(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`10 May 2007 (10.05.2007)
`
`
`
`International Patent Classification:
`C12N 15/869 (2006.01)
`A61K 35/76 (2006.01)
`
`(81)
`
`International Application Number:
`PCT/GB2006/004094
`
`International Filing Date:
`2 November 2006 (02.1 1.2006)
`
`Filing Language:
`
`Publication Language:
`
`English
`
`English
`
`(84)
`
`Priority Data:
`05224761
`
`3 November 2005 (03.1 1.2005)
`
`GB
`
`Applicant (for all designated States except US): BIOVEX
`LIMITED [GB/GB]; 70 Milton Park, Abingdon, Oxford,
`OX 14 4RX (GB).
`
`Inventor; and
`(for US only): COFFIN, Robert,
`Inventor/Applicant
`Stuart [GB/GB]; 70 Milton Park, Abingdon, Oxfordshire
`OX 14 4RX (GB).
`
`Published:
`with international search report
`
`(10) International Publication Number
`
`WO 2007/052029 A1
`
`Designated States (unless otherwise indicated, for every
`kind 9“ national protection available): AE, AG, AL, AM,
`AT,AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, GT, HN, HR, HU, in, IL, IN, IS,
`JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS,
`LT, LU, LV,LY, MA, MD, MG, MK, MN, MW, MX, MY,
`MZ, NA, NG, N1, NO, NZ, OM, PG, PH, PL, PT, R0, RS,
`RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW
`
`Designated States (unless otherwise indicated, for every
`kind 9‘ regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`2W), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV,MC, NL, PL, PT,
`RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, TG).
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`(51)
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`(21)
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`W02007/052029A1|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`Agents: TUXWORTH, Pamela, Mary et a1; J.A. Kemp
`& Co.,
`14 South Square, Gray's Inn, London WC1R 5]]
`(GB).
`
`refer to the "Guid—
`For two—letter codes and other abbreviations,
`ance Notes on Codes and Abbreviations" appearing at the beg in—
`ning qC each regular issue q” the PCT Gazette.
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`(54)
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`Title: ONCOLYTIC HERPES VIRUS VECTORS
`
`Clinical strain JS—I
`
`I::l——l::l:l’—iZZI
`
`JS-I 34.5 -47-GFP pA-
`ICP34.5
`
`/ \
`
`EX. II
`pA GFP CMV
`4—
`
`ICP34.5
`
`/ \
`4:
`
`CMV GFP pA
`—>
`
`JS-I 34.5 —47—CMV/U Sll
`
`11/111 TNFapA—
`
`ICP34 5
`
`ICP34 5
`
`
`pA hTNF 0c CMV
`pA mTNFOCCMV
`pA hTNFot USll
`pA mTNFOtUSll
`
`‘—
`
`CMV hTNFoz pA
`CMV mTNFOCpA
`USll hTNFoc pA
`USll mTNFoz pA
`
`—>
`
`ICP47
`
`ICP47
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`(57) Abstract: The present invention provides an oneolytie herpes simplex virus (HSV) comprising aheterologous gene encoding
`an immunomodulatory protein, wherein the heterologous gene is operably linked to al-lSV promoter sequence which is selectively
`active during HSV replication.
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`ONCOLYTIC HERPES VIRUS VECTORS
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`Field of the Invention
`
`The present invention relates to oncolytic herpes virus vectors and the use of the
`
`oncolytic herpes virus vectors in treating cancer.
`
`Background to the Invention
`
`Viruses have been shown to have utility in a variety of applications in
`
`biotechnology and medicine. Each use results from the unique ability of Viruses to enter
`
`cells at high efficiency followed either by viral gene expression and replication and/or
`
`expression of an inserted heterologous gene. Thus, viruses may be used to deliver and
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`express either viral or non—viral genes in cells and so are useful in, for example, gene
`
`therapy and the development of vaccines. Viruses are also useful for selectively killing
`
`tumour cells by lytic replication within infected tumour cells and/or by the action of a
`
`gene delivered to the tumour cells by the virus.
