CN102124335A - Tumor suppressor-based susceptibility of hyperproliferative cells to oncolytic viral therapy - Google Patents

Abstract

The present invention relates to methods for the treatment of cancer using oncolytic viruses such as reoviruses and myxoma virus. In particular, the invention relies on the observation that cancer cells with aberrations in a tumor suppressor are more susceptible to infection with oncolytic viruses than those that contain normal or ''wild-type'' tumor suppressors. In particular, defects associated with p53, Rb and ATM each render cancer cells susceptible to oncolytic viral therapy.

Description

Based on the hyperplasia cell of TIF susceptibility to the oncolytic virus therapy

Background of invention

The application requires to enjoy in the right of priority of the U.S. Provisional Patent Application submitted on May 22nd, 2008 number 61/055,360, and its full content is hereby incorporated by.

Technical field

The present invention relates generally to virology, molecular biology and medical domain.More particularly, it relates to the disorder of mensuration hyperplasia and comprises the susceptibility of cancer for the oncolytic virus therapy.

Background technology

Reovirus (respiratory enteric orphan virus) is a kind of ubiquitous nonenveloped virus, comprise as its genomic 10 double-stranded RNA fragments, it is normally gentle to the mankind's infection, be confined to the upper respiratory tract and intestines and stomach and be asymptomatic (Tyler, 2001) in the host of immunologic function is arranged usually.Fail about reversing the trial major part of transforming reovirus, this comprises the double-stranded RNA genome of reovirus owing to a plurality of factors.The reovirus particle of known disappearance σ 1 is FFI (Larson et al, 1994).

Importantly, reovirus is acknowledged as can presents significant cell ability (Duncan et al, 1978 of killing when it infects the transformant of particular type for many years; Hashiro et al, 1977).There is the oncolytic virus of replication capacity that noticeable anti-cancer therapies is provided.These oncolytic viruses have two major advantages.The first, different with traditional chemotherapy and radiotherapy, so because limited their energy specificity grappling cancer cells of their replication capacities in normal cell.The second, compare with the carrier of no replication capacity, the cancer cell around they can spread to from the cancer cell of initial infection, thus reach a large amount of distributions and anticancer effectively effect.

Though it is normally asymptomatic for the individuality of health to be exposed to the reovirus of wild type, but for the individuality of immunodeficiency, still can there be very big potential very serious risk, this has just limited the clinical application potential of reovirus therapy to patient, as for the cancer patient that may benefit from this therapy.Until finding to comprise the transformant of carcinogenic Ras signal path to infecting reovirus (3 type Dearing strain) especially responsive (Coffey et al, 1998 in vitro and in vivo; Strong et al, 1998; Norman et al, 2004) before, it is unknown that the ultimate principle of reovirus oncolytic ability remains.Owing to can observe Ras gene mutation (Duursma et al, 2003) usually in various human cancers, these discoveries cause the use (Norman et al, 2005) of current reovirus therapy in clinical testing.

Yet the existence of Ras gene mutation may be the partly cause of cancer cell to reovirus therapy susceptibility.Recent studies have shown that the Ras path is not unique factor (Song et al that decision allows reovirus, 2009), and demonstration can be set up the cell (Kim et al, 2007) of anti-reovirus and still keep the Ras sudden change that activates from the cell that transforms Ras.Therefore, needing improved method differentiates and screens suitable cancer cell sub-group and be used for reovirus therapy and other oncolytic virus reagent.

Summary of the invention

Summary of the invention

When the cancer cell of hyperplasia that contains specific tumor suppressor gene damaged or inactivation such as p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC and ATBF1 or conversion is exposed to reovirus in external or body, no matter this virus is to exist with wild type (3 type Dearing) or modified form form (for example: σ 1 albumen synthesizes damaged incomplete attenuated virus), and is all effective to killing and wounding these cancer cells.Therefore, the range of application of reovirus can extend to unusual with ras proto-oncogene signal path or active superfluous relevant case.Other viruses that before had been considered to mainly to reply ras, interferon or other cellular immunity signal path states also are responsive to the disappearance of tumor inhibiting factor subfunction, and their range of application can be expanded too.

Therefore,, provide a kind of definite hyperplasia disorder the method whether responsive, comprised that (a) provides a kind of hyperplasia cell the oncolysis of reovirus or myxoma virus according to the present invention; (b) mensuration is by structure, function or the expression of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC and/or the ATBF1 gene or the gene outcome of the acquisition of hyperplasia cell; And (c) relatively hyperplasia structure, function or the expression with p53 wild type, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC and/or ATBF1 gene or gene outcome cell, wherein the p53 of hyperplasia cell, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC and/or ATBF1 show that in structure, function or the defective expressed the hyperplasia disorder is responsive to the oncolysis of reovirus or myxoma virus.

This method for example may further include and treats patient by reovirus and/or myxoma virus therapy, and this patient is the source of hyperplasia cell.Reovirus therapy can be wild type reovirus therapy or attenuation type reovirus therapy, and is damaged as using σ 1 capsid protein to express, σ 1 capsid protein detect less than or do not have a reovirus of σ 1 capsid protein.This therapy can comprise with reovirus and/or myxoma virus therapy coming patient's bone marrow cell is carried out indirect interior therapeutic, perhaps can comprise giving reovirus and/or myxoma virus therapy to patient.In a junctor under the situation of therapy, this method may further include and gives anti-hyperplasia therapy for the second time to bone marrow cell, perhaps the patient is being given under the situation of virus therapy, this method may further include and gives anti-hyperplasia therapy for the second time to the patient.Anti-hyperplasia therapy can be chemotherapy, radiotherapy, hormonotherapy, gene therapy or immunotherapy for the second time.This method also may further include with the function of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC or ATBF1 or expression inhibitor and treats, this inhibitor such as albumen or polypeptide, antibody, siRNA, antisense molecule, ribozyme or micromolecule.Treatment also can comprise the anti-hyperplasia therapy of non-viral, as using chemotherapy, radiotherapy, hormonotherapy, gene therapy or immunotherapy.The hyperplasia cell can be malignant cell or benign cell.This method may further include, and in step (a) before, obtains the hyperplasia cell on one's body from patient.The mensuration of expressing can comprise Northern trace, Western trace, immunohistochemistry, RT-PCR, microarray analysis, transcribe group analysis, Proteomic analysis or metabolism group analysis.Optionally, the mensuration of structure can comprise order-checking, in situ hybridization, immunohistochemistry or structural protein group.In addition, the mensuration of function can comprise the expression or the activity of the downstream targets of evaluation p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC or ATBF1.

In another embodiment, provide the method for a kind of patient's of treatment hyperplasia disorder, comprised that (a) gives function or the expression inhibitor of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC or ATBF1 to the hyperplasia cell; And (b) give reovirus and/or myxoma virus therapy to the hyperplasia cell.Reovirus therapy can be wild type reovirus therapy or attenuation type reovirus therapy, express as σ 1 capsid protein damaged, σ 1 capsid protein detect less than or do not have a reovirus of σ 1 capsid protein.Treatment can comprise that the bone marrow cell to patient carries out indirect interior therapeutic, or patient is carried out interior therapeutic.This method may further include and gives bone marrow cell anti-hyperplasia therapy for the second time, as the anti-hyperplasia therapy second time, as chemotherapy, radiotherapy, hormonotherapy, gene therapy or immunotherapy.The hyperplasia cell can be malignant cell or benign cell.

Term in word " a " or " an " and these claims and/or this instructions " comprises " unites when using, and may refer to " one ", but also with " one or more ", the meaning of " at least one " and " or more than " is consistent.

In whole application, term " about " is used to refer to a numerical value, and this numerical value comprises the error of the equipment that is used to measure numerical value, method and the inherent variability that causes, or is present in the deviation between the research object.

For term " or ", though open text support only refer to alternative and " and/or " definition, the use of the term in claims " or " be used to refer to " and/or ", unless clearly show be only to refer to alternative or selective to as if get rid of mutually.The word that in this instructions and claims, uses " comprising " (and any form of comprising, as " comprise " and " comprises "), " having " (and any form of having, as " have " and " has "), " including " (and any form of including, as " includes " and " include ") or " containing " (and any form of containing, as " contains " and " contain ") refer to that comprise or unconfined and do not get rid of extra composition of not enumerating or method step.

Other purposes of the present invention, feature and advantage will present by following detailed description.Yet, it will be appreciated that, when pointing out specific embodiment of the present invention, describe in detail and special case only illustrates as an example because to those skilled in the art according to this detailed description to spirit of the present invention with various changes are carried out in the field and modification is conspicuous.

The description of illustrative embodiment

Cancer be a process that multistep is rapid, thereby comprise by catastrophic event and influence the accumulation of uniting that normal proto-oncogene and tumor suppressor gene cause oncogene activation and tumor suppressor gene deactivation.What is interesting is that the cell signal path that oncolytic virus utilizes different oncogene to drive comes copy choice and kills and wounds the cell that they infect.Except the oncolysis that oncogene relies on, the inventor infers that some tumor suppressor genes are promoting may also to bring into play important effect on the viral oncolysis.Therefore, they have tested various mouse and people's cancerous cell line, regulate its tumor suppressor gene and check the tumor suppressor gene dysfunction whether can influence the oncolysis of reovirus and myxoma virus.The molecule mechanism of reovirus oncolysis is to obtain advancing because of the feature of being devoted to study the reovirus cell receptor is unexpected.In 1993, two kinds of mouse cell lines that have seminar's report not express EGF-R ELISA (EGFRs) infect reovirus corresponding resistibility are arranged, yet transfection the same clone of gene of coding EGFR present obviously higher susceptibility (Strong et al, 1993).The intracellular signal transduction pathway that this results suggest reovirus utilization activates, this path is owing to the EGFR that has function on the host cell gives.Afterwards, this has proved with a collection of researchist reovirus has been infected resistive NIH3T3 cell can become responsive (Strong etal, 1996 after transforming with the EGFR downstream factor S os, the Ras that activate or verbB; Strong et al, 1998).Therefore think that the utilization of oncogene Ras signal path is the committed step in the reovirus oncolysis.

A lot of human cancers demonstrate the Ras path activation with enhancing, and this has emphasized the strong effect of this signal network in tumour takes place, and the hint reovirus may brought into play oncolytic potential widely in the kinds of tumors type.The clone above 80% that initial experiment discovery comes from the kinds of tumors type is responsive (Coffey et al, 1998) to the lethal effect that reovirus mediates.More what is interesting is, another kind of oncogene, Myc also helps the oncolysis of reovirus.The expression excessively of Myc can make the resistive cell of reovirus is become to the intrusion sensitivity (Egan et al, 2003) of reovirus.

Myxoma virus is a kind of rabbit specificity poxvirus, is considered to promising oncolytic virus platform (referring to summary Stanford and McFadden, 2007).It is height-limited in European rabbits that its host tends to, and it comprises that for the every other vertebrate kind of being tested the mankind are no pathogenicity (McFadden, 2005).Though its specificity is limited, myxoma virus can infect and kill and wound various human tumour cell lines (Sypula et al, 2004).To a great extent, the taxis of myxoma virus on cellular level be by virus adhere to and the cell in the downstream of invasion in the incident regulation and control, rather than occur on the level of specificity host receptor (McFadden, 2005).

The propelling of the molecule mechanism of myxoma virus oncolysis has benefited from the research to control poisoning intrusion human cancer cell's virus host range factor.Have report to show that the Akt of viral M-T5 host range albumen and cell interacts, this interaction has strengthened the oncolytic potential (Wang et al, 2006) of myxoma virus to the human cancer cell.Yet the myxoma virus of M-T5KO (knocking out) has been given play to the viral oncolytic potential that weakens in a class human cancer cell (being called II type cancer cell), the cancer cell that composing type Akt activates still can be invaded by the M-T5KO myxoma virus, the rise of hint Akt is being brought into play important effect to decision myxoma virus oncolytic potential, (Wang et al, 2006) are often found in the rise of this Akt in many cancer cells.

The contact of virus oncolysis and tumor suppressor gene is that the research by adenovirus advances at first.The albumen of a 55kD of human adenovirus EIB gene code, it can and make its inactivation in conjunction with cell tumour Profilin p53.The adenovirus that has the report demonstration not express the sudden change of this virus protein can be duplicated and it is killed and wounded in the handicapped human tumor cell of p53, but can not duplicate and it be killed and wounded (Bischoff et al, 1996) in the cell that the p53 function is arranged.The basic mechanism of the adenovirus oncolysis of sudden change is disappearance or the sudden change owing to p53, and the Apoptosis that the p53 that adenovirus EIA induces relies on is impaired (Bischoffet al, 1966) in the handicapped cancer cell of p53.Yet the adenovirus oncolysis that studies show that the p53 dependence afterwards is more complicated, and can make the other path in the multiple model and reply (O ' Shea et al, 2004; Abou et al, 2004; Royds et al, 2006).In fact, thus this just guides the inventor to infer that the handicapped cancer cell of p53 may be because the destruction (Carroll et al., 1999) that genomic instability has suffered cell anti-virus mechanism.

Because p53 is to keeping genomic importance, so the p53 gene is considered to defender (Gomez-Lazaro et al, 2004 of cellular genome; Vogelstein et al, 2000).Because the genome of p53 mediation is kept, the antiviral mechanism that normal cell can be kept perfectly.Yet the dysfunction of p53 can finally cause the viral susceptibility (by the disappearance of antiviral mechanism) that caused by the whole genome instability.Recent studies have shown that p53 can help intrinsic immunity not rely on function (Takaoka et al, 2003 of its pre-Apoptosis and tumor suppressor gene by the antiviral activity that strengthens the IFN dependence; Munoz-Fontela et al, 2008).The transcription of p53 is important to the activation of IFN path when the virus infections.When virus attack, p53 can activate transcribe (Munoz-Fontela et al, 2008) of IFN regulatory factor 9.In addition, when reducing in the cancer cell, being expressed in of p53 can be observed the very big raising (Dharel et al, 2008) of virus replication level.These discoveries have proved the important antiviral activity of p53.This also may exist in other cases to the important tumor suppressor gene of dna replication dna and genome stability.The inventor illustrates at this, except activating by specific oncogene signal path the susceptibility that may strengthen that causes, the handicapped cancer cell of p53, ATM or Rb all becomes responsive more to the attack of reovirus and myxoma virus, and this has and helps describe different oncolytic viruses and how to distinguish normal cell and cancer cell.The evaluation of getting in touch between the not normal and oncolytic virus susceptibility of these heredity has strengthened the application potential of oncolytic virus therapy greatly.

I. oncolytic virus

A. reovirus

Reovirus (Reoviridae) comprises naturally occurring nonenveloped virus family, and it contains double-stranded RNA (dsRNA) genome, and this genome is divided into 10 bar segment and is surrounded by two concentric icosahedron protein coats.These viruses can influence gastronintestinal system and respiratory tract.The title of reovirus derives from " respiratory enteric orphan virus ".Term " orphan " virus refers to and all unrelated virus of any known disease.Although it is relevant with multiple disease that reovirus family has been identified in recently, this original title still is used.

The infection of reovirus often takes place on mankind, but most case be gentle or clinical symptoms not obvious.This virus can be easy to be detected in ight soil, and also can obtain from pharynx or nasal discharge, urine, celiolymph and blood.Although be very easy to find reovirus in clinical sample, they are still uncertain in the effect aspect human diseases or the treatment.Reovirus does not have coating and by an icosahedral capsid (T-13) of being made up of outer and internal layer albumin layer.The viral genome of Reoviridae comprises the 10-12 bar segment, is divided three classes according to their these fragments of big young pathbreaker: L (greatly), M (in) and S (little).The sheet segment limit is about 3.9-1kB, 1-3 albumen of every bar segment coding.According to its translation source fragment certainly, reovirus protein is represented (L fragment coding λ albumen, M fragment coding μ albumen and S fragment coding σ albumen) with its corresponding Greek character.

Because these viruses have the dsRNA genome, therefore duplicate and only betide tenuigenin, and this encoding viral to the dsRNA genome duplication with change into the necessary a plurality of albumen of (+)-RNA.Virus can enter host cell by cell surface receptor.Acceptor is unknown still being considered to comprise sialic acid and being connected adhesion molecule (JAMs).Virus can partly be removed the bag quilt in endolysosome by proteinase, can further be entered cell thereby partly digest in capsid herein.Core granule enters tenuigenin by the process of present the unknown then, thereby genome is produced excessive (+) adopted chain by conservative transcribing in this process, and this (+) adopted chain is used as the mRNA template and synthesizes (-) adopted chain.Virion begins to assemble in tenuigenin after infecting 6-7 hour.

The lonely virion of infectious mammalian reovirus intestines exists with the particle that diameter is approximately 85nm.The outer capsid of virion comprises several different albumen, and wherein (σ 1,50kD) carbohydrate combination (Chappell et al, 1997 by disperseing for sigma-1; Chappell et al, 2000; Connolly et al, 2001) and virion grappling (Mah et al, 1990; Fernandes et al, 1994; Lee et al, 1994) domain mediation virus adheres to host cell surface (Lee et al, 1981; Duncan et al, 1991; Nagata et al, 1987; Turner et al, 1992).σ 1 is the product of bicistronic mRNA reovirus S1 gene, but this gene is also by the distinct overlapping reading frame coding of application non-structural protein (Ernst et al, 1985; Jacobs et al, 1985; Sarkar et al, 1985).It is reported that the reovirus particle of disappearance σ 1 is FFI (Larson et al, 1994).Reovirus S1 gene is considered to bringing into play important effect (Haller et al, 1995 on the decision reovirus is pathogenic; Wilson et al, 1994; Kaye et al, 1986; Werner et al., 1980).

