CN100379453C - Use of myxoma virus for therapeutic treatment of cancer and chronic viral infection - Google Patents
Use of myxoma virus for therapeutic treatment of cancer and chronic viral infection Download PDFInfo
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Abstract
The present invention relates to therapeutic use of Myxoma virus. Myxomas virus can selectively infect cells that have a deficient innate anti-viral response, including cells that are not responsive to interferon and can be used to treat diseases characterized by the presence of such cells, including cancer.
Description
Technical field
The present invention relates generally to the purposes of myxoma virus in treatment.
Background technology
Being used for various treatment for cancer methods at present often works by murder by poisoning or kill cancer cell.Unfortunately, cancerous cell is had toxic Therapeutic Method and generally healthy cell is also had toxicity.And, effectively treat cancer one of the main reasons---the heterologous of tumor it be unclear that.Present main flow therapy (as chemotherapy and X-ray therapy) is tended to use in narrow virulence therapeutic domain.Because being of limited application of different and these Therapeutic Method of tumor cell, the Therapeutic Method that it is believed that these types is not strong instrument.
The modern anti-cancer therapies of developing is at present attempted optionally target tumor cell, and healthy cell is had more hypotoxicity, and healthy cell is not affected.
Oncolytic therapy is intended to utilize the cell difference between tumor cell and normal cell and a kind of method of realizing treating.This method is used replication capacity, tumor is had optionally viral vector as anticarcinogen.Oncolytic virus or target cancer cell and infecting specifically, or compare with in healthy cell, duplicating, be more suitable in cancerous cell, effectively duplicating.These oncolytic viruses that replication capacity is arranged are naturally occurring, or through genetic engineering modified and become targeting allos tumor group's the high selectivity and the instrument of high efficiency.Because have and duplicate optionally that oncolytic virus does not effectively duplicate in normal cell, so particularly with respect to traditional remedies such as X-ray therapy or chemotherapy, little to patient's toxicity.
Big quantity research has been reported the oncolytic activity of various Strain, and the most promising oncolytic virus is naturally occurring or through adenovirus, herpes simplex virus 1 (" HSV1 "), reovirus, vaccinia virus, vesicular stomatitis virus (" VSV ") or the poliovirus of genetic modification.The modified oncolytic virus of studying at present as anticarcinogen comprises HSV (herpes simplex virus), adenovirus, Avian pneumo-encephalitis virus (" NDV "), reovirus, vaccinia virus, Measles virus, VSV and poliovirus.Various oncolytic viruses are in first or second stage of clinical trial, and some of them virus has demonstrated stable effect.But, do not know which virus meets the oncolytic purpose that continues to duplicate, have specificity and strong lytic activity most.Oncolytic virus candidate carrier in full force and effect is should life cycle short, form mature virion fast, can effect spread be arranged at iuntercellular, and have the bigger genome of being convenient to insert.Simultaneously, evidence suggests that suppressing early stage innate immunity, to reply and delay the development that Th1 (helper T cell 1) replys most important to the effectiveness of oncolytic therapy.For the many viruses that are intended for use to develop oncolytic virus, the high-level antibody and the t cell response that are observed from normal population are measured, and the human virus obviously has hyperimmunization originality.
Clinical position shows that present oncolytic virus is safe really, but also is not enough to have clinical effectiveness completely as the curative effect of monotherapy.Fully or effectively do not infected owing to often observe tumor cell, present trend is genetic engineering modified by candidate's virus is carried out, so that its express therapeutic transgenic, thereby improve their effectiveness.Also the above-mentioned oncolytic virus of major part is tested simultaneously in conjunction with other common oncolytic therapy.
Adenovirus can easily carry out genetic manipulation, and has the well-known function relevant with virus protein.In addition, it is relevant with quite slight disease.ONYX-015 human adenovirus (Onyx drugmaker (Onyx Pharmaceuticals Inc.)) is tried to have carried out at clinical practice the most widely one of oncolytic virus of optimizing.It is believed that it preferably duplicates in the negative tumor of p53, and in head and neck cancer patient's clinical trial, show potentiality.But, have report to show, ONYX-015 has only produced objectivity clinical response (Nemunaitis J, Khuri F, Ganly I, Arseneau J, Posner M, Vokes E, Kuhn J, McCarty T, Landers S, Blackburn A, Romel L, Randlev B, Kaye S, Kirn D in 14% treatment patient.Clinical tumor journal (J.Clin.Oncol.).January 15 calendar year 2001; 19 (2): 289～298).
WO96/03997 and WO97/26904 have described the oncolytic HSV that can suppress growth of tumour cell and neuronal cell be had specific sudden change.Other advantage of described HSV is that it can carry out genetic modification easily, and has the medicine that can close any unwanted virus replication.But the application of this common human pathogen is limited, and this is should virus because a large amount of probably crowds are contacted, and has obtained at this viral immunne response, and this will weaken the solute effect of described virus.HSV also can cause serious adverse or potential fatal disease.
III type reovirus is relevant with slight relatively disease, and its known viral gene function also solve quite clear.Oncolytic biotech company (Oncolytic Biotech) is developing III type reovirus at present by as cancer therapeutic agent, this virus has enhanced duplication characteristic in the cell of the ras oncogene of expressing sudden change, and preferentially PKR-/-cell in growth (Strong J.E. and P.W.Lee.Virus journal (J.Virology), 1996,70:612～616).But, be difficult to reovirus is carried out genetic manipulation, and its virus replication is difficult for closing.
VSV is with minor ailment is relevant relatively, and known viral gene function is also very clear.WO99/04026 has disclosed VSV purposes as carrier in gene therapy, with a lot of medicines of the various obstacles of expression treatment.But VSV exists and the same problem of reovirus: be difficult to carry out genetic manipulation, virus replication is difficult for closing.
Vaccinia virus and poliovirus are other candidate's of describing of this area oncolytic viruses, but with serious or potential fatal disease is relevant.
US 4,806, and 347 have disclosed interferon gamma and IFN γ fragment antagonism human tumor cell's purposes.WO99/18799 has disclosed the method for treatment disease in mammal, in described mammal, sick cell has the antiviral response defective of interferon mediation, and described method comprises the clone's virus that replication capacity is arranged that described administration treatment is gone up the interferon-sensitive of effective dose.It has specifically disclosed under the situation that interferon exists, and the VSV virion has toxicity to tumor cell, but the toxicity of VSV in normal cell reduces.WO99/18799 also discloses, and handles tumor cell with interferon, has observed the inductive sensitivity of NDV, and interferon is added to normal cell, can make the anti-NDV of these cells.The purpose of this method is by coming infection cell with interferon-sensitive virus, making this cell to interferon-sensitive.
Summary of the invention
On the one hand, the invention provides the method that inhibition has the cell of innate antiviral response defective, this method comprises the myxoma virus of described cell being used effective dose.
On the one hand, the invention provides the method for a kind of morbid state of treatment, there is the cell with innate antiviral response defective in being characterized as of this morbid state, and described method comprises that the patient to needs uses the myxoma virus of effective dose.
The purposes of the myxoma virus that the present invention further provides effective dose in suppressing to have the cell of innate antiviral response defective, and the purposes of medicine that is used for suppressing having the cell of innate antiviral response defective in preparation.
The present invention further provides the purposes of effective dose myxovirus in treatment disease of patient state, wherein, there is the cell with innate antiviral response defective in being characterized as of described morbid state, and is used for the treatment of purposes in the medicine of the described morbid state of patient in preparation.
