™,
`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
`1600381.6
`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 41
`
`TRANSGENE/BIOINVENT
`EXHIBIT 1009
`
`TRANSGENE/BIOINVENT
`EXHIBIT 1009
`
`Page 1 of 41
`
`

`

`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
`GB1600381.6
`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.
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`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.
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`In accordance with the rules, the words "public limited company" may be replaced by p.l.c.,
`ple, P.L.C. or PLC.
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`Re-registration under the Companies Act does not constitute a new legal entity but merely
`subjects the companyto certain additional companylaw rules.
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`Signed
`
`Aj
`
`Dated
`
`12th January 2017
`
`Page 2 of 41
`UK Intellectual Property Office is an operating name of the Patent Office
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`Page 2 of 41
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`

`

`
`Intellectual
`Property
`Office
`
`Patents Form 1
`Patents Act 1977 (Rule 72)
`
`Requestfor grant of a patent
`
`Application number
`Your reference
`
`GB 1600381.6
`
`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(ifyou 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)
`
`1200178714
`
`08/01/2016 0.00 NONE
`
`Concept House
`Cardiff Road
`Newport
`South Wales
`NP10 8QQ
`
`N406715GB
`
`Replimune Limited
`The Magdalen Centre Oxford Science Park
`Robert Robinson Avenue
`Oxford OX4 4GA
`United Kingdom
`
`11627528001
`
`VIRUS STRAIN
`
`J A Kemp
`JAKemp
`14 South Square
`Gray's Inn
`London WC1R 5JJ
`Greater London
`United Kingdom
`
`10645901001
`
`Country
`
`Application number
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`Date offiling
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`PDASAccess Code
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`Numberofearlier UK
`application
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`Dateoffiling
`(day /month /year)
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`8. Are you paying the application fee with this form?
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`No
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`No
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`(REV DECO7)
`Intellectual Property Office is an operating name of the Patent Office
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`Page 3 of 41
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`Patents Form 1(e)
`https :/Avww.gov.uk/ipo
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`Page 3 of 41
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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:
`
`28
`
`Claim(s):
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`Abstract:
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`Drawing/s):
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`6
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`1
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`2
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`If you are not filing a description, please give details of
`the previous application you are going to rely upon
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`Date offiling
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`10. If you are alsofiling any of the following, state how many
`against each item.
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`Priority documents:
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`Statement of inventorship and right to grant of a patent
`(Patents Form 7):
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`Requestfor search (Patents Form 9A):
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`Request for a substantive examination (Patents Form 10):
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`0
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`0
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`0
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`0
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`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.
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`(REV DECO7)
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`Page 4 of 41
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`Patents Form 1(e)
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`Page 4 of 41
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`

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`VIRUSSTRAIN
`
`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.
`
`Viruses also engage with various mediators of the innate immuneresponseas part
`
`15
`
`of the host response to the recognition of a viral infection throughe.g.toll-like receptors
`
`and cGAS/STINGsignalling resulting in the activation of interferon responses and
`
`inflammation which are also immunogenic signals to the host. These immune responses
`
`mayresult in the immunogenic benefit to cancer patients such that immune responses to
`
`tumorantigens provide a systemic overall benefit resulting in the treatment of tumors
`
`20
`
`which havenot been infected with the virus, including micro-metastatic disease, and
`
`providing vaccination againstrelapse.
`
`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’.
`
`25
`
`Viruses may also be usedas delivery vehicles (‘vectors’) to express heterologous
`
`genesinserted into the viral genomein infected cells. These properties make viruses
`
`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
`
`30
`
`antigens, genes intendedto increase the immunogenicity of antigens released following
`
`virus replication and cell death, to increase the general immuneactivation status of the
`
`tumor, or to increase the direct oncolytic properties (i.e. cytotoxic effects) of the virus.
