CN101560502A

CN101560502A - Herpes virus strains for gene therapy

Info

Publication number: CN101560502A
Application number: CNA2009101295146A
Authority: CN
Inventors: R·S·科芬
Original assignee: Biovex Ltd
Current assignee: Biovex Ltd
Priority date: 2000-01-21
Filing date: 2001-01-22
Publication date: 2009-10-21
Also published as: ZA200205470B; GB0001475D0; CN1865449A; ZA200205439B

Abstract

The present invention provides a herpes virus with improved oncolytic properties which comprises a gene encoding an immunomodulatory cytokine and which lacks a functional ICP34.5 gene and a functional ICP47 encoding gene.

Description

The herpes virus strains that is used for gene therapy

The application is the dividing an application that be January 22 calendar year 2001, denomination of invention the applying date for the Chinese patent application 01806743.3 of " herpes virus strains that is used for gene therapy ".

Invention field

The present invention relates to compare the herpes virus strains that on anti-tumor activity, improves to some extent with the previously-known strain.

Background of invention

Shown that in many cases virus has effectiveness in the various application of biotechnology and medical field.This is because virus has the unique ability that enters cell with high-level efficiency.This adds viral gene expression and duplicates and/or the expression of heterologous genes of inserting in such application.Thereby, the gene (perhaps virogene or other gene) that virus can or will can be used for for example gene therapy or vaccine development is delivered in the cell and at cell inner expression, and perhaps they can be used for the effect selective killing cancer cell for example by the lytic replication or the gene that transmits.

Propose hsv (HSV) and can be used for oncolytic ruling by law treatment cancer.Yet, must make virus become defective type, it is pathogenic that it is no longer had, even it can not duplicate and kill and wound non-tumor cell in non-tumor cell, but makes it still can enter tumour cell and killing tumor cell.Cancer (this therapy also can comprise the gene that transmits the described treatment effect of enhancing) is treated in ruling by law for oncolytic, many sudden changes of HSV have been identified, these sudden changes of HSV still allow virus at culture or enliven in the splitted cell (for example tumour in) in vivo and duplicate, and still stop its effectively duplicating in healthy tissues.Such sudden change comprises the encoding gene that destroys ICP34.5, ICP6 and thymidine kinase.Up to now, in these mutated viruses, the virus with ICP34.5 sudden change or ICP34.5 sudden change and for example ICP6 sudden change has shown the most favourable safe distribution plan.Shown the virus that only lacks ICP34.5 external in many tumor cell types, duplicate and mouse brain tumour the artificial induction in copy choice, and do not injure surrounding tissue.Early studies in man shows that also they are safe in the mankind.

Yet, treat cancer though be hopeful that various viruses (comprising HSV) are used for oncolytic ruling by law, the virus strain of not carrying the heterologous gene that can strengthen described anti-tumour effect is used in the overwhelming majority's work in this work always.We propose, combined utilization has the HSV of inactivation sudden change and is delivered in the encoding gene of a kind of immune modulator (as rHuGM-CSF (GM-CSF)) of encoding in this defective virus genome in the ICP34.5 encoding gene, may have best immunostimulatory properties, particularly when this viral function reduces the immunne response of the cell that is subjected to the HSV infection usually at the tumour of desire treatment.For example, the inhibition of HSVICP47 protein-specific ground is subjected to the angtigen presentation (Hill etc. 1995) of the cell of HSV infection, and UL43 gene product and vhs albumen reduce the immunostimulatory potency of the dendritic cell that is subjected to the HSV infection.Therefore, be used for a kind of oncolytic HSV mutant virus of cancer therapy, disappearance ICP47 and/or dendritic cell inactivation gene may be useful, particularly by application GM-CSF or other immunostimulating cytokines or chemokine enhancing immunity effect the time.Show that recently when expressing, rather than when being administered systemically, GM-CSF demonstrates the antineoplastic immune effect increases (Shi etc. 1999) in tumour cell.Therefore, in using like this, oncolytic HSV mutant strain can be seeded in primary tumor or the secondary tumors, destroy duplicating with oncolytic at this.Also may stimulate at the cell that infected by HSV and shift from the primary tumor position the immunne response of tumour cell at other positions.

