibit detoxification and induce apoptosis. In normal cells, myxoma virus is directly affected by PKR and eIF2α.

Anti-viral response pathways are often disrupted in cancer cells. For example, reduced or defective responses to IFNs are genetic defects that commonly occur during modification and tumorigenic processes. More than 80% of tumor cell lines do not respond to interferon or show a damaged response ( 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 Publication 2006/0263333 (supplied by reference) describes the use of myxoma virus to infect and kill cancer cells, including human tumor cells, which is hereby incorporated by reference in its entirety.

II . Tumor suppressor

Tumor suppressor genes are genes that protect cells during one stage of the cancerous process. If this gene is damaged, cells usually progress to cancer after being affected by other factors. Unlike oncogenes, tumor suppressor genes generally follow a two-hit hypothesis, meaning that alleles encoding a particular gene must be affected before the effect becomes apparent. This is because if only one allele for a gene is damaged, the second allele can still produce the correct protein. In other words, in contrast to haploinsufficient oncogenes in general, haplosufficient (a phenotype is normal even if only one gene is normal). Of course, there are obvious exceptions, such as the p53 gene product, which can exist in mutant forms that are dominant-negative for normal p53 proteins, in this case haploid-deficient.

Tumor suppressor genes, or more precisely, the proteins they encode, have either an inhibitory or inhibitory effect on cell cycle regulation or promote apoptosis, and in some cases both. The function of tumor suppressor proteins falls into several categories, including: (a) inhibiting the genes essential for the persistence of the cell cycle (if these genes are not expressed, the cell cycle does not persist and cell division is Effectively inhibited); (b) Associate the cell cycle with DNA damage. As long as there is damaged DNA in the cell, the cell cannot divide (if damage is repaired, the cell cycle can continue); (c) if damage is not repaired, apoptosis or predetermined cell death is induced, thus removing the threat taken for a better organism; And (d) some proteins involved in cell adsorption prevent tumor cells from scattering, block loss of contact inhibition, and inhibit metastasis (proteins known as metastasis inhibitors).

In contrast, oncogenes are genes that cause cancer. Many cells are supposed to die normally. In cancer, these cells survive and proliferate because of the presence of mutated carcinogenic DNA sequences. Most oncogenes require additional steps such as mutations in other genes or environmental factors such as viral infections to cause cancer. Since 1980, dozens of oncogenes have been identified in human cancer. Proto-oncogenes are normal genes that can become oncogenes due to mutation or increased expression. Proto-oncogenes encode proteins that help regulate cell growth and differentiation. Proto-genes are often involved in signal transduction and mitotic signals through their protein products. Upon activation, the proto-oncogene (or product thereof) becomes a tumor inducer, an oncogene. Examples of protogenes include RAS, WNT, MYC, ERK, and TRK.

A. p53

p53 (accession no. NM 000546) is a transcription factor that regulates the cell cycle and functions as a tumor suppressor. This is important in multicellular organisms because it helps inhibit cancer through a partial role in the regulation of cellular responses to DNA damage. p53 has been described as "the guardian of the genome", the "guardian angel gene" or the "master ranger," referring to its role in preserving stability by disrupting genomic mutations.

p53 refers to the surface molecular weight on SDS-PAGE, but is substantially 43.7 kD. This difference is due to the large number of proline residues in the p53 protein, which slow the movement of p53 on SDS-PAGE, making it appear larger. This effect is observed in p53 of various species, including species including humans, rodents, frogs and fish. The gene is located on human chromosome 17 (17pl3.1) and encodes a 393 amino acid protein with five domains: (a) the N-terminal transcription-activation domain (TAD), which is a transcription factor ( Activate residues 1-42); (b) a proline rich domain important for apoptosis activity of p53 (residues 80-94); (c) a central DNA-binding core domain (DBD) comprising one zinc atom and several arginine amino acids (residues 100-300); (d) homo-oligomerized domains (OD) (residues 307-355); And (e) the C-terminal domain (residues 356-393) involved in the down regulation of DNA binding of the central domain.

Mutations that inactivate p53 in cancer usually occur in DBDs. Most of these mutations destroy the ability of a protein to bind its target DNA sequence and thus interfere with the transcriptional activation of these genes. As such, mutations in DBDs are recessive loss-of-function mutations. P53 molecules with mutations in the OD become dimers with wild type p53, interfering with transcriptional activation. Thus, OD mutations have a dominant negative effect on p53 function.

p53 has many anticancer mechanisms. For example, it can activate the DNA repair protein if the DNA has sustained damage. It can also inhibit the cell cycle at the G ₁ / S regulatory point of DNA repair awareness (if the cell is held here for a sufficient time, the DNA repair protein can have enough time to repair damage, Will be able to sustain the cell cycle). In addition, if DNA damage proves to be irreparable, apoptosis, which is scheduled cell death, can be initiated.

p53 can be activated in response to a myriad of types of stress, including DNA damage (induced by chemicals such as UV, IR or hydrogen peroxide), oxidative stress, osmotic shock, ribonucleotide depletion, and uncontrolled tumors Gene expression includes, but is not limited to. Activation stands out with two major events. First, the half-life of the p53 protein is rapidly increased, causing rapid accumulation of p53 in stressed cells. Second, morphological changes make p53 play an active role as a transcriptional regulator in these cells. The decisive event leading to the activation of p53 is phosphorylation of the N-terminal domain of p53. The N-terminal transcriptional activation domain contains a number of phosphorylation sites and is considered the primary target for protein kinases that convert stress signals.

Protein kinases known to target the transcriptional activation domain of p53 can be roughly divided into two groups. The first group of protein kinases belong to the MAPK family (JNK1-3, ERK1-2, p38 MAPK), which respond to several types of stress, such as membrane damage, oxidative stress, osmotic shock, heat shock, etc. Known. The second group of protein kinases (ATR, ATM, Chk1, Chk2, DNA-PK, CAK) are all genomes, molecular cascades that detect and respond to some forms of DNA damage caused by genotoxic stress. It is involved in an integrity checkpoint.

In unstressed cells, p53 levels remain low through sustained degradation of p53. A protein called Mdm2 binds to p53, which transports it from the nucleus to the cytoplasm, where it is degraded by the proteasome. Mdm2-bonds are disrupted by phosphorylation of the N-terminus of p53 by the above mentioned protein kinases. Other proteins, such as Pin1, collect in p53 and further interfere with Mdm2-binding by inducing morphological changes in p53. Transcriptional coactivators such as p300 or PCAF acetylate the carboxy-terminus of p53 and the DNA binding domain of p53 is exposed, allowing p53 to activate or inhibit certain genes.

B. Rb

Retinoblastoma protein (Rb; NM 00321) is a tumor suppressor protein found in dysfunction in many different cancers. When a protein is inactivated by mutations in both alleles of the RB1 gene encoding it, it is named pRb because it results in retinoblastoma cancer. Rb is usually present within the cell as a phosphoprotein and is a target of phosphorylation by several kinases as described below. One fairly studied function of Rb is to prevent cell division or progression through the cell cycle. Thus, ineffectively in this role of Rb, mutated cells can continue to divide and become cancerous.

Rb is a member of the "pocket protein family" because it has a pocket to which proteins are bound. Such carcinogenic proteins made in cells infected with high risk human papilloma virus can bind to and inactivate Rb, which can lead to cancer. Rb prevents the damaged DNA from replicating by preventing the cell from advancing into S or the synthetic phase through the cell cycle, or to the G1 or first gap state. Rb binds to and inhibits transcription factors of the E2F family. E2F transcription factors are dimers of E2F protein and DP protein. The transcriptional activation complex of the transcriptional E2 promoter-binding-protein-dimerization partner (E2F-DP) can push the cells onto S phase. When E2F-DP is inactivated, cells remain on G1. When Rb binds to E2F, the complex acts as a growth inhibitor and prevents it from progressing through the cell cycle. The Rb-E2F / DP complex also attracts histone deacetylase (HDAC) protein to chromatin, further inhibiting DNA synthesis.

In the hypophosphorylated state, Rb is active and acts as a tumor suppressor by inhibiting cell cycle progression. Phosphorylation inactivates Rb. Rb is activated near the end of G1 when phosphatase dephosphorylates one of the residues, allowing binding to E2F. At the time when cells enter phase S, cyclin-dependent kinase (CDK) and cyclin complex phosphorylate pRb, inhibiting its activity. Initial phosphorylation is performed by Cyclin D / CDK4,6, followed by further phosphorylation performed by Cyclin E / CDK2. pRb remains phosphorylated on S, G2 and M. Phosphorylation of pRb separates E2F-DP from pRb and becomes the active form. When E2F is liberated, it activates factors such as cyclins (eg, Cyclin E and A), thereby activating the cells by activating cyclin-dependent kinases and proliferative cell nuclear antigens or molecules called PCNAs. Pushing into the cycles, thereby helping to attach polymerase to DNA, leads to rapid DNA replication and repair.

C. ATM

Capillary dilated ataxia (Ataxia telangiectasia) (AT; NM_00051) is an autosomal recessive disease caused by mutations in the ATM gene located on chromosome llq22-23. Characterized in June 1995, it consists of 66 exons spread over 150 kb of genomic DNA. It encodes an open reading frame of 9168 nucleotides and a 13 kb feature transcript. ATM protein is about 37OkDa, expressed anywhere and located in the cell nucleus. ATM proteins are giant serine-threonine kinases that are thought to play a role in the regulation of cell cycle checkpoints, double stranded DNA and meiosis (similar to the BRCS gene). ATM is also known to play a role in the regulation of p53, BRCA1 and CHEK2. Part of ATM's role in DNA repair is known as telomere repair because telomeres break down more rapidly in people affected by AT. Mutations in the ATM gene are considered to be of two types: (a) null mutations are mutations in which the protein's function is completely lost, thus inheriting in a recessive manner and causing AT; And (b) 'missense' mutations produce whole-sized proteins with reduced functions such as stable, substitutions, short in-frame insertions and deletions. These mutations mainly interfere with the normal copying of the protein. The majority of 65-70% suffering from AT have truncating mutations, with exon skipping mutations being particularly common. This leads to very low or undetectable levels of ATM protein. Missense mutations are the most common type of mutations found in breast cancer carriers. Individuals with two missense mutations appear to have a milder form of AT, which can be described as attenuated AT.

D. Other Tumor Suppressors

Various other tumor suppressors such as p53, ATM and Rb can be used in the present invention. For example, BRCA1 and BRCA2 play a critical role in the development of breast cancer. BRCA1 is a human gene that maintains genomic integrity to prevent unregulated proliferation. Multifactorial BRCA1 protein products are involved in DNA damage repair, ubiquitination, transcriptional control as well as other functions. Variations in these genes have been implicated in several genetic cancers, namely breast cancer, ovarian man and prostate cancer. The BRCA1 gene is located in the band 21 on the long (q) arm of chromosome 17 from base pair 38,449,843 to base pair 38,530,933 (map). BRCA1 protein is directly involved in the repair of damaged DNA. The exact role of this is not yet known, but the BRCA1 protein appears to interact with RAD51 during repair of the DNA double strand break. Such breaks may be caused by natural radiation or other exposures, as well as when chromosomes exchange genetic material during certain cell divisions (meiosis) that create ovaries and sperm. By influencing the repair of DNA damage, this protein plays a role in maintaining the stability of the human genome.