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`Page 5 of 41
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`It has been demonstrated that a numberofviruses including herpes simplex virus
`
`(HSV) haveutility in the oncolytic treatment of cancer. HSV for use in the oncolytic
`
`treatment of cancer must be disabled suchthat it is no longer pathogenic, but can still enter
`
`into and kill tumor cells. A numberof disabling mutations to HSV, including 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 surrounding tissue, in mouse tumor models. Clinicaltrials of
`
`10
`
`ICP34.5 deleted, or ICP34.5 and ICP6 deleted, HSV have also shownsafety and selective
`
`replication in tumortissue in man.
`
`Asdiscussed above, an oncolytic virus, including HSV, mayalso be usedto deliver
`
`a therapeutic gene in the treatment of cancer. An ICP34.5 deleted virusof this type
`
`additionally deleted for ICP47 and encoding a heterologous gene for GM-CSF hasalso
`
`15
`
`been tested in clinical trials, including a phase3 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
`
`20
`
`treatment of cancer in addition to the direct oncolytic effect. However, this and other data
`
`with oncolytic viruses generally showed that not all tumors respond to treatment andnotall
`
`patients achieve a survival advantage. Thus, improvementsto the art of oncolytic therapy
`
`and oncolytic immunotherapy are clearly needed. These mayserveto increase the direct
`
`oncolytic effects of therapy, the anti-tumor immunestimulating effects of the therapy, or
`
`25
`
`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 blockade is
`
`intended to block host immuneinhibitory mechanisms which usually serve to prevent the
`
`30
`
`occurrence of auto-immunity. However, in cancer patients these mechanisms can also
`
`serve to inhibit or block the potentially beneficial effects of any immune responses induced
`
`to tumors. Alternatively, immune responses may notbe fully potentiated due to a lack of
`
`2
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`activation or lack of full activation of immune potentiating pathways. Therefore, drugs
`
`which alleviate these blocks or stimulate immune potentiating pathways (i.e. which
`
`activate, or are ‘agonists’ of these immunepotentiating pathways) are attractive for testing
`
`and developing cancer treatments. Targets for such approved or experimental drugs
`
`include CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, VISTA, CSFIR, IDO, CEACAM1, GITR,
`
`4-1-BB. KER, SLAMEP7, OX40, CD40, [COS or CD47.
`
`For these approachesto be successful, pre-existing immune responses to tumorsare
`
`needed, so that a pre-existing immuneresponse canbe potentiated or a block to an anti-
`
`tumor immuneresponse can be relieved. The presence of an inflamed tumor micro-
`
`10
`
`environment, whichis 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 checkpoint blockade and related drugs. Only some patients may have an
`
`ongoing immuneresponse to tumorantigens including neoantigens and/or an inflamed
`
`tumor microenvironment, both of which are required for the activity of these drugs.
`
`15
`
`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
`
`potentiating drugs. This likely also explains the promising combinedanti-tumoreffects of
`
`oncolytic agents and immunecheckpoint blockade in mice and humansthat have so far
`
`20
`
`been observed.
`
`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 enhancementoflocal regulatory T cell
`
`(Treg)-mediated immunosuppression. IDO catalyses the rate-limiting step of tryptophan
`
`25
`
`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
`
`30
`
`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. IDO
`
`Page 7 of 41
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`inhibitors may also be synergistic with oncolytic immunotherapy, including together with
`
`drugs targeting other immune checkpoint or immuneco-stimulatory pathways.
`
`Summary of the Invention
`
`The invention provides improved oncolytic viruses. The improved direct oncolytic
`
`effects provided by the viruses of the invention will also lead to improved systemic anti-
`
`tumor immuneeffects. Enhancedreplication in and killing of tumorcells will result in
`
`enhanced tumorantigen release and enhanced systemic immuneresponsesto the released
`
`antigens. The expression levels of any genes inserted to augmentthe direct oncolytic
`
`10
`
`effects and/or immunestimulation will also be increased.