Summary of the invention

The invention provides the improved virus of cracking performance destruction tumour cell ability in the body.At this, adopt a kind of HSV1 or HSV2 strain to make up the hsv strain, the gene inactivation of ICP34.5 and ICP47 of wherein encoding, thereby can not express the functional protein of ICP34.5 and ICP47, described hsv strain also carries the gene of a coding immune modulator.Other gene that may relate to the inhibit feature of dendritic cell in this virus also can suddenly change, and described gene comprises the gene of UL43 gene and/or coding vhs.Therefore the invention provides the oncolytic of having the ability destroy tumour cell, and can be with the maximized virus of antineoplastic immune effect.

So, the invention provides:

-comprise the gene of a coding immune modulator and lack functional ICP34.5 and a kind of simplexvirus of ICP47 encoding gene.

-be used for by the mankind of treatment or the simplexvirus a kind of of the present invention of animal body methods of treatment.

The medicine of-application virus production treatment of the present invention cancer.

-a kind of medicinal compositions, described medicinal compositions comprise as effective constituent according to virus of the present invention and pharmaceutically acceptable carrier or thinner.

-a kind of method for the treatment of the tumour of the individuality that needs treatment, this method comprise give described individual effective dose according to virus of the present invention.

The accompanying drawing summary

Fig. 1. virus

From top to bottom, legend shows: laboratory HSV117+ strain, clinical HSV1 JS1 strain, 17+/ICP34.5-strain, JS1/ICP34.5-strain, JS1/ICP34.5-/JCP47-/hGMCSF strain, JS1/ICP34.5-/ICP47-/mGMCSF strain.

Detailed Description Of The Invention

A. viral

Herpesviral of the present invention can effectively infect target tumour cell, ICP34.5 in this virus With the encoding gene of ICP47 be inactivation. The sudden change of ICP34.5 provides selective oncolytic activity. Such sudden change is described by Chou etc. 1990 and Maclean etc. 1991, although ICP34.5 Any sudden change that loses function can be adopted. Coding ICP6 and/or thymus gland are swashed The gene inactivation of enzyme, other genes are also deactivatable, it is molten as long as these inactivations can significantly not lower The knurl effect, or as long as these disappearances strengthen described virus oncolytic characteristics or other ideals Characteristic. The effect of ICP47 usually is that blocking-up is offered by the antigen of HSV infection cell, so Its destruction causes virus can not give guarantor when being subjected to the HSV infection for infected tumour cell Protect it and avoid the characteristic that host immune system is attacked. Also encode a kind of immunity of virus of the present invention is transferred Joint albumen, GM-CSF preferably, but also other cell factors of codified, chemotactic factor (CF) as RANTES, or other immune modulators such as B7.1, B7.2 or CD40L. Can comprise list The gene of coding immune modulator solely or associating.

Change to be used for above-mentioned purpose hiv region can or (completely or partially) be eliminated or The person makes its nonfunctional, perhaps replaced by other sequences, particularly by immune modulator (as GM-CSF) gene replaces.

Virus of the present invention can derive from HSV1 or HSV2 strain, or derives from theirs Derivative, preferably HSV1. The strain of deriving comprises and containing from HSV1 strain and HSV2 (inter-type) recombinant between the type of the DNA of strain. Recombinant is retouched in this area between this type State, such as Thompson etc., 1998 and Meignier etc., 1988 have described between this type and have recombinated Body. Derive strain and HSV1 genome or the genomic sequence homology of HSV2 be preferably to Few 70%, more preferably at least 80%, even more preferably at least 90% or 95%. More preferably, The genomic sequence homogeneity of strain and HSV1 genome or HSV2 of deriving is at least 70%, More preferably at least 80% homogeneity, even more preferably at least 90%, 95% or 98% homogeneity.

For example the UWGCG program package provides the BESTFIT program that can be used for calculating homology (such as using its default setting) (Devereux etc. (1984) Nucleic Acids Research 12, the The 387-395 page or leaf). PILEUP and BLAST algorithm can be used for calculating homology or sequence is arranged Row (usually using its default setting) are for example as at Altschul (1993) J.Mol.Evol.36: 290-300; Altschul etc. (1990) J.Mol.Biol.215: described in the 403-10.