BRCA2 is the second human gene involved in repairing chromosomal damage. Although the structures of the BRCA1 and BRCA2 genes are very different, their functions appear to be similar. Proteins made by both of these genes are essential for repairing damaged DNA. To repair breaks in DNA, the BRCA2 protein binds to and regulates the protein produced by the RAD51 gene. Such breaks may be caused by natural and medical radiation or by other environmental exposures, as well as when chromosomes exchange genetic material during certain cell divisions (meiosis) that create ovaries and sperm. . BRCA1 protein also interacts with the RAD51 protein. By repairing DNA, these three proteins play a role in maintaining the stability of the human genome. Like BRCA1, BRCA2 probably regulates the activity of other genes and plays an important role in the embryonic environment. The BRCA2 gene is located from base pair 31,787,616 to base pair 31,871,804 at 12.3 (13ql2.3) on the long (q) arm of chromosome 13.

MutS is a leading member of a family or protein that supports repair mismatches in double-stranded DNA. The first step in this process is to recognize the mismatched DNA. In E. coli , MutS binds to mismatched sites in double stranded DNA and, by the cooperation of MutL and MutH proteins, targets a portion of one strand of DNA at the site of removal. Other proteins complete the repair process: the target portion is removed, degraded, and complementary strands are used as templates, patches are synthesized, and the synthesized patches are ligated at that location. And have a recovered portion of the double stranded DNA without inconsistency. Much attention is focused on human MutS homologues, since defects in some of them are responsible for some forms of genetic non-polyposis colon cancer (HNPCC) and possibly other colon cancers. Adenomatous polyposis (APC) is another tumor suppressor involved in colon cancer. This helps to determine how often the cell divides, how the cell attaches to other cells in the tissue, or whether the cell migrates in or out of the tissue, and confirms that the number of chromosomes in the cells produced through the cell division is correct Help. APC proteins accomplish this primarily through association with other proteins, particularly those involved in cell adhesion and signaling. The activity of one protein, in particular β-catenin, is controlled by the APC protein, which is part of the Wnt signaling pathway. Regulation of β-catenin prevents the gene that stimulates cell division from turning on immediately and prevents overgrowth of cells. The APC gene is located on the long q arm of chromosome 5 from base pair 112,118,468 between positions 21 and 22 to base pair 112,209,532.

AT-binding transcription factor 1, or ATBFl, is a tumor suppressor, the loss of which is involved in gastric cancer development. It is located at 16q22.3-q23.1. The overall DNA length is 261.32 kB. There are two allotropes ATBFl-A and ATBFl-B, because they use alternating promoters with alternating cuts. The protein is 3703 amino acids in size 404 kDa. This protein contains 23 zinc fingers, 4 DEAD and 1 DEAH boxes, RNA and ATP binding sites, two large RS domains, and multiple phosphorylation sites, including four homodomains and one fake zinc finger motif. The protein is nuclear localized, acts as a transcription factor that binds to the AT-rich core sequence of the enhancer element of the AFP gene, and downregulates AFP gene expression, possibly involved in neuronal differentiation. In the form of extrachromosomal double fine, non-synthetic co-amplification with myc, amplification in the initial neural crest induced cell line SJNB-12. It is observed that there is no ATBF1 expression in gastric cancer cell lines expressing α-fetoprotein. The lack of ATBFl expression is due to strong inhibition at the transcriptional level, not due to mutations, deletions or translocations.

III . Assessment of Tumor Suppressor Structure, Expression, or Function

A. Nucleic Acids Based  Diagnosis( Nucleic Acid - Based Diagnosis )

One embodiment of the present invention includes a method of detecting a variation in the expression of a tumor suppressor. The method may include determining the level of tumor suppressor or determining specific alterations in the expressed product. Standard method ( Sambrook et al , 1989 ), the nucleic acid to be used is isolated from cancer cells. The nucleic acid can be genomic DNA or fractionated or whole cell RNA. If RNA is used, it would be desirable to convert the RNA to complementary DNA. In one embodiment, the RNA is whole cell RNA, and in another embodiment, poly-A RNA. Nucleic acid is amplified normally.

Depending on the format, the particular nucleic acid of interest is identified directly in the sample using amplification, or after amplification as a second known nucleic acid. The identified product is then detected. In certain uses, detection can be performed by visual means (eg, ethidium bromide staining of the gel). Alternatively, the detection may be to identify the product indirectly via chemiluminescence, radioactive synthography or fluorescent labels of radiolabels or via electrical or thermal pulse signals ( Affymax Technology ; Bellus , 1994 ). In one embodiment, the amount of tumor suppressor expressed is determined by assessing the amount of mRNA. However, by examining various types of structural defects, changes in activity may be identified. Such defects include deletions, insertions, point mutations and duplications. Point mutations result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations occur in non-germ cell tissue and are not inherited, while germ cell mutations are inherited. Mutations within and outside the coding portion may also affect the amount of tumor suppressor produced by altering the transcription of the gene or by destabilizing the transcript (mRNA) or protein or by altering these processes.

Cells take genetic measures towards carcinogenic modifications when the allele of one of the tumor suppressor genes is inactivated due to inheritance of germline lesions or acquisition of somatic mutations. Inactivation of another allele of the gene is generally associated with somatic micromutation or chromosomal allele deletions, resulting in heterozygous loss (LOH). Alternatively, all copies of the tumor suppressor gene may be lost by homozygous deletions.

Fluorescence in situ (FISH), direct DNA sequencing, PFGE analysis, Southern or Northern blotting, single-stranded morphology analysis (SSCA), RNAse protection assay, allele-specific oligonucleotide (ASO), dot blot analysis Various assays are contemplated, including but not limited to denaturation gradient gel electrophoresis, RFLP and PCR ™ -SSCP. .

i. With primer Probe

As defined herein, a primer is meant to include any nucleic acid capable of priming initial nucleic acid synthesis in a template-dependent process. Generally, primers are oligonucleotides of 10 to 20 base pairs in length, but longer sequences can be used. Primers may be provided in double-stranded or single-stranded form, but single-stranded forms are preferred. Probes can act as primers but are defined differently. Probes can be triggered, but are designed to bind to target DNA or RNA and need not be used in the amplification process. In certain embodiments, the probe or primer is labeled with a radioactive species ( ³² P, ¹⁴ C, ³⁵ S, ³ H, or other label), fluorescent (rhodamine, floresin) or chemiluminescent (luciferase). .

ii . Template dependency amplification method

A number of template dependency processes are used to amplify marker sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (called PCR ^TM ), described in detail in US Pat. Nos. 4,683,195, 4,683,202, 4,800,159, and Innis et al, 1990, each of which is incorporated by reference in its entirety. Attached. Briefly, in PCR ™, two primer sequences are prepared that are complementary to portions on opposite complementary strands of the marker sequence. An excess of deoxynucleotide triphosphate is added to the reaction mixture with DNA polymerases such as Taq polymerase. If the marker sequence is present in the sample, the primer will bind to the merker and the polymerase will stop the primer from extending as nucleotides are added along the marker sequence. By raising and lowering the temperature of the reaction mixture, the extended primer will dissociate from the marker to form the reaction product, and excess primer will bind to the marker and the reaction product, and the process is repeated.

Reverse transcriptase PCR ™ amplification procedures can be performed to quantify the amount of mRNA amplified. Methods of reverse transcription of RNA into cDNA are well known, and Sambrook et al , described in 1989 . An alternative method for reverse transcription utilizes thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641 filed December 21, 1990. Polymerase chain reaction methods are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”), which is described in EPO No. 320 308, which is incorporated by reference in its entirety. In LCR, two complementary primer pairs are prepared and, in the presence of the target sequence, each pair will bind to opposite complementary strands of the target sequence. In the presence of ligase, two probe pairs will be connected to form a single unit. As in PCR ™, by temperature cycling, the bound ligated units dissociate from the target, acting as a “target sequence” for ligation of excess probe pairs. U.S. Patent 4,883,750 describes a method similar to LCR involving the binding of a probe pair to a target sequence.

PCT Application No. Qbeta Replicase, described in PCT / US87 / 00880, may also be used in another amplification method of the present invention. In this method, the proliferative sequence of RNA having a portion complementary to the target portion is added to the sample in the presence of RNA polymerase. This polymerase will copy the proliferative sequence to be detected.

Isothermal amplification method—This method utilizes restriction endonucleases and ligases to target molecules containing nucleotide 5 ′-[alpha-thio] -triphosphate on one strand of the restriction enzyme site. Amplification takes place-it may also be useful for amplifying the nucleic acids of the present invention ( Walker) et al , (1992) ).

Strand Displacement Amplification (SDA) is another method of performing isothermal amplification of nucleic acids, involved in multiple cycles of multiple strand substitution and synthesis, such as nick translation. A similar method called Repair Chain Reaction (RCR) involves a repair reaction where several probes are annealed in the targeted portion for amplification, followed by only two of the four bases. The other two bases will be added as biotinylated derivatives to facilitate detection. Similar methods are used for SDA. Target specific sequences may also be detected using a cyclic probe reaction (CPR). In CPR, probes with 3 'and 5' sequences of non-specific DNA and with intermediate sequences of specific RNAs are hybridized to the DNA present in the sample. In 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 native template is annealed to another circulating probe and the reaction is repeated.

GB application No. 2 202 328, and PCT application. Another amplification method described in PCT / US89 / 01025 (these documents are incorporated by reference in their entirety) can be used in accordance with the present invention. For these GB applications, "modified" primers are used for PCR ™ -like, template and enzyme-dependent synthesis. Primers can be labeled and modified with a capture moiety (eg biotin) and / or a detection moiety (eg enzyme). In the case of the international application, an excess of labeled probe is added to the sample. In the presence of the target sequence, the probe is bound and cleaved by the catalyst. After cleavage, the target sequence is released in native form bound by excess probes. Cleavage of the labeled probe indicates that the target sequence is present.

Other nucleic acid amplification processes include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR ( Kwoh et al ., 1989; Gingeras et al , PCT Application WO 88/10315 , which are incorporated by reference in their entirety). In NASBA, nucleic acids for amplification can be prepared by minispin columns for standard phenol / chloroform extraction, thermal denaturation of clinical samples, treatment with lysis buffer, and separation of guanidium chloride extracts of DNA and RNA or RNA. Such amplification techniques include annealing of primers with target specific sequences. After polymerization, the DNA / RNA hybrid is digested with RNase H, and the double stranded DNA molecules are again denatured. In either case, single stranded DNA is completely double-stranded by the addition and polymerization of a second target specific primer. Double-stranded DNA molecules are then multiple transcribed by RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, RNA is reverse transcribed into single stranded DNA, converted back into double stranded DNA, and transcribed again by an RNA polymerase such as T7 or SP6. The resulting product, whether truncated or intact, exhibits target specific sequences.

Davey et al ., EPO No. 329 822 (which is incorporated by reference in its entirety) describes a nucleic acid amplification process that involves cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA). This may be used according to the present invention. ssRNA is a template of the first primer oligonucleotide, which is stretched by reverse transcriptase (RNA-dependent DNA polymerase). RNA is then removed from the resulting DNA: RNA duplex by the action of ribonuclease H (RNase H, RNase specific to RNA, if duplexed with DNA or RNA). The resulting ssDNA is the template of the second primer and also comprises the sequence of the RNA polymerase promoter (exemplified by T7 RNA polymerase) at the 5 'position, homologous to the template. This primer is extended by a DNA polymerase (exemplified by the "Klenow" large fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA ("dsDNA") molecule, which is a primer Between them have the same sequence as the native RNA, and at one end additionally the promoter sequence, which is used by appropriate RNA polymerase to make many RNA copies of the DNA. Can reenter the cycle, leading to very rapid amplification By selecting the appropriate enzyme, such amplification can be performed isothermally without the addition of an enzyme in each cycle. The starting sequence can be selected in the form of DNA or RNA.