`
`The helpes simplex Virus (HSV) has been suggested to be of use for the
`
`oncolytic treatment of cancer. However, the HSV used in the oncolytic treatment of
`
`cancer must be disabled such that it is no longer pathogenic, i.e. does not replicate in
`
`and kill non-tumour cells, without preventing entry into and killing of tumour cells. A
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`number of disabling mutations to HSV have been identified which still allow the virus
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`to replicate in culture or in actively dividing cells such as tumour cells in viva, but
`
`which prevent significant replication in normal tissue. Such mutations include
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`disruption of the genes encoding lCP34.5, ICP6, and/or thymidinc kinasc. Viruses with
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`mutations to lCP34.5, or lCP34.5 together with mutation of lCP6 have so far shown the
`
`most favourable safety profile. Vimses deleted for only ICP34.5 have been shown to
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`replicate in many tumour cell types in vitro and to selectively replicate in artificially
`
`induced brain tumours in mice while sparing surrounding tissue. Early stage clinical
`
`trials have also shown their safety in man.
`
`The oncolytic treatment of cancer may also include the delivery of gene(s)
`
`enhancing the therapeutic effect. Genes which have been delivered to tumour cells
`
`using oncolytic viruses include GM—CSF under the control of the cytomcgalovirus
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`(CMV) immediate early promoter and IL—4 or IL—10 under the control of the Egr—l
`
`promoter to allow maximal expression of the inserted gene in dividing cells.
`
`Tumour necrosis factor alpha (TNFoc) is a multifunctional cytokine with potent
`
`anti—tumour properties. TNFOL therapy is paiticularly promising in combination with
`
`radiotherapy due to its potent radio sensitizing effects, but high systemic and local
`
`toxicity has limited its clinical use.
`
`Summary of the Invention
`
`The present inventors have shown that a heterologous gene encoding an
`
`immunomodulatory protein can be selectively expressed in cells in which an oncolytic
`
`herpes simplex virus (HSV) replicates, i.e. tumour cells. The inventors have also shown
`
`that by using a promoter that is activated only when the HSV replicates to drive
`
`expression of the heterologous gene, expression of the heterologous gene can be
`
`significantly reduced.
`
`In addition, the onset of heterologous gene expression is delayed.
`
`This was demonstrated using the HSV US 11 promoter, a true late gene HSV promoter,
`
`as an example of apromoter that is selectively active during virus replication and a
`
`TNFoc gene as an example of aheterologous gene. The properties of the HSV
`
`comprising TNFoc under the control of the USE promoter were compared to those of an
`
`HSV comprising TNFoc under the control of the CMV promoter. Toxic effects of
`
`TNFOL expression, which were seen in nude mice when TNFOL expression was driven by
`
`the CMV promoter, were absent when the U31 1promoter was used to drive expression.
`
`However, despite the reduced expression levels, USIl promoter driven TNFoc
`
`surprisingly showed better anti—tumour effects compared to the CMV promoter driven
`
`TNFoc.
`
`Accordingly the invention provides an oncolytic HSV comprising a
`
`heterologous gene encoding an immunomodulatory protein, wherein the heterologous
`
`gene is operably linked to a HSV promoter sequence which is selectively active during
`
`HSV replication. The promoter sequence is typically an HSV late promoter, such as the
`
`U31 1 promoter. The immunomodulatory protein may be a cytokine, such as TNFOL.
`
`The endogenous promoter sequence corresponding to the promoter sequence used to
`
`drive expression of the heterologous gene may be deleted to enhance stability of the
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`HSV. Where the U81 1 promoter is used, deletion of the endogenous U81 1 promoter
`
`sequence may be achieved by deleting the ICP47-encoding gene such that the US 11
`
`promoter is placed under the control of the 0047 promoter. The oncolytic HSV typically
`
`lacks functional lCP34.5—encoding and lCP47—encoding genes.
`
`In one preferred embodiment, the invention provides an oncolytic HSV
`
`comprising a TNFOL gene operably linked to a US llpromoter.
`
`In this embodiment, the
`
`HSV typically lacks functional ICP34.5—encoding and ICP47—encoding genes. The
`
`ICP47—encoding gene is typically deleted such that the endogenous USIl promoter
`
`sequence is removed and the US] 1 gene placed under the control of the (x47 promoter.