I. the application of wild type reovirus in treatment of cancer

About production and the certain methods of application of wild type reovirus in treatment of cancer (for example, United States Patent (USP) 7,300,650,7 have been described to some extent, 163,678,7,049,127,7,014,847,6,994,858,6,811,775,6,703,232,6,576,234,6,565,831,6,528,305,6,455,038,6,344,195,6,261,555,6,136,307 and 6,110,461, and U.S. Patent Publication text 2006/0165724,2006/0073166,2005/0123513,2004/0265271,2004/0126869,2004/0109878,2002/0037543,2006/0029598,2005/0026289,2002/0006398 and 2001/0048919, every patent all do not have with withdrawing a claim all be incorporated herein by reference).

Ii. the application of attenuation type reovirus in treatment of cancer

Yet, the immunodeficiency host such as new life's and SCID (severe combined immunodeficiency) animal in, the wild type reovirus is especially being brought into play important virus cause a disease function (Sabin, 1959 in nerve fiber and cardiac muscular tissue; Weiner et al, 1977; Baty et al, 1993; Loken et al, 2004).In some cases, even, comprise among the mankind wild type reovirus and virus pathogenic relevant (Terheggen et al, 2003, Hirasawa et al, 2003) the immunocompetence host.Therefore, especially in immunocompromised host or very young host, the wild type reovirus does not always work in optimum mode.For example, the host of immunocompromised host as the animal that grows up very young or immunodeficiency in, this virus also presents can infect some health tissues such as heart, liver, pancreas and neuromechanism.This important content can further be applicable to carries out a large amount of radioactive ray/chemotherapeutic cancer patient, because these cancer patients can suffer immunosupress.Therefore, clearly need to develop the more weak reovirus of toxicity and be used for viral oncolytic therapy.

In addition, the wild type reovirus has toxicity and the infectiousness that does not conform to demand in numerous occasions, and this can limit them in external potential application.Especially, with cell culture (for example, be derived from the bone marrow graft of cancer patient) be exposed to and can cause in the wild type reovirus not conforming to the killer cell of demand such as the spinoff of stem cell, stem cell is essential for a certain purposes, as recovering the immune system of cancer patient.Therefore, the toxicity of the enhancing of wild type reovirus has greatly limited the clinical potentiality of its external application, and this external application relates to cellular exposure in reovirus.

Attenuation type reovirus as described herein lacks can detected total length σ 1 capsid protein, yet unexpectedly has infectiousness.In the initial step that the virus replication sexuality is dyed, reovirus by the cell surface sialic acid residues in conjunction with and the process that adheres on the cell relate to σ 1 (Lee et a/., 1981; Duncan et a/., 1991; Nagata et al, 1987; Turner etal, 1992; Chappell et al, 1997; Chappell et al, 2000; Connolly et al, 2001).Although lack total length σ 1, attenuation type reovirus as described herein can enter host cell and carry out cytolytic virus replication.In addition, than the level of naturally occurring non-attenuation type reovirus for the CPE that non-malignant cell presented, attenuation type reovirus presents unexpected characteristic, it can cause the reduction of one or more CPE levels of non-malignant cell (that is, the reduction on the statistical significance).In view of the above and following described more details, attenuation type reovirus provided here has improved the reovirus of prior art, comprise suitable oncolytic reagent as the spinoff that does not have the demand of not meeting, for example to normal (for example, the non-pernicious) tropism of cell and spinoff of dissolving.Especially, this attenuation type reovirus can be used to external selective killing cancer or oncocyte.

In certain embodiments, as the U. S. application of submitting on August 5th, 2,006 60/704,604 and the international patent application no PCT/US2006/029881 that submits on July 31st, 2006 described in attenuation type reovirus can be used for the present invention, these patented claims all are incorporated herein by reference in this nothing with withdrawing a claim.In certain embodiments, attenuation type reovirus may lack wild type reovirus S1 gene.Attenuation type reovirus may contain the S1 gene, this S1 gene produce reduce and/or can not detected S1 gene outcome (σ 1) amount or brachymemma, sudden change and/or handicapped σ 1.Attenuation type reovirus may contain the S1 gene, compares with wild type S1 gene, and this S1 gene comprises one or more sudden changes, and sudden change wherein is that nucleotide subsitution, nucleotide deletion or nucleotide insert.

" attenuation type " as described herein reovirus comprise to host cell or in host cell its infect, duplicate and/or the cytolysis characteristic present change (that is, enhancing on the statistical significance mode or reduction) reovirus, this is for one or more these class features of the reovirus of known naturally occurring or wild type.In certain embodiments, for the wild type reovirus, attenuation type reovirus will present the potential of infectiousness, replication capacity and/or the dissolved cell of reduction.The example of the characteristic that this class changes comprises the multiple form of expression of pathological changes caused by virus effect, for example, particular host cell 8 is produced the needed infection multiplicity (MOI of infection, infect the average of the virion of each cell), the degree (further comprising apoptosis and/or necrosis) of the host cell dissolving that virus infections causes, from the titre of the host cell releasing virus of effective infection, and those of ordinary skill in the art can be used to determine virus other parameters to the activity of host cell after the virus replication of dissolved cell.The characteristic that above-mentioned this class changes can be used for differentiating present disclosed attenuated type reovirus.Other marks of CPE comprise the morphologic change of host cell, the immune system of host cell mediation causes the change of the ability of attacking, cell adhesion (adheres to substrate such as extracellular matrix protein or semisolid culturemedium, or adhere to other cells) change, the change of one or more cellular gene expression levels, the change of host cell replication capacity, and/or other changes of cell metabolic activity.

The S1 genetic fragment of considering reovirus is and the toxicity of reovirus closely-related (Weiner et al, 1977; Weiner et al, 1980; Mann et al, 2002), the inventor isolates S1 attenuation type reovirus (" AV reovirus ") (Kim et al, 2007) surprisingly from the cultured cells of persistent infection reovirus.Attenuation type reovirus strain presents greatly weaken viral pathogenic and does not damage its viral oncolysis in vivo in the animal model of immunodeficiency.Because known wild type reovirus is by retardation and suppresses blastular formation and come embryonic development (Priscott, 1983 to rat and muroid; Heggie et al, 1979) have a negative impact, so the inventor has further assessed AV reovirus pathogenic to stem cell.Like this, the inventor has compared pathogenic to embryonic stem cell (ESCs) of wild type and AV reovirus.Shown in the embodiment as described below, the wild type reovirus can be in the external growth of infecting ESCs easily and greatly suppressed stem cell in the teratoma model, yet the AV reovirus faintly infects ESCs and do not influence the growth of stem cell in the teratoma model at the external utmost point.

In different embodiment, attenuation type reovirus may comprise one or more other sudden changes.For example, in another specific embodiment, attenuation type reovirus may lack the reovirus S4 gene of wild type.Reovirus wild type S4 gene code reovirus capsid σ 3 polypeptide, this polypeptide relevant with the processing of virion when the reovirus replication host cells infected (for example, Ahmed et al, 1982; Giantini et al, 1984).

Attenuation type reovirus may include the reovirus particle of replication capacity.In addition, attenuation type reovirus may contain the hereditability sudden change (for example, displacement, insertion, disappearance) of S1 gene and/or S4 gene.The S1 gene of wild type reovirus is known to one skilled in the art.For example, the S1 gene order of wild type reovirus comprises mainly the S1 gene order of identifying out with by the naturally occurring reovirus form of separating in the breathing of infected patient or the intestinal tissue, or is derived from the conserved sequence of this type of sequence.Identify the S1 gene order of some reoviruses, comprised S1 gene order (for example, the gene pool of the human reovirus S1 gene order numbering: human 3 type reovirus S1:XO1 161 of human reovirus; Human 2 type reovirus S1:M35964; Human 1 type reovirus S1:M35963), this S1 gene order comprises the polynucleotide sequence of coding σ 1 albumen and the amino acid sequence of coding σ 1 albumen self (for example, the gene pool of main human reovirus serotype S1 gene order numbering: human 1 type reovirus strain Lang (TlL) numbering M35963; Human 2 type reovirus strain Jones (T2J) numbering M35964; Human 3 type reovirus strain Dearing (T3D) numbering XOl 161; Human 3 type reovirus strain Abney (T3A) numbering L37677).

According to certain embodiments, a kind of attenuation type reovirus is provided, the reovirus gene group that it comprises lacks wild type reovirus S1 gene, or contains reovirus S1 gene (the gene pool numbering of human reovirus S1 gene order: human 3 type reovirus S1:X01161 of reovirus σ 1 capsid protein that can not encode complete of sudden change; Human 2 type reovirus S1:M35964; Human 1 type reovirus S1:M35963).According to the disclosure and as known in the art, according to for example Ausubel et al. (1989); Ausubel et al (1993); Sambrook et al (1989); Maniatis et al. (1982); Glover (1985); Technology described in Hames and Higgins (1985) and other places identifies that by the sequence of measuring reovirus S1 gene the methodology whether the S1 gene of variation exists is conspicuous.Therefore the S1 gene of sudden change refers to contain a kind of S1 gene of polynucleotide sequence, this polynucleotide sequence inserts by one or more nucleotide subsitutions, nucleotide and nucleotide deletion changes one or more nucleotide sequences site of corresponding S 1 wild type nucleotide sequence or conserved sequence, and this can be identified easily.Hoyt etal. (2005) discloses a plurality of sudden changes of the muroid reovirus S1 gene order of coding σ 1 albumen, according to specific embodiment described in the invention here, and the clear and definite sudden change that does not comprise Hoyt et al..

As described herein with known in the art, outer capsid σ 1 albumen of reovirus can be according to its biological chemistry and/or immuno-chemical property and easily detected (for example, Mah et al, 1990; Leone et al, 1991; Chappell et al, 1997), generally be by using one or more technology, comprise that immune detection (for example, the electrophoresis of the reovirus polypeptide of σ 1 specific immunity precipitation reaction, western immunoblotting assay, immunoaffinity chromatography, immunofluorescence dyeing, immunocyte fluorescent quantitation, labelled with radioisotope, or the like), hemagglutination, and/or related side's science of law detects.Detect the method for reovirus σ 1 albumen according to these and other that set up, and in view of the discussion here, what those of ordinary skill in the art will figure out is to detect the art-recognized standard of σ 1 albumen and the sensitivity of prior art level, like this, the reovirus particle that replication capacity is arranged that lacks " can be detected " reovirus σ 1 capsid protein will be considered to comprise this type of reovirus, and complete σ 1 albumen of this reovirus can not be detected when the present traditional technology of application detects σ 1 albumen of supposition existence.

Be incorporated herein this class attenuation type reovirus is described US 11/997,537 as a reference.

Iii. the control of reovirus infection

In certain embodiments, peptidyl fluoro methyl ketone (PFMKs) can be applied in the cell composition, be used for suppressing or removing the reovirus of composition.U.S. Provisional Application sequence number 60/906,706 in a review is incorporated herein by reference its full content at this, has described peptidyl fluoro methyl ketone (PFMKs) and has been used to suppress virus replication, all is incorporated herein by reference in this nothing with withdrawing a claim.

Iv. the production of reovirus

According to certain embodiments disclosed herein, reovirus can be the reovirus in any source, it belongs to a member of Reoviridae family, and comprise the reovirus that contains various taxises and the reovirus (Tyler andFields, 1996) that can obtain by various sources.In certain embodiments, mammal reovirus and human reovirus have been looked forward to, be not subjected to so limiting though the present invention does not mean, and based on disclosure text, the technician can distinguish the situation of any specific reovirus applicable to this purpose.In certain embodiments, reovirus can be human 3 types (Dearing), 1 type (Lang), 2 types (Jones) or 3 types (Abney) reovirus, and in other certain embodiments, reovirus can be derived from one or more reoviruses that other mammal kind cells presented taxis, (for example comprise inhuman primate, chimpanzee, gorilla, macaque, monkey or the like), rodent (for example, mouse, rat, gerbil jird, hamster, rabbit, cavy or the like), dog, cat, common domestic animal (for example, ox, horse, pig, sheep or the like), perhaps, can use the reovirus (for example, birds reovirus) that contains the specificity trend.

As described herein, certain embodiments relates to implements recoverable attenuation type reovirus after the external persistent infection method, but also can consider to learn the attenuation type reovirus that obtains according to additive method, comprise persistent infection method in the body, produce and identify the damaged mutant of σ 1 defective and/or σ 1 (in certain embodiments according to molecular biology method, in addition or optionally, also comprise the generation and the evaluation of the mutant that σ 3 defectives and/or σ 3 are damaged), and also comprise mutant and/or σ 3 mutant that σ 1 defective that separating natural exists and/or σ 1 are damaged, and/or by chemistry, physics and/or genetics technology (for example, pairing recombinant reovirus gene in the host cell that effectively infects) this type of σ 1 of artificial induction (and/or σ 3) mutant.

In addition, inventor's expectation causes the phenotypic sudden change of required attenuation to find in other genes, the infectiousness of these effect gene viruses, duplicate or capacity packing, be included in arbitrary gene or its combination that virus goes up coding, as 10 genetic fragments of reovirus.For example, compare with the reovirus wild type gene, the sudden change of reovirus S1 or S4 gene can be united with 1,2,3,4,5 or more a plurality of sudden change on 0 genetic fragment of SV 12 virus.Can use several different methods to produce attenuation type reovirus, comprise the method described in the U. S. application 60/704,604 for example.

B. myxoma virus

Myxoma virus is a kind of poxvirus and contains big double-stranded DNA genome that this genome allows and may insert big (25kb) eukaryotic gene relevant with treatment.Myxoma virus is rabbit specificity virus and the mortality disease that can cause European rabbits (Oryctalagus cuniculus), is called myxomatosis.Its kind selectivity is very limited so that it is used to control Australian catastrophic hare population in generation nineteen fifty.Importantly, it is to other vertebrate kinds of test, comprise that the mankind are non-pathogenics, though it can comprise that external eternal young monkey kidney fibroblast (BGMK), IFN reply the former generation mouse cell human tumor cell different with some of hereditary detect at the specific non-rabbit cell of external effective infection.Well-known cancer cell is defective in their IFN replys.Recently, have report to show that myxoma virus is a kind of glioma of anti-experimental character in vitro and in vivo, and in a junctor oncolytic reagent (Lun et al, 2005 of anti-human malignant's glioma surgery sample; 2007; Stanford et al, 2008).It can infect and kill and wound human glioma cells, is safe through big injection of brain, and can " cure " mouse in brain is injected at human malignant's glioma model of coordination.In addition, it can infect and kill and wound all the primary neuroglial cytomas tested that directly are derived from glioma surgery sample, and its oncolysis can strengthen by rapamycin.

Myxoma virus as with other oncolytic viruses such as reovirus, need stride across the antiviral defense system that is present in the normal health cell it can be duplicated in cell.The generation of myxoma virus and other oncolytic virus inducing interferons, and normally responsive to the antivirus action of IFN path.Produce the associated protein that also mainly influences virus breeding by the IFN antiviral response and comprise PKR, OAS synzyme and ribonuclease l nuclease.PKR activates eIF2 α, causes inhibition and the cell death inducing translated.In normal cell, myxoma virus directly is subjected to the influence of PKR and eIF2 α.

The antiviral response path is destroyed in cancer cell usually.For example, IFN reply weaken or damaged be that heredity is damaged, its transform and the process of tumor development in often generation.Interferon is not replied or presents (Stojdl et al, the 2003 and references cited therein of replying of weakening above 80% tumor cell line; Wong et al, 1997; Sun et al, 1998; Matin et al, 2001; Balachandran et al, 2004).U.S. Patent application 2006/0263333 (being incorporated herein by reference) has been described myxoma infections and has been killed and wounded cancer cell, comprises human tumor cell's purposes, at this its full content is incorporated herein by reference.

II. TIF

Tumor suppressor gene is that the protection cell is not to the gene of cancer development.When this gene impairing, cell can be to cancer development, normally after it suffers the influence of other factors.Different with oncogene is that tumor suppressor gene is followed " secondary sudden change hypothesis " usually, is hinting that before effect manifests the allele of coding specific gene must all effected.If this is that second still can produce correct albumen so because this gene has only an allele impaired.Change a kind of saying, normally necessary but inadequate opposite with oncogene, TIF is normally necessary and sufficient.Certainly, the exception that should be noted that is arranged also, p53 gene outcome for example, for normal p53 albumen, it can exist with dominant negative mutant form, so it also is necessary but inadequate in such case.

Tumor suppressor gene, or their the coded albumen of saying so more accurately, the regulation and control of cell cycle or promotion Apoptosis all have both to stop or inhibiting effect sometimes.The function of TIF albumen is divided into multiple, comprises as described below: (a) suppress to keep necessary gene of cell cycle (if these genes are not expressed, the cell cycle can not continue, thereby effectively suppress cell division); (b) with the cell cycle and DNA is impaired connects.As long as damaged dna is present in the cell, cell just should not divide (if impaired can being repaired, the cell cycle just can continue); (c) cell death inducing, or apoptosis, if above-mentioned impaired could not being repaired, so in order would to make biosome better just eliminate the threat that it produced; And (d) some albumen relevant with cell adhesion prevent the tumour cell diffusion, stop the loss of contact inhibition, and suppress to shift (albumen that is called as tumor metastasis suppressor gene).

