ENGINEERED VIRUS
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`Field of the Invention
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`The invention relates to an oncolytic immunotherapeutic agent andto the use of the
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`oncolytic immunotherapeutic agent in treating cancer.
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`Backgroundto the Invention
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`Viruses have a uniqueability to enter cells at high efficiency. After entry into cells,
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`viral genes are expressed andthe virus replicates. This usually results in the death ofthe
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`10
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`infected cell and the release of the antigenic components ofthe cell as the cell rupturesasit
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`dies. As a result, virus mediated cell death tends to result in an immuneresponse 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) whichaid in the activation of the
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`15
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`immuneresponse.
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`Viruses also engage with various mediators of the innate immuneresponseaspart
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`of the host responseto the recognition ofa viral infection through e.g.
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`toll-like receptors
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`and cGAS/STINGsignalling 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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`20
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`also immunogenic signals to the host. These immuneresponses mayresult in the
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`immunogenicbenefit to cancer patients such that immune responses to tumorantigens
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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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`25
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`The combined direct (‘oncolytic’) effects of the virus, and immune responses
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`against tumorantigens (including non-self “neo-antigens’, i.e. derived from the particular
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`mutated genesin 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 genomein infected cells. These properties make viruses
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`30
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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 immuneresponsesor increase the immunogenicity of
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`TRANSGENE/BIOINVENT
`EXHIBIT 1013
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`TRANSGENE/BIOINVENT
`EXHIBIT 1013
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`antigens released following virus replication and cell death, genes intended to shape the
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`immuneresponse whichis generated, genes to increase the general immuneactivation
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`status of the tumor, or genes to increase the direct oncolytic properties (1.e. cytotoxic
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`effects) of the virus. Importantly, viruses have the ability to deliver encoded molecules
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`whichare intended to help to initiate, enhance or shape the systemic anti-tumor immune
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`responsedirectly and selectively to tumors, which may havebenefits 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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`10
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`molecules targeting the same pathways.
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`It has been demonstrated that a numberofviruses including, for example, herpes
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`simplex virus (HSV) haveutility in the oncolytic treatment of cancer. HSV for use in the
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`oncolytic treatment of cancer must be disabled such thatit is no longer pathogenic, but can
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`still enter into and kill tumorcells. A numberof disabling mutations to HSV,including
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`15
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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 preventthe virus from replicating in culture or in tumortissue in
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`vivo, 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 tumorcell types in vitro, and
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`replicate selectively in tumortissue, but not in surrounding tissue, in mouse tumor models.
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`20
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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 tumortissue in humans.
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`Asdiscussed above, an oncolytic virus, including HSV, mayalso be usedto 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-CSFhasalso
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`25
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`beentested in clinicaltrials, including a phase 3 trial in melanoma in which safety and
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`efficacy in man was shown. GM-CSFis 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-CSF is
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`important for the proliferation and maturation of antigen presenting cells, the activity of
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`30
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`whichis needed for the activation of an anti-tumor immuneresponse. Thetrial 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 uninjected 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 immunesystem 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 notall 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 shownthat oncolytic immunotherapy canresult in additive or
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`synergistic therapeutic effects in conjunction with immune checkpoint blockade(i.e.
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`inhibition or ‘antagonism’ of immune checkpoint pathways, also termed immuneco-
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`inhibitory pathways). Checkpoint (immune inhibitory pathway) blockade is intended to
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`10
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`block host immune inhibitory mechanisms which usually serve to prevent the occurrence
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`of auto-immunity. However, in cancer patients these mechanismscanalso serveto inhibit
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`the induction of or block the potentially beneficial effects of any immune responses
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`induced to tumors.
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`Systemic blockade of these pathways by agents targeting CTLA-4, PD-1 or PD-L1
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`15
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`have shownefficacy in a numberof tumor types, including melanomaandlungcancer.
