PCTIGB2017I050038
`
`Concept House
`Cardiff Road
`
`Newport
`South Wales
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`I, the undersigned, being an officer duly authorised in accordance with Section 74(1) and (4)
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`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
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`Dated
`
`12th January 2017
`
`UK Intellectual Property Office is an operating name of the Patent Office
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`

`

`
`
` .fi,
`Intellectual
`
`Property
`OffiCe
`
`Patents Form 1
`Patents Act 1977 (Rule 12)
`
`Request for grant of a patent
`
`.
`.
`Application number
`1. Your reference
`
`GB 1600382.4
`
`2.
`
`Full name, address and postcode of the applicant or of
`each applicant
`
`1200178715
`
`08/01/2016 0.00 NONE
`
`Concept House
`Cardiff Road
`Newport
`South Wales
`NP10 SQQ
`
`N406716GB
`
`Replimune Limited
`The Magdalen Centre Oxford Science Park
`Robert Robinson Avenue
`Oxford OX4 4GA
`
`United Kingdom
`
`Patents ADP number (ifyou know it)
`11627528001
`
`3. Title of the invention
`ENGINEERED VIRUS
`
`4. Name of 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)
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`Patents ADP number (ifyou know it)
`5. Priority declaration: Are you claiming priority from one or
`more earlier-filed patent applications? If so, please give
`details ofthe application(s)
`
`J A Kemp
`J A Kemp
`14 South Square
`Gray's Inn
`London WC1 R 5JJ
`Greater London
`
`United Kingdom
`
`10645901001
`
`Country
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`Application number
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`Date of filing
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`PDAS Access Code
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`(REV DEC07)
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`Description:
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`11. l/We request the grant of a patent on the basis of this application.
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`Signature: Subject: Pamela Tuxworth 23470; Issuer:
`European Patent Office, European Patent
`
`Office CA GZ
`
`08 Jan 2016
`
`Date:
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`12. Name, e-mail address, telephone, fax and/or mobile
`number, if any, of a contact point forthe applicant
`
`TUXWORTH, Ms. Pamela Mary
`Email: mail@jakemp.com
`Telephone: +44 20 3077 8600
`Fax: +44 20 7430 1000
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`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 informed if 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 which relates to military technology, or would be prejudicial to national security or the safety of the public, section 23 of
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`(REV DECO7)
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`Patents Form 1(e)
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`

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`ENGINEERED VIRUS
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`Field of the Invention
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`The invention relates to an oncolytic immunotherapeutic agent and to the use of the
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`oncolytic immunotherapeutic agent in treating cancer.
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`Background to the Invention
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`Viruses have a unique ability to enter cells at high efficiency. After entry into cells,
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`viral genes are expressed and the virus replicates. This usually results in the death of the
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`infected cell and the release of the antigenic components of the cell as the cell ruptures as it
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`dies. As a result, virus mediated cell death tends to result in an immune response to these
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`cellular components, including both those derived from the host cell and those encoded by
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`or incorporated into the virus itself and enhanced due to the recognition by the host of so
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`called damage associated molecular patterns (DAMPs) which aid in the activation of the
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`immune response.
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`Viruses also engage with various mediators of the innate immune response as part
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`of the host response to the recognition of a viral infection through e. g.
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`toll-like receptors
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`and cGAS/ STING signalling and the recognition of pathogen associated molecular patterns
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`(PAMPs) resulting in the activation of interferon responses and inflammation which are
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`also immunogenic signals to the host. These immune responses may result in the
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`immunogenic benefit to cancer patients such that immune responses to tumor antigens
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`provide a systemic overall benefit resulting in the treatment of tumors which have not been
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`infected with the virus, including micro-metastatic disease, and providing vaccination
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`against relapse.
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`The combined direct (‘oncolytic’) effects of the virus, and immune responses
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`against tumor antigens (including non—self ‘neo—antigens’, i.e. derived from the particular
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`mutated genes in individual tumors) is termed ‘oncolytic immunotherapy’.
