™,
`mY
`
`—
`
`WIPO
`WORLD
`INTELLECTUAL PROPERTY
`ORGANIZATION
`
`DOCUMENT MADE AVAILABLE UNDER THE
`PATENT COOPERATION TREATY (PCT)
`International application number:
`PCT/GB2017/050038
`
`International filing date:
`
`09 January 2017 (09.01.2017)
`
`Documenttype:
`
`Documentdetails:
`
`Certified copy of priority document
`
`Country/Office:
`Number:
`Filing date:
`
`GB
`1600380.8
`08 January 2016 (08.01.2016)
`
`Date of receipt at the International Bureau:
`
`18 January 2017 (18.01.2017)
`
`Remark: Priority document submitted or transmitted to the International Bureau in compliance with Rule
`17.1(a),(b) or (b-bis)
`
`34, chemin des Colombettes
`1211 Geneva 20, Switzerland
`
`www.wipo.int
`
`Page 1 of 35
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`TRANSGENE/BIOINVENT
`EXHIBIT 1008
`
`TRANSGENE/BIOINVENT
`EXHIBIT 1008
`
`Page 1 of 35
`
`

`

`PCT/GB2017/050038
`
`Concept House
`Cardiff Road
`Newport
`South Wales
`NP10 8QQ
`
`I, the undersigned, being an officer duly authorised in accordance with Section 74(1) and (4)
`of the Deregulation & Contracting Out Act 1994, to sign andissue certificates on behalf of
`the Comptroller-General, hereby certify that annexed hereto is a true copy of the documents
`as stored electronically on the Patents Electronic Case file System in connection with patent
`application
`GB1600380.8
`filed on 8th January 2016
`
`The Patents Electronic Case-file System is compliant with British Standard BS10008 -
`Evidential weight and legal admissibility of information stored electronically and ISO15801 -
`Electronic imaging — information stored electronically, recommendations for trustworthiness
`and reliability.
`
`In accordance with the Patents (Companies Re-registration) Rules 1982, if a company named
`in this certificate and any accompanying documents hasre-registered under the Companies
`Act 1980 with the same nameas that with whichit was registered immediately before re-
`registration save for the substitution as, or inclusionas, the last part of the name of the words
`"public limited company"or their equivalents in Welsh, references to the name of the
`companyin this certificate and any accompanying documents shall be treated as references to
`the name with whichit is so re-registered.
`
`In accordance with the rules, the words "public limited company" may be replaced by p.l.c.,
`ple, P.L.C. or PLC.
`
`Re-registration under the Companies Act does not constitute a new legal entity but merely
`subjects the companyto certain additional companylaw rules.
`
`Signed
`
`Aj
`
`Dated
`
`12th January 2017
`
`Page 2 of 35
`UK Intellectual Property Office is an operating name of the Patent Office
`
`Page 2 of 35
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`

`

`1200178709
`
`08/01/2016 0.00 NONE
`
`Concept House
`Cardiff Road
`Newport
`South Wales
`NP10 8QQ
`
`N406714GB
`
`Replimune Limited
`The Magdalen Centre Oxford Science Park
`Robert Robinson Avenue
`Oxford OX4 4GA
`United Kingdom
`
`116
`
`275280
`
`01
`
`
`Intellectual
`Property
`Office
`
`Patents Form 1
`Patents Act 1977 (Rule 72)
`
`Requestfor grant of a patent
`
`Application number
`Your reference
`
`GB 1600380.8
`
`2. Fullname, address and postcode of the applicant or of
`each applicant
`
`Patents ADP number(ifyou knowit}
`
`3. Title of the invention
`
`4. Nameof your agent(ifyou have one)
`“Address for service" to which all correspondence should
`be sent. This may be in the European Economic area or
`Channel Islands (see warning note below)
`(including the postcode)
`
`Patents ADP number (if you knowit}
`
`5. Priority declaration: Are you claiming priority from one or
`more earlier-filed patent applications? If so, please give
`details of the application(s)
`
`MODIFIED VIRUS
`
`J A Kemp
`JAKemp
`14 South Square
`Gray's Inn
`London WC1R 5JJ
`Greater London
`United Kingdom
`
`10645901001
`
`Country
`
`Application number
`
`Date offiling
`
`PDASAccess Code
`
`Numberofearlier UK
`application
`
`Dateoffiling
`(day /month /year)
`
`6. Divisionals etc: Is this application a divisional application,
`or being made following resolution of an entitlement
`dispute about an earlier application. If so, please give the
`application number andfiling date of the earlier
`application
`
`7.