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`However, unsurprisingly, based on the mechanism ofaction, 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, TIM-3,
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`20
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`VISTA, CSFIR, IDO, CEACAMI, CD47, Optimal clinical activity of these agents, for
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`example PD, PDLI, LAG-3, PIM-3, VISTA, CSFIR, IDO, CD47, CEACAM], may
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`require systemic administration or presence in all tumors due to the mechanism of action,
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`i.e. inchiding targeting of the interface of tmmnine effector cells with tumors or other
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`immune inhibitory mechanisms in/of tumors.
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`fn some cases, more localised presence in
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`25
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`&.g. Just some tumors or in some byniph nodes may also be optimallyeffective, for example
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`agents targeting CTLA-4,
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`An alternative approachto increasing the anti-tumor immune response in cancer
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`patients is to target (activate) immune co-stimulatory pathways, Le. in contrast to mbhibiting
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`wrumune co-inhibitory pathways. These pathways send activating signals into T cells and
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`30
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`other immune cells, usually resulting from the interaction ofthe relevant ligands on antigen
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`presenting cells (APCs) and the relevant receptors on the surface of T cells andother
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`immune cells. These signals, depending on the figand/receptor, can result in the increased
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`activation of T cells and/or APCs and/or NK cells and/or B ceils, including particular gub-
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`types, increased differentiation and proliferation of T cells and/or APCs and/or NK cells
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`and/or B cells, inchiding particular subtypes, or suppression ofthe activity of immane
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`inhibitory T cells such as regulatory T cells. Activation of these pathways would therefore
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`be expected to result in enhanced anti-tumor immrnneresponses, but it might also be
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`expected that systemic activation of these pathways, ic. activation of immune 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 im non-tumortissue, the degree of such off target toxicity
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`depending on the particular immune co-stimulatory pathway being targeted. Nevertheless
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`10
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`agonts (mainly agonistic antibodies, or loss frequently the soluble ligand to the receptorin
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`question} targeting immune co-stimulatory pathways, including agents targeting GITR, 4-
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`1-BB, OX40, CD46or ICOS, and intended for systemic use G.e. intravenous delivery) are
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`in or have been proposed for clinical development.
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`For many of these approachestargeting immune co-inhibitory or co-inhibitory
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`15
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`pathwaysto be successful, pre-existing immune responses to tumors are needed,i.e. so that
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`a pre-existing immuneresponse 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, whichis
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`indicative of such an ongoing response, is also needed. Pre-existing immuneresponses to
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`tumor neo-antigens appearto be particularly importantfor the activity of immuneco-
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`20
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`inhibitory pathway blockade and related drugs. Only some patients may have an ongoing
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`immuneresponse to tumorantigens 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 immuneresponses to tumorantigens,
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`including neoantigens, and/or which can induce an inflamed tumor microenvironment are
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`25
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`attractive for use in combination with immune co-inhibitory pathway blockade and
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`immunepotentiating drugs. This likely explains the promising combinedanti-tumor
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`effects of oncolytic agents and immuneco-inhibitory pathway blockade in mice and
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`humansthat have so far been observed.
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`The above discussion demonstrates that there is still much scope for improving
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`30
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`oncolytic agents and cancertherapies 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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`Summaryof the Invention
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`The invention provides oncolytic viruses expressing GM-CSFandat least one
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`molecule targeting an immuneco-stimulatory pathway. GM-CSFaidsin the induction of
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`an inflammatory tumor micro-environment andstimulates 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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`immuneresponses. These immuneresponses are amplified through activation of an
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`immuneco-stimulatory pathway or pathways using an immuneco-stimulatory pathway
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`activating molecule or molecules also delivered by the oncolytic virus.
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`10
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`The use of an oncolytic virus to deliver molecules targeting immuneco-stimulatory
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`pathways to tumors focuses the amplification of immuneeffects on anti-tumor immune
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`responses, and reduces the amplification of immuneresponses to non-tumorantigens.
