(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`26 July 2001 (26.07.2001) I||||||l|||||||||l|||llllll||l||lllllllllllllllllllllllll||||||||||l|||||l||||||
`
`(10) International Publication Number
`(43) International Publication Date
`WO 01/53505 A2
`
`(51) International Patent Classification7:
`7/01, A61K 48/00
`
`C12N 15/869,
`
`(74) Agent: WOODS, Geoffrey, Corlett; J.A. Kemp & Co., 14
`South Square, Gray’s Inn, London WC1R 5LX (GB).
`
`(21) International Application Number:
`
`PCT/GB01/00225
`
`(22) International Filing Date: 22 January 2001 (22.01.2001)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ,
`DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR,
`HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
`LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ,
`NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM,
`TR, TI‘, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.
`
`(30) Priority Data:
`00014753
`00028548
`01002880
`01004308
`
`21 January 2000 (21.01.2000)
`8 February 2000 (08.02.2000)
`5 January 2001 (05.01.2001)
`6 January 2001 (06.01.2001)
`
`GB
`GB
`GB
`GB
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
`CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`(71) Applicant (for all designated States except US): BIOVEX
`LIMITED [GB/GB]; The Windeyer Institute, 46 Cleve-
`land Street, London WlP 6DB (GB).
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): COFFIN, Robert,
`Stuart [GB/GB]; The Windeyer Institute, 46 Cleveland
`Street, London WlP 6DB (GB).
`
`For two-letter codes and other abbreviations, refer to the "Guid—
`ance Notes on Codes and Abbreviations ” appearing at the begin-
`ning ofeach regular issue of the PCT Gazette.
`
`O01/53505A2
`
`
`
`(54) Title: HERPES VIRUS STRAINS
`
`(57) 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.
`
`

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`WO 01/53505
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`PCT/GB01/00225
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`1
`
`HERPES VIRUS STRAINS
`
`Field of the Invention
`
`The present invention relates to herpes virus strains with improved anti-
`
`VI
`
`tumour activity as compared to previously known strains.
`
`Background to the Invention
`
`Viruses have been shown to have utility in a variety of applications in
`
`biotechnology and medicine on many occasions. Each is due to the unique ability of
`
`viruses to enter cells at high efficiency. This is followed in such applications by
`either virus gene expression and replication and/or expression of an- inserted
`
`heterologous gene. Thus viruses can either deliver and express genes in cells (either
`
`viral or other genes) which may be useful
`
`in for example gene therapy or the
`
`development of vaccines, or they may be useful in selectively killing cells by lytic
`
`replication or the action of a delivered gene in for example cancer.
`
`Herpes simplex virus (HSV) has been suggested to be of use for the oncolytic
`
`treatment of cancer. Here the virus must however be disabled such that it
`
`is no
`
`longer pathogenic, i.e. does not replicate in and kill non-tumor cells, but such that it
`
`can still enter and kill tumor cells. For the oncolytic treatment of cancer, which may
`
`also include the delivery of gene(s) enhancing the therapeutic effect, a number of
`
`mutations to HSV have been identified which still allow the virus to replicate in
`
`in tumors), but which prevent
`culture or in actively dividing cells in vivo (e.g.
`significant replication in normal tissue. Such mutations include disruption of the
`
`genes encoding ICP34.5,
`
`ICP6, and thymidine kinase. Of these, viruses with
`
`25
`
`mutations to ICP34.5, or ICP34.5 together with mutation of e.g. ICP6 have so far
`
`shown the most favourable safety profile. Viruses deleted for only ICP34.5 have
`
`been shown to replicate in many tumor cell types in vitro and to selectively replicate
`
`in artificially induced brain tumors in mice while sparing surrounding tissue. Early
`
`stage clinical trials have also shown their safety in man.
`
`However, while promise has been shown for various viruses including HSV
`
`for the oncolytic treatment of cancer, the majority of this work has used virus strains
`
`which do not carry a heterologous gene which may enhance the anti-tumor effect.