`
`Inventorship: (Inventors must be individuals not
`companies)
`
`Are all the applicants named abovealso inventors?
`
`8. Are you paying the application fee with this form?
`
`No
`
`No
`
`(REV DEC07)
`Intellectual Property Office is an operating name of the Patent Office
`
`Page 3 of 35
`
`Patents Form 1(e)
`https :/Avww.gov.uk/ipo
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`Page 3 of 35
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`

`

`Patents Form 1
`
`9. Accompanying documents: please enter the number of
`pages of each item accompanyingthis form.
`
`Continuation sheets of this form
`
`Description:
`
`22
`
`Claim(s):
`
`Abstract:
`
`Drawing/s):
`
`6
`
`1
`
`2
`
`If you are not filing a description, please give details of
`the previous application you are going to rely upon
`
`Country
`
`Application number
`
`Date offiling
`
`PDASAccess Code
`
`10. If you are alsofiling any of the following, state how many
`against each item.
`
`Priority documents:
`
`Statement of inventorship and right to grant of a patent
`(Patents Form 7):
`
`Requestfor search (Patents Form 9A):
`
`Request for a substantive examination (Patents Form 10):
`
`0
`
`0
`
`0
`
`0
`
`11. IAWe request the grant of a patent on the basis of this application.
`
`Any other documents(please specify): Sequence Listing
`
`Signature: Subject: Pamela Tuxworth 23470; Issuer:
`European Patent Office, European Patent
`Office CA G2
`
`Date:
`
`08 Jan 2016
`
`12. Name, e-mail address, telephone, fax and/or mobile
`number,if any, of a contact point for the applicant
`
`TUXWORTH, Ms. Pamela Mary
`Email: mail@jakemp.com
`Telephone: +44 20 3077 8600
`Fax: +44 20 7430 1000
`
`Warning
`After an application for a patent has been filed, the Comptroller will consider whether publication or communication of the
`invention should be prohibited or restricted under section 22 of the Patents Act 1977. You will be informedif it is necessary to
`prohibit or restrict your invention in this way. Furthermore, if you are resident in the United Kingdom and your application contains
`information whichrelates to military technology, or would be prejudicial to national security or the safety of the public, section 23 of
`the Patents Act 1977 prohibits you from applying for a patent abroad withoutfirst getting written permission from the Office unless
`an application has been filed at least 6 weeks beforehand in the United Kingdom for a patent for the same invention and either no
`direction prohibiting publication or communication has been given, or any such direction has been revoked. Until such time or until
`the revocation of any direction, for any such application the address for service referred to at part 4 above must bein the United
`Kingdom.
`
`Although you may have an addressfor service in the Channel Islands, any agent instructed to act for you mustreside or have a
`place of business in the European Economic Area or Isle of Man.
`
`(REV DEC07)
`
`Page 4 of 35
`
`Patents Form 1(e)
`
`Page 4 of 35
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`

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`MODIFIED VIRUS
`
`Field of the Invention
`
`The invention relates to an oncolytic immunotherapeutic agent and to the use of the
`
`oncolytic immunotherapeutic agent in treating cancer.
`
`Backgroundto the Invention
`
`Viruses have a unique ability to enter cells at high efficiency. After entry into cells,
`
`viral genes are expressed andthe virus replicates. This usually results in the death of the
`
`10
`
`infected cell and the release of the antigenic components ofthe cell as the cell rupturesasit
`
`dies. As aresult, virus mediated cell death tends to result in an immuneresponseto these
`
`cellular components, including both those derived from the host cell and those encoded by
`
`or incorporated into the virusitself. The immune responseis also enhanced dueto the
`
`recognition by the host of so called damage associated molecular patterns (DAMPs) which
`
`15
`
`aid in the activation of the immuneresponse.