Be used for carrying out the software public that BLAST analyzes can pass through National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) obtain. This algorithm bag Draw together at first be tested and appraised in search sequence with database sequence in the word string of equal length when comparing Perhaps mate or satisfy the short word string of the length W of the minimum score value T of some positive, identify High sub-sequence pairing (HSP). T is called as adjacent word string score value threshold value (Altschul etc., 1990). These original adjacent word strings are hit the seed that contains their HSP as initial search with discovery. Word string is hit with both direction and is extended along each sequence, as long as accumulated sequence comparison score value can increase Add. Hitting extension in the word string of each direction stops when running into one of following situation: the accumulation order Row comparison score value is than the low quantity X of maximum of its acquisition; The accumulation score value be zero or below, this is Because due to the accumulation of one or more negative marking residue comparisons; Or arrive the end of each sequence End. BLAST algorithm parameter W, T and X determine sensitivity and the speed of described comparison. BLAST The default word length (W) that program is used is 11, use BLOSUM62 marking matrix (referring to Henikoff and Henikoff (1992) Proc.Natl.Acad.Sci.USA89: 10915-10919) Comparison (B) is 50, and desired value (E) is 10, M=5, N=4, and compare two chains.

The BLAST algorithm carries out statistical analysis to the similitude between two sequences; Referring to for example Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA90: 5873-5787. It is minimum and probability (P (N)) that a kind of similitude that the BLAST algorithm provides is measured, and it provides The accidental index that the probability of coupling takes place between two nucleotide sequences or the amino acid sequence. Example As, if when a sequence and another sequence are compared minimum and probability less than about 1, Preferably less than about 0.1, be more preferably less than about 0.01, most preferably less than about 0.001, then think institute State First ray and second sequence similarity.

The strain of deriving can have and for example replaces 1,2 or 3 to 10,25,50 or 100 by Nucleotide and replace HSV1 genome sequence or the HSV2 genome sequences of modifying.HSV1 genome or HSV2 genome can or or be modified by one or more insertions and/or disappearance and/or by extending at arbitrary end or two ends in addition.

Virus strain of the present invention is " non-laboratory " strain.They also can be called as " clinical " strain.Those skilled in the art can be easily make a distinction laboratory strain and non-laboratory strain or clinical strain.In addition, hereinafter provided the characteristic guide that virus strain may show.

Key difference between laboratory strain and the non-laboratory strain is that laboratory strain commonly used has at present kept the long duration in culture, be many years in some cases.The cultivation of virus (for example HSV) relates to the technology that is called as continuous passage.In order to make viral growth and maintenance, with the suitable cell of virus infection, virus is in time multiplexed cell system, and results are viral then; Again infect new fresh cell then, this process constitutes the one-period of continuous passage.With regard to HSV, each such cycle may need for example several days.As discussed above, this continuous passage may cause the change of virus strain characteristic, because the selection to the characteristic (for example quick copy) that helps growing in culture takes place, rather than select and the characteristic that can be used for practical application, for example with regard to HSV with regard to, transport or infection human cell's ability along aixs cylinder for keeping.

Virus strain of the present invention can right and wrong laboratory strain, because they derive from up-to-date isolating strain from infected individual.Compare with original clinical separation strain, strain of the present invention is modified, may need to cultivate for some time, but it is shorter to cultivate used time ratio.Prepare strain of the present invention with such method, make it keep the ideal behavior of the original clinical separation strain that they originate basically.

Strain of the present invention is derived from the parental virus strain, as long as this parental virus plant mutant produces described virus.For example, virus of the present invention can be derived from clinical isolates JSI.The parental generation strain of this JSI derived virus can be JSI or by other HSV1 strains of JSI deutero-.Therefore, virus of the present invention can be the JSI virus that comprises the gene of a coding immune modulator and lack functional ICP34.5 encoding gene and functional ICP47 encoding gene.In addition, so a kind of virus can comprise any other sudden changes of mentioning herein.

Virus of the present invention can effectively infect the human cancer target cell.When a kind of so viral right and wrong laboratory or clinical strain, it will be to separate in the recent period from the individuality that infected by HSV, compare with the standard laboratory strain then, according to enhancing duplicate, the required ability of infection or killing tumor cell and/or other cells is screened in external and/or body.Compare this class virus of the present invention of improved properties with the zoo virus strain, through the engineering operation, make it lack functional ICP34.5 and ICP47 gene subsequently, and the one or more immune modulators (as GM-CSG) of under suitable promotor control, encoding.Proteic other genes (as UL43 and/or vhs) of coded interference dendritic cell function also can be inactivations.

