KR101585702B1 - 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 a tumor-soluble virus, such as a leiovirus and myxoma virus. In particular, the invention is based on the observation that cancer cells with abnormalities in tumor suppressor substances are more susceptible to infection with tumor-tolerant viruses than cells containing normal or " wild-type " tumor suppressor substances. In particular, defects associated with p53, Rb and ATM, respectively, make cancer cells sensitive to tumor soluble viral therapy.

Description

BACKGROUND OF THE INVENTION [0002] Tumor Suppressor-Based Sensitivity-Based Hypersproliferative CELLS TO ONCOLYTIC VIRAL THERAPY on Tumor-

This application claims priority to U.S. Provisional Patent Application No. 61 / 055,360, filed May 22, 2008, the full text of which is incorporated herein by reference.

1. Field of the Invention

The present invention is generally related to the fields of virology, molecular biology and medicine. More particularly, the present invention relates to a method of assessing the susceptibility of hyperproliferative diseases including cancer to chemotherapeutic viral therapy.

2. Description of Related Technology

Respiratory Enteric Orphan Virus is a genetically engineered, ubiquitous, non-enveloped virus containing 10 double-stranded RNA fragments, with generally weak human infectivity restricted to upper respiratory and gastrointestinal tracts, ( Tyler , 2001 ). Attempts to reverse-engineer rioviruses have largely failed because of several factors, including the double-stranded RNA genome of the rival virus. The lack of σ1 is recognized as being non-infectious ( Larson meat al . , 1994 ).

Importantly, it has been recognized for years that it exhibits surprising cytocidal activity when the virus is infected with certain types of transformed cells ( Duncan et al , 1978; Hashiro meat al . , 1977 ). The replication-competent anticancer virus (tumor-soluble virus) provides attractive chemotherapy. These anticancer viruses have two main advantages. First, unlike conventional chemotherapy and radiation therapy, these viruses specifically target cancer cells, which is due to their limited ability to replicate in normal cells. Second, when compared to a replication-incompetent vector, these viruses are propagated from the initially infected cancer cells to the surrounding cancer cells, resulting in a significant amount, and a potent anti-cancer effect can be obtained.

Exposure to wild-type Lyoviruses is substantially and potentially very seriously dangerous to immunocompromised individuals, although it is often asymptomatic in healthy individuals, but nonetheless, in patients like cancer patients The clinical potential of riovirus therapy is limited, and without such limitations, such therapy may be beneficial for cancer patients. Transformed cells containing the tumorigenic Ras-signaling pathwayin vitro Andin vivo The basis for the antiviral activity of lysovirus was not known until it appeared to be primarily sensitive to infection with the rio virus (type 3 Dearing strain)Coffey meat get , 1998; Strong meat get , 1998; Norman meat get , 2004). Because Ras gene mutations are frequently observed in various types of cancer in humans (Duursma meat get , 2003), And such discoveries have now led to the use of riovirus therapy in clinical practice (Norman meat get , 2005).

Nevertheless, the presence of the Ras gene mutation only partially explains the sensitivity of cancer cells to the virological therapy. In a recent study, the Ras pathway is not the only factor determining the viability of rio viruses in recent studies ( Song meat al, 2009), and is Rio viral resistant cells can be established from transfected Ras- transformed cells, while still holding the Ras mutations that are active (Kim et al., 2007 ). Thus, there is still a need for improved methods of identifying and selecting appropriate cancer cell subgroups that are to be treated with riven viruses and other anti-cancer viral agents.

SUMMARY OF THE INVENTION

Raw viruses, such as p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, and ATBFl, with wild type (Type 3 Dearing) or variant (e.g., partially attenuated virus with defective synthesis of sigma1 protein) 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 Rio virus in vivo, is effective in killing of these cancer cells. Thus, the coverage of rioviruses can extend beyond that associated with abnormal or overactive activity of the ras proto-oncohene signaling pathway. Other viruses, interferons or other cellular immune signaling pathways previously considered to have preferential reactivity to the ras state are also susceptible to loss of tumor suppressor function and can similarly be extended to their application. Thus, in accordance with the present invention there is provided a method of determining whether a hyperproliferative disease can be affected by a leiovirus or myxoma virus oncolysis, comprising: (a) providing hyperproliferative cells ; (b) assessing the structure, function or expression of the gene product of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, and / or ATBF1, gene or hyperproliferative cells; And (c) comparing the structure, function, or expression of hyperplastic cells with the wild-type structure, function, or expression of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, and / or ATBF1, The deficiency in the structure, function or expression of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC and / or ATBF1, Indicating that it may be affected by tumor dissolution.

The method can also include treating the patient from whom hyperproliferative cells have been obtained with a leiovirus and / or myxoma virus therapy. Rio virus therapy is either wild-type rivavir therapy or attenuated livovirus therapy and utilizes a siRNA capsid protein with binding, a leavirus that expresses an undetectable sigma 1 capsid protein or does not express sigma 1 capsid protein. The treatment method is to treat the bone marrow cells of the patient with ex vivo and / or myxoma virus therapy using ex vivo treatment, or administering to the patient a riow virus and / or myxoma virus therapy agent. ex In vivo therapy, the method further comprises contacting the bone marrow cells with the second anti-hyperplastic therapy, or, in a viral therapy administered as an individual, the method comprises administering to the subject a second anti-hyperplastic therapy agent Lt; / RTI > SECTION 2 - Excessive ejaculation may be chemotherapy, radiation therapy, hormone therapy, gene therapy, or immunotherapy. Such methods include, but are not limited to, inhibitors of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, APC, or ATBF1 function or expression such as proteins or peptides, antibodies, siRNA, anti- Lt; RTI ID = 0.0 > small-molecule < / RTI > The therapy may include non-viral anti-hypertrophic therapy, for example, chemotherapy, radiation therapy, hormone therapy, gene therapy, or immunotherapy. The hyperplastic cells may be malignant cells or benign cells. The method may further comprise the step of obtaining hyperproliferative cells from the patient prior to step (a). Expression evaluation can be performed using Northern blot, Western blot, immunohistochemistry, RT-PCR, microarray analysis, transcriptome analysis, proteome analysis or metabolome analysis . Alternatively, the evaluation of the structure may include sequencing, in situ hybridization, immunohistochemistry, or structural proteomics. In addition, evaluation of function may include assessing the expression or activity of downstream targets of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, or ATBF1.

In another embodiment, there is provided a method of treating a hyperproliferative disorder in a patient, the method comprising: (a) administering to the hyperproliferative cell a p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC, or ATBF1 function Or an inhibitory substance of expression; And (b) contacting the hypervirulent cells with a leavirus and / or myxoma virus therapeutic agent. The viroid therapy may be wild-type ri- viral therapy or attenuated ri- viral therapy, which is a rivirus that expresses a sigma 1 capsid protein with binding, an undetectable sigma 1 capsid protein, or a sigma 1 capsid protein. The treatment is to excrete the patient's bone marrow. vivo treatment, or in vivo treatment. This method may further include contacting the bone marrow cells with a second anti-hyperplastic therapy agent, wherein the second anti-hyperplastic therapy may be chemotherapy, radiation therapy, hormone therapy, gene therapy, or immunotherapy . Hyperplastic cells can be malignant or benign.

The indefinite article used in connection with the word " comprising " in the specification and / or the claims may mean " one ", but may also mean " one or more, " " .

The term " about " throughout this application refers to the intrinsic variation in the error of the device, the method used to determine the value, or the variable present in the study being included in the numerical value.

In the claims, " or " is used to mean " and / or " unless expressly referred to as alternatives, or alternatives are mutually exclusive, and the specification refers only to alternatives or & . It should be noted that as used in the specification and claims, the terms "comprising" (and any form such as "comprise" and "comprises"), "having" ), "Including" (and any form such as "includes" and "includes") or "contains" (including "contain" Quot;) is inclusive or open, and does 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. It should be understood, however, that the detailed description and specific examples, which are indicative of specific embodiments of the invention, are given by way of illustration only, and are not intended to be limiting of the scope of the present invention, Because it is obvious to those skilled in the art.

The following drawings are part of the specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings, along with a detailed description of the specific embodiments presented herein.
Figure 1A-B Leavirus and myxoma virus-preferential infection of L3 with p53 - / - MEF and ATM binding.
1A
p53 - / - MEF, and p53 + / + MEF (in turn, p53 knockout or wild-type p53 murine embryonic fibroblasts) is WT / AV Rio from virus or 5 MOI GFP- expressing myxoma virus (Myx-GFP at MOI 40 is Johnston et al , 2003 and is a GFP expression version of the Lausanne (ATCC) strain of MYXV). At three days post-infection, cells were fixed / infiltrated and analyzed by FACS using the rio virus antisera and the secondary FITC antiserum. In the results, both wild-type and attenuated viruses were able to preferentially infect p53 - / - MEF. In a myxoma infection study, cells were infected for 24 hours and viewed under a fluorescence microscope relative to the phase. GFP-expressing cells represent myxoma virus infection. Mock: False infection.
1B
(Lymphocyte subpopulation), BT (ATM-normal lymphoblastoid C3ABR cells; Kozlov et al, 2003) and L3 (ATM-deficient lymphocyte; Kozlov et al, 2003 ) infected with WT / AVR virus at MOI 40 At MOI 5, Myx-GFP was infected. At three days post-infection, cells were fixed / infiltrated and analyzed by FACS using the rio virus antisera and the secondary FITC antiserum. In the results, both wild-type and attenuated viruses were able to preferentially infect p53 - / - MEF. In myxoma infection studies, cells were infected for 48 hours and viewed under fluorescent microscopy relative to phase. GFP-expressing cells represent myxoma virus infection. The C35ABR (B3) and L3 cell lines, M Lavin (QueenS1and Institute of Medical Research) and Dr. Y Shiloh (Tel Aviv University). Mock: False infection.
Figures 2A-C-p 53 and Leovirus and myxoma virus preferential infection of ATM lymphoma malfunctions.
2A
p53 and ATM lack reinforcement response to IR stimulation. Mantle cell lymphoma (Granta, HBL-2 (p53 mutation, Turker et al, 2006 ), Z138C, JVM-2) and Burkitt lymphoma ( Raji, and Ramos ) were irradiated at 2 Gy for 2 hours, Cells were obtained. The cells were treated with NET-N dissolution buffer (1% NP-40) and subjected to sonication to generate whole cell lysate. 50 μg of protein was loaded onto 8% or 10% SDS-PAGE and transferred to nitrocellulose and labeled antibodies (Phospho-ATM; pSerl981 ATM and Phospho-p53; pSerl5 and p53 were obtained from Rockland Immunochemicals for Research and Cell Signaling ; ATM-specific rabbit polyclonal antibody 4BA is a gift from Dr. M Lavin). BT and L3 cells were used as reference.
2B
p53 and ATM-normal-responsive cells (BT, Z138C, JVM-2, Ramos) and p53 and / or ATM-dysfunctional-responsive cells (L3, HBL-2, Grant, Raji) Lt; RTI ID = 0.0 > viral < / RTI >One; Virus-sensitive. 0; Virus-resistant. Normal-reactive cells are resistant to viral infection, while dysfunctional cells are susceptible to viral infection.
2C
Cells were infected with WT / AV lvovirus at MOI 40 or Myx-GFP at MOI 5. In the preliminary work, Raji cells were sensitive to the reovirus and Ramos cells were found to be resistant to the rival virus ( Alain et al, 2002 ). At three days post-infection, cells were fixed / infiltrated and analyzed by FACS using the rio virus antisera and the secondary FITC antiserum. In the results, both the leiovirus and myxoma viruses 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 fluorescent microscopy relative to phase. GFP-expressing cells represent myxoma virus infection. Mock: False infection.
Figure 3 - Leovirus and myxoma virus infection of retinoblastoma cells
Retinoblastoma cells (Y79 and WERI-Rb-I, purchased from ATCC) were infected with WT / AVR virus at MOI 40 or Myx-GFP at MOI 5. At the indicated date after infection, the cells were fixed / infiltrated and analyzed by FACS, using the ryovirus antisera and the secondary FITC antiserum. In myxoma infection studies, cells were infected for 48 hours and viewed under fluorescent microscopy relative to phase. GFP-expressing cells represent myxoma virus infection. Mock: False infection.

Carcinogenesis is a multi-step process involving complex repetition of mutagenic gene activation and inactivation of tumor suppressor genes through mutational events that affect genes for normal carcinogenesis and tumor suppressor genes. Interestingly, tumor soluble viruses utilize different oncogene-driven cell-based signaling pathways to selectively replicate and kill the cells they infect. In addition to tumor gene-dependent tumor dissolution, the inventors thought that some of the tumor suppressor genes would play an important role in carrying out tumor dissolution by the virus. Thus, these inventors have tested the gene of tumor suppressor in various murine and human cancer cell lines to test whether the tumor suppressor gene function abnormality affects the dissolution of the viruses of myxoma and myxoma virus.

The molecular basis of lysovirus tumor dissolution has been progressed through efforts to characterize cell receptors for the rival virus. In 1993, two mouse cell lines without epithelial growth factor receptor (EGFRs) expression were relatively resistant to the infectious virus, and the same cell line transfected with the EGFR encoding gene exhibited significantly higher sensitivity Strong meat al . , 1993 ). As a result, it has been proposed that the lvovirus utilizes an activated cellular signal transduction pathway conferred by the presence of EGFR functioning in the host cell. Later, these same researchers could explain that NIH3T3 cells that are resistant to a leiovirus infection become susceptible when transfected with activated Sos, Ras or verbB downstream of EGFR ( Strong meat al. , 1996; Strong meat al . , 1998 ). Thus, the development of a tumorigenic Ras signaling pathway is considered to be a crucial step in lysovirus tumor dissolution.

Many cancers in humans explain enhanced Ras pathway activation, emphasizing the potential effects of such signaling networks on tumorigenesis and suggesting that the viroids will exert extensive tumor solubility in a variety of tumor types. In early experiments, more than 80% of cell lines derived from various tumor types were found to be susceptible to reovirus-mediated killing ( Coffey < RTI ID = 0.0 > meat al . , 1998 ). More interestingly, another oncogene, Myc, also contributes to lysovirus tumor dissolution. Myc overexpression permits riovirus-resistant cells to become viable ( Egan et al, 2003 ).

