™,
`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
`1600382.4
`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
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`Page 1 of 35
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`TRANSGENE/BIOINVENT
`EXHIBIT 1010
`
`TRANSGENE/BIOINVENT
`EXHIBIT 1010
`
`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
`GB1600382.4
`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 35
`UK Intellectual Property Office is an operating name of the Patent Office
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`Page 2 of 35
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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 1600382.4
`
`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)
`
`1200178715
`
`08/01/2016 0.00 NONE
`
`Concept House
`Cardiff Road
`Newport
`South Wales
`NP10 8QQ
`
`N406716GB
`
`Replimune Limited
`The Magdalen Centre Oxford Science Park
`Robert Robinson Avenue
`Oxford OX4 4GA
`United Kingdom
`
`11627528001
`ENGINEERED VIRUS
`
`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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`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
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`7.
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`Inventorship: (Inventors must be individuals not
`companies)
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`Are all the applicants named abovealso inventors?
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`8. Are you paying the application fee with this form?
`
`No
`
`No
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`(REV DEC07)
`Intellectual Property Office is an operating name of the Patent Office
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`Page 3 of 35
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`Patents Form 1(e)
`https :/Avww.gov.uk/ipo
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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:
`
`23
`
`Claim(s):
`
`Abstract:
`
`Drawing/s):
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`6
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`1
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`1
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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.
`
`(REV DEC07)
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`Page 4 of 35
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`Patents Form 1(e)
`
`Page 4 of 35
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`

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`ENGINEERED 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 virus itself and enhanced dueto the recognition by the host of so
`
`called damage associated molecular patterns (DAMPs)whichaid in the activation of the
`
`15
`
`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 e.g.
`
`toll-like receptors
`
`and cGAS/STINGsignalling and the recognition of pathogen associated molecular patterns
`
`(PAMPs)resulting in the activation of interferon responses and inflammation whichare
`
`20
`
`also immunogenic signals to the host. These immune responses may result in the
`
`immunogenic benefit to cancer patients such that immune responses to tumor antigens
`
`provide a systemic overall benefit resulting in the treatment of tumors which havenot 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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`antigens released following virus replication and cell death, genes intended to shape the
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`immune response whichis generated, genes to increase the general immuneactivation
`
`status of the tumor, or genes to increase the direct oncolytic properties (i.e. cytotoxic
`
`effects) of the virus. Importantly, viruses have the ability to deliver encoded molecules
`
`which are intended to 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
`
`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. GM-CSFis a pro-inflammatory cytokine which has multiple
`
`functions including the stimulation of monocytes to exit the circulation and migrate into
`
`tissue where they proliferate and mature into macrophages and dendritic cells. GM-CSF is
`
`important for the proliferation and maturation of antigen presentingcells, the activity of
`
`30
`
`which is needed for the activation of an anti-tumor immune response. Thetrial data
`
`demonstrated that tumor responses could be seen in injected tumors, and to a lesser extent
`
`in uninjected tumors. Responsestendedto be highly durable (months-years), and a
`
`2
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`survival benefit appeared to be achieved in responding patients. Each of these indicated
`
`engagement of the immunesystem in the 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 and notall patients achieve a survival advantage.
`
`Thus, improvementsto the art of oncolytic therapy are clearly needed.
`
`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, also termed immune co-
`
`inhibitory pathways). Checkpoint blockade is intended to block host immuneinhibitory
`
`10
`
`mechanisms which usually serve to prevent the occurrence of auto-immunity. However, in
`
`cancer patients these mechanismscanalso serve to inhibit the induction of or block the
`
`potentially beneficial effects of any immune responses induced to tumors.
`
`Systemic blockade of these pathways by agents targeting CTLA-4, PD-1 or PD-L1
`
`have shownefficacy in a number of tumor types, including melanoma and lung cancer.
`
`15
`
`However, unsurprisingly, based on the mechanism ofaction, off target toxicity can occur
`
`due to the induction of auto-immunity. Evenso, these agents are sufficiently tolerable to
`
`provide considerable clinical utility. Other immune co-inhibitory pathway andrelated
`
`targets for which agents (mainly antibodies) are in development include LAG-3, TIM-3,
`
`VISTA, CSFIR, TDO, CEACAMI, CD47. Optimal clinical activity of these agents, for
`
`20
`
`example PDI, PDL1, LAG-3, TIM-3, VISTA, CSFIR, IDO, CD47, CHACAMI, may
`
`require systemic administration or presence in all tumors due to the mechanism ofaction,
`
`i.e. including targeting of the interface of immune effector cells with tumors or other
`
`immune inhibitory mechanisms in/oftumors.
