CN102124335B - 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

The susceptibility of hyperplasia cell based on TIF to oncolytic viral therapy

Background of invention

The application requires to enjoy in the right of priority of the U.S. Provisional Patent Application number 61/055,360 of submitting on May 22nd, 2008, 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 oncolytic viral 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 in the host who has immunologic function, be asymptomatic (Tyler, 2001) conventionally.Be failed about the trial major part that reverses transformation reovirus, this comprises the double-stranded RNA genome of reovirus owing to multiple factors.The reovirus particle of known disappearance σ 1 is FFI (Larson et al, 1994).

Importantly, reovirus is acknowledged as in the time that it infects the transformant of particular type and can presents and kill significantly cell ability (Duncan et al, 1978 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 from traditional chemotherapy and radiotherapy, so due to limited their the energy specificity grappling cancer cells of their replication capacities in normal cell.The second, compared with carrier without replication capacity, they can spread to cancer cell around from the cancer cell of initial infection, thereby reach a large amount of distributions and anticancer effect effectively.

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

But the existence of Ras gene mutation may be the partly cause of cancer cell to reovirus therapy susceptibility.Recent studies have shown that Ras path is not unique factor (the Song et al that determines to allow reovirus, 2009), and show from transforming that the cell of Ras can be set up the cell (Kim et al, 2007) of anti-reovirus and the Ras sudden change that still retains activation.Therefore, need to there is improved method to differentiate and screen suitable cancer cell sub-group for reovirus therapy and other oncolytic virus reagent.

Summary of the invention

Summary of the invention

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

Therefore, according to the present invention, provide a kind of definite hyperplasia disorder the method whether responsive to the oncolysis of reovirus or myxoma virus, comprised that (a) provides a kind of hyperplasia cell; (b) structure, function or the expression of mensuration is obtained by hyperplasia cell p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC and/or ATBF1 gene or gene outcome; And (c) relatively hyperplasia structure, function or expression cell and p53 wild type, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC and/or ATBF1 gene or gene outcome, wherein the p53 of hyperplasia cell, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC and/or the ATBF1 defect in structure, function or expression shows that 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, as damaged in used σ 1 capsid protein to express, and σ 1 capsid protein can't detect or without the reovirus of σ 1 capsid protein.This therapy can comprise carries out indirect interior therapeutic to patient's bone marrow cell by reovirus and/or myxoma virus therapy, or can comprise and give reovirus and/or myxoma virus therapy to patient.The in the situation that of indirect in vivo, the method may further include and gives anti-hyperplasia therapy for the second time to bone marrow cell, or in the situation that giving virus therapy to patient, the method may further include and gives anti-hyperplasia therapy for the second time to patient.Anti-hyperplasia therapy can be chemotherapy, radiotherapy, hormonotherapy, gene therapy or immunotherapy for the second time.The method also may further include with the function of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC or ATBF1 or expression inhibiting agent and treats, and this inhibitor is as albumen or polypeptide, antibody, siRNA, antisense molecule, ribozyme or little molecule.Treatment also can comprise the anti-hyperplasia therapy of non-viral, as used chemotherapy, radiotherapy, hormonotherapy, gene therapy or immunotherapy.Hyperplasia cell can be malignant cell or benign cell.The method may further include, and in step (a) before, obtains hyperplasia cell from patient.The mensuration of expressing can comprise Northern trace, Western trace, immunohistochemistry, RT-PCR, microarray analysis, transcribe group analysis, Proteomic analysis or metabolic components and analyse.Optionally, the mensuration of structure can comprise order-checking, in situ hybridization, immunohistochemistry or structural proteomics.In addition, the mensuration of function can comprise expression or the activity of the downstream targets of evaluation p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC or ATBF1.

In another embodiment, the method of a kind of patient's for the treatment of hyperplasia disorder is provided, has comprised that (a) gives function or the expression inhibiting agent of p53, Rb, ATM, BRCA1, BRCA2, MutS, MutL, MutH, APC or ATBF1 to hyperplasia cell; And (b) give reovirus and/or myxoma virus therapy to hyperplasia cell.Reovirus therapy can be wild type reovirus therapy or attenuation type reovirus therapy, expresses damagedly as σ 1 capsid protein, and σ 1 capsid protein can't detect or without the reovirus of σ 1 capsid protein.Treatment can comprise carries out indirect interior therapeutic to patient's bone marrow cell, or patient is carried out to interior therapeutic.The method may further include and gives bone marrow cell anti-hyperplasia therapy for the second time, as anti-hyperplasia therapy for the second time, as chemotherapy, radiotherapy, hormonotherapy, gene therapy or immunotherapy.Hyperplasia cell can be malignant cell or benign cell.

Word " a " or " an " and the term in these claims and/or this instructions " comprise " combines while use, may refer to " one ", but also with " one or more ", " at least one ", and the meaning of " one or more than one " is consistent.

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

For term " or ", although open text support only refers to the definition of alternative and "and/or", the use of the term " or " in claims is used to refer to "and/or", unless be clearly shown to be only refer to alternative or selective to as if get rid of mutually.Word " comprising " (and any form of comprising using in the present specification and claims, 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 the extra composition of not enumerating or method step.

Other objects of the present invention, Characteristics and advantages will present by following detailed description.But, it will be appreciated that, in the time pointing out specific embodiment of the present invention, describe in detail and special case 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 field and modification is apparent.

The description of illustrative embodiment

Cancer be the process of a multi-step, thereby comprise by catastrophic event and affect the accumulation of combining that normal proto-oncogene and tumor suppressor gene cause oncogene activation and tumor suppressor gene deactivation.What is interesting is, the cell signal path that oncolytic virus utilizes different oncogene to drive carrys out copy choice and kills and wounds the cell that they infect.Except the oncolysis that oncogene relies on, inventor infers that some tumor suppressor genes are promoting viral oncolysis to use and may also play an important role.Therefore, they have tested various mouse and people's cancerous cell line, regulate its tumor suppressor gene to check tumor suppressor gene dysfunction whether can affect the oncolysis of reovirus and myxoma virus.The molecule mechanism of reovirus oncolysis is that unexpected the obtaining of the feature because being devoted to study reovirus cell receptor advances.In 1993, two kinds of mouse cell lines that have seminar's report not express EGF-R ELISA (EGFRs) have corresponding resistibility to reovirus infection, but 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 because the EGFR that has function on host cell gives.Afterwards, this has proved and after EGFR downstream factor S os, Ras with activating or verbB conversion, can become responsive (Strong etal, 1996 to the resistive NIH3T3 cell of reovirus infection with a collection of researchist; Strong et al, 1998).Therefore think that the utilization of oncogene Ras signal path is the committed step in reovirus oncolysis.

A lot of mankind's cancer demonstrates the Ras Pathway Activation effect with enhancing, and this has emphasized the strong effect of this signal network in tumour occurs, and hint reovirus may brought into play oncolytic potential widely in kinds of tumors type.80% the clone of exceeding that initial experiment finds to come from kinds of tumors type is responsive (Coffey et al, 1998) to the lethal effect of reovirus mediation.More what is interesting is, another kind of oncogene, Myc, also contributes to the oncolysis of reovirus.The expression excessively of Myc can make the resistive cell of reovirus become the intrusion sensitivity (Egan et al, 2003) to 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 tested every other vertebrate kind the mankind are no pathogenicity (McFadden, 2005).Although 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 is event regulation and control in the cell in the downstream that adhered to and invaded by virus, instead of occurs on the level of specificity host receptor (McFadden, 2005).

The propelling of the molecule mechanism of myxoma virus oncolysis has benefited from the research of the virus host range factor to controlling poisoning intrusion human cancer cell.Have report to show the Akt interaction of viral M-T5 host range albumen and cell, this Interaction enhanced the oncolytic potential (Wang et al, 2006) of myxoma virus to human cancer cell.But the myxoma virus of M-T5KO (knocking out) has been given play to the viral oncolysis potential weakening 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 M-T5KO myxoma virus, the rise of hint Akt plays an important role determining myxoma virus oncolytic potential, (Wang et al, 2006) are often found in the rise of this Akt in many cancer cells.

The contact of viral oncolysis effect and tumor suppressor gene is to advance by the research of adenovirus 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.There is report to show that the adenovirus of the sudden change of not expressing this virus protein can copy and be killed and wounded in the handicapped human tumor cell of p53, but can not in the cell that has p53 function, copy and be killed and wounded (Bischoff et al, 1996).The basic mechanism of the adenovirus oncolysis of sudden change is disappearance or the sudden change due to p53, and the Apoptosis that the p53 of adenovirus EIA induction relies on is impaired (Bischoffet al, 1966) in the handicapped cancer cell of p53.But the adenovirus oncolysis that studies show that p53 dependence is afterwards more complicated, and can make the other path in multiple model and reply (O ' Shea et al, 2004; Abou et al, 2004; Royds et al, 2006).In fact, thus this just guides inventor to infer that the handicapped cancer cell of p53 may suffer due to genomic instability the destruction (Carroll et al., 1999) of cell anti-virus mechanism.

Because p53 is to maintaining genomic importance, so p53 gene is considered to defender (Gomez-Lazaro et al, 2004 of cellular genome; Vogelstein et al, 2000).Because the genome of p53 mediation maintains, normal cell can keep complete Antiviral Mechanism.But the dysfunction of p53 can finally cause the viral susceptibility (by the disappearance of Antiviral Mechanism) being caused by whole genomic instability.Recent studies have shown that p53 can contribute to intrinsic immunity not rely on function (Takaoka et al, 2003 of its pre-Apoptosis and tumor suppressor gene by the antiviral activity that strengthens IFN dependence; Munoz-Fontela et al, 2008).The transcription of p53 is important to the activation of IFN path when the virus infections.In the time of virus attack, p53 can activate transcribe (Munoz-Fontela et al, 2008) of IFN regulatory factor 9.In addition in the time that reducing in cancer cell, the expression 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 to the case of DNA replication dna and the important tumor suppressor gene of genome stability at other.Inventor illustrates at this, except being activated by specific oncogene signal path the susceptibility that may strengthen causing, the handicapped cancer cell of p53, ATM or Rb all becomes more responsive to the attack of reovirus and myxoma virus, and this may contribute to describe different oncolytic viruses and how distinguish normal cell and cancer cell.The qualification contacting between the not normal and oncolytic virus susceptibility of these heredity has strengthened the application potential of oncolytic viral 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 affect 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 reovirus family in recently identified go out relevant with various diseases, this original title is still used.