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`We propose that the combined use of HSV with an inactivating mutation in the gene
`
`encoding
`
`lCP34.5
`
`together with the
`
`delivery of
`
`the
`
`gene
`
`encoding
`
`an
`
`immunomodulatory protein such as granulocyte macrophage colony stimulating
`
`factor (GM-CSF) encoded in the disabled virus genome may have optimal immune
`
`stimulating properties against the tumor to be treated, particularly if functions in the
`
`virus which usually reduce immune responses to HSV infected cells have also been
`
`inactivated.
`
`For example the HSV lCP47 protein specifically inhibits antigen
`
`presentation in HSV infected cells (Hill et al 1995), and the product of the UL43
`
`gene and the vhs protein reduce the immune-stimulating abilities of dendritic cells
`
`lO
`
`infected with HSV.
`
`ICP47 and/or dendritic cell—inactivating genes might therefore
`
`usefully be deleted from an oncolytic HSV mutant virus used for the treatment of
`
`cancer, particularly if immune effects are to be enhanced through the use of GM-CSF
`
`or other immunostimulatory cytokine or chemokine. GM-CSF has recently been
`
`shown to give an enhanced anti-tumor
`
`immune effect if expressed from within a
`
`tumor cell rather than administered systemically (Shi et al 1999). Thus in such use
`
`an oncolytic HSV mutant would be inoculated into a primary or secondary tumor
`
`where replication and oncolytic destruction of the tumor would occur.
`
`Immune
`
`responses would also be stimulated against the HSV infected cells, and also to tumor
`
`cells elsewhere which had spread from the primary tumor site.
`
`Summarv of the Invention
`
`The present invention provides viruses with improved capabilities for the
`lytic destruction of tumor cells in vivo. Here herpes simplex virus strains are
`
`constructed using a strain of HSVI or HSV2 in which the genes encoding lCP34.5
`
`and lCP47 have been inactivated such that a functional ICP34.5 or lCP47 protein
`
`cannot be expressed and which also carries a gene encoding an immunomodulatory
`
`protein. The virus may also be mutated for any additional gene(s) which may be
`
`involved in inhibiting the function of dendritic cells including the UL43 gene and/or
`
`the gene encoding vhs. The present invention therefore provides viruses capable of
`
`the oncolytic destruction of tumor cells and in which anti-tumor immune effects will
`
`have been maximised.
`
`Accordingly the invention provides:
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`

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`WO 01/53505
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`PCT/GB01/00225
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`10
`
`15
`
`20
`
`3
`
`-
`
`a
`
`herpes
`
`virus which
`
`comprises
`
`a
`
`gene
`
`encoding
`
`an
`
`immunomodulatory protein and which lacks functional lCP34.5 and ICP47 encoding
`
`genes.
`
`-
`
`a herpes virus of the invention for use in a method of treatment of the
`
`human or animal body by therapy.
`
`—
`
`use of a virus of the invention in the manufacture of a medicament for
`
`the treatment of cancer.
`
`-
`
`a pharmaceutical composition comprising as active ingredient a virus
`
`according to the invention and a pharmaceutically acceptable carrier or diluent.
`
`-
`
`a method of treating a tumour in an individual
`
`in need thereof by
`
`administering to said individual an effective amount of a virus according to the
`
`invention.
`
`Brief Description of the Drawings
`
`Fig 1. Viruses
`
`From top to bottom, diagrams show: laboratory HSVl strain 17+, , clinical HSVI
`
`strain JSl, strain l7+/ICP34.5-, strain JSl/ICP34.5-, strain J'Sl/lCP34.5-/lCP47-/
`
`hGMCSF, strain JSl/lCP34.5-/lCP47-/ mGMCSF.
`
`Detailed Description of the Invention
`
`A.