On the other hand, the invention provides a kind of pharmaceutical composition, this pharmaceutical composition contains myxoma virus and pharmaceutically acceptable carrier, this medicine is used to suppress to have the cell of innate antiviral response defective and is used for the treatment of be characterized as the morbid state that has the cell with innate antiviral response defective.
On the other hand, the invention provides the test kit that contains myxoma virus and description, this test kit is used to suppress to have the cell of innate antiviral response defective, or is used for the treatment of and is characterized as the morbid state that has the cell with innate antiviral response defective.Described morbid state comprises cancer and chronic viral infection.
The present invention further provides the method that detects the cell with innate antiviral response defective in the patient, this method comprises uses modified myxoma virus with the expression detectable label to the patient; In the patient, make described viral infection have the cell of innate antiviral response defective; With the cell that in the patient, detects the expression detectable label.
The present invention further provides the method that detects the cell with innate antiviral response defective in sample, this method comprises cultured cell; Institute's cultured cells is contacted with myxoma virus; Be subjected to infectivity with definite cell to myxoma infections.
The present invention is based on such accident finds: infectious myxoma virus can optionally infect the cell that comprises the human tumor cell, and described cell has the innate antiviral response defective, and comprises the non-cell of replying of interferon.Non-antigen-specific immune response described in the term that uses in this context " congenital ".Because myxoma virus does not effectively duplicate in human normal cell, so described virus can be used as various diseases and uncomfortable medicine, for example, oncolytic therapy medicine as cancer, there are the cell with innate antiviral response defective in described disease and uncomfortable being characterized as, comprise the non-cell of replying of interferon.Described virus can also be used to identify the cell with innate antiviral response defective, and be used for showing in vivo these cells.
For those of ordinary skills, by reading following description to specific embodiments of the present invention, and in conjunction with the accompanying drawings, others of the present invention and feature will become apparent.But should understand, detailed description when the preferred embodiment of the invention is described and specific embodiment only provide for illustrating, reason is that the variations and modifications in the spirit and scope of the present invention will become apparent for those skilled in the art from these are described in detail.
Description of drawings
The accompanying drawing of illustrating embodiment of the present invention just is used for for example, wherein:
Fig. 1 is the sketch map of the antiviral signal transduction system of inductive interferon mediation behind the virus infected cell;
Fig. 2 is that the WT rat embryo fibroblast cell (" MEFs ") of non-permission differs microphotograph in contact behind the myxoma virus, and this photo is presented at neutralizing antibody and suppresses behind interferon-ALPHA/β the MEFs permission that becomes;
Fig. 3 is the Western trace that is presented at the phosphorylation state (activation) of STAT1 and STAT2 after the myxoma infections, and this Western trace shows that the non-permission infection of MEF cell is associated with the activation of STAT1 and STAT2;
Fig. 4 is the Western trace that is presented at the phosphorylation state (non-activation) of STAT3, STAT4 after the myxoma infections, STAT5 and STAT6, and this Western trace shows that the non-permission of MEF cell is infected and can not activate any in these types;
Fig. 5 is after myxoma infections, IFN α/β R-/-MEFs and STAT1-/-MEFs and JAK1-/-MEFs and JAK2-/-MEFs differ microphotograph, this photo shows that the inactivation of IFN/STAT/JAK signal transduction makes the infection that allows the myxoma virus pair cell;
Fig. 6 is presented at after the myxoma infections Western trace of the phosphorylation state of PKR in non-permission wild type MEFs, and this Western trace shows that PKR is not activated by myxoma infections;
Fig. 7 is the Western trace that shows the phosphorylation state of PKR in wild type MEFs simulated infection or that infect in advance with myxoma virus, and this Western trace demonstration myxoma virus has been blocked the activation of PKR in the MEF cell;
Fig. 8 is the Western trace that is presented at the phosphorylation state of PERK in wild type MEFs after the myxoma infections, and this Western trace shows that myxoma virus blocked the activation of PERK in the MEF cell;
Fig. 9 be presented at PKR-behind the contact myxoma virus/-, RNase L (ribonuclease l)-/-or Mx1-/-knock out differ microphotograph, this photo is presented in the MEF cell by different approaches mediation antiviral state;
Figure 10 be presented at PKR-behind the contact myxoma virus/-, RNase L-/-and Mx1-/-triple knock out differ microphotograph;
Figure 11 be show with the neutralizing antibody of anti-IFN α/β handle and contact PKR-behind the myxoma virus/-, RNase L-/-and Mx1-/-triple knock out differ microphotograph;
After Figure 12 is the neutralizing antibody processing and contact myxoma virus that shows with anti-IFN α/β, the Western trace of eIF2 α and the PKR phosphorylation level in non-permission MEFs, the phosphorylation of this Western trace demonstration eIF2 α in nonpermissive cell carried out catalysis by the approach that is independent of PKR;
Figure 13 be show STAT1 after the myxoma infections PKR-/-, RNase L-/-and Mx1-/-the Western trace of phosphorylation state in triple the knocking out, this Western trace shows that the inductive signal transduction of normal IFN replys;
Figure 14 be illustrate the STAT1 that infects back (P.I.) 12 hours tyrosine phosphorylations nonpermissive PKR-/-, RNase L-/-or Mx1-/-Subcellular Localization in the cell differ microphotograph, this image shows, as normal IFN/STAT signal transduction being replied the prediction of being carried out, STAT is positioned nucleus;
Figure 15 be simulated infection or with the fluorescence picture of suffering from the gliomatous nude mice brain of intracranial of the dead myxoma virus of expressing GFP or the myxoma infections of living, this picture shows myxoma virus targeting neuroglial cytoma;
Figure 16 is that this image and photo show that myxoma virus only duplicates in tumor cell with the fluoroscopic image and the photo of the gliomatous slice of Mus of myxoma virus (MV GFP) infection of expressing GFP;
After Figure 17 was myxoma infections, with the HT29 human tumor cell's of X-Gal or violet staining the microphotograph that differs, this photo was presented at the example that non-permission is infected among the human cell;
After Figure 18 was myxoma infections, with the HOP92 human tumor cell's of X-Gal or violet staining the microphotograph that differs, this photo showed the example that human cell's permission is infected;
After Figure 19 was myxoma infections, with the OVCAR4 human tumor cell's of X-Gal or violet staining the microphotograph that differs, this photo showed the example that human cell's permission is infected;
After Figure 20 was myxoma infections, with the SK-MEL3 human tumor cell's of X-Gal or violet staining the microphotograph that differs, this photo showed the example that human cell's permission is infected;
After Figure 21 was myxoma infections, with the SK-MEL28 human tumor cell's of X-Gal or violet staining the microphotograph that differs, this photo showed that human tumor cell's half allows an example that infects;
After Figure 22 was myxoma infections, with the microphotograph that differs of the BGMK cell after X-Gal or the violet staining, this photo showed the example that typical permission is in contrast infected;
After Figure 23 is the proteic myxoma infections of expression LacZ that raises with concentration, with the microphotograph that differs of X-Gal painted positive control BGMK cell and human tumour cell line U87, A172 and U373, this photo shows that these human glioma cells are duplicated all myxoma virus and allows;
Figure 24 described after the myxoma infections that raises with concentration 72 hours, and the diagram of the survival rate of BGMK, U87, A172 and U373 cell, this diagram have proved that myxoma virus kills the ability of all these cells;
Figure 25 be the SF041585 astrocytoma cell that infects with MV GFP differ microphotograph and fluorescence micrograph, this differs microphotograph and fluorescence micrograph and has shown infection in the constitutional human glioma cells;
Figure 26 be with the U373 neuroglial cytoma of expressing the proteic myxoma infections of LacZ through the painted microphotograph that differs of X-Gal, this differs the infection that microphotograph has shown these human tumor cells;
Figure 27 describes to infect back 48 hours with MV GFP, and the diagram of the survival rate of SF041585 cell, this diagram have shown the situation of killing to these infected human tumor cells;
Figure 28 is that this photo has shown these human tumor cells' infection with the fluorescence micrograph of the Daoy of the myxoma infections of expressing GFP and D384 medulloblastoma system.