`
`Viruses also engage with various mediators of the innate immuneresponse as part
`
`of the host response to the recognition of a viral infection through, for example,toll-like
`
`receptors, cGAS/STINGsignalling and the recognition of pathogen associated molecular
`
`patterns (PAMPs)resulting in the activation of interferon responses and inflammation
`
`20
`
`which are also immunogenic signals to the host. These immuneresponses mayresult in
`
`the immunogenic benefit to cancer patients such that immuneresponses to tumor antigens
`
`provide a systemic overall benefit resulting in the treatment of tumors which have not been
`
`infected with the virus, including micro-metastatic disease, and providing vaccination
`
`against relapse.
`
`25
`
`The combined direct (‘oncolytic’) effects of the virus, and immune responses
`
`against tumor antigens (including non-self ‘neo-antigens’, i.e. derived from the particular
`
`mutated genes in individual tumors) is termed ‘oncolytic immunotherapy’.
`
`Viruses may also be usedas delivery vehicles (‘vectors’) to express heterologous
`
`genesinserted into the viral genomein infected cells. These properties make viruses
`
`30
`
`useful for a variety of biotechnology and medical applications. For example, viruses
`
`expressing heterologous therapeutic genes may be used for gene therapy.
`
`In the context of
`
`oncolytic immunotherapy, delivered genes may include those encoding specific tumor
`
`antigens, genes intended to induce immuneresponses or increase the immunogenicity of
`1
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`Page 5 of 35
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`antigens released following virus replication and cell death, genes intended to shape the
`
`immune response whichis generated, genes to increase the general immuneactivation
`
`status of the tumor, or genes to increase the direct oncolytic properties (1.e. cytotoxic
`
`effects) of the virus. Importantly, viruses havethe ability to deliver encoded molecules
`
`which are intendedto help to initiate, enhance or shape the systemic anti-tumor immune
`
`responsedirectly and selectively to tumors, which may have benefits of e.g. reduced
`
`toxicity or of focusing beneficial effects on tumors (including those not infected by the
`
`virus) rather than off-target effects on normal(i.e. non-cancerous) tissues as compared to
`
`the systemic administration of these same molecules or systemic administration of other
`
`10
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`molecules targeting the same pathways.
`
`It has been demonstrated that a numberofviruses including, for example, herpes
`
`simplex virus (HSV) haveutility in the oncolytic treatment of cancer. HSV for use in the
`
`oncolytic treatment of cancer must be disabled such thatit is no longer pathogenic, but can
`
`still enter into and kill tumor cells. A numberof disabling mutations to HSV, including
`
`15
`
`disruption of the genes encoding ICP34.5, ICP6, and/or thymidine kinase, have been
`
`identified which do not prevent the virus from replicating in culture or in tumortissue in
`
`vivo, but which prevent significant replication in normal tissue. HSVs in which only the
`
`ICP34.5 genes have been disrupted replicate in many tumorcell types in vitro, and
`
`replicate selectively in tumortissue, but not in surroundingtissue, in mouse tumor models.
`
`20
`
`Clinical trials of ICP34.5 deleted, or ICP34.5 and ICP6 deleted, HSV have also shown
`
`safety and selective replication in tumortissue in humans.
`
`Asdiscussed above, an oncolytic virus, including HSV, mayalso be usedto deliver
`
`a therapeutic gene in the treatment of cancer. An ICP34.5 deleted virus ofthis type
`
`additionally deleted for ICP47 and encoding a heterologous gene for GM-CSFhasalso
`
`25
`
`been tested in clinical trials, including a phase 3 trial in melanomain which safety and
`
`efficacy in man was shown. Thetrial data demonstrated that tumor responses could be
`
`seen in injected tumors, and to a lesser extent in uninjected tumors. Responses tended to
`
`be highly durable (months-years), and a survival benefit appeared to be achieved in
`
`responding patients. Each of these indicated engagement of the immunesystem in the
`
`30
`
`treatment of cancer in addition to the direct oncolytic effect. However, this and other data
`
`with oncolytic viruses generally showedthat not all tumors respond to treatment and notall
`
`patients achieve a survival advantage. Asa result, improvementsto the art of oncolytic
`
`2
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`therapy are clearly needed. These may serve to increase the direct oncolytic effects of
`
`therapy, the anti-tumor immunestimulating effects of the therapy, or both of these effects
`
`together.