The infection of reovirus often occurs 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 treatment.Reovirus is without coating and by an icosahedral capsid (T-13) being made up of outer and internal layer albumin layer.The viral genome of Reoviridae comprises 10-12 bar segment, is divided three classes according to their these fragments of large young pathbreaker: L (greatly), M (in) and S (little).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 represents (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 dsRNA genome, therefore copy and only betide tenuigenin, and this encoding viral to dsRNA genome duplication with change into the necessary multiple albumen of (+)-RNA.Virus can enter host cell by cell surface receptor.Acceptor is unknown being still considered to comprise sialic acid and being connected adhesion molecule (JAMs).Virus can be coated with by proteinase past release in endolysosome, thereby partly digested and can further enter cell in capsid herein.Then core granule enters tenuigenin by the process of current the unknown, thereby genome is produced excessive (+) adopted chain by conservative transcribing in this process, and this (+) adopted chain is used as mRNA template and synthesizes (-) adopted chain.Virion starts to assemble in tenuigenin after 6-7 hour in infection.

The particle that the lonely virion of infectious mammalian reovirus intestines is approximately 85nm with diameter exists.The outer capsid of virion comprises several different albumen, and wherein sigma-1 (σ 1,50kD) is by the carbohydrate combination (Chappell et al, 1997 that disperse; 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 determining that reovirus plays an important role (Haller et al, 1995 on pathogenic; Wilson et al, 1994; Kaye et al, 1986; Werner et al., 1980).

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

Production about wild type reovirus in treatment of cancer and the certain methods of application (are for example described to some extent, United States Patent (USP) 7, 300, 650, 7, 163, 678, 7, 049, 127, 7, 014, 847, 6, 994, 858, 6, 811, 775, 6, 703, 232, 6, 576, 234, 6, 565, 831, 6, 528, 305, 6, 455, 038, 6, 344, 195, 6, 261, 555, 6, 136, 307, with 6, 110, 461, and the open text 2006/0165724 of United States Patent (USP), 2006/0073166, 2005/0123513, 2004/0265271, 2004/0126869, 2004/0109878, 2002/0037543, 2006/0029598, 2005/0026289, 2002/0006398 and 2001/0048919, every patent is all incorporated herein by reference without withdrawing a claim).

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

But as new life's with in SCID (severe combined immunodeficiency) animal, wild type reovirus is especially being brought into play the pathogenic function of important virus (Sabin, 1959 in nerve fiber and cardiac muscular tissue immunodeficiency host; Weiner et al, 1977; Baty et al, 1993; Loken et al, 2004).In some cases, even immunocompetence host, comprise in the mankind wild type reovirus relevant to pathogenicity (Terheggen et al, 2003, Hirasawa et al, 2003).Therefore, especially, in immunocompromised host or very young host, wild type reovirus does not always work in optimum mode.For example, the host of immunocompromised host as adult animal very young or immunodeficiency in, this virus also presents can infect some health tissues as heart, liver, pancreas and neuromechanism.This important content can further be applicable to carry 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 for viral oncolysis therapy.

In addition, in numerous occasions, wild type reovirus has the toxicity and the infectiousness that do not conform to demand, and this can limit their potential application in vitro.Especially, by cell culture (for example, be derived from the bone marrow graft of cancer patient) be exposed to and in wild type reovirus, can cause the killer cell that does not conform to demand as the spinoff of stem cell, stem cell is essential for a certain purposes, as recovered 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 cell is exposed in reovirus.

Attenuation type reovirus as described herein lacks total length σ 1 capsid protein that can detect, but unexpectedly has infectiousness.In the initial step of dying in virus replication sexuality, reovirus by cell surface sialic acid residues in conjunction with and the process that adheres on 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, the level of CPE non-malignant cell being presented than naturally occurring non-attenuation type reovirus, attenuation type reovirus presents unexpected characteristic, it can cause reduction to one or more CPE levels of non-malignant cell (that is, the reduction on statistical significance).Accordingly and following described more details, attenuation type reovirus provided here has improved the reovirus of prior art, it is suitable to the oncolytic reagent of spinoff without not meeting demand to comprise, for example, for example, to normal (, the non-malignant) tropism of cell and the 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 60/704 of submitting on August 5th, 2006,604 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 are all incorporated herein by reference without withdrawing a claim at this.In certain embodiments, attenuation type reovirus may lack wild type reovirus S1 gene.Attenuation type reovirus may contain S1 gene, and this S1 gene produces S1 gene outcome (σ 1) amount or brachymemma, sudden change and/or the handicapped σ 1 that reduce and/or can not detect.Attenuation type reovirus may contain S1 gene, and compared with wild type S1 gene, this S1 gene comprises one or more sudden changes, and sudden change is wherein that nucleotide subsitution, nucleotide deletion or nucleotide insert.

" attenuation type " as described herein reovirus comprise to host cell or in host cell its infect, copy and/or cytolysis characteristic present change (that is, enhancing in 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 wild type reovirus, attenuation type reovirus will present the potential of infectiousness, replication capacity and/or dissolved cell of reduction.The example of the characteristic that this class changes comprises the many forms of pathological changes caused by virus effect, for example, particular host cell 8 is produced and infects needed infection multiplicity (MOI, infect the average of the virion of each cell), the degree (further comprising apoptosis and/or necrosis) that the host cell that virus infections causes dissolves, after the virus replication of dissolved cell, from the titre of the host cell releasing virus of effective infection, and those of ordinary skill in the art can be for determining virus other active parameters to host cell.The characteristic that above-mentioned this class changes can be used for differentiating current 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 adherence (adheres to substrate 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), 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 the pathogenicity greatly weakening and does not damage its viral oncolysis effect 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, therefore inventor has further assessed pathogenic to stem cell of AV reovirus.Like this, inventor has compared pathogenic to embryonic stem cell (ESCs) of wild type and AV reovirus.Shown in embodiment as described below, wild type reovirus can infect easily in vitro ESCs and in teratoma model, greatly suppress the growth of stem cell, however AV reovirus in vitro the utmost point faintly infect ESCs and in teratoma model, do not affect the growth of stem cell.

In different embodiment, attenuation type reovirus may comprise one or more other sudden changes.For example, in specific another 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 (for example, Ahmed et al, 1982 relevant with the processing of virion in the time of reovirus replication host cells infected; 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 the S1 gene order of mainly identifying out with the naturally occurring reovirus form of separating in the breathing by infected patient or 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 mankind's reovirus S1 gene order numbering: the mankind 3 type reovirus S1:XO1 161 of mankind's reovirus; The mankind 2 type reovirus S1:M35964; The mankind 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 mankind's reovirus serotype S1 gene order numbering: the mankind 1 type reovirus strain Lang (TlL) numbering M35963; The mankind 2 type reovirus strain Jones (T2J) numbering M35964; The mankind 3 type reovirus strain Dearing (T3D) numbering XOl 161; The mankind 3 type reovirus strain Abney (T3A) numbering L37677).

According to specific embodiment, a kind of attenuation type reovirus is provided, the reovirus gene group that it comprises lacks wild type reovirus S1 gene, or the reovirus S1 gene of reovirus σ 1 capsid protein that can not encode complete that contains sudden change (the gene pool numbering of mankind's reovirus S1 gene order: the mankind 3 type reovirus S1:X01161; The mankind 2 type reovirus S1:M35964; The mankind 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 apparent.Therefore the S1 gene of sudden change refers to the S1 gene that contains a kind of polynucleotide sequence, this polynucleotide sequence changes one or more nucleotide sequences site of corresponding S 1 wild type nucleotide sequence or conserved sequence by one or more nucleotide subsitutions, nucleotide insertion and nucleotide deletion, this can be identified out easily.Hoyt etal. (2005) discloses multiple 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 is easily detected (for example, Mah et al, 1990; Leone et al, 1991; Chappell et al, 1997), generally by applying 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, etc.), 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, those of ordinary skill in the art are to detect the art-recognized standard of σ 1 albumen and the sensitivity of prior art level by what understands, like this, the reovirus particle that has replication capacity that lacks " can detect " reovirus σ 1 capsid protein will be considered to comprise this type of reovirus, and in the time that the current traditional technology of application detects σ 1 albumen of supposition existence, complete σ 1 albumen of this reovirus can not be detected.

Be incorporated herein the US 11/997,537 of this class attenuation type reovirus of description as a reference.

Iii. the control of reovirus infection

In certain embodiments, peptidyl fluoro methyl ketone (PFMKs) can be applied in cell composition, for suppressing or remove 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) for suppressing virus replication, is all incorporated herein by reference without withdrawing a claim at this.

Iv. the production of reovirus

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

As described herein, specific embodiment relates to and implements recoverable attenuation type reovirus after external persistent infection method, but also can consider to learn according to additive method the attenuation type reovirus obtaining, comprise persistent infection method in body, according to molecular biology method produce and qualification σ 1 defect and/or the damaged mutant of σ 1 (in certain embodiments, in addition or optionally, also comprise generation and the qualification of the mutant that σ 3 defects and/or σ 3 are damaged), and also comprise separating natural exist σ 1 defect and/or damaged mutant and/or σ 3 mutant of σ 1, and/or by chemistry, physics and/or genetics technology are (for example, in the host cell effectively infecting, match recombinant reovirus gene) this type of σ 1 of artificial induction (and/or σ 3) mutant.

In addition, inventor expects to cause the phenotypic sudden change of required attenuation in other genes, to find, the infectiousness of these effect gene viruses, copy or capacity packing, be included in arbitrary gene or its combination of the upper coding of virus, as 10 of reovirus genetic fragments.For example, compared with reovirus wild type gene, the sudden change of reovirus S1 or S4 gene can with 0 genetic fragment of SV 12 virus on 1,2,3,4,5 or more sudden change associating.Can produce attenuation type reovirus by several different methods, comprise the method described in U. S. application 60/704,604 for example.

B. myxoma virus

Myxoma virus is a kind of poxvirus and contains large double-stranded DNA genome, and this genome allows likely to insert the eukaryotic gene that large (25kb) is 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, its other vertebrate kind to test, comprise that the mankind are non-pathogenics, although it can effectively infect specific non-rabbit cell in vitro, comprise that external eternal young monkey kidney fibroblast (BGMK), IFN reply the primary mouse cell human tumor cell different with some of hereditary detect.Well-known cancer cell is defective in their IFN replys.Recently, have report to show that myxoma virus is one anti-experimental character glioma in vitro and in vivo, and in a junctor oncolytic reagent (Lun et al, 2005 of anti-human class glioblastoma surgery sample; 2007; Stanford et al, 2008).It can infect and kill and wound human glioma cells, is safe through large 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 primary neuroglial cytomas tested that are directly derived from glioma surgery sample, and its oncolysis can strengthen by rapamycin.