`
`In some cases, more localised presence in
`
`€.g.
`
`just some tumors or in some lymph nodes mayalso be optimally effective, for example
`
`25
`
`agents targeting CTLA-4.
`
`An alternative approach to increasing the anti-tumor immune response in cancer
`
`patients is to target (activate) immune co-stimulatory pathways, i.e. in contrast to inhibiting
`
`immune co-inhibitory pathways. These pathways send activating signals into T cells and
`
`other immune cells, usually resulting trom the interaction of the relevant igands on antigen
`
`30
`
`presenting cells (APCs) and the relevant receptors on the surface of T cells and other
`
`iramune cells. These signals, depending on the ligand/receptor, can result in the increased
`
`activation of T cells and/or APCs and/or NK cells and/or B cells, including particular sub-
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`Page 7 of 35
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`types, increased differentiation and proliferation of T cells and/or APCs and/or NK cells
`
`and/or B cells, including particular subtypes, or suppression of the activity of immune
`
`inhibitory P cells such as regulatory T cells. Activation of these pathways would therefore
`
`be expected to result in enhanced anti-tumor immune responses, but it might also be
`
`expected that systemic activation of these pathways, i.e. activation of immune responses
`
`generally rather than anti-tumor inumume responses specifically or selectively, would result
`
`in considerable off target toxicity in non-tumortissue, the degree of such offtarget toxicity
`
`depending on the particular immune co-stimulatory pathway being targeted. Nevertheless
`
`agents (rnainly agonistic antibodies, or less frequently the soluble ligandto the receptor in
`
`10
`
`question) targeting immune co-stimulatory pathways, inchiding agents targeting GITR, 4-
`
`i-BB, OX40, CD40 or ICOS, and intended for systemic use (.e. intravenous delivery} are
`
`in or have been proposed for clinical development.
`
`For many of these approaches targeting immune co-inhibitory or co-inhibitory
`
`pathways to be successful, pre-existing immune responses to tumors are needed, so that a
`
`15
`
`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
`
`20
`
`response to tumor antigens 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 microenvironmentare
`
`attractive for use in combination with immunecheckpoint blockade and immune
`
`25
`
`potentiating drugs. This likely explains the promising combined anti-tumoreffects of
`
`oncolytic agents and immunecheckpoint blockade in mice and humansthat haveso far
`
`been observed.
`
`The above discussion demonstrates thatthere is still much scope for improving
`
`oncolytic agents and cancertherapies utilising oncolytic agents, anti-tumor immune
`
`30
`
`responses and drugs which target immune co-inhibitory or co-stimulatory pathways.
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`Summary ofthe Invention
`
`The invention provides oncolytic viruses expressing GM-CSFandat least one
`
`molecule targeting an immuneco-stimulatory pathway. GM-CSFaids in the induction of
`
`an inflammatory tumor micro-environment andstimulates the proliferation and maturation
`
`of antigen presenting cells, including dendritic cells, aiding the induction of an anti-tumor
`
`immune responses. These immuneresponsesare amplified through activation of an
`
`immune co-stimulatory pathway or pathways using an immune co-stimulatory pathway
`
`activating molecule or molecules also delivered by the oncolytic virus.
`
`The use of an oncolytic virus to deliver molecules targeting immune co-stimulatory
`
`10
`
`pathways to tumors focuses the amplification of immuneeffects on anti-tumor immune
`
`responses, and reduces the amplification of immune responses to non-tumorantigens.
`
`Thus, immunecells in tumors and tumordraining lymph nodesare selectively engaged by
`
`the molecules activating immune co-stimulatory pathways rather than immunecells in
`
`general. This results in enhanced efficacy of immune co-stimulatory pathway activation
`
`15
`
`and anti-tumor immuneresponse amplification, and can also result in reduced off target
`
`toxicity.
`
`It is also important for focusing the effects of combined systemic immune co-
`
`inhibitory pathway blockade and immune co-stimulatory pathway activation on tumors,i.e.