`
`Recently it has been shown that oncolytic immunotherapy can result in additive or
`
`synergistic therapeutic effects in conjunction with immune checkpoint blockade(i.e.
`
`inhibition or ‘antagonism’ of immune checkpoint pathways). Checkpoint blockadeis
`
`intended to block host immuneinhibitory mechanisms which usually serve to prevent the
`
`occurrence of auto-immunity. However, in cancer patients these mechanisms can also
`
`serve to inhibit the induction of or block the potentially beneficial effects of any immune
`
`10
`
`responses induced to tumors. Alternatively, immune responses may notbe fully
`
`potentiated due to a lack of activation or lack of full activation of immunepotentiating
`
`pathways. Therefore, drugs which alleviate these blocks (inhibit “immune co-inhibitory
`
`pathways’) or stimulate immune potentiating pathways (i.e. which activate, or are
`
`‘agonists’ of “immuneco-stimulatory pathways’) are attractive for testing and developing
`
`15
`
`cancer treatments. Targets for such approved or experimental drugs include CTLA-4, PD-
`
`1, PD-LL, LAG-3, TIM-3, VISTA, CSFIR, IDO, CEACAMI, GITR, 4-1-BB, KIR,
`
`SLAMP?, OX40, CD40, ICOS or CD47.
`
`For many of these approaches targeting immune co-inhibitory or co-inhibitory
`
`pathways to be successful, pre-existing immuneresponsesto tumors are needed, so that a
`
`20
`
`pre-existing immuneresponse can be potentiated or a block to an anti-tumor immune
`
`response can be relieved. The presence of an inflamed tumor micro-environment, which is
`
`indicative of such an ongoing response, is also needed. Pre-existing immune responses to
`
`tumor neo-antigens appearto be particularly important for the activity of immune
`
`checkpoint blockade and related drugs. Only some patients may have an ongoing immune
`
`25
`
`response to tumorantigens including neoantigens and/or an inflamed tumor
`
`microenvironment, both of which are required for the optimal activity of these drugs.
`
`Therefore, oncolytic agents which can induce immuneresponses to tumorantigens,
`
`including neoantigens, and/or which can induce an inflamed tumor microenvironment are
`
`attractive for use in combination with immunecheckpoint blockade and immune
`
`30
`
`potentiating drugs. This likely explains the promising combined anti-tumoreffects of
`
`oncolytic agents and immunecheckpoint blockade in mice and humansthat haveso far
`
`been observed.
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`Page 7 of 35
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`The indoleamine 2,3-dioxygenase (IDO) pathway contributes to tumor-induced
`
`tolerance by creating a tolerogenic environmentin the tumorand the tumor-draining lymph
`
`nodes, both by direct suppression of T cells and enhancement of local regulatory T cell
`
`(Treg)-mediated immunosuppression.
`
`IDOcatalyses the rate-limiting step of tryptophan
`
`degradation along the kynurenine pathway, and both the reduction in local tryptophan
`
`concentration and the production of immunomodulatory tryptophan metabolites contribute
`
`to the immunosuppressive effects of IDO. IDO is chronically activated in many cancer
`
`patients with IDOactivation correlating with more extensive disease. It can also function
`
`as an antagonist to other activators of antitumor immunity. Therefore, inhibitors of the
`
`10
`
`IDO pathwayare being developed as anticancer agents, particularly in combination with
`
`checkpoint blockade agents such as those which target CTLA-4, PD-1 or PDL-1.
`
`The above discussion demonstrates thatthere is still much scope for improving
`
`oncolytic agents and cancertherapiesutilising oncolytic agents.
`
`15
`
`Summary of the Invention
`
`The invention provides oncolytic viruses expressing a fusogenic protein andat least
`
`one immunestimulatory molecule. Oncolytic viruses of the invention provide improved
`
`treatment of cancer through improved direct oncolytic effects, viral replication and spread
`
`through tumors, mediated by the fusogenic protein, which (i) increases the amount of
`
`20
`
`tumor antigens, including neoantigens, which are released for the induction of an anti-
`
`tumor immuneresponse; and (ii) enhances the expression of the virus-encoded immune
`
`stimulatory molecule(s). Expression of the immunestimulatory molecule(s) further
`
`enhancesand potentiates the anti-tumor immuneeffect. Anti-tumorefficacy is improved
`
`whenan oncolytic virus of the invention is used as a single agent and also whenthe virusis
`
`25
`
`used in combination with other anti-cancer modalities, including chemotherapy, treatment
`
`with targeted agents, radiation, immune checkpoint blockade and/or immune potentiating
`
`drugs.