Myxoma virus, as with other oncolytic viruses as reovirus, need to stride across the antiviral defense system that is present in normal health cell can copy 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 also associated protein of major effect virus breeding by IFN antiviral response and comprise PKR, OAS synzyme and ribonuclease l nuclease.PKR activates eIF2 α, causes inhibition and the cell death inducing of translation.In normal cell, myxoma virus is directly subject to the impact of PKR and eIF2 α.

Antiviral response path is conventionally destroyed in cancer cell.For example, IFN reply weaken or damaged be that heredity is damaged, its transform and the process of tumor development in often generation.Exceed 80% tumor cell line and interferon is not replied to or present (Stojdl et al, the 2003 and references cited therein of replying of weakening; 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 Cell protection is not to the gene of cancer development.In the time of this gene impairing, cell can be to cancer development, normally after it suffers the impact of other factors.Different from oncogene, tumor suppressor gene is followed " secondary sudden change hypothesis " conventionally, is implying before effect manifests, and the allele of coding specific gene must all effected.If this is that second still can produce correct albumen so because this gene only has an allele impaired.Change a kind of saying, normally necessary but inadequate contrary with oncogene, TIF is normally necessary and sufficient.Certainly, also there is the exception should be noted that, for example p53 gene outcome, for normal p53 albumen, it can exist with dominant negative mutant form, and therefore it is also necessary but inadequate in such case.

Tumor suppressor gene, or say more accurately the albumen that they are coded, the regulation and control of cell cycle or promotion Apoptosis, sometimes all have and stop or inhibiting effect both.The function of TIF albumen is divided into multiple, comprises as described below: (a) suppress to maintain necessary gene of cell cycle (if these genes are not expressed, the cell cycle can not continue, thereby effectively suppress cell division); (b) by the cell cycle and DNA is impaired connects.As long as damaged dna is present in cell, cell just should not divide (can be repaired if impaired, the cell cycle just can continue); (c) cell death inducing, or apoptosis, if above-mentioned impaired could not being repaired, the threat producing in order to make biosome better just eliminate it so; And (d) some albumen relevant with cell adherence prevent tumour cell diffusion, stop the loss of contact inhibition, and suppress to shift (being called as the albumen of 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, and as the sudden change of another gene, or environmental factor is as virus infections.Since the 1980's, in human cancer, identify a lot of oncogene.Proto-oncogene is normal gene, and due to sudden change or expression increase, it can become oncogene.The albumen of proto-oncogene coding helps regulating cell growth and differentiation.Proto-oncogene is generally conventionally relevant with the execution of signal conduction and mitosis signal 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 regulates cell to reply DNA damage to a certain extent.The p53 that touches upon keeps the function of stability by preventing genome mutation, and it was once described to " genomic defender ", " Guardian Angel's gene ", or " guard great master ".

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

In cancer, make the sudden change of p53 inactivation usually occur in DBD district.These sudden changes of great majority have destroyed the ability of its target dna sequence of protein combination, and have therefore hindered the transcriptional activity of these genes.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, in the time that DNA is lasting impaired, it can activate DNA repair protein.In the time of identification DNA damage, it also can make the cell cycle remain on G1/S check point (if cell is remained on the sufficiently long time herein by it, DNA repair protein just can be repaired if having time damage and allow this cell to proceed the cell cycle).If when DNA damage proves unrepairable, 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 DNA damage (by UV, IR or chemical reagent are as hydrogen peroxide initiation), oxidative stress, osmotic shock, ribonucleotide loss and oncogene expression out of control.This activation is shown by two main matters.The half life period of the first, p53 albumen is increased sharply, cause be subject to stress cell in the Rapid Accumulation of p53.The second, the variation of conformation impels p53 in these cells, to exercise the active function as transcription regulaton factor.The critical event that causes p53 to activate is the phosphorylation of its N stub area.N end transcriptional activation domain comprises a large amount of phosphorylation sites and can be considered to the main target that protein kinase conduction stress signal.

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

Stress not cell in, by the lasting degraded of p53 make p53 keep low-level.Thereby the protein combination that is called Mdm2 makes it be degraded by proteasome in p53 and by it from nucleus transporte to cells matter.The phosphorylation of the N end of the p53 being caused by the above-mentioned protein kinase of mentioning has been destroyed the combination of Mdm2.Then other albumen are gone up and cause p53 conformation change as Pin1 raises p53, and this has just stoped the combination of Mdm2 more.Transcribe the co-activation factor and then make the carboxyl terminal acetylation of p53 as p300 or PCAF, 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 kinds cancer is handicapped.PRb so name is because can cause retinal glioma when thereby two allele of the RB1 gene of this albumen of encoding all suddenly change while making this albumen inactivation.Rb is present in cell usually used as phosphoric acid albumen and it is multiple kinase whose phosphorylation target as described below.A kind of function of being furtherd investigate of Rb is to stop cell division or advance the cell cycle.Therefore,, in the time of 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 prevention, or synthesis phase, thereby or advances G1 or the first gap phase to stop cellular replication damaged dna.Rb combination also suppresses E2F family transcription factor.E2F transcription factor is the binary of an 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).Once E2F-DP inactivation, cell will keep resting on the G1 phase.When Rb is incorporated into E2F, this compound will work and stop the propelling cell cycle as GIF.Rb-E2F/DP compound also can attract histone deacetylase (HDAC) albumen to chromatin, thereby it is synthetic further to suppress DNA.

In the time of phosphitylation state, Rb be activate and as TIF by suppress cell cycle progression play a role.Phosphorylation can make Rb inactivation.When phosphatase is during by a residue dephosphorylation of Rb, Rb can be activated in the time approaching G1 latter stage, thereby can be in conjunction with E2F.When cell is to will enter S phase time, the compound of cyclin-dependent kinase (CDK) and cyclin can be by 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 disintegrate down and become activity from pRb.In the time that E2F is released, it (for example can activate the factors such as 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 PCNA molecule can accelerate DNA replication dna and reparation by helping polymerase be incorporated into DNA.

C.ATM

Ataxia-telangiectasia (AT; NM_00051) be the autosome degeneration disorder being caused by the ATM gene mutation being positioned on chromosome 11q22-23.It is identified in June nineteen ninety-five, and is made up of 66 extrons that are distributed on 150kb genomic DNA.It coding 13kb the mature transcript that has 9168 nucleotide open reading frame.ATM albumen is about 370kDa, be all over express and be positioned at nucleus.ATM albumen is a kind of large serine-threonine kinase, and it is considered in cell cycle regulation checkpoint, double-stranded DNA reparation plays a role with in meiosis (similar to BRCA gene).Same known ATM plays a role on regulation and control p53, BRCA1 and CHEK2.The part effect of known ATM in DNA reparation is telomere reparation, because telomere degraded meeting is rapider in the patient who is subject to 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 short insertion and disappearance in displacement, reading frame, etc.The normal copy of albumen can be significantly disturbed in these sudden changes.Most of AT victim, 65-70%, with truncated mutant, its exon skipping sudden change is especially general.This just causes, and atm protein level is very low maybe can not be detected.Missense mutation is the modal mutation type of finding in breast cancer carrier.Be considered to the AT containing slight state with the individuality of two missense mutation, this is the case that can cause attenuation type AT.

D. other TIFs

Such as p53 of multiple other TIFs, ATM and Rb can be for the present invention.For example BRCA1 and BRCA2 are bringing into play key effect in the generation of breast cancer.Thereby BRCA1 maintains the human gene that genomic integrity stops proliferation 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.It is upper that BRCA1 gene is positioned at 21, 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.Although its accurate function is also unclear, BRCA1 albumen is considered in the time that the fracture of DNA double chain is repaired and RAD51 interacts.This class fracture can be caused by natural ray or other irradiations, but during the cell division of carrying out specific type produces sperm and ovum (meiosis), in the time of chromosome crossing over, this class fracture also can occur.By affecting DNA damage reparation, this albumen plays a role on stable maintaining human genome.

BRCA2 participates in the second human gene that chromosome damage is repaired.Although 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 BRCA2 protein combination the fracture of albumen DNA plerosis that regulates RAD51 gene to produce.This class fracture can be caused by natural and medical ray or other ambient illuminations, but during the cell division of carrying out specific type produces sperm and ovum (meiosis), in the time of chromosome crossing over, this class fracture also can occur.BRCA1 albumen also with RAD51 protein-interacting.By DNA plerosis, these three kinds of albumen play a role on stable maintaining human genome.The same with BRCA1, BRCA2 may regulate the active of other genes and in embryonic development, bring into play key effect.It is upper that BRCA2 gene is positioned at 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 mispairing or the guide member of albumen in double-stranded DNA.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, coordinates with MutL and MutH albumen, and in this site grappling DNA chain, certain fragment of a chain is with by its excision.Other albumen complete reparation process: the cut and degraded of the DNA fragmentation of grappling, is used complementary strand as the synthetic sticking patch of template, and sticking patch is connected into position produce a not double chain DNA fragment for the reparation with mispairing.A lot of interest concentrates on the MutS homologue in people source, because some such defects are to form the hereditary nonpolyposis 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.Its helps to control fissional frequency, in tissue, how to adhere to other cells or whether cell moves into or leave tissue, and it also helps to determine that whether the intracellular chromosome number producing in fission process is correct.APC albumen is mainly by completing these tasks with the combination of other albumen, and especially those relate to cell adherence and the albumen that send signal.An activity for albumen, the especially activity of β catenin are by the control of APC albumen, and it is a part for Wnt signal path.The regulation and control of β catenin stop the gene of irritation cell division opened and stop cell transition to be grown by excessively frequent.APC is positioned between 21 and 22, 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 that uses and select of selective actuation, there are two kinds of homology isomeride, ATBF1-A and ATBF1-B.This albumen has 3703 amino acid, and size is 404kDa.It comprises 4 and grows homeodomain and 23 zinc fingerses including a plan zinc-finger motif, a DEAD and a DEAH box, a RNA and an ATP-binding site, two large RS domains and multiple 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 the enhancer element of AFP gene and the expression of lowering AFP gene, may relate to neuron differentiation.Neural crest source cell is with the outer double minute form of chromosome and the non-collinear coamplification of myc in SJNB-12 in early days.In the gastric carcinoma cell lines of expressing at alpha fetal protein, can observe ATBF1 and lack expression.The shortage that ATBF1 expresses is not due to sudden change, deletion or transposition, but due to the strong inhibition of transcriptional level.