On the contrary, oncogene is the gene that causes cancer.Many cells are normally dead.In cancer, owing to there is the oncogene dna sequence dna of sudden change, those cells can be survived and breed.Most oncogene needs another step to bring out cancer, as the sudden change of another gene, or environmental factor such as virus infections.Since the 1980's, in human cancer, identified a lot of oncogene.Proto-oncogene is normal gene, because sudden change or expression increase, it can become oncogene.The proto-oncogene encoded protein helps regulating cell growth and differentiation.Proto-oncogene is generally relevant with the execution of signal conduction and mitosis signal usually by their protein product.After activating, it is oncogene that proto-oncogene (or its product) becomes TIF.The example of proto-oncogene comprises RAS, WNT, MYC, ERK and TRK.

A.p53

P53 (numbering NM 000546) is a transcription factor, and its cell cycle regulation also plays a part TIF.It is important in multicellular organisms, because it helps suppress cancer by the function that the adjusting cell is replied dna damage to a certain extent.The p53 that touches upon keeps the function of stability by preventing genome mutation, and it once was described to " genomic defender ", " Guardian Angel's gene ", or " guard great master ".

The title of p53 is relevant with its apparent molecular weight on SDS-PAGE, but in fact it actually has only 43.7kD.This is not both because a large amount of amino proline residue in the p53 albumen can make the migration of p53 on SDS-PAGE slack-off, therefore makes it seem bigger.This result can observe in the p53 of various kinds, comprises the mankind, rodent, frog and fish.This gene is positioned on the human chromosome 17 (17p13.1), and the 393 amino acids albumen of encoding, and comprises 5 zones: (a) the terminal transcriptional activation domain (TAD) of N, and can activating transcription factor (1-42 position residue); (b) the proline rich district (80-94 position residue) that the apoptosis activity of p53 is played an important role; The DNA at center comprises a zinc atom and a plurality of arginine amino acid (100-300 position residue) in conjunction with core space (DBD); With oligomerization district (OD) (307-355 position residue); And with the relevant C end (356-393 position residue) of downward modulation of the DNA binding ability of center.

In cancer, make the sudden change of p53 inactivation usually occur in the DBD district.These sudden changes of great majority have destroyed the ability of its target dna sequence of protein combination, and have therefore hindered these gene transcription activity.In this connection, the sudden change in DBD district is the sudden change of retrograde afunction.At the p53 of the p53 of OD region mutation molecule and wild type dimerization, and stop their activated transcriptions.Therefore the sudden change in OD district has significant negative effect to the function of p53.

P53 has a lot of anticancer mechanism.For example, it can activate dna repair protein when DNA is lasting impaired.It also can make the cell cycle remain on G1/S check point (if it remains on the sufficiently long time herein with cell, dna repair protein just can be repaired damage if having time and allow this cell to proceed the cell cycle) when the identification dna damage.If dna damage proves in the time of can not repairing, it also can bring out Apoptosis, i.e. apoptosis.

P53 to all kinds stress reply in be activated, these stress include but not limited to that dna damage (by UV, IR or chemical reagent cause as hydrogen peroxide), oxidative stress, osmotic shock, ribonucleotide loss and oncogene out of control expresses.This activation is shown by two main incidents.The first, the half life period of p53 albumen is increased sharply, cause be subjected to stress cell in the quick accumulation of p53.The second, the variation of conformation impels p53 to exercise active function as transcription regulaton factor in these cells.The critical event that causes p53 to activate is the phosphorylation of its N stub area.The terminal transcriptional activation domain of N comprises a large amount of phosphorylation sites and can be considered to the main target that the protein kinase conduction stress signal.

The protein kinase of known grappling p53 transcriptional activation domain can roughly be divided into two kinds.First kind of protein kinase belong to MAPK family (JNK1-3, ERK1-2, p38MAPK), known its can reply multiple stress, as membrane damage, oxidative stress, osmotic shock, heat shock or the like.Second kind of protein kinase (ATR, ATM, Chk1, Chk2, DNA-PK CAK) is contained in the genomic integrity checkpoint, promptly a kind of molecule cascade system, this system can detect and reply polytype dna damage that stress be caused by genetoxic.

Stress not cell in, the lasting degraded by p53 makes p53 keep low-level.Thereby the protein combination that is called Mdm2 makes it be degraded by proteasome in p53 and with it from nucleus transporte to cells matter.The phosphorylation of the N end of the p53 that is caused by the above-mentioned protein kinase of mentioning has been destroyed the combination of Mdm2.Other albumen such as Pin1 are raised on the p53 and are caused the p53 conformation change then, and this has just stoped the combination of Mdm2 more.Transcribe the co-activation factor such as p300 or PCAF and make the carboxyl terminal acetylation of p53 then, thereby expose the DNA land of p53, make its activation or suppress specific gene.

B.Rb

Retinoblastoma protein (Rb; NM_00321) be a kind of TIF albumen, it is found in the multiple cancer is handicapped.Thereby pRb so name is because can cause retinal glioma when two allele of the RB1 gene of this albumen of encoding all suddenly change when making this albumen inactivation.Rb is present in the cell as phosphoric acid albumen usually and it is as described below a multiple kinase whose phosphorylation target.A kind of function of being furtherd investigate of Rb is to stop cell division or advance the cell cycle.Therefore, when this disabler of Rb, the cell of sudden change will continue division and may develop into cancer.

Rb is a member of " pocket protein family ", because it contains a combinative pocket of albumen.For example those can and be made its inactivation in conjunction with Rb by excessive risk type human papilloma virus infection's the oncogene protein that cell produced, thereby can cause cancer.Rb advances the cell cycle to enter into the S phase by stoping, or synthesis phase, thereby or advances the G1 or the first gap phase to stop the cellular replication damaged dna.The Rb combination also suppresses E2F family transcription factor.The E2F transcription factor is the binary of E2F albumen and a DP albumen.Transcriptional activation compound E2 promoter can advance cell to enter the S phase in conjunction with albumen dimerization molecular chaperones (E2F-DP).In case E2F-DP inactivation, cell will keep resting on the G1 phase.When Rb is incorporated into E2F, this compound will work as GIF and stop and advance the cell cycle.The Rb-E2F/DP compound also can attract histone deacetylase (HDAC) albumen to chromatin, thereby it is synthetic further to suppress DNA.

When the phosphitylation state, Rb be activate and play a role by suppressing cell cycle progression as TIF.Phosphorylation can make the Rb inactivation.When phosphatase during with residue dephosphorylation of Rb, Rb can be activated during latter stage near G1, thereby can be in conjunction with E2F.When cell to will entering S during the phase, the compound of cyclin-dependent kinase (CDK) and cyclin can be with the pRb phosphorylation, thereby suppresses its activity.Initial phosphorylation is by Cyclin D/CDK4, and 6 carry out, afterwards by the further phosphorylation of Cyclin E/CDK2.PRb keeps phosphorylation state to run through S, G2 and M phase.The phosphorylation of pRb makes E2F-DP from the pRb activity that disintegrates down and become.When E2F is released, it (for example for example can activate the factor such as cyclin, Cyclin E and A), thereby by activation cycle protein dependent kinase and a kind of molecule that is called proliferative cell nuclear antigen or PCNA advance cell by the cell cycle, thereby the PCNA molecule can quicken dna replication dna and reparation by helping polymerase be incorporated into DNA.

C.ATM

Ataxia-telangiectasia (AT; NM_00051) be the autosome degeneration disorder that causes by the ATM gene mutation that is positioned on the chromosome 11q22-23.It is identified in June nineteen ninety-five, and is made up of 66 extrons that are distributed on the 150kb genomic DNA.It the coding 13kb the mature transcript that 9168 nucleotide open reading frame are arranged.ATM albumen is about 370kDa, is that omnipresence is expressed and is positioned at nucleus.ATM albumen is a kind of big serine-threonine kinase, and it is considered to play a role (similar to the BRCA gene) in cell cycle regulation checkpoint, double-stranded DNA reparation and meiosis.Same known ATM plays a role on regulation and control p53, BRCA1 and CHEK2.The part effect of known ATM in the DNA reparation is the telomere reparation, because telomere degraded meeting is rapider in the patient who is subjected to the AT infringement.

The sudden change of ATM gene is considered to be divided into two kinds: (a) nonsense mutation, and it can cause that protein function completely loses and thus with the heredity of degeneration mode and bring out AT; And (b) ' missense ' sudden change, it can produce the albumen of the stable total length of miopragia, for example, and displacement, the insertion and the disappearance of reading weak point in the frame, or the like.The normal copy of albumen can be significantly disturbed in these sudden changes.Most of AT victim, 65-70% has truncated mutant, and its exon skipping sudden change is especially general.This just causes, and atm protein level is very low maybe can not to be detected.Missense mutation is the modal mutation type of finding in the breast cancer carrier.Be considered to contain the AT of slight state with the individuality of two missense mutation, this is the case that can cause attenuation type AT.

D. other TIFs

Multiple other TIFs are p53 for example, and ATM and Rb can be used for the present invention.For example BRCA1 and BRCA2 are bringing into play key effect in the generation of breast cancer.Thereby BRCA1 keeps genomic integrity to stop propagation human gene out of control.Polyfactorial BRCA1 protein product participates in dna damage reparation, ubiquitination, transcriptional control and other functions.The variation of this gene relates to multiple genetic cancer, i.e. breast cancer, oophoroma and prostate cancer.The BRCA1 gene is positioned on 21 on length (q) arm of chromosome 17, and from 38,449,843 bit bases are to 38,530,933 base-pairs (collection of illustrative plates).BRCA1 albumen is participated in the reparation of damaged dna directly.Though its accurate function is also unclear, BRCA1 albumen is considered to when the dna double bound rupture is repaired and RAD51 interacts.This class fracture can be caused by natural ray or other irradiations, but during cell division generation sperm that carries out specific type and ovum (meiosis), when the chromosome crossing over, this class fracture also can take place.By influencing the dna damage reparation, this albumen plays a role on stable keeping human genome.

BRCA2 participates in second kind of human gene that chromosome damage is repaired.Though the structure of BRCA1 and BRCA2 gene is very different, it seems that their function be similar.The albumen of two kinds of gene generations is essential to repairing impaired DNA.Thereby the BRCA2 protein combination is also regulated the fracture of the albumen DNA plerosis that the RAD51 gene produces.This class fracture can be caused by natural and medical ray or other ambient illuminations, but during cell division generation sperm that carries out specific type and ovum (meiosis), when the chromosome crossing over, this class fracture also can take place.BRCA1 albumen also with the RAD51 protein-interacting.By DNA plerosis, these three kinds of albumen play a role on stable keeping human genome.The same with BRCA1, BRCA2 may regulate the active of other genes and bring into play key effect in embryonic development.The BRCA2 gene is positioned on length (q) arm 12.3 (13q12.3) of chromosome 13, and from 31,787,616 bit bases are to 31,871,804 base-pairs.

MutS helps to repair the family of the mispairing in the double-stranded DNA or the guide member of albumen.The first step of this process is the DNA of identification mispairing.In Escherichia coli, MutS is incorporated into the mispairing site of double-stranded DNA, cooperates with MutL and MutH albumen, and certain fragment of a chain is with its excision in this site grappling DNA chain.Other albumen are finished the reparation process: the cut and degraded of the dna fragmentation of grappling, and use complementary strand as the synthetic sticking patch of template, and sticking patch is connected into the double chain DNA fragment that the position produces the reparation that tape error not joins.A lot of interest concentrates on the MutS homologue in people source, because some such defectives are to form the hereditary nonpolyposis characteristic of disease colorectal cancer (HNPCC) of some form and the reason of other possible colorectal cancers.

Adenomatous polyposis coli (APC) is the TIF that another kind relates to colorectal cancer.Whether its helps the fissional frequency of control, how to adhere to other cells or cell in tissue moves into or leaves tissue, and it helps also to determine whether the intracellular chromosome number that produces is correct in fission process.APC albumen is mainly by finishing these tasks with combining of other albumen, and especially those relate to cell adhesion and the albumen that send signal.A kind of activity of albumen, the especially activity of β catenin are by the control of APC albumen, and it is the part of Wnt signal path.The regulation and control of β catenin stop the gene quilt of irritation cell division excessively frequently to be opened and stop cell transition to be grown.APC is positioned between 21 and 22 on length (q) arm of chromosome 5, and from 112,118,468 bit bases are to 112,209,532 base-pairs.

AT is a kind of TIF in conjunction with transcription factor 1 or ATBF1, and its disappearance relates to the generation of cancer of the stomach.It is positioned at 16q22.3-q23.1.Its DNA total length is 261.32kB.According to the montage of using and selecting of selective actuation, two kinds of homology isomeride are arranged, ATBF1-A and ATBF1-B.This albumen has 3703 amino acid, and size is 404kDa.It comprises 4 and grows homeodomain and comprise 23 zinc fingerses intending zinc-finger motif, a DEAD and a DEAH box, a RNA and an ATP-binding site, two big RS domains and a plurality of phosphorylation site.This albumen is positioned at nucleus and works as transcription factor, and it is incorporated on the core sequence of AT enrichment of enhancer element of AFP gene and downward modulation AFP expression of gene, may relate to the neuron differentiation.The neural crest source cell is with outer double minute form of chromosome and the non-collinear coamplification of myc among the SJNB-12 in early days.In the stomach cancer cell system that alpha fetal protein is expressed, can observe ATBF1 and lack expression.The shortage that ATBF1 expresses is not owing to sudden change, deletion or transposition, but because the strong inhibition of transcriptional level.

III. measure structure, expression or the function of TIF

A. based on the diagnosis of nucleic acid

One embodiment of the present of invention comprise the method that detects the TIF abnormal expression.This can comprise the level of measuring TIF or the special variation of measuring expression product.Used nucleic acid is to learn according to conventional methods by separating (Sambrook et al, 1989) that obtain in the cancer cell.Nucleic acid can be genomic DNA or fragmentation or whole-cell rna.In case use RNA, just may need to convert RNA to complementary DNA.In one embodiment, RNA is a whole-cell rna; In another embodiment, it is poly-A (poly-adenine) RNA.Usually, nucleic acid is the nucleic acid that is amplified.

According to formula, use the back of increasing or increase and directly identify specific nucleic acid to be measured in the sample with second kind of known nucleic acid.Then, detect the product of being identified.In specific application, can detect (for example, the gel of ethidium bromide staining) by visual method.Optionally, this detection can comprise radioactivity scintigraphy or the fluorescence labeling by chemiluminescence, labelled with radioisotope, or even identify (Affymax Technology by making electricity consumption or thermostimulation signal system carry out indirect product; Bellus, 1994).

In one embodiment, the TIF expression is to measure by the amount of measuring mRNA.Yet damaged by detecting polytype structure, also can identify active change.These are damaged to comprise deletion, insertion, point mutation and overlapping.The point mutation meeting causes terminator codon, frameshift mutation or amino acid replacement.Somatic mutation is that those betide sudden change that non-the germ cell line tissue and are hereditary, yet the sudden change of germ cell line tissue is heritable.In the code area or outer sudden change also can be by changing this gene transcription effect, the processing that perhaps changes transcript (mRNA) or albumen influences the amount that TIF produces.

When an allele of TIF because when the generation of the heredity of germ cell line or somatic mutation and inactivation, cell will be carried out hereditary process and change to carcinogenicity.The allelic inactivation of another of gene generally includes body cell micromutation or the deletion of chromosome allele that causes heterozygous deletion (LOH).Optionally, two of tumor suppressor gene copies all can be lost by homozygous deletion.

Based on this point; consider to carry out multiple different mensuration; include but not limited to; fluorescence in situ hybridization (FISH), direct dna sequencing, PFGE analysis, Southern or Northern trace, single stranded conformational analysis (SSCA), rnase protection analysis, allele specific oligonucleotide analysis (ASO), Dot blot analysis, denaturing gradient gel electrophoresis, RFLP and PCR ^TMSSCP.

I. primer and probe

Here Ding Yi term primer refers to comprise any nucleic acid that can synthesize newborn nucleic acid by the guiding of template dependence process.Typically, primer is that length is the oligonucleotides of 10-20 base-pair, but can use longer sequence.Primer can be to provide with two strands or single stranded form, though the form of preferred strand.The definition of probe is different with primer, though they also can be used as primer.Although perhaps probe can have the effect of guiding, probe is designed to combining target DNA or RNA and does not need to be used to amplification procedure.In certain embodiments, with the radioelement of band fluorophore (Dan Ruoming, fluorescein) or chemiluminescence (luciferase element) ( ³²P, ¹⁴C, ³⁵S, ³H or other marks) label probe or primer.

Ii. template dependent amplification method

Multiple template dependence program can be used for increasing and is present in the sequence of the mark in the specific template samples.A kind of foremost amplification method is that polymerase chain reaction (is called PCR ^TM), at United States Patent (USP) 4,683, have a detailed description in 195,4,683,202 and 4,800,159 and Innis et al, 1990, at this its full content is incorporated herein by reference.

In brief, at PCR ^TMIn, prepare the primer sequence of two regional complementarities on the reverse direction complementary strand with flag sequence.Add excessive deoxynucleoside triphosphate and a kind of archaeal dna polymerase, for example the Taq polymerase is in reaction mixture.If flag sequence is present in the sample, primer will be incorporated into label, and polymerase will impel primer to extend along flag sequence by adding nucleotide then.By improving and reduce the temperature of reaction mixture, the primer of extension can disintegrate down the formation reaction product from label, and excessive primer can be incorporated into label and reaction product and repetition said procedure.