`
`Accordingly, the present invention provides an oncolytic virus comprising: G)} a
`
`fusogenic protein-encoding gene; and Gi} an immune stimulatory molecule-encoding
`
`30
`
`gerne. The virus may encode more than one fusogenic protein and/or more than one
`
`immune stimulatory molecule.
`
`The fusogenic protein is preferably the glycoprotein from gibbon ape leukemia
`
`virus (GALV) and has the R transmembrane peptide mutated or removed (GALV-R-}. The
`4
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`~
`immune stimulatory molecule ts preferably GM-CSFand/or an agonist of an immune co-
`
`stimulatory pathwaysuch as GITRL, 4-1-BBL, OX40L, ICOSL or CD40L or a modified
`
`version of any thereof. Examples of modified versions include agonists of a co-stimulatory
`
`pathway that are secreted rather than being membrane bound, and/or agonists modified
`
`such that multimers of the protein are formed,
`
`The virus may be a modified clinical isolate, such as a modified clinical isolate of a
`
`virus, wherein the clinical isolate kills two or more tumorcell lines more rapidly and/orat
`
`a lower dose in vitro than one or more reference clinical isolates of the same species of
`
`Virus.
`
`10
`
`The virus is preferably a herpes simplex virus (HSV), such as HSV1. The HSV
`
`typically does not express functional 1CP34.5 and/or functional ICP47 and/or expresses the
`
`US11 gene as an immediate early gene.
`
`The invention also provides:
`
`15
`
`20
`
`-
`
`-
`
`-
`
`-
`
`-
`
`apharmaceutical composition comprising a virus of the invention and a
`
`pharmaceutically acceptable carrier or diluent;
`
`the virus of the invention for use in a method oftreating the human or animal bady
`
`bytherapy;
`
`the virus of the invention for use in a method of treating cancer, wherein the
`
`method optionally comprises administering afurther anti-cancer agent;
`
`aproduct of manufacture comprising a virus of the invention in a sterile vial,
`
`ampoule or syringe:
`
`a method of treating cancer, which comprises administering a therapeutically
`
`effective amount of a virus or a pharmaceutical composition of the invention to a
`
`patient in need thereof, wherein the methad optionally comprises administering a
`
`25
`
`further anti-cancer agent;
`
`-
`
`use of a virus of the invention in the manufacture of a medicament for use ina
`
`method of treating cancer, wherein the method optionally comprises administering
`
`a further anti-cancer agent:
`
`-
`
`amethod oftreating cancer, which comprises administering a therapeutically
`
`30
`
`effective amount of an oncolytic virus, an inhibitor of the indoleamine 2,3-
`
`dioxygenase (IDO) pathway and a further antagonist of an immune co-inhibitory
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`Page 9 of 35
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`pathway, or an agonist of an immuneco-stimulatory pathwayto a patient in need
`
`thereof.
`
`Brief Description of the Figures
`
`Figure 1 depicts the structure of an exemplary virus of the invention that comprises
`
`a gene encoding GALV-R-and a gene encoding GM-CSFinserted into the ICP34.5 gene
`
`locus, and in which the ICP47 geneis deleted such that the US11 geneis under the control
`
`of the ICP47 immediate early promoter.
`
`Figure 2 depicts the structure of an exemplary virus of the invention that comprises
`
`10
`
`a gene encoding GALV-R-, a gene encoding GM-CSFand a gene encoding CD40L.
`
`Brief Description of the Sequence Listing
`
`SEQ ID NO: 1 is the nucleotide sequence of mouse GM-CSF.
`
`SEQ ID NO: 2 is the nucleotide sequence of a codon optimized version of mouse
`
`15
`
`GM-CSF.
`
`SEQ ID NO: 3 is the nucleotide sequence of human GM-CSF.
`
`SEQ ID NO: 4 is the nucleotide sequence of a codon optimized version of human
`
`GM-CSF.
