WO 2017/118866
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`PCT/GB2017/050038
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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 ovcrall bcncfit 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 tumor, 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 vitro, 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 1CP34.5 deleted, or lCP34.5 and lCP6 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-CSF 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-CSF 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 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 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 (i.e.
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`inhibition or ‘antagonism’ of immune checkpoint pathways, also termed immune co—
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`inhibitory pathways). Checkpoint (immune inhibitory pathway) blockade is intended to
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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 mechanisms can also serve to 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-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 liAt3~3, ’"l'll‘vl —3,
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`VISTA, CSFlR, lDG, CEACAML CD47. Optimal clinical activity of these agents, fer
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`example PDl , l’Dldl , LAG-3, ’l‘ll‘vlw3, VlS'l‘A, (:Sli'l R, llIEO, CD47, CEACAM l, may
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`require systemic administration or presence in all tumors clue tn the mechanism of action,
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`l.e. including targeting el’tlie interface of immune ellecter cells with tiimers er other
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`immune inhibitory mechanisms in/ef tumnrs.
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`in some eases, mere localised presence in
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`eg. just some tumers er in seine lyl‘l’lpl‘l nedes may alse be optimally elletttive, for example
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`agents targeting C’l'LA~4.
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`An alten'iative approach, to increasing the anti~tumor immune response in cancer
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`patients is to target (activate) immune ce-stinitilateiy pathways, i.e. in centi'ast tn inhibiting
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`immune cal-inhibitory pathways. These patl'tways send activating signals into T cells and
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`other immune cells, usually resulting from the interactien of the relevant ligands on antigen
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`presenting cells (APCs) and the relevant reeeptei‘s en the surface of T cells and ether
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`immune cells. These signals, epending on the ligand/receptor, can result in the increased
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`activation oi"? cel is and/or APCs and/er NK cells and/or 8 cells, including particular sub-
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`types, increased differentiation and proliferation ofT cells and/or APCs and/or NK cells
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`andfoi' B cells, including particular subtypes, er suppressien of the activity efimniane
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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—turner innnune responses, but it might also be
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`expected that systemic activation of these patlmrayse i.e. 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 in nen~tumor tissue, the degree of such off target toxicity
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`depending on the particular immune rte—stimulatory pathway being targeted Nevertheless
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`agents (mainly agonistic antibodies, or less frequently the soluble ligand to the receptor in
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`question) targeting immune tie—stimulatory pathways, including agents targeting Gl'l‘R, 4—
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`l—BB, 0X48, CD48 or ECOS, and intended for systemic use (i.e. intravenous delivery) 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, i.e. so that
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`a 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 nee-antigens appear to be particularly important for the activity of immune co-
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`inhibitory pathway blockade and related drugs. Only some patients may have an ongoing
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`immune 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 co-inhibitory pathway blockade and
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`immune potcntiating drugs. This likely explains the promising combined anti-tumor
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`effects of oncolytic agents and immune co-inhibitory pathway blockade in mice and
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`humans that 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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`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 usc of an oncolytic virus to dclivcr molcculcs targcting immunc 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. 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—CSF 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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`immunc co-stimulatory pathway or pathways and GM-CSF. Oncolytic viruscs 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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`GM—CSF—encoding gene; and (ii) an immune constimulatory pathway activating molecule
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`or immune eo—stimulatory pathway activating moleculeueneoding gene. The virus may
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`encode more than one immune eel—stimulatory pathway activating molecule/gene.
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`The immune rte—stimulatory pathway activating molecule is preferably Tsl’l‘R l9, 4~l~
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`BBL, QX-‘QOL, lCOSL or CDK-‘lGL or a modilied version of any thereof or a protein capable
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`ot‘blocleing signaling through (Villa/5&4, for example an antibody which binds {Till/3‘4.
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`Examples of modified versions include agonists of a constimnlatory pathway that 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 i’nay be a modified clinical isolate, such as a modified clinical isolate ot‘a
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`virus, wherein the clinical isolate kills two or more tumor cell lines more rapidly an d/or at
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`a lower dose in vine than
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`_
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`a product of manufacture comprising a virus oi‘the invention in a steriie viai?
