WO2009143468A1 - Prédisposition par suppresseurs de tumeur de cellules hyperproliférantes à une thérapie virale oncolytique - Google Patents

Prédisposition par suppresseurs de tumeur de cellules hyperproliférantes à une thérapie virale oncolytique Download PDF

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WO2009143468A1
WO2009143468A1 PCT/US2009/045048 US2009045048W WO2009143468A1 WO 2009143468 A1 WO2009143468 A1 WO 2009143468A1 US 2009045048 W US2009045048 W US 2009045048W WO 2009143468 A1 WO2009143468 A1 WO 2009143468A1
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therapy
cell
cells
hyperproliferative
reovirus
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Manbok Kim
Randal N. Johnston
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Uti Limited Partnership
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Priority to CN200980126543.5A priority Critical patent/CN102124335B/zh
Priority to JP2011510734A priority patent/JP5748656B2/ja
Publication of WO2009143468A1 publication Critical patent/WO2009143468A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility

Definitions

  • the present invention relates generally to the fields of virology, molecular biology and medicine. More particularly, it concerns methods of assessing hyperproliferative disorder, including cancer, susceptibility to oncolytic viral therapies.
  • Reo virus Respiratory Enteric Orphan virus
  • Reo virus is a ubiquitous, non-enveloped virus containing 10 segments of double-stranded RNA as its genome, with human infections that are generally mild, restricted to the upper respiratory and gastrointestinal tracts and often asymptomatic, in immune functional hosts (Tyler, 2001).
  • Attempts to reverse engineer reoviruses have been largely unsuccessful due to several factors, including the double- stranded RNA genome of reoviruses.
  • Reoviral particles lacking ⁇ l have been understood to be non-infectious (Larson et al, 1994).
  • reovirus has been recognized for many years as displaying striking cytocidal activity when it infects certain types of transformed cells (Duncan et al, 1978; Hashiro et al, 1977).
  • Replication-competent oncolytic viruses provide an attractive anti- cancer therapeutic approach. These oncolytic viruses have two principal advantages. Firstly, unlike conventional chemotherapy and radiotherapy, they specifically target cancer cells because of their restricted ability to replicate in normal cells. Secondly, as compared to replication-incompetent vectors, they can propagate from initially infected cancer cells to surrounding cancer cells, thereby achieving a large volume of distribution and potent anti- cancer effects.
  • Ras gene mutations may only partially account for the susceptibility of cancer cells to reoviral therapies.
  • Recent studies showed that the Ras pathway is not the sole factor determining reovirus permissiveness (Song et al, 2009) and that reovirus resistant cells can be established from Ras-transformed cells (Kim et al, 2007) while still retaining the activating Ras mutation.
  • Ras pathway is not the sole factor determining reovirus permissiveness (Song et al, 2009) and that reovirus resistant cells can be established from Ras-transformed cells (Kim et al, 2007) while still retaining the activating Ras mutation.
  • Reovirus in either its wild-type (Type 3 Dearing) or in modified forms ⁇ e.g., a partially attenuated virus with defective synthesis of the ⁇ l protein) is effective in killing hyperproliferative or transformed cancer cells that contain certain defective or inactivated tumor suppressor genes, such as p53, Rb, ATM, BRCAl, BRCA2, MutS, MutL, MutH, APC, and ATBFl, when such cells are exposed to the virus in vitro or in vivo. Therefore, the range of application of reovirus may be extended beyond cases associated with abnormal or excess activity of the ras proto-oncogene signaling pathway.
  • a method of determining whether a hyperproliferative disorder is susceptible to reovirus or myxoma virus oncolysis comprising (a) providing a hyperproliferative cell; (b) assessing the structure, function or expression of p53, Rb, ATM, BRCAl, BRCA2, MutS, MutL, MutH, APC, and/or ATBFl, gene or gene product from the hyperproliferative cell; and (c) comparing the structure, function or expression of the hyperproliferative cell with the wild-type structure, function or expression of a p53, Rb, ATM, BRCAl, BRCA2, MutS, MutL, MutH, APC, and/or ATBFl, gene or gene product, wherein a defect in the structure, function
  • the method may further comprise treating a patient from which the hyperproliferative cell was obtained, for example, by reoviral and/or myoxma viral therapy.
  • the reoviral therapy may be wild-type reoviral therapy or attenuated reovial therapy, such as that utilizing a reovirus that expresses a defective ⁇ l capsid protein, an undetectable ⁇ l capsid protein, or no ⁇ l capsid protein.
  • the treating may comprise ex vivo treatment of bone marrow cells from the patient with the reoviral and/or myoxma viral therapy, or may comprise administration of the reoviral and/or myoxma viral therapy to the patient.
  • the method may further comprise contacting the bone marrow cells a second anti- hyperproliferative therapy, or in the context of a viral therapy administered to the subject, the method may further comprising administering to the subject a second anti -hyperproliferative therapy.
  • the second anti-hyperproliferative therapy may be chemotherapy, radiotherapy, hormone therapy, gene therapy, or immunotherapy.
  • the method may also further comprise therapy with an inhibitor of p53, Rb, ATM, BRCAl, BRCA2, MutS, MutL, MutH, APC, or ATBFl function or expression, such as a protein or peptide, an antibody, an siRNA, an antisense molecule, a ribozyme or a small molecule.
  • Treating may also comprise a non-viral anti-hyperproliferative therapy, such as with chemotherapy, radiotherapy, hormone therapy, gene therapy, or immunotherapy.
  • the hyperproliferative cell may be a malignant cell or a benign cell.
  • the method may further comprise, prior to step (a), obtaining the hyperproliferative cell from a patient.
  • Assessing expression may comprise Northern blot, Western blot, immunohistochemistry, RT-PCR, microarray analysis, transcriptome analysis, proteome analysis, or metabolome analysis.
  • assessing structure may comprise sequencing, in situ hybridization, immunohistochemistry, or structural proteomics.
  • assessing function may comprise assessing the expression or activity of a downstream target of p53, Rb, ATM, BRCAl, BRCA2, MutS, MutL, MutH, APC, or ATBFl.
  • a method of treating a hyperproliferative disorder of a patient comprising (a) contacting a hyperproliferative cell with an inhibitor of p53, Rb, ATM, BRCAl, BRCA2, MutS, MutL, MutH, APC, or ATBFl function or expression; and (b) contacting the hyperproliferative cell with a reoviral and/or myoxma viral therapy.
  • the reoviral therapy may be wild-type reoviral therapy or an attenuated reoviral therapy, such as a reo virus that expresses a defective ⁇ l capsid protein, an undetectable ⁇ l capsid protein, or no ⁇ l capsid protein.
  • Treating may comprise ex vivo treatment of bone marrow cells from the patient, or in vivo treatment of the patient.
  • the method may further comprise contacting the bone marrow cells with a second anti- hyperproliferative therapy, such as a second anti-hyperproliferative therapy, such as a chemotherapy, radiotherapy, hormone therapy, gene therapy, or immunotherapy.
  • the hyperproliferative cell may be a malignant cell or a benign cell.
  • FIGS. IA-B Reovirus and myxoma virus preferential infection of p53 -/- MEF and ATM defective L3.
  • FIG. IA p53 -/- MEF and ⁇ 53 +/+ MEF (p53 knock out or p53 wild-type murine embryonic fibroblasts respectively) were infected with WT/ AV reovirus at MOI of 40 or GFP-expressing myxoma virus (Myx-GFP, which is a GFP expressing version of the Lausanne (ATCC) strain of MYXV, has been described (Johnston et al , 2003)) at MOI of 5.
  • Myx-GFP which is a GFP expressing version of the Lausanne (ATCC) strain of MYXV
  • IB IB
  • BT ATM-normal lymphoblastoid C3ABR cells; Kozlov et al, 2003
  • L3 ATM-deficient lymphoblastoid cells; Kozlov et al, 2003
  • WT/ AV reovirus at MOI of 40 or myxoma virus (Myx-GFP) at MOI of 5.
  • Myx-GFP myxoma virus
  • FIGS. 2A-C Reovirus and myxoma virus preferential infection of p53 and ATM dysfunctional human lymphomas.
  • FIG. 2A p53 and ATM defects enhance responses to IR stimuli.
  • Mantle cell lymphomas Granta, HBL-2 (p53 mutation, Turker et al, 2006), Z138C, JVM-2)
  • Burkitt's lymphomas Raji, and Ramos
  • Whole cell lysates were produced by treatment with NET-N lysis buffer (1% NP-40) followed by sonication.
  • p53 and ATM-normal-responsive cells (BT, Z138C, JVM-2, Ramos), and p53 and/or ATM-dysfunctional-responsive cells (L3, HBL-2, Grant, Raji) were plotted with viral susceptibility to both reoviruses and myxoma virus. 1 ; virally-susceptible. 0; virally-resistant. Normally-responsive cells are resistant but dysfunctionally responsive cells are susceptible to viral infections.
  • FIG. 2C Cells were infected with WT/ AV reovirus at MOI of 40 or Myxoma virus (Myx-GFP) at MOI of 5.
  • FIG. 3 Reovirus and myxoma virus infection of retinoblastoma cells.
  • Retinoblastoma cells (Y79 and WERI-Rb-I, purchased from ATCC) were infected with WT/AV reovirus at MOI of 40 or myxoma virus (Myx-GFP) at MOI of 5.
  • WT/AV reovirus at MOI of 40
  • Myx-GFP myxoma virus
  • Carcinogenesis is a multi-step process involving the combined accumulation of oncogene activations and the inactivation of tumor suppressor genes via mutational events affecting normal proto-oncogenes and tumor suppressor genes.
  • oncolytic viruses utilize different oncogene-driven cellular signaling pathways to selectively replicate and kill the cells they infect.
  • the inventors speculated that some tumor suppressor genes may also play an important role in facilitating viral oncolysis.
  • Myxoma virus is a rabbit-specific poxvirus that is considered a promising oncolytic virus platform (reviewed by Stanford and McFadden, 2007). Its host tropism is highly restricted to European rabbits, and it is non-pathogenic for all other vertebrate species tested, including humans (McFadden, 2005). Despite this narrow specificity, myxoma virus is capable of infecting and killing a wide variety of human tumor cell lines (Sypula et ah, 2004). Myxoma virus tropism at the cellular level is largely regulated by intracellular events downstream of virus binding and entry, rather than at the level of specific host receptors (McFadden, 2005).
  • M-T5 KO (knock-out) myxoma virus exerts an attenuated viral oncolytic potential in one class of human cancer cells (designated type II cancer cells)
  • constitutively Akt activated cancer cells are still permissive to M-T5 KO myxoma virus, suggesting that upregulation of Akt, frequently found in many cancer cells, plays an important role in determining myxoma oncolytic potential (Wang et al, 2006).
  • the connection between viral oncolysis and tumor suppressor genes was initially advanced through the study of adenovirus.
  • the human adenovirus ElB gene encodes a 55 kD protein that binds and inactivates the cellular tumor suppressor protein p53. It has been shown that a mutant adenovirus that does not express this viral protein can replicate in and kill p53- dysfunctional human tumor cells, but not cells with functional p53 (Bischoff et al, 1996).
  • the underlying basis for the mutant adenovirus oncolysis is that adenoviral ElA-induced p53- dependent apoptosis is compromised in p53-dysfunctional cancer cells due to p53 deletion or mutation (Bischoff et al, 1966).
  • p53 dysfunctionality may ultimately lead to viral susceptibility (via loss of anti-viral mechanisms) caused by global genomic instability.