Miller et al , PCT application WO 89/06700 (which is incorporated by reference in its entirety), is based on hybridization of promoter / primer sequences to a single stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. Describe nucleic acid sequence amplification schemes. This process is not circulated, for example, no new template is made from the resulting RNA transcript. Other amplification methods include "RACE" and "one-sided PCR ™" ( Frohman , 1990; Ohara et al , 1989 ; Each document is incorporated by reference in its entirety.

Methods based on the ligation of two (or more) oligonucleotides in a nucleic acid having the sequence of the "di-oligonucleotides" produced thereby and amplification of the di-oligonucleotides may also be used in the amplification step of the present invention. Can Wu et al , (1989) , incorporated by reference in its entirety).

iii . Southern / Northern Blotting ( Southern Of Northern Blotting )

Blotting techniques are those well known to those skilled in the art. Southern blotting uses DNA as a target, while northern blotting involves using RNA as a target. Although each provides different forms of information, cDNA blotting is similar to blotting or RNA in many respects.

In brief, probes are used to target DNA or RNA species immobilized on appropriate matrices, often on nitrocellulose filters. Different species must be separated spatially for analysis. This is often done by gel blotting of nucleic acid species followed by “blotting” on the filter.

The blotted target is then incubated with the 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 rerenating conditions. The unbound probe is then removed and detection is performed as described above.

iv . Separation method ( Separation Methods )

In one step or another, it is usually desirable to separate the amplification product and excess primer from the template for the purpose of determining whether a particular amplification has occurred. In one embodiment, the amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods ( Sambrook et al . (1989) .

Alternatively, chromatographic techniques can be used to separate. There are many types of chromatography that can be used in the present invention: there are many specialized techniques using them, including adsorption, partitioning, ion-exchange and molecular sieve, and column, paper, thin layer and gas chromatography ( Freifelder , 1982 ).

v. Detection method ( Detection Methods )

Products can be visualized to confirm amplification of the marker sequence. One typical visualization method involves coloring the gel with ethidium bromide and visually identifying it under UV light. Alternatively, if the amplification products are labeled integrally with radioactive or fluorescently labeled nucleotides, the amplification products are visualized and separated on the x-ray film or under an appropriate stimulus spectrum.

In one embodiment, the visualization is indirect. After isolation of the amplification product, the labeled nucleic acid probe is contacted with the amplified marker sequence. Probes are preferably conjugated to chromophores that 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 is a detectable moiety.

In one embodiment, the detection is by a labeled probe. The related art is well known to those skilled in the art and can be found in many standard books on molecular protocols. For example, chromophore or radiolabeled probes or primers identify targets during or after amplification.

One example of the foregoing is the U.S. Described in patent 5,279,721, which is hereby incorporated by reference, which describes an apparatus and method for automated electrophoresis and nucleic acid transfer. This device allows for blotting without electrophoresis and external manipulation of the gel and is well suited for carrying out the methods according to the invention.

In addition, the amplification products described above may be subjected to sequencing to identify specific kinds of variations using standard sequencing techniques.

In certain methods, thorough analysis of genes is performed by sequencing using primer sets designed for optimal sequencing (Pignon et al, 1994). The present invention provides a method in which some or all of these types are used. Using the sequences described herein, oligonucleotide primers can be designed to allow amplification of sequences that can be analyzed by direct sequencing.

vi . Kit  Elements ( Kit Components )

All basic materials and reagents required for the detection and sequencing of the gene of interest can be grouped together in a kit. The kit will generally include preselected primers and probes. Also included are enzymes and buffers suitable for nucleic acid amplification including various polymerases (RT, Taq, Sequenase ™ etc.), deoxynucleotides to provide the necessary reaction mixture for amplification. Such kits will generally include, in appropriate means, separate containers for each reagent and enzyme and containers for each primer or probe.

vii . Relative quantitative RT - PCR ™ Design and Theoretical Review

Following reverse transcription of RNA to cDNA (RT), relative quantitative PCR ™ (RT-PCR ™) can be used to determine the relative concentration of specific mRNA species isolated from patients. By confirming that the concentrations of specific mRNA species vary, the gene encoding the specific mRNA species is differentially expressed.

In PCR ™, the number of molecules of amplified target DNA is increased by a factor approaching each cycle and two of the reactions until some reagents are limited. Thereafter, the amplification rate is gradually reduced until there is no increase in the amplified target between cycles. For a graph where the number of cycles is plotted on the X axis and the log concentration of the amplified target DNA is plotted on the Y axis, the plotted points are joined to form a characteristic shaped curve. At the beginning of the first cycle, the slope of the line is positive and constant. This is the linear part of the curve. After the reagents are constrained, the slope of the line begins to decrease and eventually goes to zero. At this point, the concentration of amplified target DNA is close to some fixed value. This is the plateau portion of the curve.

The concentration of target DNA in the linear portion of PCR ™ amplification is directly proportional to the starting concentration of the target prior to the start of the reaction. By determining the concentration of the amplified product of the target DNA in a PCR ™ reaction completed in the same number of cycles and within a linear range, it is possible to determine the relative concentration of a particular target sequence in the original DNA mixture. If the DNA mixture is cDNA synthesized from RNA isolated from different tissues or cells, the relative abundances of the particular mRNA from which the target sequence is derived can be determined for that tissue or cell. This direct proportion between the concentration of PCR ™ products and the relative mRNA abundances fits only in the linear range of the PCR ™ reaction.

The final concentration of target DNA in the plateau portion of the curve is determined by the reagent availability in the reaction mixture, and is independent of the native concentration of the target DNA. Thus, for the collection of RNA populations, the first condition that must be met before RT-PCR ™ is determined by the presence of mRNA species is that the concentration of amplified PCR ™ products is when the PCR ™ reaction is in the linear portion of the concentration curve. It must be sampled.

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

Most protocols for competitive PCR ™ use approximately enriched internal PCR ™ standards as targets. Such strategies are effective when the product of PCR ™ amplification is sampled on a linear phase. If the products are sampled when the reaction is close to the plateau, less abundant product will appear relatively. As in the case of examining RNA samples for differential expression, comparisons of relative abundances made for many different RNA samples are less than those that are substantially present in the relative abundance of RNA when examining RNA samples for differential expression. The differences that appear are distorted. This is not a serious problem if the internal standard is much larger than the target. If the internal standard is richer than the target, a direct linear comparison can be made between RNA samples.

The above discussion explains what should be considered theoretically in RT-PCR ™ analysis for clinically derived materials. Problems inherent to clinical samples are that the sample volume is variable (which is problematic for standardization), and that the sample quality is variable (needs to co-amplify with a reliable internal standard, preferably larger than the target). Is called. All of these problems are overcome when RT-PCR ™ is implemented with relative quantitative RT-PCR ™ with an internal standard, where the internal standard is a larger amplifiable cDNA fragment than the target cDNA fragment and the mRNA encoding the internal standard. Abundance is approximately 5-100 times higher than mRNA encoding the target. This analysis does not measure the absolute abundance of each mRNA species, but rather the relative abundance.

Other studies can also be carried out using a more convenient relative quantitative RT-PCR ™ assay in conjunction with an external standard protocol. These assays sample PCR ™ products in the linear portion of the amplification curve. The number of PCR ™ cycles that are optimal for sampling should be determined empirically for each target cDNA fragment. In addition, each RNA population reverse transcriptase product isolated from various tissue samples should be carefully standardized for the same concentration of amplifiable cDNA. This consideration is very important because the assay measures absolute mRNA abundance. Absolute mRNA abundance can be used as a measure of differential gene expression only in standardized samples. Empirical determination of the line portion of the amplification curve and standardization of cDNA preparations are a tedious and time consuming process, but consequently RT-PCR ™ analysis is superior to that derived from relative quantitative RT-PCR ™ analysis using internal standards. something to do.

One reason for this advantage is that, without internal standards / competitors, all reagents can be converted into a single PCR ™ product in the linear portion of the amplification curve, increasing the sensitivity of the assay. Another cause is just one PCR ™ product, where the display or other display method of the product on an electrophoretic gel is somewhat less complicated, has less background, and is easier to interpret.

viii . Chip technology ( Chip Technologies )

Particularly considered by the inventors is Hacia et al . (1996) and Shoemaker et al . Chip based DNA technology such as described in (1996) . In short, these techniques include quantitative methods for the rapid and accurate analysis of multiple genes. By tagging oligonucleotides into a gene or using immobilized probe arrays, one skilled in the art uses chip technology to isolate target molecules into high density arrays and screen these molecules based on hybridization. Pease et al . (1994); Odor et al . (1991) .

B. Immunodiagnosis ( Immunodiagnostics )

Antibodies of the invention are used to characterize tumor suppressor content in healthy and diseased tissues through techniques such as ELISA and western blotting. Antibodies can provide a screen to identify malignant presence or an indicator of future cancer or a strategy for predicting possible responses to exposure to oncolytic viruses in the case of the present invention.

The use of the antibodies of the invention in ELISA assays is contemplated. For example, anti-tumor inhibitor antibodies are immobilized on selected surfaces, preferably on surfaces that exhibit protein affinity, such as wells of polystyrene microtiter plates. After washing to wash off incompletely adsorbed material, the assay plate wells are bound or coated with a nonspecific protein known antigenically neutral to test antiserum, such as bovine serum albumin (BSA), casein or milk powder solution. It is preferable. This blocks non-specific adsorption sites on the immobilized surface, thereby reducing the background caused by non-specific antigen binding on the surface.

After the antibody is bound to the well, the surface to be contacted with the sample to be tested is contacted with a non-reactive material to reduce background, wash away the unbound material, and induce immune complex (antigen / antibody) formation. do.

The occurrence and amount of immunocomplex formation after specific immunocomplex formation between the test sample and the antibody bound, and subsequent washing, may be determined as a second antibody that differs from the first antibody but has specificity for the tumor suppressor. . Suitable conditions preferably include diluting the sample with dilutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered salt (PBS) / Tween®. These added materials help to reduce the non-specific background. The layered antiserum is preferably incubated at a temperature of about 25 ° C. to about 27 ° C. for about 2 to about 4 hours. After incubation, the antiserum-contacted surface is washed to remove non-immunocomplexed material. 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 may preferably have an appropriate chromogenic substrate and associated enzymes capable of developing color upon incubation. Thus, for example, one of skill in the art would know that a urease or periphery on a second antibody-bound surface is under time and conditions favorable to immune complex formation (incubation at room temperature with a PBS containing solution such as PBS / Tween® for 2 hours). Oxidase-conjugated anti-human IgG can be contacted and incubated. After incubation with the second enzyme-tagged antibody, it is washed to remove unbound material in succession, and urea, bromocresol purple or 2,2'-azino-di- (3-ethyl-benzthiazoline)- Label amounts are quantified by incubation with 6-sulfonic acid (ABTS) and enzyme labels with a chromogenic substrate such as H ₂ O _{2 for} peroxidase. Color generation is measured using a visible spectrum spectrophotometer to quantify.

The former format may be altered by first binding the sample to the assay plate. The primary antibody is then incubated with the assay plate and the bound first antibody is detected using a labeled second antibody that is specific for the primary antibody.

Antibody compositions of the invention will have significant use in immunoblot or western blot analysis. Antibodies can be used with high-affinity primary reagents for the identification of proteins immobilized on solid support matrices such as nitrocellulose, nylon or combinations thereof. In combination with immunoprecipitation, after gel electrophoresis, these antibodies can be used as a single step reagent for use in antigen detection against the second reagents used for antigen detection against causing adverse backgrounds. In this regard, it is contemplated that immunologically based detection methods that are used in combination with Western blots that include a second antibody against enzymatically radiolabeled or fluorescently-tagged tumor suppressor are used specifically.