III. measure structure, expression or the function of TIF

A. the diagnosis based on nucleic acid

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

According to formula, after application amplification or amplification, use specific nucleic acid to be measured in the nucleic acid Direct Identification sample that the second is known.Then, detect the product of identifying.In specific application, can detect by visual method (for example, the gel of ethidium bromide staining).Optionally, this detection can comprise radioactivity scintigraphy or the fluorescence labeling by chemiluminescence, labelled with radioisotope, or even by making electricity consumption or thermostimulation signal system carry out indirectly Product Identification (Affymax Technology; Bellus, 1994).

In one embodiment, TIF expression is to measure by the amount of mensuration mRNA.But by detecting polytype Structural Faults, also can identify active change.These are damaged comprises deletion, insertion, point mutation and overlapping.Point mutation meeting causes terminator codon, frameshift mutation or amino acid replacement.Somatic mutation is that those betide the sudden change of non-germ cell line tissue and are not heritable, but the sudden change of germ cell line tissue is heritable.Sudden change in or beyond code area also can pass through to change the transcription of this gene, or the processing of change transcript (mRNA) or albumen affects the amount that TIF produces.

When an allele of TIF is due to the heredity of germ cell line or the generation of somatic mutation and when inactivation, cell changes hereditary execution process to carcinogenicity.Another allelic inactivation of gene generally includes and causes the body cell micromutation of heterozygous deletion (LOH) or chromosome allele to be deleted.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 the term primer of definition, referring to comprise can be by any nucleic acid of the synthetic newborn nucleic acid of template dependence process guiding.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, although the form of preferred strand.The definition of probe is different from primer, although 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 band fluorophore (Dan Ruoming, fluorescein) or the radioelement of chemiluminescence (luciferase element) ( ³²p, ¹⁴c, ³⁵s, ³h or other marks) label probe or primer.

Ii. template dependent amplification method

Various template dependence program can be used for the sequence that amplification is present in the mark in 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 two with the reverse direction complementary strand of flag sequence on the primer sequence of regional complementarity.Add excessive deoxynucleoside triphosphate and a kind of archaeal dna polymerase, for example Taq polymerase is in reaction mixture.If flag sequence is present in sample, primer will be incorporated into label, and then polymerase will impel primer to extend along flag sequence by adding nucleotide.By improving and reduce the temperature of reaction mixture, the primer of extension can disintegrate down formation reaction product from label, and excessive primer can be incorporated into label and reaction product and repetition said procedure.

For determining the amount of the mRNA increasing, can carry out reverse transcriptase PCR ^tMamplification program.The method that is cDNA by RNA reverse transcription 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 to some extent in the WO 90/07641 submitting to Dec 21 nineteen ninety.It is well-known in the art that polymerase chain reaction method is learned.

Ligase chain reaction (" LCR ") for the another kind of method increasing, open in No. EPO 320308, at this, its full content is incorporated herein by reference.In LCR, prepare two pairs of complementary probes pair, in the time that target sequence exists, the every pair of probe can be incorporated on the rightabout complementary strand of its target contacting and adjoin each other.In the time there is 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, then as " target sequence " for the right connection of remaining probe.United States Patent (USP) 4,883,750 described the method similar to LCR for by probe to being incorporated into target sequence.

Q β replicase described in PCT application number PCT/US87/00880 also can be used as another kind of amplification method in the present invention.In the method, containing being added in sample under the existence of RNA polymerase with the rna replicon type sequence in the region of target complementation.Polymerase can copy 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 have been used to the amplification of target molecule, this target molecule comprises nucleotide 5 '-α sulfo--triphosphoric acid on a chain of restriction site, Walker etal, (1992).

Strand displacement amplification (SDA) is the another kind of method of carrying out nucleotide isothermal duplication, and it comprises many wheel strand displacements and synthetic, that is, nick translation.A kind of similarly method, is called and repairs chain reaction (RCR), comprises and makes multiple probes run through the annealing of amplification target area, repairs afterwards reaction, only has two kinds in four kinds of bases in this reaction.For easy detection, other two kinds of bases can be used as biotinylation derivant and add.A kind of similar method is applied in SDA.Also can react by circle probe (CPR) and detect target particular sequence.In CPR, on the DNA containing the Probe Hybridization of the intermediate sequence of 3 of non-specific DNA ' and 5 ' sequence and special RNA in being present in sample.After hybridization, carry out processing reaction with ribonuclease H, 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 having another kind of amplification method can be that the present invention is used, and it describes to some extent in GB application number 2202328 and PCT application number PCT/US89/01025, 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 caught component (for example, biotin) and/or detected components (for example, enzyme) mark and be modified by use.After application in, excessive label probe is added in sample.In the time that target sequence exists, probe combination and catalyzed cutting.After cutting, target sequence is by complete release and by excessive probe combination.The cutting of label probe is the signal that target sequence exists.

Other nucleic acid amplification program comprises the amplification system (TAS) based on transcribing, and 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 pass through conventional phenol/chloroform extraction, the thermal denaturation of clinical sample, processes or the guanidine hydrochloride of RNA extracts preparing nucleic acid for amplification with the little centrifugal column of lysis buffer and DNA isolation and RNA.These amplification techniques comprise the primer annealing making containing target distinguished sequence.After polymerization, 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 by adding Article 2 target special primer, carry out afterwards polymerization.Then double chain DNA molecule is transcribed as T7 or SP6 at double by RNA polymerase.In isothermal circulation reaction, RNA is inverted record for single stranded DNA, and single stranded DNA changes into double-stranded DNA subsequently, then transcribes again as T7 or SP6 by RNA polymerase.The product producing, and though be brachymemma or complete, all feeling the pulse with the finger-tip mark distinguished sequences.

Davey et al., No. EPO 329 822 (its full content being incorporated herein by reference at this) discloses one and comprised the synthesizing single-stranded RNA of circulation (" ssRNA "), the nucleic acid amplification method of ssDNA and double-stranded DNA (dsDNA), the method can be used for the present invention.SsRNA is the template of Article 1 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 producing subsequently.The ssDNA producing is the template of Article 2 primer tasteless nucleotide, and it also comprises rna polymerase promoter (as the t7 rna polymerase) sequence that is positioned at template homologue 5 ' end.By archaeal dna polymerase (as large " Klenow " fragment of e. coli dna polymerase I), primer is extended subsequently, produce double-stranded DNA (" dsDNA ") molecule, it contains the sequence that original RNA between one section and primer is identical and at one end contains other promoter sequence.This promoter sequence can be by suitable RNA polymerase numerous RNA of this DNA of generation used copy.Thereby these copies can reenter to circulate and cause very fast amplification subsequently.By the selecting properly of enzyme, this amplification can be carried out and need not add enzyme in each circulation by isothermal.Due to the cycle characteristics of the 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, the method is upper based on promoter/primer sequence being hybridized in target single stranded DNA (" ssDNA "), transcribes afterwards numerous RNA copies of this sequence.The method does not circulate, and for example, new template is not synthetic by the rna transcription thing producing.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).

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

Iii.Southern/Northern trace

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

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

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

Iv. separation method

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

Optionally, can reach separation object with chromatographic technique.There is the multiple chromatography method of the present invention that can be used for: 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 to be visualized.A kind of typical method for visualizing comprises with ethidium bromide gel-colored and estimate under ultraviolet ray.Optionally, if amplified production has integrally been labeled the nucleotide of radioactivity or fluorophotometric mark, after separation, amplified production can be exposed to subsequently on X-ray film or under suitable excitation spectrum and manifest.

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

In one embodiment, detected by the probe of mark.The technology relating 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, is introduced into as a reference at this, and this patent discloses for automatically

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

In addition, can above-mentioned amplified production be identified to specific variation type by sequential analysis with conventional sequence analysis method.In specific method, use the primer sets that is designed for best order-checking, by sequential analysis, gene is carried out to detailed analysis (Pignon et al, 1994).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 design oligonucleotides primer, can carry out analysing amplified sequence by direct Sequencing subsequently.

Vi. kit composition

For detection of with measure genes of interest the needed all base stocks of sequence and reagent can together be assembled in a kit.Conventionally this kit can comprise primer and the probe selected in advance.Also the enzyme that is applicable to amplification of nucleic acid be can comprise, multiple polymerase (RT, Taq, Sequenase comprised ^tMetc.), deoxynucleotide and damping fluid, for providing 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 in suitable mode conventionally.

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

Be the PCR that cDNA carries out relative quantification afterwards by RNA reverse transcription ^tM(RT-PCR ^tM) can be for measuring the relative concentration of the various specific mRNA that separate from patient.By measuring the variation of various specific mRNA concentration, the differential expression of gene that can the specific mRNA kind of code displaying.

At PCR ^tMin, each circulation of reaction is increased to the quantity of the molecule of the target dna of amplification and approaches 2 times, until some reagent become limited.Thereafter, amplification rate becomes more and more slowly until the target increasing between circulation does not increase.If drawn a diagram, X-axis is period, and Y-axis is the concentration logarithm of the target dna of amplification, will form the curve of peculiar shape by the point of drafting is coupled together.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 starts decline and finally become 0.At this moment the concentration of the target dna of amplification 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 starting to reaction is proportional.Same loop number of times the PCR in its range of linearity are completed by measuring each ^tMthe concentration of target dna amplified production in reaction, is just likely determined at the relative concentration of particular target sequence in original DNA potpourri.If DNA potpourri is the synthetic cDNA of RNA by different tissues or cell separation gained,, so just can measure the relative abundance of the specific mRNA of corresponding tissue and cell, mRNA is wherein the source of target sequence.PCR ^tMdirect proportionality between the abundance of the concentration of product and relatively mRNA is only applicable at PCR ^tMin the range of linearity of reaction.

Ultimate density at the target dna of the steady part of curve is to be determined by the availability of the reagent in reaction mixture, and does not rely on the original concentration of target dna.Therefore, pass through RT-PCR ^tMto gather RNA sample population carry out various mRNA relative abundance measure must be satisfied the most important condition be, the PCR of amplification ^tMthe sample of the concentration of product must be at PCR ^tMwhile reacting the linear segment in their curves, gather.

Pass through RT-PCR ^tMtesting and successfully measuring condition that second of relative abundance of various specific mRNA must be satisfied is that the relative concentration of the cDNA that can increase must be standardized as certain independently standard.RT-PCR ^tMthe object of experiment is to measure the abundance of the various specific mRNA for the average abundance of all various mRNA in sample.