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`ampouie or syringe;
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`a method of treating cancer, which con’iprises administering a therapeuticaiiy
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`effective amount ot‘a virus or a pharmaceutical composition of the invention to a
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`patient in need thereof, wherein the method optionaiiy comprises administering a,
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`further anti-cancer agent which is optionaiiy an antagonist of an immune com
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`inhibitory pathway; or an agonist of an immune eo~stimuiatory pathway;
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`—
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`use of a virus of the invention in the manufacture of a 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 antiwcaneer agent which is optionaiiy an antagonist of an immune co—
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`inhihitory pathway; or an agonist of an immune cowstimuiatory pathway;
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`—
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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 fiirther 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 CD40L.
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`F i gure 2 shows the differential abilities of the eight top ranking HSVI ciinicai
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`isoiate strains as assessed by crystal violet staining 24 hours or 48 hours after infection
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`with a MOE of (it 9 {3,9} or 0.801 as indicated in the Figure to kit} Fadu. SK.~i'nel~28, AME}.
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`H’I‘itiéit}, MtAwPAwCA—l H129 and MBA—M8331 human tumor ceii iines. The virus
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`strains ranked first and second on each ceii tine are indicated. The virus Rim} 8A was
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`ranked first on each of the Fadu, HTMESG, MEAJ’AJJA—E and H129 ecii lines and second
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`on each of the SKsmei—Zhg A549 and MDA~MB~231 celi hnes, Ri’iBQIE-A was ranked joint
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`first with RHG'S 8A and RHIMSA on the H1339 ceil tine, first on the SKnmei-Zti and A549
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`eeii tines and second on the Fade ceii tine. RHGZSA was ranked first on the MBA—Wigs
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`231 eeii tine and second on the HTIGBO eeii iine, RE—iOBiA was ranked seeond on each of
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`the hiIA—PA~CA—2 and HT29 ceil tines. RHGKEGA was "aiihedjoint second on the PIT/'39
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`Figure 3 shows a comparison between strain RHOl 8A, the strain ranked lirst of all
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`the strains tested, with an ‘average" strain from the screen (ie. strain RHGéSA).
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`Approximately 10 fold less of strain Ri—i’Ol 8A was needed to ltill an equal proportion ef
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`cells than was needed of strain RHGéSA as shown by crystal violet staining 24 or 48 hours
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`post infection with MOis ofthi, 0.01 and 0.001 in SK—n’iel—‘ZX, HTlOSQ, MDA—MBQS i ,
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`Fadu, MlA--PA--C.I—\--2 and A549 celi lines.
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`Figures 4 and 5 depict structures ul’HSVi viruses modified by the deletion of
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`1(33845 and lCP47 such that the USE '3, gene is under control of the ECP457 immediate
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`early promoter and containing heterologous genes in the lCF345 lncus, The viruses were
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`constructed using the RHOESA strain unless otherwise stated in the Figure.
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`Figure 6 shows the results of an ELISA to detect expression of human or mouse
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`GM-CSF in supematants from BHK cells 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 between the cell-killing abilities of strain RH018A in
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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 between the cell-killing abilities of strain RH018A in
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`which ICP34.5 and ICP47 are deleted and which expresses GALVR- and GM-CSF (virus
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`17) with a prior art strain with the same modifications as determined by crystal violet
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`staining in four cell lines.
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`Figure 9 shows the effectiveness of Virus 16 (ICP34.5 and ICP47 deleted
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`expressing GALVR— and mGM—CSF) in treating mice harbouring A20 lymphoma tumors
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`in both flanks. Tumors on the right flanks were injected with the virus or vehicle and the
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`effects on tumor size was observed for 30 days. The virus was effective against both
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`inj cctcd tumors and non-injected tumors.
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`Figure 10 demonstrates the effects of Virus 15 (ICP34.5 and ICP47 deleted
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`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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`(Virus 15) showed enhanced killing of rat 9L cells in vitro as compared to a virus which
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`does not express GALV (Virus 24).
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`Figure 11 shows the antitumor effects of Virus 16 in Balb/c mice harboring mouse
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`CT26 tumors in 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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`(mRPl) injected in the right flank tumor every other day; anti-mouse PD1 alone (10mg/kg
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`i.p. 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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`CTLA4 together with Virus 16; 1—methyl trypotophan (I—MT; IDO inhibitor (5mg/ml in
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`drinking water)); anti—mouse PD1 together with 1—methyl trypotophan; or anti—mouse PD1
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`together with l-methyl trypotophan and Virus 16. Effects on tumor size were observed for
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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 has a better anti-tumor effect than using
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`either anti-PD1 or the virus alone. Figure 11B shows that the anti-tumor effect ofVirus 16
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`in combination with anti-CTLA-4 was better than the anti-tumor effect of either Virus 16
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`or anti-CTLA-4 alone. Figure 11C shows that enhanced tumor reduction was observed
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`using Virus 16 together with both anti-PD1 and IDO inhibition as compared to anti-PD1
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`and 1-MT inhibition in the absence of the virus.