The specific embodiment
The inventor finds that the myxoma virus of common infected rabbits can selectively infect and kill the cell (comprising the human cell) with innate antiviral response defective, for example, and to the non-cell of replying of interferon.On the other hand, myxoma virus can not effectively duplicate in human normal cell.Because have cell with innate antiviral response defective such as a lot of diseases such as cancer or uncomfortable being characterized as, comprise the non-cell of replying of interferon, so myxoma virus can be used for the treatment of described disease and the discomfort that comprises cancer, and low to normal healthy cell toxicity.Myxoma virus also can be used for treating chronically infected cell, because these cells have the innate antiviral response defective.For example, a lot of encoding viral gene outcomes, the function of this product are to suppress the antiviral property interferon response of cell.Myxoma virus can optionally infect these cells.
Myxoma virus (" MV ") is the pathogen that causes myxomatosis in rabbit.The hare poxvirus (Leporipoxvirus) that MV belongs to the Poxviridae of maximum in the DNA viruses belongs to.MV causes benign disease in its natural host America cottontail.But, it is strong and the host specificity poxvirus, in European rabbits, cause fatal disease, it is characterized in that the general pathological changes, especially (Cameron C, Hota-Mitchell S, Chen L, Barrett J, Cao JX, Macaulay C, Willer D, Evans D, McFadden G, virusology (Virology) near the mucosal areas, 1999,264 (2): 298～318; Kerr P﹠amp; McFadden G, virological immunology (ViralImmunology), 2002,15 (2): 229～246).
MV virus is the genomic big virus of the double-stranded DNA of a kind of 163kb of having, and this genome duplicates (B.N.Fields, D.M.Knipe in the Cytoplasm of infected cell, P.M.Howley compiles, virusology, Lippincott Raven publishing house, New York, second edition, 1996).The many secretory proteins relevant of known MV coding with cell, the downward modulation of this albumen participation host immune and inflammatory reaction and to the inhibition of virus infected cell apoptosis.MV can be absorbed by all human somatic cells.But except normal rabbit somatic cell, if described cell has normal innate antiviral response, described virus just can not effectively infect described cell, means not reproducible and cause cell death of described virus.
Interferon (" IFN ") is for replying the excretory cytokine family of various stimulations.Interferon is attached to cell surface receptor, the activation signal transductory cascade, and this signal transduction cascade causes numerous cell responses, comprises inducing of antiviral response and growth inhibited and/or apoptotic signal.Interferon is divided into I type or II type.I type interferon comprises interferon-alpha, interferon-, τ interferon and omega interferon, and all is monomer; II type IFN has only one kind of IFN γ, is dimer.12 kinds of different subtypes of IFN α are produced by 14 genes, but all other interferon all is (Arduini etc., 1999) of monogenic.Interferon is brought into play direct anti-tumor activity by regulating oncogene expression.The forfeiture of crossing expression or tumor inhibitor oncogene of growth stimulation oncogene can cause vicious transformation.P53, Rb, PC, NF1, WT1 and DCC relate to some genes that cancer takes place.
Myxoma virus is the same with VSV as reovirus with other oncolytic virus, all needs to pass existing antiviral protection in the normal health cell, could duplicate in cell.The generation of MV and other oncolytic virus inducing interferon, general antivirus action sensitivity to the IFN approach.Comprise PKR, OAS synzyme and RNase L nuclease by inductive associated protein of IFN antiviral response and the main associated protein that influences virus multiplication.PKR activates eIF2 α, causes suppressing translation and apoptosis-induced.What describe in Fig. 1 is the sketch map of IFN response pathway.In normal cell, MV is subjected to the direct influence of PKR and eIF2 α.
Anti-viral response pathways often is damaged in cancerous cell.For example, to weakening of replying of IFN or invalid be the hereditary detect that often occurs in conversion process or in the tumor development.Tumor cell line above 80% does not produce interferon replys or replys impaired (Stojdl etc., cancerous cell (Cancer Cell), (2003) 4:263～275 and the list of references of wherein being quoted; Wong etc., Biochemical Journal (J.Biol Chem) (1997) 272 (45): 28779～28785; Sun etc., blood (blood), (1998) 91 (2): 570～576; Matin etc., cancer research (Cancer Res.) (2001) 61 (5): 2261～2266; Balachandran etc., cancerous cell (2004) 5 (1): 51～65).The inventor is surprised to find MV and can infects and kill cancer cell, comprises the human tumor cell, and is not subjected to the restriction of any particular theory, it is believed that it is because there is the innate antiviral response defective in these cells that MV can infect these cells.
Evidence suggests that suppressing early stage innate immunity, to reply and delay development that Th1 replys be important to the efficient of oncolytic therapy.Though myxoma virus is a kind of potent virus, it has host specificity, and has very narrow host range; It is infected person or Mus not.Be not bound to any specific theory, it is believed that because of myxoma virus right and wrong human virus, it should not run into the immunity identification that is pre-existing in the mankind.Therefore, it will be subjected to less infringement as the potentiality of oncolytic virus; To the admissibility tumor cell, myxoma virus will provide the more effective infection than natural human virus, can provide effective oncolytic therapy for cancer in view of the above.
Therefore, in one embodiment, provide the method that the cell with innate antiviral response defective is suppressed, this method comprises the myxoma virus of described cell being used effective dose.In one embodiment, described cell does not produce interferon and replys.
In specific embodiments, described cell is a mammalian cancer cells.In one embodiment, described cell is the human cancer cell, comprises the human entity oncocyte.
In another embodiment, with the viral chronic infection of described cell.
Here the term of Shi Yonging " cell with innate antiviral response defective " is meant such cell: after invading when contact virus or by virus, this cell not inducing anti-disease poison defense mechanism (comprise suppress virus replication, produce interferon, the inducing interferon response pathway and by or can't help the interferon mediated Apoptosis), and therefore described cell can be subjected to infecting of MV virus.Described term comprises, with respect to normal cell (as cell or the non-cancerous cell that is not infected), the cell that has that weaken or invalid innate antiviral response when contacting with virus or being infected by the virus.This comprises non-cell of replying of interferon and apoptotic response or apoptosis-induced approach weakens or invalid cell.Described defective may be caused by multiple reason, comprise infection, genetic defect or environmental effect.But should be understood that when defective be when causing by the infection that is pre-existing in, can not comprise the superinfection that causes by MV, the technical staff can easily differentiate this situation.The technical staff can easily utilize suitable experiment to detect any particular cell types whether to have the innate antiviral response defective thereby can be infected by myxoma virus.For example, VSV is often used in the antiviral response that detects cell.