`
`such that the amplified immune responses from which co-inhibitory blocks are released are
`
`antitumor immuneresponses rather than responses to non-tumorantigens.
`
`20
`
`The invention utilizes the fact that, when delivered by an oncolytic virus, the site of
`
`action of co-stimulatory pathway activation and of GM-CSFexpression is in the tumor
`
`and/or tumor draining lymph node, but the results of such activation (an amplified systemic
`
`anti-tumor-immuneresponse) are systemic. This targets tumors generally, and not only
`
`tumors to which the oncolytic virus has delivered the molecule or molecules targeting an
`
`25
`
`immune co-stimulatory pathway or pathways and GM-CSF. Oncolytic viruses of the
`
`invention therefore provide improved treatment of cancer through the generation of
`
`improved tumor focused immuneresponses. The oncolytic virus of the invention also
`
`offers improved anti-tumor immunestimulating effects such that the immune-mediated
`
`effects on tumors whichare not destroyed by oncolysis, including micro-metastatic
`
`30
`
`disease, are enhanced, resulting in more effective destruction of these tumors, and more
`
`effective long term anti-tumor vaccination to prevent future relapse and improve overall
`
`survival.
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`Anti-tumorefficacy is improved when an oncolytic virus of the invention is used as
`
`a single agent and also whenthe virus is used in combination with other anti-cancer
`
`modalities, including chemotherapy, treatment with targeted agents, radiation and, in
`
`preferred embodiments, immune checkpoint blockade drugs(i.e. antagonists of an immune
`
`co-inhibitory pathway) and/or agonists of an immuneco-stimulatory pathway.
`
`Accordingly, the present invention provides an oncolytic virus comprising: (} a
`
`GM-CSF-encoding gene; and Gi) an immune co-stimulatory pathway activating molecule
`
`or unmune co-stimulatory pathwayactivating molecule-encoding gene. The virus may
`
`encode more than one inumune co-stimulatory pathwayactivating molecule/gene.
`
`10
`
`The immume co-stimulatory pathway activating molecule is preferably GITRL, 4-1-
`
`BBL, OX40L, ICOSL or CD40Lor a moditied version of any thereof. Examples of
`
`modified versions include agonists of a co-stimulatory pathwaythat are secreted rather
`
`than being membrane bound, and/or agonists modified such that multimers of the protein
`
`are formed.
`
`15
`
`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 tumor cell lines more rapidly and/orat
`
`a lower dose in vitro than one or more reference clinical isolates of the same species of
`
`Virus.
`
`The virus is preferably a herpes simplex virus (HSV), such as HSV1. The HSV
`
`20
`
`typically does not express functional 1CP34_8 and/or functional ICP47 and/or expresses the
`
`US11 gene as an immediate early gene.
`
`The invention also provides:
`
`25
`
`30
`
`-
`
`-
`
`-
`
`-
`
`a pharmaceutical 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
`
`by therapy;
`
`the virus of the invention for use in a method of treating cancer, wherein the
`
`method optionally comprises administering a further anti-cancer agent,
`
`aproduct of manufacture comprising a virus of the invention in a sterile vial,
`
`ampoule or syringe,
`
`amethod of treating cancer, which comprises administering a therapeutically
`
`effective amount of a virus or a pharmaceutical composition of the invention to a
`
`Page 10 of 35
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`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 oftreating cancer, wherein the method optionally comprises administering
`
`a further anti-cancer agent;
`
`a methodoftreating 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 an agonist of an immuneco-stimulatory pathway to a patient in need
`
`10
`
`thereof.
`
`Brief Description of the Figures
`
`Figure 1 depicts the structure of an exemplary virus of the invention that comprises
`
`a gene encoding GM-CSFanda gene encoding CD40L.
`
`15
`
`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
`
`GM-CSF.
`
`20
`
`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.
`
`SEQ ID NO: 6 is the amino acid sequence of human GM-CSF.
`
`25
`
`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-
`
`R-.
`
`SEQ ID NO: 9 is the amino acid sequence of GALV-R-.
`
`SEQ ID NO:10 is the nucleotide sequence of a codon optimized version of a
`
`30
`
`human/mouse hybrid membrane bound version of CD40L.
`
`SEQ ID NO: 11 is the amino acid sequence of a human/mouse hybrid membrane
`
`bound version of CD40L.