For the amount of definite mRNA that increases, can carry out reverse transcriptase PCR ^TMAmplification program.With the RNA reverse transcription is that the method for cDNA is well-known and at Sambrook et al, describes to some extent in 1989.The method of selectable reverse transcription is used heat-resisting RNA dependent dna-polymerases.These methods are described in the WO 90/07641 that submits to Dec 21 nineteen ninety to some extent.It is well-known that polymerase chain reaction method is learned in the present technique field.

The another kind of method that is used to increase is ligase chain reaction (" LCR "), and is open in EPO numbers 320308, at this its full content is incorporated herein by reference.In LCR, the probe of preparing two pairs of complementations is right, and when target sequence existed, the every pair of probe can be incorporated on the rightabout complementary strand of its target that contacts and adjoin each other.When having ligase, two pairs of probes are connected to form single unit to meeting.By as PCR ^TMIn temperature cycles, in conjunction with connection unit just disintegrate down from target, be used for the right connection of remaining probe as " target sequence " then.United States Patent (USP) 4,883,750 have described with the LCR similar methods and have been used for probe being incorporated into target sequence.

Q β replicase described in the PCT application number PCT/US87/00880 also can be used as another kind of amplification method in the present invention.In the method, the rna replicon type sequence that contains with the zone of target complementation is added in the sample in the presence of RNA polymerase.Polymerase can copy the science sequence, and this sequence can be detected subsequently.

A kind of isothermal amplification method also can be used to amplification of nucleic acid in the present invention, in the method, restriction endonuclease and ligase are used to finish the amplification of target molecule, this target molecule on a chain of restriction site, comprise nucleotide 5 '-α sulfo--triphosphoric acid, Walker etal, (1992).

Strand displacement amplification (SDA) is the another kind of method of carrying out the nucleotide isothermal duplication, and it comprises many wheel strand displacements and synthetic, that is, nick translation.A kind of similar method is called and repairs chain reaction (RCR), comprises making a plurality of probes run through the annealing of amplification target area, repairs reaction afterwards, only has two kinds in four kinds of bases in this reaction.For easy detection, other two kinds of bases can be used as the biotinylation derivant and add.A kind of similar methods is applied among the SDA.Also can react (CPR) by circle probe and detect the target particular sequence.In CPR, contain 3 of non-specific DNA ' and the DNA of probe hybridization in being present in sample of the intermediate sequence of 5 ' sequence and special RNA on.After the hybridization, come processing reaction with ribonuclease H, the probe product is accredited as special product, and this product is released after digestion.Primary template is annealed on another circle probe and repeats above-mentioned reaction.

Also have the another kind of amplification method can be used for the present invention, it be described in GB application number 2202328 and PCT application number PCT/US89/01025 to some extent, at this its full content is incorporated herein by reference.In application before, " modification " primer is used to similar PCR ^TMTemplate and enzyme dependent synthetic.This primer can be by being modified with catching component (for example, biotin) and/or detected components (for example, enzyme) mark.After application in, excessive label probe is added in the sample.When target sequence exists, probe in conjunction with and by catalyze cleavage.After cutting, target sequence is by complete release and by the excessive probes combination.The cutting of label probe is the signal that target sequence exists.

Other nucleic acid amplification program comprises that based on the amplification system of transcribing (TAS) this system comprises amplification (NASBA) and 3SR (Kwoh et al., 1989 based on nucleotide sequence; Gingeraset al, PCT applies for WO 88/10315, at this its full content is incorporated herein by reference).In NASBA, can be by conventional phenol/chloroform extraction, the thermal denaturation of clinical sample extracts preparing nucleic acid to be used for amplification with the little centrifugal post processing of lysis buffer and DNA isolation and RNA or the guanidine hydrochloride of RNA.These amplification techniques comprise makes the primer annealing that contains the target distinguished sequence.After the polymerization, the DNA/RNA crossbred by rnase digestion and double chain DNA molecule by thermal denaturation again.In each case, make single stranded DNA become complete two strands, carry out polymerization afterwards by adding second target special primer.Double chain DNA molecule is transcribed at double by RNA polymerase such as T7 or SP6 then.In the isothermal circular response, RNA is inverted record for single stranded DNA, and single stranded DNA changes into double-stranded DNA subsequently, transcribes again by RNA polymerase such as T7 or SP6 then.The product that produces, and though be brachymemma or complete, equal feeling the pulse with the finger-tip mark distinguished sequences.

Davey et al., EPO number 329 822 (its full content being incorporated herein by reference at this) disclose a kind of circulation synthesizing single-stranded RNA (" ssRNA ") that comprises, the nucleic acid amplification method of ssDNA and double-stranded DNA (dsDNA), this method can be used for the present invention.SsRNA is the template of article one primer tasteless nucleotide, extends (RNA dependent dna-polymerases) by reverse transcriptase.By the effect of ribonuclease H (ribonuclease H, specific effect is in forming a kind of ribonuclease of the RNA of binary with DNA or RNA) RNA is removed from the DNA:RNA binary that produces subsequently.The ssDNA that produces is the template of second primer tasteless nucleotide, and it also comprises rna polymerase promoter (as the t7 rna polymerase) sequence that is positioned at template homologue 5 ' end.Make primer extension by archaeal dna polymerase (as big " Klenow " fragment of e. coli dna polymerase I) subsequently, produce double-stranded DNA (" dsDNA ") molecule, its contain one section with primer between the identical sequence of original RNA and at one end contain other promoter sequence.This promoter sequence can be by numerous RNA copies of used this DNA of generation of suitable RNA polymerase.Thereby these copies can reenter to circulate and cause very fast amplification subsequently.By the correct selection of enzyme,, this amplification isothermal do not need all to add enzyme in each circulation but carrying out.Owing to the cycle characteristics of this method, can select the homing sequence of DNA or rna form.

Miller et al, PCT application WO 89/06700 (its full content being incorporated herein by reference at this) discloses a kind of amplification of nucleic acid sequences method, this method is transcribed numerous RNA copies of this sequence afterwards based on promoter/primer sequence being hybridized on target single stranded DNA (" ssDNA ").This method is not a round-robin, and for example, new template is not synthetic by the rna transcription thing that produces.Other amplification method comprises " RACE " and " monolateral PCR ^TM" (Frohman, 1990; Ohara et al, 1989; At this its full content is incorporated herein by reference).

In the presence of the nucleic acid of " dual oligonucleotide " sequence that contains generation, two (or more) oligonucleotides connect, and the dual oligonucleotide that increases thus also can be used for amplification step of the present invention based on above method.Wu et al, (1989) are incorporated herein by reference its full content at this.

The iii.Southern/Northern trace

Engram technology is well known to those skilled in the art.The Southern trace comprises use DNA as target, and the Northern trace comprises that use RNA is as target.Every kind provides different kinds of information, though the cDNA trace is similar to the trace of RNA kind aspect a lot.

In brief, probe is used for various DNA or the RNA that are fixed on the suitable matrix of grappling, and this matrix is nitrocellulose filter normally.Different kinds should be carried out apart and analyze so that help.Normally " trace " finished to the film after the gel electrophoresis by various nucleic acid in this separation.

Subsequently, the target of usefulness probe (normally mark) incubation trace under the condition that promotes sex change and hybridization once more.Because probe is designed to form base-pair with target, so probe will be incorporated into the part of target sequence under the condition of sex change.Unconjugated subsequently probe is removed, and by finishing detection as mentioned above.

Iv. separation method

In order to determine whether specific amplification takes place, need amplified production be separated from template and excessive primer one or another stage usually.In one embodiment, use conventional method amplified production to be separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis.Referring to Sambrook et al. (1989).

Optionally, can use chromatographic technique to reach the separation purpose.The multiple chromatography method of the present invention that can be used for is arranged: absorption, distribution, ion-exchange and molecular sieve, use the multiple technical skill of these methods to comprise pillar, paper, thin layer and gas chromatography (Freifelder, 1982).

V. detection method

In order to determine the amplification of flag sequence, product need be by visual.A kind of typical method for visualizing comprises with ethidium bromide gel-colored and estimate under ultraviolet ray.Optionally, if amplified production integrally has been labeled the nucleotide of radioactivity or fluorophotometric mark, after separation, amplified production can be exposed to subsequently on the X-ray film or under suitable excitation spectrum and manifest.

In one embodiment, can reach visual indirectly.After separating amplified production, use the nucleic acid probe of tape label to contact with the flag sequence of amplification.Probe preferably with the chromophore coupling, but can be labelled with radioisotope.In another embodiment, probe and binding partners coupling as antibody or biotin, are combined into another right member and carry detectable component.

In one embodiment, the probe by mark detects.The technology that relates to is well-known to those skilled in the art and can finds in a lot of conventional books about molecular method.Referring to Sambrook et al. (1989).For example, among amplification or afterwards, the probe of chromophore or labelled with radioisotope or primer are differentiated target.

An above-mentioned embodiment is at United States Patent (USP) 5,279, describes to some extent in 721, be introduced into as a reference at this, this patent disclosure be used for automatically

The instrument and the method for electrophoresis and nucleic acid migration.This instrument do not need to allow extra gel operation and carries out electrophoresis and trace, and is ideally suited and is applied to the present invention.

In addition, can use conventional sequence analysis method that above-mentioned amplified production is identified specific variation type by sequential analysis.In specific method, use the primer sets that is designed for best order-checking gene to be carried out detailed analysis (Pignon et al, 1994) by sequential analysis.The invention provides the method for the analysis that can use arbitrary or all these types.By using sequence disclosed herein, can carry out sequence amplification by the design oligonucleotides primer, can come analysing amplified sequence by direct order-checking subsequently.

Vi. kit composition

The needed all base stocks of sequence and the reagent that are used for detecting and measure genes of interest can be assembled in a kit together.Usually this kit can comprise primer and the probe of selecting in advance.Also the enzyme that is applicable to amplification of nucleic acid be can comprise, multiple polymerase (RT, Taq, Sequenase comprised ^TMOr the like), deoxynucleotide and damping fluid are used to provide amplification necessary reaction mixture.In suitable method, this class kit also can comprise the special container that loads every kind of reagent and enzyme and every kind of primer or probe with suitable manner usually.

Vii. to the RT-PCR of relative quantification ^TMDesign and theoretical consider

With the RNA reverse transcription is the PCR that cDNA carries out relative quantification afterwards ^TM(RT-PCR ^TM) can be used to measure the relative concentration of the various specific mRNA that separate from patient on one's body.By measuring the variation of various specific mRNA concentration, but the differential expression of the gene of the specific mRNA kind of code displaying.

At PCR ^TMIn, each circulation of reaction increases near 2 times the quantity of molecule of the target dna of amplification, becomes limited until some reagent.Thereafter, amplification rate becomes does not increase until the target that increases between circulation more and more slowly.If draw a diagram, X-axis is a period, and Y-axis is the concentration logarithm of target dna of amplification, couples together the curve that will form peculiar shape by the point that will draw.From first circulation, the degree of tilt of line is positive number and is constant.This is called as the linear segment of curve.Become in limited time at reagent, the degree of tilt of line begins to descend and finally become 0.At this moment the concentration of Kuo Zeng target dna graduates into certain fixed value.This is called the steady component of curve.

At PCR ^TMIn the linear segment of amplification, the concentration of target dna is that the initial concentration of the target before directly beginning with reaction is proportional.By measuring the PCR that each has been finished the same loop number of times and has been in its range of linearity ^TMThe concentration of target dna amplified production in the reaction just might be determined at the relative concentration of particular target sequence in the original DNA potpourri.If the DNA potpourri is by the synthetic cDNA of the RNA of different tissues or cell separation gained,, so just can measure the relative abundance of the specific mRNA of corresponding tissue and cell, mRNA wherein is the source of target sequence.PCR ^TMDirect proportionality between the abundance of the concentration of product and relative mRNA only is applicable at PCR ^TMIn the range of linearity of reaction.

In the ultimate density of the target dna of the steady part of curve is by the availability decision of the reagent in the reaction mixture, and does not rely on the original concentration of target dna.Therefore, pass through RT-PCR ^TMThe relative abundance that the RNA sample population of gathering is carried out various mRNA is measured the most important condition that must satisfy and is amplification PCR ^TMThe sample of the concentration of product must be at PCR ^TMGather when reacting the linear segment that is in their curves.

Pass through RT-PCR ^TMSecond condition that must satisfy of testing the relative abundance of successfully measuring various specific mRNA is that the relative concentration of the cDNA that can increase must be standardized as certain independent standard.RT-PCR ^TMThe purpose of experiment is to measure the abundance of the various specific mRNA for the average abundance of all various mRNA in the sample.

Competitive PCR ^TMMost of method use inherent PCR ^TMMaster sample, this master sample is approximately the same with target sample abundant.If at PCR ^TMThe linear phase of reaction is to PCR ^TMAmplified production is sampled, and these methods are effective so.If product is to sample near the stage of stable development in reaction, it is too high relatively that the abundance of so more low-abundance product can seem.The relative abundance of relatively a lot of different RNA samples of measuring, as in the case of the differential expression that detects the RNA sample, the distortion that can become in one aspect makes the seeming and will lack of their reality of diversity ratio of RNA relative abundance.If the inherent standard sample is much abundanter than target sample, this is not a major issue just so.If the inherent standard sample is abundanter than target sample, can between the RNA sample, carry out direct linear ratio so.

Above discussion has been described detecting the RT-PCR of clinical material ^TMTheory consider.Intrinsic problem in clinical sample is their quantitative change (generation standardization issue), and their qualitative change (needing the coamplification of believable inherent check sample, preferably bigger than target sample fragment inherent check sample).If RT-PCR ^TMAs the relative quantification RT-PCR that has the inherent standard sample ^TMMeasure, then the problem of above-mentioned quantitative change and qualitative change can be readily solved, wherein said inherent standard sample is the cDNA fragment that increases bigger than target cDNA fragment, and the abundance of the mRNA of the inherent standard sample of wherein encoding approximately than the high 5-100 of mRNA of coding target sample doubly.This analysis is relative abundance rather than an absolute abundance of measuring various mRNA.

Can use the more conventional relative quantification RT-PCR that has external master sample scheme ^TMAnalyze and carry out other researchs.These are analyzed at PCR ^TMThe linear segment of the amplification curve of product is to PCR ^TMProduct is sampled.Must rule of thumb determine the PCR of the optimum sampling of each target cDNA fragment ^TMPeriod.In addition, the reverse transcription product for each RNA sample population of being come by the different tissues sample separation must carefully be standardized as it cDNA that increases with isoconcentration.Because this analysis is an absolute abundance of measuring mRNA, so this consideration is very important.The mRNA absolute abundance can only be used for the gene differential expression of the sample of bioassay standardization.Though rule of thumb determining the standardization of the range of linearity of amplification curve and cDNA goods is tediously long and time-consuming process, consequent RT-PCR ^TMAnalysis can surpass the relative quantification RT-PCR of band inherent standard sample ^TMAnalyze.

A reason of this advantage is that all reagent can be converted into the single PCR in the range of linearity of amplification curve if there is not inherent standard sample/rival ^TMProduct, thus the sensitivity of analyzing improved.Another reason is if having only a kind of PCR ^TMProduct, on running gel or in the another kind of display packing, it is so not complicated that the demonstration of product just becomes, and background dies down and is easier to and explains.

Viii. chip technology

The inventor the special dna technique of considering that is based on chip, as Hacia et al. (1996) and Shoemaker et al. (1996) described those.In brief, these technology comprise the method for a large amount of genes of quantitative test exactly that reaches fast.By with the oligonucleotides marker gene or use fixing probe array, can the application chip technical point from the target molecule of high density arrays and by these molecules of screening by hybridization.Also referring to Pease et al. (1994); Fodor etal. (1991).

B. immunodiagnostics

Can be by such as the technology of ELISA and Western trace antibody of the present invention being used to characterize the TIF content of healthy and illing tissue.This can provide a kind of existence or non-existent method of screening malignant tumour, perhaps as the dopester of following cancer, perhaps in the present invention as prediction to being exposed to the method that may reply of oncolytic virus.

Consideration is applied to antibody of the present invention in the ELISA experiment.For example, the antibody of antitumor inhibiting factor is fixed on the selected surface, preferably presents the surface of protein affinity, as the hole of polystyrene microtiter plates.Behind the material that wash-out not exclusively adsorbs, need hole with nonspecific proteins combination or coating assay plate, known this nonspecific proteins is an antigen neutrality to the antiserum of test, as bovine serum albumin(BSA) (BSA), casein or milk power solution, this just can block the non-specific adsorption site of fixed surface, is incorporated into the background that the surface is produced thereby reduce by antigen non-specific.

Thereby antibodies is reduced background and wash-out not after the bound substances in the hole, with the coating of the material of anergy, immobilized surface is contacted with testing sample with a kind of method of immune complex (antigen/antibody) formation that helps.

Between the antibody of testing sample and combination, form after specific immunity compound and the wash-out subsequently, can resist and measure generation that the specific immunity compound forms and equal amounts by adding two, two is anti-to resisting different TIFs that specificity is arranged with one.Appropriate condition preferably includes with dilution such as BSA, ox gamma Globulin (BGG) and phosphate buffer (PBS)/tell

Dilute sample.The reagent of these interpolations also helps to help to reduce non-specific background.Subsequently with the antiserum of stratification about 2～4 hours of incubation under about 25 ℃～27 ℃ suitable temperature.After the incubation, thereby non-immune complex material is removed on the sero-fast surface of flushing contact.Preferred elution program comprises using such as PBS/ to be told

Or the solution of borate buffer solution carries out wash-out.