`
`Virus species naturally exist in a range of variants (strains) within the natural
`
`population which may differ by a small or larger numberof nucleotides while still
`
`retaining the antigenic characteristics and sufficient sequence identity to still be recognized
`
`as the same species. Thesestrains, dueto their differing sequences, may exhibit a range of
`
`15
`
`differing properties, including properties which have been selected for by natural selection
`
`in their natural host or hosts (for example the ability to infect or replicate in the target cell
`
`types of the virus in question, spread betweenthesecells, or to evade the host innate or
`
`adaptive immunesystem, or to spread between infected individuals of the host species) and
`
`properties which have not been specifically selected for (e.g. the ability to replicate in and
`
`20
`
`kill or spread between cell types which are not the natural targets of the virus in question,
`
`including tumoror other non-target cell types or tissues). The inventors have recognised
`
`that sampling a range ofviral strains of a particular viral species which are present in the
`
`natural host population (in the case of viruses infecting humans, here termed ‘clinical
`
`isolates’) and comparing these to each other to select for the strain with the best properties
`
`25
`
`for the intended purpose for which itis to be used(e.g. infection and killing of tumorcells)
`
`can be used to identify a virus with optimal properties for that purpose. A virus identified
`
`by this approachis likely to have more optimal properties for the intended purpose than a
`
`‘prototype’ or ‘laboratory’ virus strain or a clinical strain which has not been selected for
`
`the required property or properties from a broad group ofviral strains. This is because the
`
`30
`
`full biological complexity in the natural population, particularly with respect to the
`
`particular desirable property or properties, is unlikely to have been sampled through taking
`
`a narrow approachto screening for the desired property or properties, bearing in mind the
`
`degree of sequence variation present in natural virus populations. In particular, these may
`4
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`vary in sequence within an infected host (as is often the case with RNAorretroviral
`
`populations where so-called quasi-species are often present), between individual infected
`
`hosts, or between different geographically separated viral populations.
`
`Viruses of the invention have therefore been selected by sampling a rangeofviral
`
`strains present in the natural population of a particular viral species and testing these
`
`against each other for the desired property or properties (e.g. the ability to infect and kill
`
`tumorcells). The virus strain or strains with the best properties for the intended purpose
`
`are used for further development.
`
`Where the intended useis oncolytic viral therapy, taking such an approach provides
`
`10
`
`an improved starting point for development of an oncolytic agent, which requires further
`
`manipulation of the advantageousvirusstrains. Such manipulation includes the deletion of
`
`viral genes to provide, for example, tumorselectivity, and/or the insertion of exogenous
`
`genes to improve oncolytic or immunepotentiating properties further.
`
`The viruses of the invention therefore include novel clinical isolates of a viral
`
`15
`
`species that have better anti-tumoreffects than the otherclinical isolates to which they
`
`were compared and through which comparison they wereidentified.
`
`In particular, the
`
`clinical isolates of the invention kill tumorcell lines #7 vitro more quickly and/or at a lower
`
`dose than these reference clinical isolates of the same virus type. Typically, a clinical
`
`isolate of the invention will have been identified through comparison of >5 clinical isolates
`
`20
`
`of a viral species for the required property or properties, preferably through comparison of
`
`>10 clinical isolates of the viral species, and more preferably through comparison of >20
`
`clinical isolates of the viral species. A clinical isolate of the invention typically shows
`
`better tumorcell killing activity than 3/5, 6/10 or 11/20ths, preferably better than 4/5, 8/10
`
`or 17/20ths, more preferably better than 9/10 or 19/20ths ofthe viral strains tested.
`
`25
`
`Typically, a clinical isolate of the invention can kill two or more tumorcell lines in
`
`vitro within 24 to 48 hoursafter infection at a multiplicity of infection (MODof 0.01 to
`
`0.001 orless.
`
`Theclinical isolates of the invention may be modified to further enhance their anti-
`
`tumoreffects. The genomeofa clinical isolate of the invention may be modified to delete
`
`30
`
`or alter expression of one or more viral genes, and/or the genomeoftheclinical isolate
`
`may be modified to express one or more heterologous genes, such as genes encoding a
`
`fusogenic protein and/or an immunestimulatory molecule or molecules.