IV . Anti-hyperproliferative treatment ANTI - HYPERPROLIFERATIVE THERAPIES )

Hyperproliferative diseases are a group of diseases characterized by abnormal or uncontrolled cell proliferation. These diseases generally fall into two categories: more generally cancer or “malignant disease” and less common benign hyperproliferative diseases such as benign prostatic hyperplasia, benign epithelial hyperplasia of the breast, endometrial hyperplasia, hyperthyroidism, and skin Or epidermal hyperplasia of the dermis. While many aspects of the following discussion focus on cancer therapy, it will be appreciated that the same appropriate methods can be applied to benign diseases.

In general, in contrast to benign diseases with abnormal cell numbers, cancer refers to the presence of dysplastic, neoplastic, or otherwise proliferative, carcinogenic, and / or transformed cells in an individual. , Tumors, non-contact inhibited or carcinogenic transformed cells or similar known in the art and with established criteria for diagnosis and classification (eg melanoma, adenocarcinoma, squamous cell carcinoma, small cell carcinoma, Carcinomas such as oat cell carcinoma, sarcoma such as fibrosarcoma, chondrosarcoma, osteosarcoma, liver tumor, neuroblastoma, melanoma, lymphoma, hematopoietic cancer such as leukemia, myeloma, etc.). Without wishing to be bound by any theory, the oncolytic properties of rioviruses are defined herein or in environments susceptible to, for example, damaged PKR phosphorylation in Ras-activated cells, as described in the art. As can be seen, it can be derived from viral preference for malignantly transformed cells in cooperation with an environment having defective tumor suppressor function.

A. Oncolytic virus treatment Oncolytic Viral Therapies )

Specific methods related to the production and use of wild type lioviruses in cancer treatment have been described (e.g., US Pat. , 6,136,307, and 6,110,461, and US Patent Publications 2006/0165724, 2006/0073166, 2005/0123513, 2004/0265271, 2004/0126869, 2004/0109878, 2002/0037543, 2006/0029598, 2005/0026289, 2002/0006398, And 2001/0048919, each of which is incorporated by reference in its entirety without waiver. Myxoma virus therapy is described in US Patent Publication 2006/0263333, incorporated by reference.

B. Removal Therapies ( Purging Therapies )

The present invention, in certain embodiments, provides a method for purging a cell population of cancer cells. For example, individuals who have undergone bone marrow removal treatment with hematopoietic or solid cancer may then receive hematopoietic stem cell rescue using removed bone marrow tissue collected from the subject prior to chemotherapy. In these embodiments, the bone marrow can be treated with an oncolytic virus to remove or kill neoplastic cells while reducing or eliminating damage to hematopoietic stem cells.

Patient-specific adult stem cells can be used for cell therapy to avoid problems such as host immunity, graft-versus-host disease, and ethical debate. Human adult stem cells have been widely evaluated for a variety of tissue regeneration treatment modalities. However, the adult stem cells in the It has been reported that spontaneous transformation can occur during in vitro culture expansion. ( Tolar et al ., 2007; Romano , 2005 ). Therefore, an appropriate purging strategy is needed to remove spontaneously transformed cells present in cultured expanded adult stem cells for safer use in transplantation and tissue regeneration. Wild type riovirus in In vitro , it has been useful for the removal of cancer cells in autologous hematopoietic stem cell populations. ( Thirukkumaran et al ., 2003; Thirukkumaran et al ., 2005 ). Attenuated reovirus and myxoma virus can also be used in a similar manner. The inventors predict that the present invention may utilize virtually any adult stem or embryonic stem cell line now known or discovered in the future.

In certain embodiments, it may be desirable to remove the liovirus from the cell composition after a sufficient time to allow the killing of neoplastic cells (if present). For example, the bone marrow may be taken from an individual and treated with riovirus to destroy neoplastic cells, but, before re-transplantation of the bone marrow in the individual (eg, a cancer patient who does not function immune), It may be desirable to destroy, eliminate, and / or remove viruses. In these embodiments, anti-viral material may be added to the cell composition. These substances include inhibitors of essential viral enzymes such as anti-riovirus antibodies and complements, RNA-induced RNA polymerase inhibitors, or substances that interfere with the release from successful virus packages, assemblies or infected cells. do.

Additional purification steps may be used, including washing and / or centrifuging the cells to further remove virus and / or killed cells from the cell composition. Such methods are known and include methods for enhancing the desired cell type, for example, methods based on fluorescence activated cell sorting or adsorption, which allows positive selection of the intended cells, such as stem cell populations. .

C. Combination Therapy Combination Therapy )

To increase the effect of selective killing of cancer cells by oncolytic viruses, in certain embodiments, using chemotherapy agents, radiotherapy, immunotherapy, hormone therapy, toxin therapy, another natural virus or engineered virus It would be desirable to contact additional cells with cells such as therapy or gene therapy.

Chemotherapeutic agents include 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambutyl, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, and estrogen receptors. Binding agents, etoposide (VP 16), farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide, mecloethamine, melfaran, mitomycin, nabelbin, nitrosurea, flicomycin, procar Include, but are not limited to, vagin, raloxyphene, tamoxyphene, taxol, temazolomide (a water soluble form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative of the foregoing. Do not. These substances or drugs are classified according to their mode of activity in the cell, for example, depending on which stage affects the cell cycle and on which stage. Alternatively, a substance may be characterized based on its ability to cross-link directly to DNA, to be inserted into DNA or to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolic agents, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrozoreas, hormonal substances, other substances, and any analogs or derivatives thereof. Variants.

Oncolytic therapy may be carried out before or next at intervals of several minutes to several weeks, concurrent with the application of the other substance (s) to the cancer cells. For example, in such cases, one of skill in the art can contact multiple modalities substantially simultaneously (eg, within about 1 minute) with a lyovirus attenuated to cancer cells, tissues, organs or organisms. In another aspect, the one or more substances are about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 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 Time, 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 42 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, 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks In a period of about 6 weeks, about 7 or about 8 weeks or more and within or substantially simultaneously with any range derivable therefrom, or before and / or after attenuated or wild-type type riovirus May be administered.

Various complex regimes of attenuated or wild type riovirus and one or more substances may be used. Non-limiting examples of such complexes are shown below, wherein the oncolytic virus is labeled "A" and the anticancer agent is labeled "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 / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

i. Chemotherapy

Various chemotherapeutic agents can be used in the present invention. "Chemotherapy" refers to the use of drugs to treat cancer. Chemotherapeutic agents (“chemotherapeutic agents”) are used to refer to compounds or compositions administered in cancer treatment. These substances or drugs are classified according to their mode of activity in the cell, for example, depending on which stage affects the cell cycle and on which stage. Alternatively, a substance may be characterized based on its ability to cross-link directly to DNA, to be inserted into DNA or to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylates, anti-metabolic agents, anti-tumor antibiotics, mitosis inhibitors, and nitrozourea.

Alkylating Alkylating Materials agents ) . Alkylated substances are drugs that interact directly with genomic DNA to prevent cancer cells from proliferating. Chemotherapy drugs in this category are substances that affect all phases of the cell cycle, ie, are not phase-specific. Alkylated materials can be used to treat chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, certain cancers of the breast, lung and ovaries. Such drugs include busulfan, chlorambutyl, cisplatin, cyclophosphamide (cytooxane), dacarbazine, ifosfamide, meclomethamine (mustargen) and melparan. Troglitazaone can be used in combination with one or more alkylating agents to treat cancer, some of which are discussed below.

Busulfan (also known as milleran) is a bifunctional alkylating substance. Busulfan is known as 1,4-butanediol dimethanesulfonate.

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

Busulfan is used for palliative treatment of chronic myeloid (myeloid, myelocytic) leukemia. Although not curative, boospan reduces total granulocyte mass, relieves symptoms of the disease, and improves the clinical condition of the patient. About 90% of previously untreated adult patients with chronic myeloid leukemia will get hematopoietic remission, such as degeneration or stabilization of organomegaly after the use of sulphate. Survival and hemoglobin levels were better than splenic irradiation and were found to be equivalent to irradiation in controlling long-term hypertrophy.

Chlorambutyl (also known as leukeran) is a bifunctional alkylating agent of the nitrogen mustard type and is active against selected human renal tissue diseases. Chlorambutyl is known by chemical nomenclature as 4- [bis (2-chloroethyl) amino] benzenebutanoic acid.

Chlorambutil is available as a tablet for oral administration. Absorbed quickly and completely by the gastrointestinal tract. After a single oral dose of 0.6-1.2 mg / kg, chlorambutyl plasma peak (peak) levels are reached within one hour, and the terminal half-life of the parent drug is estimated to be about 1.5 hours. 0.1 to 0.2 mg / kg / day or 3 to 6 mg / m 2 / day or alternatively 0.4 mg / kg may be used for anti-neoplastic treatment. Treatment regimens are well known to those skilled in the art, "Physicians Desk Reference "and"Remington's Pharmaceutical Sciences. "

Chlorambuchil is prescribed for the treatment of chronic lymphocytic (lymphocytic) leukemia, lymphosarcoma, malignant lymphoma including giant follicular lymphoma and Hodgkin's disease. There is no cure for these diseases, but clinically useful relief may be obtained. Therefore, it can be used in combination with troglitazone in cancer treatment.

Cisplatin has been widely used to treat cancers such as metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is used alone or in combination with other substances, and the effective dose for clinical use is 15-20 mg / m 2 over three days, for a total of three weeks. Exemplary dosages are 0.50 mg / m 2, lOmg / m 2, 1.50 mg / m 2, 1.75 mg / m 2, 2.0 mg / m 2, 3.0 mg / m 2, 4.0 mg / m 2, 5.0 mg / m 2, 10 mg / m 2. Of course, all of these doses are exemplary, and any dose between these doses is expected to be used in the present invention.

Cisplatin is not absorbed orally and must be given by injection intravenously, subcutaneously, intratumorally or peritoneally.

Cyclophosphamide is 2 H -l, 3,2-oxazaphosphorin-2-amine, N, N -bis (2-chloroethyl) tetrahydro-, 2-oxide, monohydrate; In Mead Johnson under the name Cytoxan; And in Adria, it is available under the name Neosar. Cyclophosphamide is a mixture of 3-amino-1-propanol with N, N -bis (2-chloroethyl) phosphoramide dichloride [(ClCH ₂ CH ₂ ) ₂ N- in dioxane solution under the catalytic effect of triethylamine. -POCl ₂ ] to prepare. Condensation is carried out in duplicate, involving both hydroxyl and amino groups, affecting the cyclization.

Unlike other β-chloroethylamido alkylates, the active ethyleneimmonium is not immediately cyclized until activated by liver enzymes. Thus, the substance is stable in the gastrointestinal tract, resistant, and effective in both the oral and extranasal routes, does not cause local vesication, necrosis, phlebitis and pain.

Suitable dosages for adults are orally 1 to 5 mg / kg / day (usually in combination) or 1 to 2 mg / kg / day, depending on gastrointestinal resistance; Intravenously, divided into 2 to 5 days divided by 40 to 50 mg / kg or 10 to 15 mg / kg every 7 to 10 days or 3 to 5 mg / kg or 1.5 to 3 mg / kg twice a week Include / day A dose of 250 mg / kg / day may be administered as an anti-neoplastic agent. Because of gastrointestinal side effects, loading is preferably intravenous. A white blood cell count of 3000-4000 / mm <3> is normally preferred during maintenance. The drug is also sometimes administered by infusion into the muscle or into the body cavity. When used as injectable doses of 100 mg, 200 mg and 500 mg and tablets of 25 mg and 50 mg, one of ordinary skill in the art will understand that Remington's Pharmaceutical Sciences "15 th Edition , chapter 61 ”, which is attached as a reference.