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`Viruses may also be used as delivery vehicles (‘vectors’) to express heterologous
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`genes inserted into the viral genome in infected cells. These properties make viruses
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`useful for a variety of biotechnology and medical applications. For example, viruses
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`expressing heterologous therapeutic genes may be used for gene therapy. In the context of
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`oncolytic immunotherapy, delivered genes may include those encoding specific tumor
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`antigens, genes intended to induce immune responses or increase the immunogenicity of
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`antigens released following virus replication and cell death, genes intended to shape the
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`immune response which is generated, genes to increase the general immune activation
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`status of the tum or, or genes to increase the direct oncolytic properties (i.e. cytotoxic
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`effects) of the virus. Importantly, viruses have the ability to deliver encoded molecules
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`which are intended to help to initiate, enhance or shape the systemic anti-tumor immune
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`response directly and selectively to tumors, which may have benefits of e.g. reduced
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`toxicity or of focusing beneficial effects on tumors (including those not infected by the
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`virus) rather than off-target effects on normal (i,e. non-cancerous) tissues as compared to
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`the systemic administration of these same molecules or systemic administration of other
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`molecules targeting the same pathways.
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`It has been demonstrated that a number of viruses including, for example, herpes
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`simplex virus (HSV) have utility in the oncolytic treatment of cancer. HSV for use in the
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`oncolytic treatment of cancer must be disabled such that it is no longer pathogenic, but can
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`still enter into and kill tumor cells. A number of disabling mutations to HSV, including
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`disruption of the genes encoding ICP34.5, ICP6, and/or thymidine kinase, have been
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`identified which do not prevent the virus from replicating in culture or in tumor tissue in
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`viva, but which prevent significant replication in normal tissue. HSVs in which only the
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`ICP34.5 genes have been disrupted replicate in many tumor cell types in vilro, and
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`replicate selectively in tumor tissue, but not in surrounding tissue, in mouse tumor models.
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`Clinical trials of ICP34.5 deleted, or ICP34.5 and ICP6 deleted, HSV have also shown
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`safety and selective replication in tumor tissue in humans.
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`As discussed above, an oncolytic virus, including HSV, may also be used to deliver
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`a therapeutic gene in the treatment of cancer. An ICP34.5 deleted virus of this type
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`additionally deleted for ICP47 and encoding a heterologous gene for GM-CSF has also
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`been tested in clinical trials, including a phase 3 trial in melanoma in which safety and
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`efficacy in man was shown. GM-C SF is a pro-inflammatory cytokine which has multiple
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`functions including the stimulation of monocytes to exit the circulation and migrate into
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`tissue where they proliferate and mature into macrophages and dendritic cells. GM-C SF is
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`important for the proliferation and maturation of antigen presenting cells, the activity of
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`which is needed for the activation of an anti-tumor immune response. The trial data
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`demonstrated that tumor responses could be seen in injected tumors, and to a lesser extent
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`in uninj ected tumors. Responses tended to be highly durable (months-years), and a
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`survival benefit appeared to be achieved in responding patients. Each of these indicated
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`engagement of the immune system in the treatment of cancer in addition to the direct
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`oncolytic effect. However, this and other data with oncolytic viruses generally showed
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`that not all tumors respond to treatment and not all patients achieve a survival advantage.
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`Thus, improvements to the art of oncolytic therapy are clearly needed.
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`Recently it has been shown that oncolytic immunotherapy can result in additive or
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`synergistic therapeutic effects in conjunction with immune checkpoint blockade (ie.
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`inhibition or ‘antagonism’ of immune checkpoint pathways, also termed immune co-
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`inhibitory pathways). Checkpoint blockade is intended to block host immune inhibitory
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`mechanisms which usually serve to prevent the occurrence of auto-immunity. However, in
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`cancer patients these mechanisms can also serve to inhibit the induction of or block the
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`potentially beneficial effects of any immune responses induced to tumors.