For a kind of detection method is provided, preferred two resist the enzymes that contain combination, and after with suitable chromophoric substrate incubation, this enzyme will produce color development.Therefore, for example, wish to promote to produce under the condition that the specific immunity compound forms, use in conjunction with the anti-human IgG of urase or peroxidase and (for example, at room temperature tell with contacting with incubation a period of time at solution that contains PBS such as PBS/ in conjunction with two anti-surfaces

Middle incubation 2 hours).

Educating with two temperature resistances of enzyme labeling and subsequently behind the unconjugated material of wash-out, peroxidase as enzyme target situation under, the amount of label is by coming quantitatively with the chromophoric substrate incubation, chromophoric substrate such as urea and bromcresol purple or 2,2 '-azo group-two-(3-ethyl-benzothiazole quinoline)-6-sulfonic acid (ABTS) and H ₂O ₂Finish quantitatively by the degree of measuring the color generation subsequently, for example, use the visible spectrum spectrophotometric determination.

Above-mentioned form can change into and earlier sample being incorporated on the assay plate, and then, with anti-and an assay plate incubation, using afterwards anti-ly has two of specific mark to resist to detect the anti-of combination to one.

Antibody compositions of the present invention is extensive use of in Western blotting or Western engram analysis.Antibody can be used to discern the albumen that is fixed on the solid supported matrix as the main agents of high-affinity, and the solid supported matrix for example is cellulose nitrate, polyamide fibre or its composition.Gel electrophoresis is carried out in the binding immunoassay precipitation reaction subsequently, and these antibody can be used as one step reagent and be used to detect antigen, and the second kind of reagent that is used to detect antigen causes the disadvantageous background of antigen.Based on this point, consider that specific use is used in combination with the Western trace based on immunologic detection method, comprise that enzyme, radioactive label or fluorescently-labeled antitumor inhibiting factor two are anti-.

IV. anti-hyperplasia therapy

The hyperplasia disorder is an a kind of disease family, is characterized as unusual or uncontrolled hyperplasia.These diseases are divided into two classes usually: typical cancer or " pernicious " disorder, and more not general optimum excessively proliferative disease, as optimum hypertrophy of the prostate, optimum galactophore epithelial cell hyperplasia, endometrial hyperplasia, thyroid gland hypertrophy and epidermis or corium epithelial hyperplasia.Though a lot of aspects of Tao Luning are to concentrate on the cancer afterwards, it will be appreciated that method identical when suitable also is applicable to benign disease.

With typically a benign disease unusual on cell quantity is different, cancer refers to exist dysplastic on one's body the patient, form tumour or inappropriate hyperplasia, cancer and/or cell transformed, comprise for example dysplastic, tumour, noncontact cell that suppress or neoplastic transformation or the like (for example, melanoma, cancer such as gland cancer, squamous cell carcinoma, small cell carcinoma, oat-cell carcinoma or the like, sarcoma such as fibrosarcoma, chondrosarcoma, osteosarcoma or the like, hepatoma, neuroblastoma, melanoma, hemopoietic system malignant tumour such as lymthoma, leukaemia, myeloma or the like), these are for known in the art, and have set up the standard of diagnosis and classification for it.Be not subjected to the restriction of any theory, the oncolytic characteristic of reovirus may be derived to the hiv chemokine of the malignant conversioning cell relevant with the intracellular environment of sensitivity to, for example, as the PKR phosphorylation that weakens in the described Ras active cell in this area, or contain damaged tumor inhibiting factor subfunction as herein defined.

A. oncolytic virus therapy

In treatment of cancer, have description (for example, United States Patent (USP) 7,300,650,7 about the generation of wild type reovirus and the ad hoc approach of use, 163,678,7,049,127,7,014,847,6,994,858,6,811,775,6,703,232,6,576,234,6,565,831,6,528,305,6,455,038,6,344,195,6,261,555,6,136,307 and 6,110,461, and U.S. Patent Publication text 2006/0165724,2006/0073166,2005/0123513,2004/0265271,2004/0126869,2004/0109878,2002/0037543,2006/0029598,2005/0026289,2002/0006398 and 2001/0048919, its full content is incorporated herein by reference in this nothing) with withdrawing a claim.The myxoma virus therapy is described in U.S. Patent Publication text 2006/0263333 to some extent, also is incorporated herein by reference.

B. purify therapy

The present invention provides the method that purifies the cell population of cancer cell in a particular embodiment.For example, can accept the candidate stem cell treatment by using the myeloid tissue that purifies subsequently with the band hemopoietic system of clear marrow sex therapy treatment or the individuality of solid carcinoma, the myeloid tissue of purification is by obtaining in the individuality before chemotherapy.In these embodiments, thus can with oncolytic virus handle that marrow is removed or killing tumor cell to reduce or to remove damage to candidate stem cell.

Use patient's oneself ripe stem cell is used for cell therapy and can avoids problems such as host immune, graft versus host disease and ethics subject under discussion.Therefore people's ripe stem cell has extensive value in various regeneration therapies.Yet, it is reported, spontaneous nuclear transformation (Tolar et al., 2007 can take place in the cultured and amplified in vitro of ripe stem cell; Romano, 2005).Therefore, for safer use in transplanting and regeneration, need suitable purification strategy remove the cell that is present in the spontaneous nuclear transformation in the ripe stem cell of cultivating amplification.Report shows that the wild type reovirus can effectively remove cancer cell (Thirukkumaran et al., 2003 that are present in self hematopoietic stem cell populations external; Thirukkumaran et al., 2005).Attenuation type reovirus and myxoma virus can be used in a similar manner.The inventor expect the present invention can be actually with any maturation at present known or that find afterwards or embryonic stem cell line use.

In a particular embodiment, may need from cell composition, to remove reovirus, for example, come killing tumor cell (if existence) through one section time enough after.For example, thus marrow can be by obtaining and handle with reovirus to destroy tumour cell in the individuality; Yet, before being implanted to marrow in the individuality again (for example, the cancer patient of immunodeficiency), may need to destroy, eliminate and/or remove the virus in the composition.In these embodiments, can add Anti-virus agent in cell composition.These reagent comprise the antibody and the complement of anti-reovirus, the RNA polymerase inhibitor that the inhibitor of necessary viral enzyme such as RNA instruct, or the viral interference reagent successfully packing, assemble or discharge from infection cell.

Other purification step comprises wash-out and/or centrifugal separating cell, can be used to further remove from cell composition virus and/or dead cell.These methods are known in the art and comprise the method for the cell of the required type of enrichment, comprise based on fluorescence activated cell or based on the method that adheres to, this method can be given the positive-selecting of target cell such as stem cell colony.

C. conjoint therapy

In order to improve effectiveness by oncolytic virus selective killing cancer cell, may need in a particular embodiment to come exposing cell, as chemotherapeutant, radiotherapy, immunotherapy, hormonotherapy, toxinotherapy, with another kind of natural or therapy or gene therapy that engineering virus carries out with other antitumor and anticancer agent.

Chemotherapeutant includes but not limited to 5 FU 5 fluorouracil, bleomycin, busulfan, camptothecine, NSC-241240, Chlorambucil, neoplatin (CDDP), endoxan, dactinomycin D, daunorubicin, adriamycin, the estrogen receptor binding reagents, etoposide (VP16),, farnesyl protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, new mould acid amides, nitroso ureas, plicamycin, procarbazine, Raloxifene, tamoxifen, taxol, Temozolomide (liquid DTIC), cis-platinum, vincaleukoblastinum and methotrexate (MTX), vincristine or above-mentioned any analog or derivant variant.According to them in intracellular active mode with these reagent or classification of drug, for example, whether and in which they influence the cell cycle in stage.Perhaps, can based on it directly with DNA is crosslinked, insertion DNA or synthesize and cause the feature that the unusual ability of chromosome and mitosis is represented reagent by influencing nucleic acid.Most of chemotherapeutant can be divided into following kind: alkylating reagent, metabolic antagonist, antitumor antibiotics, corticosteroid hormone, mitotic inhibitor and nitroso ureas, hormone reagent, mix reagent and any analog or derivant variant.

Oncolytic therapy can with other agent administration in cancer cell, carry out afterwards or before, the time interval be several minutes to several weeks.For example, in this class embodiment,, can relate to various modes and basically side by side (for example, be less than within 1 minute) contact cancer cell, tissue, organ or biosome approximately as the attenuated type reovirus.In other respects, can be before giving attenuation type or wild type reovirus and/or about afterwards 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 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks or about 8 weeks or more, and any by in its time range of deriving out, or side by side give one or more reagent.

Attenuation type reovirus or wild type reovirus can be got up use with one or more agent combination.The non-limiting example that below shows this class combination, wherein oncolytic virus is " A " and antitumor and anticancer agent is " B ":

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

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

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

I. chemotherapy

Various chemotherapeutants can be used for the present invention.Term " chemotherapy " refers to use medicine to treat cancer." chemotherapeutant " is used in reference to a kind of compound or the composition that gives in the treatment cancer.According to them in intracellular active mode with these reagent or classification of drug, for example, whether and in which they influence the cell cycle in stage.Perhaps, can based on it directly with DNA is crosslinked, insertion DNA or synthesize and cause the feature that the unusual ability of chromosome and mitosis is represented reagent by influencing nucleic acid.Most of chemotherapeutant can be divided into following kind: alkylating reagent, metabolic antagonist, antitumor antibiotics, mitotic inhibitor and nitroso ureas.

Alkylating reagent.Alkylating reagent is direct and genomic DNA interacts and the medicine of prevention cancer cell hyperplasia.This class chemotherapeutant representative influences the reagent in cell cycle in all stages, that is to say that they are not phase specificities.Alkylating reagent can be used to treat chronic leukemia, non-Hodgkin lymphoma, hodgkin's disease, Huppert's disease, and the particular cancers of mammary gland, lung and ovary.They comprise: busulfan, Chlorambucil, neoplatin, endoxan (cyclophosphamide), dacarbazine, ifosfamide, mechlorethamine (mustargen) and melphalan.Troglitazaone can with these alkylating reagents in any one or more kinds of the associating be used for the treatment of cancer, some of them are discussed below.

Busulfan (being also referred to as the bridle orchid) is a kind of difunctional alkylating reagent.Busulfan chemically is being called as 1, myleran.

Busulfan is not the analogue of mustargen.Busulfan is used for oral administration with tablet form.Each scored tablet comprises 2mg busulfan and non-active ingredient dolomol and sodium chloride.

Busulfan is used to the leukemic mitigation treatment of chronic marrow (marrow, myelocytic, granulocytic).Though be not curing property, busulfan has reduced granulocytic sum, has alleviated the symptom of disease, and has improved patient's clinical symptoms.About 90% adult who had not before treated chronic myelogenous leukemia can obtain that the organomegaly is disappeared or stable hematologic response after using busulfan.With regard to survival period and pigment horizontal stable, its curative effect is better than the irradiation spleen, and is equating with curative effect with radiation exposure aspect the control spleen enlargement.

Chlorambucil (being also referred to as chlorambucil) is a kind of alkylating reagent of bifunctional mustargen type, and being found on the non preference human tumor disease has activity.The chemical name of Chlorambucil is two (2-chloroethyl) amino of 4-[] phenylbutyric acid.

Chlorambucil is used for oral administration with tablet form.It can be absorbed by intestines and stomach quick and completely.After giving single oral dose 0.6-1.2mg/kg, blood Chlorambucil level can arrive the peak in 1 hour, and the half life period of final original medicine is estimated as 1.5 hours.0.1-0.2mg/kg/day or 3-6mg/m ²/ day or optionally 0.4mg/kg can be used for antineoplaston.Methods of treatment is well known to those skilled in the art and can finds in " Physicians Desk Reference " and " Remington ' sPharmaceutical Sciences " that quoted here.

Chlorambucil is used to treat chronic lymphatic (lymphocytic) leukaemia, malignant lymphoma comprises lymphosarcoma, giant follicular lymphoma and hodgkin's disease.It is not a curing property in these illnesss any, but can produce effective mitigation clinically.Therefore, it can unite use with troglitazone in treatment of cancer.

Neoplatin is widely used in treating cancer such as metastatic testis or oophoroma, late period carcinoma of urinary bladder, head or neck cancer, cervix cancer, lung cancer or other tumours.Neoplatin can use separately or unite use with other reagent, and its effective dose in clinical practice is 15-20mg/m ², per three weeks administration 5 days, three courses of treatment altogether.Exemplary dosage can be 0.50mg/m ², 1.0mg/m ², 1.50mg/m ², 1.75mg/m ², 2.0mg/m ², 3.0mg/m ², 4.0mg/m ², 5.0mg/m ², 10mg//m ²Certainly, these all dosage all are exemplary, and any dosage that also expects between these points can be used for the present invention.

Neoplatin can not be by oral absorption, therefore must be by in vein, subcutaneous, the knurl or intraperitoneal injection.

Endoxan is 2H-1,3, and 2-oxynitride phosphor-2-amine, N, two (2-chloroethyl) tetrahydrochysenes of N-, 2-oxygen, monohydrate; Be called cyclophosphamide, can obtain from Mead Johnson company; Be called Neosar, can obtain from Adria company.Under the catalytic action of triethylamine with amino 1-propyl alcohol of 3-and N, two (2-chloroethyl) phosphinylidyne dichloro [(ClCH of N- ₂CH ₂) ₂N--POCl ₂] condensation prepared endoxan in dioxane solution.Condensation reaction is double, relates to hydroxyl and amino group, thereby realizes cyclisation.

Different with the amino alkylating agent of other β-ethyl chloride, it was not cyclized into activated azacyclopropane form easily before being activated by the liver enzyme.Therefore, this material is stable in intestines and stomach, is well-tolerated and effective by oral and parent's administration, and do not cause to blister in the part, gangrenous, phlebitis even pain.

The proper dosage that is used to be grown up comprises that oral, 1-5mg/kg/day (administering drug combinations usually) depends on gastrointestinal tolerant power; Or 1-2mg/kg/day; Intravenous injection, initial in 2-5 days time divided dose injection 40-50mg/kg or per 7 to 10 days injection 10-15mg/kg or inject twice 3-5mg/kg weekly or 1.5-3mg/kg/day.The dosage that can give 250mg/kg/day is as antitumor.Because to the detrimental effect of stomach and intestine, intravenous approach is preferred the use.In stationary phase, need 3000-4000/mm usually ³The leucocyte amount.Sometimes also carry out administered intramuscular by soaking into or entering body cavity.Injection 100,200 and 500mg and tablet 25 and 50mg are feasible dosage forms, and with reference to " Remington ' s Pharmaceutical Sciences " the 15th edition, 61 chapters are introduced as a reference at this about the professional technique of the details of dosage.

Melphalan is also referred to as melophalan, Phenylalanin-Lost, melphalan, L-PAM or Phenylalanin-Lost, is the phenylalanine derivative of mustargen.Melphalan is a kind of difunctional alkylating reagent, and it has activity on non preference human tumor disease.Its chemical name is two (2-ethyl chloride) amino of 4-[]-the L-phenylalanine.

Melphalan is this compound activated L type isomeride and is synthetic by Bergel and Stock in nineteen fifty-three at first; D type isomeride is called d-sarcolysine, and the activity on specific animal tumor is lower, and produce to render a service required dosage than required big of L type isomeride on chromosome.The form of racemic (DL-) is called as melphalan or sarcolycin.Melphalan is insoluble in water, its pKa ₁Value is for～2.1.Melphalan can the tablet form oral medication and is used for the treatment of Huppert's disease.

Available evidence shows that about 1/3rd to 1/2nd myelomatosis multiplex people presents favourable reaction to oral this medicine.

Melphalan is used to treat the ovarian epithelial cell cancer.A kind of normally used method that is used for the treatment of oophoroma is the dosed administration with 0.2mg/kg melphalan every day, and administration 5 days is as a course of treatment.According to the blood tolerance, (Smith and Rutledge, 1975 repeat a treatment in per four-five weeks; Young et al, 1978).Perhaps the using dosage of melphalan can be low to moderate 0.05mg/kg/day or high to 3mg/kg/day, or any dosage between these dosage or that be higher than these dosage.According to patient's to be treated situation, on the dosage some variations must be arranged.Under any circumstance, should be to the suitable dosage of individual patient by the reaction decision of human body to administration.

Metabolic antagonist.Metabolic antagonist destroys the synthetic of DNA and RNA.Different with alkylating reagent, they influence the cell cycle in S phase specificity.Except mammary gland, ovary and gastroenteric tumor, they also are used to resist chronic leukemia.Metabolic antagonist comprises 5 FU 5 fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine and methotrexate.

The chemical name of 5 FU 5 fluorouracil (5-FU) be 5-fluoro-2,4 (1H, 3H)-hybar X.Its mechanism of action is considered to be undertaken by the methylation reaction that the blocking-up deoxyuridylic acid becomes thymidylic acid.Therefore, 5-FU disturbs the synthetic of DNA (deoxyribonucleic acid) (DNA) and suppresses the formation of RNA (ribonucleic acid) (RNA) on than low degree.Because DNA and RNA are essential for cell division and propagation, thereby so the effect of 5-FU be considered to produce the thymidine defective and cause cell death.Therefore, 5-FU can be applicable in the cell with metastatic cancer feature of division rapidly.

