(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
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`(43) International Publication Date
`13 July 2017 (13.07.2017)
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`WIPOI PCT
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`\9
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`(10) International Publication Number
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`WO 2017/118866 A1
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`(51)
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`International Patent Classification:
`CIZN 7/00 (2006.01)
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`(21)
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`International Application Number:
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`PCT/GB2017/050038
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`(22)
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`International Filing Date:
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`9 Januaiy 2017 (09.01.2017)
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`(25)
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`(26)
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`(30)
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`(71)
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`(72)
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`(74)
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`(81)
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`Filing Language:
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`Publication Language:
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`Priority Data:
`16003808
`16003816
`16003824
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`8 January 2016 (08.01.2016)
`8 Janualy 2016 (08.01.2016)
`8 January 2016 (08.01.2016)
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`English
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`English
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`GB
`GB
`GB
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`Applicant: REPLIMUNE LIMITED [GB/GB]; 69 In-
`novation Centre, Milton Park, Oxford 0X14 4RQ (GB).
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`Inventor: COFFIN, Robert; c/o Replimune Limited, 69
`Innovation Drive, Milton Park, Abingdon Oxfordshire
`OX14 4RQ (GB).
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`Agent: TUXWORTH, Pamela Mary; J A Kemp & Co, 14
`South Square, Gray's Inn, London Greater London WC1R
`5]] (GB).
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`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, G11, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN,
`KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA,
`MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG,
`N1, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS,
`RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY,
`TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN,
`ZA,ZM, ZW.
`
`(84)
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`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
`TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, KM, ML, MR, NE, SN, TD, TG).
`Published:
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`with international search report (Art. 21(3))
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`with (an) indication(s) in relation to deposited biological
`material furnished under Rule 13bis separately from the
`description (Rules 13bis.4(d)(i) and 48.2(a)(viii))
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`with sequence listing part ofdescription (Rule 5.2(a))
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`(54) Title: ENGINEERED VIRUS
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`(57) Abstract: The present invention relates to oncolytic virus comprising: (i) a GM—CSF—encoding gene; and (ii) an immune co—
`stimulatory pathway activating molecule or an immune co-stimulatory pathway activating molecule-encoding gene.
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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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`Backoround t0 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 immunogcnic signals to the host. Thcsc immune responses may result in thc
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`immunogenic benefit to cancer patients such that immune responses to tumor antigens
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`provide a systemic overall benefit resulting in the treatment of tumors which have not been
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`infected with the virus, including micro-metastatic disease, and providing vaccination
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`against relapse.
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`The combined direct (‘oncolytic’) effects of the virus, and immune responses
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`against tumor antigens (including non—self ‘neo—antigens’, i.e. derived from the particular
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`mutated genes in individual tumors) is termed ‘oncolytic immunotherapy’.
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`Viruses may also be used as delivery vehicles (‘vectors’) to express heterologous
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`genes inserted into the Viral genome in infected cells. These properties make viruses
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`useful for a variety ofbiotechnology 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 pathogcnic, 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 ICP34.5 deleted, or ICP34.5 and ICP6 deleted, HSV have also shown
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`safety and selective replication in tumor tissue in humans.
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`As discussed above, an oncolytic virus, including HSV, may also be used to deliver
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`a therapeutic gene in the treatment of cancer. An ICP34.5 deleted virus ofthis 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 LAfi-3, 'HM —3,
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`VISTA, CSFlR, lDG, CEACAMl, CD47. Optimal clinical activity of these agents, for
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`example Pill , PDlsl, LAG—3, 'l‘l lVl—3, VlS’l‘A, CS? l R, lDO, 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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`i.e. including targeting et't‘ne intertaee at” immune ellecter cells with tinners er ether
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`immune inhibitory mechanisms iii/0f tumers.
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`ln some cases, mere localised presence in
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`eg. just some tnrners er in seine lyinpli netles may also be optimally et‘lee'tive, for example
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`agents targeting C’l'LAJl.