Myxoma virus is a rabbit-specific poxvirus considered to be a promising tumor-soluble virus base ( Stanford and McFadden , 2007 ). Its host tropism is highly restricted to European rabbits and non-pathogenic to all other vertebrate species including humans ( McFadden , 2005 ). Despite these limited specificities, myxoma viruses can infect and kill a wide variety of human tumor cell lines ( Sypula < RTI ID = 0.0 > meat al . , 2004 ). At the cellular level, myxoma virus affinity is largely regulated by downstream intracellular events of viral binding and entry, rather than being regulated at specific afferent receptor levels ( McFadden , 2005 ).

Through the study of virus host range factors that control virus tolerance in human cancer cells, the molecular basis of myxoma virus tumor dissolution has been advanced. Viral M-T5 host range protein interacts with the cellular Akt, this interaction enhances the solubility myxoma tumor capabilities in a human cancer cells (Wang meat al , 2006 ). M-T5 KO (green-out) myxoma viruses exhibited attenuated viral tumor solubility in a single class of human cancer cells (Type II cancer cells), and constitutively Akt-activated cancer cells exhibited M-T5 KO myxoma virus , Suggesting that upregulation of Akt, which is frequently found in many cancer cells, may play an important role in determining myxoma tumor solubility ( Wang meat al . , 2006 ).

Adenovirus studies have for the first time led to a relationship between viral tumor dissolution 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. Mutant adenoviruses that do not express such viral proteins are replicated in p53-dysfunctional human tumor cells and can kill the cells, but not functional p53 ( Bischoff < RTI ID = 0.0 > meat al . , 1996 ). The rationale for lysis of mutated adenovirus tumors is that adenovirus E1A-induced p53-dependent apoptosis does not function in p53 dysfunctional cancer cells due to p53 deletion or mutation ( Bischoff meat al . , 1966 ). However, further studies have shown that p53-dependent adenoviral tumor dissolution is more complex in a variety of models and is more reactive to additional pathways ( O'Shea meat al. , 2004; Abou meat al. , 2004; Royds et al , 2006 ). In fact, because of this, the inventors thought that p53 dysfunctional cancer cells could destroy the cellular antiviral mechanism through genomic instability. ( Carroll et al ., 1999 ). Because of the importance of genomic retention by p53, the p53 gene is considered to be the recipient of the cellular genome ( Gomez - Lazaro meat al. , 2004; Vogelstein meat al . , 2000 ). Because of the maintenance of the p53-mediated genome, normal cells may be able to maintain their unique anti-viral mechanism. However, p53 dysfunction will ultimately be induced in viral susceptibility (through the loss of existing anti-viral) caused by global genomic instability. In a recent study, p53 has been shown to contribute to intrinsic immunogenicity by enhancing IFN-dependent anti-viral activity as a pro-apoptotic and tumor suppressor gene ( Takaoka meat al. , 2003; Munoz - Fontela meat al . , 2008 ). The p53 transcriptional role is important in activating the IFN pathway in viral infections. p53 may activate the transcription of IFN regulatory factor 9 upon virus challenge ( Munoz- Fontela et < RTI ID = 0.0 & gt; al . , 2008 ). Moreover, when p53 expression was reduced in cancer cells, a significantly higher level of viral replication was observed ( Dharel meat al . , 2008 ). These findings demonstrate the important antiviral activity of p53. This may be true even in the case of other tumor suppressor genes that are important for DNA repair and genomic stability. Here, the inventors have shown that cancer cell dysfunction in p53, ATM or Rb is highly sensitive to both the virologic and myxoviral challenge, which may be enhanced by activation of a specific oncogene signaling pathway In addition to this, it can be helpful to explain how the tumor-soluble virus distinguishes between normal and cancer cells. Identification of linkage between such genetic abnormalities and tumor-soluble virus susceptibility greatly increases the likelihood of applying tumor-soluble virus therapy.

I. Tumor Soluble Virus

A. rio virus ( Resoviridae )

The virus contains a naturally occurring, non-enveloped virus family with a double-stranded RNA (dsRNA) genome, divided into 10 fragments and surrounded by two concentric icosahedral protein capsids. These viruses can affect the gastrointestinal system and the respiratory tract. The name Leovirid comes from the "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 a variety of diseases, the original name is still used.

Although rio virus infection is common in humans, it is mostly mild or subclinical. The virus can be easily detected in the stool, but it can also be recovered from the throat or nasal secretions, urine, cerebrospinal fluid, and blood. Despite the fact that they can easily be found in clinical specimens, their role in human disease or treatment remains unclear. The virus has an icosahedral capsid (T-13) composed of the outer and inner protein shells. The virus genome of Riovidade contains 10-12 fragments, which are divided into three classes according to their size: L (large), M (middle), S (small). Fragments range from ~ 3.9-1 kB, with each fragment encoding 1-3 proteins. The ribaviride protein is represented by the Greek letter corresponding to the fragment to which this protein is to be decoded (the L fragment encodes the lambda protein, the M fragment encodes the mu protein, and the S fragment encodes the [sigma] protein ).

Because these viruses have the dsRNA genome, replication occurs exclusively in the cytoplasm, and the virus encodes several proteins necessary for replication and conversion of the dsRNA genome into (+) - RNAs. The virus can enter the host cell through a receptor on the cell surface. Although the receptor is not known, it is considered to include sialic acid and conjugative adsorbent molecules (JAM). The virus is partially cleaved by the protease in the endolysosome, and the capsid in the endolysosomes is partially digested to further enter the cell. The core particles then enter the cytoplasm by an unseen process, the genome conservatively transcribed from the cytoplasm, and the (+) sense strand becomes redundant, which becomes an mRNA template and the (-) sense strand is synthesized do. The virus particles start assembly in the cytoplasm within 6-7 hours of infection.

Infectious mammalian virus virus virions are produced with particles of approximately 85 nm in diameter. The virion outer capsid contains several distinct protein species, sigma-1 (σ1, 50 kDa), through individual carbohydrate bonds ( Chappell meat al. , 1997; Chappell et al., 2000; Connolly meat al , 2001 ), and the virion-annealing domain ( Mah meat al. , 1990; Fernandes et al. , 1994; Lee meat al. , 1994 ), mediating the attachment of virus to the surface of host cells ( Lee meat al. , 1981; Duncan meat al. , 1991; Nagata meat al. , 1987; Turner meat al . , 1992 ). 1 is the product of the bicistronic Lyovirus S1 gene, which also encodes a non-structural protein named sigma 1s using a separate yet overlapping reading frame ( Ernst meat al. , 1985; Jacobs meat al. , 1985; Sarkar meat al . , 1985 ). Viral particles with no sigma 1 have been reported as non-infectious ( Larson meat al . , 1994 ). The R1 virus S1 gene appears to play a significant role in determining the virulence of the virus ( Haller meat al. , 1995; Wilson et al., 1994; Kaye meat al. , 1986; Werner meat al . , 1980 ).

i. Wild type in cancer therapy Rio virus

Specific methods for the production and use of wild-type reovirus in cancer therapy have been described (see, for example, US Pat. Nos. 7,300,650, 7,163,678, 7,049,127, 7,014,847, 6,994,858, 6,811,775, 6,703,232, 6,576,234, 6,565,831, 6,528,305, 6,455,038, 6,344,195, 6,261,555, 6,136,307, and 6,110,461, and U.S. 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 a waiver).

ii . In cancer therapy Attenuated Rio virus

In hosts that fail immune system function, such as in newborn animals and in SCID (severe combined immunodeficiency) animals, wild-type viruses exert a particularly viral virulence in nervous tissue and cardiac muscle tissue ( Sabin , 1959; Weiner meat al. , 1977; Baty meat al. , 1993; Loken meat al . , 2004 ). In some cases, immune-functioning hosts, including humans, have been linked to viral etiology ( Terheggen meat al , 2003, Hirasawa meat al . , 2003 ). Thus, in immunocompromised or very young hosts, the wild-type virus does not always act in a favorable manner. For example, in a host immune system, such as a very young or immunodeficient adult, the virus has been shown to infect some healthy tissues such as the heart, liver, pancreas, and nervous system. These issues may be further applied to cancer patients treated with potent radiation therapy / chemotherapy, which should be immunosuppressed. Thus, there is a clear need to develop leukemia with reduced virulence for viral tumor solubility therapy.

Moreover, in many cases, wild-type ryoviruses possess undesirable virulence and infectivity that limit their use in vitro . In particular, exposure of a cell culture (e. G., A bone marrow transplant taken from a cancer patient) to a wild-type virus may cause undesirable effects of the cancer patient's immune system to re-kill cells such as bone marrow cells. Thus, the enhanced outbreak of the wild-type rio virus greatly limits the clinical availability in vitro associated with exposing the cell to the rio virus.

The attenuated Rioviruses described here are deficient in the sigma1 capsid protein of the detectable battlefield, but are still unexpectedly infectious, and < RTI ID = 0.0 > sigma1 < / RTI > ( Lee < RTI ID = 0.0 > meat al ., 1981; Duncan meat al ., 1991; Nagata meat al. , 1987; Turner et al. , 1992; Chappell meat al. , 1997; Chappell meat al. , 2000; Connolly meat al . , 2001 ). Despite the insufficiency of σ1 of the total length, the attenuated rhovinus described herein is capable of host cell entry and cytolytic viral replication. Additionally, the attenuated virus may have one or more of the non-malignant cells when compared to the level of cytopathic effect exhibited against naturally occurring non-malignant cells by non-attenuated viruses Lt; RTI ID = 0.0 > (e. G., A statistically significant decrease) in the cytopathic effect. Thus, as described in more detail below, the attenuated ryoviruses provided herein provide improvements over existing rio viruses, the improvement being both tropism for normal cells (e.g., non-malignant cells) Suitable for use as a tumor soluble material without undesirable side effects such as dissolution. In particular, such attenuated ryoviruses can be used for the selective killing of cancerous or neoplastic cells in may be used in vitro .

In certain embodiments, U.S. Pat. The attenuated ryoviruses described in application 60 / 704,604 and described in international application PCT / US2006 / 029881, filed July 31, 2006 (these references being incorporated by reference in their entirety) can be used in the present invention. In certain embodiments, the attenuated lyovirus is deficient in the wild-type rhovinid S1 gene. The attenuated virus may have a reduced and / or undetectable amount or a truncated, mutated, and / or over-functioning S1 gene of the S1 gene product (sigma 1). The attenuated virus may have an S1 gene comprising one or more mutations when compared to the wild-type S1 gene, wherein the mutation is a nucleotide substitution, a nucleotide deletion, or a nucleotide insertion.

As used herein, an " attenuated " ryovirus may be considered to be an " attenuated " virus when compared to the level of one or more infectious, replication and / or hemolytic properties, Such properties include those that have been modified (e. G., Reduced or increased in a statistically significant manner). In certain embodiments, the attenuated rio virus will exhibit reduced infectivity, replication and / or hemolytic potential compared to wild-type rio virus. Examples of such altered properties that can identify the attenuated viroids presented herein include a variety of manifestations of the viral cytopathic effects, including, for example, the need for a productive infection of a given host cell The multiplicity of infection (MOI, the average number of virions infecting each cell), the degree of host cell lysis induced by virus infection (including addition of poppostocide and / or necrosis) Other variables that are well known in the art that can be used to measure viral titer released from sexually infected host cells and viral activity against host cells. Other manifestations of the cytopathic effects include altered host cell morphology, altered ability to induce an attack by host cell-mediated immune system, (modified to a substance such as extracellular matrix protein or semi-solid growth medium, or to other cells) Cell adhesion, altered expression levels of one or more cellular genes, altered ability of the host cell to replicate, and / or other modifications in cellular metabolic activity.

Given that the S1 gene fragment of the rio virus is strongly associated with the toxicity (virulence) of the rio virus ( Weiner meat al. , 1977; Weiner meat al. , 1980; Mann meat al, 2002), the inventors have surprisingly attenuated S1 from the continuous Rio viral infection of cultured cells Rio virus ( "AV Rio virus") was isolated (Kim meat al . , 2007 ). The attenuated leiovirus strains are in exhibit significantly reduced viral pathogenesis in immune-deficient animal models without impairing its viral tumor-solvating activity in vivo . Since wild-type rifampin is known to disadvantage the development of murine and murine embryos by retarding blastocytst development and inhibiting formation in mouse and murine embryos ( Priscott , 1983; Heggie meat al. , 1979 ), the inventors further evaluated the virulence of AVIVO virus on stem cells. Thus, the inventors compared the pathogenicity of wild-type and avio viruses on embryonic stem cells (ESCs). As shown in the Examples below, the wild-type virus in Rio Although ESCs were easily infected in vitro and significantly inhibited stem cell development in teratoma models, AVV infects at least ESCs in vitro and does not affect stem cell development in teratoma models.

In various embodiments, the attenuated virus may comprise one or more additional mutations. For example, in a further specific embodiment, the attenuated ryovirus may be deficient in the wild-type ryvovirus S4 gene. The lvovirus wild-type S4 gene encodes a virus envelope sigma3 polypeptide associated with virion processing during a lvovirus proliferative infection of the host cell (e. G. , Ahmed meat al. , 1982; Giantini et al , 1984 ).

The attenuated virus may include a replication competent virus virus. Moreover, the attenuated virus may have inherited mutations (e.g., substitutions, insertions, deletions) in the S1 gene and / or the S4 gene. The wild-type Riovirus S1 gene is known to those skilled in the art. For example, the wild-type S1 gene sequence comprises or comprises the S1 gene sequence identified in the predominant forms of the naturally-occurring lvovirus isolated from the respiratory or enteric tissues of the infected individual, or a common sequence derived from such sequence consensus sequences. The amino acid sequence of the encoded σ1 protein itself (eg, Genbank Accession numbers for the major human leiovirus serotype S1 gene sequence: human type 1 riovirus strain Lang (TlL) Ace. No. M35963; (T3A) Ace. No. L37677), as well as the human type 3 rio virus strain Dealing (T3D) Ace. No. XOl 161, (Including, for example, the GenBank accession numbers for human leiovirus S1 gene sequences, human type 3 viruses S1: XOl 161, human type < RTI ID = 0.0 > 2 < / RTI > viruses S1: M35964; human type 1 viruses S1: M35963).