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`Thus, immunecells in tumors and tumordraining lymph nodesare selectively engaged by
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`the molecules activating immune co-stimulatory pathways rather than immunecells in
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`15
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`general. This results in enhanced efficacy of immuneco-stimulatory pathway activation
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`and anti-tumor immuneresponse amplification, and can also result in reduced off target
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`toxicity. It is also important for focusing the effects of combined systemic immune co-
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`inhibitory pathway blockade and immuneco-stimulatory pathway activation on tumors,1.e.
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`such that the amplified immune responses from which co-inhibitory blocks are released are
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`20
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`antitumor immuneresponsesrather than responses to non-tumorantigens.
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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-CSFexpressionis in the tumor
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`and/or tumordraining 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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`25
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`tumors to which the oncolytic virus has delivered the molecule or molecules targeting an
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`immuneco-stimulatory pathway or pathways and GM-CSF. 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 immunestimulating effects such that the immune-mediated
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`30
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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 whenthe 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: (4) 4
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`GM-CSF-encading gene; and (ii) an mmmnime co-stimulatory pathway activating molecule
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`10
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`or immune co-stimulatory pathway activating moalecule-encoding gene. The virus may
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`encode more than one immune co-stimulatory pathwayactivating molecule/gene,
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`The immune co-stimulatory pathway activating molecule is preferably GITRL, 4-1-
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`BBL, OX40L, ICOSL or CD40L or a modified version of any thereof or a protein capable
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`of blocking signaling through CTLA-4, for example an antibody which binds CTLA-4.
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`15
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`Examples of modified versions include agonists of a co-stimulatory pathwaythat are
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`secreted rather than being membrane bound, and/or agonists modified such that multimers
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`of the protein are formed.
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`The virus may be a modified clinical isolate, such as a modified clinical isolate ofa
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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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`20
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`a lower dose in vitro than one or more reference clinical isolates of the same species of
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`VITUS.
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`The virus is preferably a herpes simplex virus (HSV), such as HSVi. The HSV
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`typically does not express functional ICP34.5 and/or functional ICP47 and/or expresses the
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`UST gene as an immediate carly gene.
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`25
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`The inventionalso provides:
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`-
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`~
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`-
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`30
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`a pharmaceutical composition comprising a virus of the invention and a
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`pharmaceutically acceptable carrier or dilaent:
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`the virus of the invention for use in a method of treating the human or animal body
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`bytherapy;
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`the virus of the invention for use in a methodoftreating cancer, wherein the
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`method optionally comprises administering a further anti-cancer agent;
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`~
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`-
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`aproduct of manufacture comprising a viras of the invention in a sterile vial,
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`ampoule or syringe:
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`amethod of treating cancer, which comprises administering a therapeutically
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`effective amount of a virus or a pharmaceutical composition of the invention to a
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`patient in need thereof, wherein the method optionally comprises administering a
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`further anti-cancer agent which is optionally an antagonist of an immmine co-
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`inhibitory pathway, or an agonist of an immuneco-sliroulatory pathway;
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`-
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`use ofa virus of the invention in the manufacture of a medicament for use ina
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`method of treating cancer, wherein the method optionally comprises administering
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`10
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`a further anti-cancer agent which is optionally an antagonist of an immmine co-
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`inhibitory pathway, or an agortist of an immrune co-stimulatory pathway;
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`-
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`amethodoftreating cancer, which comprises administering a therapeutically
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`effective amountof 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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`15
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`pathway, or an agonist of an immuneco-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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`20
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`a gene encoding GM-CSFanda gene encoding CD40L.