`
`The invention also provides:
`
`—
`
`an oncolytic HSV according to the invention for use in a method of treating the
`
`human or animal body by therapy;
`
`—
`
`use of an HSV according to the invention in the manufacture of a medicament
`
`for treating cancer;
`
`—
`
`a method of treating a subj ect having a tumour, the method comprising
`
`administering to the subject a therapeutically effective amount of an HSV according to
`
`the invention;
`
`-
`
`apharmaceutical composition comprising an HSV according to the invention
`
`and a pharmaceutically acceptable carrier or diluent; and
`
`-
`
`products containing an HSV according to the invention and a chemotherapeutic
`
`agent as a combined preposition for simultaneous, separate or sequential use in the
`
`treatment of cancer.
`
`Methods of treating cancer according to the invention may comprise
`
`administering the HSV before, during or after chemotherapy or radiotherapy.
`
`Brief Description of the Drawings
`
`Figure l is a schematic representation of virus vectors used in the Examples.
`
`HSVl strain JSl was isolated by taking a swab from a cold sore of an otherwise healthy
`
`volunteer (Liu et ah, 2002). JSl 34.5-47-pA- has two deletions. The first involves
`
`removal of the coding region of the ICP34.5 gene (nucleotides 124948-125713 based on
`
`the sequence HSVl strain 17+). The second involves a 280bp deletion of ICP47
`
`(nucleotides 145570—145290 based on the sequence of HSVl strain 17+) (Liu et al,
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`2002).
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`.131 34.5—47—pA— was then further mutated to express human or mouse TNFa
`
`under the control of either the CMV promoter or the US llpromoter as illustrated in the
`
`bottom panel.
`
`Figure 2 shows that expression of TNFoc from the U81 lpromoter (B) results in
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`reduced and delayed kinetics of expression as compared to expression from the CMV
`
`promoter (A).
`
`Figure 3 shows that acyclovir has a greater relative effect on expression level
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`when TNFoc is driven by the US] lpromoter than the CMV promoter in Fadu cells (A)
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`and BHK cells (B).
`
`Figure 4 shows the relative expression levels of TNFoc are reduced in non—
`
`permissive cells using the U81 1 promoter (B) as compared to the CMV promoter (A).
`
`The expression levels of TNFoc from the US llpromoter are shown as a percentage of
`
`TNFCX. expression levels from the CMV promoter (C).
`
`Figure 5 shows that similar tumour shrinkage effects of injected tumours were
`
`observed with all tested viruses (A), but that US 11 driven TNFa viruses had a better
`
`systemic anti-tumour effect on non-injected tumours (B).
`
`Figure 6 shows that the U81 1 driven TNFoz vector showed better tumour
`
`shrinkage effect than CMV driven TNFa vector on a Fadu, human head and neck
`
`tumour model.
`
`Figure 7 shows that a skin rash (D) and tumour ulcer (B) were seen on nude
`
`mice injected with CMV driven TNFa vector 29 days after the last of three injections,
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`while no such toxicity was seen on nude mice injected with US 11 driven TNFa vector
`
`(A and C).
`
`Brief Description of the Seguences
`
`SEQ ID NO: 1 is the sequence of the HSV USl lpromoter.
`
`SEQ 1]) NOS: 2 to 5 are the sequences of the primers and oligonucleotides used
`
`in the Examples.
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`Detailed Description of the Invention
`
`A.
`
`Viruses
`
`A herpes simplex Virus (HSV) of the invention is oncolytic. Oncolytic viruses
`
`infect and replicate in tumour cells, subsequently killing the tumour cells. Thus, an
`
`HSV of the invention is replication competent. Preferably, the l-lSV is selectively
`
`replication competent in tumour cells. This means that either it replicates in tumour
`
`cells and not in non—tumour cells, or that it replicates more effectively in tumour cells
`
`than in non—tumour cells. The replicative ability of an HSV can be determined using
`
`techniques well known in the art.
`
`An HSV of the invention is capable of efficiently infecting and replicating in
`
`target tumour cells, such as human tumour cells, but is incapable of efficiently infecting
`
`and replicating in neurons, such as human neurons. Selective oncolytic activity is
`
`typically the result of a mutation to one or more Viral genes that inhibit replication in
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`neuronal tissue but not in dividing tumour cells. For example, the ICP34.5, ICP6 and/or
`
`thymidine kinase encoding genes may be mutated such that no functional ICP34.5,
`
`ICP6 and/or thymidine kinase is produced by the Virus.