According to a particular embodiment, an attenuated reovirus comprising the genome of the genome of the genome of the genome is provided comprising an attenuated virus comprising the genome of the genome, The attenuated rio virus is provided (human leiovirus S1 gene sequence Genbank Accession numbers: human type 3 leiovirus S1: X01161; human type 2 reovirus S1: M35964; human type 1 reovirus S1: M35963). Methods for determining whether a mutated S1 gene is present, by sequencing the Riovirus S1 gene, are apparent from this disclosure and are described in Ausubel et al. (1989); Ausubel et al (1993); Sambrook et al (1989); Maniatis et al. (1982); Glover (1985); Hames and Higgins (1985) ; Are known in the art according to techniques described in other documents. Thus, the mutated S1 gene may be modified by one or more nucleotide substitutions, nucleotide insertions, and nucleotide deletions (such as can be readily determined) from the nucleotide sequence of the corresponding S1 wild-type or common sequence, such that one or more nucleotide sequences are different Refers to the S1 gene having a polynucleotide sequence. Several mutations within the murine riV1 gene encoding the sigma 1 protein have been described by Hoyt et al. (2005), and in accordance with certain embodiments of the invention described herein, the mutations of Hoyt et al are expressly excluded.

As described herein, and as is known in the art, the lvovirus outer capsid sigma 1 protein is based on its biochemical and / or immunochemical properties (see, for example, Mah et al, 1990; Leone et al, 1991; Chappell immunoaffinity chromatography, immunocytofluorimetry, electrophoresis of radiolabeled lvovirus polypeptides, and immunoprecipitation (e. g., < RTI ID = 0.0 > Etc.), hemagglutination reactions, and / or related methods. Such means and other means for detecting the leavirus sigma 1 protein have been established and by the techniques described herein those of skill in the art will recognize what is acceptable in the art for detecting < RTI ID = 0.0 > And the unique σ1 protein is not detected when, for example, the current routine procedure is used to determine the σ1 protein (if present) in the clone-competent vi virus virion lacking the "detectable" viable σ1 capsid protein You will understand that it does not include the rio virus.

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

iii . Rio virus  Control of infection

In certain embodiments, peptidyl fluoromethyl ketones (PFMKs) can be used in cell compositions to inhibit or remove the leavirus from the composition. Co-pending US Application No. 60 / 906,706 (which is incorporated by reference in its entirety) describes peptidyl fluoromethyl ketones (PFMKs) for inhibiting viral replication, which are incorporated herein by reference in their entirety without any waiver.

iv . Rioovirus  production

According to the specific embodiment described herein, a ryovirus refers to a member of the levoviridae (family), including rio viruses, which can be derived from any ryovirus and can be obtained from a variety of sources ( Tyler and Fields , 1996 ). In certain embodiments, mammalian leioviruses and human leioviruses are considered, but the present invention is not so limited, and based on the present disclosure, one of ordinary skill in the art will recognize that any particular leiovirus may also be desirable for such purposes. In certain embodiments, the reovirus may be human Type 3 (Dealing), Type 1 (Lang), Type 2 (Jones), or Type 3 (Abney) (Eg, chimpanzees, gorillas, macaques, monkeys, etc.), rodents (eg, mice, rats, gerbils rats, hemsters, rabbits, guinea pigs, etc.), dogs, cats, Derived from one or more rioviruses that exhibit tropism for cells of other mammalian species, including, for example, cows, horses, horses, pigs, Avian reovirus) may be used.

As described herein, certain embodiments relate to attenuated rhovinus, which can be recovered after a continuous infectious regimen in vitro , but the attenuated rhovinus is in vivo persistent infection regimens, generation and identification of σ1-deficient and / or σ1-deficient mutants by molecular biology (and in certain embodiments, additionally or alternatively, σ3-deficiency and / or σ3-deficiency (And / or < RTI ID = 0.0 > 3) < / RTI > mutants and / or σ3 mutants having naturally occurring σ1- and / or σ1- It is contemplated that the mutant can be derived in accordance with other methods, including artificial induction by chemical, physical and / or genetic techniques (such as assortative recombination of the viroid gene in a proliferating infected host cell).

Furthermore, the inventors believe that the preferred attenuation phenotype resulting from mutation is a function of virus infectivity, replication or packaging ability, including any gene or combination of genes encoded on the virus, such as ten gene fragments of the virus To be observed in other genes. For example, mutations in the Rio virus S1 or S4 gene, when compared to the wild-type gene of the riVovirus, can result in one, two, three, four, five or more mutations on ten gene fragments of the riVovirus . In order to make the attenuated virus, various methods including those described in US application 60 / 704,604 can be used.

B. myxoma virus ( Myxoma Virus )

Myxoma virus is a poxvirus and has a large double-stranded DNA genome that allows for the large (25 kb), therapeutically relevant, potential insertion of a eukaryotic gene. Myxoma viruses are rabbit-specific viruses and cause fatal diseases of myxomatosis in the European rabbit ( Oryctalagus cuniculus ). This species specificity was so limited that it was used to control a group of rabbit rabbits that caused disaster in Australia in 1950. Immunized baby monkey kidney fibroblasts (BGMK), genetically deficient primary murine cells in the IFN response, and in lt; RTI ID = 0.0 > in- vitro < / RTI > It is possible to proliferate infect cells in vitro except for certain rabbits, but importantly, it is non-pathogenic to all other tested vertebrate animals including humans. Cancer cells are well known for lack of IFN response. Recently, myxoma virus has been used in human malignant glioma surgical specimens in vitro , in vivo and ex is a tumor-soluble substance for experimental glioma in vivo ( Lun meat al. , 2005; 2007; Stanford meat al . , 2008 ). Myxoma virus can infect human glioma cells, kill them, be safe when administered into the brain, and "treat" mice administered in tumors in orthotopic human malignant glioma models. Furthermore, all primary tested glioma cells obtained directly from glioma surgical specimens are infected and killed, and their tumor solubility activity is enhanced by rapamycin.

With other tumor-soluble viruses, such as rio viruses, myxoma viruses need to bypass anti-viral defense in normal healthy cells so that they can replicate in cells. Myxoma viruses and other tumor-soluble viruses induce interferon production and are generally susceptible to anti-viral effects of the IFN pathway. Related proteins that are induced by IFN anti-viral reactions and primarily affect the virus increase include PKR, OAS synthase and Rnase L nucleases. PKR activates eIF2α, inhibits detoxification, and induces apoptosis. In normal cells, myxoma viruses are directly affected by PKR and eIF2α.

The anti-viral pathway often collapses in cancer cells. For example, a reduced or defective response to IFN is a genetic defect that is common during transformation and tumorigenesis. More than 80% of tumor cell lines do not respond to interferon or show impaired response ( Stojdl meat al. , 2003 and the references cited therein; Wong meat al. , 1997; Sun meat al. , 1998; Matin meat al. , 2001; Balachandran meat al . , 2004 ). US Patent Publication 2006/0263333 (incorporated by reference) describes the use of myxoma viruses to infect and kill cancer cells, including human tumor cells, which are incorporated by reference.

II . Tumor suppressor

The tumor suppressor gene is a gene that protects cells at a stage in the process of progressing to cancer. When this gene is damaged, the cell usually undergoes cancer, after being influenced by other factors. Unlike oncogenes, tumor suppressor genes generally follow a two-hit hypothesis, which means that all alleles encoding a particular gene must be affected before the effect becomes apparent. This is because if only one allele of the gene is damaged, the second allele can still produce the correct protein. In other words, haplosufficient (the phenotype is normal even if only one gene is normal), as opposed to the tumor gene, which is generally haploinsufficient. There is, of course, a clear exception, such as the p53 gene product, which may exist in a dominant-negative mutant form for normal p53 protein, in which case it is haploid-insoluble.

Tumor suppressor genes, or more precisely, the proteins they encode, have a damping or inhibitory effect on the regulation of the cell cycle or promote, and in some cases both, of the poppostis. The function of the tumor suppressor protein belongs to several categories, including: (a) inhibiting genes essential for the cell cycle to persist (if these genes are not expressed, the cell cycle is not sustained; Effectively inhibited); (b) associating the cell cycle with DNA damage. As long as there is damaged DNA in the cell, the cell can not be cleaved (once the damage is restored, the cell cycle can last); (c) If damage is not restored, apoptosis or prospective apoptosis is induced, thus eliminating the threat posed for a better organism; And (d) some proteins involved in cell adhesion prevent tumor cell migration, block contact inhibition, and inhibit metastasis (a protein known as a transit inhibitor).

In contrast, oncogenes are genes that cause cancer. Many cells are expected to die normally. In cancer, because of the presence of mutated carcinogenic DNA sequences, these cells survive and multiply. Most oncogenes require additional steps, such as mutations, in environmental factors such as another gene or virus infection to cause cancer. Since 1980, dozens of tumor genes have been identified in human cancers. Raw cancer is a normal gene that can become a tumor gene due to mutation or increased expression. Raw cancer genes encode proteins that help regulate cell growth and differentiation. Raw carcinomas are often involved in signal transduction and mitosis signals through their protein products. Upon activation, the native cancer gene (or its product) becomes a tumor-inducing agent, a tumor gene. Examples of native cancer genes 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 a "guardian of the genome", "guardian angel gene" or "master patrolman", referring to its role in preserving stability by interfering with genomic mutations.

p53 is the name referring to the surface molecular weight on SDS-PAGE, but is actually 43.7 kD. This difference is due to the large number of amino acids in the p53 protein, the proline residue, which makes the migration of p53 slower on SDS-PAGE, making it appear larger. Such effects are observed in p53 of a variety of species including, for example, humans, rodents, frogs and fish. The gene is located on human chromosome 17 (17pl3.1) and encodes 393 amino acid proteins with five domains: (a) N-terminal transcription-activation domain (TAD) Residue 1-42); (b) a proline-rich domain (residues 80-94) important for the apoptotic activity of p53; (c) a central DNA-binding core domain (DBD) (residue 100-300) comprising one zinc atom and several arginine amino acids; (d) homolog-oligomerization domain (OD) (residues 307-355); And (e) the C-terminal domain (residues 356-393) involved in down-regulating the DNA binding of the central domain.

Mutations that deactivate p53 in cancers usually occur in DBD. Most of these mutations disrupt the ability of proteins to bind to its target DNA sequence and thus prevent transcriptional activation of these genes. Thus, a mutation in DBD is a recessive loss of function. P53 molecules with mutations in OD are dimerized with wild-type p53, interfering with transcriptional activation. Thus, the OD mutation has a dominant negative effect on p53 function.

p53 has many antitumor mechanisms. For example, it can activate DNA repair proteins if the DNA has sustained damage. In addition, the cell cycle can be inhibited at the G ₁ / S regulatory point of DNA repair cognition (if we hold the cells here for a sufficient amount of time, the DNA repair protein can have enough time to repair the damage, Will be able to sustain the cell cycle). In addition, if the DNA damage is proved to be irreversible, it may initiate a predetermined apoptosis, apoptosis.

p53 can be activated in response to a myriad of types of stresses, including but not limited to DNA damage (induced by chemicals such as UV, IR or hydrogen peroxide), oxidative stress, osmotic shock, ribonucleotide depletion, Including but not limited to gene expression. Activation is highlighted by two major events. First, the half-life of p53 protein is rapidly increased, and p53 accumulates rapidly in stressed cells. Second, morphological changes lead p53 to play an active role as transcriptional regulator in these cells. The crucial event that drives the activation of p53 is the 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 belongs to the MAPK family (JNK1-3, ERK1-2, p38 MAPK), which responds to several types of stresses, such as membrane damage, oxidative stress, osmotic shock, It is known. The second group of protein kinases (ATR, ATM, Chk1, Chk2, DNA-PK, and CAK) detects several forms of DNA damage due to genotoxic stress and detects the molecular cascade, Integrity Checkpoint is involved.

In unstressed cells, p53 levels remain low through continued degradation of p53. A protein called Mdm2 binds to p53 and carries it from the nucleus to the cytoplasm and is degraded by the proteasome in the cytoplasm. The Mdm2-linkage in the phosphorylation of the N-terminus of p53 is disrupted by the above-mentioned protein kinases. Other proteins, such as Pin1, accumulate in p53 and induce morphological changes in p53, further interfering with Mdm2-binding. Transcriptional coactivators, such as p300 or PCAF, are acetylated at the carboxy-terminus of p53 and exposed to the DNA-binding domain of p53, allowing p53 to activate or inhibit specific genes.

B. Rb

Retinal subpopulation protein (Rb; NM 00321) is a tumor suppressor protein that is found to be dysfunctional in a variety of cancers. If the protein is inactivated by mutation in both alleles of the RB1 gene encoding it, it is named pRb because it causes retinitis subcarcinoma. Rb is usually present as a phosphorylation protein in cells and is a target of phosphorylation by several carboxases as described below. One feature that has been studied extensively in Rb is that it prevents cell division or progression through the cell cycle. Thus, in this role of Rb ineffectively, 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 papillomavirus can bind to Rb and deactivate it, which can be linked to cancer. Rb prevents replication of the damaged DNA by preventing the cell from progressing through the cell cycle to S or synthetically, or by blocking the progression to G1 or the first gap state. Rb binds to and inhibits the transcription factors of the E2F family. E2F transcription factors are dimers of E2F and DP proteins. The transcriptional activation complex of the transcriptional E2 promoter-binding-protein-dimerization partner (E2F-DP) can push the cell to the S phase. When E2F-DP is inactivated, the cells remain on G1. When Rb is bound 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 and DNA synthesis is further inhibited.

In the hypo phosphorylated state, Rb is active and plays a role as a tumor suppressor by inhibiting cell cycle progression. Phosphorylation deactivates Rb. Rb is activated near the end of G1 to allow binding to E2F when one of the phosphatase residues is diphosphorylated. At the time of cell entry into the S phase, the cyclin-dependent kinase (CDK) and cyclin complexes phosphorylate pRb and its activity is inhibited. Initial phosphorylation is carried out by Cyclin D / CDK4,6, followed by additional phosphorylation performed by Cyclin E / CDK2. pRb remains phosphorylated on S, G2, and M. Phosphorylation of pRb separates E2F-DP from pRb, resulting in an active form. Once E2F is liberated, it activates factors such as cyclins (e.g., Cyclin E and A), thereby activating cells called cyclin-dependent kinases, and proliferating cell nuclear antigens, or PCNA, So that DNA replication and repair are rapidly progressing by helping to attach polymerases to the DNA.