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`Figure 2 shows the differential abilities of the eight top ranking HSV1 clinical
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`isolate strains as assessed bycrystal violet staining 24 hours or 48 hours after mfection
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`with a MOT of 0.1, 6.01 or 0.001 as indicated in the Figure to kill Fadu, SK-mel-28, A549,
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`HT1080, MIA-PA-CA-?, HT29 and MDA-MB-231 human tumor cell lines. The virus
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`25
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`strains ranked first and second on each cell line are indicated. The viras RHOISA was
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`ranked first on cach of the Fadu, HTP1080, MIA-PA-CA-2? and HT29 cell lines and second
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`on each of the SK-mel-28, 4549 and MDA-MB-231 cell lines. RHO04A was ranked joint
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`first with RHOTSA and RHOISA on the HT29 cell line, first on the SK-mel-28 and AS49
`
`cell lines and second on the Fada cell line. RHOZ3A was ranked first on the MDA-MB-
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`30
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`231 cell line and second on the HT1O80 cell line. RHO3LA was ranked second on each of
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`the MIA-PA-CA-2 and HT29 cell lines. RHO40A was ranked joint second on the HT29
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`cell line.
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`Figure 3 shows a comparison between strain RHOISA,the strain ranked first of all
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`the strains tested, with an ‘average’ strain from the screen (ic. stra RHOGSA).
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`Approximately 10 fold less of strain RHOISA was needed to kill an equal proportion of
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`cells than was needed of strain RHOGSA as shownbycrystal violet staining 24 or 48 hours
`
`post infection with MOls of 0.1, 0.01 and 0.001 in SK-mel-28, HT1080, MDA-MB-231,
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`Fadu, MIA-PA-CA-2 and AS49 cell lines.
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`Figures 4 and § depict structares of HSV1 viruses modified by the deletion of
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`ICP34.5 and iCP47 such that the USI] gene is under control of the ICP457 immediate
`
`carly promoter and containing heterologous genes in the ICP34.5 locus, The viruses were
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`10
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`constructed using theRHGI8Astrain unless otherwise stated in the Figure.
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`Figure 6 showsthe results of an ELISA to detect expression of human or mouse
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`GM-CSFin supernatants from BHKcells infected with virus 16 (mGM-CSF and GALVR-
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`), virus 17 (hGM-CSF and GALVR-) and virus 19 (mGM-CSF).
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`Figure 7 is a comparison betweenthe cell-killing abilities of strain RHO18A in
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`15
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`which ICP34.5 is deleted and which expresses GALVR- and GFP(virus 10) with a virus
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`that expresses only GFP (virus 12) as determined by crystal violet staining in three cell
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`lines at low magnification.
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`Figure 8 is a comparison betweenthe cell-killing abilities of strain RHO18A in
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`which ICP34.5 and ICP47 are deleted and which expresses GALVR- and GM-CSF(virus
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`20
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`17) with a prior art strain with the same modifications as determined by crystal violet
`
`staining in four cell lines.
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`Figure 9 showsthe effectiveness of Virus 16 (ICP34.5 and ICP47 deleted
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`expressing GALVR- and mGM-CSF)in treating mice harbouring A20 lymphomatumors
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`in both flanks. Tumors onthe right flanks were injected with the virus or vehicle and the
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`25
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`effects on tumorsize was observed for 30 days. The virus waseffective against both
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`injected tumors and non-injected tumors.
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`Figure 10 demonstrates the effects of Virus 15 (ICP34.5 and ICP47 deleted
`
`expressing GALVR- and GFP) and Virus 24 (ICP34.5 and ICP47 deleted expressing GFP)
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`on rat 9L cells in vitro as assessed by crystal violet staining. The virus expressing GALV
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`30
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`(Virus 15) showed enhancedkilling of rat 9L cells in vitro as comparedto a virus which
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`does not express GALV (Virus 24).