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`An alternative approach to increasing the anti~tunior immune response in cancer
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`patients is tn target (activate) immune co-stimnlateiy pathways, i.e. in contrast to inhibiting
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`iininnitie co~inhihitery pathways. These pathways send activating signals into T cells and
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`other immune cells, usually resulting freni the interactien of the relevant ligands on antigen
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`presenting cells (Al’Cs) and the relevant receptors on the surface of’l‘ cells and, other
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`immune cells. These signals, depending on the ligandfreceptor, can result in the increased
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`activation of '1“ cells and/"or APCs and/or NK cells and/or l3 cells, including particular suh-
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`types, increased differentiation and proliferation ofT cells and/or APCS anti/or NE: cells
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`and/or B cells, including particular subtypes. or suppression of the activity ol‘inininne
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`inhibitory T cells such as regulatory T cells. Activation of these pathways would therefore
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`he expected to result in enhanced anti—turner immune responses, hut it might also be
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`expected that systemic activation of these pathways, i.e. activation of immune responses
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`generally rather than anti—tun‘ior immune responses specifically or selectively, would result
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`in considerable off target toxicity in nonwtunior 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 rte—stimulatory pathways, including agents targeting GiTR, Zl—
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`l~i333, 0X48, {23340 or EGGS, 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 neo—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 potentiating 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 use of an oncolytic virus to deliver molecules targeting immune co-stimulatory
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`pathways to tumors focuses the amplification of immune effects on anti—tumor immune
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`responses, and reduces the amplification of immune responses to non—tumor antigens.
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`Thus, immunc cclls 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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`immune co-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 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—C SAP—encoding gene; and (ii) an immune constimulatory pathway activating molecule
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`or immune co—stimulatory pathway activating moleculeueneodina gene. "l‘he virus may
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`encode more than one immune eat—stimulatory pathway activating molecule/gene.
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`The immune rte—stimulatory pathway activating molecule is preferably Gl’l‘Rld, 4—i—
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`BBL, OX-‘QOL, lCOSL or CDz-‘lvGL or a modified version of any thereof or a protein capable
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`ol’blocltiug signaling through Cl‘liAsél, for example an antibody which hinrls C'l‘chJl.
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`Examples of modified versions include agonists of a co—stimulatory pathway that are
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`secreted rather than heing membrane bound, anal/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 ol‘a
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`virus, wherein the clinical isolate ltills two or more turner cell lines more rapidly anti/or at
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`a lower dose in vino than one or more reference clinical isolates of the same species of
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`virus.
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`The virus is preferably a herpes simplex virus (HSV), such as HSVl. The HSV
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`typically does not express functional lC 134.5 anti/or lunctional lCP47 anti/or expresses the
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`USl l gene as an immediate early gene.
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`The invention also prrwitles:
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`—
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`--
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`a
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`a pharmaceutical composition comprising a virus of the invention and a
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`pharmaceutically acceptahle carrier or rliluent;
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`the virus of the in tention for use in a method of treating the human or animal body
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`by therapy;
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`the virus of the invention for use in a method of treating cancer, wherein the
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`method optionally comprises administering a further anti—cancer agent;
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`-
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`—
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`a product ofntanutacture comprising a virus ofthe invention in a steriie viai,
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`ainpouie or syringe;
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`a method of treating cancer, which comprises administering a therapeuticaliy
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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 optionaily comprises administering a
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`further anti—cancer agent which is optionaiiy an antagonist of an immune cow
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`inhihitory pathway; or an agonist of an immune cmstimnEatery 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 optionaiiy comprises administering
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`a further antiwcaneer agent which is optionaiiy an antagonist of an immttne c0»
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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 indoleaminc 2,3-
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`dioxygenase (lDO) pathway and a filrther 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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`Figure ’2 shows the difierential abilities of the eight top ranking HS V 1 ciinicai
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`isoiate strains as assessed by crystal vioiet staining 24 hours or 48 hours after infection
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`with a MGE oft}! , 6,0? or one: as indicated in the Figure to kit} Fadu, SK angst—28, A549,
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`HTMEBG, M EAmPAwCA—Q, H’TZ‘) and MDA—h’iB-"’31 human tumor ceii iines. The virus
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`strains ranked first and second on each ceii iine are indicated The virus Ri'im 8A was
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`ranked first on each of the Fadtt, H'fi‘ifiiiii, M EAwi’AmCA-Z and HT29 ceii lines and second
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`on each of the SKsi’nei—ZSR, A549 and MDA—h/‘iB—ZS] celi tines, RHOQltA was rankedjoint
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`first with RHO'E 8A and RHGiSA on the H7529 ceii tine: first on the SK—meE-QS and A549
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`ceii tines and second on the Fade ceii line. RHGZSA was ranked first. on the MDAnMB«
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`23,1 ccii iinc and second on the i-i'i‘iQSO ecii iine.