In order to determine whether specific cell type (as specific cancerous cell type) has the innate antiviral response defective, the technical staff can obtain explant, make its some cells in growth in vitro, and determine by VSV or replaceability ground, by the infectivity of myxoma infections.
Employed term in whole description " to the non-cell of replying of the interferon " meaning is meant such cell, this cell does not produce the antiviral of interferon activity such as interferon or anti-tumor activity and replys, perhaps, this cell does not have normal interferon response, for example, that weaken or invalid interferon response, perhaps, this cell is for example used the phosphorylation of signaling molecule such as transcription factor (as STAT1) or activation to detect to have abnormal interferon signal transduction.For example include but not limited to, do not stand to breed the cell of inhibition, maybe do not have killed cell, and the level of described interferon can be enough to cause described reaction to interferon in the aitiogenic cell when the contact interferon.May there be defective in by normal activated approach in the intracellular signal transduction approach or in responsive cell to the non-cell of replying of interferon.Usually, the susceptibility that VSV is infected shows replys interferon is non-, the technical staff can detect easily that specific cells is had the ability or not having ability that interferon is produced replys, infect in the presence of interferon, to suppress VSV, perhaps use other interferon activity labelling known in the art, for example, the gene of the IFN of expression stimulation such as PKR, STAT, OAS, MX.
Employed term " has replication capacity " be meant that virus can infect and duplicate in particular host cell in whole description.
Here the term of Shi Yonging " cell " comprises unicellularly, also comprises many cells or cell mass.Pair cell use medicament comprise external use with body in use.
Here the term of Shi Yonging " effective dose " meaning is can effectively obtain the necessary amount of required result over a period to come with certain dosage.
Here the term of Shi Yonging " animal " comprises all members of regnum animale, comprises the mankind.
The cell that term " inhibition " has the innate antiviral response defective comprises the cell death that causes by dissolution or apoptosis, or other machine-processed cell death, also comprises in addition making described cell not grow or to divide.
Myxoma virus can be any virus that belongs to the tularemia seed culture of viruses of the poxvirus that replication capacity is arranged.Described myxoma virus can be the wild strain system of myxoma virus, or the genetic modification strain of myxoma virus system.
The known standard molecular biological technique of operation technique personnel, can easily modify to express the therapeutic transgenic of one or more kinds the myxoma virus genome, ((2001) molecular cloning: laboratory manual (Molecular Cloning:a LaboratoryManual) such as Sambrook for example, the third edition, publishing house of cold spring harbor laboratory (Cold Spring Harbour LaboratoryPress)) technology described in.The technical staff can easily determine myxoma virus genomic which the part can delete, make this virus still can effectively infect.For example, compare by virus genome sequence and other viral genome that has fully been characterized that will announce, can infer resectable virus genomic inessential part (for example referring to C.Cameron, S.Hota-Mitchell, L.Chen, J.Barrett, J.-X.Cao, C.Macaulay, D.Willer, D.Evans and G.McFadden, virusology (1999) 264:298～318).
Here the term of Shi Yonging " therapeutic genes " or " therapeutic transgenic " are intended to describe widely any gene, and its expression can influence required result, for example, and antiviral effect.For example, virus is modified make it carry the gene that can improve with the anticancer effect of this viral therapy.This gene can be the gene that participates in causing apoptosis, or participate in the targeting infected cell to carry out immune destruction, as repair the gene of interferon response defective, or the result causes expressing the gene of the cell surface marker (for example bacterial cell surface antigen) that stimulates antibody response.Described virus can also be modified, and participates in being closed into the propagation of oncocyte or cancerous cell and the gene of growth to express, and stops the division of described cell in view of the above.Simultaneously, described virus can also be modified to comprise therapeutic genes, as participate in the synthetic gene of chemotherapeutant, perhaps, described virus can be modified to have the levels of replication of raising in specific cell type such as human cell, derives the cell that desire suppresses or kills from described cell type.Can be inserted in the myxoma virus adenoviral gene that comprises the coding proteic human gene of TRAK or coding E4 or f4 polypeptide with the object lesson of the gene that improves anticancer effect, the albumen of two genoids all participates in killing the human tumor cell.
Should be appreciated that the therapeutic effect of myxoma virus can obtain by the dissolving or the delivery of therapeutic product of viral pair cell.
Can be by standard technique preparation virus known in the art.For example, virus can infect the rabbit cell cultivated with the myxoma strains that desire is used, make to infect and continue development so that described virus is duplicated and obtained in institute's cultured cells, and the standard method of available destruction cell surface known in the art discharges, thereby discharges the virion that is used to gather.In case after gathering, promptly measure viral titre, and carry out plaque check (seeing (1996) such as Mossman, virusology 215:17～30) by the level ground of converging of infected rabbits cell.
In a scheme, provide and in the patient of needs treatment, treated the method that is characterized as the morbid state that has cell with innate antiviral response defective, this method comprises the myxoma virus of described patient being used effective dose.Described patient can be any animal, comprises mammal, comprising the mankind.
" being characterized as the morbid state that has the cell with innate antiviral response defective " of here using is meant any relevant with the cell with innate antiviral response defective, that relate to this cell or having disease, obstacle or the discomfort of this cell as feature, and can treat described disease, obstacle, discomfort or its symptom by killing these cells.For example, described morbid state can be a cancer.Described morbid state also can comprise viral chronic infection.
" treatment " morbid state is meant and is used to obtain useful or required result, comprises the method for clinical effectiveness.Useful or required result can include but not limited to, alleviate or improve that one or more are planted symptom or discomforts, reduce disease degree, stable disease state, the development that wards off disease, the propagation that wards off disease, postpone or slow down disease process, postpone or slow down disease outbreak, improve or palliate a disease state and (partially or completely) recovery, and no matter whether this useful or required result can detect obtain." treatment " can also refer to prolong patient's life span, and this life span is longer than the life span of being predicted without treatment." treatment " can also refer to suppress disease process, temporarily slows down disease process, although more preferably, comprises and ends disease process enduringly.Just as understood as technical staff, if a kind of the treatment improved specific morbid state, but this treatment will be better than any benefit that this treatment obtained to the unfavorable effect of patient's generation of treatment, and then the possibility of result is not useful or required.
In one embodiment, described morbid state is a cancer.Described cancer can be the cancer of any kind, and wherein, although need not to be whole cells, at least some cells have the innate antiviral response defective.In one embodiment, described cancer can be that wherein at least some cells are to the non-cancer of replying of interferon.Here the term of Shi Yonging " tumor ", " tumor cell ", " cancer " and " cancerous cell " (can exchange use) are meant the cell that shows irregular growth, and it is obviously out of hand to it is characterized by cell proliferation, or by the cell of immortalization.Term " cancer " or " tumor " comprise transitivity and non-metastatic cancer or tumor." the one-tenth tumor " or " tumor " general reference propagation here used do not have a cell or a plurality of cell that normal growth suppresses mechanism, so comprise benign tumor, also comprise cancerous cell and abnormal development cell or proliferative cell.
Cancer comprises the existence of malignant tumor, can diagnose with the generally accepted standard in this area.