C. ATM

Ataxia telangiectasia (AT; NM_00051) is an autosomal recessive disorder due to mutations in the ATM gene located on chromosome llq22-23. It was characterized in June 1995 and consists of 66 exons spanning 150 kb of genomic DNA. It encodes an open reading frame of 9168 nucleotides and a 13kb mature transcript. ATM protein is about 37OkDa, expressed everywhere, and located in the cell nucleus. The ATM protein is a giant serine-threonine kinase that is thought to play a role in the regulation of cell cycle checkpoints, double-stranded DNA repair and meiosis (similar to the BRCS gene). ATM is also known to play a role in the regulation of p53, BRCA1 and CHEK2. In DNA repair, part of the ATM role is known as telomere repair, because terminal ploys are more rapidly degraded 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 function of the protein is completely lost, thus being inherited in a recessive fashion and causing AT; And (b) a 'missense' mutation produces a full-length protein with reduced functions such as stable, substitution, short in-frame insertion and deletion. These mutations mainly interfere with the normal copying of proteins. The majority of 65-70% of AT patients have truncating mutations, and exon skipping mutations are particularly common. This results in a very low or undetectable level of ATM protein. Missense mutations are the most common type of mutation found in breast cancer carriers. Individuals with two missense mutations appear to have a milder form of AT, which can be explained by the attenuated AT.

D. Other Tumor Inhibitors

In the present invention, various other tumor suppressing substances such as p53, ATM and Rb can be used. For example, BRCA1 and BRCA2 play a crucial role in the development of breast cancer. BRCA1 is a human gene that maintains genomic integrity to prevent uncontrolled proliferation. Most important tribal BRCA1 protein products are involved in DNA damage repair, ubiquitination, and enterprise control as well as other functions. Mutations in these genes have been implicated in several genetic cancers, namely breast, ovarian, and prostate cancer. The BRCA1 gene is located on bands 21 on the long (q) arm of chromosome 17 from base pairs 38,449,843 to base pairs 38,530,933 (map). The BRCA1 protein is directly involved in the repair of damaged DNA. Although the exact role of this is still unknown, the BRCA1 protein appears to interact with RAD51 during the repair of DNA double-strand breaks. Such braking may be caused by natural radiation or other exposures, as well as when chromosomes exchange genetic material during specific cell division (meiosis) to create ovaries and sperm. By affecting DNA repair, this protein plays a role in maintaining the stability of the human genome.

BRCA2 is a second human gene involved in chromosome repair. The structure of the BRCA1 and BRCA2 genes is very different, but their function seems to be similar. Proteins made by both of these genes are essential for repairing damaged DNA. To restore braking in DNA, the BRCA2 protein binds to and regulates proteins made by the RAD51 gene. Such braking may be caused by natural and medical radiation or other environmental exposures, as well as when chromosomes exchange genetic material during specific cell division (meiosis) to create ovaries and sperm . The BRCA1 protein also interacts with the RAD51 protein. By restoring 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 at 12.3 (13ql2.3) on the long (q) arm of chromosome 13 from base pairs 31,787,616 to base pairs 31,871,804.

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 inconsistent DNA. In E. coli , MutS binds to the mismatched site in the double-stranded DNA and targets a portion of one strand of the DNA strand at the deletion site, in cooperation with the MutL and MutH proteins. Other proteins complete the repair process: the target portion is removed, degraded, complementary strands are used as a template, a patch is synthesized, and the synthesized patch is ligated ), Resulting in a fragment recovered from the double-stranded DNA without inconsistency. Much attention is focused on the human MutS homologue because defects in some of these are responsible for some forms of genetic non-polyposis colorectal cancer (HNPCC) and possibly other colon cancer. Adenocarcinoma of the stomach (APC) is another tumor suppressor involved in colon cancer. It helps to determine how often a cell divides, how it attaches to other cells in the tissue, how it moves in or out of tissue, and how accurate the number of chromosomes in cells produced through cell division Help. APC proteins do this primarily through association with other proteins, particularly proteins involved in cell attachment and signaling. The activity of a protein, in particular, beta -catenin, is regulated by APC proteins, which are part of the Wnt signaling pathway. Control of beta -catenin prevents the gene that immediately stimulates cell division from turning on and prevents overgrowth of cells. The APC gene is located on the long (q) arm of chromosome 5 from base pairs 21 and 22 to base pairs 112, 118, 468 to base pairs 112,

AT-binding transcription factor 1, or ATBF1, is a tumor suppressor, and its loss is implicated in stomach cancer development. It is located at 16q22.3-q23.1. The overall DNA length is 261.32 kB. There are two allotypes ATBFl-A and ATBFl-B, because they use an alternating promoter using alternating cleavage. The protein is 3703 amino acids and has a size of 404 kDa. This protein contains 23 zinc fingers, one DEAD and one DEAH box, an RNA and ATP binding site, two large RS domains and multiple phosphorylation sites, including four homologous domains and one spurious zinc finger motif. The protein is nuclear localized, acting as a transcription factor that binds to the AT-rich core sequence of the enhancer element of the AFP gene, and possibly down-regulates the AFP gene expression involved in neuron differentiation. Amplification in the neural crest, in the form of extrachromosomal double-fine, non-synthetic co-amplification with myc induced cell line SJNB-12. It is observed that ATBF1 expression is not observed in the gastric cancer cell line expressing? -fetic protein. The lack of ATBFl expression is due to strong inhibition at the transcriptional level, not due to mutations, defects or translocation (tranS1ocation).

III . Assessment of tumor suppressor substance structure, expression or function

A. Nucleic acid Grounded  Diagnosis( Nucleic Acid - Based Diagnosis )

One embodiment of the invention includes a method of detecting a variation in the expression of a tumor suppressor. The method may include determining the level of the tumor suppressor or determining alterations in the expressed product. According to standard methods ( 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 may 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. The nucleic acid is amplified normally.

Depending on the format, a particular nucleic acid of interest is identified directly in the sample using amplification, or is identified as a second known nucleic acid after amplification. The identified product is then detected. In certain applications, detection can be performed by visual means (e.g., ethidium bromide staining of the gel). Alternatively, the detection may be chemiluminescence, radioactive scintillation of a radioactive label or indirect labeling of the product through a fluorescent label or through an electrical or thermal pulse signal ( Affymax Technology ; Bellus , 1994 ). In one embodiment, the amount of tumor suppressor material expressed is determined by evaluating the amount of mRNA. However, by examining various types of structural defects, changes in activity may be identified. Such defects include defects, insertions, point mutations and redundancies. Point mutations result in stop codons, frame shift mutations or amino acid substitutions. Somatic mutations occur in non-germ cell tissues and are not inherited, whereas germ cell mutations are inherited. Mutations within and outside the coding region can also affect the amount of tumor suppressor material produced by altering the transcription of a gene or destabilizing a transcript (mRNA) or protein or modifying these processes.

The cell takes genetic measures towards carcinogenic transformation when one of the tumor suppressor genes is inactivated due to the genetic or somatic mutation of the germline lesion. Inactivation of another allele of that gene is generally associated with somatic cell mutations (micromutation) or chromosomal allele deficiency, resulting in loss of heterozygosity (LOH). Alternatively, all copies of the tumor suppressor gene gene may be lost by homozygous deletion.

(ASO), Dot Blot analysis (FBS), direct DNA sequencing, PFGE analysis, Sudden or Northern blotting, Single-stranded form analysis , Denaturing gradient gel electrophoresis, RFLP, and PCR ™ -SSCP. .

i. Primer and Probe

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

ii . Template-dependent amplification method

A number of template-dependent 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 (referred to as PCR ^TM ), described in detail in US Pat. Nos. 4,683,195, 4,683,202, 4,800,159 and Innis et al, 1990, Is attached. Briefly, in PCR ™, two primer sequences complementary to the fragments on the opposite complementary strand of the marker sequence are prepared. An excess of deoxynucleotide triphosphate is added to the reaction mixture along with a DNA polymerase, such as Taq polymerase. If a marker sequence is present in the sample, the primer will be bound to the marker, and the polymerase will stop extending the primer as the nucleotide is 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 a reaction product, the excess primer will bind to the marker and the reaction product, and the process is repeated.

The reverse transcriptase PCR (TM) amplification procedure can be performed to quantify the amount of amplified mRNA. Methods for reverse transcribing RNA into cDNA are well known, and Sambrook meat al. , & lt ; / RTI & gt ; 1989 . Alternative methods for reverse transcription use 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" 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 be bound to the opposite complementary strand of the target sequence. In the presence of ligase, the two probe pairs will be connected to form a single unit. By temperature cycling, as in PCR < (TM) >, bound ligated units are dissociated from the target and serve as " target sequences " for ligation of excess probe pairs. U.S.A. Patent 4,883,750 describes a method similar to LCR in connection with the binding of a probe pair to a target sequence.

PCT application no. The 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 the RNA bearing the complementary portion to the target portion is added to the sample in the presence of the RNA polymerase. This polymerase will copy the proliferative sequence to be detected.

Isothermal Amplification Method - This method utilizes a restriction endonuclease and a ligase to amplify the target molecules containing the nucleotide 5 ' - [alpha-thio] -triphosphate on one strand of the restriction enzyme site Amplification is carried out - it may also be useful for nucleic acid amplification of the present invention ( Walker meat al. , (1992) ).

Strand Displacement Amplification (SDA) is another method of performing isothermal amplification of nucleic acids, involving multiple rounds of multiple strand displacement and synthesis, such as nick decoding. A similar method, called Repair Chain Reaction (RCR), involves a repair reaction in which several probes are annealed to the targeted portion for amplification followed by only two of the four bases. The other two bases will be added as biotinylated derivatives for ease of detection. A similar method is used for SDA. The target specific sequence may 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 RNA are hybridized to DNA present in the sample. At hybridization, the reaction is treated with RNase H, and the product of the probe is identified as a specific product released after digestion. The inherent 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 herein by reference) can be used in accordance with the present invention. For the GB application, "modified" primers are used for PCR ™ -like, template and enzyme-dependent synthesis. Primers can be labeled and modified with capture moieties (e.g., biotin) and / or detection moieties (such as enzymes). In the case of the above international application, an excess of labeled probes is added to the sample. In the presence of the target sequence, the probe is bound and cleaved by a catalyst. After cleavage, the target sequence is released in its native form bound by an excess of probe. Cleavage of the labeled probe indicates that the target sequence is present.

Other nucleic acid amplification procedures include a nucleic acid sequence-based amplification (NASBA) and a transcription-based amplification system (TAS) including 3SR ( Kwoh meat al ., 1989; Gingeras meat al , PCT Application WO 88/10315 , all of which are incorporated herein by reference). In NASBA, amplification nucleic acids can be prepared by standard phenol / chloroform extraction, thermal denaturation of clinical samples, treatment with lysis buffer, and minispin columns for the isolation of guanidium extracts of DNA and RNA or RNA. Such amplification techniques include annealing primers with a target specific sequence. After polymerization, the DNA / RNA hybrid is digested with RNase H and the double stranded DNA molecules are thermally denatured again. In either case, the single stranded DNA is fully double-stranded by the addition and polymerization of a second target specific primer. The double-stranded DNA molecules are then multiply transcribed by a RNA polymerase such as T7 or SP6. In the isothermal circulation reaction, RNA is reverse transcribed into single stranded DNA, again converted to double stranded DNA, and re-transcribed by RNA polymerases such as T7 or SP6. The resultant product exhibits a target specific sequence, whether it is truncated or intact.

Davey meat al ., EPO No. 329 822 (which is incorporated by reference in its entirety) describes a nucleic acid amplification process comprising cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA) May be used according to the present invention. The ssRNA is a template of the first primer oligonucleotide and is extended by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA: RNA duplex by the action of a ribonuclease H (RNase H, RNA-specific RNase when duplexing with DNA or RNA). The resulting ssDNA is the template of the second primer and also contains 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, With a promoter sequence at one end and an additional promoter sequence at the other end that is used by the appropriate RNA polymerase to make a number of RNA copies of the DNA. It is possible to re-enter the circulation to induce a very rapid amplification. With the choice of the appropriate enzyme, such amplification can be carried out isothermally without the addition of enzymes in each cycle. , The starting sequence may be selected in the form of DNA or RNA.

Miller meat al. , PCT application WO 89/06700 (which is incorporated by reference in its entirety) discloses the hybridization of a promoter / primer sequence to a single stranded DNA ("ssDNA") that is the target followed by the transcription of many RNA copies of the sequence Describe the nucleic acid sequence amplification scheme. This process is not cyclic, for example, a new template is not made from the resulting RNA transcript. Other amplification methods include "RACE" and "one-sided PCR ™" ( Frohman , 1990; Ohara meat al. , 1989 ; Each of which is incorporated by reference in its entirety).

Methods based on ligation of two (or more) oligonucleotides in a nucleic acid having a sequence of the resulting "di-oligonucleotide " and thereby amplification of a de-oligonucleotide can also be used in the amplification step of the invention Can ( Wu meat al. , (1989) , incorporated herein by reference).

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

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

Briefly, probes are used to target DNA or RNA species immobilized on a suitable matrix, often a nitrocellulose filter. The different species must be spatially separated for analysis. This is often done by gel electrophoresis of the nucleic acid species followed by " blotting " on the filter.

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

iv . Separation method ( Separation Methods )

In one step or another step, it is usually desirable to separate the amplification product from the template and the excess primer, 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 meat al . (1989) ).

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

v. Detection method ( Detection Methods )

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

In one embodiment, visualization is done temporally. After isolation of the amplification product, the labeled nucleic acid probe is contacted with the amplified marker sequence. The probe is preferably conjugated to a chromophore that may be radioactively labeled. 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, detection is by a labeled probe. Related art is well known to those skilled in the art and can be found in many standard books on molecular protocols (see Sambrook et al. (1989) ). For example, chromophore or radioactively labeled probes or primers identify the target during or after amplification.

One example of the foregoing is U.S. Pat. Is described in patent 5,279,721, which is incorporated by reference, which describes an apparatus and method for automated electrophoresis and nucleic acid transfer. The device is capable of blotting without electrophoresis and external manipulation of the gel and is well suited for practicing the methods according to the present invention.

In addition, the amplification products described above may be subjected to sequencing to identify certain types of mutations using standard sequence analysis techniques.

In a particular method, a thorough analysis of the gene is performed by sequencing using a set of primers 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 the basic substances and reagents required for the detection and sequencing of genes 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 an essential reaction mixture for amplification. Such kits will generally include separate containers for each reagent and enzyme and containers for each primer or probe within suitable means.

vii . Relatively quantitative RT - PCR ™ Design and Theoretical Considerations

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

In PCR ™, the number of molecules of amplified target DNA is increased by a factor of two and a cycle of reaction until some reagent is restricted. Thereafter, the amplification rate is gradually reduced until there is no increase in the amplified target between cycles. In the case of plotting the number of cycles on the X-axis and plotting the log concentration of the amplified target DNA on the Y-axis, connecting the plotted points results in a characteristic curve. At the beginning of the first cycle, the slope of the line is positive and constant. This becomes a linear part of the curve. After the reagent is limited, the slope of the line begins to decrease and eventually becomes zero. At this point, the concentration of the amplified target DNA is close to some fixed value. This becomes the plateau part of the curve.