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`Systemic blockade of these pathways by agents targeting CTLA-4, PD-l or PD-Ll
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`have shown efficacy in a number of tumor types, including melanoma and lung cancer.
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`However, unsurprisingly, based on the mechanism of action, off target toxicity can occur
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`due to the induction of auto-immunity. Even so, these agents are sufficiently tolerable to
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`provide considerable clinical utility. Other immune co-inhibitory pathway and related
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`targets for which agents (mainly antibodies) are in development include LAG-3, TIRE—3,
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`VES'E'A, (ESP 1R, HM), {IlEix‘sCAh/ll , CD47. Optimal clinical activity of these agents, for
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`example PDl, PDLl, LAG—3, TIE/L3, V lS'i'A, CSFlR, IDS, CE47, CEACAl‘V/ll, may
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`require systemic administration or presence in all tumors tine to the mechanism of action,
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`iie. including targeting of the interface of immune effector cells with tumors or other
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`immune inhibitory mechanisms iii/of tumors.
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`in same cases, more localised presence in
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`e. g, just some turners or in some lymph nodes may also he optimally efective, for example
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`agents targeting CTillA—s’l.
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`An alternative approach to increasing the antimturnor immune response in cancer
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`patients is to target (activate) immune co—stirnulatory pathways, ice. in contrast to ll’ll’llbll‘ilig
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`immune cominhihitrny pathways. These pathways send activating signals into T cells and
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`other immune cells, usually resulting from the interaction of the relevant ligands on antigen
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`presenting cells (Alfiis) and the relevant receptors on the surface of '5‘ cells and other
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`immune cells, These signals, depending on the ligand/receptor, can result in the increased
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`activation of T cells and/or APCS and/or NK cells and/or 8 cells, including particular suh~
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`types, increased differentiation and proliferation of T cells and/0r APCs and/or NK cells
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`and/or 8 cells, including particular subtypes, 0r suppression of the activity (if immune
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`inhibitoiy T cells such as regulatery T cells. Activatien at“ these pathways would therefore
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`he expected to result in enhanced anti—tumor immune respeuses, but it might also he
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`expected that systemic activation of these pathways, i.e. aetivation of ii’nmune responses
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`generally rather than anti-tumor immune responses specifically or selectively, would result
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`in considerable off target toxicity in non—tumor tissue, the degree of such off target toxicity
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`depending on the particular immune ce—stiuuilatoiy pathway being targeted Nevertheless
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`agents (mainly agonistic antibodies, or less frequently the scluhle ligand te the recepter in
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`question) targeting immune cry—stimulatory pathways, including agents targeting Ts‘lTR, 4~
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`l-BB, 0X40, CD40 or ICQS, and intended fer systemic use tin intravenous deliveiy) are
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`in or have been proposed for clinical development.
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`For many of these approaches targeting immune co-inhibitory or co-inhibitory
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`pathways to be successful, pre-existing immune responses to tumors are needed, so that a
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`pre-existing immune response can be potentiated or a block to an anti-tumor immune
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`response can be relieved. The presence of an inflamed tumor micro-environment, which is
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`indicative of such an ongoing response, is also needed. Pre-existing immune responses to
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`tumor neo-antigens appear to be particularly important for the activity of immune
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`checkpoint blockade and related drugs. Only some patients may have an ongoing immune
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`response to tumor antigens including neoantigens and/or an inflamed tumor
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`microenvironment, both of which are required for the optimal activity of these drugs.
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`Therefore, oncolytic agents which can induce immune responses to tumor antigens,
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`including neoantigens, and/or which can induce an inflamed tumor microenvironment are
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`attractive for use in combination with immune checkpoint blockade and immune
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`potentiating drugs. This likely explains the promising combined anti-tumor effects of
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`oncolytic agents and immune checkpoint blockade in mice and humans that have so far
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`been observed.