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`Figure 11 showsthe antitumoreffects of Virus 16 in Balb/c mice harboring mouse
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`CT26 tumorsin the left and right flanks. Groups of 10 mice were then treated with:
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`Vehicle (3 injections into right flank tumors every other day); 5x10exp6 pfu of Virus 16
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`(mRP1) injected in the right flank tumor every other day; anti-mouse PD1 alone (10mg/kg
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`ip. every three days, BioXCell clone RMP1-14); anti-mouse CTLA-4 (3mg/kg i.p every
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`three days, BioXCell clone 9D9); anti-mouse PD1 together with Virus 16; anti-mouse
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`CTLA4together with Virus 16; 1-methyl trypotophan (I-MT; IDO inhibitor (Smg/mlin
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`drinking water)); anti-mouse PD1 together with 1-methyl trypotophan;or anti-mouse PD1
`
`together with 1-methyl trypotophan and Virus 16. Effects on tumor size were observed for
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`10
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`a further 30 days. Greater tumor reduction was seen in animals treated with combinations
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`of virus and checkpoint bockade than with the single treatment groups. Figure 11A shows
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`that using Virus 16 and anti-PD1 in combination hasa better anti-tumoreffect than using
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`either anti-PD1 or the virus alone. Figure 11B showsthat the anti-tumoreffect of Virus 16
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`in combination with anti-CTLA-4 was better than the anti-tumoreffect of either Virus 16
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`15
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`or anti-CTLA-4 alone. Figure 11C showsthat enhanced tumor reduction was observed
`
`using Virus 16 together with both anti-PD1 and IDOinhibition as compared to anti-PD1
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`and 1-MTinhibition in the absence of the virus.
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`Figure 12 showsthe enhanced anti-tumoractivity of Virus 16 in combination with
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`immunecheckpoint blockade in mouse A20 tumorsin both flanks of Balb/c mice as
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`20
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`comparedto either virus alone or checkpoint blockade alone (anti-PD1).
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`Figure 13 showsthe structure of ICP34.5 and ICP47 deleted viruses expressing
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`GALVR-, GM-CSFand codon optimized anti-mouse or anti-human CTLA-4 antibody
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`constructs (secreted scFv molecules linked to human or mouse IgG1 Fe regions). The
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`scFvs contain the linked ([G,S],) light and heavy variable chains from antibody 9D9
`
`25
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`(US2011044953: mouse version) and from ipilimumab (US20150283234; humanversion).
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`The resulting structure of the CTLA-4 inhibitor is also shown.
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`Figure 14 showsanti-tumoreffects of Virus 16 and Virus 19 in a human xenograft
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`model (A549). There were three injections of Virus 16, Virus 19 or of vehicle over one
`
`weekat three different dose levels (N=10/group). The doses of the viruses used is
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`30
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`indicated. The anti-tumoreffects of Virus 16 which expresses GALV were better than
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`those of Virus 19 which does not express GALV.
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`Figure 15 demonstrates the effects of viruses of the invention expressing GALVR-
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`on 9L cells in the flanks of Fischer 344 rats. The following treatments were administered
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`to groups of rats (ten per group), into one flank of each rat only three times per week for
`three weeks: 50ul of vehicle; SOul of 10’ pfu/ml of Virus 19 (expresses mGM-CSF but not
`GALVR-); or SOul of 107 pfu/ml of Virus 16 (expresses both mouse GM-CSF and GALV-
`
`R-). Effects on tumor growth were then observed for a further 30 days. Superior tumor
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`control and shrinkage was observed with the virus expressing GM-CSF and GALV-R-as
`
`comparedto the virus expressing GM-CSFalone.
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`Figure 16 showsthe anti-tumoreffects of viruses expressing anti-mCTLA-4(virus
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`10
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`27), mCD40L(virus 32), mOX4OL(virus 35), m4-2BBL(virus 33), , each also with
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`mGM-CSF and GALV-R- compared to virus 16 (expresses GALV and mGM-CSF).
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`Brief Description of the Sequence Listing
`
`SEQ ID NO: 1 is the nucleotide sequence of mouse GM-CSF.