`
`Viruses
`
`A herpes virus of the invention is capable of efficiently infecting target tumor
`
`cells and the genes encoding lCP34.5 and lCP47 are inactivated in the virus.
`
`Mutation of ICP34.5 allows selective oncolytic activity.
`
`Such mutations are
`
`described in Chou et al 1990 and Maclean et al 1991, although any mutation in
`
`which ICP34.5 is non-functional may be used. The genes encoding lCP6 and/or
`
`thymidine kinase may additionally be inactivated, as may other genes if such
`
`inactivation does significantly reduce the oncolytic effect, or
`
`if such deletion
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`20
`
`enhances oncolytic or other desirable properties of the virus. 1CP47 usually functions
`
`to block antigen presentation in HSV-infected cells so its disruption leads to a virus
`
`that does not confer on infected tumour cells properties that might protect them from
`
`the host’s immune system when infected with HSV. Viruses of the invention
`
`additionally encode an irnrnunomodulatory protein, preferably GM-C SF, but may
`
`also encode other cytokines. chemokines such as RANTES, or other immune-
`
`modulatory
`
`proteins
`
`such
`
`as B7. 1,
`
`87.2
`
`or CD40L. Genes
`
`encoding
`
`irnmunomodulatory proteins may be included individually or in combination.
`
`Viral
`
`regions altered for
`
`the purposes described above may be either
`
`eliminated (completely or partly), or made non—functional, or substituted by other
`sequences, in particular by a gene for an immunornodulatory- protein such as GM-
`CSF .
`
`The virus of the invention may be derived from a HSVl or HSV2 strain, or
`
`from a derivative thereof, preferably HSVl. Derivatives include inter-type
`
`recombinants containing DNA from HSVl and HSVZ strains. Such inter-type
`
`recombinants are described in the art, for example in Thompson et al, 1998 and
`
`Meignier et al, 1988. Derivatives preferably have at least 70% sequence homology
`
`to either the HSVl or HSV2 genomes, more preferably at least 80%, even more
`
`preferably at least 90 or 95%. More preferably, a derivative has at least 70%
`
`sequence identity to either the HSVl or HSVZ genome, more preferably at least 80%
`
`identity, even more preferably at least 90%, 95% or 98% identity.
`
`For example the UWGCG Package provides the BESTFIT program which
`can be used to calculate homology (for example used on its default settings)
`
`(Devereux er a1. (1984) Nucleic Acids Research 12, p3 87—395). The PILEUP and
`
`BLAST algorithms can be used to calculate homology or line up sequences (typically
`
`on their default settings), for example as described in Altschul (1993) J . Mol. Evol.
`
`3_6_:290-300; Altschul er a1. (1990) J. Mol. Biol. £2403-10.
`
`Software for performing BLAST analyses is publicly available through the
`
`National Centre for Biotechnology Information (http://www.ncbi.nlrn.nih.gov/).
`
`3O
`
`This algorithm involves first identifying high scoring sequence pair (HSPS) by
`
`identifying short words of length W in the query sequence that either match or satisfy
`
`some positive-valued threshold score T when aligned with a word of the same length
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`in a database sequence. T is referred to as the neighbourhood word score threshold
`
`(Altschul er al, 1990). These initial neighbourhood word hits act as seeds for
`
`initiating searches to find HSPs containing them. The word hits are extended in both
`
`directions along each sequence for as far as the cumulative alignment score can be
`
`increased. Extensions for the word hits in each direction are halted when:
`
`the
`
`cumulative alignment score falls off by the quantity X from its maximum achieved
`
`value; the cumulative score goes to zero or below, due to the accumulation of one or
`
`more negative-scoring residue alignments; or the end of either sequence is reached.
`
`The BLAST algorithm parameters W, T and X determine the sensitivity and speed of
`
`the alignment. The BLAST program uses as defaults a word length (W) of l l, the
`
`BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. A cad
`
`Sci. USA fl: 10915-10919) alignments (B) of 50, expectation (E) of 10. M=5, N=4,
`
`and a comparison of both strands.