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`The above discussion demonstrates that there is still much scope for improving
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`oncolytic agents and cancer therapies utilising oncolytic agents, anti-tumor immune
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`responses and drugs which target immune co-inhibitory or co-stimulatory pathways.
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`Summary of the Invention
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`The invention provides oncolytic viruses expressing GM-CSF and at least one
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`molecule targeting an immune co-stimulatory pathway. GM-CSF aids in the induction of
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`an inflammatory tumor micro-environment and stimulates the proliferation and maturation
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`of antigen presenting cells, including dendritic cells, aiding the induction of an anti-tumor
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`immune responses. These immune responses are amplified through activation of an
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`immune co-stimulatory pathway or pathways using an immune co-stimulatory pathway
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`activating molecule or molecules also delivered by the oncolytic virus.
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`The use of an oncolytic virus to deliver molecules targeting immune co-stimulatory
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`pathways to tumors focuses the amplification of immune effects on anti-tumor immune
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`responses, and reduces the amplification of immune responses to non-tumor antigens.
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`Thus, immune cells in tumors and tumor draining lymph nodes are selectively engaged by
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`the molecules activating immune co-stimulatory pathways rather than immune cells in
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`general. This results in enhanced efficacy of immune co-stimulatory pathway activation
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`and anti-tumor immune response amplification, and can also result in reduced off target
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`toxicity.
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`It is also important for focusing the effects of combined systemic immune co-
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`inhibitory pathway blockade and immune co-stimulatory pathway activation on tumors, i.e.
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`such that the amplified immune responses from which co-inhibitory blocks are released are
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`antitumor immune responses rather than responses to non-tumor antigens.
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`The invention utilizes the fact that, when delivered by an oncolytic Virus, the site of
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`action of co-stimulatory pathway activation and of GM-C SF expression is in the tumor
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`and/or tumor draining lymph node, but the results of such activation (an amplified systemic
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`anti-tumor-immune response) are systemic. This targets tumors generally, and not only
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`tumors to which the oncolytic virus has delivered the molecule or molecules targeting an
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`immune co-stimulatory pathway or pathways and GM-C SF. Oncolytic viruses of the
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`invention therefore provide improved treatment of cancer through the generation of
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`improved tumor focused immune responses. The oncolytic Virus of the invention also
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`offers improved anti-tumor immune stimulating effects such that the immune-mediated
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`effects on tumors which are not destroyed by oncolysis, including micro-metastatic
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`disease, are enhanced, resulting in more effective destruction of these tumors, and more
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`effective long term anti-tumor vaccination to prevent future relapse and improve overall
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`survival .
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`Anti-tumor efficacy is improved when an oncolytic virus of the invention is used as
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`a single agent and also when the virus is used in combination with other anti-cancer
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`modalities, including chemotherapy, treatment with targeted agents, radiation and, in
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`preferred embodiments, immune checkpoint blockade drugs (i.e. antagonists of an immune
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`co-inhibitory pathway) and/or agonists of an immune co-stimulatory pathway.
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`Accordingly, the present invention provides an oncolytic virus comprising: (i) a
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`Gl‘t/LCSFeencodin g gene; and (ii) an immune too—stimulatory pathway activating molecule
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`or immune era-stimulatory pathway activating moleculesencoding gene, The virus may
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`encode more than one immune CDmSilfl‘ltllaiGi‘y pathway activating molecule/gene
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`The immune co~stimulatory pathway activating molecule is preferably GTTRL, 4~l ~
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`BBL, OXAl-OL, lCQSL or CD401. or a modified version of any thereof. Examples of
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`modified versions include agonists of a co—stimulatory pathway that are secreted rather
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`than being membrane bound, and/or agonists modified such that inultimers of the protein
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`are formed
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`The virus may he a modified clinical isolate, such as a modified clinical isolate of a
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`virus, wherein the. clinical isolate kills two or more tumor cell lines more rapidly and/or at
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`a lower dose in ixz’z‘ro than one or more reference clinical isolates or" the same species or"
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`virus.