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`15
`
`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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`20
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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-(the first three nucleotides are optional)
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`25
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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 membrane boundversion of CD40L.
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`SEQ ID NO: 11 is the amino acid sequence of a human membrane
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`boundversion of CD40L.
`
`30
`
`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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`10
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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
`
`5
`
`mouse CD40L.
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`SEQ ID NO: 16 is a codon optimized version of the nucleotide sequence of wild-
`
`type human CD40L.
`
`SEQ ID NO: 17 is the amino acid sequence of wild-type human CD40L.
`
`SEQ ID NO: 18 is a codon optimized version of the nucleotide sequence of wild-
`
`10
`
`type mouse CD40L.
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`SEQ ID NO: 19 is the amino acid sequence of wild-type mouse CD40L.
`
`SEQ ID NO: 20 is the nucleotide sequence of a codon optimized version of murine
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`4-1BBL.
`
`SEQ ID NO: 21 is the nucleotide sequence of a codon optimized version of human
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`4-1BBL.
`
`SEQ ID NO: 22is the nucleotide sequence of a codon optimized version of
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`secreted mouse 4-1BBL.
`
`SEQ ID NO: 23 is the nucleotide sequence of a codon optimized version of human
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`secreted 4-1BBL.
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`SEQ ID NO: 24is the nucleotide sequence of a codon optimized version of murine
`
`GITRL.
`
`SEQ ID NO: 25 is the nucleotide sequence of a codon optimized version of human
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`GITRL.
`
`SEQ ID NO: 26 is the nucleotide sequence of a codon optimized version of
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`secreted murine GITRL.
`
`SEQ ID NO: 27 is the nucleotide sequence of a codon optimized version of
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`secreted human GITRL.
`
`SEQ ID NO: 28 is the nucleotide sequence of a codon optimized version of murine
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`OX40L.
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`SEQ ID NO: 29 is the nucleotide sequence of a codon optimized version of human
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`OX40L.
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`SEQ ID NO: 30 is the nucleotide sequence of a codon optimized version of
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`secreted murine OX40L.
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`SEQ ID NO: 31 is the nucleotide sequence of a codon optimized version of
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`secreted human OX40L.
`
`SEQ ID NO: 32 is the nucleotide sequence of a codon optimized version of murine
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`ICOSL.
`
`ICOSL.
`
`SEQ ID NO: 33 is the nucleotide sequence of a codon optimized version of human
`
`SEQ ID NO: 34is the nucleotide sequence of a murine scFv CTLA-4antibody.
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`The first six and last cight nucleotides are restriction sites added for cloning purpases.
`
`SEQ ID NO: 35 is the nucleotide sequence of a murine scFv CTLA-4 antibody,
`
`The first six and last eight nacleotides are restriction sites added for cloning purposes.
`
`SEQ ID NO: 36 is the nucleotide sequence of the CMV promoter.
`
`SEQ ID NO: 37 is the nucleotide sequence of the RSV promoter.
`
`SEQ ID NO: 38 is the nucleotide sequence of BGH polyA.
`
`SEQ ID NO: 39 is the nucleotide sequence of SV40 late polyA.
`
`SEQ ID NO: 40 is the nucleotide sequence of the SV40 enhancer promoter.
`
`SEQ ID NO: 41 is the nucleotide sequence of rabbit beta-globulin (RBG) polyA.
`
`SEQ ID NO: 42 is the nucleotide sequence of GFP.
`
`SEQ ID NO: 43 is the nucleotide sequence of the MoMuLV LTRpromoter.
`
`
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`20
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`SEQ ID NO: 44is the nucleotide sequence of the EFla promoter.
`
`SEQ ID NO: 45 is the nucleotide sequence of HGH polyA.
`
`Detailed Description of the Invention
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`Qneolytic Virus
`
`The virus of the invention is oncolytic. An oncolytic virus is 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
`
`competent in tumortissue. A virusis selectively replication competent in tumortissueifit
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`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 techniquesintheart.