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`Figure 12 shows the enhanced anti-tumor activity of Virus 16 in combination with
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`immune checkpoint blockade in mouse A20 tumors in both flanks of Balb/c mice as
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`compared to either virus alone or checkpoint blockade alone (anti-PD1).
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`Figure 13 shows the structure of ICP34.5 and ICP47 deleted viruses expressing
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`GALVR—, GM—CSF and codon optimized anti—mouse or anti—human CTLA—4 antibody
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`constructs (secreted scFv molecules linked to human or mouse IgG1 Fc regions). The
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`scFvs contain the linked ([G4S]3) light and heavy variable chains from antibody 9D9
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`(US2011044953: mouse version) and from ipilimumab (U820150283234; human version).
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`The resulting structure of the CTLA—4 inhibitor is also shown.
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`Figure 14 shows anti—tumor effects 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
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`week at three different dose levels (N=10/group). The doses of the viruses used is
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`indicated. The anti-tumor effects 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
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`three weeks: 50ul of vehicle; 50ul of 107 pfu/ml of Virus 19 (expresses mGM-CSF but not
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`GALV R—); or Soul of 107 pfu/ml of Virus 16 (expresses both mouse GM-CSF and GALV-
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`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
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`compared to the virus expressing GM—CSF alone.
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`Figure 16 shows the anti-tumor effects of viruses expressing anti-mCTLA-4 (virus
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`27), mCD4OL (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 Seguence Listing
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`SEQ ID \IO: 1 is the nucleotide sequence ofmouse GM-CSF.
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`SEQ ID NO: 2 is the nucleotide sequence of a codon optimized version of mouse
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`GM-CSF.
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`SEQ ID NO: 3 is the nucleotide sequence of human GM-CSF.
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`SEQ ID NO: 4 is the nucleotide sequence of a codon optimized version of human
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`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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`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 bound version of CD40L.
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`SEQ ID NO: 11 is the amino acid sequence of a human membrane
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`bound version of CD40L.
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`SEQ ID NO: 12 is the nucleotide sequence of a codon optimized version of a
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`multimeric secreted version of human CD40L.
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`SEQ ID NO: 13 is the amino acid sequence of a multimeric secreted version of
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`4-1BBL.
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`4-1BBL.
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`SEQ ID \IO: 21 is the nucleotide sequence ofa eodon optimized version of human
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`human CD40L.
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`SEQ ID NO: 14 is the nucleotide sequence ofa codon optimized version ofa
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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 multimerie secreted version of
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`5
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`mouse CD40L.
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`SEQ ID NO: 16 is a codon optimized version of the nucleotide sequence of Wild—
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`type human CD40L.
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`SEQ ID NO: 17 is the amino acid sequence of Wild—type human CD40L.
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`SEQ ID NO: 18 is a codon optimized version of the nucleotide sequence of Wild-
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`type mouse CD40L.
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`SEQ ID NO: 19 is the amino acid sequence of Wild-type mouse CD40L.
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`SEQ ID NO: 20 is the nucleotide sequence ofa codon optimized version of murine
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`SEQ ID NO: 22 is the nucleotide sequence of at 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: 24 is the nucleotide sequence of a codon optimized version of murine
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`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 ofa codon optimized version of murine
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`OX40L.
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`30
`
`SEQ ID NO: 29 is the nucleotide sequence of a eodon 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.
`
`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.
`
`SEQ ID NO: 33 is the nucleotide sequence of a codon optimized version of human
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`ICOSL.
`
`SEQ. ID NO: 34 is the nucleotide sequence ofa murine seFv CTLA~4 antibedy.
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`The first six and iast eight nucleotides are restriction sites added. for Cloning purposes.
`
`SEQ iD NO: 35 is the nucleotide sequence of a murine scFv C'I‘LA—4 antibody,
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`The first six and East eight nucleotides are restriction sites added for ciening purpeses.
`
`SEQ ID NO: 36 is the nucleotide sequence of the CMV promoter.
`
`SEQ ID \IO: 37 is the nucleotide sequence of the RSV promoter.