  • Recent studies showed that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene (Takaoka et al, 2003; Munoz-Fontela et al, 2008).
  • p53 transcriptional role is important in activation of the IFN pathway upon viral infections.
  • p53 can activate transcription of IFN regulatory factor 9 upon viral challenges (Munoz-Fontela et al, 2008).
  • significantly higher levels of viral replication were observed when p53 expression was reduced in cancer cells (Dharel et al, 2008).
  • the reoviruses ⁇ Reoviridae comprise a family of naturally-occurring, non-enveloped viruses having a double-stranded RNA (dsRNA) genome that is divided into ten segments and enclosed by two concentric icosahedral protein capsids. These viruses can affect the gastrointestinal system and respiratory tract.
  • dsRNA double-stranded RNA
  • the name Reoviridae is derived from "respiratory enteric orphan viruses.”
  • the term "orphan" virus means a virus that is not associated with any known disease. Even though viruses in the Reoviridae family have more recently been identified with various diseases, the original name is still used.
  • Reovirus infection occurs often in humans, but most cases are mild or subclinical.
  • the virus can be readily detected in feces, and may also be recovered from pharyngeal or nasal secretions, urine, cerebrospinal fluid, and blood.
  • Reoviruses are non-enveloped and have an iscohedral capsid (T- 13) composed of an outer and inner protein shell.
  • T- 13 iscohedral capsid
  • the genomes of viruses in Reoviridae contain 10-12 segments which are grouped into three categories corresponding to their size: L (large), M (medium) and S (small). Segments range from -3.9-1 kB and each segment encodes 1-3 proteins.
  • Reoviridae proteins are denoted by the Greek character corresponding to the segment it was translated from (the L segment encodes for ⁇ proteins, the M segment encodes for ⁇ proteins and the S segment encodes for ⁇ proteins).
  • viruses Since these viruses have dsRNA genomes, replication occurs exclusively in the cytoplasm and the virus encodes several proteins which are needed for replication and conversion of the dsRNA genome into (+)-RNAs.
  • the virus can enter the host cell via a receptor on the cell surface. The receptor is not known but is thought to include sialic acid and junctional adhesion molecules (JAMs).
  • JAMs junctional adhesion molecules
  • the virus is partially uncoated by proteases in the endolysosome, where the capsid is partially digested to allow further cell entry.
  • the core particle then enters the cytoplasm by a yet unknown process where the genome is transcribed conservatively causing an excess of (+) sense strands, which are used as mRNA templates to synthesize (-) sense strands. Viral particles begin to assemble in the cytoplasm 6-7 hours after infection.
  • Infectious mammalian reovirus virions occur as particles of approximately 85 nm in diameter.
  • the virion outer capsid includes several distinct protein species, among them sigma-1 ( ⁇ l, 50 kDa) which mediates viral attachment to host cell surfaces (Lee et ah, 1981; Duncan et al, 1991; Nagata et al, 1987; Turner et al, 1992) via discrete carbohydrate- binding (Chappell et al, 1997; Chappell et al, 2000; Connolly et al, 2001) and virion- anchoring (Mah et al, 1990; Fernandes et al, 1994; Lee et al, 1994) domains, ⁇ l is a product of the bicistronic reoviral Sl gene, which also encodes a non-structural protein designated ⁇ ls using a distinct but overlapping reading frame (Ernst et al, 1985; Jacobs et al, 1985; Sarkar
  • Reoviral particles that lack ⁇ l have been reported to be noninfectious (Larson et al, 1994).
  • the reoviral Sl gene has been believed to play a significant role in determining reoviral pathogenesis (Haller et al, 1995; Wilson et al, 1994; Kaye et al, 1986; Werner et ⁇ /., 1980).
  • wild-type reovirus In immune compromised hosts such as newborn and SCID (severe combined immunodeficiency) animals, however, the wild-type reovirus exerts significant viral pathogenesis especially in neural tissue and cardiac muscle tissue (Sabin, 1959; Weiner et al, 1977; Baty et al, 1993; Loken et al, 2004). In some cases, even in immune-competent hosts including humans, wild-type reovirus has been associated with viral pathogenesis (Terheggen et al, 2003, Hirasawa et al, 2003). Therefore, especially in immune-compromised or very young hosts, wild-type reovirus does not always act in a benign manner.
  • a cell culture e.g., a bone marrow transplant taken from a cancer patient
  • wild-type reoviruses may result in the undesirable side effect of killing cells such as stem cells which would be needed for a purpose such as repopulating the immune system of a cancer patient.
  • the increased virulence of wild-type reoviruses present significant limitations for the clinical potential of in vitro applications involving exposure of cells to reoviruses.
  • a herein described attenuated reovirus lacks detectable full-length ⁇ l capsid protein yet is, unexpectedly, infectious, ⁇ l has been implicated in reoviral binding and attachment to cells via cell surface sialic acid residues in an initial step of viral replicative infection (Lee et a/., 1981; Duncan et a/., 1991; Nagata et al, 1987; Turner et al, 1992; Chappell et al, 1997; Chappell et al, 2000; Connolly et al, 2001).
  • the attenuated reovirus described herein is capable of host cell entry and cytolytic viral replication.
  • the attenuated reovirus exhibits the surprising property of inducing a decreased (i.e., reduced with statistical significance) level of one or more cytopathic effects toward a non-malignant cell relative to the level of the cytopathic effect that is exhibited toward the non-malignant cell by a naturally occurring, non-attenuated reovirus.
  • the attenuated reovirus provided herein offers improvements over reoviruses of the prior art, including suitability for use as an oncolytic agent without undesirable side-effects such as tropism for, and cytolysis of, normal (e.g., non-malignant) cells.
  • this attenuated reovirus may be used in vitro for the selective killing of cancerous or neoplastic cells.
  • an attenuated reovirus as described in U.S. Application 60/704,604, filed August 5, 2006, and International Patent Application No. PCT/US2006/029881, filed July 31, 2006, which are incorporated by reference herein in its entirety without disclaimer, may be used with the present invention.
  • the attenuated reovirus may lack a wild-type reovirus Sl gene.
  • the attenuated reovirus may possess a Sl gene which produces a reduced and/or undetectable amount of the Sl gene product ( ⁇ l) or a truncated, mutated, and/or dysfunctional ⁇ l.
  • An attenuated reovirus may have a Sl gene that contains one or more mutations, as compared to a wild-type Sl gene, wherein the mutation is a nucleotide substitution, a nucleotide deletion or a nucleotide insertion.
  • "Attenuated" reo viruses described herein include reo viruses that exhibit altered (i.e., increased or decreased in a statistically significant manner) infective, replicative and/or lytic properties toward or in a host cell, relative to levels of one or more such properties that are exhibited by known, naturally occurring or wild-type reoviruses.
  • the attenuated reovirus will exhibit decreased infectivity, replicative ability and/or lytic potential, relative to a wild-type reovirus.
  • altered properties by which one may discern an attenuated reovirus as presently disclosed include various manifestations of viral cytopathic effects, for instance, the multiplicity of infection (MOI, the average number of virions that infect each cell) required for productive infection of a given host cell, the degree of host cell cytolysis induced by viral infection (further including apoptosis and/or necrosis), the titer of viruses released from a productively infected host cell following cytolytic viral replication, and other parameters by which those familiar with the art can determine viral activities toward host cells.
  • MOI multiplicity of infection
  • the degree of host cell cytolysis induced by viral infection further including apoptosis and/or necrosis
  • the titer of viruses released from a productively infected host cell following cytolytic viral replication and other parameters by which those
  • indicia of cytopathic effects include altered host cell morphology, altered capacity to elicit attack by the host cell-mediated immune system, altered cell adhesion (to substrates such as extracellular matrix proteins or semisolid growth media, or to other cells), altered expression levels of one or more cellular genes, altered ability of host cells to replicate, and/or other alterations in cellular metabolic activity.
  • AV reovirus Sl attenuated reovirus
  • wild-type reovirus is known to adversely affect the development of rat and murine embryos by retarding development and inhibiting blastocytst formation (Priscott, 1983; Heggie et al, 1979), the inventors further evaluated the pathogenicity of AV reovirus on stem cells. Thus, the inventors compared the pathogenicity of wild-type and AV reovirus on embryonic stem cells (ESCs). As shown in the below examples, wild-type reovirus readily infects ESCs in vitro and significantly suppresses stem cell development in a teratoma model, whereas AV reovirus minimally infects ESCs in vitro and does not affect stem cell development in a teratoma model.
  • ESCs embryonic stem cells
  • the attenuated reovirus may comprise one or more additional mutations.
  • the attenuated reovirus may lack a wild-type reovirus S4 gene.
  • the reovirus wild-type S4 gene encodes a reovirus capsid ⁇ 3 polypeptide involved in virion processing during reoviral replicative infection of a host cell (e.g., Ahmed et al, 1982; Giantini et al, 1984).
  • the attenuated reovirus may comprise a replication-competent reovirus virion. Further, an attenuated reovirus may have a heritable mutation (e.g., substitution, insertion deletion) in the Sl gene and/or the S4 gene.
  • the wild-type reovirus Sl gene is known to those of skill in the art. For example, wild-type Sl gene sequences include the Sl gene sequences identified in predominant forms of naturally-occurring reoviruses isolated from respiratory or enteric tissues of infected subjects, or consensus sequences derived from such sequences.
  • Sl gene sequences for a number of reoviruses, including the human reoviruses have been determined, (e.g., Genbank Accession numbers for human reovirus Sl gene sequences: human type 3 reovirus Sl : XOl 161; human type 2 reovirus Sl: M35964; human type 1 reovirus Sl : M35963) including polynucleotide sequences encoding ⁇ l proteins as well as the amino acid sequences of the encoded ⁇ l proteins themselves, (e.g., Genbank Accession numbers for major human reovirus serotype Sl gene sequences: human type 1 reovirus strain Lang (TlL) Ace. No.
  • an attenuated reovirus comprising a reovirus genome that lacks a wild-type reovirus Sl gene, or that comprises a mutated reovirus Sl gene that is incapable of encoding an intact reovirus ⁇ l capsid protein (Genbank Accession numbers for human reovirus Sl gene sequences: human type 3 reovirus Sl : X01161; human type 2 reovirus Sl : M35964; human type 1 reovirus Sl : M35963).
  • a mutated Sl gene thus refers to an Sl gene having a polynucleotide sequence that differs at one or a plurality of nucleotide sequence positions from the nucleotide sequence of a corresponding Sl wild-type or consensus sequence by one or more of a nucleotide substitution, a nucleotide insertion, and a nucleotide deletion, as can be readily determined.
  • Several mutations in a murine reoviral Sl gene sequence encoding a ⁇ l protein are disclosed by Hoyt et al. (2005) and according to certain embodiments of the invention described herein the mutations of Hoyt et al. are expressly excluded.
  • the reo virus outer capsid ⁇ l protein may be readily detected on the basis of its biochemical and/or immunochemical properties (e.g., Mah et ah, 1990; Leone et ah, 1991; Chappell et ah, 1997), typically by employing one or more techniques including immunodetection (e.g., ⁇ l -specific immunoprecipitation, western immunoblot analysis, immunoaffmity chromatography, immunofluorescent staining, immunocytofluorimetry, electrophoresis of radiolabeled reovirus polypeptides, etc.), hemagglutination, and/or related methodologies.