Preferred virus strain of the present invention is separated its not modified clinical precursor strain and is counted and experience below 3 years or 3 years in culture from its host.Strain more preferably of the present invention is experience below a year or a year in culture, for example below nine months or nine months, six months or below six months, three months or below three months or below two months or two months, one month or below one month, two weeks or two weeks are following or week or below the week.These definition of time are meant the time actual used in culture in culture.Therefore, for example, common way is that virus strain is freezing, so that preserve them.Obviously, preserve by freezing preservation or with equivalent way and do not regard as and in culture, keep strain.Thereby, used time of freezing preservation or preserve the used time with other method and be not included in the scope of above-mentioned definition of used time of culture.Normally actual experience used time of continuous passage, the i.e. time during can selecting undesired feature of used time in culture.Preferred virus strain of the present invention is separated its not modified clinical precursor strain and is counted and experienced 1,000 or continuous passage cycle still less from its host.More preferably it experienced 500 or still less, 100 or still less, 90 or still less, 80 or still less, 70 or still less, 60 or still less, 50 or still less, 40 or still less, 30 or still less, 20 or still less or 10 or still less such cycle.

Preferably, learn check according to canonical statistics and measure, to carry out some ability that can be used for the function of application at present strong than having the Reference Lab strain that is equal to modification for non-zoo virus.With regard to the oncolytic virus that is used for oncotherapy, preferred non-zoo virus strain of the present invention than have the Reference Lab virus strain infection tumour cell that is equal to modification or duplicate therein, the ability of killing tumor cell or the propagation of the iuntercellular in tissue is strong.More preferably this stronger ability is the stronger ability of tool significance,statistical.For example, according to the present invention, with regard to characteristic to be measured, the ability of non-zoo virus strain of the present invention can be 1.1 times at the most, 1.2 times, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times or 100 times of ability of described reference strain.

Can use standard test, for example t-check, ANOVA or χ2Jian Yan carry out statistical analysis to characteristic described herein.Usually, the significance,statistical level of measurement is p=0.05 (5%), more preferably p=0.01, p=0.001, p=0.0001, p=0.000001.

Virus infection tumour cell of the present invention and duplicating therein then kills these tumour cells.Therefore, such virus has replication.Preferably they have the copy choice ability in tumour cell.This means or they duplicate in tumour cell, and in non-tumor cell, do not duplicate, or their duplicating efficiencies in tumour cell are than the duplicating efficiency height in non-tumor cell.The cell that virus can be duplicated within it is to allow cell.Adopt the detection method of duplicating that is used for detecting described herein, can detect the copy choice ability, if necessary, also can adopt and mention statistical technique analysis at this with the tumor cytotoxicity ability.

Preferred virus of the present invention than have not modified former generation virus strain infection's tumour cell or duplicate therein, the ability of killing tumor cell or the propagation of the iuntercellular in tissue is strong.More preferably this stronger ability is the stronger ability of tool significance,statistical.For example, according to the present invention, with regard to characteristic to be measured, the ability of virus of the present invention can be 1.1 times at the most, 1.2 times, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times or 100 times of ability of described not modified former generation strain.

The characteristic of this virus strain aspect tumour cell can adopt any manner known in the art to measure.For example, the ability of virus infection tumour cell can be measured the required viral dosage of given cell percentage (for example 50% or 80% cell) and carries out quantitatively by being measured as.The ability of duplicating in tumour cell can be measured by growth, the mensuration of being carried out in an embodiment for example is for example by measuring in the time cycle of 6 hours, 12 hours, 24 hours, 36 hours, 48 hours or 72 hours or longer time at intracellular viral growth.

The ability of virus killing tumor cell can be roughly quantitative by naked eyes, and is perhaps quantitative more accurately by the viable count that keeps under point and the MOI in preset time for given cell type is at the appointed time counted.For example, can and use any known cancer cell type to compare in 24 hours, 48 hours or 72 hours.Specifically, can use HT29 colorectum adenocarcinoma cell, LNCaP.FGC prostate cancer cell, MDA-MB-231 breast cancer cell, SK-MEL-28 malignant melanoma cell or U-87 MG glioblastoma astrocytoma cell.Any combination of any or these cell type in these cell types can be used, also other tumor cell type can be used.May preferably make up the one group of standard tumor cell type that is used for this purpose.For the viable count that keeps at preset time point is counted, can count the number of the cell (being viable cell) of getting rid of Trypan Blue.Also available fluorescence amplifying cell separator (FACS) or MTT analyze and carry out quantitative assay.Measure the tumor cytotoxicity ability in also can body, for example by measuring because the reducing of the gross tumor volume that specific virus causes.