Melparan (also known as alkeran, L-phenylalanine mustard, phenylalanine mustard, L-PAM, or L-sarcorycin) is a phenylalanine derivative of nitrogen mustard. Melparan is a bifunctional alkylating agent with selective activity against human neoplastic disease. The chemical name is 4- [bis (2-chloroethyl) amino] -L-phenylalanine.

Melparan is the active L-isomer of the compound, first synthesized by Bergel and Stock in 1953; D-isomers (known as medphalans) are less active against certain tumors in animals, and the required dose to effect on chromosomes is greater than for L-isomers. The racem (DL-) form is known as mulfaran or sarcoricin. Melparan is insoluble in water, and the value of pK _a1 is -2.1. Melparan is used as a tablet for oral administration and has been used to treat multiple myeloma.

According to available evidence, about one third to one half of patients with multiple myeloma show a favorable response to oral administration of this drug.

Melparan has been used to treat epithelial ovarian carcinoma. One common regimen used for the treatment of ovarian carcinoma is the administration of melparan at a dose of 0.2 mg / kg daily for five days in a single procedure. The procedure is repeated every four to five weeks depending on liver tolerance ( Smith and Rutledge , 1975; Young et al , 1978 ). Alternatively, the dosage of melparan used may be as low as 0.05 mg / kg / day or as high as 3 mg / kg / day or any dosage between these dosage ranges or any dosage above these dosage ranges. have. Some changes in dosage may necessarily occur depending on the condition of the subject to be treated. In either case, the person responsible for the medication will determine the appropriate dose for the individual patient.

Antimetabolites (Antimetabolites). Antimetabolic compounds disrupt DNA and RNA synthesis. Unlike alkylating substances, anti-metabolites specifically affect the cell cycle during the S phase. In addition to tumors of the breast, ovary and gastrointestinal tract, it has been used to treat chronic leukemia. Antimetabolic agents include 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine and methotrexate.

The chemical name of 5-fluorouracil (5-FU) is 5-fluoro-2,4 (lH, 3H)-pyrimidinedione. Its mechanism of action appears to block deoxyuridic acid methylation reaction to thymidylic acid. Thus, 5-FU interferes with the synthesis of deoxyribonucleic acid (DNA) and inhibits the synthesis of ribonucleic acid (RNA) to a somewhat weaker level. DNA and RNA are essential for cell division and proliferation, and the effect of 5-FU is to produce thymidine deficiency, which induces cell death. Thus, the effect of 5-FU can be seen in cells that are rapidly dividing (characteristic of metastatic cancer).

Antitumor antibiotics (Antitumor Antibiotics ) . Antitumor antibiotics have both antimicrobial and cytotoxic activity. These drugs can interfere with DNA by chemically inhibiting enzymes and mitosis or by altering cell membranes. Because these substances are not phase specific, they act on all phases of the cell cycle. Therefore, it is used for various cancers. Examples of antitumor antibiotics include bleomycin, dactinomycin, daunorubicin (Adriamycin) and idarubicin, some of which are discussed in more detail below. With widespread use in clinical settings for the treatment of neoplasia, these compounds are in the dosage range of 25-75 mg / m 2 to 35-100 mg / m 2 (in oral 21-day intervals of adriamycin, intravenously; etoposide Oral vein or orally) intravenously).

Doxorubicin hydrochloride, 5,12-naphthaesenedione, (8s- cis ) -10-[(3-amino-2,3,6-trideoxy-aL-lyxo-hexopyranosyl) oxy] -7, 8,9,10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl) -methoxy-hydrochloride (hydroxydaunorubicin hydrochloride, adriamycin) has extensive anti-neoplastic properties Used in the spectrum. Binds to DNA, inhibits nucleic acid synthesis, inhibits mitosis and promotes chromosomal aberrations.

When administered alone, it is a first-choice drug for the treatment of thyroid adenoma and primary hepatocellular carcinoma. Ovarian, endometrial and breast tumors, bronchial oat-cell carcinoma, non-small cell lung carcinoma, gastric adenocarcinoma, retinoblastoma, neuroblastoma, mycosis fungal carcinoma, pancreatic carcinoma, prostate carcinoma, bladder carcinoma, myeloma, diffuse histologic lymphoma, Wilms Wilms is a component of 31 primary selection drug combinations for the treatment of tumors, Hodgkin's disease, adrenal tumors, osteosarcoma soft tissue sarcoma, Ewing's sarcoma, rhabdomyosarcoma and acute lymphocytic leukemia. It is an alternative for the treatment of islet (iS1et) cells, cervical, testicular and adrenal cortex cancer. This drug is also immunosuppressive.

Doxorubicin is poorly absorbed and must be administered intravenously. Pharmacopoeia is multicompartmental. The distribution phase has a half life of 12 minutes and 3.3 hours. Elimination half-life is about 30 hours. 40-50% are secreted into bile. Most of the remainder is metabolized in the liver and partially metabolized to the active metabolite (doxorubicinol), but some is excreted in the urine. In case of liver damage, the dose should be reduced.

Suitable dosages are 25 to 30 mg / m 2 daily, or 20 mg / m 2 once a week, for two or three consecutive days, repeated at 60 to 75 mg / m 2 or 3 or 4 week intervals at 21 day intervals for adult, intravenous administration. For older patients, the lowest dose should be used when there is existing bone marrow suppression or neoplastic bone marrow infiltration caused by existing chemotherapy, or when used in combination with other myeloid hematopoietic inhibitory drugs. The dose should be reduced by 50% when serum bilirubin is between 1.2 and 3 mg / dL, and 75% when above 3 mg / dL. The lifetime dose should not exceed 550 mg / m 2 in patients with normal cardiac function and 400 mg / m 2 in patients undergoing mediastinal irradiation. Alternatively, the amount of 30 mg / m 2 is repeated every four weeks for three consecutive days. Exemplary dosages are 10 mg / m 2, 20 mg / m 2, 30 mg / m 2, 50 mg / m 2, 100 mg / m 2, 150 mg / m 2, 175 mg / m 2, 200 mg / m 2, 225 mg / m 2, 250 mg / m 2, 275 mg / m 2, 300 mg / m 2, 350 mg / m 2, 400 mg / m 2, 425 mg / m 2, 450 mg / m 2, 475 mg / m 2, 500 mg / m 2. Of course, both of these dosages are exemplary and it is expected that any dosage between these ranges may also be used in the present invention.

Daunorubicin hydrochloride, 5,12-naphthaesenedione, (8S- cis ) -10-[(3-amino-2,3,6-trideoxy-aL-lyxo-hexanopyranosyl) oxy]- 7,8,9,10-tetrahydro-6,8,11-trihydroxy-10-methoxy-hydrochloride; It is also named cerubidine and is available from Wyeth. Daunorubicin interrupts DNA, blocks DNA-induced RNA polymerase and inhibits DNA synthesis. This can prevent cell division at doses that do not interfere with nucleic acid synthesis.

In combination with other drugs, it is included in the first-line chemotherapy of acute phases of acute myeloid leukemia (to induce relief), acute myeloid leukemia and chronic myelogenous leukemia in adults. Oral absorption is poor and should be given 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 metabolized mostly in the liver and is also excreted in bile (ca 40%). In case of liver failure or kidney failure, the dose should be reduced. Appropriate doses (base basis), young adults under 60 years old, 45 mg / m 2 daily (30 mg / m 2 if age 60 or older) or every 3 or 4 days intravenously every 3 or 4 weeks, 1, 2 or 3 days 0.8 mg / kg daily, 3 to 6 days per week; No more than 550 mg / m2 should be given during life, and no more than 450 mg / m2 if there was a chest scan; For children, 25 mg / m 2 once a week if the body surface is less than 0.5 m if less than 2 years old or if a weight-based adult schedule is used. Injectable dosage forms (equivalent bases) are available at 20 mg (bases equivalent to 21.4 mg of hydrochloride). Exemplary dosages are 10 mg / m 2, 20 mg / m 2, 30 mg / m 2, 50 mg / m 2, 100 mg / m 2, 150 mg / m 2, 175 mg / m 2, 200 mg / m 2, 225 mg / m 2, 250 mg / m 2, 275 mg / m M 2, 300 mg / m 2, 350 mg / m 2, 400 mg / m 2, 425 mg / m 2, 450 mg / m 2, 475 mg / m 2, 500 mg / m 2. Of course, both of these dosages are exemplary and it is expected that any dosage between these ranges may also be used in the present invention.

Mitomycin (also known as mutamycin and / or mitomycin C) is an anti-tumor activity of Streptomyces Streptomyces. caespitosus ) is an antibiotic isolated from broth. This compound is heat stable, has a high melting point, and freely dissolves in organic solvents. Mitomycin selectively inhibits the synthesis of deoxyribonucleic acid (DNA). Guanine and cytosine content is related to the degree of mitomycin-induced linkage. At high concentrations of the drug, cellular RNA and protein synthesis are also inhibited.

In humans, mitomycin is rapidly removed from serum after intravenous administration. The time required for the serum concentration to decrease by about 50% after 30 mg intravenous injection is 17 minutes. 30 mg, 20 mg, or 10 mg I.V. After injection, the maximum serum concentrations were in turn 2.4 mg / ml, 1.7 mg / ml, and 0.52 mg / ml. It is mainly eliminated by metabolism in the liver, but of course also in other tissues. The clearance rate is inversely proportional to the maximum serum concentration, which is believed to be due to saturation of the degradable pathway. About 10% of the mitomycin dose is released into the urine unchanged. Because metabolic pathways are saturated at relatively low doses, increasing doses increases the proportion of dose released into urine. In children, the release of intravenously administered mitomycin is similar.

Actinomycin D (dactinomycin) [50-76-0]; C ₆₂ H ₈₆ N ₁₂ O ₁₆ (1255.43) is an anti-neoplastic drug that inhibits DNA-dependent RNA polymerase. The drug is a component of the first-selective combination in the treatment of chorionic carcinoma, embryonic rhabdomyosarcoma, testicular tumor, and Wilms' tumor. Tumors that are ineffective in systemic therapy sometimes respond to local irrigation. Dactinomycin increases the effect of radiotherapy. It is a secondary (centrifugal) immunosuppressant.

Actinomycin D is used in combination with primary surgery, radiotherapy, and other drugs, especially vincristine and cyclophosphamide. Anti-neoplastic activity has also been recorded in Ewing's tumor, Kapus sarcoma, and Soft tissue sarcoma. Dactinomycin is effective in women with advanced chorionic carcinoma. In addition, in patients with metastatic testicular carcinoma, there is a sustained response in combination with chlorambutyl and methotrexate. Responses can sometimes be observed in patients with Hodgkin's disease and non-Hodgkin's lymphoma. Dactinomycin has also been used to inhibit immune responses, particularly kidney transplant rejection.

Half of the dose is excreted in the bile in its own form and 10% is excreted in the urine; The half life is about 36 hours. The drug does not cross the brain-vascular wall. Actinomycin D is supplied in lyophilized powder (0/5 mg in each vial). Typical daily dosages are 10-15 mg / kg; Given intravenously for 5 days; There were no toxic manifestations and additional courses could be provided at intervals of three to four weeks. Children were given 100 to 400 mg by injection daily for 10 to 14 days; In other regimens, 3 to 6 mg / kg, total 125 mg / kg, and weekly maintenance dose were 7.5 mg / kg. It would be safer to administer the drug via an intravenous infusion tube, but be careful to treat the needle used to withdraw the drug from the vial, directly by intravenous injection to avoid a subcutaneous reaction. Exemplary dosages are 100 mg / m 2, 150 mg / m 2, 175 mg / m 2, 200 mg / m 2, 225 mg / m 2, 250 mg / m 2, 275 mg / m 2, 300 mg / m 2, 350 mg / m 2, 400 mg / m 2, 425 mg / m 2, 450 mg / m 2, 475 mg / m 2, 500 mg / m 2. Of course, both of these dosages are exemplary and it is expected that any dosage between these ranges may also be used in the present invention.