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

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

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

Most protocols for competitive PCR ™ utilize abundant internal PCR ™ standards as targets. These strategies are effective when the product of PCR ™ amplification is linearly sampled. When the products are sampled when the reaction is close to the plateau, the less abundant products become relatively more visible. Comparison of the relative abundance created for many different RNA samples, such as when examining an RNA sample for differential expression, indicates that when examining an RNA sample for differential expression, less than substantially It is distorted in such a way that the emerging difference occurs. This is not a serious problem if the internal standard is much larger than the target. If the internal standard is more abundant than the target, a direct linear comparison between the RNA samples can be made.

The above discussion illustrates what should be considered theoretically in RT-PCR analysis of clinically derived materials. Problems inherent in clinical samples are that the amount of sample is variable (which is problematic for standardization) and that the sample quality is variable (needs to be co-amplified with a reliable internal standard of larger size, preferably larger than the target) . When RT-PCR ™ is performed with relative quantitative RT-PCR ™ with internal standards, all of these problems are overcome, wherein the internal standard is a larger amplifiable cDNA fragment than the target cDNA fragment, and the mRNA encoding the internal standard The abundance is approximately 5-100 times higher than the mRNA encoding the target. This analysis measures the relative abundance, not the absolute presence of each mRNA species.

Other studies can also be performed using the more convenient relative quantitative RT-PCR ™ analysis with external standard protocols. These assays sample the PCR products in the linear portion of the amplification curve. The number of PCR ™ cycles 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 is very important because the assay measures the absolute mRNA presence. Absolute mRNA abundance can be used as a measure of differential gene expression only in standardized samples. The empirical determination of the linear portion of the amplification curve and the normalization of cDNA preparations are tedious and time consuming processes, but consequently RT-PCR ™ analysis is superior to that derived from relative quantitative RT-PCR ™ analysis using internal standards something to do.

One of the advantages of this is that all reagents can be converted into a single PCR product in the linear portion of the amplification curve, without internal standard / competitive material, thus increasing the sensitivity of the assay. Another cause is only one PCR ™ product, the display of the product on an electrophoresis gel or other display method is somewhat less complex, less background, and easier to interpret.

viii . Chip Technology ( Chip Technologies )

Particularly considered by the present inventors is Hacia meat al . (1996) and Shoemaker meat al . (1996) . ≪ / RTI > Briefly, these techniques include quantitative methods for rapid and accurate analysis of multiple genes. By tagging oligonucleotides in the gene or using a fixed probe sequence, one skilled in the art will be able to isolate the target molecules into a high density array using chip technology and screen these molecules based on hybridization. Pease meat al . (1994); Fodor meat al . (1991) .

B. Immunodiagnosis Immunodiagnostics )

The antibodies of the invention are used to characterize the tumor inhibiting material content of healthy and diseased tissues through techniques such as ELISA and Western blotting. Antibodies can provide a screen to identify malignant neoplasms or strategies for predicting future cancerous indications or possible response to exposure to tumor-soluble virus in the case of the present invention.

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

After the antibody is bound to the wells, the immobilized surface is contacted with the sample to be tested in such a way that it is coated with a non-reactive material to reduce the background and to wash out the unbound material and induce the formation of an immunocomplex (antigen / antibody) do.

The occurrence and amount of immune complex formation following the formation of a specific immune complex between the test sample and the antibody bound and subsequent to washing may differ from the first antibody but can be determined as the second antibody having specificity for the tumor suppressor . Under appropriate conditions, the sample is preferably diluted with a diluent such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS) / Tween®. These added materials help to reduce the non-specific background. The layered antiserum is incubated preferably at a temperature of about 25 [deg.] C to about 27 [deg.] C for about 2 to about 4 hours. After incubation, the antiserum-contacted surface is washed to remove unimmunocomplexed material. Preferred washing procedures include washing with a solution such as PBS / Tween®, or borate buffer.

To provide a means of detection, the second antibody may preferably have a suitable chromogenic substrate and associated enzyme capable of developing color upon incubation. Thus, for example, those skilled in the art will appreciate that, under conditions and conditions favorable for the formation of immune complexes (incubation at room temperature for 2 hours with a PBS containing solution such as PBS / Tween), the second antibody- An oxydase-conjugated anti-human IgG may be contacted and a warming treatment may be required. After incubation with the second enzyme-tagged antibody, the column was washed to remove unbound material continuously and incubated with urea, bromocresol purple or 2,2'-azino-di- (3-ethyl-benzthiazoline) In the case of 6-sulfonic acid (ABTS) and enzyme label peroxidase, the amount of label is quantified by incubation with a chromogenic substrate such as H ₂ O ₂ . The degree of color generation is measured and quantified using a visible spectrum spectrophotometer.

The previous format may be modified by first binding the sample to the assay plate. The primary antibody is then incubated with the assay plate and the bound primary antibody is detected using a labeled secondary antibody having specificity for the primary antibody.

The antibody compositions of the present invention will have significant utility in immunoblot or Western blot analysis. The antibody can be a high-affinity primary reagent for the identification of a protein immobilized on a solid support matrix, such as nitrocellulose, nylon or a combination thereof. Along with immunoprecipitation, after gel electrophoresis, these antibodies can be used as a single-step reagent for use in antigen detection against the causing an adverse background of the second reagents used for antigen detection. In this regard, immunologically-based detection methods that are used in conjunction with a western blot that encompasses a second antibody for enzymatically, radiolabeled, or fluorescently-tagged tumor suppressor will be particularly useful.

IV . Anti-hypertrophic therapy ANTI - HYPERPROLIFERATIVE THERAPIES )

Hypertrophic diseases are a group of diseases characterized by abnormal or uninhibited cell proliferation. These diseases are generally divided into two categories: more commonly cancerous or "malignant disease" and a somewhat less common benign hyperplastic disease, such as benign prostatic hypertrophy, breast benign epithelial hyperplasia, endometrial hyperplasia, thyroid hyperplasia, Or epithelial hyperplasia of the dermis. Many aspects of the following discussion will focus on cancer therapy, but will recognize that the same appropriate methodology can be applied to benign diseases as well.

Cancer refers to the presence of dysplastic, neoplastic, or otherwise proliferative, carcinogenic, and / or transformed cells in an individual, as opposed to an abnormal benign disease, in general, , Tumors, non-contact inhibited or carcinogenic transformed cells, or similar (e.g., melanoma, adenocarcinoma, squamous cell carcinoma, small cell carcinoma, Sarcoma such as carcinomas such as ovarian carcinoma, fibrosarcoma, chondrosarcoma, osteosarcoma, hepatoma, neuroblastoma, melanoma, lymphoma, leukemia, hematopoietic cancer such as myeloma, etc.). Without being bound by any theory, the tumor solubility properties of the lvovirus may be determined in an environment susceptible to, for example, an environment such as damaged PKR phosphorylation in Ras-activated cells, as described herein, , Can be derived from the viral orientations of malignantly transformed cells in cooperation with an environment having a defective tumor suppressor function.

A. Tumor Soluble Virus Therapy ( Oncolytic Viral Therapies )

Certain methods related to the production and use of wild-type reovirus in cancer therapy have been described (see, for example, US patents 7,300,650, 7,163,678, 7,049,127, 7,014,847, 6,994,858, 6,811,775, 6,703,232, 6,576,234, 6,565,831, 6,528,305, 6,455,038, 6,344,195, 6,261,555 , 6,136,307, and 6,110,461, and US Patent 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 any waiver). Myxoma virus therapy is described in U.S. Patent Application Publication 2006/0263333 and is incorporated by reference.

B. Removal Therapy ( Purging Therapies )

In certain embodiments, the invention provides a method of purging a population of cancer cells. For example, individuals who have hematopoietic or solid cancers may receive a hematopoietic stem cell rescue using the removed bone marrow tissue collected from the subject prior to chemotherapy. In these embodiments, the bone marrow can be treated with a tumor-soluble virus, in order to eliminate or kill neoplastic cells while reducing or eliminating damage to hematopoietic stem cells.

For cell therapy, patient-specific adult stem cells can be used to avoid problems such as host immunity, graft-versus-host disease, and ethical debate. Human adult stem cells have been extensively evaluated for a variety of tissue regenerative therapy modalities. However, the adult stem cells in the It has been reported that spontaneous transformation can occur during the in vitro culture expansion. ( Tolar meat al ., 2007; Romano , 2005 ). Therefore, a proper purging strategy is needed to remove spontaneously transformed cells present in cultured expanded adult stem cells for safer use in transplantation and tissue regeneration. The wild-type virus, in In vitro , it was useful for the removal of cancer cells present in a population of autologous hematopoietic stem cells. ( Thirukkumaran meat al ., 2003; Thirukkumaran meat al ., 2005 ). Attenuated Rioviruses and Myxoma viruses can also be used in a similar manner. The present invention predicts that virtually any adult stem cell or embryonic stem cell line currently known or to be discovered in the future can be used.

In certain embodiments, it may be desirable to remove the leavirus from the cell composition after a sufficient time that the killing of the neoplastic cells (if present) is allowed. For example, it may be treated with a virus to take a bone marrow from an individual and destroy the neoplasia, but before re-transplanting the bone marrow in an individual (e.g., a patient who does not have immune function) It may be desirable to destroy, remove and / or clear the virus. In these embodiments, the anti-viral material may be added to the cell composition. These materials include inhibitors of essential viral enzymes, such as anti-rifovirus antibodies and complements, RNA-induced RNA polymerase inhibitors, or substances that interfere with successful viral packaging, assembly, or release from infected cells do.

Further purification steps may be employed, including washing and / or centrifugation of cells to further remove viruses and / or killed cells from the cell composition. Such methods are well known and include methods based on methods for enhancing the desired cell type, for example, fluorescence activated cell sorting or adsorption capable of positive screening of intended cells, such as stem cell populations .

C. Combination Therapy ( Combination Therapy )

To increase the effect of selective killing of cancer cells by the tumor soluble virus, in a particular embodiment, a chemotherapeutic agent, radiation therapy, immunotherapy, hormone therapy, toxin therapy, another natural state virus or engineered virus It may be desirable to contact the cells with additional anti-cancer agents such as therapy or gene therapy.

Chemotherapeutic agents include, but are not limited to, 5-fluorouracil, bleomycin, vesulfate, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor Binding agent, etoposide (VP 16), parnesyl-protein transferase inhibitor, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelin, nitrosourea, But are not limited to, but not limited to, alginic acid, fenugreek, tamoxifen, tamoxifen, taxol, thezolozide (a water soluble form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, Do not. These substances or drugs are classified according to their mode of activity in the cell, for example, depending on whether they affect the cell cycle and on which stage. Alternatively, the substance may be characterized based on its ability to cross-link directly to DNA, its ability to be inserted into DNA, or its ability to induce chromosomal and mitotic anomalies by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone substances, other substances and any analogues or derivatives thereof Mutant.

Tumor solubility therapy may be conducted either before or after the application of the other substance (s) to the cancer cells, at a time interval of several minutes to several weeks. For example, in such cases, one of ordinary skill in the art will be able to contact multiple forms at substantially the same time (e.g., within about 1 minute) with a leavirus attenuated in cancer cells, tissues, organs, or organisms. In another aspect, the one or more materials may be administered for 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 About 17 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 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 About 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 42 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 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 hours About 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 1 day, about 8 days, about 9 days, about 10 days, About 5 days, about 5 weeks, 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, , Within about 7 or about 8 weeks or more of the period and within any range that can be derived therefrom, or substantially simultaneously, or may be administered before and / or after the attenuation or administration of the wild-type Tyrian virus .

A variety of complex regimens of attenuated or wildtype tyrosinavirus and one or more substances can be used. A non-limiting example of such a composite is shown below wherein the tumor-soluble virus is labeled "A" and the anticancer agent is labeled "B"

A / B / A B / A / B B / B / A / A / B / B /

B / B / B / A / B / A / B / A / B / A /

B / A / B / AB / A / A / B / A / A / B / A /

i. Chemotherapy

Various chemotherapeutic agents may be used in the present invention. "Chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used when referring to a compound or composition to be administered in the treatment of cancer. These substances or drugs are classified according to their mode of activity in the cell, for example, depending on whether they affect the cell cycle and on which stage. Alternatively, the substance may be characterized based on its ability to cross-link directly to DNA, its ability to be inserted into DNA, or its ability to induce chromosomal and mitotic anomalies by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

Alkylated material (Alkylating agents). Alkylating agents are drugs that interact directly with genomic DNA to prevent cancer cells from multiplying. Chemotherapeutic drugs in this category are substances that affect all phases of the cell cycle, i. E., Phase-specific. Alkylating agents may be used to treat certain cancers of chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, breast, lung and ovary. Such drugs include, but are not limited to, vanillin, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ipospamide, mechlorethamine (muscarinic) and melphalan. Troglitazaone can be combined with one or more alkylating agents to treat cancer, some of which are discussed below.

The substrate (also known as Milleran) is a bifunctional alkylating material. The substrate is known as 1,4-butanediol dimethanesulfonate.

The substrate is not a structural analogue of nitrogen mastad. The tablet is used in tablet form for oral administration. Each of the scored tablets contains 2 mg substrate and magnesium stearate and sodium chloride inactive ingredients.

The vascular plate is used for the palliative treatment of myeloid, myelocytic, granulocytic leukemia. There is no healing power, but the vascular plate reduces the amount of total granulocytes, alleviates the symptoms of the disease, and improves the patient's clinical condition. Approximately 90% of patients with previously untreated chronic myelogenous leukemia will receive hematopoietic mitigation, such as regression or stabilization of organomegaly after use of the vascular plate. Survival time and hemoglobin level maintenance were superior to spleen irradiation and were comparable to control in controlling long term hypertrophy.

Chlorambucil (also known as luceran) is active against selected human renal tissue diseases as a bimolecular type of bifunctional alkylating agent. Chlorambucil is known by its chemical nomenclature as 4- [bis (2-chloroethyl) amino] benzene butanoic acid.

Chlorambucil is available as a tablet for oral administration. It is rapidly and completely absorbed in the gastrointestinal tract. After a single oral dose of 0.6-1.2 mg / kg, the plasma peak (peak) level of chlorambucil is 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-sinus therapy. Therapeutic regimens are well known to those skilled in the art and can be found in " Physicians Desk Reference " and "Remington's Pharmaceutical Sciences ".