In order to measure the characteristic of virus of the present invention, generally speaking, preferably use standard laboratory reference strain to compare.Can use any suitable standard laboratory reference strain.With regard to HSV, preferably use among HSV1 strain 17+, HSV1 strain F or the HSV1 strain KOS one or more.Described reference strain has the modification that is equal to the present invention's strain to be measured usually.Therefore, described reference strain has the modification of being equal to usually, and for example genetically deficient and/or heterologous gene insert.With regard to virus of the present invention, ICP34.5 and ICP47 encoding gene are become under the non-functional situation, it is non-functional then in the reference strain they to be become.Can be identical to the modification that the reference strain carries out with the modification that strain of the present invention is carried out.For this reason, mean that gene disruption in the reference strain is arranged in the position that the gene disruption with strain of the present invention is equal to fully, for example disappearance is for identical size and be positioned at same position.Equally, in these embodiments, heterologous gene can insert in same position, be driven or the like by identical promoters.Yet, not necessarily need to carry out identical modification.Importantly the reference gene has the modification that is equal on the function, for example makes identical gene become non-functional and/or inserts one or more identical heterologous genes.

B. mutation method

Can make described various virogene not have the active gene of function by multiple technologies well-known in the art.For example, can or insert by disappearance, replacement, preferably make their no function activity by disappearance.Disappearance can be removed the one or more partial or complete gene of gene.For example, can carry out the only disappearance of a Nucleotide, cause frameshit.Yet, preferably carry out bigger disappearance, at least 25%, more preferably at least 50% (perhaps, using absolute term, at least 10 Nucleotide, more preferably at least 100 Nucleotide, at least 1000 Nucleotide most preferably) of for example whole coding and non-coding sequence.Preferred especially described complete gene and some flanking sequence removed.When two or more gene copies when viral genome occurs, preferably make two copies all not have the function activity.

According to homologous recombination method well known to those skilled in the art, described simplexvirus is suddenlyd change.For example, with the HSV genomic dna with comprising in abutting connection with the carrier of the mutant nucleotide sequence of HSV homologous sequence, preferably plasmid vector transfection.Described mutant nucleotide sequence can comprise disappearance, inserts or replace, and all these mutant nucleotide sequences can make up by routine techniques.Insertion can comprise selectable marker gene, and for example lacZ or green fluorescent protein (GFP) are used for by for example betagalactosidase activity or fluorescent screening recombinant virus.

C. heterologous gene and promotor

Can modify virus of the present invention, make it to carry the heterologous gene of a coding immune modulator.Preferred this immune modulator can strengthen this viral anti-tumor activity.More preferably this albumen is GM-CSF or other cytokines, chemokine such as RANTES or other immune modulatory molecules such as B7.1, B7.2 or CD40L.Most preferably immune modulatory molecules is GM-CSF.Described immunomodulatory gene can be arbitrary allelic variation body of wild type gene, maybe can be mutator gene.This immunomodulatory gene derives from mammals, preferably derives from rodents or primates, more preferably derives from the mankind.This immunomodulatory gene preferably effectively is connected with one section control sequence of allowing that described gene is expressed in the cell in vivo.Virus of the present invention thereby can be used in vivo described immunomodulatory gene being passed to cell and express therein.

Available any appropriate technology, as, the HSV strain with the carrier (such as plasmid) that has the described gene that carries flank connection HSV sequence carries out homologous recombination, and this immunomodulatory gene is inserted in the viral genome.Adopt clone technology known in the art, GM-CSF gene or other immunomodulatory genes can be incorporated in the suitable plasmid vector that comprises the simplexvirus sequence.This gene can be inserted into any position in the viral genome, as long as still keep its oncolytic characteristic.Immunomodulatory gene can be inserted into virus genomic a plurality of site.For example, 2-5 gene can be inserted in the genome.