`
`The BLAST algoritth performs a statistical analysis of the similarity
`
`between two sequences; see e.g., Karlin and Altschul ( 1993) Proc. Natl. Acad. Sci.
`
`USA 90: 5873-5 787. One measure of similarity provided by the BLAST algorithm
`
`is the smallest sum probability (P(N)), which provides an indication of the
`
`probability by which a match between two nucleotide or amino acid sequences
`
`would occur by chance. For example, a sequence is considered similar to another
`
`sequence if the smallest sum probability in comparison of the first sequence to the
`
`second sequence is less than about 1, preferably less than about 0.1, more preferably
`
`less than about 0.01, and most preferably less than about 0.001.
`
`A derivative may have the sequence of a HSVl or HSV2 genome modified
`
`by nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100
`
`substitutions. The HSVl or HSV2 genome may alternatively or additionally be
`
`modified by one or more insertions and/or deletions and/or by an extension at either
`
`or both ends.
`
`Virus strains of the invention may be "non-laboratory” strains. These can
`
`also be referred to as “clinical” strains. A person of skill in the art will readily be
`
`able to distinguish between a laboratory strain and a non-laboratory, or clinical,
`
`strain. Further guidance on the properties likely to be exhibited by virus strains is
`
`given below.
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`

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`WO 01/53505
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`PCT/GB01/00225
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`6
`
`The key distinction between a laboratory and non-laboratory strain is that
`
`laboratory strains currently in common use have been maintained for long periods,
`
`many years in some cases, in culture. The culture of viruses such as HSV involves a
`
`technique known as serial passage. To grow and maintain viruses, suitable cells are
`
`1.1-
`
`infected with the virus, the virus replicates within the cell and the virus is then
`
`harvested; fresh cells are then re-infected, this process constitutes one cycle of serial
`
`passage. Each such cycle may take, for example, a few days in the case of HSV. As
`
`discussed above, such serial passaging may lead to changes in the properties of the
`
`virus strain, in that selection takes places for properties that favour growth in culture
`
`10
`
`(eg. rapid replication), as opposed to properties useful for practical applications, eg.
`maintenance of the capacity to travel along axons in the case of HSV or to infect
`
`human cells.
`
`Virus strains of the invention are may be non-laboratory strains in that they
`
`are derived from strains recently isolated from infected individuals. Strains of the
`
`invention are modified compared to the original clinical isolates, and may have spent
`
`a time in culture, but any time spent in culture will be comparatively short. Strains
`
`of the invention are prepared in such a manner as to retain substantially the desirable
`
`properties of the original clinical isolates from which they are derived.
`
`A virus strain of the invention is derived from a parental virus strain if the
`
`20
`
`parental virus strain is mutated to produce the virus. For example, a virus of the
`
`invention may be derived from the clinical isolate JSl. The parental strain of such a
`
`JSl-derived virus may be JSI or another HSVl strain derived from J SI. Thus a virus
`of
`the
`invention may
`be
`a
`JSI
`virus
`comprising
`a gene
`encoding an
`
`irnrnunomodulatory protein and which lacks a functional ICP34.5 encoding gene and
`
`Ix) UI
`
`a functional ICP47 encoding gene.
`
`In addition, such a virus may contain any other
`
`mutation as mentioned herein.
`
`A virus of the invention is capable of efficiently infecting target human
`
`cancer cells. When such a virus is a non-laboratory or clinical strain it will have
`
`been recently isolated from an HSV infected individual and then screened for the
`
`0
`
`desired ability of enhanced replication, infection or killing of tumour and/or other
`
`cells in vitro and/or in vivo in comparison to standard laboratory strains.
`
`Such
`
`viruses of the invention with improved properties as compared to laboratory virus
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`U!