Competitive PCR ^tMmost of method use inherent PCR ^tMmaster sample, this master sample is approximately equally abundant with target sample.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 approach the stage of stable development in reaction to sample, it is relatively too high 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 differential expression that detects RNA sample, can become distortion in one aspect, make RNA relative abundance their reality of diversity ratio seem to lack.If inherent standard sample is much abundanter than target sample, this is not just a major issue so.If inherent standard sample is abundanter than target sample, can between RNA sample, carry out direct linear ratio so.

The RT-PCR to detecting clinical material has been described in above discussion ^tMtheory consider.Intrinsic problem in clinical sample is their quantitative change (generation standardization issue), and their qualitative change (need to have the coamplification of believable inherent check sample, preferably the inherent check sample larger than target sample fragment).If RT-PCR ^tMas the relative quantification RT-PCR with inherent standard sample ^tMmeasure, 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 larger 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 instead of the absolute abundance of measuring various mRNA.

Can use the more conventional relative quantification RT-PCR with 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,, for the reverse transcription product of each RNA sample population of being come by different tissues sample separation, must be carefully standardized as the cDNA increasing with isocyatic.Because this analysis is the absolute abundance of measuring mRNA, therefore this consideration is very important.MRNA absolute abundance can only be used for the gene differential expression of the sample of bioassay standard.Although rule of thumb determine that the range of linearity of amplification curve and the standardization of cDNA goods are tediously long and time-consuming process, consequent RT-PCR ^tManalysis can surpass the relative quantification RT-PCR with 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 no inherent standard sample/rival ^tMproduct, thus the sensitivity of analyzing improved.Another reason is if only have a kind of PCR ^tMproduct, on running gel or in 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

What the inventor considered especially is the DNA technique based on chip, as Hacia et al. (1996) and Shoemaker et al. (1996) described those.In brief, these technology comprise fast and the method for quantitative test lots of genes exactly.By by oligonucleotides marker gene or use fixing probe array, can 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 antibody of the present invention be used for characterizing by the technology such as ELISA and Western trace the TIF content of healthy and illing tissue.This can provide a kind of existence or non-existent method of screening malignant tumour, or as the dopester of following cancer, or in the present invention as prediction to being exposed to the method that may reply of oncolytic virus.

Consider antibody of the present invention to be applied in ELISA experiment.For example, the antibody of antitumor inhibiting factor is fixed on selected surface, preferably presents the surface of protein affinity, as the hole of polystyrene microtiter plates.After the material not exclusively adsorbing at wash-out, need to use the hole of nonspecific proteins combination or coating assay plate, known this nonspecific proteins is 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 thereby reduce by antigen non-specific the background that surface produces.

Thereby antibody is incorporated into hole, reduces background and wash-out not in conjunction with after material with the material coating of anergy, immobilized surface is contacted by the method that one contributes to immune complex (antigen/antibody) to form with testing sample.

Form specific immunity compound and wash-out subsequently between testing sample and the antibody of combination after, can be by adding two to resist to measure generation that specific immunity compound forms and equal amounts, two resist the TIF different from primary antibodie are had to specificity.Suitable condition preferably includes with dilution as BSA, ox gamma Globulin (BGG) and phosphate buffer (PBS)/tell dilute sample.The reagent of these interpolations also contributes to help to reduce non-specific background.Subsequently by the antiserum of stratification incubation approximately 2～4 hours at the suitable temperature of approximately 25 DEG C～27 DEG C.After incubation, remove nonimmune compound material thereby rinse the sero-fast surface of 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, the preferably two anti-enzymes that contain combination, after the chromophoric substrate incubation with suitable, this enzyme will produce color development.Therefore, for example, wish promoting to produce under the condition that specific immunity compound forms, use in conjunction with the anti-human IgG of urase or peroxidase with (for example, at room temperature tell as PBS/ at the solution containing PBS in conjunction with two anti-Surface Contacts and incubation a period of time middle incubation 2 hours).

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

Above-mentioned form can change into first sample is incorporated in assay plate, then, by primary antibodie and assay plate incubation, uses afterwards and has two of specific mark to resist to detect the primary antibodie of combination to primary antibodie.

Antibody compositions of the present invention is widely used in Western blotting or Western engram analysis.Antibody can be as the main agents of high-affinity for identifying the albumen being fixed on solid supported matrix, and solid supported matrix is for example cellulose nitrate, polyamide fibre or its composition.Binding immunoassay precipitation reaction, carries out gel electrophoresis subsequently, and these antibody can be used as one step reagent for detection of antigen, causes the disadvantageous background of antigen for detection of the second reagent of antigen.Based on this point, consider that specific use is combined with 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

Hyperplasia disorder is a kind of disease family, is characterized as abnormal or uncontrolled hyperplasia.These diseases are divided into two classes conventionally: more 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.Although a lot of aspects of discussing are afterwards to concentrate in cancer, it will be appreciated that method identical when suitable is also applicable to benign disease.

From typically a benign disease abnormal on cell quantity is different, cancer refers to exist dysplastic with it patient, form tumour or inappropriate hyperplasia, cell cancer and/or that transform, comprise for example dysplastic, tumour, noncontact cell that suppress or neoplastic transformation etc. (for example, melanoma, cancer is as gland cancer, squamous cell carcinoma, small cell carcinoma, oat-cell carcinoma etc., sarcoma is as fibrosarcoma, chondrosarcoma, osteosarcoma etc., hepatoma, neuroblastoma, melanoma, Hematopoietic Malignancies is as lymthoma, leukaemia, myeloma etc.), these are for known in the art, and for it has set up the standard of diagnosis and classification.Be not bound by any theory restrictions, the oncolytic characteristic of reovirus may be derived to the hiv chemokine of the malignant conversioning cell relevant with responsive intracellular environment to, for example, the PKR phosphorylation weakening in Ras active cell as described in this area, or contain as herein defined damaged tumor inhibiting factor subfunction.

A. oncolytic viral therapy

In treatment of cancer, (for example have description about the generation of wild type reovirus and the ad hoc approach of use, United States Patent (USP) 7, 300, 650, 7, 163, 678, 7, 049, 127, 7, 014, 847, 6, 994, 858, 6, 811, 775, 6, 703, 232, 6, 576, 234, 6, 565, 831, 6, 528, 305, 6, 455, 038, 6, 344, 195, 6, 261, 555, 6, 136, 307 and 6, 110, 461, and the open text 2006/0165724 of United States Patent (USP), 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 without withdrawing a claim at this).Myxoma virus therapy is described to some extent in the open text 2006/0263333 of United States Patent (USP), is also 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, accept candidate stem cell treatment by the myeloid tissue that the individuality with hemopoietic system or solid carcinoma of clear marrow sex therapy treatment can purify by use subsequently, the myeloid tissue of purification is by obtaining in the individuality before chemotherapy.In these embodiments, thus can with oncolytic virus process 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 can avoid the problems such as host immune, graft versus host disease and ethics subject under discussion for cell therapy.Because this person's ripe stem cell has extensive value in various regeneration therapies.But, it is reported, in the cultured and amplified in vitro of ripe stem cell, can there are spontaneous nuclear transformation (Tolar et al., 2007; 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 wild type reovirus can effectively remove the cancer cell (Thirukkumaran et al., 2003 that are present in self hematopoietic stem cell populations in vitro; Thirukkumaran et al., 2005).Attenuation type reovirus and myxoma virus can be applied in a similar manner.Inventor expect the present invention can be actually with any maturation at present known or that find afterwards or embryonic stem cell line together with use.

In a particular embodiment, may from cell composition, remove reovirus, for example, come through one section of time enough after killing tumor cell (if existence).For example, thus marrow can by individuality obtain and with reovirus process destroy tumour cell; But, in marrow is implanted to individuality again before (for example, the cancer patient of immunodeficiency), may need to destroy, eliminate and/or remove the virus in composition.In these embodiments, can add Anti-virus agent in cell composition.These reagent comprise antibody and the complement of anti-reovirus, the RNA polymerase inhibitor that the inhibitor of necessary viral enzyme instructs as RNA, 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 from cell composition, remove 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 that the method can be given target cell as the positive-selecting of stem cell colony based on fluorescence activated cell or the method based on adhering to.

C. conjoint therapy

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

Chemotherapeutant includes but not limited to 5 FU 5 fluorouracil, bleomycin, busulfan, camptothecine, NSC-241240, Chlorambucil, neoplatin (CDDP), endoxan, dactinomycin D, daunorubicin, adriamycin, estrogen receptor binding reagents, etoposide (VP16), , farnesyl protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, NA, 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 by these reagent or classification of drug, for example, whether and in which they affect the cell cycle in stage.Or, directly, insertion DNA crosslinked with DNA that can be based on it or synthesize to cause the feature that chromosome and the abnormal ability of mitosis represent reagent by affecting 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 by 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, as attenuated type reovirus, can relate to various modes and substantially side by side (for example, approximately be less than within 1 minute) contact cancer cell, tissue, organ or biosome.In other respects, can be before or after giving attenuation type or wild type reovirus approximately 1 minute, approximately 5 minutes, approximately 10 minutes, approximately 20 minutes, approximately 30 minutes, approximately 45 minutes, approximately 60 minutes, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 25 hours, approximately 26 hours, approximately 27 hours, approximately 28 hours, approximately 29 hours, approximately 30 hours, approximately 31 hours, approximately 32 hours, approximately 33 hours, approximately 34 hours, approximately 35 hours, approximately 36 hours, approximately 37 hours, approximately 38 hours, approximately 39 hours, approximately 40 hours, approximately 41 hours, approximately 42 hours, approximately 43 hours, approximately 44 hours, approximately 45 hours, approximately 46 hours, approximately 47 hours, approximately 48 hours, approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 14 days, approximately 15 days, approximately 16 days, approximately 17 days, approximately 18 days, approximately 19 days, approximately 20 days, approximately 21 days, approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks or approximately 8 weeks or more, and in any time range of being derived by it, 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.Below show the non-limiting example of 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 in treatment cancer, give.According to them in intracellular active mode by these reagent or classification of drug, for example, whether and in which they affect the cell cycle in stage.Or, directly, insertion DNA crosslinked with DNA that can be based on it or synthesize to cause the feature that chromosome and the abnormal ability of mitosis represent reagent by affecting 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 affects 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 in these alkylating reagents any one or more kinds of associating be used for the treatment of cancer, some of them are discussed below.