`
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`Oncolytic viruses of the invention provide improved treatment of cancer through
`
`improved direct oncolytic effects, viral replication and spread through tumors, which(1)
`
`increases the amount of tumorantigens, including neoantigens, which are released for the
`
`induction of an anti-tumor immune response; and(i1) enhancesthe expression of the virus-
`
`encoded immunestimulatory molecule(s). Expression of immune stimulatory molecule(s)
`
`by the virus can further enhanceandpotentiate the anti-tumor immuneeffect. Expression
`
`of fusogenic protein(s) by the virus can further enhanceviral spread through tumors.
`
`Anti-tumorefficacy of an oncolytic virus of the invention is achieved when the
`
`virus is used as a single agent and also whenthe virus is used in combination with other
`
`10
`
`anti-cancer modalities, including chemotherapy, treatment with targeted agents, radiation,
`
`immune checkpoint blockade(i.e. administration of one or more antagonist of an immune
`
`co-inhibitory pathway) and/or immune potentiating drugs (e.g.one or more agonists of an
`
`immune co-stimulatory pathway). The improved direct oncolytic effects (i.e. virus
`
`replication in, spread between, and direct killing of tumorcells) and improved systemic
`
`15
`
`anti-tumor immuneeffects of the viruses of the invention improve on the combined
`
`benefits of oncolytic therapy and immuneco-inhibitory pathway blockade and/or immune
`
`co-stimulatory pathway activation.
`
`Accordingly, the present invention provides an oncolytic virus whichis, oris
`
`derived from, aclinical isolate which has been selected by comparing the abilities of a
`
`20
`
`panel of three or more clinical isolates of the same viral species to kill tumorcelis of two
`
`or more tumorcell lines /# vitro and selecting a clinical isolate which is capable of kidling
`
`cells of two or more tumor cell lines more rapidly and/or at a lower dose in vitro than one
`
`or more of the other clinical isolates in the panel. The clinical isolate may be modified. A
`
`modified clinical isolate may have mutations, such as deletions in the viral genome and/or
`
`25
`
`may express one or more heterologous genes.
`
`The virus may be a strain of any virus species which may be used for the oncolytic
`
`treatment of cancer, inchiding strains a herpes virus, pox virus, adenovirus, retrovirus,
`
`rhabdovirus, paramyxovirusor reovirus. The virus is preferably a herpes simplex virus
`
`(HSV), such as HSV1. The HSVtypically does not express functional [CP34.5 and/or
`
`30
`
`functional [CP47 and/or expresses the US11 gene as an immediate early gene.
`
`The virus may comprise G) a fusogenic protein-encoding gene; and/or Gi) an
`
`immune stimulatory molecule or an immune stimulatory molecule-encoding gene. The
`
`Page 10 of 41
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`Page 10 of 41
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`virus may encode more than one fhsogentc 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 immune stimulatory molecule is preferably GM-CSFand/or an agonist
`
`ofan immune co-stimulatory pathway including GITRL, 4-1-BBL, OX40L, ICOSLor
`
`CD40L or a modified version in each case thereof.
`
`The invention also provides:
`
`-
`
`-
`
`-
`
`-
`
`-
`
`apharmaceutical composition comprising a virus of the invention and a
`
`pharrnaceutically acceptable carrier or diluent;
`
`the virus of the invention for use in a method oftreating the human or animal body
`
`bytherapy;
`
`the virus of the invention for use in a method oftreating cancer, wherein the
`
`method optionally comprises administering a further anti-cancer agent;
`
`aproduct of manufacture comprising a virus of the invention in asterife vial,
`
`ampoule or syringe;
`
`arnethod 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 method optionally comprises administering a
`
`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
`
`effective amount of an oncolytic virus, an inhibitor of the indoleamine 2,3-
`
`dioxygenase (IDO) pathwayand a further antagonist of an immune co-inhibitory
`
`pathway, or agonist of an immune co-stimulatory pathway to a patient in need
`
`thereof; and
`
`-
`
`arnethod of selecting an oncolytic virus, the method comprising:
`
`Gi)
`
`comparing the abilities of a panel of three or more clinical isolates of the
`
`same viral strain to kill humor cells of two or more tumor cell lines fvitro;
`
`scoring the abilities of each of the panel of viruses to kill tumorcells;
`
`selecting a virus which has one ofthe best scores;
`
`Gp
`
`Git}
`
`Page 11 of 41
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`Page 11 of 41
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`Gv}
`
`(v)
`
`optionally modifying the virus to inactivate one or more viral genes; and/or
`
`optionally modifying the virus to express one or rnore immune stimulatory
`
`molecule encoding genes and/or one or more fusogentc protein-encoding
`
`genes.