Chlorambucil is a chronic lymphoid (lymphoid) leukemia; Lymphoma, lymphoid sarcoma, giant follicular lymphoma and Hodgkin's disease. There is no cure for these diseases, but clinically useful mitigation will 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 may be used alone or in combination with other substances, with a dose effective for clinical use of 15-20 mg / m 2 for a total of three courses of 3 weeks for 5 days. Exemplary dosage amounts are 0.50 mg / m2, 1.0 mg / m2, 1.50 mg / m2, 1.75 mg / m2, 2.0 mg / m2, 3.0 mg / m2, 4.0 mg / m2, 5.0 mg / m2 and 10 mg / m2. Of course, all these dosages are exemplary and any dosage between these doses is expected to be useful in the present invention.

Cisplatin should not be taken orally, but should be delivered via intravenous, subcutaneous, intratumoral, or peritoneal injection.

Cyclophosphamide is 2 H -l, 3,2-oxazaposporin-2-amine, N, N -bis (2-chloroethyl) tetrahydro-, 2-oxide, monohydrate; Mead Johnson in the name of Cytoxan; It is available in the name of Neosar in Adria. Cyclophosphamide is prepared by reacting 3-amino-1-propanol with N, N -bis (2-chloroethyl) phosphoramid dichloride [(ClCH ₂ CH ₂ ) ₂ N- -POCl ₂ ]. Condensation is carried out with the dual involvement of both hydroxyl and amino groups, affecting cyclization.

Unlike other? -Chloroethylamido alkylates, the active ethylene monomer does not cyclize directly into the monomorphic form until it is activated by the liver enzyme. Therefore, it is stable in the gastrointestinal tract, resistant, effective for both oral and extra-alimentary routes, and does not cause vesication, necrosis, phlebitis or pain.

Suitable dosage for adults is 1 to 5 mg / kg / day (usually complex) or 1 to 2 mg / kg / day, orally, depending on gastrointestinal tolerance; Intravenously at an initial dose of 40-50 mg / kg for 2-5 days, or 10-15 mg / kg for 7-10 days or 3-5 mg / kg or 1.5-3 mg / kg twice a week / day. A dose of 250 mg / kg / day may be administered as an anti-neoplastic agent. Because of gastrointestinal side effects, loading is preferred to a venous route. While maintained, a typical number of leukocyte counts is usually 3000 to 4000 / min. The drug is also sometimes administered by infusion into the muscle or by the body. Those skilled in the art will refer to Remington ' s Pharmaceutical Sciences 15th Edition, chapter 61, for a dosage of 100 mg, 200 mg, and 500 mg, and 25 mg and 50 mg tablets,

Mel-paran (also known as alkenol, L-phenylalanine mutant, phenylalanine mutant, L-PAM, or L-saccharin) is a phenylalanine derivative of nitrogen mastad. Melphalan is a bifunctional alkylating agent with selective activity against human renal disease. The chemical name is 4- [bis (2-chloroethyl) amino] -L-phenylalanine.

Melphalan is the active L-isomer of the compound, first synthesized by Bergel and Stock in 1953; The D-isomer (known as medfaran) is less active for certain tumors in animals, and the dose required to effect on the chromosome is greater than for the L-isomer. The form of rhamnem (DL-) is known as mulberry or sarcoricin. Mel-blue is insoluble in water, and the value of pK _a1 is ~ 2.1. Melalon is used as a tablet for oral administration and has been used for the treatment of 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.

Melphalan was used for the treatment of epithelial ovarian carcinoma. One common regimen used to treat ovarian carcinoma is a single course of melphalan at a daily dose of 0.2 mg / kg for five days. The process is repeated every 4 to 5 weeks according to liver tolerance ( Smith and Rutledge , 1975; Young meat al . , 1978 ). Alternatively, the dosage of melphalan used may be as low as 0.05 mg / kg / day, or as high as 3 mg / kg / day, or any dose between these dose ranges or any dose beyond these dose ranges have. Some changes in dosage can necessarily occur depending on the condition of the individual being treated. In any case, the person responsible for the medication will determine the appropriate dosage for the individual patient.

Antimetabolites (Antimetabolites). Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylated materials, antimetabolites specifically affect the cell cycle during the S phase. In addition to tumors of the breast, ovary and gastrointestinal tract, it was used to treat chronic leukemia. Antimetabolites include 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine and methotrexate.

The chemical name for 5-fluorouracil (5-FU) is 5-fluor-2,4 (lH, 3H) -pyrimidinedione. Its mechanism of action seems to be to block methylation of deoxyuridyl acid to timimidyl acid. Thus, 5-FU interferes with the synthesis of deoxyribonucleic acid (DNA) and inhibits the synthesis of ribonucleic acid (RNA) to a somewhat weaker extent. DNA and RNA are essential for cell division and proliferation, and the effect of 5-FU is to produce thymidine deficiency, leading to apoptosis. Thus, the effect of 5-FU can be seen in cells that are rapidly cleaved (a feature of metastatic cancer).

Antitumor antibiotics (Antitumor Antibiotics ) . Antitumor antibiotics have both antibacterial and cytotoxic activities. These drugs can chemically inhibit enzymes and mitosis or alter cell membranes to interfere with DNA. Because these materials 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 dirubicin, some of which are discussed in more detail below. With widespread use in clinical settings for neoplasia therapy, these compounds can be administered at doses ranging from 25-75 mg / m 2 to 35-100 mg / m 2 (at intervals of 21 days of adriamycin, intravenously; By oral < / RTI > intravenous injection).

(8- cis ) -10 - [(3-amino-2,3,6-trideoxy-aL-ribso-hexopyranosyl) oxy] -7, 8,9,10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl) -l-methoxy-hydrochloride (hydroxydodorubicin hydrochloride, adriamycin) It is used for spectrum. It binds to DNA, inhibiting nucleic acid synthesis, inhibiting mitosis, and promoting chromosomal anomalies.

Administered alone, is the primary choice drug for the treatment of thyroid adenoma and primary HCC. Ovary, endometrial and breast tumor, bronchial oat-cell carcinoma, non-small cell lung carcinoma, gastric adenocarcinoma, retinoblastoma, neuroblastoma, mycosis fungoides, pancreatic carcinoma, prostate carcinoma, bladder carcinoma, myeloma, diffuse histiocytic lymphoma, Wilms' (Wilms) tumor, Hodgkin's disease, adrenal tumor, osteogenic sarcoma soft tissue sarcoma, Ewing sarcoma, rhabdomyosarcoma, and acute lymphoblastic leukemia. It is an alternative drug for treating islet (iS1et) cells, cervical, testicular, and adrenocortical cancers. This drug is also immunosuppressive.

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

A suitable dosage is 25-30 mg / m 2 per day for 2 or 3 consecutive days or 20 mg / m 2 once a week, repeated 60-75 mg / m 2 or every 3 or 4 weeks at 21 day interval for adult, intravenous administration. In older patients, when there is existing bone marrow suppression or renal bone marrow infusion caused by conventional chemotherapy, or when used in combination with other bone marrow antihypertensive drugs, the lowest dose should be used. If serum bilirubin is between 1.2 and 3 mg / dL, the dosage should be reduced by 50%, and if it is above 3 mg / dL, it should be reduced by 75%. In patients with normal cardiac function, the total dose should not exceed 550 mg / m2 for a lifetime and should not exceed 400 mg / m2 for patients receiving mediastinal irradiation. Alternatively, an amount of 30 mg / m 2 per day for three consecutive days is repeated every 4 weeks. Exemplary dosage amounts are 10 mg / m2, 20 mg / m2, 30 mg / m2, 50 mg / m2, 100 mg / m2, 150 mg / m2, 175 mg / m2, 200 mg / m2, 225 mg / m2, 450 mg / m2, 475 mg / m2, and 500 mg / m2, respectively. Of course, all of these dosage forms are illustrative, and any dosage between these ranges is also expected to be used in the present invention.

(8S- cis ) -8-acetyl-10 - [(3-amino-2,3,6-trideoxy-aL-Rxy-hexanopyranosyl ) Oxy] -7,8,9,10-tetrahydro-6,8,11-trihydroxy-10-methoxy-hydrochloride; It is also called serubidine and can be used in Wyeth. Daunorubicin interferes with DNA, blocks DNA-induced RNA polymerase, and inhibits DNA synthesis. This can inhibit cell division at doses that do not interfere with nucleic acid synthesis.

In combination with other drugs, in acute myelogenous leukemia in adults (to induce mitigation), acute myelogenous leukemia and acute phase of chronic myelogenous leukemia. Oral absorption is poor and should be provided intravenously. The distribution half-life is 45 minutes and the elimination half-life is about 19 hours. The half-life of daunorubicinol, its active metabolite, is about 27 hours. Daunorubicin is metabolized mostly in the liver and is also released into the bile (ca 40%). In the case of hepatic failure or renal failure, the dose should be reduced. A dose of 45 mg / m2 (30 mg / m2 for 60 years of age or more) or 3 weeks or 4 weeks for every 3 or 4 weeks, 1, 2 or 3 days on a vein Week, 0.8 mg / kg daily for 3 to 6 days; No more than 550 mg / m2 should be provided for a lifetime, and no more than 450 mg / m2 if thoracic irradiation has been performed; For children, if an adult schedule is used that is not under 2 years of age or based on weight, the body surface less than 0.5 m is 25 mg / m2 once a week. It is available in 20 mg (equivalent to 21.4 mg hydrochloride) for the injectable (equivalent base). M 2, 200 mg / m 2, 225 mg / m 2, 250 mg / m 2, 275 mg / m 2, 20 mg / m 2, 30 mg / M2, 300 mg / m2, 350 mg / m2, 400 mg / m2, 425 mg / m2, 450 mg / m2, 475 mg / m2 and 500 mg / m2. Of course, all of these dosage forms are illustrative, and any dosage between these ranges is also expected to be used in the present invention.

Mitomycin (mitomycin Muta and / or mitomycin also known as C) is shown a case wherein Streptomyces MRS-tumor activity Pitot Versus (Streptomyces caespitosus ) Antibiotics isolated from broth. This compound is thermally stable, has a high melting point, and is freely soluble in organic solvents. Mitomycin selectively inhibits the synthesis of deoxyribonucleic acid (DNA). The guanine and cytosine contents are related to the degree of mitomycin-induced association. At high concentrations of this drug, cellular RNA and protein synthesis is also inhibited.

In humans, after intravenous administration, mitomycin is rapidly removed from the serum. The time required to reduce the serum concentration 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 2.4 mg / ml, 1.7 mg / ml, and 0.52 mg / ml, respectively. It is largely eliminated by metabolism in the liver, but metabolism occurs in other tissues as well. The removal rate is inversely proportional to the maximum serum concentration, probably due to saturation of the degradation pathway. Approximately 10% of the mitomycin dose is released into the urine without change. As the metabolic pathway is saturated at relatively low doses, the proportion of the dose released to urine increases as the dosage is increased. 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 agent that inhibits DNA-dependent RNA polymerase. This drug is a component of primary selection for the treatment of choriocarcinoma, embryonic rhabdomyosarcoma, testicular tumors, and Wilms' tumor. Tumors that are ineffective for systemic treatment often respond to localization. Dactinomycin increases the effectiveness of radiation therapy. It is a secondary (effervescent) immunosuppressant.

Actinomycin D is used in combination with primary surgery, radiation therapy, and other medications, particularly vincristine and cyclophosphamide. Antinociceptive activity was also recorded in Ewing's tumor, Kaposi's sarcoma, soft tissue sarcoma. Dactinomycin is effective in women with advanced chorionic villages. It also shows a continuous response in combination with chlorambucil and methotrexate in patients with metastatic testicular carcinoma. In patients with Hodgkin's disease and non-Hodgkin's lymphoma, reactions may sometimes be observed. Dactinomycin has also been used to inhibit immune responses, particularly renal transplant rejection.

Half of the dose is excreted in its native form into the bile, 10% is excreted in the urine; The half-life is about 36 hours. Drugs can not pass through the brain-blood vessel wall. Actinomycin D is supplied as a lyophilized powder (0/5 mg for each vial). A typical daily dose is 10 to 15 mg / kg; It is delivered intravenously for 5 days; There was no toxicity manifestation and additional procedures could be provided at intervals of three to four weeks. 100 to 400 mg for the children were injected daily for 10 to 14 days; In another regimen, 3 to 6 mg / kg, a total of 125 mg / kg, and a weekly maintenance dose of 7.5 mg / kg. Although it would be safer for the drug to be administered via the IV tube, it is injected directly into the vein to avoid subcutaneous reactions, taking care to dispose of the needle used when removing the drug from the vial. Exemplary dosage amounts range from 100 mg / m2, 150 mg / m2, 175 mg / m2, 200 mg / m2, 225 mg / m2, 250 mg / m2, 275 mg / m2, 300 mg / m2, 350 mg / m2, 425 mg / m2, 450 mg / m2, 475 mg / m2, and 500 mg / m2. Of course, all of these dosage forms are illustrative, and any dosage between these ranges is also expected to be used in the present invention.

Bleomycin is Streptomyces MRS suberic tichil Ruth (Streptomyces verticillus ) which is a cytotoxic glycopeptide antibiotic. Although the precise criteria for the action of bleomycin are not known yet, according to available evidence, the main mode of action is inhibition of DNA synthesis, and in some evidence, RNA and protein synthesis are also inhibited to some extent.

In mice, high concentrations of bleomycin are found in skin, lung, kidney, peritoneum and lymphatic glands. Skin and lung tumor cells were found to have a high concentration of bleomycin, in contrast to the low levels found in hematopoietic tissues. Low levels of bleomycin found in bone marrow may be associated with the bleomycin degrading enzyme found in this tissue.

The final elimination half-life of bleomycin in serum or plasma is about 115 minutes in patients who are removed from creatinine> 35 ml per minute (> 35 ml). In patients with less than 35 ml (<35 ml) creatinine removed, the final elimination half-life of serum or plasma bromomycin 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 can be provided via intramuscular, intravenous or subcutaneous routes. It is freely soluble in water.

Bleomycin should be considered a palliative treatment. Such as squamous cell carcinomas such as head and neck (including mouth, tongue, one way, nasopharynx, medullar pharynx, hole, palate, lips, oral mucosa, gums, epiglottis and larynx), skin, penis, Has proven to be useful in regulating nephrolithiasis in a proven combination with other approved chemotherapeutic agents. Lymphoma and testicular carcinoma.

With the potential for anaphylactoid reaction, patients with lymphoma should be treated with less than two units or the first two doses. If an acute response does not occur, then the normal dose can be followed.