Preferably the transcription sequence with described immunomodulatory gene effectively is connected with the control sequence that allows described gene to express in tumour cell.Term " effectively connect " be meant a kind of side by side, the relation between the wherein said component allows them to work in the mode of its plan." effectively is connected " mode of connection of control sequence with encoding sequence, makes that the expression of described encoding sequence is achieved under the condition of mating with described control sequence.

Described control sequence comprises described immunomodulatory gene expression promoter of permission and the signal that is used to stop transcribing.Described promotor is selected from the promotor that function Mammals, is preferably arranged in the human tumor cell.Described promotor can derive from the promoter sequence of eukaryotic gene.For example, promotor can derive from the cell of described allogeneic gene expression, preferably mammalian cell, the genome of people's cell preferably wherein will take place.With regard to eukaryotic promoter, they can be the promotors (for example promotor of beta-actin, tubulin) that works in ubiquitous mode, or the promotor that works in the tumour-specific mode.They also can be the promotors that particular stimulation is replied, for example in conjunction with the promotor of steroid hormone receptor.Also can use viral promotors, for example Moloney muroid leukosis virus long terminal repeat (MMLV LTR) promotor or other reverse transcription disease virus promoter-people or murine cytomegalovirus (CMV) IE

Promotor or hsv gene promotor comprise driving the hsv gene promotor of expressing latency-associated transcript.

Adopt conventional clone technology well known by persons skilled in the art (referring to for example Sambrook etc., 1989, Molecular Cloning-A laboratory manual; Cold Spring HarborPress), can make up expression cassette and other the suitable construct that comprises described immunomodulatory gene and control sequence.

Described promotor is inducible promoter preferably, makes that the expression level of described immunomodulatory gene can be regulated in the lifetime of cell.Being meant of induction type can be regulated the employing expression level that described promotor obtained.For example, virus of the present invention can also be included in the heterologous gene of the coding tet repressor/VP16 transcription activating protein fusion rotein under strong promoter (for example CMV IE promotor) control, and promotor (Gossen and Bujard that described immunomodulatory gene can be replied the tet repressor VP16 transcription activating protein fusion rotein of previous report, 1992, Grossen etc., 1995) under the control.Therefore, in this embodiment, the expression of described immunomodulatory gene will depend on whether there is tsiklomitsin.

In the hsv gene group, can hold a plurality of heterologous genes.Therefore, virus of the present invention can comprise two or more immunomodulatory genes, for example 2 to 3,4 or 5 immunomodulatory genes.Can on virus genomic single site or a plurality of site, will introduce in the specific HSV strain with relevant control sequence above a gene.Perhaps, can use paired promotor (identical or different promotor), shown in promotor each other facing to opposite direction, drive a kind of expression of immunomodulatory gene respectively.

D. treatment is used

Virus of the present invention can be used for the cancer treatment method of the mankind or animal body.Particularly, virus of the present invention can be used for the oncolytic treatment of cancer, perhaps extra prodrugs therapy or the stimulation anti-tumor immune response of combination or debond.Virus of the present invention can be used for therapeutic treatment mammals, human any solid tumor preferably.For example, virus of the present invention can suffer from the curee of following cancer: prostate cancer, breast cancer, lung cancer, liver cancer, carcinoma of endometrium, bladder cancer, colon or cervical cancer, gland cancer, melanotic cancer, lymphoma, neurospongioma or sarcoma be soft tissue and osteosarcoma for example.

E. administration

Virus of the present invention can be used for the patient of needs treatment, preferably human patients.Needing the patient of treatment is the individuality of suffering from cancer, preferably patients with solid tumor.Therapeutic purpose are to improve patient's illness.Usually carry out the symptom that therapeutic treatment can be alleviated cancer with virus of the present invention.Comprise and give the virus of the present invention that the cancer patients treats significant quantity by a kind of method for cancer for the treatment of of the present invention.Give tumour patient oncolytic virus of the present invention, understand killing tumor cell usually, thereby tumour is diminished and/or prevent that malignant cell from spreading from tumour.

A method of drug treatment relates to described virus and pharmaceutically acceptable carrier or mixing diluents, produces medicinal compositions.Suitable carriers and thinner comprise isotonic saline solution, for example phosphate buffered saline(PBS).