`
`SEQ ID NO: 5 is the amino acid sequence of mouse GM-CSF.
`
`20
`
`SEQ ID NO: 6 is the amino acid sequence of human GM-CSF.
`
`SEQ ID NO: 7 is the nucleotide sequence of GALV-R-.
`
`SEQ ID NO: 8 is the nucleotide sequence of a codon optimized version of GALV-
`
`SEQ ID NO: 9 is the amino acid sequence of GALV-R-.
`
`25
`
`SEQ ID NO:10 is the nucleotide sequence of a codon optimized version of a
`
`human/mouse hybrid membrane bound version of CD40L.
`
`SEQ ID NO:11 is the amino acid sequence of a human/mouse hybrid membrane
`
`boundversion of CD40L .
`
`SEQ ID NO:12 is the nucleotide sequence of a codon optimized version of a
`
`30
`
`multimeric secreted version of human CD40L.
`
`SEQ ID NO:13 is the amino acid sequence of a multimeric secreted version of
`
`human CD40L.
`
`Page 10 of 35
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`SEQ ID NO: 14 is the nucleotide sequence of a codon optimized version of a
`
`multimeric secreted version of mouse CD40L.
`
`SEQ ID NO:15 is the amino acid sequence of a multimeric secreted version of
`
`mouse CD40L.
`
`SEQ ID NO: 16 is the nucleotide sequence of wild-type human CD40L.
`
`SEQ ID NO:17 is the amino acid sequence of wild-type human CD40L.
`
`SEQ ID NO:18 is the nucleotide sequence of wild-type mouse CD40L.
`
`SEQ ID NO:19 is the aminoacid sequence of wild-type mouse CD40L.
`
`10
`
`Detailed Description of the Invention
`
`Oncolvtic Virus
`
`The virus of the invention is oncolytic. An oncolytic virusis a virus that infects
`
`and replicates in tumorcells, such that the tumorcells are killed. Therefore, the virus of
`
`the invention is replication competent. Preferably, the virus is selectively replication
`
`15
`
`competent in tumortissue. A virusis selectively replication competent in tumortissueif it
`
`replicates more effectively in tumortissue than in non-tumortissue. The ability of a virus
`
`to replicate in different tissue types can be determined using standard techniquesin theart.
`
`Oncolytic effects rely on the virus replicating in and killing initially infected cells,
`
`and progeny virions going on to infect and kill other tumorcells, spreading within the
`
`20
`
`tumor as a result. Thus, the ability of the virus of the invention to effectively kill tumor
`
`cells and spread within tumorsresults in optimal direct anti-tumoreffects. Efficient spread
`
`and virus replication associated lysis of tumor cells also maximises the amount of tumor
`
`antigen released and therefore also maximises the potency of the anti-tumor immune
`
`response induced.
`
`25
`
`The virus of the invention may be any virus which hasthese properties, including a
`
`herpes virus, pox virus, adenovirus, retrovirus, rhabdovirus, paramyxovirus or reovirus, or
`
`any species or strain within these larger groups. Viruses of the tavention may be wild type
`
`G.e. unaltered from the parental virus species}, or with gene disruptions or gene additions.
`
`Which of these is the case will depend on the virus species to be used. Preferably the virus
`
`30
`
`is a species of herpes virus, more preferablya strain of HSV, including strains of HSVI
`
`and HSV2, and is most preferably a strain of HSV1. In particularly preferred embodiments
`
`the virus of the invention is based on a clinical isolate of the virus species to be used. The
`
`Page 11 of 35
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`clinical isolate may have been selected on the basis of tt having particular advantageous
`
`properties for the treatmertt of cancer.
`
`The virus may be a modified clinical isolate, wherein the clinical isolate kills pwo
`
`or more tumor cell lines more rapidly and/or at a lower dose in vitro than one or more
`
`reference clinical isolate of the same species of virus. Typically, the clinical isolate will
`
`kul two or more tumor cell lines within 48 hours, preferably within 24 hours, of infection
`
`at roultiplicities of infection (MODofless than or equal to 0.1. Preferably the clinical
`
`isolate will kill a broad range of tumorcell fines, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or, for
`
`example, all of the following human tumor cell lines: U87MG (glioma), HT29 (colorectal),
`
`10
`
`LNCaP(prostate), MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu (squamouscell
`
`carcinoma), MCF7 (breast), A549 (lung), MIAPACA-2 (pancreas), CAPAN-1(pancreas),
`
`HT1080 (fibrosarcoma).