  • immunodetection e.g., ⁇ l -specific immunoprecipitation, western immunoblot analysis, immunoaffmity chromatography, immunofluorescent staining, immunocytofluorimetry, electrophoresis of radiolabeled reovirus polypeptides, etc.
  • peptidyl fiuoromethylketones may be applied to the cellular composition to inhibit or eliminate reovirus from the composition.
  • PFMKs peptidyl fiuoromethylketones
  • the reovirus may be derived from any reovirus, which refers to a member of the Family Reoviridae and includes reoviruses having a variety of tropisms and which may be obtained from a variety of sources (Tyler and Fields, 1996).
  • reovirus refers to a member of the Family Reoviridae and includes reoviruses having a variety of tropisms and which may be obtained from a variety of sources (Tyler and Fields, 1996).
  • mammalian reoviruses and human reoviruses are envisioned, although the invention is not intended to be so limited, and based on the present disclosure the skilled artisan will recognize situations where any particular reovirus may be desirable for such purposes.
  • the reovirus may be human Type 3 (Dealing), Type 1 (Lang), Type 2 (Jones), or Type 3 (Abney) reoviruses, while in certain other embodiments, the reovirus may be derived from one or more reoviruses displaying tropisms toward cells of other mammalian species, including non-human primates (e.g., chimpanzee, gorilla, macaque, monkey, etc.), rodents (e.g., mice, rats, gerbils, hamsters, rabbits, guinea pigs, etc.), dogs, cats, common livestock (e.g., bovine, equine, porcine, caprine, etc.), or alternatively, reo viruses having distinct tropisms (e.g., avian reo viruses) may be used.
  • non-human primates e.g., chimpanzee, gorilla, macaque, monkey, etc.
  • rodents e.g., mice
  • Attenuated reoviruses that may be recovered following persistent infection regimens in vitro, but attenuated reoviruses are also contemplated that may be derived according to other methodologies, including persistent infection regimens in vivo, generation and identification of ⁇ l -deficient and/or ⁇ l -defective mutants (and in certain embodiments also including, additionally or alternatively, generation and identification of ⁇ 3 -deficient and/or ⁇ 3 -defective mutants) by molecular biological approaches, and also including isolation of naturally occurring ⁇ l -deficient and/or ⁇ l- defective mutants and/or ⁇ 3 mutants, and/or artificial induction of such ⁇ l (and/or ⁇ 3) mutants by chemical, physical and/or genetic techniques (e.g., assortative recombination of reoviral genes in a productively infected host cell).
  • persistent infection regimens in vivo generation and identification of ⁇ l -deficient and/or ⁇ l -defective
  • mutations resulting in a desirable attenuation phenotype may be observed in other genes that affect viral infectivity, replicative or packaging ability, including any or a combination of the genes encoded on the virus, such as the ten gene segments of a reovirus.
  • mutation of a reovirus Sl or S4 gene as compared to the reovirus wild-type gene, may be coupled with 1, 2, 3, 4, 5 or more mutations on the ten gene segments of the reovirus.
  • Various methods may be used to generate attenuated reoviruses, including methods, e.g., described in U.S. Application 60/704,604.
  • Myxoma virus is a poxvirus and has a large double-stranded DNA genome that allows for the potential insertion of large (25 kb), therapeutically relevant, eukaryotic genes.
  • Myxoma virus is a rabbit-specific virus and causes a lethal disease termed myxomatosis in the European rabbit (Oryctalagus cuniculus). Its species selectivity is so narrow that it was used to control the disastrous feral rabbit population in Australia in the 1950's.
  • non-pathogenic for all other vertebrate species tested including humans, though it can productively infect certain non-rabbit cells in vitro including immortalized baby monkey kidney fibroblasts (BGMK), primary murine cells genetically deficient in IFN responses, and a number of different human tumor cells in vitro. Cancer cells are well known to be deficient in their IFN responses. Recently, myxoma virus has been show to be an oncolytic agent against experimental gliomas in vitro, in vivo, and ex vivo against human malignant glioma surgical specimens (Lun et al, 2005; 2007; Stanford et al, 2008).
  • Myxoma virus as with other oncolytic viruses such as reovirus, needs to bypass the anti-viral defenses that exist in normal healthy cells in order to be able to replicate within cells.
  • Myxoma virus and other oncolytic viruses induce interferon production, and are generally sensitive to the anti-viral effect of the IFN pathway.
  • Relevant proteins induced by the IFN anti-viral response, and which principally affect virus multiplication include PKR, OAS synthetase and Rnase L nuclease.
  • PKR activates eIF2 ⁇ , leading to inhibition of translation and induction of apoptosis.
  • myxoma virus is directly affected by PKR and eIF2 ⁇ .
  • Anti-viral response pathways are often disrupted in cancerous cells. For example, reduced or defective response to IFN is a genetic defect that often arises during the process of transformation and tumour evolution. Over 80% of tumour cell lines do not respond to, or exhibit impaired responses to, interferon. (Stojdl et al, 2003 and references cited therein; Wong et al, 1997; Sun et al, 1998; Matin et al, 2001; Balachandran et al, 2004).
  • U.S. Patent Publication 2006/0263333 (incorporated by reference) describes the use of myxoma virus to infect and kill cancer cells, including human tumor cells, and is hereby incorporated by reference in its entirety.
  • a tumor suppressor gene is a gene that protects a cell from one step on the path to cancer. When this gene is damaged, the cell can progress to cancer, usually after it is subject to other factors. Unlike oncogenes, tumor suppressor genes generally follow the "two-hit hypothesis," which implies that both alleles that code for a particular gene must be affected before an effect is manifested. This is due to the fact that if only one allele for the gene is damaged, the second can still produce the correct protein. In other words, tumor suppressors are usually haplosufficient as opposed to oncogenes which are generally haploinsufficient. Of course, there are notable exceptions such as the p53 gene product, which can exist in a mutated form that is dominant-negative with respect to the normal p53 protein, which is therefore also haploinsufficient in such cases.
  • Tumor suppressor genes or more precisely, the proteins for which they code, either have a dampening or repressive effect on the regulation of the cell cycle or promote apoptosis, and sometimes do both.
  • the functions of tumor suppressor proteins fall into several categories including the following: (a) repression of genes that are essential for the continuing of the cell cycle (if these genes are not expressed, the cell cycle will not continue, effectively inhibiting cell division); (b) coupling the cell cycle to DNA damage.
  • oncogenes are genes that cause cancer. Many cells are normally destined to die. In cancer, because of the presence of a mutated oncogenic DNA sequence, those cells survive and proliferate. Most oncogenes require an additional step, such as mutations in another gene, or environmental factors such as viral infection, to cause cancer. Since the 1980s, dozens of oncogenes have been identified in human cancer.
  • a proto-oncogene is a normal gene that can become an oncogene due to mutations or increased expression.
  • Proto- oncogenes code for proteins that help to regulate cell growth and differentiation. Proto- oncogenes are often involved in signal transduction and execution of mitogenic signals, usually through their protein products. Upon activation, a proto-oncogene (or its product) becomes a tumor inducing agent, an oncogene. Examples of proto-oncogenes include RAS, WNT, MYC, ERK and TRK.
  • p53 p53 (accession no. NM 000546) is a transcription factor that regulates the cell cycle and functions as a tumor suppressor. It is important in multicellular organisms as it helps to suppress cancer, in part through its role in coordinating the cellular response to damage to DNA. p53 has been described as "the guardian of the genome,” “the guardian angel gene,” or the “master watchman,” referring to its role in conserving stability by preventing genome mutation.
  • p53 is in reference to its apparent molecular mass on SDS-PAGE, but in fact it is actually only 43.7 kD. This difference is due to the high number of amino-acid proline residues in the p53 protein which slow p53's migration on SDS-PAGE, thus making it appear larger. This effect is observed with p53 from a variety of species, including humans, rodents, frogs, and fish.
  • the gene is located on the human chromosome 17 (17pl3.1), and encodes a 393 amino acid protein with five domains: (a) an N-terminal transcription- activation domain (TAD), which activates transcription factors (residues 1-42); (b) a proline rich domain important for the apoptotic activity of p53 (residues 80-94); a central DNA- binding core domain (DBD) containing one zinc atom and several arginine amino acids (residues 100-300); a homo-oligomerization domain (OD) (residues 307-355); and a C- terminal involved in downregulation of DNA binding of the central domain (residues 356- 393).
  • TAD N-terminal transcription- activation domain
  • DBD central DNA- binding core domain
  • OD homo-oligomerization domain
  • C- terminal involved in downregulation of DNA binding of the central domain (residues 356- 393).
  • Mutations that deactivate p53 in cancer usually occur in the DBD. Most of these mutations destroy the ability of the protein to bind to its target DNA sequences, and thus prevents transcriptional activation of these genes. As such, mutations in the DBD are recessive loss-of-function mutations. Molecules of p53 with mutations in the OD dimerise with wild-type p53, and prevent them from activating transcription. Therefore OD mutations have a dominant negative effect on the function of p53. p53 has many anti-cancer mechanisms. For example, it can activate DNA repair proteins when DNA has sustained damage.
  • p53 becomes activated in response to a myriad of stress types, which include but is not limited to DNA damage (induced by either UV, IR or chemical agents, such as hydrogen peroxide), oxidative stress, osmotic shock, ribonucleotide depletion and deregulated oncogene expression. This activation is marked by two major events.
  • the critical event leading to the activation of p53 is the phosphorylation of its N-Terminal domain.
  • the N-Terminal transcriptional activation domain contains a large number of phosphorylation sites and can be considered as the primary target for protein kinases transducing stress signals.
  • a first group of protein kinases belongs to the MAPK family (JNK1-3, ERK1-2, p38 MAPK), which is known to respond to several types of stress, such as membrane damage, oxidative stress, osmotic shock, heat shock, etc.
  • a second group of protein kinases (ATR, ATM, Chkl, Chk2, DNA-PK, CAK) is implicated in the genome integrity checkpoint, a molecular cascade that detects and responds to several forms of DNA damage caused by genotoxic stress.
  • Mdm2 binds to p53 and transports it from the nucleus to the cytosol where it becomes degraded by the proteasome. Phosphorylation of the N-terminal end of p53 by the above mentioned protein kinases disrupts Mdm2 -binding. Other proteins, such as Pinl, are then recruited to p53 and induce a conformational change in p53 which prevents Mdm2- binding even more. Trancriptional coactivators, like p300 or PCAF, then acetylate the carboxy-terminal end of p53, exposing the DNA binding domain of p53, allowing it to activate or repress specific genes.
  • Trancriptional coactivators like p300 or PCAF, then acetylate the carboxy-terminal end of p53, exposing the DNA binding domain of p53, allowing it to activate or repress specific genes.
  • the retinoblastoma protein (Rb; NM 00321) is a tumor suppressor protein found to be dysfunctional in a number of types of cancer. pRb was so named because retinoblastoma cancer results when the protein is inactivated by a mutation in both alleles of the RBl gene that codes for it. Rb is usually present as a phosphoprotein inside cells and is a target for phosphorylation by several kinases as described below. One highly studied function of Rb is to prevent the cell from dividing or progressing through the cell cycle. Thus, when Rb is ineffective at this role, mutated cells can continue to divide and may become cancerous.
  • Rb is a member of the "Pocket protein family," because it has a pocket to which proteins can bind. Oncogenic proteins such as those produced by cells infected by high-risk types of human papillomaviruses can bind and inactivate Rb, which can lead to cancer. Rb prevents the cell from replicating damaged DNA by preventing its progression through the cell cycle into its S, or synthesis phase or progressing through Gl, or first gap phase. Rb binds and inhibits transcription factors of the E2F family. E2F transcription factors are dimers of an E2F protein and a DP protein. The transcription activating complexes of E2 promoter- binding-protein-dimerization partners (E2F-DP) can push a cell into S phase.