Improvement of Hodgkin's disease and testicular tumors is immediate and occurs within 2 weeks. If no improvement is seen by this time, there is no possibility of improvement. Squamous cell carcinomas respond more slowly and, in some cases, require about three weeks for any improvement to occur.

Mitotic inhibitors (Mitotic Inhibitors). Mitotic 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. Mitotic inhibitors include dodecaxel, etoposide (VP16), paclitaxel, taxol, taxotere, vinblastine, vincristine and vinorelbine.

VP16, also known as etoposide, is compounded with bleomycin and cisplatin and is mainly used in the treatment of testicular tumors, and is used in the treatment of small cell carcinoma of the lung with cisplatin. It is also active against non-Hodgkin lymphoma, acute non-lymphoid 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. In the case of small cell carcinoma of the lung, intravenous dose (in combination therapy) is as high as 100 mg / m 2, or as low as 2 mg / m 2, usually from 35 mg / m 2 per day for 4 days to 50 mg / May be used. When given orally, the dosage should be doubled. The dose of small cell lung carcinoma should be as high as 200-250 mg / ㎡. Venous doses for testicular cancer (with combined therapy) range from 50 to 100 mg / m2 daily for 5 days or alternately 100 mg / m2 for 3 doses. It is common that the therapy cycle is repeated every 3 to 4 weeks. Perhaps the drug should be given slowly during 30 to 60 minutes of infusion to avoid hypotension and bronchospasm due to the solvent used in the formulation.

Taxus is a Western ash tree ( Taxus brevifolia ) shells. Taxol binds to tubulin (at a site separate from that used by vinca alkaloids) to facilitate assembly of microtubules. Taxol is currently clinically evaluated; Taxol has activity on malignant melanoma and ovarian carcinoma. The maximum dose is 30 mg / m 2 per day for 5 days or 210 to 250 mg / m 2 every 3 weeks. Of course, all of these dosage forms are illustrative, and any dosage between these ranges is also expected to be used in the present invention.

Vinblastin is another example of a plant alkyloid used in combination with troglitazone for the treatment of cancer and precancer. When the cells are incubated with vinblastine, microvessel dissociation occurs.

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

After intravenous injection, vinblastine shows the removal of multimeric patterns from plasma: After distribution, the drug disappears from the plasma with a half-life of about 1 and 20 hours. Vinblastin is metabolized in the liver to biologically activate desacetylbindarastine. Approximately 15% of the administered dose is detected in the urine in its native state, and approximately 10% is recovered from the stool after bile discharge. In patients with liver dysfunction, dosage should be reduced. If the plasma has a bilirubin concentration above 3 mg / dl (approximately 50 mM), the dose should be reduced by at least 50%.

Vinblastine sulfate is used as a injectable preparation. This drug is delivered intravenously; Particular attention should be given to subcutaneous eruptions because this drug can cause painful irritation and ulceration. This drug should not be injected into the affected limbs. At a single dose of 0.3 mg / kg, bone marrow suppression is maximal within 7 to 10 days. If moderate leukocyte depletion (about 3000 cells / dose) is not obtained, the dose may be increased incrementally by 0.05 mg / kg per week. In regimens for the treatment of testicular cancer, 0.3 mg / kg Vinblastine is used every three weeks, regardless of blood cell count or toxicity. The most important clinical use of vinblastine is in the treatment of metastatic testicular tumors in combination with bleomycin and cisplatin. Reactions beneficial in a variety of lymphomas, particularly Hodgkin's disease, have been reported, and significant improvement can be noted at 50-90% in Hodgkin's disease. If the disease is refractory to the alkylating agent, the effect of binaphylaxis at high lymphoma rates is not reduced. Kaposi sarcoma, neuroendocrine tumors, Letterer-Siwe disease (histiocytosis X), as well as breast and choriocarcinomas in women.

The dosage of vinblastine may be determined by the clinician depending on the individual patient needs. 0.1 to 0.3 mg / kg may be administered, or 1.5 to 2 mg / m 2 may be administered. M2, 0.1 mg / m2, 0.15 mg / m2, 0.2 mg / m2, 0.25 mg / m2, 0.5 mg / m2, 1.0 mg / m2, 1.2 mg / m2, 1.4 mg M 2, 2 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 and 20 mg / It is possible. Of course, all of these dosage forms are illustrative, and any dosage between these ranges is also expected to be used in the present invention.

Vincristine blocks mitosis and produces a metaphase arrest. Most of the biological activity of this drug appears to be explained as binding specifically to tubulin and blocking the ability of the protein to polymerize into microtubules. Through the microtubule disruption of the mitotic apparatus, cell division is inhibited in the metaphase. If the chromosomes are not correctly separated during mitosis, the cells will probably die.

The relatively low toxicity of vincristine in normal bone marrow cells and epithelial cells is made especially among anti-neoplasms and in combination with other bone marrow suppressants.

Unexpected adsorption has been reported following oral administration of vinblastine or vincristine. At normal clinical doses, the peak concentration of each drug in plasma is about 0.4 mM.

Vinblastine and vincristine bind to plasma proteins. They are fairly concentrated in the platelets, and are somewhat weak in leukocytes and red blood cells.

Vincristine has a multiphasic elimination pattern from plasma: the terminal half-life is about 24 hours. Drugs are metabolized in the liver, but biologically active derivatives have not been identified. In patients with liver dysfunction, dosage should be reduced. At the plasma level of bilirubin greater than 3 mg / dl (about 50 mM), at least 50% of the dose is prescribed.

Vincristine sulfate is used as an intravenous solution (1 mg / ml). Vincristine, used in conjunction with corticosteroids, is currently a selective treatment that induces mitigation in childhood leukemia; The optimal dose of these drugs is vincristine, intravenously, weekly, 2 mg / m 2 per body surface dose; And prednisone, oral administration, daily, 40 mg / m 2. Adult patients with Hodgkin's disease or non-Hodgkin's lymphoma usually receive vincristine as part of a combined protocol. When vincristine is used in a MOPP regimen, the recommended dose of vincristine is 1.4 mg / m 2. A high dose of vincristine appears to be more resistant to children with leukemia than adults, and adults can experience severe neurotoxicity. If the drug is administered more frequently than every seven days, or if administered at higher doses, the toxicity manifestation appears to be increased without a proportional improvement in the rate of the reaction. Preventive measures should be used to avoid eruptions during intravenous administration of vincristine. Vincristine (and vinblastine) is comparable toxicity and may be injected into the tumor's arterial blood supply at doses several times higher than can be administered intravenously. Vincristine was effective in Hodgkin's disease and other lymphomas. (III and IV) of Hodgkin's disease, when used in combination with mechlorethamine, prednisone, and procarbazine (MOPP regimen), although slightly less beneficial than vinblastine when used alone in Hodgkin's disease, &Lt; / RTI &gt; In non-Hodgkin's lymphoma, vincristine is an important substance and is particularly important when used with cyclophosphamide, bleomycin, doxorubicin, and prednisone. Vincristine is more useful than Vinblastine in lymphoid leukemia. A beneficial response has been reported in patients with a variety of other tumors (neoplasms), particularly Wilms tumor, neuroblastoma, brain tumors, rhabdomyosarcoma, breast, bladder, and gynecological carcinomas.

The amount of vincristine used will depend on the individual patient in need. 0.01 to 0.03 mg / kg may be administered, or 0.4 to 1.4 mg / m 2 may be administered, or 1.5 to 2 mg / m 2 may be administered. M 2, 0.14 mg / m 2, 0.15 mg / m 2, 0.2 mg / m 2, 0.06 mg / m 2, 0.07 mg / / M2, 0.25 mg / m &lt; 2 &gt; may be provided by continuous intravenous infusion. Of course, all of these dosage forms are illustrative, and any dosage between these ranges is also expected to be used in the present invention.

Camphothecine is a Chinese tree ( Camptotheca acuminata Decne. &Lt; / RTI &gt; Camptothecin and its derivatives stabilize the covalent reaction intermediate termed a "cleavable complex" and are unique in its ability to inhibit DNA topoisomerase, resulting in the killing of tumor cells. Many people believe that camptothecin analogues exhibit excellent anti-tumor and anti-leukemic activity. Clinical use of camphothecin is limited because of serious side effects and poor water solubility. Currently, some camptothecin analogs (topotecan; irinotecan), which are synthetic or semi-synthetic, have been applied to cancer therapy and show satisfactory clinical efficacy. The molecular formula of camphothecin is C ₂₀ H ₁₆ N ₂ O ₄ , and the molecular weight is 348.36. Yellow powder and can be dissolved in clear yellow solution at 50 mg / ml in DMSO 1N sodium hydroxide. In a dry, closed, light-resistant environment, it is stable for at least 2 years if stored at 2-8 ° C.

Nitro jaws LEA (Nitrosureas). Like nitrosylated materials, nitrosourea inhibits DNA repair proteins. In addition to brain-tumors, it is used in the treatment of non-Hodgkin's lymphoma, multiple myelogenous and malignant melanoma. Exemplary materials include carmustine and rosuvastatin.

Carmustine (sterilized Carmustine) is one of the Nitrozoureas used in the treatment of certain renal diseases. The chemical name is l, 3 bis (2-chloroethyl) -l-nitrosourea. Upon lyophilization, it becomes a pale yellow piece or a solidified mass, with a molecular weight of 214.06. It is very soluble in alcohol and lipids, and does not dissolve well in water. Carmustine is administered by intravenous infusion after reconstitution as recommended. Sterile carmustine is commonly used as a 100 mg single dose vial of lyophilized material.

Carmustine appears to alkylate DNA and RNA, but does not have cross-resistance with other alkylators. Together with other Nitrosoureas, several key enzymatic processes may be restricted by carbamoylation of amino acids in proteins.

Carmustine is prescribed as a palliative therapy with a single agent or with other chemotherapeutic agents approved in brain tumors such as glioma, glioma, medullobladyoma, astrocytoma, ventricular cell tumor, and metastatic brain tumor. It is prescribed in combination therapy. It has also been used in combination with Fred Nizhny for the treatment of multiple myeloma. Carmustine is useful as a second-line therapy in combination with other drugs approved for patients who have recurred during treatment with Hodgkin's disease and non-Hodgkin's lymphoma during treatment with primary therapy or who have not responded to the primary regimen Proved.

The recommended dose of carmustine used as a single substance in patients who have not been previously treated is intravenous, 150 to 200 mg / m2, every 6 weeks. May be provided in a single dose or in divided doses daily from 75 to 100 mg / m &lt; 2 &gt; for two consecutive days. When carmustine is used in combination with other myelosuppressive drugs or in patients with depleted bone marrow reserve, the dosage should be adjusted accordingly. The dose after the first dose should be adjusted according to the patient's hematological response to the preceding dose. Of course, other dosage amounts may be used in the present invention, for example, 10 mg / m2, 20 mg / m2, 30 mg / m2, 40 mg / m2, 50 mg / m2, 60 mg / M 2, 80 mg / m 2, 90 mg / m 2 or 100 mg / m 2. Those skilled in the art "Remington's Pharmaceutical Sciences, "15 th Edition , Chapter 61 . Depending on the condition of the subject being treated, some changes in the dose may be necessary. The person responsible for the administration will be able to determine the appropriate dosage for the subject to be treated.

Rumustine is one of the Nitrozoureas used in the treatment of certain neoplasia. The chemical name is l- (2-chloro-ethyl) -3-cyclohexyl-1-nitrozoarea. 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. Rumustine is dissolved in 10% ethanol (0.05 mg per mL) and dissolved in pure alcohol (70 mg per ml). Rumutin is relatively insoluble in water (<0.05 mg per ml). It is not relatively ionized at physiological pH. The inactive ingredients in the rosemastin capsule are magnesium stearate and mannitol. It is generally recognized that rosuvastatin alkalates DNA and RNA, but does not have cross-resistance with other alkylators. Together with other Nitrosoureas, several key enzymatic processes may be restricted by carbamoylation of amino acids in proteins.

Romestin will be provided orally. When orally administered with a dose ranging from 30 mg / m 2 to 100 mg / m 2 of radioactive actinomestin, about half of the provided radioactivity is released in the form of a degradation product within 24 hours. The serum half-life of the metabolite is 16 hours to 2 days. Tissue levels comparable to plasma levels at 15 minutes after intravenous administration.

It has been shown that rosuvastatin is useful as a single substance in both primary and metastatic brain tumors, in addition to other forms of treatment, or in established combination therapies, in patients who have already undergone appropriate surgical procedures and / or have undergone radiotherapy procedures. Combined with other approved drugs in patients who relapsed during the first course of therapy or who did not respond to the first regimen, it proved to be effective as a second-line therapy for Hodgkin's disease.

The recommended dose for rosuvastatin for adults and children is 6 weeks and the dose for oral single dose is 130 mg / m2 as a single substance in previously untreated patients. The dosage of non-immune bone marrow patients should be reduced to 100 mg / m2 every 6 weeks. When rosmustine is used in combination with other myelosuppressive drugs, the dosage should be adjusted accordingly. Other dosage amounts 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 &lt; 2 &gt;, 100 mg / m &lt; 2 &gt;, 120 mg / m &lt; 2 &gt;, or any range between the above values which can be determined essentially by the clinician depending on the individual to be treated.

Other substances . Other substances may be used, including Avastin, Iressa, Erbitus, Belcade and Gleevec. In addition, a growth factor inhibitor and a small molecule kinase inhibitor are also useful in the present invention. All described therapies are described in Cancer : Principles and Practice of Oncology (2001) , which is incorporated by reference. The following additional treatments are also included.

ii . Immunotherapy immunotherapy )

Immunotherapy is generally the use of immune effect substance cells and molecules to target and destroy cancer cells. The immune effect substance can be, for example, an antibody specific for some of the markers on the tumor cell surface. The antibody alone may act as an effective agent for therapy, or may recruit other cells that substantially affect cell death. The antibody may also be conjugated to a drug or toxin (chemotherapy, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) or may simply act as a targeting agent. Alternatively, the effect agent may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. Thus, immunotherapy may be used as part of a combination therapy in combination with a lvovirus or other tumor soluble virus therapy. Common methods for combination therapy are discussed below. In general, tumor cells are not present in most of the other cells and must have some markers that enable targeting. There are many tumor markers, some of which will be suitable for targeting consistent with the present invention. Typical tumor markers include cancer embryonic antigens, prostate-specific antigens, urologic tumor associated antigens, fetal antigens, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, Receptor, erb B, and p155. In addition, tumor cells or other hyperproliferative cells infected with a lvovirus or other tumor-soluble virus can express viral antigens on the cell surface and thus become susceptible to attack by the immune system.