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`7
`
`strains are then engineered such that they lack functional ICP34.5 and ICP47 genes
`
`and encode a gene(s) for an immunomodulatory protein(s) such as GM-CSF under
`
`the control of a suitable promoter(s). Other genes encoding proteins which interfere
`
`with the function of dendritic cells such as UL43 and/or vhs may also be inactivated.
`
`Preferably, a non-laboratory virus strain of the invention has undergone three
`
`years or less in culture since isolation of its unmodified clinical precursor strain from
`
`its host. More preferably, the strain has undergone one year or less in culture, for
`
`example nine months or less, six months or less, three months or less, or two months
`
`or less, one month or less,
`
`two weeks or less, or one week or less. By these
`
`definitions of time in culture, is meant time actually spent in culture. Thus, for
`example,
`it is a common practice to freeze virus strains in order to preserve them.
`
`Evidently, preserving by freezing or in an equivalent manner does not qualify as
`
`maintaining the strain in culture. Thus, time spent frozen or otherwise preserved is
`
`not included in the above definitions of time spent in culture. Time spent in culture
`
`is typically time actually spent undergoing serial passage, i.e. time during which
`
`selection for undesirable characteristics can occur.
`
`Preferably, a non-laboratory virus strain has undergone 1,000 or less cycles or
`
`serial passage since isolation of its unmodified clinical precursor strain from its host.
`
`More preferably, it has undergone 500 or less, 100 or less, 90 or less, 80 or less, 70
`
`or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less such
`
`cycles.
`
`Preferably, a non-laboratory virus has a greater ability, as measured by
`standard statistical
`tests,
`than a reference laboratory strain with the equivalent
`
`modifications to perform certain functions useful in the application at hand.
`
`In the
`
`case of an oncolytic virus for tumour treatment, a non-laboratory virus strain of the
`
`invention will preferably have a greater ability than a reference laboratory strain with
`
`equivalent modifications to infect or replicate in tumour cells, to kill tumour cells or
`
`to spread between cells in tissue. More preferably, such greater ability is a
`
`statistically significantly greater ability. For example, according to the invention, a
`
`non-laboratory strain of the invention may have up to 1.1 fold, 1.2 fold, 1.5 fold, 2
`
`fold, 5 fold, 10 fold, 20 fold, 50 fold, or 100 fold the capacity of the reference strain
`
`in respect of the property being tested.
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`WO 01/53505
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`U-
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`
`[\J (1|
`
`8
`
`Statistical analysis of the properties described herein may be carried out by
`
`standard tests, for example,
`
`t-tests, ANOVA, or Chi squared tests.
`
`Typically,
`
`statistical significance will be measured to a level of p = 0.05 (5%), more preferably
`
`p = 0.01p, p = 0.001, p = 0.0001, p = 0.000001.
`
`Viruses of the invention infect and replicate in tumour cells, subsequently
`
`killing the tumour cells. Thus, such viruses are replication competent. Preferably,
`
`they are selectively replication competent in tumour cells. This means that either
`
`they replicate in tumour cells and not in non-tumour cells, or that they replicate more
`
`effectively in tumour cells than in non—tumour cells. Cells in which the virus is able
`
`to replicate are permissive cells. Measurement of selective replication competence
`can be carried out by the tests described herein for measurement of replication and
`
`tumour cell-killing capacity, and also analysed by the statistical techniques
`
`mentioned herein if desired.
`
`A virus of the invention preferably has a greater ability than an unmodified
`
`parent strain to infect or replicate in a tumour cell, to kill tumour cells or to spread
`
`between cells in tissues. Preferably this ability is a statistically significant greater
`
`ability. For example, a virus according to the invention may have up to 11 fold, 1.2
`
`fold, 1.5 fold, 2 fold, 5 fold, 10 fold, 20 fold, 50 fold or 100 fold the capacity of the
`
`unmodified parent strain in respect of the property being tested.