Antitumor antibiotics.Antitumor antibiotics contains antibiotic property and two kinds of activity of cytotoxicity.These medicines are also by in chemically inhibitory enzyme and mitosis or change cell membrane and disturb DNA.These reagent are not phase specificities, so they all worked in all stages of cell cycle.Therefore, they are widely used in various cancers.Antitumor antibiotics comprises bleomycin, dactinomycin D, daunorubicin, adriamycin (adriamycin) and jaundice element, and some of them can discuss in more detail below.These compounds are widely used in clinical setting and are used for the treatment of tumour, and dosage range gives the 25-75mg/m of adriamycin from passing through the administration of vein bolus injection with 21 days intervals ², to the 35-100mg/m of intravenous injection or orally give etoposide ²

ADMh; 5,12-naphthalenedione, (8s-is suitable)-10-[(3-amino-2; 3; 6-three deoxidations-a-L-lysol-own pyranose) oxygen]-7,8,9; 10-tetrahydro-6; 8,11-trihydroxy-8-(glycolyl)-1-methoxyl-hydrochloride (hydroxydaunomycin hydrochloride, adriamycin) is used as broad-spectrum anti-cancer drug.It is incorporated into DNA and suppresses nucleic acid synthetic, suppresses mitosis and impels chromosome aberration.

When individually dosed, it is the choice drug that is used for the treatment of thyroid adenoma and early hepatocyte cancer.It is 31 kinds of a kind of compositions in the first-selected combination, is used for the treatment of ovary, endometrium and mastadenoma, bronchus green oats cell cancer, ridicules cell lung cancer, sdenocarcinoma of stomach, retinoblastoma, neuroblastoma, mycosis fungoides, cancer of pancreas, prostatitis adenosquamous carcinoma, carcinoma of urinary bladder, myeloma, more warm nature histocytic lymphoma, Wilms' tumor, hodgkin's disease, suprarenoma, osteogenic sarcoma, soft tissue sarcoma, Ewing's sarcoma, rhabdomyosarcoma and acute lymphatic leukemia.It is a kind of alternative drugs that is used for the treatment of islet-cell carcinoma, cervix cancer, testis and adrenocortical carcinoma.It also is a kind of immunodepressant.

The adriamycin malabsorption must intravenous administration.Pharmacokinetics is a multicell.The half life period of allocated phase is 12 minutes and 3.3 hours.The half life period of getting rid of is about 30 hours.40-50% is secreted in the bile.The metabolism in liver of most residue, part metabolism become activated metabolin (adriamycinol), but have some number percents to be secreted in the urine.When having hepar damnification, dosage need reduce.

Adult's intravenous suitable dosage is to give 60-75mg/m with 21 days intervals ², or repeated to give 25-30mg/m in every continuous 2 or 3 days with 3 or 4 weekly intervals ², or give 20mg/m weekly ²When having the early stage bone marrow suppression that causes by early stage chemotherapy or the invasion of tumour marrow, perhaps when this medicine is united use with other myelocytes generation suppressive drug, should use lowest dose level to aged patient.If serum bilirubin is at 1.2-3mg/dL, dosage should reduce by 50% so, if be higher than 3mg/dL, should reduce by 75% so.Should not surpass 550mg/m to the accumulated dose of patient between the operating period that the normal heart function is arranged ², and should not surpass 400mg/m to the patient who has accepted mediastinum radiotherapy ²Perhaps, whenever give 30mg/m for three days on end ², whenever repeat once all around.Exemplary dosage can be 10mg/m ², 20mg/m ², 30mg/m ², 50mg/m ², 100mg/m ², 150mg/m ², 175mg/m ², 200mg/m ², 225mg/m ², 250mg/m ², 275mg/m ², 300mg/m ², 350mg/m ², 400mg/m ², 425mg/m ², 450mg/m ², 475mg/m ², 500mg/m ²Certainly, these all dosage all are exemplary, and any dosage that also expects between these points can be used for the present invention.

Daunorubicin hydrochloride, 5,12-naphthalenedione, (8s-is suitable)-10-[(3-acetyl group-2,3, the 6-three deoxidations-own pyranose of a-L-lysol lyxo-) oxygen]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-10-methoxyl-hydrochloride; Be also referred to as daunorubicin, can obtain from Wyeth company.Daunorubicin inserts DNA, and the RNA polymerase that blocking dna instructs also suppresses DNA and synthesizes.It can be with the not synthetic dosage prevention mitosis of interfere RNA.

With the other drug associating, it can be used for the acute stage as first-selected chemotherapy treatment adult's acute myelocytic leukemia (being used for promoting to slow down), acute lymphatic leukemia and chronic granulocytic leukemia.The daunorubicin oral absorption is bad, and it must intravenous administration.Distributing the half life period is 45 minutes and get rid of the half life period and be about 19 hours.The half life period of its active metabolite daunorubicinol is about 27 hours.The metabolism and also be secreted into (ca 40%) in the bile in liver of daunorubicin major part.Dosage must reduce in the incomplete individuality of liver or renal function.

The intravenous suitable dosage (radix equivalence) of being grown up is: patient below 60 years old per 3 or 4 weeks injection 45mg/m ²(patient more than 60 years old is 30mg/m to/day ²) 1,2 or 3 day, perhaps per 3 or 4 weeks injection 0.8mg/kg/day 3-6 days; If except having carried out the chest irradiation can only be 450mg/m ²Outside, administration during use should not surpass 550mg/m ²For children, unless the age be less than 0.5m less than 2 years old or health area ², be weekly 25mg/m ², to use adult's table in this case based on body weight.Injectable dosage form (radix equivalence) 20mg (radix is equivalent to the hydrochloride of 21.4mg) is feasible.Exemplary dosage can be 10mg/m ², 20mg/m ², 30mg/m ², 50mg/m ², 100mg/m ², 150mg/m ², 175mg/m ², 200mg/m ², 225mg/m ², 250mg/m ², 275mg/m ², 300mg/m ², 350mg/m ², 400mg/m ², 425mg/m ², 450mg/m ², 475mg/m ², 500mg/m ²Certainly, these all dosage all are exemplary, and any dosage that also expects between these points can be used for the present invention.

Mitomycin (being also referred to as mutamycin and/or Mitomycin-C) is the microbiotic of separating in a kind of culture by the head streptomysin, and it has antitumor activity.This compound is heat-staple, and the fusing point height can be dissolved in the organic solvent in a large number.

The mitomycin selectivity suppresses the synthetic of DNA (deoxyribonucleic acid) (DNA).The content of guanine and cytimidine is relevant with the crosslinking degree that mitomycin is induced.Under this medicine of high concentration, cell RNA and albumen are synthetic also to be downtrod.

In human body, behind intravenous administration, mitomycin can promptly be removed from serum.To reduce for 50% required time be 17 minutes to serum-concentration after the bolus injection 30mg.After intravenous injection 30mg, 20mg or 10mgI.V., maximum serum concentration is respectively 2.4mg/ml, 1.7mg/ml, and 0.52mg/ml.Remove the influence that mainly is subjected to metabolism in the liver, but metabolism can occur in its hetero-organization also.Owing to thinking the saturated of degraded path, the speed and the maximum serum concentration of removing are inverse ratio.The mitomycin of about 10% dosage is not secreted in the urine with changing.Because metabolic pathway is saturated under low relatively dosage, the percentage that is secreted into the mitomycin in the urine increases along with the increase of dosage.For children, the secretion of the mitomycin of intravenous administration is similar therewith.

Actinomycin D (dactinomycin D) [50-76-0]; C ₆₂H ₈₆N ₁₂O ₁₆(1255.43) be a kind of antineoplastic, it suppresses the RNA polymerase that DNA relies on.It is a kind of composition that is used for the treatment of in the first-selection combination of choriocarcinoma, embryonal rhabdomyosarcoma, testicular tumor, Wilms' tumor.Tumour to systematization treatment answer failed is replied local perfusion therapy sometimes.Dactinomycin D can strengthen radiotherapy.It is Secondary cases (spreading out of a property) immunodepressant.

Actinomycin D and main especially vincristine and cyclophosphamide combined use of operation, radiotherapy and other drug.Its antitumor activity shows in Ewing's tumor, Kaposi and soft tissue sarcoma significantly.Dactinomycin D late among the women of choriocarcinoma case effectively.It is united use with Chlorambucil and methotrexate (MTX) the patient of metastatic carcinoma of testis is also produced identical reaction.Observe on one's body the patient of hodgkin's disease and non-Hodgkin lymphomas sometimes and reply.Dactinomycin D also is used to suppress immunological response, especially to the repulsion of kidney transplant.

One half-value dose intactly is secreted in the bile and 10% in urine; Half life period is about 36 hours.Medicine does not pass through blood brain barrier.Actinomycin D is with the form supply (every bottle 0/5mg) of freeze-dried powder.Usually the dosage of every day is 10-15mg/kg; Intravenous injection 5 days; If do not have the toxigenicity performance, can give a course of treatment again with the time interval in 3-4 week.Inject 100-400mg every day for children, continue 10-14 days; In additive method, use 3-6mg/kg, 125mg/kg altogether, and fixed dosage is 7.5mg/kg weekly.Though safer in the conduit with medicine afferent veins infusion, but give direct intravenous injection, for avoiding subcutaneous reaction, its preventive measure are discarded syringe needles that are used for extracting from bottle medicine.Exemplary dosage can be 100mg/m ², 150mg/m ², 175mg/m ², 200mg/m ², 225mg/m ², 250mg/m ², 275mg/m ², 300mg/m ², 350mg/m ², 400mg/m ², 425mg/m ², 450mg/m ², 475mg/m ², 500mg/m ²Certainly, these all dosage all are exemplary, and any dosage that also expects between these points can be used for the present invention.

Bleomycin is the potpourri by the cytotoxin glycopeptide antibiotic of separating in the streptomyces verticillus.Though definite mechanism of action the unknown of bleomycin has available evidence to show and it seems that its main mechanism is suppress DNA synthetic, also has some evidences to show the synthetic of less important inhibition RNA and albumen.

In mouse, find that the bleomycin of high concentration is present in skin, lung, kidney, peritonaeum and the lymphatic vessel.Opposite with the low concentration in hematopoietic tissue, can find that the tumour cell of skin and lung contains the bleomycin of high concentration.The bleomycin of the low concentration of finding in the marrow may be relevant with the high-caliber bleomycin digestive enzyme of discovery in these tissues.

In the patient of creatinine removing＞per minute 35ml, the half life period that serum or blood plasma are finally got rid of bleomycin approximately is 115 minutes.Remove among the patient of per minute＜35ml at creatinine, the final half life period of getting rid of of blood plasma or serum is along with weakening of creatinine removing is exponential increase.In human body, the dosage that 60%-70% gave is recovered in the urine with the form of active bleomycin.Can give bleomycin by muscle, vein or subcutaneous route.It can dissolve in water in large quantities.

Bleomycin should be considered for the therapy of releiving.The report demonstration is arranged, and it can effectively treat following tumour with single reagent form or with confirmed forms with other authorized chemotherapeutants combinations: squamous cell carcinoma such as head and neck (comprising mouth, tongue, tonsillotome, nasopharynx, oropharynx, hole, maxilla, lip, mucous membrane of mouth, gums, epiglottis, larynx), skin, penis, cervix and vaginal orifice.It also is used to treat lymthoma and carcinoma of testis.

Because the lymthoma patient has anaphylactoid possibility, should or still less treat patient with 2 units for initial two doses.If acute reaction does not take place, so just can follow conventional dose form.

Can be rapidly in two weeks and significantly improve hodgkin's disease and carcinoma of testis.If do not see improvement in this time, the possibility of Gai Shaning is just little so.The squamous cell carcinoma response is slower, needs sometimes to reach and 3 weeks just can see some improvement.

Mitotic inhibitor.Mitotic inhibitor comprises plant alkaloid and other natural agents, and it can suppress the synthetic or mitosis of the required albumen of cell division.They work at the particular stage of cell cycle.Mitotic inhibitor comprises docetaxel, etoposide (VP16), taxol, taxol, taxotere, vincaleukoblastinum, vincristine and vinorelbine.

VP16 is also referred to as etoposide, and its main associating with bleomycin and neoplatin is used for the treatment of orchioncus, unites with neoplatin and is used for the treatment of small-cell carcinoma of the lung.It also has the activity of anti-non-Hodgkin lymphomas, acute nonlymphocytic leukemia, breast cancer and the Kaposi relevant with aids (AIDS).

VP16 can be used for intravenous administration and be used for oral administration with the form of the liquid filled capsules of 50mg with solution (20mg/ml) form.For small cell carcinoma of lung, intravenous injection dosage (in conjoint therapy) can be up to 100mg/m ²Or be low to moderate 2mg/m ², also can use administration every day 35mg/m usually ²Continuous administration 4 days-every day 50mg/m ²Continuous 5 days.When oral administration, dosage need double.Therefore for small-cell carcinoma of the lung, dosage can be up to 200-250mg/m ²The intravenous injection dosage (at conjoint therapy) that is used for carcinoma of urinary bladder is administration every day 50-100mg/m ²Continuous 5 days, or administration 100mg/m every other day ²Continuous three doses.Common every 3-4 week of treatment cycle repeats once.Should be in 30-60 minute slowly the infusion administration thereby to avoid may be because hypopiesia and the bronchial spasm that the solvent in the prescription causes.

Taxol is an antimitotic reagent experimentally, by the bark separation of ash tree Chinese yew Taxusbrevifolia.It is incorporated into tubulin (with vinca alkaloids used site completely different) and promotes the microtubule assembling.Carry out clinical evaluation over against taxol at present; It has the activity of anti-chromoma and oophoroma.Maximum dose is administration every day 30mg/m ²Continuous 5 days or per 3 all administration 210-250mg/m ²Certainly, all these dosage all are exemplary, and any dosage that also expects between these points also can be used for the present invention.Vincaleukoblastinum is another example of plant alkaloid, and it can be united with troglitazone and be used for the treatment of cancer and precancer.When with the vincaleukoblastinum cultured cell, the disintegration of microtubule can take place.

It is reported behind the vincaleukoblastinum or vincristine of orally give, have unexpected absorption.After giving conventional clinical dosage, the maximum concentration of every kind of medicine is approximately 0.4mM in the blood plasma.Vincaleukoblastinum and vincristine are incorporated into plasma proteins.They extensively concentrate on blood platelet and are present on than low degree in leucocyte and the red blood cell.

After intravenous injection, vincaleukoblastinum has the multiple form of removing from blood plasma; After distribution, the half life period that medicine disappears from blood plasma is approximately 1 and 20 hours.Vincaleukoblastinum is metabolised to the derivant desacetyl vinblastine of biologic activity in liver.After choleresis, about 15% dosage can detect in urine intactly, and 10% be recycled in the excrement.Patient for hepatosis should reduce dosage.If the bilirubinic concentration of blood plasma need reduce by 50% dosage so at least greater than 3mg/dl (about 50mM).

The form that vinblastine sulfate provides is the goods that can be used for injecting.This medicine passes through intravenous administration; Need take special preventive measure to resist subcutaneous oozing of blood, because this can cause pain stimulation and ulcer.Medicine should be expelled to the health end that circulation weakens.Give single dosage 0.3mg/kg by body weight after, bone marrow suppression reaches its mxm. in 7-10 days.If can not obtain mitigation level (about 3000cells/mm of leukopenia ³), dosage weekly can progressively improve by the increment of body weight with 0.05mg/kg.In the therapy that is designed for the treatment carcinoma of urinary bladder, the using dosage of vincaleukoblastinum need not be considered blood cell count or toxicity for per 3 all 0.3mg/kg.

The most important clinical practice of vincaleukoblastinum is to be used from curative therapy metastatic bladder knurl with bleomycin and neoplatin one.It is reported in multiple lymthoma, to have favourable replying, especially hodgkin's disease, in this case of 50-90%, can see significant improvement.When disease was difficult to be cured by alkylating reagent, the effectiveness of vincaleukoblastinum in a high proportion of lymthoma did not reduce.It is also to Kaposi, neuroblastoma and Letterer-Siwe disease (histiocytosis X), and women's breast cancer and choriocarcinoma work.

The dosage of vincaleukoblastinum is to be determined according to each patient's needs by the clinician.0.1-0.3mg/kg can be given or also 1.5-2mg/m can be given ²Optionally, can give 0.1mg/m ², 0.12mg/m ², 0.14mg/m ², 0.15mg/m ², 0.2mg/m ², 0.25mg/m ², 0.5mg/m ², 1.0mg/m ², 1.2mg/m ², 1.4mg/m ², 1.5mg/m ², 2.0mg/m ², 2.5mg/m ², 5.0mg/m ², 6mg/m ², 8mg/m ², 9mg/m ², 10mg/m ², 20mg/m ²Certainly, these all dosage all are exemplary, and any dosage that also expects between these points can be used for the present invention.

Vincristine is blocked mitosis and is caused that stop metaphase.As if most of biologic activity of this medicine can specificity be incorporated into tubulin and blocks protein by it and is aggregated to the ability of microtubule and explains probably.By destroying the microtubule of mitosis organ, cell division will stop at metaphase.In mitosis, can not correctly separate chromosome and may cause cell death.