  • E2F-DP The transcription activating complexes of E2 promoter- binding-protein-dimerization partners
  • E2F- DP As long as E2F- DP is inactivated, the cell remains stalled in the Gl phase.
  • the complex acts as a growth suppressor and prevents progression through the cell cycle.
  • the Rb- E2F/DP complex also attracts a histone deacetylase (HDAC) protein to the chromatin, further suppressing DNA synthesis.
  • HDAC histone deacetylase
  • Rb In the hypophosphorylated state, Rb is active and carries out its role as tumor suppressor by inhibiting cell cycle progression. Phosphorylation inactivates Rb.
  • Rb is activated near the end of Gl phase when a phosphatase dephosphorylates one of its residues, allowing it to bind E2F.
  • CDK cyclin-dependent kinases
  • cyclins phosphorylate pRb, inhibiting its activity.
  • the initial phosphorylation is performed by Cyclin D/CDK4,6 and followed by additional phosphorylation by Cyclin E/CDK2.
  • pRb remains phosphorylated throughout S, G2 and M phases. Phosphorylation of pRb allows E2F-DP to dissociate from pRb and become active.
  • E2F When E2F is freed it activates factors like cyclins (e.g., Cyclin E and A), which push the cell through the cell cycle by activating cyclin-dependent kinases, and a molecule called proliferating cell nuclear antigen, or PCNA, which speeds DNA replication and repair by helping to attach polymerase to DNA.
  • factors like cyclins (e.g., Cyclin E and A), which push the cell through the cell cycle by activating cyclin-dependent kinases, and a molecule called proliferating cell nuclear antigen, or PCNA, which speeds DNA replication and repair by helping to attach polymerase to DNA.
  • cyclins e.g., Cyclin E and A
  • PCNA proliferating cell nuclear antigen
  • ATM Ataxia telangiectasia (AT; NM_00051) is an autosomal recessive disorder caused by mutations in the ATM gene located on chromosome l lq22-23. It was characterised in June of 1995 and is made up of 66 exons spread across 150kb of genomic DNA. It encodes a 13kb mature transcript with an open reading frame of 9168 nucleotides.
  • the ATM protein is about 37OkDa and is ubiquitously expressed and is localised to the cell nucleus.
  • the ATM protein is a large serine-threonine kinase thought to play a role in regulating cell cycle checkpoints, repair of double stranded DNA and meiosis (similar to the BRCA genes).
  • ATM is also known to play a role in regulating p53, BRCAl and CHEK2.
  • Part of ATM's role in DNA repair is known to be that of telomere repair as telomeres degrade more rapidly in people affected with AT.
  • Mutations in the ATM gene are thought to come in two types: (a) null mutations are those which cause complete loss of function of the protein and are therefore inherited in a recessive manner and cause AT; and (b) 'missense' mutations which produce stable, full sized protein with reduced function, e.g., substitutions, short in-frame insertions and deletions, etc. These mutations act by dominantly interfering with the normal copy of the protein.
  • BRCAl and BRCA2 play a critical role in the development of breast cancer.
  • BRCAl is a human gene that maintains genomic integrity to prevent uncontrolled proliferation.
  • the multifactorial BRCAl protein product is involved in DNA damage repair, ubiquitination, transcriptional regulation as well as other functions. Variations in the gene have been implicated in a number of hereditary cancers, namely breast, ovarian and prostate.
  • the BRCAl gene is located on the long (q) arm of chromosome 17 at band 21, from base pair 38,449,843 to base pair 38,530,933 (map).
  • the BRCAl protein is directly involved in the repair of damaged DNA.
  • the BRCAl protein is thought to interact with RAD51 during repair of DNA double-strand breaks. These breaks can be caused by natural radiation or other exposures, but also occur when chromosomes exchange genetic material during a special type of cell division that creates sperm and eggs (meiosis). By influencing DNA damage repair, this protein plays a role in maintaining the stability of the human genome.
  • BRCA2 is a second human gene involved in the repair of chromosomal damage. Although the structures of the BRCAl and BRCA2 genes are very different, their functions appear to be similar. The proteins made by both genes are essential for repairing damaged DNA.
  • the BRCA2 protein binds to and regulates the protein produced by the RAD51 gene to fix breaks in DNA. These breaks can be caused by natural and medical radiation or other environmental exposures, but also occur when chromosomes exchange genetic material during a special type of cell division that creates sperm and eggs (meiosis).
  • the BRCAl protein also interacts with the RAD51 protein. By repairing DNA, these three proteins play a role in maintaining the stability of the human genome.
  • BRCA2 probably regulates the activity of other genes and plays a critical role in embryo development.
  • the BRCA2 gene is located on the long (q) arm of chromosome 13 at position 12.3 (13ql2.3), from base pair 31,787,616 to base pair 31,871,804.
  • MutS is the lead member of a family or proteins that assist in the repair mismatches in double-stranded DNA.
  • the first step in this process is the recognition of the mismatched DNA.
  • MutS binds to the site of a mismatch in double-stranded DNA and, with the cooperation of the MutL and MutH proteins, targets a section of one of the DNA strands at that location for removal.
  • Other proteins complete the repair process: the section of DNA that has been targeted is removed and degraded, a patch is synthesized using the complementary strand as a template, and the patch is ligated into place resulting in a repaired section of double-stranded DNA without mismatches.
  • Adenomatosis polyposis coli is another tumor suppressor implicated in colorectal cancer. It helps control how often a cell divides, how it attaches to other cells within a tissue, or whether a cell moves within or away from a tissue, and also helps ensure that the chromosome number in cells produced through cell division is correct.
  • the APC protein accomplishes these tasks mainly through association with other proteins, especially those that are involved in cell attachment and signaling.
  • the activity of one protein in particular, ⁇ -catenin is controlled by the APC protein, which part of the Wnt signaling pathway. Regulation of ⁇ -catenin prevents genes that stimulate cell division from being turned on too often and prevents cell overgrowth.
  • the APC gene is located on the long (q) arm of chromosome 5 between positions 21 and 22, from base pair 112,118,468 to base pair 112,209,532.
  • AT-binding transcription factor 1 is a tumor suppressor, the loss of which is implicated in the development of gastric cancer. It is located at 16q22.3-q23.1. The overall DNA length is 261.32 kB. There are two isoforms, ATBFl-A and ATBFl-B, due to alternative promoter usage combined with alternate splicing.
  • the protein is 3703 amino acids and 404 kDa in size. It contains four homeodomains and 23 zinc fingers including 1 pseudo zinc finger motif, one DEAD and one DEAH box, a RNA and an ATP binding site, two large RS domains and multiple phosphorylation sites.
  • the protein is nuclear localized and acts as a transcription factor that binds to the AT-rich core sequence of the enhancer element of the AFP gene and downregulates AFP gene expression, possibly involved in neuronal differentiation.
  • Amplification in one early neural crest derived cell line SJNB- 12 under the form of extrachromosomally double minutes, non-syntenic co-amplification with myc. Absence of ATBFl expression is observed in ⁇ -fetoprotein-expressing gastric cancer cell lines. The lack of ATBFl expression not due to mutation, deletion or translocation, but to strong repression at the transcriptional level.
  • Nucleic Acid-Based Diagnosis comprises a method for detecting variation in the expression of a tumor suppressor. This may comprise determining the level of a tumor suppressor or determining specific alterations in the expressed product.
  • Nucleic acid used is isolated from cancer cells according to standard methodologies (Sambrook et al, 1989). The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA. In one embodiment, the RNA is whole cell RNA; in another, it is poly-A RNA. Normally, the nucleic acid is amplified.
  • the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification.
  • the identified product is detected.
  • the detection may be performed by visual means ⁇ e.g., ethidium bromide staining of a gel).
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).
  • the amount of tumor suppressor expressed is measured by assessing the amount of an mRNA. However, by examining various types of structural defects, one can also identify alteration in activity.
  • Point mutations result in stop codons, frameshift mutations or amino acid substitutions.
  • Somatic mutations are those occurring in non- germline tissues and are not heritable, whereas germ-line tissue mutations can be inherited. Mutations in and outside the coding region also may affect the amount of a tumor suppressor produced, both by altering the transcription of the gene or in destabilizing or otherwise altering the processing of either the transcript (mRNA) or protein.
  • a cell takes a genetic step toward oncogenic transformation when one allele of a tumor suppressor gene is inactivated due to inheritance of a germline lesion or acquisition of a somatic mutation.
  • the inactivation of the other allele of the gene usually involves a somatic micromutation or chromosomal allelic deletion that results in loss of heterozygosity (LOH).
  • LHO heterozygosity
  • both copies of a tumor suppressor gene may be lost by homozygous deletion.
  • FISH fluorescent in situ hybridization
  • PFGE direct DNA sequencing
  • SSCA single-stranded conformation analysis
  • ASO allele-specif ⁇ c oligonucleotide
  • dot blot analysis denaturing gradient gel electrophoresis, RFLP and PCRTM-SSCP.
  • primer as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.
  • Probes are defined differently, although they may act as primers. Probes, while perhaps capable of priming, are designed to binding to the target DNA or RNA and need not be used in an amplification process.
  • the probes or primers are labeled with radioactive species ( P, 14 C, 35 S, 3 H, or other label), with a fluorophore (rhodamine, fluorescein) or a chemillumiscent (luciferase).
  • radioactive species P, 14 C, 35 S, 3 H, or other label
  • fluorophore rhodamine, fluorescein
  • chemillumiscent luciferase
  • PCRTM polymerase chain reaction
  • the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a reverse transcriptase PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989.
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641 filed December 21, 1990. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha- thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et ah, (1992).
  • Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • a similar method, called Repair Chain Reaction (RCR) involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present.
  • CPR cyclic probe reaction
  • a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
  • the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • primers are used in a PCRTM-like, template- and enzyme-dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference in their entirety).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR 3SR
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
  • the single stranded DNA is made fully double-stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • an RNA polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into single-stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al, PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and "one-sided PCRTM" (Frohman, 1990; Ohara et al, 1989; each herein incorporated by reference in their entirety).
  • Blotting techniques are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species.
  • a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose.
  • a suitable matrix often a filter of nitrocellulose.
  • the different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter.
  • the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will binding a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above. iv. Separation Methods
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods.
  • chromatographic techniques may be employed to effect separation.
  • chromatography There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography (Freifelder, 1982).
  • Products may be visualized in order to confirm amplification of the marker sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
  • detection is by a labeled probe.
  • the techniques involved are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al. (1989). For example, chromophore or radiolabel probes or primers identify the target during or following amplification.
  • amplification products described above may be subjected to sequence analysis to identify specific kinds of variations using standard sequence analysis techniques.
  • exhaustive analysis of genes is carried out by sequence analysis using primer sets designed for optimal sequencing (Pignon et al, 1994).
  • the present invention provides methods by which any or all of these types of analyses may be used.
  • oligonucleotide primers may be designed to permit the amplification of sequences that may then be analyzed by direct sequencing.
  • kits This generally will comprise preselected primers and probes. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, SequenaseTM etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • RT polymerases
  • Taq Taq
  • SequenaseTM deoxynucleotides
  • buffers to provide the necessary reaction mixture for amplification.
  • kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.