The cancer types that can treat according to the present invention includes but not limited to, comprises hematopoietic cancer, colon cancer, pulmonary carcinoma, renal carcinoma, cancer of pancreas, carcinoma of endometrium, thyroid carcinoma, oral cancer, ovarian cancer, laryngeal carcinoma, hepatocarcinoma, cancer of biliary duct, squamous cell carcinoma, carcinoma of prostate, breast carcinoma, cervical cancer, colorectal carcinoma, melanoma and other any tumor of leukemia and lymphoma.Solid tumor such as sarcoma and cancer include but not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcoma, synovioma, mesothelioma, outstanding Yin Shi tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymph sample malignant tumor, cancer of pancreas, breast carcinoma, pulmonary carcinoma, ovarian cancer, carcinoma of prostate, hepatocarcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, mastoid process sample adenocarcinoma, medullary carcinoma, lung bronchogenic carcinoma, the kidney cell cancer, hepatoma, cancer of biliary duct, choriocarcinoma, Wilms' tumor, cervical cancer, tumor of testis, bladder cancer and central nerve neuroma are (as glioma, astrocytoma, medulloblastoma, craniopharyngioma (craniopharyogioma), ependymoma, pinealoma, hemangioblastoma, the auditory nerve tumor, oligodendroglioma, meninges tumor (menangioma), melanoma, neuroblastoma and retinoblastoma).
In another scheme, described morbid state is a chronic viral infection.
Thereby described chronic infection virus can be anyly to infect for a long time with continuous fashion in zooblast and duplicate the virus that causes pathological state.Described chronic infection virus can be relevant with the development of cancer or relevant virus.
The virus chronic infection can be diagnosed by standard method known in the art.For example, chronic viral infection can detect by the existence of antiviral antibody among the patient, perhaps detects by the tests positive that exists to viral RNA or DNA in patient's cell.
Can use the medication of standard that the patient is used described virus.In one embodiment, using of virus is to be administered systemically.In the another one embodiment, use virus by injecting in the affected part.In a particular, described morbid state is a solid tumor, and by using virus in the tumor locus injection.In many embodiments, using of virus is to be undertaken by oral or parenteral, perhaps adopts standard method known in the art to carry out.
When using to the patient, the effective dose of virus is with in certain dosage is during sufficiently long, alleviates, improves, relaxes, improves, stablizes, prevents to propagate, delay or postponement process or the needed amount of cure diseases.For example, effective dose can be the amount that is enough to obtain following effect: reduce cancerous cell or become the number of oncocyte or with its destruction; Perhaps reduce the number of viral chronic infection cell or with its destruction; The growth and/or the propagation that perhaps suppress described cell.
The effective dose that the patient is used can change according to many factors, and described factor is the drug effect characteristic of virus for example; Administering mode; Patient's age, health status and body weight; The type (if any) of the nature and extent of morbid state, the frequency of treatment, the treatment carried out simultaneously, and the toxicity and the titre of virus.
Those skilled in the art can be based on the suitable consumption of above-mentioned factor decision.According to patient's clinical response, the right quantity of viral initial application can be adjusted as required.Can be according to the maximum virus quantity of safely use and the required result's of generation minimum virus quantity, the effective dose of by virtue of experience decision virus.
When virus is administered systemically, identical in order to make its clinical effectiveness with the clinical effectiveness that is obtained at the affected part injecting virus, need use obviously more virus quantity.But suitable dosage level should be the minimum that can obtain required result.
The concentration of virus to be administered is decided by the toxicity of the myxoma virus particular strain desiring to use and the character of target cell.In one embodiment, human patients is used be lower than about 10
9The dosage of individual plaque forming unit (" pfu ").In various embodiments, can use about 10
2Pfu～about 10
9Pfu, about 10
2Pfu～about 10
7Pfu, about 10
3Pfu～about 10
6Pfu or about 10
4Pfu～about 10
5The single dose of pfu.
According to the effect of initial therapeutic modality, can repeat to provide the virus of effective dose.Generally be cyclical administration, monitor any reaction that is produced simultaneously.According to the selected route of administration of time of administration harmony in the exterior, the technical staff can use and be below or above dosage as implied above.
Described virus can be individually dosed, perhaps carries out administration in conjunction with other Therapeutic Method (comprising chemotherapy, X-ray therapy or other antiviral therapy).For example, can before or after the surgical resection primary tumor, use virus, perhaps before X-ray therapy or traditional chemical medicine are used, use or use simultaneously or use afterwards.In one embodiment, described virus can or be used in turn with other oncolytic virus while, and described other oncolytic virus can demonstrate specificity at different tumor cell types.
In order to help administration, described virus can be able to be made a component of pharmaceutical composition.Therefore, in an other embodiment, provide the pharmaceutical composition that contains myxoma virus and pharmaceutically acceptable diluent.Therefore, one aspect of the present invention also comprises described pharmaceutical composition, and this pharmaceutical composition is used to suppress to have congenital disease-resistant cell or treatment of replying defective and is characterized as the morbid state that has the cell with innate antiviral response defective.Described pharmaceutical composition contains salt, buffer agent, antiseptic and the various compatible carrier of pharmaceutically acceptable concentration usually.For sending of form of ownership, described recombinant virus can be prepared into physiological solt solution.
Described pharmaceutical composition can also contain other therapeutic agent, as anticarcinogen.In one embodiment, described compositions comprises chemotherapeutant.For example, described chemotherapeutant can be in fact at patient's cancerous cell or become any medicament of the oncolytic effect that oncocyte shows and do not suppress or do not reduce any medicament of described viral kill tumor effect.For example chemotherapeutant can be but be not limited to, anthracycline, alkylating agent, alkylsulfonate, ethylene imine, aziridine, methylmelamine, chlormethine, nitroso ureas, antibiotic, antimetabolite, folacin, purine analogue, pyrimidine analogue, enzyme, podophyllotoxin, platiniferous agent or cytokine.Preferably, described chemotherapeutant is known effective antagonism cancerous cell or the chemotherapeutant that becomes these particular cell types of oncocyte.
The ratio of pharmaceutically acceptable diluent and kind depend on selected route of administration, with the compatibility of live virus and the pharmacy convention of standard.Usually, pharmaceutical composition adopts the component of the described live virus biological nature of not appreciable impact to prepare.
Pharmaceutical composition can be prepared by known preparation method, and this known preparation method is fit to the pharmaceutically acceptable compositions that preparation is used to the patient, makes and the active substance and the pharmaceutically acceptable carrier of effective dose can be combined into mixture.The carrier that is fit to, for example, in the pharmacy science of Remington, be described (Remington ' s Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., USA 1985).Based on this, described compositions includes, but not limited to plant pharmaceutically acceptable carrier or the bonded viral solution of diluent with one or more, and be contained in have suitable pH's and with the isoosmotic buffer of physiological fluid in.
Those skilled in the art should understand, and according to selected route of administration, pharmaceutical composition can be used the patient with various forms.Compositions of the present invention can be passed through oral or parenteral.Parenteral comprises that intravenous, peritoneal cavity are interior, subcutaneous, intramuscular, pass in epithelium, intranasal, the lung, in the sheath, rectum and local mode administration.Can carry out parenteral by in selected a period of time, importing continuously.
Described pharmaceutical composition can be with inert diluent or Orally administered with assimilable carrier, and perhaps, it can be wrapped in the hard or soft shelly gelatine capsule and use, and perhaps is pressed into tablet and uses.For oral therapeutic administration, described virus can with mixed with excipients, and use can absorb tablet, buccal tablet, lozenge, capsule, elixir, suspending agent, syrup and eye disc forms such as (wafers).