Vincristine makes this reagent unusual in antineoplastic to normal marrow cell with epithelial relative low toxicity, and it usually and other myelosuppressives unite use.

It is reported behind orally give vincaleukoblastinum or vincristine, have unexpected absorption.After giving conventional clinical dosage, the maximum concentration of every kind of medicine is approximately 0.4mM in the blood plasma.

Vincaleukoblastinum and vincristine are incorporated into plasma proteins.They extensively concentrate on blood platelet and are present on than low degree in leucocyte and the red blood cell.

Vincristine contains the multiple form of removing from blood plasma; About 24 hours of its final half life period.This medicine by metabolism, does not still identify the derivant that it has biologic activity in liver.Patient for hepatosis should reduce dosage.If the bilirubinic concentration of blood plasma need reduce by 50% dosage so at least greater than 3mg/dl (about 50mM).

Vincristine sulphate can solution form (1mg/ml) be used for intravenous injection.It is to be used to impel the optional therapy of alleviation leukemia of children at present that vincristine and corticosteroid are united use; It seems that the optimal dose of these medicines be by body surface area intravenous injection vincristine 2mg/m weekly ², and every day orally give prednisone 40mg/m ²Hodgkin's disease or the lymphadenomatous adult patient of Fei Huoqijinshi disease accept the part of vincristine as conjoint therapy usually.When using in the MOPP therapy, the RD of vincristine is 1.4mg/m ²Leukemic children seem better than the adult to the tolerance of the vincristine of high dose, and the adult may stand serious neurotoxicity.Seem to improve the toxicity performance and also disproportionate improvement on reactive ratio than once more frequent administration in 7 days or higher dosage.When intravenous injection gives vincristine, also should take preventive measures and avoid oozing of blood.Under the similar condition of toxicity, the dosage ratio that vincristine (and vincaleukoblastinum) can be infused in the tumour artery supply blood can be by the high several times of dosage of intravenous administration.

Vincristine has effect in hodgkin's disease and other lymthomas.Though when it was used for hodgkin's disease separately, it seemed lower a little than the effectiveness of vincaleukoblastinum, when uniting use with mustargen, prednisone and procarbazine (so-called MOPP therapy), it is the first-selected therapy of these terminal stages of a disease (III and IV).In the non-Hodgkin lymphomas, vincristine is a kind of important reagent, especially when uniting use with endoxan, bleomycin, adriamycin and prednisone.In lymphocytic leukemia, vincristine is more effective than vincaleukoblastinum.It is reported in the patient of multiple other tumours, to have favourable replying, especially Wilms' tumor, neuroblastoma, brain tumor, the reproductive system cancer of rhabdomyosarcoma and mammary gland, bladder and masculinity and femininity.

The employed dosage of vincristine is according to each patient's needs and definite by the clinician.Can give 0.01-0.03mg/kg or 0.4-1.4mg/m ², perhaps also can give 1.5-2mg/m ²Optionally, can continue venoclysis and give 0.02mg/m ², 0.05mg/m ², 0.06mg/m ², 0.07mg/m ², 0.08mg/m ², 0.1mg/m ², 0.12mg/m ², 0.14mg/m ², 0.15mg/m ², 0.2mg/m ², 0.25mg/m ²Certainly, these all dosage all are exemplary, and reckon with that also any dosage between these points can be used for the present invention.

Camptothecine is a kind of alkaloid that is derived from the tree camplotheca acuminata of China.Camptothecine and derivant thereof have unique ability, can suppress the DNA topoisomerase by the stable covalent reaction intermediate product that is called " can shear compound ", finally cause death of neoplastic cells.Generally believe that camptothecin analogues presents significant antitumor and anti-leukocythemia liveness.Because its serious adverse and bad water-soluble, camptothecine application clinically is limited.At present, some camptothecin analogues (Hycamtins; CPT-11), no matter that synthesize or semisynthetic, be used to treatment of cancer and demonstrated gratifying clinical effectiveness.The molecular formula of camptothecine is C ₂₀H ₁₆N ₂O ₄, molecular weight is 348.36.It is supplied with the yellow powder form, and can be dissolved as the limpid yellow solution of 50mg/ml in DMSO 1N NaOH.If be stored in 2-8 ⁰In the environment of the drying sealing lucifuge of X, it can be stablized 2 years at least.

Nitroso ureas.Nitroso ureas, the same with alkylating reagent, suppress dna repair protein.They are used to treat non-Hodgkin lymphomas, Huppert's disease, chromoma and brain tumor.Embodiment comprises Carmustine and lomustine.

Carmustine (aseptic Carmustine) is to be used for the treatment of a kind of in the nitroso ureas of specific tumors disease.It is 1,3 pair of (2-ethyl chloride)-1-nitroso ureas.It is faint yellow freeze-drying sheet or freeze-drying piece, and molecular weight is 214.06.Its solubleness height in alcohol and lipid, indissoluble in water.The administration of suggestion Carmustine is to restore the back at it to give by venoclysis.Aseptic Carmustine provides with the form of the freeze dried substance of the single dosage bottle of 100mg usually.

Though it has been generally acknowledged that Carmustine alkylation DNA and RNA, it does not intersect resists other alkylating reagents.With other nitroso ureas, it also can suppress the enzymatic process of a plurality of keys by making amino acid carbamoylation in the albumen.

Now show, Carmustine can single reagent form, or is used for the treatment of brain tumor such as spongioblastoma, brain stem glioma, medulloblastoma, astrocytoma, ependymoma and metastatic brain tumor with the conjoint therapy form set up with other authorized chemotherapeutants as slowing down therapy.It is also united with prednisone and is used for the treatment of Huppert's disease.Proved that Carmustine is effectively on treatment hodgkin's disease and non-Hodgkin lymphomas, and with other authorized medication combined uses as the recurrence or to the unresponsive patient of therapy first after the therapy first of secondary therapy for treating.

Carmustine is per 6 all intravenous injection 150-200mg/m as the formerly untreated patient of single reagent RD on one's body ²This administration can be used as single dosage and gives or be divided into every day injection as 75-100mg/m, injects continuously 2 days.Unite when using or being used in patient that the marrow deposit exhausts on one's body when Carmustine and other myelosuppressives, should correspondingly adjust dosage.Should reply the dosage of adjusting behind the predose to the blood of above-mentioned dosage according to patient.Certainly it should be understood that other dosage also can be used for the present invention, for example 10mg/m ², 20mg/m ², 30mg/m ², 40mg/m ², 50mg/m ², 60mg/m ², 70mg/m ², 80mg/m ², 90mg/m ², or 100mg/m ²Please the professional and technical personnel with reference to " Remington ' s Pharmaceutical Sciences " the 15th edition, 61 chapters.According to the patient's who treats situation, dosage must change to some extent.Human body is to the suitable dosage that can determine individual patient in any case that is reflected at of administration.

Lomustine is to be used for the treatment of a kind of in the nitroso ureas of specific tumor disease.It is 1-(2-ethyl chloride)-3-cyclohexyl-1 nitroso ureas.It is a yellow powder, and chemical formula is C ₉H ₁₆ClN ₃O ₂, molecular weight is 233.71.Lomustine dissolves in 10% the ethanol (0.05mg/mL) and straight alcohol (70mg/mL).Lomustine in water be insoluble relatively (＜0.05mg/mL).It is non-ionized relatively under the physiological pH value.Non-active ingredient in the lomustine capsule is: dolomol and sweet mellow wine.

Make DNA and RNA alkylation though generally believe lomustine, it does not intersect resists other alkylating reagents.With other nitroso ureas, it also can suppress the enzymatic process of a plurality of keys by making amino acid carbamoylation in the albumen.

Lomustine can be taken orally.At orally give 30mg/m ²-100mg/m ²Behind radioactive lomustine of dosage, only about half of radioactivity of giving form with catabolite in 24 hours is secreted.The serum half-life of metabolin is 16 hours-2 days.After 15 minutes, it is suitable organizing level and blood plasma level at intravenous administration.

It is reported, lomustine is except other therapies, can be used as single reagent or use, be used for the treatment of the patient's who accepts suitable surgical intervention and/or radiotherapy program primary and metastatic brain tumor with the form of the conjoint therapy set up with other authorized chemotherapeutants.Also confirmed it and other authorized medication combined in secondary treatment patient's hodgkin's disease effectively, this patient recurrence or therapy first do not replied after therapy first.

Lomustine as the RD of single reagent in adult and children is: formerly untreated patient is per 6 all 130mg/m on one's body ²As single oral dose.For the individuality that marrow function weakens, its dosage should reduce to per 6 all 100mg/m ²When lomustine and the medication combined use of other myelosuppressives, should corresponding adjustment dosage.Should be understood that other dosage also can use, for example 20mg/m ², 30mg/m ², 40mg/m ², 50mg/m ², 60mg/m ², 70mg/m ², 80mg/m ², 90mg/m ², 100mg/m ², 120mg/m ²Or any dosage between these data, this is according to the required decision of the individuality for the treatment of by the clinician.

Other reagent.Operable other reagent comprise A Wasiting, Iressa, erbitux, bortezomib and Gleevec.In addition, growth factor receptor inhibitors and micromolecule inhibitors of kinases are also useful in the present invention.At this all therapies described in Cancer:Principles and Practice of Oncology (2001) are incorporated herein by reference.Also comprise other following therapies.

Ii. immunotherapy

Immunotherapy depends on usually uses immune effector cell and molecule and destruction of cancer cells to target.Immune effector molecule can be, for example, and the specific antibody of tumor cell surface particular marker.Independent antibody can be used as the effector molecule of treatment, and perhaps it can be raised other cells and realize killing and wounding of cell veritably.Antibody also can with medicine or toxin (chemotherapeutic, radioactive nuclide, ricin A chain, cholera toxin, pertussis toxin, or the like) in conjunction with and as just destination agent.Optionally, effector molecule can be to carry directly or indirectly and the lymphocyte of the interactional surface molecular of tumour cell target.Various effector molecule cells comprise cytotoxin T cell and NK cell.

Therefore, immunotherapy can be used as the part of conjoint therapy, combines with reovirus or other oncolytic virus therapies.Conventional steps at following discussion conjoint therapy.Usually, tumour cell must carry the particular marker that is applicable to grappling, that is this label does not exist in most other cells.Tumor marker has a variety of, and any one of these labels can be applicable to grappling in content of the present invention.Common tumor marker comprises carcinomebryonic antigen, prostate specific antigen, urinary system tumor associated antigen, embryonal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialylated Louis's antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.In addition, infecting the tumour cell of reovirus or another kind of oncolytic virus or other hyperplasia cells can be at the cell surface expression viral antigen, thereby also makes them present susceptibility to immune system attack.

TNF is a kind of glycoprotein, this glycoprotein kills and wounds the cancer cell of some kind, active cell factor product, activating macrophage and endothelial cell, promote the generation of collagen and clostridiopetidase A, it is that a kind of inflammatory mediator also is the medium of infectious shock, and promotes kalabolism, heating and sleep.The stimulation that some infectious reagent produce by TNF causes the tumour decline.When using separately with effective dose, TNF is very virose, and therefore best therapy may be united use with lower dosage and other drug with it.Its immunosuppressive action can be strengthened by gamma interferon, so conjoint therapy is proemial.The potpourri of TNF and interferon-' alpha ' also is found has antitumaous effect.

Iii. hormonotherapy

According to method as described herein sex hormone is used for the treatment of cancer.Though method as described herein is not limited to the treatment of particular cancers, the use of hormone is useful to mammary gland, prostate and endometrium (inner membrance in uterus) cancer.The example of these hormones has estrogen, antiestrogenic, progesterone and androgen.

The corticosteroid hormone is effective on the cancer (lymthoma, leukaemia and Huppert's disease) of some type of treatment.The corticosteroid hormone can improve the effectiveness of other chemotherapeutants, and therefore, they are often used in the therapeutic alliance.The example of corticosteroid hormone has prednisone and dexamethasone.

Iv. radiotherapy

Radiotherapy is also referred to as radiotherapy, is the method with ionization radiation therapy cancer and other diseases.The energy that ionising radiation is accumulated makes the impossible continued growth of these cells by destroying the cell that its inhereditary material damaged or damaged the zone of being treated.Though radiation damage cancer cell and normal cell, the latter can self repair and correct functionating.Radiotherapy can be used to treat the solid tumor of localization, as skin, tongue, larynx, brain, mammary gland or cervix cancer.It also can be used to treat leukaemia and lymthoma (being respectively cancer and lymphoid cancer that blood forms cell).

Can include but not limited to according to radiotherapy used in the present invention, tumour is used gamma-radiation, X-ray and/or radioactive isotope is directly delivered to tumour cell.Also can consider to utilize other forms of dna damage factor, as microwave and UV irradiation.Most possible is all of these factors taken together can duplicating and reparation and chromosomal assembling and keep the damage that produces wide scope DNA, DNA precursor, DNA.The dosage range of X-ray is used the single dose of 50-200 roentgen's dosage to 2000-6000 roentgen from the every day of long period (3-4 week).Radioisotopic dosage range alters a great deal, and it depends on the intensity and the kind of the radioactive ray of isotopic half life period, emission, and the absorption of tumour cell.

Radiotherapy can comprise uses radiolabeled antibody dose radiation directly to be sent to the position (radioimmunotherapy) of cancer.Antibody is high specific albumen, and it is produced replying of antigen (is allogenic material by immune system recognition) existence by body.Some tumour cells comprise specific antigen, can cause the generation of tumor specific antibody.These a large amount of antibody can and be incorporated into radiomaterial (being called radiolabeled process) in prepared in laboratory.In case be injected in the body, antibody will be found out cancer cell energetically, and the cell killing (cytotoxicity) by radioactive ray is used for destruction of cancer cells.This method can make healthy cell by the risk minimization of radiation damage.

It is linear accelerator that conformal radio therapy equally uses identical radiotherapy instrument with conventional radiotherapy, thereby but places the form fit that shape that derby changes it makes itself and cancer on the path of X ray light beam.This just guarantees to give tumour higher dose radiation.Healthy peripheral cell and near structure are accepted the radioactive ray than low dosage, so the possibility of spinoff has just reduced.Now developed a kind of substitute that is called the device of multi-diaphragm collimator and can be used as derby.Multi-diaphragm collimator comprises that some are fixed in the sheet metal on the linear accelerator.Thereby can adjust each lamella makes the radiotherapy light beam can mate the shape of area for treatment and does not need derby.The accurate location of radiotherapy instrument is very important for conformal radio therapy, and in each beginning for the treatment of, can use a special scanner to check the position of your inherent organ.

The high resolving power intensity modulated radiation therapy is also used multi-diaphragm collimator.In this treatment, the lamella of multi-diaphragm collimator is removed when treating.This method may make the shape of treatment light beam reach even be more accurate and make radiotherapeutic dosage keep constant in whole area for treatment.

Though investigation shows conformal radio therapy and intensity modulated radiation therapy and can reduce radiotherapeutic spinoff, the area for treatment that might so accurately formalize just can stop cancer cell small outside area for treatment destroyed.This just means that these professional radiotherapy technology of use can make cancer higher in the risk of recurrence in future.Three-dimensional located irradiation therapy is used to treat brain tumor.This technology is carried out radiotherapy from a lot of different angles, thereby makes the dosage that enters tumour very high and to influence the dosage of surrounding health tissue very low.Before treatment, thereby guarantee the accurate grappling of radiotherapy, and when accepting radiotherapy, patient's head is fixed in the framework of a special manufacturing by a plurality of scannings of Computer Analysis.Give a plurality of dosage.

The three-dimensional located irradiation operation (γ cutter) that brain tumor is carried out is not to use cutter, and is to use the very accurately fixed gammatherapy light beam from hundreds of different angles of target.Only need the primary emission therapy, approximately need four to five hours.For this treatment, the metal frame that has a special manufacturing connects your head.Repeatedly scan then with X ray and seek the accurate zone that needs treatment.In radiotherapy, patient bed and their head in a big helmet, thereby have hundreds of holes that the radiotherapy light beam can be passed through on the helmet.

Scientist also seeks and improves the approach that radiotherapy is renderd a service.They have studied two kinds of research property medicines to standing the effect of the cell behind the radioactive ray.Radiosensitizer makes the ruined possibility of tumour cell bigger, and radiosensitizer can protect normal structure to avoid the influence of radioactive ray.People also studied the hyperthermia that uses heat make tissue to radioactive ray the effect aspect responsive.

V. row operation again

About 60% cancer patient can experience the operation of some type, comprises the operation of preventative, diagnostic or interim, curative and slowing down property.Therapeutic operative treatment cancer, it can combine with other therapies, as therapy of the present invention, chemotherapy, radiotherapy, hormonotherapy, gene therapy, immunotherapy and/or selectivity therapy.

The therapeutic operation comprises resection, and cancerous tissue all or a part is by physical removal, excision and/or destruction in resection.Tumorectomy refers to that physical removal is to the small part tumour.Except tumorectomy, operative treatment comprises laser surgey, cryosurgery, electrosurgery and micro-control operation (mohs' technique).Can further contemplate that the present invention can with shallow cancer, precancer, or use is united in the removal of the accidental amount of normal structure.

After cut-out or whole cancer cell, tissue or tumour, can form a cavity in vivo.Can with other anti-cancer therapies by the perfusion, directly the injection or this zone of local coating finish treatment.The treatment of this class can be for example per 1,2,3,4, and 5,6 or 7 days, perhaps per 1,2,3,4 and 5 weeks or per 1,2,3,4,5,6,7,8,9,10,11 or repeat once Dec.These treatments also can be used different dosage.