Bleomycin is a Streptomyces verticillus ) is a cytotoxic glycopeptide antibiotic isolated from. The exact criteria for the action of bleomycin are not yet known, but evidence suggests that the main mode of action is inhibition of DNA synthesis, and some evidence shows that RNA and protein synthesis are also somewhat inhibited.

In mice, high concentrations of bleomycin are found in the skin, lungs, kidneys, peritoneum and lymph glands. Tumor cells in the skin and lungs were found to have high concentrations of bleomycin, in contrast to the low concentrations found in hematopoietic tissues. Low concentrations of bleomycin found in bone marrow may be associated with bleomycin degrading enzymes found in these tissues.

The final elimination half life of serum or plasma bleomycin in patients with greater than 35 ml per minute (> 35 ml) of creatinine removed is about 115 minutes. In patients with less than 35 ml (<35 ml) creatinine removed, the final elimination half-life of bleomycin in serum or plasma is exponentially increased when creatinine clearance is reduced. In humans, 60% to 70% of the administered dose is recovered from urine with active bleomycin. Bleomycin may be provided via an intramuscular, intravenous or subcutaneous route. Freely soluble in water

Bleomycin should be considered as a palliative treatment. As a single substance in squamous cell carcinoma such as head and neck (including mouth, tongue, tonsils, nasopharynx, central pharynx, hole, palate, lips, oral mucosa, gums, epiglottis, larynx), skin, penis, cervical and vulva Proven combinations with other approved chemotherapeutic agents have been shown to be useful in regulating neoplasia. It has also been used to treat lymphoma and testicular carcinoma.

Because of the possibility of an anaphylactoid reaction, lymphoma patients should be treated in two units or in doses less than the first two doses. If an acute reaction does not occur, then regular dosages may be followed.

Improvement of Hodgkin's disease and testicular tumors is immediate and appears within two weeks. If no improvement is seen by this time, there is no possibility of improvement. Squamous cell cancers respond more slowly and in some cases require up to three weeks for any improvement to occur.

Mitotic inhibitors (Mitotic Inhibitors). Mitosis inhibitors include plant alkaloids and other natural substances that inhibit proteins required for cell division or mitosis. These inhibitors act on specific phases during the cell cycle. Mitosis inhibitors include docetaxel, etoposide (VP16), paclitaxel, taxol, taxotere, vinblastine, vincristine and vinorelbine.

VP16, also known as etoposide, is commonly used in the treatment of testicular tumors in combination with bleomycin and cisplatin, and in combination with cisplatin to treat small-cell carcinoma of the lung. It is also active against non-Hodgkin's lymphoma, acute non-lymphocytic leukemia, breast carcinoma and Kaposi's sarcoma associated with acquired immunodeficiency syndrome (AIDS).

VP16 is used as a solution for intravenous administration (20 mg / ml) or as a 50 mg capsule filled with an oral liquid. For small cell carcinoma of the lung, the intravenous dose (in combination therapy) is as high as 100 mg / m 2, or as low as 2 mg / m 2, typically 35 mg / m 2 per day for 4 days to 50 mg / m 2 per day for 5 days. This may be used. When given orally, the dose should be doubled. Doses of small cell lung carcinoma should be as high as 200-250 mg / m 2. Intravenous doses for testicular cancer (in combination therapy) are between 50 and 100 mg / m 2 daily for 5 days or alternately 100 mg / m 2 for three doses. The therapy cycle is generally repeated every three to four weeks. The drug should be administered slowly during the 30 to 60 minute infusion, presumably to avoid hypotension and bronchial spasm due to the solvent used in the formulation.

Taxol is the ash tree ( Taxus) brevifolia ) is an experimental antimitotic substance isolated from the bark. Taxol binds to tubulin (in a separate site from that used by vinca alkaloids), facilitating the assembly of microtubules. Taxol is currently being evaluated clinically; Taxol is active against malignant melanoma and ovarian carcinoma. The maximum dose is 30 mg / m 2 daily for 5 days or 210 to 250 mg / m 2 once every three weeks. Of course, both of these dosages are exemplary and it is expected that any dosage between these ranges may also be used in the present invention.

Vinblastine is another example of plant alkyloids used in combination with troglitazone for the treatment of cancer and precancerous. Incubation of cells with vinblastine results in microtubule dissociation.

Unexpected absorption after oral administration of vinblastine or vincristine has been reported. In typical clinical doses, the peak concentration of each drug in plasma is about 0.4 mM. Vinblastine and vincristine bind to plasma proteins. They are highly concentrated in platelets and somewhat weakly in leukocytes and red blood cells.

After intravenous injection, vinblastine shows a multiphasic pattern of elimination from the plasma: after distribution, the drug disappears from the plasma with a half-life of about 1 and 20 hours. Vinblastine is metabolized in the liver to biologically activate desacetylbindlastine. About 15% of the dose administered is inherently detected in urine and about 10% is recovered from feces after bile excretion. In patients with liver dysfunction, the dose should be reduced. If the bilirubin concentration in plasma is above 3 mg / dl (about 50 mM), the dose should be reduced by at least 50%.

Vinblastine sulfate is used in the preparation of injectables. This drug is given intravenously; Special attention should be given to subcutaneous ejection, as this drug can cause painful irritation and ulcers. This drug should not be injected into the limbs with impaired circulation. At a single dose of 0.3 mg / kg, myelosuppression peaks within 7 to 10 days. If moderate leukopenia (approximately 3000 cells / dL) is not obtained, the dose per week may be increased in increments of 0.05 mg / kg. In the regimen for treating testicular cancer, 0.3 mg / kg of vinblastine is used every 3 weeks, regardless of blood cell count or toxicity. The most important clinical use of vinblastine is in combination with bleomycin and cisplatin in the treatment of metastatic testicular tumors. Beneficial responses have been reported in various lymphomas, in particular Hodgkin's disease, and significant improvements can be noted in 50-90% in Hodgkin's disease. If the disease is refractory to alkylating agents, the effect of vinblastine at high lymphoma rates is not reduced. It is active in Kaposi's sarcoma, neuroblastoma, Letterer-Siwe disease (histocytosis X), as well as breast and choriocarcinoma in women.

The dosage of vinblastine may be determined by the clinician depending on the individual patient in need. 0.1 to 0.3 mg / kg may be administered or 1.5 to 2 mg / m 2 may be administered. Alternatively, 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, 0.5 mg / m 2, 1.0 mg / m 2, 1.2 mg / m 2, 1.4 mg / M 2, 1.5 mg / m 2, 2.0 mg / m 2, 2.5 mg / m 2, 5.0 mg / m 2, 6 mg / m 2, 8 mg / m 2, 9 mg / m 2, 10 mg / m 2, 20 mg / m 2 It may be. Of course, both of these dosages are exemplary and it is expected that any dosage between these ranges may also be used in the present invention.

Vincristine blocks mitosis and produces metaphase arrest. Most of the biological activity of the drug appears to be explained by its specific binding to tubulin and blocking the ability of proteins to polymerize into microtubules. Through microtubule disruption of the mitotic device, cell division is inhibited at metaphase. If chromosomes cannot be separated correctly during mitosis, the cells are probably killed.

The relatively low toxicity of vincristine in normal bone marrow cells and epithelial cells makes it special among anti-neoplastic drugs and is included in combination with other myelosuppressive substances.

Unpredictable adsorption has been reported after oral administration of vinblastine or vincristine. In conventional clinical doses, the peak concentration of each drug in plasma is about 0.4 mM.

Vinblastine and vincristine bind to plasma proteins. They are highly concentrated in platelets and somewhat weaker in leukocytes and red blood cells.

Vincristine has a multiphasic removal pattern from plasma: the terminal half-life is about 24 hours. The drug is metabolized in the liver, but no biologically active derivatives have been identified. In patients with liver dysfunction, the dose should be reduced. If the bilirubin concentration in plasma is above 3 mg / dl (about 50 mM) at least 50% of the dose is prescribed for reduction.

Vincristine sulfate is used as an intravenous solution (1 mg / ml). Vincristine used with corticosteroids is currently a selective treatment that induces remission in childhood leukemia; Optimal doses of these drugs were vincristine, intravenously, weekly, 2 mg / m 2 per body surface area; And predinison, oral, daily, 40 mg / m 2. Adult patients with Hodgkin's disease or non-Hodgkin's lymphoma are usually given vincristine as part of a complex protocol. If vincristine is used in the MOPP regimen, the recommended dose of vincristine is 1.4 mg / m 2. High doses of vincristine appear to be more resistant to children with leukemia than adults, and adults may experience severe neurotoxicity. When the drug is administered more frequently than every seven days or at higher doses, toxicity manifestations seem to increase without proportional improvement in reaction rate. Precautions should be used to avoid eruption during intravenous administration of vincristine. Vincristine (and vinblastine) may be injected into the arterial blood supply of the tumor in doses of comparable toxicity, several times higher than can be administered intravenously. Vincristine was effective against Hodgkin's disease and other lymphomas. When used alone with Hodgkin's disease, it is somewhat less beneficial than vinblastine, but when used in combination with mecloethamine, pdenisone and procarbazine (MOPP regimen), advanced stages of Hodgkin's disease (III and IV) is a preferred treatment. Vincristine is an important substance in non-Hodgkin's lymphomas, especially when used with cyclophosphamide, bleomycin, doxorubicin and prednisone. Vincristine is more useful than vinblastine in lymphocytic leukemia. Beneficial responses have been reported in patients with various other tumors (neoplastics), in particular Wilms' tumor, neuroblastoma, brain tumor, rhabdomyosarcoma, breast, bladder and male and female reproductive carcinoma.

The dosage of vincristine used will depend on the individual patient in need. 0.01 to 0.03 mg / kg may be administered, 0.4 to 1.4 mg / m 2 or 1.5 to 2 mg / m 2 may be administered. Alternatively, 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 can be given by continuous intravenous infusion. Of course, both of these dosages are exemplary and it is expected that any dosage between these ranges may also be used in the present invention.

Camptothecin is a Chinese tree ( Camtotheca acuminata Decne ) is an alkaloid derived from. Campfotothecin and its derivatives are unique in their ability to stabilize covalent reaction intermediates, termed "cleavable complexes," thereby inhibiting DNA topoisomerase, resulting in the death of tumor cells. Many people believe that camptothecin analogs show excellent anti-tumor and anti-leukemic activity. The use of camptothecin in the clinic is limited because of severe side effects and poor solubility in water. Currently, some camptothecin analogs (topotecan; irinotecan), either synthetic or semi-synthesized, have been applied to cancer therapy and show satisfactory clinical effects. The molecular formula of camptothecin is C ₂₀ H ₁₆ N ₂ O ₄ , and has a molecular weight of 348.36. It is provided as a yellow powder and can be dissolved in a clear yellow solution at 50 mg / ml in DMSO 1N sodium hydroxide. Stable for at least 2 years if stored at 2-8 ° C in a dry, closed, light-resistance environment.

Nitro jaws LEA (Nitrosureas). Like the ylchelating agent, nitrozourea inhibits DNA repair proteins. In addition to brain-tumors, it is used for the treatment of non-Hodgkin's lymphoma, multiple myeloid, malignant melanoma. Exemplary materials include carmustine and romustine.

Carmustine (sterile carmustine) is one of the nitrozoreas used to treat certain neoplastic diseases. The chemical name is l, 3bis (2-chloroethyl) -l-nitrozourea. Lyophilization results in pale yellow flakes or condensed mass with a molecular weight of 214.06. Very soluble in alcohols and lipids, not very well in water. Carmustine is administered by intravenous infusion after reconstitution as recommended. Sterile carmustine is commonly used in 100 mg single dose vials of lyophilized material.