`
`In one preferred embodiment, selective oncolytic activity is achieved by
`
`mutating the ICP34.5—encoding genes. Suitable mutations of the ICP34.5—encoding
`
`genes are described in Chou et a]. (1990) Science 250:1262—1266, Maclean er a]. (1991)
`
`J. Gen. Virol. 722631—639 and Liu et al. (2003) Gene Therapy 102292—303 which are
`
`incorporated herein by reference. The genes encoding ICP6 and/or thymidine kinase
`
`may additionally be inactivated, as may other genes if such inactivation does not
`
`significantly reduce the oncolytic effect, or if such deletion enhances oncolytic or other
`
`desirable properties of the Virus.
`
`Placing a heterologous gene in an HSV which is selectively replication
`
`competent in tumour cells under the control of apromoter sequence which allows
`
`selective expression of the heterologous gene when the HSV is actively replicating
`
`results in the heterologous gene being expressed selectively in tumour cells. Selective
`
`expression in tumour cells occurs where expression is achieved predominantly in
`
`tumour cells compared to other cells in the body, such as healthy cells, e.g. neurons.
`
`Thus, selective expression occurs where expression is maximal in tumour cells and
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`minimal or non—existent in other cells of the body. Accordingly, in one aspect, the
`
`invention provides benefits of localised heterologous gene expression combined with
`
`the anti—tumour effect provided by oncolytic virus replication.
`
`An HSV of the invention may comprise one or more further mutations to
`
`improve the anti-tumour effects of the HSV. For example, ICP47 usually functions to
`
`block antigen presentation in HSV—infected cells. Mutation of the ICP47—encoding gene
`
`is, therefore, desirable because it results in 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.
`
`In addition, deletion of the ICP47—encoding gene can lead to the
`
`increased expression of the U81 1 gene such that replication in tumour cells is enhanced
`
`(see Liu et al, 2003).
`
`In another embodiment of the invention, the HSV may contain a further
`
`mutation which enhances replication of the HSV in tumour cells.
`
`In this embodiment,
`
`the resulting enhancement of a viral replication in tumour cells also enhances the level
`
`of heterologous gene expression.
`
`Replication in tumour cells of an oncolytic HSV, in particular an HSV lacking
`
`functional ICP34.5 encoding genes, may be enhanced by mutating the HSV such that
`
`the U81 l gene is expressed at early time points during infection. The USl
`
`l gene is an
`
`HSV late gene that is normally selectively expressed during HSV replication.
`
`It has
`
`been shown that placing the US 11 gene under the control of an immediate early gene
`
`promoter which allows expression of U311 before HSV replication occurs enhances
`
`replication of an HSV lacking functional ICP34.5-encoding genes. Accordingly, in one
`
`embodiment the HSV of the invention comprises a U31 1 gene operably linked to a
`
`promoter, wherein the activity of the promoter is not dependent on viral replication.
`
`Suitable promoters include HSV early (E) promoters, HSV immediate early (1E)
`
`promoters and non—HS V promoters which are active in mammalian, such as human,
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`tumour cells. The promoter may, for example, be derived from a eukaryotic promoter
`
`sequence. For example, the promoter may be derived from the genome of a mammal,
`
`such as a human. The eukaryotic promoter may be a promoter that functions in a
`
`ubiquitous manner (such as promoters of B-actin or tubulin) or, alternatively, in a cell—
`
`speeifie, such as tumour—specific, manner. Viral promoters may also be used, for
`
`example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR)
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`promoter or the human or mouse cytomegalovirus (CMV) IE promoter. HSV early and
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`immediate early promoters are well known in the art. These include, for example, the
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`000, 064, 0622, all and 0(47 IE promoters and the tk, gD, ICP36, ICP8 and ICP6 E
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`promoters.