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`The virus is preferably a herpes simplex virus til-18V), such as l-TS‘v’l. The lEiSV
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`typically does not express functional lCl’345 and/or functional {(3347 and/or expresses the
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`U 8 ll gene as an immediate early gene,
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`The invention also provides:
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`—
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`—
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`-
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`—
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`~
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`a pharmaceutical composition comprising a virus of the invention and a
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`pharmaceutically acceptable carrier or diluent;
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`the virus of the invention for use in a method ol‘treating the human or anim al body
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`by therapy;
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`the virus of the invention for use in a method of treating cancer, wherein the
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`method optionally comprises administering a further anti—cancer agent;
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`a product of manufacture comprising a virus of the invention in a sterile vial,
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`ainpoule or syringe;
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`a method of treating cancer, which comprises administering a therapeutically
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`effective amount or" a virus or a pharmaceutical composition of the invention to a
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`patient in need thereof, wherein the method optionaily comprises administering a
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`further anti—cancer agent;
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`use ofa virus ofthe invention in the manufacture ofa medicament, for use in a
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`method of treating cancer; wherein the method optionally comprises administering
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`a further anti—can oer agent;
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`a method of treating cancer, which comprises administering a therapeutically
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`effective amount of an oncolytic virus, an inhibitor of the indoleamine 2,3-
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`dioxygenase (IDO) pathway and a further antagonist of an immune co-inhibitory
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`pathway, or an agonist of an immune co-stimulatory pathway to a patient in need
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`thereof.
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`Brief Description of the Figures
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`Figure 1 depicts the structure of an exemplary virus of the invention that comprises
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`a gene encoding GM-CSF and a gene encoding CD4OL.
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`Brief Description of the Seguence 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-C SF.
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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-C SF.
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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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`R-.
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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 ofa multimeric secreted version of
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`human CD40L.
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`SEQ ID 1\O: 14 is the nucleotide sequence of a codon optimized version of a
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`multimeric secreted version of mouse CD4OL.
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`SEQ ID 1\O:15 is the amino acid sequence ofa multimeric secreted version of
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`mouse CD4OL.
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`
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`SEQ ID 1\O: 16 is the nucleotide sequence of wild-type human CD40L.
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`SEQ ID 1\O: 17 is the amino acid sequence of Wild-type human CD40L.
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`SEQ ID 1\O:18 is the nucleotide sequence of wild-type mouse CD4OL.
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`SEQ ID 1\O: 19 is the amino acid sequence of wild-type mouse CD4OL.
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`Detailed Description of the Invention
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`Oneoéylic 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 tumor cells, such that the tumor cells 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 tumor tissue. A virus is selectively replication competent in tumor tissue if it
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`replicates more effectively in tumor tissue than in non-tumor tissue. The ability of a virus
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`to replicate in different tissue types can be determined using standard techniques in the art.
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`The virus of the invention may be any virus which has these 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 oi’tlie invention may be wild type
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`(ie. unaltered from the parental virus species), or with gene disruptions er gene additions.
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`W’l’iicb oftliese 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 HS‘V, including strains 01” HSV'E
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`and HSVLZ, and is must preferably a strain 0f HSVl. In particularly preferred ernbedinients
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`the virus nl‘tlie invention is based on a clinical isniate nftlte virus species to be used. The
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`elinieal iselate may have been selected on the basis of it having particular advantageeus
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`properties fer the treatment or“ cancer.