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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
`
`any species or strain within these larger groups. Viruses ofthe invention may be wild type
`
`G.e. unaltered from the parental virus species), or with gene disruptions or gene additions.
`
`Whichofthese is the case will depend onthe virus species to be used. Preferably the virus
`
`is a species of herpes virus, more preferablya strain of HSV, including strains of HSV1
`
`and HSV2, and is most preferablya strain of HSV1.
`
`In particularly preferred embodiments
`
`the virus of the invention is based on a clinical isolate of the virus species to be used. The
`
`clinical isolate may have been selected on the basis of it having particular advantageous
`
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`properties for the treatment of cancer.
`
`The clinical isolate may have surprisingly good anti-tumor effects comparedto
`
`other strains of the same virus isolated from other patients, wherein a patient is an
`
`individual harbouring the virus species to be tested. The virus strains used for comparison
`
`to identify viruses of the invention may be isolated from a patient or an otherwise healthy
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`(i.e. other than harboring the virus species to be tested) volunteer, preferably an otherwise
`
`healthy volunteer. HSV1 strains used to identify a virus of the invention are typically
`
`isolated from cold sores of individuals harboring HSV 1, typically by taking a swab using
`
`e.g. Virocult (Sigma) brand swab/container containing transport media followed by
`
`transport to 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
`
`typically prepared, for example by growingthe isolated viruses on BHKorverocells.
`
`Preferably, this is done following no more than 3 cycles of freeze thaw between taking the
`
`sample and it being grown on, for example, BHKorvero cells to prepare the virus stock
`
`for further use. More preferably the virus sample has undergone2 or less than 2 cycles of
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`25
`
`freeze thaw prior to preparation of the stock for further use, more preferably one cycle of
`
`freeze thaw, most preferably no cycles of freeze thaw. Lysates from the cell lines infected
`
`with the viruses prepared in this way after isolation are compared, typically by testing for
`
`the ability of the virus to kill tumorcell lines in vitro. Alternatively, the viral stocks may
`
`be stored under suitable conditions, for example by freezing, prior to testing. Viruses of
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`
`the invention have surprisingly good anti-tumor effects compared to otherstrains of the
`
`samevirus isolated from other individuals, preferably when comparedto those isolated
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`from >5 individuals, more preferably >10 other individuals, most preferably >20 other
`
`individuals.
`
`Thestocksof the clinical isolates identified for modification to produce viruses of
`
`the invention (i.e. having surprisingly good properties for the killing of tumorcells as
`
`comparedto otherviral strains to which they were compared) may be stored undersuitable
`
`conditions, before or after modification, and used to generate further stocks as appropriate.
`
`A chnical isolate is astrain of a virus species which has beenisolated frorn its
`
`natural host. The clinical isolate has preferably been isolated for the purposes of testing
`
`and comparing the clinical isolate with other clinical isolates of that virus species for a
`
`10
`
`desired property, in the case of viruses of the invention that being the ability to kill human
`
`tumor cells. CHnical isolates which may be used for comparison also include those from
`
`clinical samples present in clinical repositories, Le. previously collected for clinical
`
`diagnostic or other purposes. In cither case the clinical isolates used for comparison and
`
`identification of viruses of the invention will preferably have undergone minimal culture ia
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`15
`
`vitre prior to being tested for the desired property, preferably having only undergone
`
`sufficient culture to enable generation of sufficient stocks for comparative testing purposes.
`
`As such, the viruses used for comparisonto identify viruses of the invention may also
`
`include deposited strains, wherein the deposited strain has been isolated from a patient,
`
`preferably an HSV1 strain isolated from the cold sore ofa patient.
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`
`The virus may be a modified clinical isolate, wherein the clinical isolate kills two
`
`or more turnorcell fines more rapidly and/or at a lower dose in vitro than one or more
`
`reference clinical isolate of the sarn