`
`The various genes referred to above may be rendered functionally inactive by
`
`several techniques well known in the art. For example, they may be rendered
`
`functionally inactive by deletion(s), substitution(s) or insertion(s). As used herein, the
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`term "gene" is intended to mean the nucleotide sequences encoding a protein,
`
`i.e. the
`
`coding sequences of the gene. The various genes referred to above may be rendered
`
`non—functional by mutating the gene itself or the control sequences flanking the gene,
`
`for example the promoter sequence. Deletions may remove one or more portions of the
`
`gene, the entire gene or the entire gene and all or some of the control sequences. For
`
`example, deletion of only one nucleotide within the gene may be made, resulting in a
`
`frame shift. However, preferably a larger deletion(s) is (are) made, for example at least
`
`about 25%, more preferably at least about 50% of the total coding and non—coding
`
`sequence (or alternatively,
`
`in absolute terms, at least about 10 nucleotides, at least about
`
`100 nucleotides or at least about 1000 nucleotides.
`
`In one preferred embodiment,
`
`the
`
`gene(s) being rendered functionally inactive is (are) deleted. For example, the entire
`
`gene and some of the flanking sequences may be removed from the virus. Where two
`
`or more copies of the gene are present in the viral genome both copies of the gene are
`
`rendered functionally inactive.
`
`A gene may be inactivated by substituting other sequences, for example by
`
`substituting all or part of the endogenous gene with aheterologous gene and optionally
`
`apromoter sequence. Where no promoter sequence is substituted, the heterologous
`
`gene may be inserted such that it is controlled by the promoter of the gene being
`
`rendered non—functional.
`
`In one preferred embodiment,
`
`the ICP34.5 encoding—genes are rendered non-I
`
`functional by the insertion of a heterologous gene and a promoter sequence operably
`
`linked thereto into each the ICP34.5—encoding gene loci.
`
`Mutations may be made in the herpes simplex viruses by any suitable method.
`
`Homologous recombination methods, in particular, are well known to those skilled in
`
`the art. For example, HSV genomic DNA may be transfected together with a vector,
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`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, for example lacZ or green fluorescent protein (GFP),
`
`for screening recombinant viruses, for example, B—galactosidase activity or
`
`fluorescence.
`
`The herpes simplex virus of the invention may be derived from a‘HSVl or
`
`HSV2 strain, or from a derivative thereof. Derivatives include inter—type recombinants
`
`containing DNA from HSVl and HSV2 strains. Such inter—type recombinants are
`
`described in the art, for example in Thompson et a]. (1998) Virus Genes l(3):275-286
`
`and Meignier er al. (1988) Infect. Dis. 159:602—614, the disclosures of which are
`
`incorporated herein by reference. Derivatives may have at least about 70% sequence
`
`homology to either the HSVl or HSV2 genomes, for example, at least about 80%, such
`
`as at least about 90%, about 95% or about 98%. Thus, a derivative may have at least
`
`about 70% sequence identity to either the HSVl or HSV2 genome, at least about 80%,
`
`about 90%, about 95% or about 98% identity.
`
`Sequence homologies and identities may be determined by any suitable method.
`
`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 a]. (1984)
`
`Nucleic Acids Research 12, p387-395). 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. 36:290—300; Altschul et al.
`
`(1990) J. Mol. Biol. Ziz403—10.
`
`Software for performing BLAST analyses is publicly available through the
`
`National Centre for Biotechnology Information ghttp://www.ncbi.nlm.nih.gov/ 2. 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 in a
`
`database sequence. T is referred to as the neighbourhood word score threshold
`
`(Altschul et 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.
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`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 aword length (W) of 11, the
`
`BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sd.
`
`USA &: 10915—10919) alignments (B) of 50, expectation (E) of 10, M25, N24, and a
`
`comparison of both strands.
`
`The BLAST algorithm perfo Tins a statistical analysis of the similarity between
`
`two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sd. USA fl:
`
`5873—5787. 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 0r 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 about 5, such as 1, 2 or 3, to about 10, aboth
`
`25, about 50 or about 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
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`extension at either or both ends.
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`An HSV of the invention may be based on any HSVl or HSV2 strain. HSVl
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`strains in common usage include strain 17+, strain F and strain KOS. Available HSV2
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`strains include HG52, MS, strain 333, curtis, strain 12, strain G and strain 186. An
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`HSV of the invention may, in one embodiment, be derived from an HSVl or HSV2
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`clinical isolate, i.e. a strain isolated from apatient, for example from a cold sore which
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`may be a frequently reactivating cold sore.