Busulfan (also referred to as bridle orchid) is a kind of difunctional alkylating reagent.Busulfan is chemically being called as Isosorbide-5-Nitrae-bis-methylsulfonyl butane.

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).Although be not cure 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 previously treated chronic myelogenous leukemia, after using busulfan, can obtain that organomegaly is disappeared or stable hematologic response.With regard to survival period and pigment horizontal stable, its curative effect is better than irradiation spleen, and is equating with the curative effect with radiation exposure aspect the enlargement of control spleen.

Chlorambucil (also referred to as chlorambucil) is a kind of alkylating reagent of bifunctional mustargen type, is found in non preference human tumor disease and 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 quick and complete by intestines and stomach.After giving single oral dose 0.6-1.2mg/kg, blood Chlorambucil level can arrive 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 for antineoplastons.Methods for the treatment of is well known to those skilled in the art and can in " Physicians Desk Reference " and " Remington ' sPharmaceutical Sciences " that quoted here, finds.

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

Neoplatin is widely used in treating cancer as metastatic testis or oophoroma, advanced bladder carcinoma, head or neck cancer, cervix cancer, lung cancer or other tumours.Neoplatin can use separately or combine use with other reagent, and its effective dose in clinical practice is 15-20mg/m ², every three weeks administration 5 days, altogether three courses for the treatment of.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 ².Certainly, these all dosage are all exemplary, and any dosage also expecting between these points can be for the present invention.

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

Endoxan is 2H-1,3,2-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 by amino 3-1-propyl alcohol and N, two (2-chloroethyl) phosphinylidyne dichloro [(ClCH of N- ₂cH ₂) ₂n--POCl ₂] in dioxane solution condensation prepare endoxan.Condensation reaction is double, relates to hydroxyl and amino group, thereby realizes cyclisation.

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

Comprise for the suitable dosage of being grown up, oral, 1-5mg/kg/day (administering drug combinations conventionally), depends on gastrointestinal tolerant power; Or 1-2mg/kg/day; Intravenous injection, initial within the time of 2-5 days divided dose injection 40-50mg/kg or every 7 to 10 days injection 10-15mg/kg or inject weekly twice 3-5mg/kg or 1.5-3mg/kg/day.Can give the dosage of 250mg/kg/day as antitumor.Due to the detrimental effect to stomach and intestine, intravenous approach is preferably to use.In stationary phase, conventionally need 3000-4000/mm ³leucocyte amount.Sometimes also carry out administered intramuscular by infiltrating or entering body cavity.Injection 100,200 and 500mg and tablet 25 and 50mg are feasible dosage forms, and about the professional technique of the details of dosage, with reference to " Remington ' s Pharmaceutical Sciences " the 15th edition, 61 chapters, introduce in this as list of references.

Melphalan, 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 in non preference human tumor disease.Its chemical name is two (2-ethyl chloride) amino of 4-[]-L-Phe.

Melphalan is the activated L-type isomeride of this compound 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 on chromosome, produces required the wanting greatly of the required dose ratio L-type isomeride of effect.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 tablet form oral medication and is used for the treatment of Huppert's disease.

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

Melphalan is used to treat Epithelial ovarian tumor.The 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 for the treatment of.According to blood tolerance, (Smith and Rutledge, 1975 repeat a treatment for every four-five weeks; Young et al, 1978).Or the using dosage of melphalan can be low to moderate 0.05mg/kg/day or high to 3mg/kg/day, or between these dosage or higher than any dosage of these dosage.According to patient's to be treated situation, on dosage, must there be some variations.Under any circumstance, should be determined the reaction of administration by human body the suitable dosage of individual patient.

Metabolic antagonist.Metabolic antagonist destroys the synthetic of DNA and RNA.Different from alkylating reagent, they affect the cell cycle in S phase specificity.Except mammary gland, ovary and gastroenteric tumor, they are also 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) is fluoro-2,4 (1H, the 3H)-hybar X of 5-.The methylation reaction that its mechanism of action is considered to become thymidylic acid by blocking-up deoxyuridylic acid carries out.Therefore, 5-FU disturbs the synthetic of DNA (deoxyribonucleic acid) (DNA) and suppresses the formation of RNA (ribonucleic acid) (RNA) on compared with low degree.Because DNA and RNA are essential for cell division and propagation, thereby therefore the effect of 5-FU is considered to produce thymidine defect and causes cell death.Therefore, 5-FU can be applicable in the cell with metastatic cancer feature of division rapidly.

Antitumor antibiotics.Antitumor antibiotics is containing antibiotic property and two kinds of activity of cytotoxicity.These medicines are also by 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, from passing through the administration of vein bolus injection with 21 days intervals, gives the 25-75mg/m of adriamycin ², to intravenous injection or the oral 35-100mg/m that gives etoposide ².

ADMh; 5,12-naphthalenedione, (8s-is suitable)-10-[(3-amino-2; 3; 6-tri-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 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 is also a kind of immunodepressant.

Adriamycin malabsorption, must intravenous administration.Pharmacokinetics is 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 bile.The metabolism in liver of most residue, part metabolism becomes activated metabolin (adriamycinol), but has some number percents to be secreted in urine.In the time there is hepar damnification, dosage needs to reduce.

Adult's intravenous suitable dosage is to give 60-75mg/m with 21 days intervals ², or often within continuous 2 or 3 days, repeat to give 25-30mg/m with 3 or 4 weekly intervals ², or give weekly 20mg/m ².When existing while invade by early stage chemotherapy or tumour marrow the early stage bone marrow suppression causing, or in the time that this medicine and other myelocytes generation suppressive drug are combined use, should use lowest dose level to aged people.If serum bilirubin is at 1.2-3mg/dL, dosage should reduce by 50% so, if higher than 3mg/dL, should reduce by 75% so.To there being the accumulated dose between patient's operating period of normal heart function should not exceed 550mg/m ², and should not exceed 400mg/m to the patient who has accepted mediastinal irradiation ².Or, often give for three days on end 30mg/m ², every surrounding repeats once.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 ².Certainly, these all dosage are all exemplary, and any dosage also expecting between these points can be for the present invention.

Daunorubicin hydrochloride, 5,12-naphthalenedione, (8s-is suitable)-10-[(3-acetyl group-2, the own pyranose of 3,6-, tri-deoxidations-a-L-lysol lyxo-) oxygen]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-10-methoxyl-hydrochloride; 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.

Combine with other drug, it can be for the acute stage of acute myelocytic leukemia (slowing down for promotion), acute lymphatic leukemia and the chronic granulocytic leukemia for the treatment of adult in the chemotherapy as first-selected.Daunorubicin oral absorption is bad, and it must intravenous administration.Distribute the half life period be 45 minutes and get rid of the half life period be about 19 hours.The half life period of its active metabolite daunorubicinol is about 27 hours.The metabolism and be also secreted into (ca 40%) in bile in liver of daunorubicin major part.In liver or the incomplete individuality of renal function, dosage must reduce.

The intravenous suitable dosage (radix equivalence) of being grown up is: 60 years old following every 3 or 4 weeks injection 45mg/m of patient ²(within 60 years old, above patient is 30mg/m to/day ²) 1,2 or 3 day, or every 3 or 4 weeks injection 0.8mg/kg/day 3-6 days; If can only be 450mg/m except having carried out chest irradiation ²outside, administration during use should not exceed 550mg/m ²; For children, unless the age is less than 2 years old or health area is less than 0.5m ², be weekly 25mg/m ², to use in this case the adult's table 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 ².Certainly, these all dosage are all exemplary, and any dosage also expecting between these points can be for the present invention.

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

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

In human body, after 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 impact that is mainly subject to metabolism in liver, but metabolism also can occur in its hetero-organization.Owing to thinking the saturated of degraded path, speed and the maximum serum concentration of removing are inverse ratio.The mitomycin of about 10% dosage does not change and is secreted in urine.Because metabolic pathway is saturated under relatively low dosage, the percentage that is secreted into the mitomycin in 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 being used for the treatment of in the first-selection combination of choriocarcinoma, embryonal rhabdomyosarcoma, testicular tumor, Wilms' tumor.The tumour of Systematic therapy answer failed is replied to local perfusion therapy sometimes.Dactinomycin D can strengthen radiotherapy.It is Secondary cases (spreading out of property) immunodepressant.

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

One half-value dose is intactly secreted in 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.Conventionally the dosage of every day is 10-15mg/kg; Intravenous injection 5 days; If do not produce toxicity performance, can give again a course for the treatment of with the time interval in 3-4 week.Inject 100-400mg every day to children, continue 10-14 days; In additive method, use 3-6mg/kg, 125mg/kg altogether, and fixed dosage is 7.5mg/kg weekly.Although by safer in the conduit of medicine afferent veins infusion, but give direct intravenous injection, for avoiding subcutaneous reaction, its preventive measure are syringe needles of discarding for extract medicine from bottle.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 ².Certainly, these all dosage are all exemplary, and any dosage also expecting between these points can be for the present invention.

Bleomycin is the potpourri by the cytotoxin glycopeptide antibiotic of separating in streptomyces verticillus.Although definite mechanism of action the unknown of bleomycin, has available evidence to show 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 lymphatic vessel.Contrary with the low concentration in hematopoietic tissue, can find the bleomycin that the tumour cell of skin and lung contains high concentration.The bleomycin of the low concentration of finding in marrow may with in these tissues, find that high-caliber bleomycin digestive enzyme is relevant.

Remove in the patient of > per minute 35ml at creatinine, the half life period that serum or blood plasma are finally got rid of bleomycin is approximately 115 minutes.Remove in 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% gives is recovered in urine with the form of active bleomycin.Can give bleomycin by muscle, vein or subcutaneous route.It can dissolve in large quantities in water.

Bleomycin should be considered for the therapy of releiving.It can effectively treat following tumour with single reagent form or with confirmed with the forms of other authorized chemotherapeutants combinations report demonstration: squamous cell carcinoma is 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 is also used to treat lymthoma and carcinoma of testis.

Because Lymphoma patients has anaphylactoid possibility, should or still less treat patient with 2 units for initial two doses.If there is not acute reaction, so just can follow conventional dose form.

In two weeks, can improve rapidly and significantly hodgkin's disease and carcinoma of testis.If do not see improvement within this time, the possibility of improving is so just little.Squamous cell carcinoma response is slower, sometimes needs to reach 3 weeks and 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 it is mainly combined and be used for the treatment of orchioncus with bleomycin and neoplatin, combines be used for the treatment of small-cell carcinoma of the lung with neoplatin.It also has the activity of anti-non-Hodgkin's lymphomas, acute nonlymphocytic leukemia, breast cancer and the Kaposi's sarcoma relevant to aids (AIDS).