Tumor necrosis factor is a glycoprotein that kills some kinds of cancer cells, activates cytokine production, activates macrophages and endothelial cells, promotes collagen and collagenase production, is an inflammatory mediator, It is an intermediary substance, and promotes catabolism, fever and sleep. Some infectious agents cause tumor regression through stimulation of TNF production. Because TNF is highly toxic when used alone as an effective dose, the optimal regimen is likely to be combined with other drugs and used at lower doses. Immunosuppression is enhanced by gamma-interferon, and combination therapy is potentially dangerous. Hybrids of TNF and interferon-alpha have been found to possess anticancer activity.

iii . Hormone therapy Hormonal Therapy )

Sex hormones are used according to the methods described in cancer therapy. Although the methods described herein are not limited to specific cancer therapies, the use of such hormones has an effect on breast, prostate, and endometrial (lining) cancer. Examples of these hormones are estrogens, anti-estrogens, progesterone and androgens.

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

iv . Radiation therapy Radiotherapy )

Radiation therapy (also known as radiation therapy) is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation impairs the cellular genetic material that keeps these cells growing by injecting energy that damages or destroys the cells in the treated area. Radiation damages all cancer cells and normal cells, but normal cells can recover themselves and function properly. Radiation therapy can be used to treat localized fidelity tumors, such as skin, tongue, larynx, brain, breast or neck cancers. Leukemia and lymphoma (in turn, blood forming cells and lymphoid cancers).

Radiation therapy used in accordance with the present invention includes, but is not limited to, the use of gamma-rays, X-rays, and / or direct delivery of radioisotopes to tumor cells. Other types of DNA damage factors such as microwave and UV-irradiation may be considered. All of these factors affect extensive damage to DNA, DNA precursors, replication and repair of DNA, and assembly and maintenance of chromosomes. The dosage range of X-rays ranges from a daily dose of 50-200 roentgens to a single dose range of 2000-6000 roentgens for a considerable period of time (3-4 weeks). The range of radioisotope dose ranges is very wide and depends on the half-life of the isotope, the intensity and type of radiated radiation, and the intake by neoplastic cells.

Radiotherapy may involve the use of radioactively labeled antibodies (radioimmunotherapy) to deliver radiation doses directly to the cancer site. Antibodies are highly specific proteins made in the body in response to the presence of an antigen (a substance that is recognized as an exogenous substance 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 (processes known as radioactive labeling). Once injected into the body, the antibody actively searches for cancer cells, and cancer cells are destroyed by the action of radioactive cell-killing (cytotoxic) action. Such a method can minimize radiation damage to healthy cells.

Conformal radiotherapy uses the same radiotherapy equipment and linear accelerator as normal radiotherapy, but the metal block is placed in the path of the x-ray beam and deforms to fit the shape of the cancer. This allows a higher dose of radiation to be delivered to the tumor. Structures that are close to healthy surrounding cells are provided with a lower dose of radiation, reducing the potential for side effects. A device called a multi-leaf collimator has been developed and can be used as a substitute for a metal block. The multicolor collimate consists of a number of metal sheets fixed to a linear accelerator. Each layer is adjusted so that radiation therapy rays can be formed to the treatment site without the aid of metal blocks. Accurate positioning of stereotactic radiotherapy devices is very important, and the position of the internal organs of the patient can be checked at the start of each treatment using a specific scanning device.

High-Resolution Intensity Controlled Radiation Therapy also uses multicolor collimates. During this treatment, the layers of the polyglutarimide are migrated to provide treatment. This method provides a more accurate form of therapeutic light and provides a certain amount of radioactivity across the entire treatment area.

In the study, it was shown that stereotyped radioactivity and intensity-modulated radiotherapy could reduce the side effects of radiation therapy. However, if the treatment site is formed precisely, small cancer cells just outside the treatment site to be destroyed may be disturbed . This means that such specialized radiotherapy techniques may have a higher risk of cancer recurrence in the future. Stereotactic radiation therapy is used to treat brain tumors. This technique sends radiation therapy from many different angles, so the dose to the tumor is very high, and the dose that affects the surrounding healthy tissue is very low. Before treatment, several scans were analyzed by the computer to determine whether the radiotherapy was precisely targeted and whether the patient's head was within the precisely framed frame during radiotherapy. Several doses are provided.

Stereotactic radiosurgery (gamma knife) for brain tumors does not use a knife, but highly accurately targeted rays of gamma radiation therapy are used from hundreds of different angles. A session of radiotherapy requires only about 4 to 5 hours. For such treatment, a specially designed metal frame will be placed on the patient's head. Then, several scans and x-rays are run to find the exact site that needs treatment. During radiotherapy, the patient lays his head in a large helmet, and the helmet has hundreds of holes through which the radiation passes. Scientists are also looking for ways to increase the effectiveness of radiotherapy.

Two types of research drugs are being investigated for effects on cells receiving radiotherapy. Radiosensitizers make tumor cells more destructive, and radioprotectants protect normal tissues from radiation effects. Hyperthermia using heat has also been studied for its effect in making tissue sensitive to radiation.

v. Follow-up Surgery ( Subsequent Surgery )

Approximately 60% of cancer patients receive some form of surgery, which includes prophylactic, diagnostic, or phased, therapeutic, and palliative surgery. Curative surgery is a cancer therapy used in combination with other therapies such as chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapies such as the treatment of the present invention.

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

When cutting off all cancer cells, tissues or portions of tumors, cavities may form in the body. Treatment may be done locally, at the site under direct injection or additional chemotherapy. Such treatments may be repeated on a daily, 2, 3, 4, 5, 6 or 7 day or every week, every other week, every 3 weeks, 4 weeks and 5 weeks or every month, 2 months, 3 Month, 4 month, 5 month, 6 month, 7 month, 8 month, 9 month, 10 month, 11 month or 12 month. In such a treatment, the dose may also be changed.

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 invention and, therefore, should be construed as being constructed in an optimal manner for its implementation. However, it will be appreciated by those skilled in the art that many changes may be made in the particular embodiments described, and that such changes may also achieve similar results, without departing from the scope and spirit of the invention, in accordance with the description of the invention.

Example  1- Materials and Methods

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

Wild type and attenuated Preparation of rio virus . The wild-type virus strain T3D used in this study was proliferated in L929 cells and purified as previously described. The attenuated rA virus induced in HTR1 cultures was purified by the same method used for wild-type rio virus preparation, but the avirius virus was propagated in HT1080 and L929 cells. The virus was added to the cells at an MOI of 5-10 and maintained at 37 ° C for 48-72 hours. After the virus denaturation effect was observed (generally 20-30% cell lysis), the virus was purified from the pelleted cells. CsCl centrifugation of the virus was performed at 35,000 rpm using a SW41 rotor for 7-8 hours. The band forming virus was collected and actively dialyzed against 150 mM NaCl, 10 mM MgCl ₂ , and 10 mM Tris (pH 7.5). For the wild-type virus titer Rio, HEK 293 cells were plated per well in 6-well plates at 2 x 10 ⁵ cells. After 2 hours of adsorption at 37 ° C, the inoculum was removed. The cell monolayers were covered with 1% agar and fresh medium. Flack was counted 5-7 days after infection.

In the case of attenuated virus, the same procedure was carried out except that the agar was removed 5-7 days after the infection, and the cytotoxicity of the cytotoxic virus (Cytofix / Cytoperm (BD Bioscience) Were fixed and / or permeabilized. The avioVirus flap was identified through immunofluorescence detection. As an alternative to the attenuated Rovovirus, standard flock analysis on L929 cells was also performed. At three days post-infection, the agar was covered with neural red, and the cell layers were examined 24 hours later for flock formation. For harvesting ripe viruses attenuated from HT1080 and HTR1 infected supernatants, high speed centrifugation at 35,000 rpm was used and the viruses were pelleted.

Myxoma virus preparation. Myx-GFP of myxoma virus (strain Lausanne ), made by intergenic insertion of green fluorescent protein (GFP) driven by the synthetic latex early / late promoter, was used for infection studies. Myx-GFP was propagated and as previously described ( Opgenorth meat al. , 1992 ) and titrated with focus formation on BGMK cells.

FACS analysis. For flow cytometry analysis, cells were trypsinized and fixed using a cytofix / cytoperm solution (PharMingen, San Diego, Calif.). The 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 - Results

Leovirus and myxoma viruses preferentially infect p53 or ATM deficient cells . In order to examine whether the modulation of the tumor suppressor gene affects tumor susceptibility, the inventors have chosen p53 and ATM tumor suppressor genes that frequently mutate in a variety of cancers. p53 is a prototypical tumor suppressor gene that is most frequently mutated in a variety of cancer cells and has p53 mutations in over 50% of cancers ( White, 1994; Morris, 2002 ). The ATM (mutated vasodilatation 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 a rare symptomatic vasodilatatory ataxia characterized by cancer tendency, radiation sensitivity and neurodegeneration ( Kitagawa et al, 2005 ). Thus, the inventors examined tumor susceptibility virus susceptibility using p53 - / - MEF (mouse embryo fibroblast) and ATM deficient L3 lymphocyte ( Kozlov et al, 2003 ). As shown in Figures 1A-B, when compared to the MEF (p53 normal) and BT (ATM normal) criteria, as shown by microscopic detection of FACS or fluorescent positive cells (FITC + or GFP + cells) Kim et al., 2007 ) and myxoma viruses preferentially infected p53 - / - MEF or L3 (ATM-deficient) cells.

The leiovirus and myxoma viruses preferentially infect human lymphoma with p53 or ATM function. Because the rio virus and myxoma virus preferentially infect p53 or ATM-deficient cells, then the inventors then examined whether the rio virus and myxoma virus preferentially infected human lymphoma with p53 or ATM deficiency. The inventors tested six different lymphomas (HBL-2, Granta, Z138C, JVM2, Raji and Ramos) showing various cellular responses resulting from p53 and ATM-dependent pathways during genotoxic attack 2A). The functional status of p53 and ATM was evaluated by detection of p53 and ATM phosphorylation by IR irradiation and Western blotting. BT (ATM-normal) and L3 (ATM-deficient) were used as reference in the ATM response by IR. HBL-2 and Raji account for dysfunctional p53 responses when there is genotoxic stress, as evidenced by constitutive phosphorylation of serine-15 of p53 ( Li et al, 2006 ) (Fig. 2A). Granta cells showed ATM function abnormalities when genotoxic stress was present, and ATM was not activated in ionizing radiation (IR) stimulation (FIG. 2A). In the case of Z138C, JVM2 and Ramos cells, p53 and ATM responses were normal after genotoxic stress, as can be seen by over-phosphorylation of p53 and ATM by IR stimulation (Fig. 2A). Interestingly, as seen in FASC or microscopic detection of fluorescence-positive cells (FITC + or GFP + cells) as compared to ATM- and p53-reactive lymphomas (Z183C, JVM-2, Ramos) Dysfunctional lymphoma (HBL2, Granta, and Raji) [HBL-2 and Raji showed constitutive p53 activation, and Granta showed ATM deficiency at IR stimulation] were favored for both the rioovirus and myxoma virus infections (Fig. 2C). As can be seen in FIG. 2B, the ATM or p53 dysfunction of the cells is significantly associated with both leiovirus and myxoma virus susceptibility. This strongly suggests that ATM and p53 both participate in establishing cellular resistance to viral infections, while defects in either of these genes or defects in one of its pathways can confer virus sensitivity (Figure 2A) .

Retinoblastoma cells are susceptible to infectious viruses such as leiovirus and myxoma virus. Because the rio virus and myxoma virus preferentially infect p53 and ATM dysfunctional cancer cells, the inventors have also examined whether RB defective cancer cells are collectively sensitive to infections of the leiovirus and myxoma viruses. As can be seen in Figure 3, two different human retinal subpopulations are susceptible to infectious viruses and myxoma virus infections, as can be seen by FACS or microscopic detection of fluorescent positive cells (FITC + or GFP + cells). These two cells contain defects in the Rb gene ( Reid meat al ., 1974 )

All of the methods described and claimed herein can be made and practiced without undue experimentation based on the description of the invention. It will be apparent to those skilled in the art that the methods and compositions of the present invention have been described in terms of preferred embodiments and that various changes can be made in the methods, method steps or the procedures described herein without departing from the concept, spirit and scope of the invention. More specifically, it is clear that certain substances chemically and physiologically related can be substituted for the materials described herein, and that the same or similar results can be obtained. 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 incorporated by reference in providing exemplary process or other supplementary material to those set forth herein.

Claims (34)

delete delete A composition for treating a patient suffering from a hyperproliferative disorder accompanied by a mutation in at least one of tumor suppressor genes selected from the group consisting of p53, Rb and ATM genes, wherein the composition is selected from the group consisting of a lvoviral pathogen and a myxoma viral pathogen At least one, and a pharmaceutically acceptable excipient, wherein said viral pathogen is selected from the group consisting of p53, Rb and ATM genes, a differential infection of hyperplastic cells with a mutation in one or more of the genes selected from the group consisting of p53, Rb and ATM genes Wherein the composition exhibits a tumor solubility effect. 4. The composition of claim 3, further comprising a non-viral cancer therapeutic agent. 5. The composition according to claim 4, wherein the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent, a hormone therapy agent, a gene therapy agent, an immunotherapeutic agent, an adult stem cell agent, and an inhibitor of p53, Rb or ATM function or expression. The composition according to any one of claims 3 to 5, wherein the patient is suffering from cancer. The composition according to any one of claims 3 to 5, wherein the rhioviral pathogen is a wild type virus. The composition according to any one of claims 3 to 5, wherein the rhioviral pathogen is an attenuated ryovirus. 9. The composition of claim 8, wherein the attenuated virus is a defective sigma 1 capsid protein, a sigma 1 capsid protein that can not be detected, or a lyovirus that does not express sigma 1 capsid protein. delete The composition according to any one of claims 3 to 5, further comprising a second agent-hyperproliferative agonist. delete 12. The composition of claim 11, wherein said second anti-hyperprolactic agent is selected from the group consisting of 5-fluorouracil, bleomycin, bisulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, (VP 16), a parnesyl-protein transferase inhibitor, gemcitabine, isoparasimide, mechloetamine, melphalan, mitomycin, navelbine, (Water soluble form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any of the foregoing substances, or any combination thereof. Analog or derivative thereof. The composition according to any one of claims 3 to 5, wherein the hyperplastic cell is a malignant cell or a benign cell. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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