`
`An HSVofthe invention is capable of replicating selectively in tumors, such as
`
`human tumors. Typically, the HSV replicates efficiently in target tumors but does not
`
`15
`
`replicate efficiently in non-tumortissue. This HSV may comprise one or more mutations
`
`in one or moreviral genes that inhibit replication in normaltissue butstill allow replication
`
`in tumors. The mutation may, for example, be a mutation that prevents the expression of
`
`functional ICP34.5, ICP6 and/or thymidine kinase by the HSV.
`
`In one preferred embodiment, the ICP34.5-encoding genes are mutated to confer
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`20
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`selective oncolytic activity on the HSV. Mutations of the ICP34.5-encoding genesthat
`
`prevent the expression of functional ICP34.5 are described in Chouef al. (1990) Science
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`250:1262-1266, Maclean ef a/. (1991) J. Gen. Virol. 72:631-639 and Liu et al. (2003) Gene
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`Therapy 10:292-303, which are incorporated herein by reference. The ICP6-encoding
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`gene and/or thymidine kinase-encoding gene mayalso be inactivated, as may other genes
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`25
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`provided that such inactivation does not prevent the virus infecting or replicating in
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`tumors.
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`The HSV maycontain a further mutation or mutations which enhancereplication of
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`the HSV in tumors. The resulting enhancementofviral replication in tumors not only
`
`results in improved direct ‘oncolytic’ tumorcell killing by the virus, but also enhancesthe
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`30
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`level of heterologous(i.e. a gene inserted into the virus, in the case of viruses of the
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`invention genes encoding fusogenic protein(s) and an immune modulatory molecule(s))
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`gene expression and increases the amount of tumor antigen released as tumorcells die,
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`both of which may also improve the immunogenic properties of the therapy for the
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`treatment of cancer. For example, in a preferred embodiment of the invention, deletion of
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`the ICP47-encoding gene in a mannerthat places the US11 gene underthe control of the
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`immediate early promoter that normally controls expression of the ICP47 encoding gene
`
`leads to enhancedreplication in tumors(see Liu ef al., 2003, which is incorporated herein
`
`by reference).
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`Other mutations that place the US11 coding sequence, which is an HSVlate gene,
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`underthe control of a promoter that is not dependent on viral replication may also be
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`introduced into a virus of the invention. Such mutations allow expression of US11 before
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`10
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`HSVreplication occurs and enhanceviral replication in tumors. In particular, such
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`mutations enhancereplication of an HSV lacking functional ICP34.5-encoding genes.
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`Accordingly, in one embodiment the HSV of the invention comprises a US11 gene
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`operably linked to a promoter, wherein the activity of the promoter is not dependent on
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`viral replication. The promoter may be an immediate early (IE) promoter or a non-HSV
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`15
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`promoter which is active in mammalian, preferably human, tumor cells. The promoter
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`may, for example, be a eukaryotic promoter, such as a promoter derived from the genome
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`of a mammal, preferably a human. The promoter maybe a ubiquitous promoter (such as a
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`promoter of B-actin or tubulin) or a cell-specific promoter, such as tumor-specific
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`promoter. The promoter maybe a viral promoter, such as the Moloney murine leukaemia
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`20
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`virus long terminal repeat (MMLV LTR) promoter or the human or mouse
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`cytomegalovirus (CMV) IE promoter. HSV immediate early (IE) promoters are well
`
`knownin the art. The HSV IE promoter may be the promoter driving expression of ICPO,
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`ICP4, ICP22, ICP27 or ICP47.
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`The genesreferred to above, the functional inactivation of which provides the
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`25
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`property of tumorselectivity to the virus, may be rendered functionally inactive by any
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`suitable method, for example by deletion or substitution of all or part of the gene and/or
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`control sequence of the gene or by insertion of one or more nucleic acids into or in place of
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`the gene and/or the control sequence of the gene. For example, homologous recombination
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`methods, which are standard in the art, may be used to generate the virus of the invention.