`
`The properties of the virus strain in respect of tumour cells can be measured
`
`in any manner known in the art. For example, the capacity of a virus to infect a
`
`tumour cell can be quantified by measuring the dose of virus required to measure a
`given percentage of cells, for example 50% or 80% of cells. The capacity to
`
`replicate in a tumour cell can be measured by growth measurements such as those
`
`carried out in the Examples, e.g. by measuring virus growth in cells over a period of
`
`6, 12, 24, 36, 48 or 72 hours or longer.
`
`The ability of a virus to kill tumour cells can be roughly quantitated by eye or
`
`more exactly quantitated by counting the number of live cells that remain over time
`
`for a given time point and M01 for given cell type. For example, comparisons may
`
`be made over 24, 48 or 72 hours and using any known tumour cell type.
`
`In
`
`particular, HT29 colorectal adenocarcinoma, LNCaP.FGC prostate adenocarcinoma,
`
`MDA-MB-231 breast adenocarcinoma, SK-MEL-28 malignant melanoma or U-87
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`MG glioblastoma astrocytoma cells can be used. Any one of these cell types or any
`
`combination of these cell types can be used, as may other tumour cell types.
`
`It may
`
`be desirable to construct a standard panel of tumour cell types for this purpose. To
`
`count the number of live cells remaining at a given time point, the number of trypan
`
`blue-excluding cells (i.e. live cells) can be counted. Quantitation may also be carried
`
`out by fluorescence activated cell sorting (FACS) or MTT assay. Tumour cell-
`
`killing ability may also be measured in viva, e.g. by measuring the reduction in
`
`tumour volume engendered by a particular virus.
`
`In order to determine the properties of viruses of the invention, it will
`
`generally be desirable to use a standard laboratory reference strain for comparison.
`Any suitable standard laboratory reference strain may be used. in the case of HSV, it
`
`is preferred to use one or more of HSVl strain 17+, HSVl strain F or HSVl strain
`
`KOS. The reference strain will typically have equivalent modifications to the strain
`
`of the invention being tested. Thus, the reference strain will typically have
`
`equivalent modifications gene deletions and, such as heterologous gene insertions. In
`
`the case of a virus of the invention, where the ICP34.5 and ICP47—encoding genes
`
`have been rendered non-functional, then they will also have been rendered non-
`
`functional in the reference strain. The modifications made to the reference strain
`
`may be identical to those made to the strain of the invention. By this, it is meant that
`
`the gene disruptions in the reference strain will be in exactly equivalent positions to
`
`those in the strain of the invention, e.g. deletions will be of the same size and in the
`
`same place. Similarly, in these embodiments, heterologous genes will be inserted in
`
`the same place, driven by the same promoter, etc. However, it is not essential that
`
`identical modifications be made. What is important is that the reference gene has
`
`functionally equivalent modifications, e.g. that the same genes are rendered non-
`
`functional and/or the same heterologous gene or genes is inserted.
`
`U.
`
`10
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`15
`
`I\) U!
`
`B.
`
`Methods of mutation
`
`The various genes referred to may be rendered functionally inactive by
`
`several
`
`techniques well known in the art.
`
`For example,
`
`they may be rendered
`
`fiinctionally inactive by deletion(s), substitution(s) or insertion(s), preferably by
`
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`deletion. Deletions may remove one or more portions of the gene or the entire gene.
`
`For example, deletion of only one nucleotide may be made, resulting in a frame shift.
`
`However, preferably a larger deletion() is made, for example at least 25%, more
`
`preferably at least 50% of the total coding and non-coding sequence (or alternatively,
`
`in absolute terms, at least 10 nucleotides, more preferably at least 100 nucleotides,
`
`most preferably, at least 1000 nucleotides).
`
`It is particularly preferred to remove the
`
`entire gene and some of the flanking sequences. Where two or more copies of the
`
`gene are present in the viral genome it is preferred that both copies of the gene are
`
`rendered functionally inactive.