`
`Page 11 of 35
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`Page 11 of 35
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`SEQ ID NO: 12 is the nucleotide sequence of a codon optimized version of a
`
`multimeric secreted version of human CD40L.
`
`SEQ ID NO:13 is the amino acid sequence of a multimeric secreted version of
`
`human CD40L.
`
`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.
`
`10
`
`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 amino acid sequence of wild-type mouse CD40L.
`
`Detailed Description of the Invention
`
`15
`
`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
`
`20
`
`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.
`
`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
`
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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 ofthese is the case will depend on the virus species to be used. Preferably the virus
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`iS a spectes 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 HSV1. In particularly preferred embodiments
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`the virus of the invention is based on a clinical isolate of the virus species to be used. The
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`clinical isolate may have been selected on the basis of it having particular advantageous
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`properties for the treatment of cancer.
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`The virus may be a modified clinical isolate, wherein the clinical isolate kills pwo
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`or more turnor cell lines more rapidly and/or at a lower dose in vitro than ore or more
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`reference clinical isolate of the same species of virus. Typically, the clinical isolate will
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`kill two or more tumorcell lines within 48 hours, preferably within 24 hours, of infection
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`at multiplicities of infection (MODof tess than or equal to 0.1. Preferably the climeal
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`isolate will kil a broad range of tumor cell lines, such as 2, 3, 4, 5, 6, 7,8, 9, 10 or, for
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`example, all of the following human tumorcell lines: U87MG (glioma), HT29 (colorectal),
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`LNCaP(prostate), MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu (squamouscell
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`carcinoma), MCF7 (breast), A549 (lung), MIAPACA-2 (pancreas), CAPAN-1(pancreas),
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`HT1080 (fibrosarcoma).
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`An HSVofthe invention is capable of replicating selectively in tumors, such as
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`human tumors. Typically, the HSV replicates efficiently in target tumors but does not
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`replicate efficiently in non-tumortissue. This HSV may comprise one or more mutations
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`in one or more viral genes that inhibit replication in normaltissue butstill allow replication
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`in tumors. The mutation may, for example, be a mutation that prevents the expression of
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`functional ICP34.5, ICP6 and/or thymidine kinase by the HSV.
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`In one preferred embodiment, the ICP34.5-encoding genes are mutated to confer
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`selective oncolytic activity on the HSV. Mutations of the ICP34.5-encoding genesthat
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`prevent the expression of functional ICP34.5 are described in Chouef al. (1990) Science
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`20
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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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`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
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`results in improved direct ‘oncolytic’ tumorcell killing by the virus, but also enhancesthe
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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 GM-CSFand an immuneco-stimulatory pathway activating
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`30
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`molecule(s)) gene expression and increases the amount of tumor antigen released as tumor
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`cells die, both of which may also improve the immunogenic properties of the therapy for
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`the treatment of cancer. For example, in a preferred embodimentof the invention, deletion
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`of 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
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`leads to enhancedreplication in tumors(see Liu ef al., 2003, which is incorporated herein
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`by reference).
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`Other mutations that place the US11 coding sequence, which is an HSV late gene,
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`underthe control of a promoter that is not dependenton 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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`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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`10
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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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`promoter which is active in mammalian, preferably human, tumorcells. 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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`promoterof 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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`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
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`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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`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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`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
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`30
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`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,
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`resulting in a frame shift. However, a larger deletion may be made, for example at least
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`about 25%, more preferably at least about 50% of the total coding and/or non-coding
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`sequence. In one preferred embodiment, the gene being rendered functionally inactive is
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`deleted. For example, the entire gene and optionally someofthe flanking sequences may
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`be removed from the virus. Where two or more copies of the gene are present in the viral
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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
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`substituting all or part of the endogenousgene with a heterologous gene and optionally a
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`promoter sequence. Where no promoter sequenceis substituted, the heterologous gene
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`may be inserted suchthat it is controlled by the promoter of the gene being rendered non-
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`functional. In an HSV ofthe inventionit is preferred that the ICP34.5 encoding-genesare
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`rendered non-functional by the insertion of a heterologous gene or genes and a promoter
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`15
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`sequence or sequences operably linked thereto, and optionally other regulatory elements
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`such as polyadenylation sequences, into each t