VP16 can be used for oral administration for intravenous administration and 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, conventionally also can use administration every day 35mg/m ²continuous administration 4 days-every day 50mg/m ²continuous 5 days.In the time of oral administration, dosage need to double.Therefore for small-cell carcinoma of the lung, dosage can be up to 200-250mg/m ².Intravenous injection dosage (at conjoint therapy) 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 for the treatment of cycle repeats once.Should be in 30-60 minute slowly infusion administration thereby to avoid may be hypopiesia and the bronchial spasm causing due to the solvent in formula.

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

It is reported after the oral vincaleukoblastinum or vincristine giving, have unexpected absorption.Giving after conventional clinical dosage, in blood plasma, the maximum concentration of every kind of medicine is approximately 0.4mM.Vincaleukoblastinum and vincristine are incorporated into plasma proteins.They extensively concentrate on blood platelet and on compared with low degree, are present in leucocyte and 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 intactly in urine, and 10% is recycled in excrement.Patient for hepatosis should reduce dosage.If the concentration of plasma bilirubin is greater than 3mg/dl (about 50mM), so at least need to reduce 50% dosage.

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

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 and in multiple lymthoma, there is favourable replying, especially hodgkin's disease, in this case of 50-90%, can see significant improvement.In the time that disease is difficult to be cured by alkylating reagent, the effect of vincaleukoblastinum in a high proportion of lymthoma does not reduce.It is also to Kaposi's sarcoma, 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 sick human needs by clinician.Can give 0.1-0.3mg/kg or also can give 1.5-2mg/m ².Optionally, 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 ².Certainly, these all dosage are all exemplary, and any dosage also expecting between these points can be for the present invention.

Vincristine is blocked mitosis and is caused that stop metaphase.Seeming most of biologic activity of this medicine probably can be aggregated to the ability of microtubule and explain in tubulin blocks protein by specific binding by it.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 combines use with other myelosuppressives conventionally.

It is reported and after vincaleukoblastinum or vincristine, have unexpected absorption oral giving.Giving after conventional clinical dosage, in blood plasma, the maximum concentration of every kind of medicine is approximately 0.4mM.

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

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

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

Vincristine has effect in hodgkin's disease and other lymthomas.Although when it is during separately for hodgkin's disease, it seems lower a little than the effect of vincaleukoblastinum, but when combine 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 non-Hodgkin's lymphomas, vincristine is a kind of important reagent, especially when combine use with endoxan, bleomycin, adriamycin and prednisone.In lymphocytic leukemia, vincristine is more effective than vincaleukoblastinum.It is reported and in the patient of multiple other tumours, there is favourable replying, especially Wilms' tumor, neuroblastoma, brain tumor, rhabdomyosarcoma, and the reproductive system cancer of mammary gland, bladder and masculinity and femininity.

The dosage that vincristine uses by clinician according to each sick human needs and definite.Can give 0.01-0.03mg/kg or 0.4-1.4mg/m ², or also can give 1.5-2mg/m ².Optionally, can give 0.02mg/m by Intravenous Infusion 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 ².Certainly, these all dosage are all exemplary, and also expect that any dosage between these points can be for the present invention.

Camptothecine is a kind of alkaloid that is derived from Chinese tree camplotheca acuminata.Camptothecin analogues has unique ability, can, by the stable covalent reaction intermediate product that is called " can shear compound ", suppress DNA topoisomerase, finally causes death of neoplastic cells.Generally believe, camptothecin analogues presents significant antitumor and anti-leukocythemia liveness.Due to its serious spinoff and bad water-soluble, camptothecine application is clinically 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 yellow powder form, and can in DMSO 1N NaOH, be dissolved as the limpid yellow solution of 50mg/ml.If be stored in 2-8 ⁰in the environment of the dry sealing lucifuge of X, it can at least be stablized 2 years.

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

Carmustine (aseptic Carmustine) is the one being used for the treatment 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 in alcohol and lipid is high, indissoluble in water.The administration of suggestion Carmustine is to give by venoclysis after it restores.Aseptic Carmustine provides with the form of the freeze dried substance of the single dosage bottle of 100mg conventionally.

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

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

Carmustine is every 6 weeks intravenous injection 150-200mg/m as single reagent at the RD of previous untreated patient ².This administration can be used as single dosage and gives or be divided into and inject as 75-100mg/m every day, injects continuously 2 days.Combine with other myelosuppressives when using or being used in the patient that marrow deposit exhausts when Carmustine, should correspondingly adjust dosage.Should reply to adjust the dosage after predose to the blood of above-mentioned dosage according to patient.Certainly it should be understood that other dosage also can be 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 ².Please professional and technical personnel with reference to " Remington ' s Pharmaceutical Sciences " the 15th edition, 61 chapters.According to the patient's for the treatment of situation, dosage must change to some extent.Human body under any circumstance can determine the suitable dosage of individual patient to the reaction of administration.

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

Although generally believe that lomustine makes DNA and RNA alkylation, it does not intersect resists other alkylating reagents.Together with other nitroso ureas, it also can be by making amino acid carbamoylation in albumen suppress the enzymatic process of multiple keys.

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

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

As single reagent, the RD in adult and children is lomustine: be every 6 weeks 130mg/m at previous untreated patient ²as single oral dose.The individuality weakening for marrow function, its dosage should reduce to every 6 weeks 100mg/m ².In the time of lomustine and other myelosuppressive Drug combinations, 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 individual required decision for the treatment of by clinician.

Other reagent.Operable other reagent comprise Arastin, Iressa, erbitux, bortezomib and Gleevec.In addition, growth factor receptor inhibitors and little molecule 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 conventionally depends on target is used to immune effector cell and molecule and destruction of cancer cells.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 for the treatment of, or it can be raised other cells and realizes veritably killing and wounding of cell.Antibody also can with medicine or toxin (chemotherapeutic, radioactive nuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) 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 a part for conjoint therapy, is combined with reovirus or other oncolytic viral therapies.At the following conventional steps that conjoint therapy is discussed.Conventionally, 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 in content of the present invention, any one of these labels can be applicable to grappling.Common tumor marker comprises carcinomebryonic antigen, prostate specific antigen, Patients with Urinary System Tumors related 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 cell surface expression viral antigen, thereby also makes them present the 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 is also the medium of infectious shock, and promotes kalabolism, heating and sleep.The stimulation that some infectious reagent produce by TNF causes tumour decline.In the time using separately with effective dose, TNF is very virose, and therefore best therapy may be combined use with lower dosage with other drug by it.Its immunosuppressive action can be strengthened by gamma interferon, and therefore conjoint therapy is proemial.The potpourri of TNF and interferon-' alpha ' is also found to have antitumaous effect.

Iii. hormonotherapy

According to method as described herein, sex hormone is used for the treatment of to cancer.Although 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.

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

Iv. radiotherapy

Radiotherapy, also referred to as radiotherapy, is the method with ionizing radiation treatment cancer and other diseases.The energy that ionising radiation is accumulated damages or damages the cell in the region being treated by destroying its inhereditary material, make the impossible continued growth of these cells.Although radiation damage cancer cell and normal cell, the latter can self repair and correct functionating.Radiotherapy can be used to the solid tumor for the treatment 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 the lymphoid cancer of blood formation cell).

Can include but not limited to according to radiotherapy used in the present invention to, 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 copying and reparation and chromosomal assembling and maintain the damage that produces wide scope DNA, DNA precursor, DNA.The dosage range of X-ray uses 50-200 roentgen's dosage to 2000-6000 roentgen's single dose from the every day of long period (3-4 week).Radioisotopic dosage range alters a great deal, and it depends on intensity and the kind of the radioactive ray of isotopic half life period, transmitting, and the absorption of tumour cell.

Radiotherapy can comprise the position (radioimmunotherapy) that with radiolabeled antibody, dose radiation is directly sent to cancer.Antibody is high specific albumen, and replying that it is existed antigen (being allogenic material by immune system recognition) by body produces.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 laboratory preparation.Once be injected in body, antibody will be found out cancer cell energetically, is used for destruction of cancer cells by the cell killing (cytotoxicity) of radioactive ray.This method can make healthy cell by the risk minimization of radiation damage.

With conventional radiotherapy, equally to use identical radiotherapy instrument be linear accelerator to conformal radio therapy, thereby but on the path of X ray light beam, place shape that derby changes it and make the form fit of itself and cancer.This just ensures to give the dose radiation that tumour is higher.Healthy peripheral cell and near structure are accepted compared with the radioactive ray of low dosage, so the possibility of spinoff has just reduced.Now develop 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 on the sheet metal on linear accelerator.Thereby can adjust each lamella makes 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 check with a special scanner position of your inherent organ.

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

Although investigation shows that conformal radio therapy and intensity modulated radiation therapy can reduce radiotherapeutic spinoff, the area for treatment that likely so accurately formalizes just can stop cancer cell small outside area for treatment destroyed.This just means that the risk that uses these professional radiotherapy technology can make cancer recur in future is higher.Three-dimensional located irradiation therapy is used to treat brain tumor.This technology is carried out radiotherapy from a lot of different angles, thereby make to enter, the dosage of tumour is very high and to affect the dosage of surrounding health tissue very low.Before treatment, thereby guarantee the accurate grappling of radiotherapy by the multiple scanning of Computer Analysis, and in the time accepting radiotherapy, patient's head is fixed in the framework of a special manufacture.Give multiple dosage.

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

Scientist also finds the approach that improves radiotherapy effect.They have studied two kinds of research medicines to standing the effect of the cell after radioactive ray.Radiosensitizer makes the destroyed possibility of tumour cell larger, and radiosensitizer can protect normal structure to avoid the impact of radioactive ray.People have also studied the hyperthermia that uses heat makes tissue to the effect aspect radioactive ray sensitivity.

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 be combined with other therapies, as therapy of the present invention, chemotherapy, radiotherapy, hormonotherapy, gene therapy, immunotherapy and/or selectivity therapy.

Therapeutic operation comprises resection, and in resection, cancerous tissues all or a part are by physical removal, excision and/or destruction.Tumorectomy refers at least part of tumour of physical removal.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 combined in the removal of the accidental amount of normal structure.