  • RT Reverse transcription
  • RT-PCRTM relative quantitative PCRTM
  • PCRTM the number of molecules of the amplified target DNA increase by a factor approaching two with every cycle of the reaction until some reagent becomes limiting. Thereafter, the rate of amplification becomes increasingly diminished until there is no increase in the amplified target between cycles.
  • a graph is plotted in which the cycle number is on the X axis and the log of the concentration of the amplified target DNA is on the Y axis, a curved line of characteristic shape is formed by connecting the plotted points. Beginning with the first cycle, the slope of the line is positive and constant. This is said to be the linear portion of the curve. After a reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero.
  • concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.
  • concentration of the target DNA in the linear portion of the PCRTM amplification is directly proportional to the starting concentration of the target before the reaction began.
  • concentration of the amplified products of the target DNA in PCRTM reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundances of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCRTM products and the relative mRNA abundances is only true in the linear range of the PCRTM reaction.
  • the final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the first condition that must be met before the relative abundances of a mRNA species can be determined by RT-PCRTM for a collection of RNA populations is that the concentrations of the amplified PCRTM products must be sampled when the PCRTM reactions are in the linear portion of their curves.
  • the second condition that must be met for an RT-PCRTM experiment to successfully determine the relative abundances of a particular mRNA species is that relative concentrations of the amplifiable cDNAs must be normalized to some independent standard.
  • the goal of an RT-PCRTM experiment is to determine the abundance of a particular mRNA species relative to the average abundance of all mRNA species in the sample.
  • RT-PCRTM assay for clinically derived materials.
  • the problems inherent in clinical samples are that they are of variable quantity (making normalization problematic), and that they are of variable quality (necessitating the co-amplification of a reliable internal control, preferably of larger size than the target). Both of these problems are overcome if the RT-PCRTM is performed as a relative quantitative RT-PCRTM with an internal standard in which the internal standard is an amplifiable cDNA fragment that is larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5-100 fold higher than the mRNA encoding the target.
  • This assay measures relative abundance, not absolute abundance of the respective mRNA species.
  • RT-PCRTM assays can be superior to those derived from the relative quantitative RT- PCRTM assay with an internal standard.
  • chip-based DNA technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al. (1994); Fodor et al. (1991). B. Immunodiagnostics
  • Antibodies of the present invention can be used in characterizing the tumor suppressor content of healthy and diseased tissues, through techniques such as ELISAs and Western blotting. This may provide a screen for the presence or absence of malignancy or as a predictor of future cancer or in the present case as a strategy to predict likely responses to exposure to an oncolytic virus.
  • anti-tumor suppressor antibodies are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a non-specific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of powdered milk.
  • BSA bovine serum albumin
  • casein casein
  • the immobilizing surface After binding of antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the sample to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for a tumor suppressor that differs the first antibody.
  • Appropriate conditions preferably include diluting the sample with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween ® . These added agents also tend to assist in the reduction of nonspecific background.
  • BSA bovine gamma globulin
  • PBS phosphate buffered saline
  • the layered antisera is then allowed to incubate for from about 2 to about 4 hr, at temperatures preferably on the order of about 25° to about 27 0 C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween ® , or borate buffer.
  • the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • one will desire to contact and incubate the second antibody-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hr at room temperature in a PB S -containing solution such as PBS/Tween ® ).
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl- benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl- benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label.
  • Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • the preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with specificity for the primary antibody.
  • the antibody compositions of the present invention will find great use in immunoblot or Western blot analysis.
  • the antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof.
  • a solid support matrix such as nitrocellulose, nylon or combinations thereof.
  • immunoprecipitation followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background.
  • Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the tumor suppressor are considered to be of particular use in this regard.
  • Hyperproliferative disorders are a family of diseases that are characterized by abnormal or otherwise unchecked cell proliferation. These disease generally fall into two categories: the more typical cancers, or "malignant" disorders, and the less prevalent benign hyperproliferative diseases, such as benign prostatic hyperplasia, benign epithelial hyerplasia of the breast, endometrial hyperplasia, thyroid hyperplasia and epithelial hyperplasia of the skin or dermis. Though many aspects of the following discussion focus on cancer therapy, where appropriate the same approach should be understood as applicable to benign disease as well.
  • cancer refers to the presence of dysplastic, neoplastic or otherwise inappropriately proliferating, cancerous and/or transformed cells in the subject, including, for example neoplastic, tumor, non-contact inhibited or oncogenically transformed cells, or the like (e.g., melanoma, carcinomas such as adenocarcinoma, squamous cell carcinoma, small cell carcinoma, oat cell carcinoma, etc., sarcomas such as fibrosarcoma, chondrosarcoma, osteosarcoma, etc., hepatoma, neuroblastoma, melanoma, hematopoietic malignancies such as lymphoma, leukemia, myeloma, etc.), which are known to the art and for which criteria for diagnosis and classification are established.
  • neoplastic e.g., melanoma, carcinomas such as adenocarcinoma, squamous cell carcinoma, small cell carcinoma, o
  • the oncolytic properties of reo viruses may derive from viral tropism for malignantly transformed cells in concert with a susceptible intracellular environment, for example, impaired PKR phosphorylation in Ras-activated cells as described in the art, or having defective tumor suppressor function, as now defined herein.
  • Myxoma virus therapy is described in U.S. Patent Publication 2006/0263333, also incorporated by reference.
  • the present invention provides, in certain embodiments, methods for purging cell populations of cancer cells.
  • individuals with hematopoietic or solid cancers that are treated by myeloablative therapy may subsequently receive hematopoietic stem cell rescue using purged bone marrow tissue that was harvested from the individual prior to chemotherapy.
  • the bone marrow may be treated with an oncolytic virus in order to remove or kill neoplastic cells while reducing or eliminating damage to hematopoietic stem cells.
  • a reovirus may be removed from a cellular composition, for example, after a period of time sufficient to allow for killing of a neoplastic cell (if present).
  • bone marrow may be taken from an individual and treated with a reovirus to destroy neoplastic cells; however, prior to re-implanting the bone marrow in the individual (e.g., an immunocompromised cancer patient) it may be desirable to destroy, eliminate and/or remove the virus from the composition.
  • an anti-viral agent may be added to the cellular composition. These agents include anti-reovirus antibodies and complements, inhibitors of essential viral enzymes such as RNA-directed RNA polymerase inhibitors, or agents that interfere with successful viral packaging, assembly or release from infected cells.
  • Additional purification steps including washing and/or centrifuging the cells may be employed to further remove virus and/or dead cells from a cellular composition.
  • These methods are known in the art and include methods that would enrich for desirable cell types, including fluorescence activated cell sorting or adhesion based methods that would enable positive selection of intended cells, such as stem cell populations.
  • an additional anti-cancer agent such as a chemotherapeutic agent, radiotherapy, immunotherapy, hormone therapy, toxin therapy, therapy with another natural or engineered virus, or gene therapy.
  • Chemotherapeutic agents include, but are not limited to, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP 16), farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative variant of the foregoing.
  • CDDP chlorambucil
  • cyclophosphamide cyclophosphamide
  • agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle.
  • an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations 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 agents, miscellaneous agents, and any analog or derivative variant thereof.
  • the oncolytic therapy may be concurrent with, follow or precede the application of other agent(s) to a cancer cell by intervals ranging from minutes to weeks. For example, in such instances, it is contemplated that one may contact a cancer cell, tissue, organ or organism, with multiple modalities substantially simultaneously (i.e., within less than about a minute) as the attenuated reovirus.
  • one or more agents may be administered within or from substantially simultaneously, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 2 hours
  • chemotherapeutic agents may be used in accordance with the present invention.
  • the term "chemotherapy” refers to the use of drugs to treat cancer.
  • a "chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer.
  • agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle.
  • an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.
  • Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific.
  • Alkylating agents can be implemented to treat chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, and particular cancers of the breast, lung, and ovary. They include: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan. Troglitazaone can be used to treat cancer in combination with any one or more of these alkylating agents, some of which are discussed below.
  • Busulfan (also known as myleran) is a bifunctional alkylating agent. Busulfan is known chemically as 1 ,4-butanediol dimethanesulfonate.
  • Busulfan is not a structural analog of the nitrogen mustards. Busulfan is available in tablet form for oral administration. Each scored tablet contains 2 mg busulfan and the inactive ingredients magnesium stearate and sodium chloride. Busulfan is indicated for the palliative treatment of chronic myelogenous (myeloid, myelocytic, granulocytic) leukemia. Although not curative, busulfan reduces the total granulocyte mass, relieves symptoms of the disease, and improves the clinical state of the patient. Approximately 90% of adults with previously untreated chronic myelogenous leukemia will obtain hematologic remission with regression or stabilization of organomegaly following the use of busulfan. It has been shown to be superior to splenic irradiation with respect to survival times and maintenance of hemoglobin levels, and to be equivalent to irradiation at controlling splenomegaly.
  • Chlorambucil (also known as leukeran) is a bifunctional alkylating agent of the nitrogen mustard type that has been found active against selected human neoplastic diseases. Chlorambucil is known chemically as 4-[bis(2-chlorethyl)amino] benzenebutanoic acid.
  • Chlorambucil is available in tablet form for oral administration. It is rapidly and completely absorbed from the gastrointestinal tract. After single oral doses of 0.6-1.2 mg/kg, peak plasma chlorambucil levels are reached within one hour and the terminal half-life of the parent drug is estimated at 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 antineoplastic treatment. Treatment regimes are well know to those of skill in the art and can be found in the "Physicians Desk Reference" and in "Remington's Pharmaceutical Sciences" referenced herein.
  • Chlorambucil is indicated in the treatment of chronic lymphatic (lymphocytic) leukemia, malignant lymphomas including lymphosarcoma, giant follicular lymphoma and
  • Hodgkin's disease It is not curative in any of these disorders but may produce clinically useful palliation. Thus, it can be used in combination with troglitazone in the treatment of cancer.
  • 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 can be used alone or in combination with other agents, with efficacious doses used in clinical applications of 15-20 mg/m 2 for 5 days every three weeks for a total of three courses. Exemplary doses may be 0.50 mg/m 2 , l.Omg/m 2 , 1.50 mg/m 2 , 1.75 mg/m 2 , 2.0 mg/m 2 , 3.0 mg/m 2 , 4.0 mg/m 2 , 5.0 mg/m 2 , 10mg//m 2 . Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
  • Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
  • Cyclophosphamide is 2H-l,3,2-Oxazaphosphorin-2-amine, NJV-bis(2- chloroethyl)tetrahydro-, 2-oxide, monohydrate; termed Cytoxan available from Mead Johnson; and Neosar available from Adria.
  • Cyclophosphamide is prepared by condensing 3- amino-1-propanol with iV r /V-bis(2-chlorethyl) phosphoramidic dichloride [(C1CH 2 CH 2 ) 2 N ⁇ POCl 2 ] in dioxane solution under the catalytic influence of triethylamine. The condensation is double, involving both the hydroxyl and the amino groups, thus effecting the cyclization.
  • the substance Unlike other ⁇ -chloroethylamino alkylators, it does not cyclize readily to the active ethyleneimonium form until activated by hepatic enzymes. Thus, the substance is stable in the gastrointestinal tract, tolerated well and effective by the oral and parental routes and does not cause local vesication, necrosis, phlebitis or even pain.