Can adopt and to be injected directly into target tissue (described target tissue can be tumour or the blood vessel of giving tumor feeding) by the virus composition, carry out therapeutic treatment.With regard to HSV, the virus quantity scope that gives is 10 ⁴-10 ¹⁰Pfu, preferred 10 ⁵-10 ⁸Pfu, more preferably from about 10 ⁶-10 ⁸Pfu.Basically the injection consumption of the medicinal compositions of forming by described virus and a kind of pharmaceutically acceptable suitable carrier or thinner 500 μ l, 1-200 μ l, preferred 1-10 μ l usually at the most usually.Yet, treat application for some oncolytic ruling by law, according to described tumour and inoculation position, also can use the comparatively large vol of 10ml at the most.

Because the technician can easily determine best route of administration and dosage, so described route of administration and dosage are only as guide.Described dosage can be according to various parameters, especially determine according to knub position, tumour size, patient's age to be treated, body weight and illness and route of administration.Preferably, give described virus by being injected directly in the tumour.Also can system give described virus, or give in the blood vessel of described tumor feeding by described virus injection is arrived.The suitableeest route of administration will depend on the position and the size of described tumour.

Following examples are explained the present invention.

Before shown that wherein neurovirulence factor ICP34.5 was by the I type herpes simplex of inactivation

Virus (HSV1) all instructs tumour-specific lysis in two kinds of tumor models in vitro and in vivo.In the I clinical trial phase that this viroid carries out in by the brain that is injected directly into neuroglia cancer patient in late period, it is safe also demonstrating.

The laboratory strain isolated (deriving from the virus of HSV1 strain 17+ or HSV1 strain F) that connects the HSV1 that goes down to posterity is used in early stage research always, compares with up-to-date clinical separation strain, can expect that the cracking ability of described virus aspect human tumor cells weakened.

In target is to produce in the research of the ICP34.5 absence type HSV with enhanced oncolytic potential and antitumor potential, and we make HSV1 strain JS1 lack ICP47 and ICP34.5, and have inserted the immunomodulatory gene of GM-CSF.

Virus formulation (referring to Fig. 1)

Used virus or based on HSV1 strain 17+ (standard laboratory strain) or based on the person's (frequent re-activator) that derives from the frequent recurrence herpes labialis HSV1 clinical separation strain.This clinical or " non-laboratory " strain called after JS1.Make strain 17+ and JS1 lack ICP34.5 and insertion CMV-GFP box fully.By inserting human GM-CSF (hGM-CSF) or mouse GM-CSF (mGM-CSG),, also carry out further engineered to JS1 so that replace the ICP34.5 gene and make the ICP47 disappearance.The JS1 discussed in this article also right and wrong laboratory strain of strain of deriving, i.e. the non-laboratory strain that the present invention is modified.

The cracking ability of virus

In all tested tumor cell lines, strengthen cracking (cell killing) ability with the non-laboratory strain of JS1 deutero-derived virus.More particularly, with reference to Fig. 3, JS1/ICP34.5-virus, promptly remove the JS1 of ICP34.5, be presented at cracking ability enhancing in the following cell: HT29 colorectum gland cancer, LNCaP.FGC prostate cancer, MDA-MB-231 mammary cancer, SK-MEL-28 malignant melanoma and U-87MG glioblastoma astrocytoma by disappearance.

Therefore, when in human patients, being used for the treatment of cancer, in all cases, kill more tumour cell than laboratory strain isolated 17+ with clinical separation strain virus BL1 and JS1, reaching oncolytic activity increases, therefore use up-to-date clinical disease strain may strengthen this class modified with

Obtain the anti-tumor capacity that tumor-selective duplicates the virus of (for example by disappearance ICP34.5).

Further enhanced anti-tumor activity

If these viruses are used for transmitting the gene with anti-tumor activity, then also can expect further enhanced activity.Such gene comprises coding prodrug activator or the proteic gene of immunostimulation.

Made up the ICP34.5 absence type clinical separation strain of the HSV1 of a kind of expressing human GM-CSF or mouse GM-CSF by JS1.GM-CSF is a kind of effective immunostimulant.This virus design strengthens anti-tumor immune response in order to injection back in tumour.When producing these viruses in bhk cell is cultivated, (Biotrak Amersham) has proved these expressing virals mankind or mouse GM-CSF to adopt the elisa assay test kit.After 24 hours, each hole of six orifice plates produces 0.56 or 0.54 milligram of mankind or muroid GM-CSF respectively at the bhk cell that converges with the MOI=0.5 infection.