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`In one embodiment, a virus of the invention is derived from the clinical isolate
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`JSl which was deposited at the European Collection of Cell Cultures (ECACC) CAMR,
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`Salisbury, Wiltshire, SP4 ()JG, United Kingdom, on 2 January 2001 under accession
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`number 01010209. Thus, a virus of the invention may be a JSl virus which lacks a
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`functional ICP34.5 encoding gene. Li addition, such a virus may contain any other
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`mutation or combination of mutations as mentioned herein.
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`In one embodiment, an HSV of the invention comprises one or more
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`heterologous gene wherein at least one of the heterologous genes encodes an
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`immunomodulatory protein, such as TNFoc. At least the heterologous gene encoding an
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`immunomodulatory protein is operably linked to a promoter sequence which is
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`selectively active during HSV replication. Viruses of the invention may thus be used to
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`deliver one or more heterologous genes to a tumour cell in viva such that the
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`heterologous gene is expressed when the virus replicates.
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`In one embodiment, the
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`heterologous gene is expressed in the tumour cells at a relatively low level, particularly
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`when compared to the level of expression observed when the heterologous gene is
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`placed under the control of apromoter, such as the CMV promoter, which does not
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`depend on viral replication.
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`The promoter sequence which is selectively active during HSV replication is
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`generally a HSV promoter sequence. HSV genes can be divided into distinct temporal
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`classes that are regulated in a cascade fashion. Five immediate early (IE) genes, also
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`known as 61 genes, are transcribed first. The proteins encoded by the IE genes activate
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`the early (E) genes, also known as [3 genes. The E genes encode the viral DNA
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`replicative machinery. Finally, expression of the late genes occurs. Expression of the
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`leaky late (71) genes is induced prior to viral replication with expression of these genes
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`increasing when viral replication begins. The true late (72) genes are activated only
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`after DNA replication has commenced.
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`hi one embodiment of the invention, the promoter sequence is an HSV late
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`promoter. Suitable HSV true late (1/2) promoters include the USl 1, UL44 (glycoprotein
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`C), UL38, UL4, UL9.5, UL14, UL20.5, UL25, UL27.5, UL31, UL32, UL35, UL49.5,
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`UL56 and U82 promoters. Suitable HSV leaky late (71) promoters include the 'yl34.5,
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`UL8.5, UL18 (VP23), UL19 (VPS), UL21, UL34, UL37, UL41 (vhs), UL47 (VP13/14),
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`UL55, US8 (gE) and USlO promoters. These promoters are well known in the art. See,
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`for Example, Chapter 72 of Herpes Simplex Viruses and their Replication. Bernard
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`Roizman and David M. Knipe. Fields Virology 2001. eds David M. Knipe, Peter M.
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`Howley er al. Lippicott Williams & Wilkins which is incorporated herein by reference.
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`Expression of the heterologous gene is generally more restricted when a true late (72)
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`promoter is used.
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`It has been shown that the structural features of a late promoter that are
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`sufficient to restrict gene transcription to actively replicating HSV genomes are a TATA
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`box and an RNA cap site. See Kibler et a]. (1991) J. Virol. 65:6749—6760, Hama et al.
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`(1988) Genes Dev. 2:40—53, Flanagan er a]. (1991) J. Virol. 65:769—786 and Johnson &
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`Everett (1986) Nucl. Acid. Res. 14:8247—8264 the disclosures of which are incorporated
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`herein in their entirety.
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`An HSV early promoter may be converted into a promoter that is active only
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`during viral replication by deleting regulatory sequences distal to the TATA box
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`(Johnson & Everett (1986)). Accordingly, the promoter which is selectively active
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`during HSV replication included in an HSV vector of the invention may comprise the
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`TATA box and RNA cap site from an HSV leaky late promoter, an HSV true late
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`promotcr or an HSV early promoter. Where sequences from an early promoter are used,
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`the control sequences distal to the TATA box are absent. Thus, the promoter may
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`consist of, or consist essentially of, the TATA box and RNA cap site of any HSV
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`promoter.
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`HSV promoters are well known in the art and the skilled person would readily
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`be able to determine the location of the TATA box and RNA cap site of each promoter.