Carmustines appear to alkylate DNA and RNA, but do not have cross resistance with other alkylators. Along with other nitrozoreas, carbamoylation of amino acids in proteins may limit some key enzymatic processes.

Carmustine is a single substance that has been prescribed in palliative therapy or established with other chemotherapeutic agents approved in brain tumors such as gliomas, brain stem gliomas, medullobladyoma, astrocytoma, ventricular cell tumor and metastatic brain tumors. It is prescribed in combination therapy. It has also been used in combination with prednisone for the treatment of multiple myeloma. Carmustine has been shown to be useful as a second line therapy in combination with other drugs approved in patients who have relapsed or who have failed to respond to the first line of therapy in treatment of Hodgkin's disease and non-Hodgkin's lymphoma. Proved.

The recommended dose of carmustine, used as a single substance in previously untreated patients, is every 6 weeks, intravenously, 150-200 mg / m 2. It can be provided in a single dose or divided into daily injections at 75-100 mg / m 2 for two consecutive days. If carmustine is used in combination with other bone marrow suppressing drugs or in patients depleted of bone marrow stock, the dosage should be adjusted accordingly. Doses after the initial dose should be adjusted according to the patient's hematological response to the preceding dose. Of course, other dosages may also be used in the present invention, for example, 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. Those skilled in the art will understand "Remington's Pharmaceutical Sciences, "15 th Edition , Chapter 61 . Depending on the condition of the individual being treated, some changes in dosage may be necessary. The person in charge of administration will in any case be able to determine the appropriate dosage for the individual to be treated.

Romustine is one of the nitrozoreas used to treat certain neoplastic diseases. The chemical name is l- (2-chloro-ethyl) -3-cyclohexyl-l nitrozourea. The actual molecular formula is C ₉ H ₁₆ ClN ₃ O ₂ , which is a yellow powder, with a molecular weight of 233.71. Romustine is dissolved in 10% ethanol (0.05 mg per mL) and in pure alcohol (70 mg per ml). Romustine is relatively insoluble in water (<0.05 mg per ml). Relatively no ionization at physiological pH. Inactive ingredients in romustine capsules are magnesium stearate and mannitol. It is generally recognized that romustine alkylates DNA and RNA, but it does not have cross resistance with other alkylators. Along with other nitrozoreas, carbamoylation of amino acids in proteins may limit some key enzymatic processes.

Romustine will be given orally. When radioactive romustine is administered orally in doses ranging from 30 mg / m 2 to 100 mg / m 2, about half of the provided radioactivity is released in the form of degradation products within 24 hours. The serum half life of the metabolite is 16 hours to 2 days. Tissue levels are comparable to plasma levels 15 minutes after intravenous administration.

Romustine has been shown to be useful as a single substance for both primary and metastatic brain tumors, in addition to other treatment modalities, or in established combination therapies in patients who have already undergone adequate surgery and / or radiotherapy procedures. In combination with other drugs approved in patients who have relapsed or did not respond to the primary therapy during treatment with the primary therapy, it has been shown to be effective as a secondary therapy for Hodgkin's disease.

As a single substance in patients who have not been previously treated, the recommended dose of romustine for adults and children is every 6 weeks, and the oral single dose is 130 mg / m 2. The dose of bone marrow patients who are not immune should be reduced to 100 mg / m 2 every 6 weeks. If romustine is used in combination with other myelosuppressive drugs, the dosage will have to be adjusted accordingly. Other dosages may be used, for example 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, 120 mg / m 2, or any dosage range between these values, which may be essentially determined by the clinician, depending on the individual to be treated.

Other substances . Other materials can be used, including Avastin, Iressa, Herbitus, Velcarde and Gleevec. Growth factor inhibitors and small molecule kinase inhibitors are also useful in the present invention. All therapies described are Cancer : Principles and Practice of It is described in Oncology (2001) and is incorporated by reference. The following additional therapies are also included.

ii . Immunotherapy ( immunotherapy )

Immunotherapy is generally the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of the tumor cell. Antibodies alone can recruit other cells that act as an effective agent of therapy or that substantially affect cell death. Antibodies may also be conjugated to drugs or toxins (chemotherapy, radionuclides, Lykin A chains, cholera toxins, whooping cough toxins, etc.) and may simply serve as targeting agents. Alternatively, the effector may be a lymphocyte carrying a surface molecule that directly or indirectly interacts with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. Thus, immunotherapy can be used as part of a combination therapy combined with riovirus or other oncolytic virus therapy. General methods for combination therapy are discussed below. In general, tumor cells are not present in most other cells and must have some marker that enables targeting. There are many tumor markers, some of which will be suitable for targeting consistent with the teachings of the present invention. Common tumor markers include cancer embryo antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin Receptors, erb B and p155. In addition, tumor cells or other hyperproliferative cells infected with lyoviruses or other oncolytic viruses can express viral antigens on the cell surface and thus be attacked by the immune system.

Tumor necrosis factor is a glycoprotein that kills some types of cancer cells, activates cytokine production, activates macrophages and endothelial cells, and promotes collagen and collagenase production. It is an intermediary and promotes catabolism, fever and sleep. Some infectious agents cause tumor regression through stimulation of TNF production. Because TNF is so toxic when used alone in an effective amount, the optimal regimen is probably combined with other drugs and used in lower doses. Immunosuppressive action is enhanced by gamma-interferon, so combination therapy is potentially dangerous. Hybrids of TNF and interferon-α have been shown to possess anticancer activity.

iii . Hormone therapy ( Hormonal Therapy )

Sex hormones are used according to the methods described in Cancer Treatment. Although the methods described herein are not limited to specific cancer treatments, the use of such hormones is effective for breast cancer, prostate cancer and endometrial (intrauterine lining) cancer. Examples of these hormones are estrogens, anti-estrogens, progesterones and androgens.

Corticosteroid hormones are useful for the treatment of some forms of cancer (lymphoma, leukemia and multiple myeloma). Corticosteroid hormones can increase the effectiveness of other chemotherapeutic agents and, as a result, are commonly used in combination therapy. Prednisone and dexamethasone are examples of corticosteroid hormones.

iv . Radiotherapy Radiotherapy )

Radiation therapy (also called radiotherapy) is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation inflicts damage to the genetic material of cells that injects energy that damages or destroys the cells at the treatment site to keep them growing. Radiation damages cancer cells and normal cells, but normal cells can repair themselves and function properly. Radiotherapy can be used to treat localized solid tumors such as cancers of the skin, tongue, larynx, brain, breast or neck. Leukemias and lymphomas (in turn hematopoietic cells and lymphoid cancer) can also be used to treat.

Radiation therapy used in accordance with the present invention includes, but is not limited to, the use of γ-rays, X-rays, and / or direct delivery of radioisotopes to tumor cells. Other forms of DNA damage factors such as microwave and UV-irradiation may also be considered. Both of these factors affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dosage range of X-rays ranges from 50 to 200 roentgens per day, ranging from 2000 to 6000 roentgens, for a significant time (3 to 4 weeks). The dosage range of radioisotopes is very broad and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by neoplastic cells.

Radiotherapy may include the use of radiolabeled antibodies to deliver radiation doses directly to the cancer site (radioimmunotherapy). Antibodies are very specific proteins made in the body in response to the presence of antigens (substances recognized as foreign by the immune system). Some tumor cells contain specific antigens that trigger the production of tumor-specific antibodies. Large amounts of these antibodies are made in the laboratory and attached to radioactive materials (a process known as radiolabeling). Once injected into the body, the antibody actively searches for cancer cells and destroys them by radioactive cell-killing (cytotoxic) action. Such a method can minimize the damage of healthy cells to radiation.

Conformal radiotherapy uses the same radiotherapy devices, linear accelerators as normal radiotherapy treatments, but a metal block is placed in the path of the x-ray beam to modify its shape to match the shape of the cancer. This allows higher doses of radiation to be provided to the tumor. Structures in close proximity to healthy surrounding cells receive lesser doses of radiation, reducing the likelihood of side effects. A device called multi-leaf collimator has been developed and can be used as a substitute for metal blocks. Multileaf collimates consist of a number of metal sheets fixed to a linear accelerator. Each layer is adjusted so that the radiotherapy beam can be tailored to the treatment site without the aid of a metal block. Accurate positioning of stereoscopic radiotherapy devices is very important, and a specific scanning device can be used to check the position of the patient's internal organs at the start of each treatment.

High-resolution intensity controlled radiotherapy also uses multi-leaf collimates. During this treatment, treatment is provided as the layers of polychloride collimates are moved. This method yields a more accurate form of the treatment beam and allows a constant amount of radiation to be provided over the entire treatment site.

Investigations have shown that stereoscopic radiotherapy and intensity-controlled radiotherapy can reduce the side effects of radiotherapy, but precisely forming treatment sites can interfere with small cancer cells just outside the treatment area that needs to be destroyed. There is this. This means that such specialized radiotherapy techniques may present a higher risk of cancer recurrence in the future. Stereotactic radiotherapy is used to treat brain tumors. This technique sends radiotherapy from many different angles, so the dose to the tumor is very high, and the dose to affect the surrounding healthy tissue is very low. Before the treatment, several scans are analyzed by a computer to confirm that the radiotherapy is correctly targeted and that the patient's head is in the frame correctly made during the radiation therapy. Several dosages are provided.

Stereotactic radiosurgery (gamma knives) for brain tumors utilizes a highly precisely targeted beam of gamma radiotherapy from hundreds of different angles without using a knife. One session of radiotherapy requires only about 4 to 5 hours. For this treatment, a specially constructed metal frame will be placed on the patient's head. Next, several scans and x-rays are performed to find the exact area requiring treatment. During radiotherapy, the patient lies head down in a large helmet, which has hundreds of holes through which the radiation beam passes. Scientists are also looking for ways to increase the effectiveness of radiotherapy.

Two types of research drugs are being studied for their effects on cells undergoing radiation therapy. Radiosensitizers make tumor cells more disruptive, and radioprotectors protect normal tissues from radiation effects. Hyperthermia, which uses heat, has also been investigated for its effect on tissue sensitivity to radiation.

v. Follow-up surgery ( Subsequent Surgery )

About 60% of cancer patients undergo some form of surgery, which includes prophylactic, diagnostic or staged, therapeutic and palliative surgery. Curative surgery is a cancer treatment used in combination with other therapies, such as chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy and / or replacement therapy, such as the treatment of the present invention.

Therapeutic surgery involves ablation that physically removes, cuts and / or destroys all or part of cancerous tissue. Tumor resection refers to physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatment includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). In addition, the present invention can be used for the removal of epidermal cancer, pre-cancer, or the removal of incidental amounts of normal tissue.

When cutting all cancer cells, tissues or portions of a tumor, a cavity may form in the body. Treatment may be performed locally at the site of irrigation, direct injection, or additional chemotherapy. Such treatment may be repeated, every day, two days, three days, four days, five days, six days or seven days, or every week, every other week, three weeks, four weeks and five weeks or monthly, two months, three It can be repeated every month, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months. Dosage may also vary in such treatments.

V. Example

The following examples illustrate preferred embodiments of the present invention. Those skilled in the art will recognize that the techniques described below are techniques developed by the inventors to function well in the practice of the present invention, and therefore should be considered to be configured in an optimal manner for implementation. However, one of ordinary skill in the art will recognize that, in accordance with the teachings of the present invention, there can be many variations to the specific embodiments described, and such variations may yield similar results without departing from the scope and spirit of the invention.