`
`The further anti-cancer agent may be an antagonist of an immuneco-inhibitory
`
`pathwayor an agonist of an immune co-stinvulatory pathway
`
`Brief Description of the Figures
`
`Figure 1 depicts the structure of an exemplary virus of the invention that comprises
`
`10
`
`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 gene is underthe control
`
`of the ICP47 immediate early promoter.
`
`Figure 2 depicts the structure of an exemplary virus of the invention that comprises
`
`a gene encoding GALV-R-, a gene encoding GM-CSFand a gene encoding CD40L.
`
`15
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`Brief Description of the Sequence Listing
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`SEQ ID NO: 1 is the nucleotide sequence of mouse GM-CSF.
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`SEQ ID NO: 2 is the nucleotide sequence of a codon optimized version of mouse
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`GM-CSF.
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`SEQ ID NO: 3 is the nucleotide sequence of human GM-CSF.
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`SEQ ID NO: 4 is the nucleotide sequence of a codon optimized version of human
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`GM-CSF.
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`SEQ ID NO: 5 is the amino acid sequence of mouse GM-CSF.
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`SEQ ID NO: 6 is the amino acid sequence of human GM-CSF.
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`SEQ ID NO: 7 is the nucleotide sequence of GALV-R-.
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`SEQ ID NO: 8 is the nucleotide sequence of a codon optimized version of GALV-
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`SEQ ID NO: 9 is the amino acid sequence of GALV-R-.
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`SEQ ID NO:10 is the nucleotide sequence of a codon optimized version of a
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`human/mouse hybrid membrane bound version of CD40L.
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`SEQ ID NO:11 is the amino acid sequence of a human/mouse hybrid membrane
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`bound version of CD40L.
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`SEQ ID NO:12 is the nucleotide sequence of a codon optimized version of a
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`multimeric secreted version of human CD40L.
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`SEQ ID NO:13 is the amino acid sequence of a multimeric secreted version of
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`human CD40L.
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`SEQ ID NO: 14 is the nucleotide sequence of a codon optimized version of a
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`multimeric secreted version of mouse CD40L.
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`SEQ ID NO:15 is the amino acid sequence of a multimeric secreted version of
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`mouse CD40L.
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`SEQ ID NO: 16 is the nucleotide sequence of wild-type human CD40L.
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`SEQ ID NO:17 is the amino acid sequence of wild-type human CD40L.
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`SEQ ID NO:18 is the nucleotide sequence of wild-type mouse CD40L.
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`SEQ ID NO:19 is the aminoacid sequence of wild-type mouse CD40L.
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`Detailed Description of the Invention
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`Oncolptie Virus
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`The virus of the invention is oncolytic. An oncolytic virus is a virus that infects
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`and replicates in tumorcells, such that the tumorcells are killed. Therefore, the virus of
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`the invention is replication competent. Preferably, the virus is selectively replication
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`competent in tumortissue. A virusis selectively replication competent in tumortissueif it
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`replicates more effectively in tumortissue than in non-tumortissue. The ability of a virus
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`to replicate in different tissue types can be determined using standard techniquesin theart.