Preservation information

HSV1 strain JS-1 is preserved in European cell culture preservation center (ECACC) January 2 calendar year 2001, CAMR, and Salisbury, Wiltshire SP4 0JG, Britain, interim preserving number is V01010209.

Bibliography

Hill etc., 1995, Nature 375:411-415

Shi etc., 1999, Cancer-Gene-Ther 6:81-88

Chou etc., 1990, Science 250:1262-1266

Maclean etc., 1991, J.Gen.Virol.72:631-639

Gossen M and Bujard H, 1992, PNAS 89:5547-5551

Gossen M etc., 1995, Science 268:1766-1769

Thompson etc., 1998, Virus Genes 1 (3): 275-286

Meignier etc., 1988, Infect.Dis.159:602-614

Claims

1. simplexvirus, described virus comprises a kind of gene of the immune modulator of encoding, and lacks functional ICP34.5 encoding gene and functional ICP47 encoding gene, and replication is arranged in tumour cell.

2. the virus of a claim 1, wherein said immunomodulatory gene is the albumen that cytokine, chemokine maybe can be regulated T cell proliferation.

3. the virus of a claim 2, wherein said cytokine is GM-CSF, described chemokine is that RANTES or described albumen are B7.1, B7.2 or CD40L.

4. the virus of aforementioned each claim, two or more immune modulators of described encoding viral.

5. the virus of aforementioned each claim, described virus also lacks the functioning gene of encode ICP6, glycoprotein h or thymidine kinase.

6. the virus of aforementioned each claim, described virus also lacks the gene that coding can suppress the proteic functional copy of dendritic cell function.

7. the virus of a claim 6, the wherein said albumen that can suppress the dendritic cell function is UL43 or vhs.

8. the virus of aforementioned each claim, described virus is a kind of non-zoo virus strain.

9. simplexvirus, described virus comprises the gene of GM-CSF of encoding, and lacks functional ICP34.5 encoding gene, and described virus is a kind of non-zoo virus strain and replication is arranged in tumour cell.

10. the virus of aforementioned each claim, described virus is the strain of 1 type or herpes simplex types 2 virus.

11. each virus among the claim 8-10, wherein said non-zoo virus strain:

(a) from its host, separate its not modified precursor strain and count, in culture, experienced below 1 year or 1 year, or

(b) from its host, separate its not modified precursor strain and count, experienced 100 continuous passage cycles or 100 following continuous passage cycles, or

(c) than have the Reference Lab virus strain infection tumour cell that is equal to modification or in tumour cell, duplicate, ability that killing tumor cell or the iuntercellular in tissue are propagated is strong, or

(d) with regard to one or more characteristics of definition in (c), has the ability of its not modified precursor strain basically.

12. the virus of a claim 11 wherein is the stronger ability of tool significance,statistical in ability stronger described in (c); Be identical ability or the ability that does not have significant difference basically wherein perhaps in ability identical described in (d).

13. the virus of a claim 11 or 12, wherein said non-zoo virus strain is a kind of HSV strain, and the reference strain that limits in the claim 11 is to have the HSV1 strain 17 that is equal to modification with described non-zoo virus strain ⁺, HSV1 strain F and HSV1 strain KOS.

14. the virus of aforementioned each claim, described virus are derived from the European cell culture preservation center (ECAAC) that is deposited in, preserving number is 01010209 HSV1 JS1 temporarily.

15. virus that is used for aforementioned each claim of the method by treating human body or animal body.

16. the purposes of the virus of aforementioned each claim, described virus are used for production treatment for cancer medicine.

17. the purposes of claim 16, wherein said medicine are used for inoculation in the direct tumour.

18. a pharmaceutical composition, described pharmaceutical composition comprise as each virus and a kind of pharmaceutically acceptable carrier or thinner among the claim 1-14 of effective constituent.

19. medicine that is used for the treatment of cancer, described medicine comprises a kind of simplexvirus, and described simplexvirus contains a kind of gene of the immunomodulatory cytokine of encoding and lack functional ICP34.5 encoding gene and functional ICP47 encoding gene and replication is arranged in tumour cell.

20. be deposited in European cell culture preservation center (ECAAC), interim preserving number and be 01010209 HSV1 strain JS1, or its deutero-HSV1 strain.