  • Suitable doses for adults include, orally, 1 to 5 mg/kg/day (usually in combination), depending upon gastrointestinal tolerance; or 1 to 2 mg/kg/day; intravenously, initially 40 to 50 mg/kg in divided doses over a period of 2 to 5 days or 10 to 15 mg/kg every 7 to 10 days or 3 to 5 mg/kg twice a week or 1.5 to 3 mg/kg/day .
  • a dose 250 mg/kg/day may be administered as an antineoplastic. Because of gastrointestinal adverse effects, the intravenous route is preferred for loading. During maintenance, a leukocyte count of 3000 to 4000 /mm 3 usually is desired. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.
  • Melphalan also known as alkeran, L-phenylalanine mustard, phenylalanine mustard, L-PAM, or L-sarcolysin, is a phenylalanine derivative of nitrogen mustard.
  • Melphalan is a bifunctional alkylating agent which is active against selective human neoplastic diseases. It is known chemically as 4-[bis(2-chloroethyl)amino]-L-phenylalanine.
  • Melphalan is the active L-isomer of the compound and was first synthesized in 1953 by Bergel and Stock; the D-isomer, known as medphalan, is less active against certain animal tumors, and the dose needed to produce effects on chromosomes is larger than that required with the L-isomer.
  • the racemic (DL-) form is known as merphalan or sarcolysin.
  • Melphalan is insoluble in water and has a pKaj of -2.1. Melphalan is available in tablet form for oral administration and has been used to treat multiple myeloma.
  • melphalan has been used in the treatment of epithelial ovarian carcinoma.
  • One commonly employed regimen for the treatment of ovarian carcinoma has been to administer melphalan at a dose of 0.2 mg/kg daily for five days as a single course. Courses are repeated every four to five weeks depending upon hematologic tolerance (Smith and Rutledge, 1975; Young et al, 1978).
  • the dose of melphalan used could be as low as 0.05mg/kg/day or as high as 3mg/kg/day or any dose in between these doses or above these doses. Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, they specifically influence the cell cycle during S phase. They have been used to combat chronic leukemias in addition to tumors of breast, ovary and the gastrointestinal tract. Antimetabolites include 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate. 5-Fluorouracil (5-FU) has the chemical name of 5-fluoro-2,4(lH,3H)- pyrimidinedione.
  • 5-FU interferes with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibits the formation of ribonucleic acid (RNA). Since DNA and RNA are essential for cell division and proliferation, it is thought that the effect of 5-FU is to create a thymidine deficiency leading to cell death. Thus, the effect of 5-FU is found in cells that rapidly divide, a characteristic of metastatic cancers.
  • Antitumor Antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific so they work in all phases of the cell cycle. Thus, they are widely used for a variety of cancers. Examples of antitumor antibiotics include bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), and idarubicin, some of which are discussed in more detail below.
  • these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m 2 at 21 day intervals for adriamycin, to 35- 100 mg/m 2 for etoposide intravenously or orally.
  • Doxorubicin hydrochloride 5,12-Naphthacenedione, (8s-c ⁇ )-10-[(3-amino-2,3,6- trideoxy-a-L-lyxo-hexopyranosyl)oxy]-7,8,9, 10-tetrahydro-6,8, 11 -trihydroxy-8- (hydroxyacetyl)-l-methoxy-hydrochloride (hydroxydaunorubicin hydrochloride, Adriamycin) is used in a wide antineoplastic spectrum. It binds to DNA and inhibits nucleic acid synthesis, inhibits mitosis and promotes chromosomal aberrations.
  • Administered alone it is the drug of first choice for the treatment of thyroid adenoma and primary hepatocellular carcinoma. It is a component of 31 first-choice combinations for the treatment of ovarian, endometrial and breast tumors, bronchogenic oat-cell carcinoma, non-small cell lung carcinoma, gastric adenocarcinoma, retinoblastoma, neuroblastoma, mycosis fungoides, pancreatic carcinoma, prostatic carcinoma, bladder carcinoma, myeloma, diffuse histiocytic lymphoma, Wilms' tumor, Hodgkin's disease, adrenal tumors, osteogenic sarcoma soft tissue sarcoma, Ewing's sarcoma, rhabdomyosarcoma and acute lymphocytic leukemia. It is an alternative drug for the treatment of islet cell, cervical, testicular and adrenocortical cancers. It is also an immunosuppressant.
  • Doxorubicin is absorbed poorly and must be administered intravenously.
  • the pharmacokinetics are multicompartmental. Distribution phases have half-lives of 12 minutes and 3.3 hr. The elimination half-life is about 30 hr. Forty to 50% is secreted into the bile. Most of the remainder is metabolized in the liver, partly to an active metabolite (doxorubicinol), but a few percent is excreted into the urine. In the presence of liver impairment, the dose should be reduced.
  • Appropriate doses are, intravenous, adult, 60 to 75 mg/m 2 at 21 -day intervals or 25 to 30 mg/m 2 on each of 2 or 3 successive days repeated at 3- or 4-wk intervals or 20 mg/m 2 once a week.
  • the lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.
  • the dose should be reduced by 50% if the serum bilirubin lies between 1.2 and 3 mg/dL and by 75% if above 3 mg/dL.
  • the lifetime total dose should not exceed 550 mg/m 2 in patients with normal heart function and 400 mg/m 2 in persons having received mediastinal irradiation. Alternatively, 30 mg/m 2 on each of 3 consecutive days, repeated every 4 wk.
  • Exemplary doses may be 10 mg/m 2 , 20 mg/m 2 , 30 mg/m 2 , 50 mg/m 2 , 100 mg/m 2 , 150 mg/m 2 , 175 mg/m 2 , 200 mg/m 2 , 225 mg/m 2 , 250 mg/m 2 , 275 mg/m 2 , 300 mg/m 2 , 350 mg/m 2 , 400 mg/m 2 , 425 mg/m 2 , 450 mg/m 2 , 475 mg/m 2 , 500 mg/m .
  • all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
  • Daunorubicin hydrochloride 5,12-Naphthacenedione, (8S-m)-8-acetyl-10-[(3-amino- 2,3 ,6-trideoxy-a-L-lyxo-hexanopyranosyl)oxy]-7,8,9, 10-tetrahydro-6,8, 11 -trihydroxy- 10- methoxy-, hydrochloride; also termed cerubidine and available from Wyeth.
  • Daunorubicin intercalates into DNA, blocks DAN-directed RNA polymerase and inhibits DNA synthesis. It can prevent cell division in doses that do not interfere with nucleic acid synthesis.
  • Exemplary doses may be 10 mg/m 2 , 20 mg/m 2 , 30 mg/m 2 , 50 mg/m 2 , 100 mg/m 2 , 150 mg/m 2 , 175 mg/m 2 , 200 mg/m 2 , 225 mg/m 2 , 250 mg/m 2 , 275 mg/m 2 , 300 mg/m 2 , 350 mg/m 2 , 400 mg/m 2 , 425 mg/m 2 , 450 mg/m 2 , 475 mg/m 2 , 500 mg/m 2 .
  • all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
  • Mitomycin also known as mutamycin and/or mitomycin-C
  • mutamycin and/or mitomycin-C is an antibiotic isolated from the broth of Streptomyces caespitosus which has been shown to have antitumor activity.
  • the compound is heat stable, has a high melting point, and is freely soluble in organic solvents.
  • Mitomycin selectively inhibits the synthesis of deoxyribonucleic acid (DNA).
  • DNA deoxyribonucleic acid
  • the guanine and cytosine content correlates with the degree of mitomycin-induced cross-linking. At high concentrations of the drug, cellular RNA and protein synthesis are also suppressed.
  • mitomycin is rapidly cleared from the serum after intravenous administration. Time required to reduce the serum concentration by 50% after a 30 mg. bolus injection is 17 minutes. After injection of 30 mg, 20 mg, or 10 mg I. V., the maximal serum concentrations were 2.4 mg/ml, 1.7 mg/ml, and 0.52 mg/ml, respectively. Clearance is effected primarily by metabolism in the liver, but metabolism occurs in other tissues as well. The rate of clearance is inversely proportional to the maximal serum concentration because, it is thought, of saturation of the degradative pathways. Approximately 10% of a dose of mitomycin is excreted unchanged in the urine. Since metabolic pathways are saturated at relatively low doses, the percent of a dose excreted in urine increases with increasing dose. In children, excretion of intravenously administered mitomycin is similar.
  • Actinomycin D (Dactinomycin) [50-76-0]; C 62 H 86 N 12 O 16 (1255.43) is an antineoplastic drug that inhibits DNA-dependent RNA polymerase. It is a component of first- choice combinations for treatment of choriocarcinoma, embryonal rhabdomyosarcoma, testicular tumor and Wilms' tumor. Tumors that fail to respond to systemic treatment sometimes respond to local perfusion. Dactinomycin potentiates radiotherapy. It is a secondary (efferent) immunosuppressive. Actinomycin D is used in combination with primary surgery, radiotherapy, and other drugs, particularly vincristine and cyclophosphamide.
  • Dactinomycin can be effective in women with advanced cases of choriocarcinoma. It also produces consistent responses in combination with chlorambucil and methotrexate in patients with metastatic testicular carcinomas. A response may sometimes be observed in patients with Hodgkin's disease and non-Hodgkin's lymphomas. Dactinomycin has also been used to inhibit immunological responses, particularly the rejection of renal transplants.
  • Half of the dose is excreted intact into the bile and 10% into the urine; the half-life is about 36 hr.
  • the drug does not pass the blood-brain barrier.
  • Actinomycin D is supplied as a lyophilized powder (0/5 mg in each vial).
  • the usual daily dose is 10 to 15 mg/kg; this is given intravenously for 5 days; if no manifestations of toxicity are encountered, additional courses may be given at intervals of 3 to 4 weeks.
  • Daily injections of 100 to 400 mg have been given to children for 10 to 14 days; in other regimens, 3 to 6 mg/kg, for a total of 125 mg/kg, and weekly maintenance doses of 7.5 mg/kg have been used.
  • Exemplary doses may be 100 mg/m 2 , 150 mg/m 2 , 175 mg/m 2 , 200 mg/m 2 , 225 mg/m 2 , 250 mg/m 2 , 275 mg/m 2 , 300 mg/m 2 , 350 mg/m 2 , 400 mg/m 2 , 425 mg/m 2 , 450 mg/m 2 , 475 mg/m 2 , 500 mg/m 2 .
  • All of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
  • Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus. Although the exact mechanism of action of bleomycin is unknown, available evidence would seem to indicate that the main mode of action is the inhibition of DNA synthesis with some evidence of lesser inhibition of RNA and protein synthesis.
  • mice high concentrations of bleomycin are found in the skin, lungs, kidneys, peritoneum, and lymphatics. Tumor cells of the skin and lungs have been found to have high concentrations of bleomycin in contrast to the low concentrations found in hematopoietic tissue.
  • the low concentrations of bleomycin found in bone marrow may be related to high levels of bleomycin degradative enzymes found in that tissue.
  • the serum or plasma terminal elimination half-life of bleomycin is approximately 115 minutes, hi patients with a creatinine clearance of ⁇ 35 ml per minute, the plasma or serum terminal elimination half-life increases exponentially as the creatinine clearance decreases.
  • 60% to 70% of an administered dose is recovered in the urine as active bleomycin.
  • Bleomycin may be given by the intramuscular, intravenous, or subcutaneous routes. It is freely soluble in water.