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`The virus may be a modifi ed clinical isolate, wherein the clinical isolate kills two
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`or more tumor cell lines more rapidly and/or at a lower dose in vino than one or more
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`reference clinical isolate ofthe same species ofvirus. Typically, the clinical isolate will
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`kill two or more tumor cell lines within 48 hours, preferably within 2.4 hours, of infection
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`at n’iultiiplicities of infection (Ev/{01) of less 11h an or equal to 01. Preferably the clinical
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`isolate will kill 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 tumor cell lines: U87MG (glioma), HT29 (colorectal),
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`LNCaP (prostate), lV[DA—lV[B-231 (breast), SK-MEL-28 (melanoma), Fadu (squamous cell
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`carcinoma), MCF7 (breast), A549 (lung), MIAPACA—2 (pancreas), CAPAN—1(pancreas),
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`10
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`HT1080 (fibrosarcoma).
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`An HSV of the 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-tumor tissue. This HSV may comprise one or more mutations
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`in one or more viral genes that inhibit replication in normal tissue but still 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-enc0ding genes that
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`prevent the expression of functional ICP34.5 are described in Chou el al. (1990) Science
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`250:1262-1266, Maclean er a]. (1991) J. Gen. Virol. 721631-639 and Liu et a]. (2003) Gene
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`Therapy 101292-303, which are incorporated herein by reference. The ICP6-encoding
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`gene and/or thymidine kinase-encoding gene may also 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 may contain a fuIther mutation or mutations which enhance replication of
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`the HSV in tumors. The resulting enhancement of viral replication in tumors not only
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`results in improved direct ‘oncolytic’ tumor cell killing by the virus, but also enhances the
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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-CSF and an immune co-stimulatory pathway activating
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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 embodiment of the invention, deletion
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`15
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`20
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`25
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`30
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`9
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`

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`of the ICP47-encoding gene in a manner that places the US 11 gene under the control of the
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`immediate early promoter that normally controls expression ofthe ICP47 encoding gene
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`leads to enhanced replication in tumors (see Liu et al., 2003, which is incorporated herein
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`by reference).
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`Other mutations that place the USll coding sequence, which is an HSV late gene,
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`under the 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 US] 1 before
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`HSV replication occurs and enhance viral replication in tumors.
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`In particular, such
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`mutations enhance replication of an HSV lacking functional ICP34.5-encoding genes.
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`Accordingly, in one embodiment the HSV of the invention comprises a US 11 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, 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 may be 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 may be a viral promoter, such as the Moloney murine leukaemia
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`virus long terminal repeat (MIVJLV 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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`known in the art. The HSV IE promoter may be the promoter driving expression of ICPO,
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`ICP4, ICPZZ, ICP27 or ICP47.
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`The genes referred to above the filnctional inactivation of which provides the
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`property of tumor selectivity 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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`As used herein, the term “gene” is intended to mean the nucleotide sequence
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`encoding a protein, i.e. the coding sequence of the gene. The various genes referred to
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`above may be rendered non-functional by mutating the gene itself 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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`10
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`10
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`15
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`20
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`25
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`30
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`

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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
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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 some ofthe 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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`genome both copies of the gene are rendered functionally inactive.
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`A gene may be inactivated by substituting other sequences, for example by
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`substituting all or part of the endogenous gene with a heterologous gene and optionally a
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`promoter sequence. Where no promoter sequence is substituted, the heterologous gene
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`may be inserted such that it is controlled by the promoter of the gene being rendered non-
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`functional. In an HSV of the invention it is preferred that the ICP34.5 encoding-genes are
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`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
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`such as polyadenylation sequences, into each the ICP34.5-encoding gene loci.
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`A virus of the invention is used to express GM-CSF and an immune co-stimulatory
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`pathway activating molecule in tumors. This is typically achieved by inserting a
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`heterologous gene encoding GM-CSF and a heterologous gene encoding the immune co-
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`stimulatory pathway activating molecule in the genome of a selectively replication
`
`competent virus wherein each gene is under the control of a promoter sequence. As
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`replication of such a virus will occur selectively in tumor tissue, expression of the GM-
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`CSF and the immune co-stimulatory activating protein by the virus is also enhanced in
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`tumor tissue as compared to non-tumor tissue in the body. Enhanced expression occurs