Prepare in the buffer that described viral solution can suit on the physiology.Under common storage and service condition, these prepared products contain antiseptic, and preventing microbial growth, but described antiseptic can not make described live virus inactivation.Those skilled in the art will be appreciated that how to prepare appropriate formulations.Select and prepare the traditional handicraft of suitable preparation and component for example, in the pharmacy science of Remington and the American Pharmacopeia of publishing in 1999: be described in the NF (The United StatesPharmacopeia:The National Formulary, USP 24NF 19).
In different embodiments, described compositions is by using such as affected part direct injection such as tumor locus (subcutaneous (subcuteanously), intravenous, intramuscular etc.), and is perhaps Orally administered, as that selects, applied dermally.
Be fit to medicine composition dosage form that injection uses comprise aseptic aqueous solution or dispersion liquid and be used for aseptic Injectable solution of interim preparation or dispersion liquid sterile powder, wherein, the aseptic myxoma virus alive that does not prolong and desire to use of term itself.In all cases, described dosage form must be aseptic, and flowability must reach its degree of injection easily that makes.
The dosage of the pharmaceutical composition that desire is used depends on the characteristic (if any) of the specified disease of being treated, severity of disease, patient's individual parameter (comprising age, physical qualification, height and body weight), the course of treatment, the therapy of carrying out simultaneously, concrete route of administration, and in health worker's knowledge and other the similar factor in the professional skill scope.These factors are known to those skilled in the art, and can obtain by a small amount of normal experiment.
Described myxoma virus or the pharmaceutical composition that contains myxoma virus also can be packaged into test kit, this test kit contains the operation instructions of described virus, described use comprises, the cell that uses myxoma virus to suppress to have the innate antiviral response defective, perhaps, in the patient of needs, use myxoma virus to treat and be characterized as the morbid state that has cell with innate antiviral response defective.Described morbid state can be a cancer, perhaps can be chronic viral infection.
The present invention has also considered the purposes of myxoma virus in suppressing to have the cell of innate antiviral response defective.In one embodiment, described cell is replied interferon is non-.The present invention also provides, and in the patient of needs, myxoma virus is characterized as purposes in the morbid state that has the cell with innate antiviral response defective in treatment.In one embodiment, described morbid state is a cancer.The present invention also provides the purposes of myxoma virus in medication preparation, described medicine is used for suppressing having the cell of innate antiviral response defective or the patient of needs, treatment is characterized as the morbid state that has the cell with innate antiviral response defective.
The ability that selectivity that MV is had in the animal except that its natural host infects the cell of innate antiviral response defective provides MV to detect purposes in the cell with innate antiviral response defective in animal, and described cell comprises the non-cell of replying of interferon.Otherwise these cells may be not easy to detect, and for example, in animal, also do not develop into palpable tumor or also do not cause some cancerous cell of discernable disease.
Thereby, an embodiment, the invention provides the method that in the patient, detects cell with innate antiviral response defective, this method comprises uses modified myxoma virus with the expression detectable label to described patient; In the patient, make described viral infection have the cell of innate antiviral response defective; With the cell that in the patient, detects the described detectable label of expression.
The cell that can use the visual any traditional technique in measuring of diagnostic image to infect.Described detection method depends on employed specific detectable label.For example, can use the fluorescence digital imaging microscope to detect by the cell of process genetic modification with the myxoma infections of express fluorescent protein.Other method comprises computer control x-ray tomography art (CT), whole body scanning such as Positron Emission Tomography (PET), nuclear magnetic resonance (MRI) and ultrasonography.Those of skill in the art can determine to be used to detect the proper method of specific detectable label.
Detectable label includes but not limited to, any labelling, and it is used for expressing or synthetic gene can insert the myxoma virus genome, to express in by the cell of modified viral infection or synthetic described labelling.For example, in one embodiment, detectable label can be a fluorescin.After the patient used described modification virus, detect infected cells at interval with reasonable time, so that any cell with innate antiviral response defective of viral infection, and make the expression of detectable label in described cell reach detectable level.For example, 2～20 days whenever detect after genetic modification is with the virus of express fluorescent protein can used to the patient.
Detection method can repeat in the patient by phased manner, the existing of cell that has the innate antiviral response defective with monitoring in described patient.For example, the method of the described cell of myxoma virus is used in detection, if necessary, can carry out in the patient by the interval of 6 months, 1 year or 2 years, this depends on the characteristic of the cell with innate antiviral response defective and by the characteristic of any morbid state that existence caused of described cell in the patient.Repeat described method a period of time, with the development or the alleviation of monitor disease states, or disease is in the intravital diffusion of patient.
Myxoma virus can infect the cell with innate antiviral response defective by selectivity, and can be used as the indicant of described defective in cell.Therefore, use method of the present invention, can carry out the innate antiviral response defects detection the cell that from the patient, takes out.When combining with other indicant, this detection can indicate the patient may suffer from specific morbid state, for example, and cancer.
Therefore, in one embodiment, the invention provides the method that detects the cell with innate antiviral response defective in sample, this method comprises cultivates described cell; Institute's cultured cells is contacted with myxoma virus; And the detection cell is subjected to the infectivity of myxoma infections.
Described cell can be obtained from comprise human object of study by known biopsy method.Described biopsy method is decided by the position, place and the type of cell to be checked.
According to known culture technique cultured cell, and contact with MV by adding live virus in the culture medium.Infection multiplicity (" MOI ") can be different because of best MOI, density and the culture technique of particular cell types, use behind the known contact MV can the infected cells culture as positive control.
Institute's cultured cells can be passed through the whole bag of tricks known to the skilled by the infectivity that MV infects, and comprises that the deadly ability of MV pair cell detects.Also can comprise reagent is added described cell culture, to finish the enzymatic or the chemical reaction of itself and expressing viral product.Described expressing viral product can be expressed by the reporter gene that is inserted in the MV genome.
In one embodiment, MV can be modified, so that the detection of Infection Status is become easy.For example, can carry out genetic modification with presentation markup to MV, this labelling can easily detect by phase contrast microscopy, fluorescence microscopy or radiophotography.Described labelling can be participate in the fluorescin after the expression of chrominance response or radio-labeled reaction or express after enzyme.In another embodiment, described labelling is a gene outcome of destroying or suppress to be examined the cell specific function.
All lists of references of here quoting are introduced by reference fully.
Embodiment:
Strain
Employed Strain comprises wild type MV, modified MV with expressing green fluorescent protein (" GFP ") or beta galactosidase (" LacZ "), and the MV of be killed (" extremely ").With standard technique virus is prepared and titration.
Cell line
Utilize mouse embryo fibroblasts (" MEFs ") to carry out mouse test, described mouse embryo fibroblasts from wild-type mice and through below the mice that knocks out: IFN α/beta receptor isozygoty knock out, STAT1 isozygoty knock out, the PKR heterozygosis knocks out, RNase L heterozygosis knocks out, the Mx1 heterozygosis knocks out, triple the isozygotying of PKR/RNase L/Mx1 knocks out.
Carry out mankind's test with BGMK control cells and human tumour cell line, described human tumour cell line is HT29, HOP92, OVCAR4, OVCAR5, SK-MEL3, SK-MEL28, M14, SKOV3, PC3, DU145, CAKI-1,786-0, T47D, MDAMB 435, SF04, U87, A172, U373, Daoy and D384, as (cancerous cell (CancerCell), (2003) 4:263～275) as described in Stojdl etc.
Method
Usually, analyze and test as Lalani etc. at virusology (1999) 256:233～245 and Johnston etc. at viral journal (2003) 77 (13): carry out described in 7682～7688.