`
`Mutations are made in the herpes viruses by homologous recombination
`methods well known to those skilled in the art. For example, HSV genomic DNA is
`
`transfected together with a vector, preferably a plasmid vector, comprising the
`
`mutated sequence flanked by homologous HSV sequences. The mutated sequence
`
`may comprise a deletion(s),
`
`insertion(s) or substitution(s), all of which may be
`
`constructed by routine techniques.
`
`Insertions may include selectable marker genes,
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`for example lacZ or green fluorescent protein (GFP), for screening recombinant
`
`viruses, for example, B-galactosidase activity or fluorescence.
`
`C.
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`Heterologous genes and promoters
`
`The viruses of the invention may be modified to carry a heterologous gene
`
`encoding an immunomodulatory protein. Preferably the immunomodulatory protein
`
`will enhance the anti-tumour activity of the virus. More preferably the protein is
`
`GM-CSF or another cytokine,
`
`a chemokine such as RANTES, or another
`
`immunomodulatory molecule such as B7.l, B72 or CD4OL. Most preferably the
`
`immunomodulatory molecule is GM—CSF. The immunomodulatory gene may be any
`
`allelic variant of a wild-type gene, or
`
`it may be
`
`a mutant gene.
`
`The
`
`immunomodulatory gene will be derived from a mammal, preferably a rodent or
`
`primate, more preferably a human. The immunomodulatory gene is preferably
`
`operably linked to a control sequence permitting expression of said gene in a cell in
`
`vivo. Viruses of the invention may thus be used to deliver the immunomodulatory
`
`gene (or genes) to a cell in vivo where it will be expressed.
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`The immunomodulatory gene may be inserted into the viral genome by any
`
`suitable technique such as homologous recombination of HSV strains with, for
`
`example, plasmid vectors carrying the gene flanked by HSV sequences. The GM-
`
`_CSF gene, or other immunomodulatory gene, may be introduced into a suitable
`
`plasmid vector comprising herpes viral sequences using cloning techniques well-
`
`known in the art. The gene may be inserted into the viral genome at any location
`
`provided that oncolytic properties are still retained.
`
`Immunomodulatory genes may
`
`be inserted at multiple sites within the virus genome. For example, from 2 to 5 genes
`
`may be inserted into the genome.
`
`The transcribed sequence of the immunomodulatory gene is preferably
`operably linked to a control sequence permitting expression of the gene in a tumour
`
`cell. The term “operably linked” refers to a juxtaposition wherein the components
`
`described are in a relationship permitting them to function in their intended manner.
`
`A control sequence “operably linked” to a coding sequence is ligated in such a way
`
`that expression of the coding sequence is achieved under conditions compatible with
`
`the control sequence.
`
`The control sequence comprises a promoter allowing expression of the
`
`immunomodulatory gene and a signal for termination of transcription. The promoter
`
`is selected from promoters which are functional in mammalian, preferably human
`
`tumour cells. The promoter may be derived from promoter sequences of eukaryotic
`
`genes. For example, the promoter may be derived from the genome of a cell in
`
`which expression of the heterologous gene is to occur, preferably a mammalian,
`preferably a human tumour cell. With respect to eukaryotic promoters, they may be
`
`promoters that function in a ubiquitous manner (such as promoters of [E-actin,
`
`Ix) U:
`
`tubulin) or, alternatively, in a tumour-specific manner. They may also be promoters
`
`that respond to specific stimuli, for example promoters that bind steroid hormone
`
`receptors. Viral promoters may also be used, for example the Moloney murine
`
`leukaemia virus long terminal repeat (MMLV LTR) promoter or other retroviral
`
`promoters, the human or mouse cytomegalovirus (CMV) IE promoter, or promoters
`
`of herpes virus genes including those driving expression of the latency associated
`
`transcripts.