  • Bleomycin should be considered a palliative treatment. It has been shown to be useful in the management of the following neoplasms either as a single agent or in proven combinations with other approved chemotherapeutic agents in squamous cell carcinoma such as head and neck (including mouth, tongue, tonsil, nasopharynx, oropharynx, sinus, palate, lip, buccal mucosa, gingiva, epiglottis, larynx), skin, penis, cervix, and vulva. It has also been used in the treatment of lymphomas and testicular carcinoma. Because of the possibility of an anaphylactoid reaction, lymphoma patients should be treated with two units or less for the first two doses. If no acute reaction occurs, then the regular dosage schedule may be followed.
  • head and neck including mouth, tongue, tonsil, nasopharynx, oropharynx, sinus, palate, lip, buccal mucosa, gingiva, epiglottis, larynx
  • Mitotic Inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors comprise docetaxel, etoposide (VP 16), paclitaxel, taxol, taxotere, vinblastine, vincristine, and vinorelbine.
  • VP 16 is also known as etoposide and is used primarily for treatment of testicular tumors, in combination with bleomycin and cisplatin, and in combination with cisplatin for small-cell carcinoma of the lung. It is also active against non-Hodgkin's lymphomas, acute nonlymphocytic leukemia, carcinoma of the breast, and Kaposi's sarcoma associated with acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • VP 16 is available as a solution (20 mg/ml) for intravenous administration and as 50- mg, liquid-filled capsules for oral use.
  • the intravenous dose in combination therapy
  • the intravenous dose is can be as much as 100 mg/m 2 or as little as 2 mg/m 2 , routinely 35 mg/m 2 , daily for 4 days, to 50 mg/m 2 , daily for 5 days have also been used.
  • the dose should be doubled.
  • the doses for small cell lung carcinoma may be as high as 200-250 mg/m .
  • the intravenous dose for testicular cancer (in combination therapy) is 50 to 100 mg/m 2 daily for 5 days, or 100 mg/m 2 on alternate days, for three doses. Cycles of therapy are usually repeated every 3 to 4 weeks.
  • the drug should be administered slowly during a 30- to 60-minute infusion in order to avoid hypotension and bronchospasm, which are probably due to the solvents used in the formulation.
  • Taxol is an experimental antimitotic agent, isolated from the bark of the ash tree, Taxus brevifolia. It binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules. Taxol is currently being evaluated clinically; it has activity against malignant melanoma and carcinoma of the ovary. Maximal doses are 30 mg/m per day for 5 days or 210 to 250 mg/m given once every 3 weeks. Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
  • Vinblastine is another example of a plant aklyloid that can be used in combination with troglitazone for the treatment of cancer and precancer. When cells are incubated with vinblastine, dissolution of the microtubules occurs.
  • vinblastine After intravenous injection, vinblastine has a multiphasic pattern of clearance from the plasma; after distribution, drag disappears from plasma with half-lives of approximately 1 and 20 hours. Vinblastine is metabolized in the liver to biologically activate derivative desacetylvinblastine. Approximately 15% of an administered dose is detected intact in the urine, and about 10% is recovered in the feces after biliary excretion. Doses should be reduced in patients with hepatic dysfunction. At least a 50% reduction in dosage is indicated if the concentration of bilirubin in plasma is greater than 3 mg/dl (about 50 mM). Vinblastine sulfate is available in preparations for injection.
  • the drug is given intravenously; special precautions must be taken against subcutaneous extravasation, since this may cause painful irritation and ulceration.
  • the drug should not be injected into an extremity with impaired circulation. After a single dose of 0.3 mg/kg of body weight, myelosuppression reaches its maximum in 7 to 10 days. If a moderate level of leukopenia (approximately 3000 cells/mm 3 ) is not attained, the weekly dose may be increased gradually by increments of 0.05 mg/kg of body weight. In regimens designed to cure testicular cancer, vinblastine is used in doses of 0.3 mg/kg every 3 weeks irrespective of blood cell counts or toxicity.
  • vinblastine The most important clinical use of vinblastine is with bleomycin and cisplatin in the curative therapy of metastatic testicular tumors. Beneficial responses have been reported in various lymphomas, particularly Hodgkin's disease, where significant improvement may be noted in 50 to 90% of cases.
  • the effectiveness of vinblastine in a high proportion of lymphomas is not diminished when the disease is refractory to alkylating agents. It is also active in Kaposi's sarcoma, neuroblastoma, and Letterer-Siwe disease (histiocytosis X), as well as in carcinoma of the breast and choriocarcinoma in women.
  • 0.1 to 0.3 mg/kg can be administered or 1.5 to 2 mg/m 2 can also be administered.
  • Vinblastine and vincristine bind to plasma proteins. They are extensively concentrated in platelets and to a lesser extent in leukocytes and erythrocytes.
  • Vincristine has a multiphasic pattern of clearance from the plasma; the terminal half- life is about 24 hours.
  • the drug is metabolized in the liver, but no biologically active derivatives have been identified. Doses should be reduced in patients with hepatic dysfunction. At least a 50% reduction in dosage is indicated if the concentration of bilirubin in plasma is greater than 3 mg/dl (about 50 niM).
  • Vincristine sulfate is available as a solution (1 mg/ml) for intravenous injection. Vincristine used together with corticosteroids is presently the treatment of choice to induce remissions in childhood leukemia; the optimal dosages for these drugs appear to be vincristine, intravenously, 2 mg/m 2 of body-surface area, weekly, and prednisone, orally, 40 mg/m 2 , daily.
  • Adult patients with Hodgkin's disease or non-Hodgkin's lymphomas usually receive vincristine as a part of a complex protocol. When used in the MOPP regimen, the recommended dose of vincristine is 1.4 mg/m 2 .
  • Vincristine (and vinblastine) can be infused into the arterial blood supply of tumors in doses several times larger than those that can be administered intravenously with comparable toxicity. Vincristine has been effective in Hodgkin's disease and other lymphomas.
  • vincristine is an important agent, particularly when used with cyclophosphamide, bleomycin, doxorubicin, and prednisone. Vincristine is more useful than vinblastine in lymphocytic leukemia.
  • neoplasms particularly Wilms' tumor, neuroblastoma, brain tumors, rhabdomyosarcoma, and carcinomas of the breast, bladder, and the male and female reproductive systems.
  • Doses of vincristine for use will be determined by the clinician according to the individual patients need. 0.01 to 0.03 mg/kg or 0.4 to 1.4 mg/m 2 can be administered or 1.5 to 2 mg/m 2 can also be administered.
  • 0.02 mg/m 2 , 0.05 mg/m 2 , 0.06 mg/m 2 , 0.07 mg/m 2 , 0.08 mg/m 2 , 0.1 mg/m 2 , 0.12 mg/m 2 , 0.14 mg/m 2 , 0.15 mg/m 2 , 0.2 mg/m 2 , 0.25 mg/m can be given as a constant intravenous infusion.
  • all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
  • Camptothecin is an alkaloid derived from the Chinese tree Camptotheca acuminata Decne. Camptothecin and its derivatives are unique in their ability to inhibit DNA Topoisomerase by stabilizing a covalent reaction intermediate, termed "the cleavable complex," which ultimately causes tumor cell death. It is widely believed that camptothecin analogs exhibited remarkable anti-rumour and anti-leukaemia activity. Application of camptothecin in clinic is limited due to serious side effects and poor water-solubility. At present, some camptothecin analogs (topotecan; irinotecan), either synthetic or semi- synthetic, have been applied to cancer therapy and have shown satisfactory clinical effects.
  • camptothecin analogs topotecan; irinotecan
  • the molecular formula for camptothecin is C 20 H 16 N 2 O 4 , with a molecular weight of 348.36. It is provided as a yellow powder, and may be solubilized to a clear yellow solution at 50 mg/ml in DMSO IN sodium hydroxide. It is stable for at least two years if stored at 2-8 0 X in a dry, airtight, light-resistant environment. Nitrosureas. Nitrosureas, like alkylating agents, inhibit DNA repair proteins. They are used to treat non-Hodgkin's lymphomas, multiple myeloma, malignant melanoma, in addition to brain tumors. Examples include carmustine and lomustine.
  • Carmustine (sterile carmustine) is one of the nitrosoureas used in the treatment of certain neoplastic diseases. It is l,3bis (2-chloroethyl)-l -nitrosourea. It is lyophilized pale yellow flakes or congealed mass with a molecular weight of 214.06. It is highly soluble in alcohol and lipids, and poorly soluble in water. Carmustine is administered by intravenous infusion after reconstitution as recommended. Sterile carmustine is commonly available in 100 mg single dose vials of lyophilized material.
  • carmustine alkylates DNA and RNA it is not cross resistant with other alkylators. As with other nitrosoureas, it may also inhibit several key enzymatic processes by carbamoylation of amino acids in proteins.
  • Carmustine is indicated as palliative therapy as a single agent or in established combination therapy with other approved chemotherapeutic agents in brain tumors such as glioblastoma, brainstem glioma, medullobladyoma, astrocytoma, ependymoma, and metastatic brain tumors. Also it has been used in combination with prednisone to treat multiple myeloma. Carmustine has proved useful, in the treatment of Hodgkin's Disease and in non-Hodgkin's lymphomas, as secondary therapy in combination with other approved drugs in patients who relapse while being treated with primary therapy, or who fail to respond to primary therapy.
  • the recommended dose of carmustine as a single agent in previously untreated patients is 150 to 200 mg/m intravenously every 6 weeks. This may be given as a single dose or divided into daily injections such as 75 to 100 mg/m on 2 successive days.
  • the doses should be adjusted accordingly. Doses subsequent to the initial dose should be adjusted according to the hematologic response of the patient to the preceding dose. It is of course understood that other doses may be used in the present
  • 9 O O 9 0 0 invention for example 10 mg/m , 20 mg/m , 30 mg/m , 40 mg/m , 50 mg/m , 60 mg/m , 70 mg/m 2 , 80 mg/m 2 , 90 mg/m 2 or 100 mg/m 2 .
  • the skilled artisan is directed to "Remington's Pharmaceutical Sciences," 15th Edition, Chapter 61. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • Lomustine is one of the nitrosoureas used in the treatment of certain neoplastic diseases. It is l-(2-chloro-ethyl)-3-cyclohexyl-l nitrosourea. It is a yellow powder with the empirical formula Of CgHi 6 ClN 3 O 2 and a molecular weight of 233.71. Lomustine is soluble in 10% ethanol (0.05 mg per mL) and in absolute alcohol (70 mg per ml). Lomustine is relatively insoluble in water ( ⁇ 0.05 mg per ml). It is relatively unionized at a physiological pH. Inactive ingredients in lomustine capsules are: magnesium stearate and mannitol.
  • lomustine alkylates DNA and RNA it is not cross resistant with other alkylators. As with other nitrosoureas, it may also inhibit several key enzymatic processes by carbamoylation of amino acids in proteins.
  • Lomustine may be given orally. Following oral administration of radioactive lomustine at doses ranging from 30 mg/m 2 to 100 mg/m 2 , about half of the radioactivity given was excreted in the form of degradation products within 24 hours. The serum half-life of the metabolites ranges from 16 hours to 2 days. Tissue levels are comparable to plasma levels at 15 minutes after intravenous administration.
  • Lomustine has been shown to be useful as a single agent in addition to other treatment modalities, or in established combination therapy with other approved chemotherapeutic agents in both primary and metastatic brain tumors, in patients who have already received appropriate surgical and/or radiotherapeutic procedures. It has also proved effective in secondary therapy against Hodgkin's Disease in combination with other approved drugs in patients who relapse while being treated with primary therapy, or who fail to respond to primary therapy.