For mice study in the body, human intracranial glioma U87 is implanted nude mice.Implanted back 15 days, with live or dead MV GFP mice is carried out injecting in the tumor, titre is 5 * 10
6, or carry out simulated infection.Infected back 72 hours, kill animals is taken out brain, imbed the best cut warm complex (Optimal Cutting Temperature compound, OCT), the cutting frozen section.Can see myxoma virus-GFP in the whole brain sections by fluorescence microscopy.Fixing then section, and use H﹠amp; E (hematoxylin and eosin) dyeing makes tumor visual.
Analyze for the human tumor cell, operation one finishes promptly to carry out tumor trypsinized (trypsonized) and bed board, and second day, infect with virus, MOI is 0.1,1.0 or 10.Infection back 24 hours and 48 hours, use phase microscope art and fluorescence microscopy to collect the data of cells involved toxicity and expressing viral respectively.Utilize yellow tetrazolium salts MTT to detect, with the cell survival rate % (being the percentage ratio of the cell of simulated infection survival) of quantitative infection back 48 hours, 72 hours or 96 hours.
Myxoma virus-GFP with 10M.O.I infects human pediatric medulloblastoma cell line Daoy and D384.Infected back 72 hours, and detected the survival ability of cell with MTT.
The infection of mouse cell lines
Studies show that in the past, can be with some mice 3T3 cell clone of chemokine receptors transfection by myxoma infections, other clone then can not.Whether depend on any special receptor in order to study the tropism of myxoma virus in other mouse cell, inventor's research is from the elementary mouse embryo fibroblasts (MEFs) of wild type (WT) mice and range gene knock-out mice.
Because IFN carries out playing a crucial role in the antiviral response in preparation, so inventor's supposition, restrictive phenotype is relevant with " antiviral state " of IFN mediation.With in the antibody and circulation IFN, perhaps produce the mice that pathway gene in IFN receptor negative mice or the signal transduction born of the same parents has lacked, and the event chain of destruction IFN system, with the resistance of grievous injury host to myxoma virus, described myxoma virus does not generally infect the normal mouse cell.
In order to check this hypothesis, the inventor need prove whether myxoma virus non-infectious in nonpermissive cell is because the antivirus action of IFN.Bucketing is tested except one or more participate in the proteic various MEF cell types that the IFN signal transduction is replied in the born of the same parents, to understand the influence of MV infection to the IFN approach.
Experiment showed, that to what elementary MEFs carried out wild type (" WT ") MEFs is not by myxoma infections.When blocking the IFN approach, can use myxoma infections MEFs (Fig. 2) fully with the neutralizing antibody of anti-IFN α/β.But the MEFs that contacts with anti-IFN γ neutralizing antibody still keeps non-permission.This just understands IFN α/β on the whole, rather than IFN γ, is being that myxoma virus Infection in Vitro MEFs creates the importance that allows in the environment.Different IFN α/β and IFN γ intracellular signal transduction approach have been determined in the literature.But different with the fibroblast of being cultivated, IFN α/β and IFN γ all may play an important role in infected host.The inventor infers, existing the human tumor of defective to infect in will be to the body of myxoma virus in IFN α/β and/or IFN γ approach is susceptibility.
The inventor has measured STAT1 and the activity of STAT2 in the non-permission WT MEFs that MV infects.Result displayed shows in Fig. 3, and STAT1 and STAT2 are activated.Further studies show that STAT3, STAT4 and STAT5 be not activated (Fig. 4).
In order to determine the importance of approach in keeping the non-enable state of MEFs in IFN α/β born of the same parents, carry out gene delection research, so that cascade is damaged in IFN α/beta receptor and the born of the same parents.Carry out the gene delection of IFN receptor or JAK1 or STAT1.With MV infect WT MEFs, IFN α/β R-/-MEFs and STAT1-/-MEFs.IFN α/β R-/-MEFs and STAT1-/-MEFs allows MV, and it is crucial (Fig. 5) that the signal transduction cascade that proves IFN α/β and STAT1 is infected MV
Protein kinase R (PKR) is the inductive enzyme that is subjected to IFN α/β in numerous cells.This kinases, when existing, stand the autophosphorylation effect at dsRNA (double-stranded RNA), then a plurality of cell proteins of phosphorylation, comprise eukaryotic protein synthesis initiator (eIF-2 α), the phosphorylation of this eukaryotic protein synthesis initiator can be induced the inhibition of protein translation and apoptosis.PKR also indicates the activation of RNase L.The inventor has detected MV and has infected the activation of back PKR in non-permission MEFs.In having set up the non-permission MEFs of good antiviral state, PKR is not by phosphorylation (Fig. 6).And MV infects the phosphorylation (Fig. 7) that has suppressed PKR.In addition, after the myxoma infections, PERK (, ER kinases similar) with PRK in elementary WT MEFs not by phosphorylation (Fig. 8).
Infect the MEFs (Fig. 9) that PKR, RNase L or Mx1 single-gene knock out with MV.Find that PKR, RNase L or Mx1 are nonessential to keeping the non-admissibility for myxoma infections.In order further to confirm the nonessential property of PKR, RNase L or Mx1, carry out PKR-/-, RNase L-/-and Mx1-/-triple knocking out.PKR-/-, RNase L-/-and Mx1-/-triplely knock out the infection (Figure 10) that does not help myxoma virus, but, handle the MEFs of PKR, RNase L and the triple KO of Mx1 (triple knocking out) with the neutralizing antibody of anti-interferon alpha/β, to myxoma infections permissions (contrasting Figure 10 and Figure 11) that become.These experiment showed, that PKR, RNaseL and Mx1 are optional to the admissibility of MV for mediation MEFs.
For after detecting MV and infecting, eIF-2 α and PKR the IFN of nonpermissive wild type MEFs and permission α/β R-/-MEFs and STAT1-/-activation among the MEFs, carried out further experiment.After MV infects, although PKR allow with nonpermissive MEFs in not by phosphorylation, eIF-2 α is in both cases all by phosphorylation (Figure 12).This proof does not have the participation of PKR and PERK, and antiviral state is by the approach mediation that causes eIF2 α phosphorylation in addition.
Behind the nonpermissive PKR of myxoma infections, RNase L and Mx1 is triple after knocking out the MEFs, STAT1 is subjected to serine phosphorylation and tyrosine phosphorylation (Figure 13) simultaneously.After also having shown myxoma infections, the STAT1 of tyrosine phosphorylation non-permission PKR-/-+RNase L-/-+Mx1-/-Subcellular Localization (Figure 14) among the MEFs.
In a word, these results show that the antiviral pathway that parallel PKR/PERK independently relates to IFN/STAT1 is crucial to the poxvirus tropism.And eIF2 α phosphorylation is the best labelling of IFN antivirus action.
Human tumor research
The inventor has studied the ability that MV infects the human tumor cell in the system in vivo.Inject nude mice with human glioma cells, form the intracranial glioma subsequently.Live virus can infect these human tumor cells, but does not infect peripheral cell (Figure 15).Show that in Figure 16 the fluorescence signal from GFP is positioned tumor.
Suppose that many human tumors reply interferon is non-, and, to compare with the IFN signal cascade of being found at the normal human subject cell, tumor cell does not have normal IFN signal cascade, and the inventor has carried out research to explore the influence of myxoma virus to human tumor.The result is summarized as follows.