`
`SEQ ID NO: 38 is the nucleotide sequence of BGH polyA.
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`SEQ ID NO: 39 is the nucleotide sequence of SV40 late polyA.
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`
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`SEQ ID NO: 40 is the nucleotide sequence of the SV40 enhancer promoter.
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`SEQ ID NO: 41 is the nucleotide sequence of rabbit beta-globulin (RBG) polyA.
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`SEQ ID NO: 42 is the nucleotide sequence of GFP.
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`SEQ ID NO: 43 is the nucleotide sequence of the MoMuLV LTR promoter.
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`SEQ ID NO: 44 is the nucleotide sequence of the EF1a promoter.
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`15
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`20
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`SEQ ID NO: 45 is the nucleotide sequence of HGH polyA.
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`Detailed Description of the Invention
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`25
`
`Oncoivfic Virus
`
`The virus of the invention is oncolytic. An oncolytic virus is a virus that infects
`
`and replicates in tumor cells, such that the tumor cells are killed. Therefore, the virus of
`
`the invention is replication competent. Preferably, the virus is selectively replication
`
`competent in tumor tissue. A virus is selectively replication competent in tumor tissue ifit
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`replicates more effectively in tumor tissue than in non-tumor tissue. The ability of a virus
`
`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
`
`herpes virus, pox virus, adenovirus, retrovirus, rhabdovirus, paramyxovirus or reovirus, or
`
`any species or strain within these larger groups. Viruses of the invention may be wild type
`
`(ie. unaltered from the parental virus species), or with gene disruptions er gene additions.
`
`Which of these is the case will depend on the virus species to be used. Preferably the virus
`
`is a species ef herpes virus, more preferably a strain of HSV, including strains ef HSVE
`
`and HSVZ, and is most preferably a strain of i’i-SV l .
`
`in particularly preferred embodiments
`
`the virus ef the in rentien is based on a clinical isetate cf the virus species te he used. The
`
`clinical iselate may have been selected on the basis of it having particular advantageous
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`preperties fer the treatment of cancer.
`
`The clinical isolate may have surprisingly good anti-tumor effects compared to
`
`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
`
`(i.e. other than harboring the virus species to be tested) volunteer, preferably an otherwise
`
`healthy volunteer. HSVl strains used to identify a virus of the invention are typically
`
`isolated from cold sores of individuals harboring HSVl, 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 further testing.
`
`After isolation of viruses to be compared from individuals, stocks of the viruses are
`
`typically prepared, for example by growing the isolated viruses on BHK or vero cells.
`
`Preferably, this is done following no more than 3 cycles of freeze thaw between taking the
`
`sample and it being grown on, for example, BHK or vero cells to prepare the virus stock
`
`for filrther use. More preferably the virus sample has undergone 2 or less than 2 cycles of
`
`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 tumor cell lines in vitro. Alternatively, the viral stocks may
`
`be stored under suitable conditions, for example by freezing, prior to testing. Viruses of
`
`the invention have surprisingly good anti-tumor effects compared to other strains of the
`
`same virus isolated from other individuals, preferably when compared to those isolated
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`from >5 individuals, more preferably >10 other individuals, most preferably >20 other
`
`individuals.
`
`The stocks of the clinical isolates identified for modification to produce Viruses of
`
`the invention (i.e. having surprisingly good properties for the killing of tumor cells as
`
`compared to other Viral strains to which they were compared) may be stored under suitable
`
`conditions, before or after modification, and used to generate further stocks as appropriate.
`
`A clinical isolate is a strain ofa virus species which has been isolatetl from 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
`
`desired property, in the case of viruses of the invention that being the ability to kill human
`
`tumor cells. Clinical 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 either case the clinical isolates used for comparison and
`
`identification of viruses of the invention will preferably have undergone l’l‘lll‘lllllal culture in
`
`'viiro 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 comparison to identify viruses of the invention may also
`
`include deposited strains, wherein the deposited strain has been isolated from a patient,
`
`preferably an HSVl strain isolated from the cold sore of a patient.
`
`The virus may he a modified clinical isolate, wherein the clinical isolate kills two
`
`or more tumor cell lines more rapidly anti/"or at a lower close in virm than one or more
`
`reference clinical isolate of the same species of virus. Typically, the clinical isolate will
`
`kill two or more tinnor cell lines within '72 hours, preferably within 48 hours, more
`
`pre "erably within 24 hours, of infection at multiplicities of infection (MOI) of less than or
`
`equal to