Description of drawings

The following drawings forms a part of the present invention and is used for further proving specific part of the present invention by being comprised.One or more by reference accompanying drawings also can make the present invention be easier to understand in conjunction with the detailed description of specific embodiment shown here.

Figure 1A-B reovirus and myxoma virus preferably infect p53-/-MEF and the damaged L3 of ATM.(Figure 1A) myxoma virus (Myx-GFP of the expression GFP of the WT/AV reovirus of usefulness 40MOI or 5MOI, the GFP that is Lausanne (ATCC) strain of MYXV expresses version, this is described referring to (Johnston et al, 2003)) infect p53-/-MEF and p53+ /+MEF (be respectively p53 knocks out or the rat embryo fibroblast cell of p53 wild type).Infect after 3 days, pair cell fixes/permeates and use reovirus antiserum and the 2nd FITC antiserum by FACS they to be analyzed.The result show wild type and attenuation type reovirus all can preferably infect p53-/-MEF.For the research of myxoma infections, infection cell was observed under phase correlation microscope and fluorescent microscope after 24 hours.The cell of expressing GFP presents myxoma infections.Mock: simulated infection.(Figure 1B) infect BT (the normal lymphoblastoid C3ABR of ATM cell with the WT/AV reovirus of 40MOI or the myxoma virus (Myx-GFP) of 5MOI; Kozlov et al, 2003) and L3 (the lymphoblastoid cell of ATM defective; Kozlov et al, 2003).Infect after 3 days, pair cell fixes/permeates and use reovirus antiserum and the 2nd FITC antiserum by FACS they to be analyzed.The result show wild type and attenuation type reovirus all can preferably infect p53-/-MEF.For the research of myxoma infections, infection cell was observed under phase correlation microscope and fluorescent microscope after 48 hours.The cell of expressing GFP presents myxoma infections.C35ABR (B3) and L3 clone are respectively to be provided by doctor MLavin (Queensland state Institute for Medical Research) and Y doctor Shiloh (Tai Laweifu university) friendship.Mock: simulated infection.

Fig. 2 A-C reovirus and myxoma virus preferably infect the human lymphoma of p53 and atm feature obstacle.(Fig. 2 A) p53 and ATM defective have strengthened replying that IR stimulates.With 2Gy ionising radiation lymphoma mantle cell (Granta, HBL-2 (p53 sudden change, Turker et al, 2006) Z138C, JVM-2)) and Burkitt lymphoma (Raji and Ramos) 2 hours harvesting then.With the full cell pyrolysis liquid of NET-N lysis buffer (1%NP-40) Processing of Preparation, follow by ultrasonication.Then 50 μ g albumen are carried out 8% or 10%SDS-PAGE, it is gone to nitrocellulose filter and surveys (Phospho-ATM with labelled antibody, pSerl981 ATM and Phospho-p53, pSerl5 and p53 are available from RocklandImmunochemicals for Researchand Cell Signaling; ATM specificity rabbit polyclonal antibody 4BA is given by M doctor Lavin friendship).BT and L3 cell are in contrast.(Fig. 2 B) p53 and ATM reply normal cell (BT, Z138C, JVM-2, Ramos) and p53 and/or ATM answering obstacle cell (Grant is Raji) to the viral susceptibility diagram of reovirus and myxoma virus for L3, HBL-2.1; Responsive to virus.0; Virus there is resistibility.Reply that normal cell has resistibility to virus infections and the cell of answering obstacle to the virus infections sensitivity.(Fig. 2 C) uses the WT/AV reovirus of 40MOI or myxoma virus (Myx-GFP) infection cell of 5MOI.The previous Raji cell that studies show that is responsive and the Ramos cell is resistive (Alain et al, 2002) to reovirus to reovirus.Infect after 3 days, with cell fixation/infiltration and use reovirus antiserum and the 2nd FITC antiserum is analyzed it by FACS.The result shows that reovirus and myxoma virus all preferably infect the unresponsive lymthoma of p53 and/or ATM.For the research of myxoma infections, infection cell was observed under phase correlation microscope and fluorescent microscope after 48 hours.The cell of expressing GFP presents myxoma infections.Mock: simulated infection.

Fig. 3 reovirus and myxoma infections retinoblastoma cell.Infect retinoblastoma cell (Y79 and WERI-Rb-1 are available from ATCC) with the WT/AV reovirus of 40MOI or the myxoma virus (Myx-GFP) of 5MOI.After infecting specified number of days, cell fixation/infiltration and use reovirus antiserum and the 2nd FITC antiserum are analyzed it by FACS.For the research of myxoma infections, infection cell was observed under phase correlation microscope and fluorescent microscope after 48 hours.The cell of expressing GFP presents myxoma infections.Mock: simulated infection.

Embodiment

Following examples are used to the preferred embodiments of the present invention of demonstrating.Those skilled in the art will be appreciated that the technology that on behalf of the inventor, disclosed technology disclose in following examples, go on well in the operation of the present invention, therefore can consider to use it for the preferred version of formulating operation.Yet, under the guide of disclosure text, it should be recognized by those skilled in the art that and do not departing under the spirit and scope of the present invention, in disclosed specific embodiment, can carry out many changes and still can obtain same or analogous result.

Embodiment 1-material and method

Clone.P53 ^-/-The L3 cell of MEF, BT and ATM defective, and human lymphoma HBL2, Granta, Z138C, Raji, Ramos and JVM2 cell, and cancer eye clone (Y-19 and WERI-Rb-1) is available from American Type Culture Collection.Cell maintains RPMI 1640, among the 10%FBS.

The preparation of wild type and attenuation type reovirus.The wild type reovirus T3D strain described purifying that carries out before breeding of L929 cell and basis that is used for these researchs.Except the AV reovirus is in HT10801 and L929 cell the breeding, the method for attenuation type reovirus that purifying is derived from the HTR1 nutrient culture media is identical with the method for preparing the wild type reovirus.The reovirus of 5-10 MOI joined in the cell and at 37 ℃ kept 48-72 hour.Observing viral oncolysis (typically 20-30% lysis) afterwards, purified virus from the cell of sedimentation.With 35,000rpm carried out the centrifugal 7-8 of CsCl hour virus with the SW41 rotary head.Collect the virus of layering and use 150mM NaCl, 10mMMgCl2 and 10mM Tris (pH 7.5) extensively dialyse to it.For carrying out the titration of wild type reovirus, HEK 293 cells are laid on 6 orifice plates, every hole is 2x10 ⁵Individual cell.After 2 hours, remove inoculum 37 ℃ of absorption.Cover cell monolayer with 1% agar and fresh culture then.Infect and calculate the plaque number after 5-7 days.

For carrying out the titration of attenuation type reovirus, except after infecting 5-7 days, removing agar and cell monolayer being fixed/permeate with cell fixation/osmotic agent box (BD Bioscience), so that antiserum and the 2nd FITC antibody with reovirus carry out outside the immunostaining it, all the other steps are identical with wild type.Detect the plaque of identifying the AV reovirus by immunofluorescence.Optionally, for attenuation type reovirus, also on the L929 cell, carry out conventional plaque experiment.Infected back 3 days, agar covers with dimethyl diaminophenazine chloride, and detects cell monolayer after plaque forms 24 hours.For for the attenuation type reovirus of collecting the supernatant of HT1080 and HTR1 infection, use 35, the high speed ultracentrifugation of 000rpm makes viral sedimentation.

The preparation of myxoma virus.Use the Myx-GFP of myxoma virus (Lausanne strain) to carry out infection research, Myx-GFP produces by inserting green fluorescent protein (GFP) expression cassette in the gene, and this expression cassette is driven by synthetic vaccinia virus morning/late promoter.On the BGMK cell, concentrate formation to breed by foregoing with titration Myx-GFP (Opgenorth et al, 1992).

Facs analysis.For flow cytometry analysis, (PharMingen, San Diego CA) make cell trypsinized and fixing with cell fixation/penetrating fluid.(Cedarlane, Ontario) incubation is together analyzed by flow cytometer then for the cell of this is fixing and infiltration and the anti-rabbit igg of first reovirus antiserum and the 2nd FITC covalency.

Embodiment 2-result

Reovirus and myxoma virus preferably infect the cell of p53 or ATM defective.For whether the adjusting that detects tumor suppressor gene influences the susceptibility of oncolytic virus, the inventor has selected in multiple cancer p53 and the ATM tumor suppressor gene through regular meeting's sudden change.P53 be former suppressor oncogene and in multiple cancer cell sudden change the most frequent, it is reported that cancer 50% or more keeps p53 suddenly change (White, 1994; Morris, 2002).ATM (the ataxia telangiectasia sudden change) is a kind of serine threonine protein kinase, is activated when dna damage.Two of the ATM gene copies are dysfunction in ataxia telangiectasia, ataxia telangiectasia is a kind of rare syndrome, show as cancer tendentiousness, radiosusceptibility and neurodegeneration (Kitagawa et al, 2005).Therefore the inventor use p53-/-the L3 lymphoid stem cells (Kozlov et al, 2003) of MEF (mouse embryo fibroblasts) and ATM defective detects the susceptibility of oncolytic virus.Shown in Figure 1A-B, show by FACS or microscopic examination fluorescence positive cells (cell of FITC+ or GFP+), for the control cells of MEF (p53 is normal) and BT (ATM is normal), reovirus (WT and AV reovirus; Kim et al, 2007) and myxoma virus all preferably infect p53 ^-/-The cell of MEF or L3 (ATM defective).

Reovirus and myxoma virus preferably infect the human lymphoma of p53 or atm feature obstacle.Because reovirus and myxoma virus preferably infect the cell of p53 or ATM defective, the inventor has detected reovirus subsequently and can myxoma virus preferably infect the human lymphoma that carries p53 or ATM defective.The inventor has tested 6 kinds of lymthomas (HB L-2, Granta, Z138C, JVM2, Raji and Ramos), and when attacking in the face of genotoxicity, these lymthomas present the different cell response (Fig. 2 A) from p53 and ATM dependence path.By the IR radiation with identify the functional status of p53 and ATM with the phosphorylation that the western trace detects p53 and ATM.BT (ATM is normal) and L3 (ATM defective) are used as the contrast that the ATM that caused by IR replys.HBL-2 and Raji reply the p53 dysfunction that stress present of genotoxicity, and this constitutive character phosphorylation by 15 serines of p53 is proved (Liet al, 2006) (Fig. 2 A).The Granta cell stress present that the atm feature obstacle is replied and ATM be not activated (Fig. 2 A) under ionising radiation (IR) stimulates to genotoxicity.At Z138C, in the case of JVM2 and Ramos cell, p53 and ATM to genotoxicity stress reply normal, shown in the hyperphosphorylation that stimulates the p53 that causes and ATM by IR (Fig. 2 A).What is interesting is, show by FACS or microscopic examination fluorescence positive cells (cell of FITC+ or GFP+), the lymthoma of replying with respect to ATM-and p53-(Z183C, JVM-2, Ramos), p53 and/or atm feature obstacle lymthoma (HBL2, Granta, and Raji)-promptly when IR stimulates, the p53 that HBL-2 and Raji present constitutive character activates, and Granta to present ATM defective-to reovirus and myxoma infections be preferred responsive (Fig. 2 C).Shown in Fig. 2 B, it is susceptibility height correlation with reovirus and myxoma virus that the ATM of cell or the dysfunction of p53 are replied.This proves that forcefully p53 and ATM all participate in setting up the resistibility of cell to virus infections, and its viral susceptibility (Fig. 2 A) is given in gene or its damaged meeting of replying path.

The cancer eye cell is responsive to the infection of reovirus and myxoma virus.Because reovirus and myxoma virus preferably infect p53 and atm feature obstacle cancer cell, the inventor has also detected whether also related infection sensitivity to reovirus and myxoma virus of the damaged cancer cell of RB.As shown in Figure 3, show that two kinds of different human cancer eye cells are responsive to the infection of reovirus and myxoma virus by FACS or microscopic examination fluorescence positive cells (cell of FITC+ or GFP+).Two kinds of cells all contain damaged Rb gene (Reid et al., 1974).

****************************

According to present disclosed content, all disclosed and claimed here methods all can carry out and finish, and need not carry out excessive experiment.Though described the compositions and methods of the invention according to preferred embodiment, obviously, for a person skilled in the art, under the situation that does not depart from content of the present invention, spirit and scope, can to method as described herein with and step or method in the order of step change.More particularly, apparent, as long as obtain same or analogous result, chemically with physiology on related particular agent can substitute reagent as described herein.Substitute that all these classes are similar and variant obviously should be thought to those skilled in the art in spirit of the present invention, content and scope as appended claim definition.

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Claims (34)

1. whether a definite hyperplasia disorder to the method for oncolytic reovirus and/or myxoma virus sensitivity, comprising:

(a) provide the hyperplasia cell;

(b) measure the p53 that is derived from described hyperplasia cell, Rb, and/or the structure of ATM gene or gene outcome, function or expression; And

(c) p53 of the said structure of more described hyperplasia cell, function or expression and wild type, Rb, and/or the structure of ATM gene or gene outcome, function or expression,

P53 wherein, Rb, and/or ATM shows that in structure, function or the defective expressed above-mentioned hyperplasia disorder is responsive to the oncolysis of reovirus or myxoma virus.

2. method as claimed in claim 1 further comprises the treatment patient, and this patient is the source that obtains described hyperplasia cell.

3. method as claimed in claim 2, wherein therapy comprises reovirus therapy.

4. method as claimed in claim 3, wherein said reovirus therapy are the wild type reovirus therapy.

5. method as claimed in claim 3, wherein said reovirus therapy are the reovirus therapy of attenuation type.

6. method as claimed in claim 5, wherein said attenuation type reovirus therapy use a kind of σ 1 capsid protein express damaged, σ 1 capsid protein detect less than or do not have a reovirus of σ 1 capsid protein.

7. method as claimed in claim 3, wherein treatment comprises with described reovirus therapy coming the bone marrow cell from described patient is carried out indirect interior therapeutic.

8. method as claimed in claim 3, wherein treatment comprises and gives described reovirus therapy to described patient.

9. method as claimed in claim 7 further comprises with anti-hyperplasia therapy for the second time contacting described bone marrow cell.

10. method as claimed in claim 8 further comprises giving anti-hyperplasia therapy for the second time to described patient.

11. as the method for claim 9 or 10, the wherein said second time, anti-hyperplasia therapy was chemotherapy, radiotherapy, hormonotherapy, gene therapy or immunotherapy.

12. method as claimed in claim 3, wherein treatment comprises the therapy with the inhibitor of p53, Rb or atm feature or expression.

13. as the method for claim 12, wherein said inhibitor is albumen or peptide, antibody, siRNA, antisense molecule, ribozyme or micromolecule.

14. method as claimed in claim 2, wherein treatment comprises the myxoma virus therapy.

15. method as claimed in claim 2, wherein treatment comprises the anti-hyperplasia therapy of non-viral.

16. as the method for claim 15, wherein said non-viral cancer therapy is chemotherapy, radiotherapy, hormonotherapy, gene therapy or immunotherapy.

17. method as claimed in claim 1, wherein said hyperplasia cell is malignant cell or benign cell.

18. method as claimed in claim 1 further comprises from patient in step (a) obtaining described hyperplasia cell on one's body before.

19. method as claimed in claim 1 is wherein measured expression and is comprised Northern trace, Western trace, immunohistochemistry, RT-PCR, microarray analysis, transcribes group analysis, Proteomic analysis or metabolism group analysis.

20. method as claimed in claim 1 is wherein measured structure and is comprised order-checking, in situ hybridization, immunohistochemistry or structural proteins group.

21. method as claimed in claim 1, wherein measurement function comprises expression or the activity of measuring p53, Rb or ATM downstream targets.

22. the method for treatment patient hyperplasia disorder comprises:

(a) inhibitor with p53, Rb or atm feature or expression contacts excessive proliferative cell; And

(b) contact described hyperplasia cell with reovirus or myxoma virus therapy.

23. as the method for claim 22, wherein said patient treats with reovirus therapy.

24. as the method for claim 23, wherein said reovirus therapy is the wild type reovirus therapy.

25. as the method for claim 24, wherein said reovirus therapy is an attenuation type reovirus therapy.

26. as the method for claim 25, wherein said attenuation type reovirus therapy use a kind of σ 1 capsid protein express damaged, σ 1 capsid protein detect less than or do not have a reovirus of σ 1 capsid protein.

27. as the method for claim 22, wherein said patient uses the myxoma virus therapy for treating.

28. as the method for claim 22, wherein treatment comprises the bone marrow cell from described patient is carried out indirect interior therapeutic.

29. as the method for claim 22, wherein treatment comprises described patient is carried out interior therapeutic.

30., further comprise with anti-hyperplasia therapy for the second time contacting described bone marrow cell as the method for claim 28.

31., further comprise giving anti-hyperplasia therapy for the second time to described patient as the method for claim 29.

32. as the method for claim 30, the wherein said second time, anti-hyperplasia therapy was chemotherapy, radiotherapy, hormonotherapy, gene therapy or immunotherapy.

33. as the method for claim 31, anti-hyperplasia therapy was chemotherapy, penetrated therapy, hormonotherapy, gene therapy or immunotherapy the wherein said second time.

34. as the method for claim 22, wherein said hyperplasia cell is malignant cell or benign cell.

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