  • the recommended dose of lomustine in adults and children as a single agent in previously untreated patients is 130 mg/m as a single oral dose every 6 weeks. In individuals with compromised bone marrow function, the dose should be reduced to 100 mg/m every 6 weeks. When lomustine is used in combination with other myelosuppressive drugs, the doses should be adjusted accordingly. It is understood that other doses may be used for example, 20 mg/m 2 30 mg/m 2 , 40 mg/m 2 , 50 mg/m 2 , 60 mg/m 2 , 70 mg/m 2 , 80 mg/m 2 , 90 mg/m 2 , 100 mg/m , 120 mg/m or any doses between these figures as determined by the clinician to be necessary for the individual being treated.
  • agents that may be used include Avastin, Iressa, Erbitux, Velcade, and. Gleevec.
  • growth factor inhibitors and small molecule kinase inhibitors have utility in the present invention as well. All therapies described in Cancer:
  • Immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • Immunotherapy could be used as part of a combined therapy, in conjunction with reoviral or other oncolytic viral therapy.
  • the general approach for combined therapy is discussed below.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
  • tumor cells or other hyperproliferative cells infected with reovirus or another oncolytic virus may express viral antigens on the cell surface, thereby also rendering them 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 the production of collagen and collagenases, is an inflammatory mediator and also a mediator of septic shock, and promotes catabolism, fever and sleep. Some infectious agents cause tumor regression through the stimulation of TNF production. TNF can be quite toxic when used alone in effective doses, so that the optimal regimens probably will use it in lower doses in combination with other drugs. Its immunosuppressive actions are potentiated by gamma- interferon, so that the combination potentially is dangerous. A hybrid of TNF and interferon- ⁇ also has been found to possess anti-cancer activity.
  • sex hormones according to the methods described herein in the treatment of cancer. While the methods described herein are not limited to the treatment of a specific cancer, this use of hormones has benefits with respect to cancers of the breast, prostate, and endometrial (lining of the uterus). Examples of these hormones are estrogens, anti-estrogens, progesterones, and androgens.
  • Corticosteroid hormones are useful in treating some types of cancer (lymphoma, leukemias, and multiple myeloma). Corticosteroid hormones can increase the effectiveness of other chemotherapy agents, and consequently, they are frequently used in combination treatments. Prednisone and dexamethasone are examples of corticosteroid hormones.
  • Radiotherapy also called radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly. Radiotherapy may be used to treat localized solid tumors, such as cancers of the skin, tongue, larynx, brain, breast, or cervix. It can also be used to treat leukemia and lymphoma (cancers of the blood-forming cells and lymphatic system, respectively).
  • Radiation therapy used according to the present invention may include, but is not limited to, the use of ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • Radiotherapy may comprise the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmuno therapy).
  • Antibodies are highly specific proteins that are made by the body in response to the presence of antigens (substances recognized as foreign by the immune system). Some tumor cells contain specific antigens that trigger the production of rumor-specific antibodies. Large quantities of these antibodies can be made in the laboratory and attached to radioactive substances (a process known as radiolabeling). Once injected into the body, the antibodies actively seek out the cancer cells, which are destroyed by the cell-killing (cytotoxic) action of the radiation. This approach can minimize the risk of radiation damage to healthy cells.
  • Conformal radiotherapy uses the same radiotherapy machine, a linear accelerator, as the normal radiotherapy treatment but metal blocks are placed in the path of the x-ray beam to alter its shape to match that of the cancer. This ensures that a higher radiation dose is given to the tumor. Healthy surrounding cells and nearby structures receive a lower dose of radiation, so the possibility of side effects is reduced.
  • a device called a multi-leaf collimator has been developed and can be used as an alternative to the metal blocks.
  • the multi-leaf collimator consists of a number of metal sheets which are fixed to the linear accelerator. Each layer can be adjusted so that the radiotherapy beams can be shaped to the treatment area without the need for metal blocks. Precise positioning of the radiotherapy machine is very important for conformal radiotherapy treatment and a special scanning machine may be used to check the position of your internal organs at the beginning of each treatment.
  • High-resolution intensity modulated radiotherapy also uses a multi-leaf collimator. During this treatment the layers of the multi-leaf collimator are moved while the treatment is being given. This method is likely to achieve even more precise shaping of the treatment beams and allows the dose of radiotherapy to be constant over the whole treatment area.
  • Radiotherapy is used to treat brain tumours. This technique directs the radiotherapy from many different angles so that the dose going to the tumour is very high and the dose affecting surrounding healthy tissue is very low.
  • scans are analysed by computers to ensure that the radiotherapy is precisely targeted, and the patient's head is held still in a specially made frame while receiving radiotherapy. Several doses are given.
  • Stereotactic radio-surgery for brain tumors does not use a knife, but very precisely targeted beams of gamma radiotherapy from hundreds of different angles. Only one session of radiotherapy, taking about four to five hours, is needed. For this treatment you will have a specially made metal frame attached to your head. Then several scans and x- rays are carried out to find the precise area where the treatment is needed. During the radiotherapy, the patient lies with their head in a large helmet, which has hundreds of holes in it to allow the radiotherapy beams through.
  • scientists also are looking for ways to increase the effectiveness of radiation therapy.
  • Radiosensitizers make the tumor cells more likely to be damaged, and radioprotectors protect normal tissues from the effects of radiation.
  • Hyperthermia the use of heat, is also being studied for its effectiveness in sensitizing tissue to radiation.
  • Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy.
  • Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
  • These treatments may be of varying dosages as well.
  • p53 "A MEF, BT, and ATM deficient L3 cells, plus human lymphoma HBL2, Granta, Z138C, Raji, Ramos, and JVM2 cells, plus retinoblastoma cell lines (Y -19 and WERI-Rb-I) were purchased from the American Type Culture Collection. The cells were maintained in RPMI 1640 in 10% FBS.
  • Wild-type and attenuated Reovirus preparation The wild-type reovirus T3D strain used in these studies was propagated in L929 cells and purified as described previously. Attenuated reovirus derived from the HTRl culture was purified by the same method used in wild-type reovirus preparation except AV reovirus was propagated in HTl 080 and L929 cells. Reovirus was added to cells at a MOI of 5-10 and these were maintained at 37°C for 48-72 hrs. After viral cytopathic effects were observed (typically 20-30% cell lysis), virus was purified from pelleted cells. CsCl centrifugation of virus was performed using an SW41 rotor at 35,000 rpm for 7-8 hrs.
  • HEK 293 cells were plated in 6-well plates at 2 x 10 5 cells per well. After 2 hr of adsorption at
  • reovirus titration For attenuated reovirus titration, the same procedure was followed except after 5-7 days of infection, the agar was removed and cell monolayers were fixed/permeabilzed with cytofix/cytoperm (BD Bioscience) for immunostaining with reovirus antiserum and secondary
  • AV reovirus plaques were identified by immunofluorescence detection.
  • Attenuated reovirus a standard plaque assay was also performed on L929 cells. Three days following infection, the agar was overlayed with neutral red, and the cell monolayers were examined for plaque formation 24 hrs later. For attenuated reovirus collection from HTl 080 and HTRl infected supernatants, high speed ultracentrifugation at HTl 080 and HTRl infected supernatants, high speed ultracentrifugation at
  • Myxoma virus preparation Myx-GFP of myxoma virus (strain Lausanne), created by intergenic insertion of a green fluorescent protein (GFP) cassette driven by a synthetic vaccinia virus early/late promoter, was used for infection studies. Myx-GFP was propagated and titrated by focus formation on BGMK cells as described previously
  • FACS analysis For flow cytometry analysis, cells were trypsinized and fixed using cytofix/cytoperm solution (PharMingen, San Diego, CA). The fixed and permeabilized cells were incubated with primary reovirus antiserum and secondary FITC conjugated anti-rabbit
  • IgG (Cedarlane, Ontario), then analyzed by flow cytometry.
  • Reovirus and myxoma virus preferentially infect p53 or ATM deficient cells.
  • p53 and ATM tumor suppressor genes that are frequently mutated in various cancers.
  • p53 is the prototypical tumor suppressor gene and most commonly mutated in various cancer cells, and it has been shown that over 50% of cancers retain p53 mutations (White, 1994; Morris, 2002).
  • ATM Alaxia telangiectasia mutated
  • IA-B, reoviruses (WT and AV reovirus; Kim et al, 2007) and myxoma virus each preferentially infected p53 -/- MEF or L3 (ATM-deficient) cells as shown by FACS or microscopic detection of fluorescence positive cells (FITC+ or GFP+ cells) compared to the MEF (p53 normal) and BT (ATM normal) control cells.
  • Reovirus and myxoma virus preferentially infect p53 or ATM dysfunctional human lymphomas.
  • reovirus and myxoma virus preferentially infect p53 or ATM- deficient cells
  • the inventors then examined whether reovirus and myxoma virus can preferentially infect human lymphomas that carry p53 or ATM deficiencies.
  • the inventors tested 6 different lymphomas (HB L-2, Granta, Z138C, JVM2, Raji and Ramos) that exhibit variable cellular responses arising from p53 and ATM-dependent pathways upon genotoxic challenge (FIG. 2A).
  • the functional status of p53 and ATM was evaluated by IR irradiation and detection of p53 and ATM phosphorylation by western blotting.
  • BT ATM-normal
  • L3 ATM-deficient
  • HBL-2 and Raji demonstrated a dysfunctional p53 response upon genotoxic stress as evidenced by the constitutive phosphorylation of serine-15 of p53 (Li et al, 2006) (FIG. 2A).
  • Granta cells exhibited ATM dysfunctional response upon genotoxic stress and ATM is not activated up ionizing radiation (IR) stimuli (FIG. 2A).
  • IR ionizing radiation
  • p53 and ATM responses were normal following genotoxc stress as shown by hyper- phosphorylation of p53 and ATM by IR stimuli (FIG. 2A).
  • Retinoblastoma cells are sensitive to reovirus and myxoma virus infection. Because reovirus and myxoma virus preferentially infect p53 and ATM dysfunctional cancer cells, the inventors also examined whether RB defective cancer cells are also jointly susceptible to reovirus and myxoma virus infection. As shown in FIG. 3, two different human retinoblastoma cells are susceptible to reoviruses and myxoma virus infection as shown by FACS or microscopic detection of fluorescence positive cells (FITC+ or GFP+ cells). Both cells contain deficiencies of the Rb gene (Reid et al., 191 A)

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Abstract

La présente invention concerne des procédés pour le traitement du cancer à l'aide de virus oncolytiques tels que des réovirus et un virus myxome. En particulier, l'invention s'appuie sur l'observation selon laquelle des cellules cancéreuses ayant des aberrations dans un suppresseur de tumeur sont davantage prédisposées à l'infection avec des virus oncolytiques que celles qui contiennent des suppresseurs de tumeur normaux ou du "type sauvage". En particulier, les défauts associés à p53, Rb et ATM rendent chacun les cellules cancéreuses prédisposées à une thérapie virale oncolytique.
PCT/US2009/045048 2008-05-22 2009-05-22 Prédisposition par suppresseurs de tumeur de cellules hyperproliférantes à une thérapie virale oncolytique WO2009143468A1 (fr)

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KR102401077B1 (ko) * 2019-01-25 2022-05-24 바이로큐어 주식회사 다람쥐 섬유종 바이러스 및 리오바이러스를 포함하는 암 예방 또는 치료용 약학적 조성물
KR20220090452A (ko) 2020-12-22 2022-06-29 바이로큐어 주식회사 신규한 재조합 믹소마바이러스 및 이의 용도
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