At first, the use myxoma virus is studied infectivity and the molten effect of born of the same parents to various contrasts and human tumour cell line BGMK, HT29, HOP92, OVCAR4, SK-MEL3 and SK-MEL28.MV demonstrates various infectivities and the molten effect of born of the same parents: HT29 (Figure 17), HOP92 (Figure 18), OVCAR4 (Figure 19), SK-MEL3 (Figure 20), SK-MEL28 (Figure 21) and BGMK (Figure 22).
Other tumor cell is checked, and following table 1 is divided into non-permission, permission or half permission with the various tumor types of being checked.Half allows expression, and with respect to allowing cell line, myxoma virus can grade and moderate infection.
Table 1 myxoma virus is to human tumor cell's tropism
| Cell | Type | Non-permission | Allow | Half allows |
| HT29 | Colon | X | ||
| HOP92 | Lung | X | ||
| M14 | Melanoma | X | ||
| SK-MEL3 | Melanoma | X | ||
| SK-MEL28 | Melanoma | X | ||
| OVCAR4 | Ovary | X | ||
| OVCAR5 | Ovary | X | ||
| SKOV3 | Ovary | X | ||
| PC3 | Prostate | X | ||
| DU145 | Prostate | X | ||
| CAKI-1 | Kidney | X | ||
| 786-0 | Kidney | X | ||
| T47D | Mammary gland | X | ||
| MDAMB 435 | Mammary gland | X |
Along with the rising of MV-LacZ concentration, various human tumors system is also different to infecting the shown reaction that goes out.For example, the U373 cell needs higher virus titer could obtain U87 in the level (Figure 23 and Figure 24) of hanging down the cell killing that titre promptly obtains.Myxoma virus infects astrocytoma cell (Figure 25) and neuroglial cytoma (Figure 26) effectively.Myxoma virus 48 I after infection are effectively killed human astrocytoma glucagonoma cell and pediatric medulloblastoma cell (Figure 27 and Figure 28).
It will be appreciated by those skilled in the art that and to carry out many modifications to exemplary as described herein.And the present invention includes modification as in claims institute limited range all.
Claims (36)
1. the purposes of the myxoma virus of effective dose in medication preparation, described medicine is used to suppress to have the cell of innate antiviral response defective.
2. purposes as claimed in claim 1, wherein, described cell is replied interferon is non-.
3. purposes as claimed in claim 2, wherein, described cell demonstrates abnormal interferon signal transduction.
4. as each described purposes of claim 1～3, wherein, described cell is a mammalian cancer cells.
5. purposes as claimed in claim 4, wherein, described cell is the human cancer cell.
6. purposes as claimed in claim 5, wherein, described cell is lung carcinoma cell, melanoma cells, kidney cancer cell, neuroglial cytoma or astrocytoma cell.
7. purposes as claimed in claim 5, wherein, described myxoma virus is a wild-type virus.
8. purposes as claimed in claim 7, wherein, described myxoma virus is through genetic modification.
9. purposes as claimed in claim 8, wherein, described myxoma virus process genetic modification is with the express therapeutic gene.
10. purposes as claimed in claim 1, wherein, described cell is subjected to the chronic infection of virus.
11. the purposes of the myxoma virus of effective dose in medication preparation, described medicine is used for the treatment of the disease of patient state, and wherein, there is the cell with innate antiviral response defective in being characterized as of described morbid state.
12. purposes as claimed in claim 11, wherein, described morbid state is a cancer.
13. purposes as claimed in claim 12, wherein, described cancer is a solid tumor.
14. purposes as claimed in claim 12, wherein, described cancer is hematopoietic cancer, colon cancer, pulmonary carcinoma, renal carcinoma, cancer of pancreas, carcinoma of endometrium, thyroid carcinoma, oral cancer, ovarian cancer, laryngeal carcinoma, hepatocarcinoma, cancer of biliary duct, squamous cell carcinoma, carcinoma of prostate, breast carcinoma, cervical cancer, colorectal carcinoma or melanoma.
15. purposes as claimed in claim 14, wherein, described hematopoietic cancer is leukemia or lymphoma.
16. purposes as claimed in claim 12, wherein, described cancer is pulmonary carcinoma, melanoma, ovarian cancer, carcinoma of prostate, renal cancer, glioma or astrocytoma.
17. purposes as claimed in claim 11, wherein, described morbid state is a chronic viral infection.
18. as each described purposes of claim 11～17, wherein, described patient is human.
19. as each described purposes of claim 11～17, wherein, described myxoma virus is a wild-type virus.
20. as each described purposes of claim 11～17, wherein, described myxoma virus is through genetic modification.
21. purposes as claimed in claim 20, wherein, described myxoma virus process genetic modification is with the express therapeutic gene.
22. being used for the treatment of, a pharmaceutical composition that contains myxoma virus and pharmaceutically acceptable carrier, this pharmaceutical composition be characterized as the morbid state that has cell with innate antiviral response defective.
23. pharmaceutical composition as claimed in claim 22, wherein, described morbid state is a cancer.
24. pharmaceutical composition as claimed in claim 23, wherein, described cancer is a solid tumor.
25. pharmaceutical composition as claimed in claim 23, wherein, described cancer is hematopoietic cancer, colon cancer, pulmonary carcinoma, renal carcinoma, cancer of pancreas, carcinoma of endometrium, thyroid carcinoma, oral cancer, ovarian cancer, laryngeal carcinoma, hepatocarcinoma, cancer of biliary duct, squamous cell carcinoma, carcinoma of prostate, breast carcinoma, cervical cancer, colorectal carcinoma or melanoma.
26. pharmaceutical composition as claimed in claim 25, wherein, described hematopoietic cancer is leukemia or lymphoma.
27. pharmaceutical composition as claimed in claim 23, wherein, described cancer is pulmonary carcinoma, melanoma, ovarian cancer, carcinoma of prostate, renal cancer, glioma or astrocytoma.
28. pharmaceutical composition as claimed in claim 27, this pharmaceutical composition also contains therapeutic agent.
29. pharmaceutical composition as claimed in claim 28, wherein, described therapeutic agent is a chemotherapeutant.
30. pharmaceutical composition as claimed in claim 27, this pharmaceutical composition are suitable for injecting at tumor locus.
31. pharmaceutical composition as claimed in claim 22, wherein, described morbid state is a chronic viral infection.
32. a test kit that contains myxoma virus, this test kit is used to suppress to have the cell of innate antiviral response defective.
33. test kit as claimed in claim 32, wherein, described cell is replied interferon is non-.
34. a test kit that contains myxoma virus, this test kit are used for treating cancer the patient of needs.
35. a test kit that contains myxoma virus, this test kit are used for treating chronic viral infection the patient of needs.
36. a method that detects the cell with innate antiviral response defective in sample, this method comprises: cultivate described cell; Institute's cultured cells is contacted with myxoma virus; And definite cell is subjected to the infectivity of myxoma infections.
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| US45252103P | 2003-03-07 | 2003-03-07 | |
| US60/452,521 | 2003-03-07 | ||
| US60/455,393 | 2003-03-18 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2001004318A2 (en) * | 1999-07-12 | 2001-01-18 | Viron Therapeutics, Inc. | Myxoma virus genes for immune modulation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2001004318A2 (en) * | 1999-07-12 | 2001-01-18 | Viron Therapeutics, Inc. | Myxoma virus genes for immune modulation |
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| CN1756563A (en) | 2006-04-05 |
| ZA200506461B (en) | 2007-01-31 |
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