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`30
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`Alternatively bacterial artificial chromosome (BAC)-based approaches may be used.
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`Asused herein, the term “gene” 1s intended to mean the nucleotide sequence
`
`encoding a protein, i.e. the coding sequence of the gene. The various genesreferred to
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`above may be rendered non-functional by mutating the geneitself or the control sequences
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`flanking the gene, for example the promoter sequence. Deletions may remove one or more
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`portions of the gene, the entire gene or the entire gene and all or some of the control
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`sequences. For example, deletion of only one nucleotide within the gene may be made,
`
`resulting in a frame shift. However, a larger deletion may be made, for example at least
`
`about 25%, more preferably at least about 50% ofthe total coding and/or non-coding
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`sequence. In one preferred embodiment, the gene being rendered functionally inactive is
`
`deleted. For example, the entire gene and optionally someofthe flanking sequences may
`
`be removed from the virus. Where two or more copies of the gene are presentin the viral
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`10
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`genomeboth copies of the gene are rendered functionally inactive.
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`A gene may beinactivated by substituting other sequences, for example by
`
`substituting all or part of the endogenousgene with a heterologous gene andoptionally a
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`promoter sequence. Where no promoter sequenceis substituted, the heterologous gene
`
`may be inserted suchthat it is controlled by the promoter of the gene being rendered non-
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`15
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`functional. In an HSV ofthe inventionit is preferred that the ICP34.5 encoding-genesare
`
`rendered non-functional by the insertion of a heterologous gene or genes and a promoter
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`sequence or sequences operably linked thereto, and optionally other regulatory elements
`
`such as polyadenylation sequences, into each the ICP34.5-encoding geneloci.
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`A virus of the invention is used to express a fusogenic protein and an immune
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`20
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`stimulatory protein in tumors. This is typically achieved by inserting a heterologous gene
`
`encoding the fusogenic protein and a heterologous gene encoding the immunestimulatory
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`protein in the genomeofa selectively replication competent virus wherein each geneis
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`underthe control of a promoter sequence. As replication of such a virus will occur
`
`selectively in tumortissue, expression of the fusogenic protein and immunestimulatory
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`25
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`protein by the virus is also enhanced in tumortissue as compared to non-tumortissue in the
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`body. Enhanced expression occurs where expression is greater in tumors as compared to
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`other tissues of the body. Accordingly, the invention provides benefits of expression of
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`both a fusogenic protein and an immunestimulatory protein selectively in tumors
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`combined with the anti-tumoreffect provided by oncolytic virus replication.
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`30
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`The virus of the invention may comprise one or more further heterologous genes in
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`addition to the fusogenic protein and an immunestimulatory protein, including further
`
`fusogenic or immunestimulatory proteins.
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`10
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`Fusogenic protein
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`The virus of the invention comprises a gene encoding a fusogenic protein. The
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`fusogenic protein may be any heterologous protein capable of promoting fusion ofa cell
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`infected with the virus of the invention to another cell. A fusogenic protein, preferably a
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`wild type or modified viral glycoprotein (i.e. modified to increase its fusogenic properties),
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`is a protein which is capable in inducing the cell to cell fusion (syncitia formation)of cells
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`in which it is expressed. Examples of fusogenic glycoproteins include VSV-G,syncitin-1
`
`(from human endogenousretrovirus-W (HERV-W)) or syncitin-2 (from HERVFRDE1),
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`10
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`paramyxovirus SV5-F, measles virus-H, measles virus-F, RSV-F, the glycoprotein from a
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`retrovirus or lentivirus, such as gibbon ape leukemia virus (GALV), murine leukemia virus
`
`(MLV), Mason-Pfizer monkey virus (MPMV)and equine infectious anemia virus (EIAV)
`
`with the R transmembrane peptide removed (R- versions). In a preferred embodiment the
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`fusogenic protein is from GALV andhasthe R- peptide removed (GALV-R-).
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`15
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`The virus of the invention may comprise multiple copies of the fusogenic protein-
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`encoding gene, preferably 1 or 2 copies. The virus may comprise two or more different
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`fusogenic proteins, including any of the fusogenic proteins listed above.
`
`The fusogenic protein or prote