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`15
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`Expression
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`cassettes
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`and
`
`other
`
`suitable
`
`constructs
`
`comprising
`
`the
`
`immunomodulatory gene and control sequences can be made using routine cloning
`
`techniques known to persons skilled in the art (see, for example, Sambrook er a[.,
`
`1989, Molecular Cloning — A laboratory manual; Cold Spring Harbor Press).
`
`It may also be advantageous for the promoters to be inducible so that the
`
`levels of expression of the immunomodulatory gene(s) can be regulated during the
`
`life-time of the tumour cell.
`
`Inducible means that the levels of expression obtained
`
`using the promoter can be regulated. For example, a virus of the invention may
`
`further comprise a hererologous gene encoding the tet repressorNP16 transcriptional
`
`activator fusion protein under the control of a strong promoter (e.g.
`the CMV IE
`promoter) and the immunomodulatory gene may be under the-control of a promoter
`
`responsive to the tet
`
`repressor VP16 transcriptional activator
`
`fusion protein
`
`previously reported (Gossen and Bujard, 1992, Gossen er al, 1995). Thus, in this
`
`example, expression of the immunomodulatory gene would depend on the presence
`
`or absence of tetracycline.
`
`Multiple heterologous genes can be accommodated in the herpes virus
`
`genome.
`
`Therefore, a virus of the invention may comprise two or more
`
`immunomodulatory genes, for example from 2 to 3, 4 or 5 immunomodulatory
`
`genes. More than one gene and associated control sequences could be introduced
`
`into a particular HSV strain either at a single site or at multiple sites in the virus
`
`genome. Alternatively pairs of promoters (the same or different promoters) facing in
`
`opposite orientations away from each other, each driving the expression of an
`
`immunomodulatory gene may be used.
`
`D.
`
`Therapeutic uses
`
`Viruses of the invention may be used in methods of cancer therapy of the
`
`human or animal body.
`
`In particular, viruses of the invention may be used in the
`
`oncolytic treatment of cancer, either with or without additional pro—drug therapy or
`
`stimulation of an anti-tumour immune response. Viruses of the invention may be
`
`used in the therapeutic treatment of any solid tumour in a mammal, preferably in a
`
`human. For example viruses of the invention may be administered to a subject with
`
`prostate, breast,
`
`lung,
`
`liver, endometrial, bladder, colon or cervical carcinoma;
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`adenocarcinoma; melanoma; lymphoma; glioma; or sarcomas such as soft tissue and
`
`bone sarcomas.
`
`E.
`
`Administration
`
`The viruses of the invention may be used in a patient, preferably a human
`
`patient, in need of treatment. A patient in need of treatment is an individual suffering
`
`from cancer, preferably an individual with a solid tumour, The aim of therapeutic
`
`treatment is to improve the condition of a patient. Typically therapeutic treatment
`
`using a virus of the invention allieviates the symptoms of the cancer. A method of
`
`10
`
`treatment of cancer
`
`according to the
`
`invention comprises
`
`administering a
`
`therapeutically effective amount of a virus of the invention to a patient suffering
`
`from cancer. Administration of an oncolytic virus of the invention to an individual
`
`suffering from a tumour will typically kill the cells of the tumour thus decreasing the
`
`size of the tumour and/or preventing spread of malignant cells from the tumour.
`
`One method of administering therapy involves combining the virus with a
`
`pharmaceutically acceptable
`
`carrier or diluent
`
`to produce
`
`a pharmaceutical
`
`composition.
`
`Suitable carriers and diluents include isotonic saline solutions, for
`
`example phosphate-buffered saline.
`
`Therapeutic treatment may be carried out following direct injection of the
`
`20
`
`virus composition into target tissue which may be the tumour or a blood vessel
`
`supplying the tumour. The amount of virus administered is in the case of HSV in the
`
`range of from 104 to 1010 pfu, preferably from 105 to 108 pfu, more preferably about
`
`106 to lo8 pfu. Typically up to soopl, typically from 1 to zoopl preferably from 1 to
`
`lOul of
`
`a pharmaceutical composition consisting essentially of the