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

Brief description of the drawings

The following drawings forms a part of the present invention and is included to further prove specific part of the present invention.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 use WT/AV reovirus of 40MOI or the expression GFP of 5MOI, the GFP that is Lausanne (ATCC) strain of MYXV expresses version, this description is 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, be fixed/permeate and use reovirus antiserum and the 2nd FITC antiserum by FACS, they to be analyzed to cell.Result shows that wild type and attenuation type reovirus all can preferably infect p53-/-MEF.For the research of myxoma infections, infection cell was observed after 24 hours under phase correlation microscope and fluorescent microscope.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 defect; Kozlov et al, 2003).Infect after 3 days, be fixed/permeate and use reovirus antiserum and the 2nd FITC antiserum by FACS, they to be analyzed to cell.Result shows that wild type and attenuation type reovirus all can preferably infect p53-/-MEF.For the research of myxoma infections, infection cell was observed after 48 hours under phase correlation microscope and fluorescent microscope.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 defect have strengthened replying that IR is stimulated.With 2Gy ionising radiation lymphoma mantle cell (Granta, HBL-2 (p53 sudden change, Turker et al, 2006) Z138C, JVM-2)) and then harvesting of Burkitt lymphoma (Raji and Ramos) 2 hours.Process the full cell pyrolysis liquid of preparation with NET-N lysis buffer (1%NP-40), follow by ultrasonication.Then 50 μ g albumen are carried out to 8% or 10%SDS-PAGE, gone to nitrocellulose filter and surveyed (Phospho-ATM with labelled antibody, pSerl981 ATM and Phospho-p53, pSerl5 and p53 are purchased 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,, and p53 and/or ATM answering obstacle cell (L3, HBL-2 Ramos), Grant, Raji) the viral susceptibility of reovirus and myxoma virus is illustrated.1; Responsive to virus.0; Virus is had to resistibility.Reply that normal cell has resistibility to virus infections and the cell of answering obstacle to virus infections sensitivity.(Fig. 2 C) uses the WT/AV reovirus of 40MOI or myxoma virus (Myx-GFP) infection cell of 5MOI.Previous studies show that Raji cell is responsive and Ramos cell is resistive (Alain et al, 2002) to reovirus to reovirus.Infect after 3 days, cell is fixed/permeated and use reovirus antiserum and the 2nd FITC antiserum by FACS, they to be analyzed.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 after 48 hours under phase correlation microscope and fluorescent microscope.The cell of expressing GFP presents myxoma infections.Mock: simulated infection.

Fig. 3 reovirus and myxoma infections retinoblastoma cell.With the WT/AV reovirus of 40MOI or the myxoma virus (Myx-GFP) of 5MOI infection retinoblastoma cell (Y79 and WERI-Rb-1, purchased from ATCC).Infecting after specified number of days, cell is fixed/is permeated and use reovirus antiserum and the 2nd FITC antiserum by FACS, they to be analyzed.For the research of myxoma infections, infection cell was observed after 48 hours under phase correlation microscope and fluorescent microscope.The cell of expressing GFP presents myxoma infections.Mock: simulated infection.

Embodiment

Following examples are used for the preferred embodiments of the present invention of demonstrating.Those skilled in the art will be appreciated that in following examples, disclosed technology represents the technology that inventor discloses, and go on well in the operation of the present invention, therefore can consider to use it for the preferred version of formulating operation.But, 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 published specific embodiment, can carry out many changes and still can obtain same or analogous result.

Embodiment 1-materials and methods

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

The preparation of wild type and attenuation type reovirus.For the wild type reovirus T3D strain of these researchs the breeding of L929 cell and according to before described in carry out purifying.Except AV reovirus is in HT10801 and L929 cell breeding, the method for attenuation type reovirus that purifying is derived from HTR1 nutrient culture media is identical with the method for preparing wild type reovirus.The reovirus of 5-10 MOI is joined in cell and at 37 DEG C and maintains 48-72 hour.Observing viral oncolysis effect (typically 20-30% lysis) afterwards, purified virus from the cell of sedimentation.With 35,000rpm, virus is carried out to the centrifugal 7-8 hour of CsCl with SW41 rotary head.Collect the virus of layering and use 150mM NaCl, 10mMMgCl2, and 10mM Tris (pH 7.5) extensively dialyses 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 DEG C of absorption.Then cover cell monolayer with 1% agar and fresh culture.Infect and calculate plaque number after 5-7 days.

For carrying out the titration of attenuation type reovirus, except removing agar after 5-7 days and cell monolayer fixed/permeated with fix/osmotic agent of cell box (BD Bioscience) infecting, to it is carried out outside immunostaining with antiserum and the 2nd FITC antibody of reovirus, all the other steps are identical with wild type.Detect the plaque of qualification AV reovirus by immunofluorescence.Optionally, for attenuation type reovirus, also on L929 cell, carry out conventional plaque experiment.Infect latter 3 days, agar covers by dimethyl diaminophenazine chloride, and after 24 hours, detects cell monolayer at plaque test.For for the attenuation type reovirus of collecting the supernatant of HT1080 and HTR1 infection, use the high speed ultracentrifugation of 35,000rpm to make 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 gene, and this expression cassette is driven by the vaccinia virus morning/late promoter synthesizing.On BGMK cell, concentrate formation to breed with titration Myx-GFP (Opgenorth et al, 1992) by foregoing.

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

Embodiment 2-result

Reovirus and myxoma virus preferably infect the cell of p53 or ATM defect.Whether affect the susceptibility of oncolytic virus in order to detect the adjusting of tumor suppressor gene, inventor has selected p53 and the ATM tumor suppressor gene that in kinds cancer, often can suddenly change.P53 be former suppressor oncogene and in multiple cancer cell sudden change the most frequent, it is reported that more than 50% cancer retains p53 sudden change (White, 1994; Morris, 2002).ATM (ataxia telangiectasia sudden change) is a kind of serine threonine protein kinase, in the time of DNA damage, is activated.In ataxia telangiectasia, two of ATM gene copies are dysfunction, ataxia telangiectasia is a kind of rare syndrome, show as cancer tendentiousness, radiosusceptibility and neurodegeneration (Kitagawa et al, 2005).Therefore inventor uses the L3 lymphoid stem cells (Kozlov et al, 2003) of p53-/-MEF (mouse embryo fibroblasts) and ATM defect to detect the susceptibility of oncolytic virus.As shown in Figure 1A-B, cell (cell of FITC+ or GFP+) by FACS or the microscopic examination fluorescence positive shows, 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 defect).

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 defect, inventor has detected subsequently reovirus and can myxoma virus preferably infect the human lymphoma that carries p53 or ATM defect.Inventor has tested 6 kinds of lymthomas (HB L-2, Granta, Z138C, JVM2, Raji and Ramos), and in the time attacking in the face of genotoxicity, these lymthomas present the different cell response (Fig. 2 A) from p53 and ATM dependence path.By IR radiation with identify the functional status of p53 and ATM by the phosphorylation that western trace detects p53 and ATM.The contrast that the ATM that BT (ATM is normal) and L3 (ATM defect) are used as being 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).Granta cell stress present that 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, as shown in the p53 causing as stimulated by IR and the hyperphosphorylation of ATM (Fig. 2 A).What is interesting is, cell (cell of FITC+ or GFP+) by FACS or the microscopic examination fluorescence positive shows, lymthoma (the Z183C replying with respect to ATM-and p53-, JVM-2, Ramos), p53 and/or atm feature obstacle lymthoma (HBL2, Granta, and Raji)-in the time that IR stimulates, the p53 that HBL-2 and Raji present constitutive character activates, and Granta presents ATM defect-be preferably responsive (Fig. 2 C) to reovirus and myxoma infections.As shown in Figure 2 B, to reply be the susceptibility height correlation with reovirus and myxoma virus for the ATM of cell or the dysfunction of p53.This proves that p53 and ATM all participate in setting up the resistibility of cell to virus infections forcefully, and its viral susceptibility (Fig. 2 A) is given in gene or its damaged meeting of replying path.

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, whether also inventor has also detected the damaged cancer cell related infection sensitivity to reovirus and myxoma virus of RB.As shown in Figure 3, show by the cell (cell of FITC+ or GFP+) of FACS or the microscopic examination fluorescence positive, two kinds of different mankind's cancer eye cells are responsive to the infection of reovirus and myxoma virus.Two kinds of cells all contain damaged Rb gene (Reid et al., 1974).

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

According to current disclosed content, disclosed and claimed all methods all can be carried out and complete here, and need not carry out excessive experiment.Although described the compositions and methods of the invention according to preferred embodiment, obviously, for a person skilled in the art, in the situation that not departing 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.The 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 the scope of the claim definition as appended.

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Claims (11)

1. the application of at least one in reovirus reagent and myxoma virus reagent in patient's the medicine for the preparation for the treatment of with hyperplasia disorder, wherein said viral reagent has the effect of oncolytic to the hyperplasia cell of tumor suppressor gene sudden change, and wherein said hyperplasia cell is p53, ATM or the handicapped cell of Rb.

2. application as claimed in claim 1, wherein, described medicine further comprises non-viral cancer therapy reagent.

3. application as claimed in claim 2, wherein said non-viral cancer therapy reagent is selected from the group being made up of the inhibitor of chemotherapy reagent, radiotherapy reagent, hormonotherapy reagent, gene therapy reagent, immunotherapy reagent and p53, Rb or atm feature or expression.

4. the application as described in any one of claim 1-3, wherein said patient suffers from cancer.

5. the application as described in any one of claim 1-3, wherein said reovirus reagent is wild type reovirus.

6. the application as described in any one of claim 1-3, wherein said reovirus reagent is attenuation type reovirus.

7. application as claimed in claim 6, wherein said attenuation type reovirus uses a kind of σ 1 capsid protein to express that damaged, σ 1 capsid protein can't detect or without the reovirus of σ 1 capsid protein.

8. the application as described in any one of claim 1-3, wherein said myxoma virus reagent is the myxoma virus that GFP expresses.

9. application as claimed in claim 1, wherein, described medicine further comprises the second anti-hyperplasia reagent.

10. application as claimed in claim 9, wherein, described the second anti-hyperplasia reagent is 5 FU 5 fluorouracil, bleomycin, busulfan, camptothecine, NSC-241240, Chlorambucil, neoplatin, endoxan, dactinomycin D, daunorubicin, adriamycin, estrogen receptor binding reagents, etoposide, farnesyl protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, NA, nitroso ureas, plicamycin, procarbazine, Raloxifene, tamoxifen, taxol, Temozolomide, cis-platinum, vincaleukoblastinum, methotrexate (MTX), the derivant of vincristine or above-mentioned substance.

11. application as described in any one of claim 1-3, wherein said hyperplasia cell is malignant cell or benign cell.