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`The virus of the invention may be any virus which hasthese properties, including a
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`herpes virus, pox virus, adenovirus, retrovirus, rhabdovirus, paramyxovirus or reovirus, or
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`any species or strain within these larger groups. Viruses of the invention may be wild type
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`(..e. unaltered from the parental virus species), or with gene disruptions or gene additions.
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`Which of these is the case will depend on the virus species to be used. Preferably the virus
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`iS a species of herpes virus, more preferably a strain of HSV, including strains of HSV1
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`and HSV2, and is most preferably a strain of HSVi. The virus of the invention is based on
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`a clinical isolate of the virus species to be used. The clinical isolate is selected on the basis
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`of it having particular advantageous properties for the treatment of cancer. The virus ofthe
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`invention has surprisingly good anti-tumor effects comparedto otherstrains of the same
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`virus isolated from other patients. The virus strains used for comparisonto identify viruses
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`of the invention may be isolated from a patient or an otherwise healthy (i.e. other than
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`harboring the virus species to be tested) volunteer, preferably an otherwise healthy
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`volunteer. HSV1 strains used to identify a virus of the invention are typically isolated
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`from cold sores of individuals harboring HSV1, typically by taking a swab using e.g.
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`Virocult (Sigma) brand swab/container containing transport media followed by transport to
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`the facility to be used for furthertesting.
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`After isolation of viruses to be compared from individuals, stocks of the viruses are
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`typically prepared, for example by growing the isolated viruses on BHK orverocells.
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`This is done following no morethan 3 cycles of freeze thaw between taking the sample and
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`it being grown on, for example, BHK orvero cells to prepare the virus stock for further
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`use. Preferably the virus sample has undergone 2 or less than 2 cycles of freeze thaw prior
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`to preparation of the stock for further use, more preferably one cycle of freeze thaw, most
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`preferably no cycles of freeze thaw. Lysates from the cell lines infected with the viruses
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`prepared in this way after isolation are compared,typically by testing for the ability of the
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`virus to kill tumorcell lines in vitro. Alternatively, the viral stocks may be stored under
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`suitable conditions, for example by freezing, prior to testing. Viruses of the invention have
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`surprisingly good anti-tumor effects comparedto otherstrains of the samevirus isolated
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`from other individuals, preferably when comparedto those isolated from >5 individuals,
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`more preferably >10 other individuals, most preferably >20 other individuals.
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`The stocks of the clinical isolates identified as viruses of the invention (i.e. having
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`surprisingly good properties for the killing of tumor cells as comparedto otherviral strains
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`to which they were compared) may be stored undersuitable conditions, before or after
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`modification, and used to generate further stocks as appropriate.
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`A clinical isolate is a strain of a virus species which has been isolated fromm its
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`natural host. The clinical isolate has preferably been isolated for the purposes oftesting
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`and comparing the clinical isolate with other clinical isolates of that virus species for a
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`desired property, in the case of viruses of the invention that being the abilityto kill human
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`tumor cells. Clinical isolates which maybe used for comparison also include those from
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`clinical samples present in clinical repositories, i.e. previously collected for clinical
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`diagnostic or other purposes.
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`In either case the clinical isolates used for comparison and
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`identification of viruses of the invention will preferably have undergone minimal culture in
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`vitro prior to being tested for the desired property, preferably having only undergone
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`10
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`sufficient culture to enable generation of sufficient stocks for comparative testing purposes.
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`As such, the viruses used for comparisonto identify viruses of the invention may also
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`include deposited strains, wherein the deposited strain has been isolated from a patient,
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`preferably an HSV1 strain isolated from the cold sore ofa patient.
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`The virus of the invention is an oncolytic virus whichis, or is derived from, a
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`clinical isolate which has been selected by comparing the abilities ofa panel of three or
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`more clinical isolates of the same viral species to lall tumor cells of two or more tumorcell
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`lines ivitro and selecting a clinical isolate which is capable of killing cells of two or more
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`tumor cell lines more rapidly and/or at a lower dose #7 vitro than one or more of the other
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`clinical isolates in the panel. Thus, the virus is a