Example  1- Materials and Methods

Cell line . p53 ^+/- MEF, BT, ATM deficient L3 cells, plus human lymphoma HBL2, Granta, Z138C, Raji, Ramos, JVM2 cells, plus retinoblastoma cell lines (Y-19 and WERI-Rb-I) from American Type Culture Collection Purchased. Cells were maintained in RPMI 1640/10% FBS

Wild type and damped Rio virus preparation. The wild type liovirus T3D strain used in this study was grown in L929 cells and purified as previously described. Attenuated reovirus induced in HTR1 culture was purified by the same method used for wild type reovirus preparation, but AV reovirus was propagated in HT1080 and L929 cells. Lyovirus was added to the cells at MOI 5-10 and maintained at 37 ° C. for 48-72 hours. After virus denaturing effects were observed (typically 20-30% cell lysis), the virus was purified from pelleted cells. CsCl centrifugation of the virus was performed at 35,000 rpm using SW41 rotor for 7-8 hours. Banded viruses were collected and actively dialyzed against 150 mM NaCl, 10 mM MgCl ₂ , and 10 mM Tris (pH 7.5). For wild type riovirus titers, HEK 293 cells were plated in 6-well plates at 2 × 10 ⁵ cells per well. After 2 hours adsorption at 37 ° C., the inoculum was removed. Cell monolayers were coated with 1% agar and fresh medium. Flock counted 5-7 days after infection.

In the case of attenuated riovirus titers, the same procedure is performed, except that agar is removed 5-7 days after infection, and cell monolayers with Cytofix / Cytoperm (BD Bioscience) for riovirus antiserum and secondary FITC antibodies and immunostaining. Were fixed / permeabilze. AV riovirus flocs were identified through immunofluorescence detection. Alternatively, in the case of attenuated riovirus alternatively, standard floc assays were also performed on L929 cells. Three days after infection, the agar was covered with neutral red, and cell monolayers were examined 24 hours later for floc formation. For attenuated lyovirus harvest from HT1080 and HTR1 infected supernatants, high speed centrifugation was used at 35,000 rpm so that the virus was pelleted.

Myxoma virus preparation. Myx-GFP of myxoma virus (strain Lausanne ), made by intergenic insertion of green fluorescent protein (GFP) driven by synthetic vaccinia virus early / late promoters, was used for infection studies. Myx-GFP has been breeding and as previously described ( Opgenorth et al , 1992 ), titration was achieved by focus formation on BGMK cells.

FACS analysis. For flow cytometry analysis, cells were trypsinized and fixed using cytofix / cytoperm solution (PharMingen, San Diego, Calif.). Fixed, osmotic cells were incubated with primary riovirus antiserum and secondary FITC conjugated anti-rabbit IgG (Cedarlane, Ontario) and then analyzed with a flow cytometer.

Example  2-result

Rioviruses and myxoma viruses preferentially infect p53 or ATM deficient cells. To examine whether the regulation of tumor suppressor genes affects oncolytic virus susceptibility, the inventors have selected p53 and ATM tumor suppressor genes that are frequently mutated in various cancers. p53 is a prototypical tumor suppressor gene, most commonly mutated in various cancer cells, and has been shown to have p53 mutations in more than 50% of cancers ( White , 1994; Morris , 2002 ). The ATM (mutated vasodilatory ataxia) gene is a serine threonine protein kinase that is activated in response to DNA damage. Both copies of the ATM gene are dysfunctional in rare symptoms of vasodilatory ataxia, characterized by cancer tendency, radiation sensitivity, and neurodegeneration ( Kitagawa). et al , 2005 ). Thus, we found that p53 − / − MEF (mouse embryonic fibroblasts) and ATM deficient L3 lymphoblasts ( Kozlov et al. al , 2003 ) were used to examine oncolytic virus susceptibility. As shown in FIGS. 1A-B, as shown by microscopic detection of FACS or fluorescence positive cells (FITC + or GFP + cells), lyovirus (WT and WT and compared with MEF (p53 normal) and BT (ATM normal) criteria AV Reovirus; Kim et al , 2007 ) and myxoma viruses each preferentially infected p53 − / − MEF or L3 (ATM-deficient) cells. Rioviruses and myxoma viruses preferentially infect p53 or ATM dysfunctional human lymphomas. Rioviruses and myxoma viruses preferentially infect p53 or ATM-deficient cells, and the inventors then examined whether riovirus and myxoma viruses preferentially infect human lymphomas with p53 or ATM deficiency. The inventors tested six different lymphomas (HB L-2, Granta, Z138C, JVM2, Raji and Ramos) with various cellular responses resulting from p53 and ATM-dependent pathways in genotoxic attacks ( Fig. 2A). Functional status of p53 and ATM was assessed by detection of p53 and ATM phosphorylation by IR irradiation and western blotting. BT (ATM-normal) and L3 (ATM-deficiency) were used as reference in the ATM response by IR. HBL-2 and Raji account for dysfunctional p53 responses in the presence of genotoxic stress, as evidenced by the constitutive phosphorylation of serine-15 of p53 ( Li et al , 2006 ) (FIG. 2A). Granta cells showed ATM dysfunction in the presence of genotoxic stress, and ATM is not activated in ionizing radiation (IR) stimulation (FIG. 2A). For Z138C, JVM2 and Ramos cells, p53 and ATM responses were normal after genotoxic stress, as seen by over-phosphorylation of p53 and ATM by IR stimulation (FIG. 2A). Interestingly, p53 and / or ATM, as seen in FASC or microscopic detection of fluorescent positive cells (FITC + or GFP + cells) when compared to ATM- and p53-reactive lymphomas (Z183C, JVM-2, Ramos). Dysfunctional lymphomas (HBL2, Granta, and Raji) [HBL-2 and Raji showed constitutive p53 activation, and Granta showed ATM deficiency upon IR stimulation] are preferred for both liovirus and myxoma virus infections Sensitive (FIG. 2C). As can be seen in FIG. 2B, the ATM or p53 dysfunctional response of the cells is significantly associated with both lyovirus and myxoma virus susceptibility. This strongly suggests that both ATM and p53 participate in establishing cell resistance to viral infections, whereas defects in either of these genes or in one of its response pathways can confer viral susceptibility (FIG. 2A). .

Retinoblastoma cells are sensitive to Rio virus and myxoma virus infection. Since riovirus and myxoma virus preferentially infect p53 and ATM dysfunctional cancer cells, the inventors also examined whether RB-deficient cancer cells are jointly susceptible to riovirus and myxoma virus infection. As can be seen in FIG. 3, two different human retinoblastoma cells are susceptible to lyovirus and myxoma virus infection, as seen by FACS or microscopic detection of fluorescent positive cells (FITC + or GFP + cells). These two cells contain a defect in the Rb gene ( Reid et al ., 1974 )

All of the methods described and claimed herein can be made and implemented without undue experimentation based on the description of the invention. While the methods and compositions of the present invention have been described in preferred embodiments, it will be apparent to those skilled in the art that various changes may be made in the methods, steps or order of the methods described herein without departing from the spirit, scope, and scope of the invention. More specifically, it is apparent that certain materials that are chemically and physiologically related may be substituted for the materials described herein and that the same or similar results may be obtained. All such similar substitutions and modifications apparent to those skilled in the art are within the spirit, scope and concept of the invention as defined by the claims.

references

The following references are hereby incorporated by reference to provide exemplary procedures or other supplements to those set forth herein.

Claims (34)

In a method of determining whether a hyperproliferative disease is sensitive to oncolytic reovirus and / or myxoma virus,
(a) providing a hyperproliferative cell;
(b) assessing the structure, function or expression of the p53, Rb and / or ATM gene or gene product from said hyperproliferative cells; And
(c) the structure, function, or expression of the hyperproliferative cell comprises comparing to the wild-type structure, function or expression of the p53, Rb or ATM gene or gene product, wherein the structure of p53, Rb and / or ATM, The defect in function or expression is characterized in that the hyperproliferative disease is sensitive to oncolytic riovirus and / or myxoma virus. The method of claim 1, further comprising treating the patient from which the hyperproliferative cells are obtained. The method of claim 2, wherein the treatment comprises lyovirus therapy. The method of claim 3, wherein the riovirus therapy is wild type riovirus therapy. The method of claim 3, wherein the riovirus therapy is attenuated riovirus therapy. The method of claim 5, wherein the attenuated riovirus therapy utilizes a riovirus that expresses a defective σ1 capsid protein, an undetectable σ1 capsid protein, or does not express σ1 capsid protein. The method according to claim 3, wherein the treatment is ex as the Rio virus therapy bone marrow cells of a patient A method comprising treating in vivo . 4. The method of claim 3, comprising administering the riovirus therapy to a patient. 8. The method of claim 7, further comprising contacting the bone marrow cells with a second anti-hyperproliferation regimen. The method of claim 8, further comprising administering a second anti-hyperproliferation regimen to the patient. The method of claim 9 or 10, wherein the second anti-hyperproliferative therapy is chemotherapy, radiotherapy, hormonal therapy, gene therapy or immunotherapy. The method of claim 3, wherein the treatment further comprises treating with an inhibitor of p53, Rb or ATM function or expression. The method of claim 12, wherein the inhibitor is a protein or peptide, antibody, siRNA, antisense molecule, ribozyme or small molecule. The method of claim 2, wherein the treatment comprises myxoma virus therapy. The method of claim 2, wherein the treatment comprises non-viral anti-hyperproliferative therapy. The method of claim 15, wherein the non-viral cancer therapy is chemotherapy, radiotherapy, hormonal therapy, gene therapy or immunotherapy. The method of claim 1, wherein the hyperproliferative cells are malignant cells or benign cells. The method of claim 1, further comprising, prior to step (a), obtaining a hyperproliferative cell from the patient. The method of claim 1, wherein the expression evaluation is Northern blot, Western blot, Immunohistochemistry, RT-PCR, Microarray analysis, Transcriptome analysis, Proteome analysis or Meta A method comprising a metabolome. The method of claim 1, wherein the evaluation of the structure is sequenced, in situ hybridization, immunohistochemistry or structural proteomics. The method of claim 1, wherein the evaluation of the function comprises assessing the expression or function of a target downstream of p53, Rb or ATM. In the treatment of hyperproliferative diseases in patients:
(a) contacting hyperproliferative cells with inhibitors of p53, Rb or ATM function or expression; And
(b) contacting the hyperproliferative cell with a lyovirus or myxoma virus therapy. The method of claim 22, wherein the patient is treated with riovirus therapy. The method of claim 23, wherein the riovirus therapy is wild type riovirus therapy. The method of claim 24, wherein the riovirus therapy is attenuated riovirus therapy. The method of claim 25, wherein the attenuated riovirus therapy utilizes a riovirus that expresses a defective σ1 capsid protein, an undetectable σ1 capsid protein, or does not express σ1 capsid protein. The method of claim 22, wherein the patient is treated with myxoma virus therapy. The method of claim 22, wherein the treatment ex exerts bone marrow cells of the patient. A method comprising treating in vivo . The method of claim 22, wherein the treatment is in the bone marrow cells of the patient in A method comprising treating in vivo . The method of claim 28, further comprising contacting the bone marrow cells with a second anti-hyperproliferation therapy. The method of claim 29, further comprising administering a second anti-hyperproliferation regimen to the patient. 31. The method of claim 30, wherein the second anti-hyperproliferative therapy is chemotherapy, radiotherapy, hormonal therapy, gene therapy or immunotherapy. The method of claim 31, wherein the second anti-hyperproliferative therapy is chemotherapy, radiotherapy, hormonal therapy, gene therapy or immunotherapy. The method of claim 22, wherein the hyperproliferative cells are malignant cells or benign cells.

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