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`By way of example the sequence of the US 11 promoter is shown in SEQ ID NO: 1 and
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`the minimal sequences required for promoter activity are indicated.
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`hi a further aspect, the endogenous sequence corresponding to the HSV
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`promoter sequence used to drive expression of thc hctcrologous gene is deleted from the
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`HSV. Alternatively, the heterologous gene may be placed under the control of the
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`endogenous promoter sequence. Homologous recombination between the endogenous
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`promoter sequence in the virus and the inserted promoter sequence used to drive
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`heterologous gene expression is thus prevented. This enhances the stability of the HSV.
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`The endogenous promoter sequence is a promoter sequence in its naturally occurring
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`position in the HSV genome.
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`In one preferred embodiment, the promoter sequence used to drive expression of
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`the heterologous gene is the U31 1promoter. The US] l promoter may be deleted by
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`removing the ICP47—encoding gene. This has the added advantage of placing the U31 1
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`gene under the control of the 0047 promoter such that replication of the HSV in tumour
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`cells is enhanced.
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`The heterologous gene typically encodes an immunomodulatory protein. The
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`immunomodulatory protein may be any gene encoding a protein that is capable of
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`modulating an immune response. The protein capable of modulating an immune
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`response may be a cytokine, such as GM—CSF, TNFOC, an interleukin (for example
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`lLlZ), a chemokine such as RANTES or a macrophage inflammatory protein (for
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`example MIP—3) or another immunomodulatory molecule such as B7.l, B7.2 or CD4OL.
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`The protein capable of causing cell to cell fusion may also be capable of modulating an
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`immune response. For example, GALV is capable of modulating an immune response.
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`Multiple heterologous genes can be accommodated in the herpes virus genome.
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`Therefore, a virus of the invention may comprise two or more heterologous genes which
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`may be the same or different, for example from 2 to 3, 4 or 5 therapeutic genes. More
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`than one gene and associated promoter sequences could be introduced into a particular
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`HSV strain either at a single site or at multiple sites in the virus genome.
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`The additional heterologous genes typically encode therapeutic proteins which
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`enhance the anti—tumour activity of the virus. For example, the therapeutic protein may
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`be a second or further immunomodulatory protein, a fusogenic or cell death promoting
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`protein or pro—drug activating enzyme. The heterologous gene may be any allelic
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`variant of a wild-type gene, or it may be a mutant gene.
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`The prodrug activating protein may be a cytosine deaminase enzyme. Cytosine
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`deaminase genes are capable of converting the inactive prodrug 5—fluorocytosine to the
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`active drug S—flurouracil. Various cytosine deaminase genes are available including
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`those of bacterial origin and of yeast origin. A second gcnc, typically a gene encoding
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`a second enzyme, may be used to enhance the prodrug conversion activity of the
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`cytosine deaminase gene. For example, the second gene may encode a uracil
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`phosphoribosyltransferase.
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`Any suitable fusogenic gene encoding a protein capable of causing cell to cell
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`fusion and hence cell death may be used. Preferably the protein capable of causing cell
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`to cell fusion is selected from a modified retroviral envelope glycoprotein, such as an
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`envelope glycoprotein derived from gibbon ape leukaemia virus (GALV) or human
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`endogenous retrovirus W, a fusogenic F or H protein from measles virus and the
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`vesicular stomatitis virus Gprotein. More preferably, the protein capable of causing
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`cell to cell fusion is a GALV fusogenic glycoprotein.
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`Controlling expression of aheterologous gene is of special benefit when the
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`heterologous gene is potentially harmful. For example, the heterologous gene may have
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`toxic effects when expressed at ahigh level or when expressed in non—target cells, such
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`as non—tumour cells. Thus, in one embodiment, the oncolytic HSV vector of the
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`invention provides a vector for the controlled expression of a heterologous gene with
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`potentially toxic properties. Selectively expressing such a heterologous gene in tumour
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`cells and/or reducing the spread of the heterologous gene products from the tumour cells
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`has the advantage of reducing side effects associated with heterologous gene expression.
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`In one of the preferred embodiments of the invention, one or more of the
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`heterologous gene may encode aprotein(s) that has