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`iii-{031135) was ranked second on each of
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`the MiA~PA~CA—2 and H129 ceil iines. RfiUéiUA was ranked joint second on the H129
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`Figure 3 shows a comparison between strain Rl-lm 8A, the strain ranked tirst of all
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`the strains tested, with an ‘average" strain frem the screen (ie. strain RHGéSA).
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`Approximately it) told less of strain Ri—iOl 8A was needed to ltill an equal proportion of
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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 Mills of 8.1, {3.01 and 8.003 in, SK—ntel—‘Z‘é, HTlOSO, PV’lDA“l\’i,8~23 l ,
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`Fadu, Mlfi‘i—FA—CA—Z and A549 cell lines
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`Figures 4 and 5 depict. structures ol’HSVl viruses modified by the deletion of
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`:ECF‘345 and ICP47 such that the USl l gene is under control of the lCP457 immediate
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`early promoter and containing heterologous genes in the lCP34.5 lecus. The viruses were
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`constructed using the RHQiSA 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 RHOlSA 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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`injected 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 PDl alone (lOmg/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 PD] together with Virus 16; anti-mouse
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`CTLA4 together with Virus 16; 1-methyl trypotophan (I-MT; IDO inhibitor (Sing/1111 in
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`drinking water)); anti-mouse PDl together with 1-methyl trypotophan; or anti-mouse PDl
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`together with 1-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—PDl in combination has a better anti—tumor effect than using
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`either anti-PDl or the virus alone. Figure 11B shows that thc anti-tumor effect of Virus 16
`
`in combination with anti-CTLA-4 was better than the anti-tumor effect of either Virus 16
`
`or anti-CTLA-4 alone. Figure 11C shows that enhanced tumor reduction was observed
`
`using Virus 16 together with both anti-PDl and IDO inhibition as compared to anti-PDl
`
`and 1-MT inhibition in the absence of the virus.
`
`Figure 12 shows the enhanced anti-tumor activity of Virus 16 in combination with
`
`immune checkpoint blockade in mouse A20 tumors in both flanks of Balb/c mice as
`
`compared to either virus alone or checkpoint blockade alone (anti—PD1).
`
`Figure 13 shows the structure of ICP34.5 and ICP47 deleted viruses expressing
`
`GALVR-, GM-C SF and codon optimized anti-mousc or anti-human CTLA-4 antibody
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`constructs (secreted scFv molecules linked to human or mouse IgGl Fc regions). The
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`scFvs contain the linked ([G4S]3) light and heavy variable chains from antibody 9D9
`
`(US2011044953: mouse version) and from ipilimumab (US20150283234; human version).
`
`The resulting structure of the CTLA-4 inhibitor is also shown.
`
`Figure 14 shows anti-tumor effects of Virus 16 and Virus 19 in a human xenograft
`
`model (A549). There were three injections of Virus 16, Virus 19 or of vehicle over one
`
`week at three different dose levels (N=10/group). The doses of the viruses used is
`
`indicated. The anti—tumor effects of Virus 16 which expresses GALV were better than
`
`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-
`
`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; 50ul of 107 pfu/ml of Virus 19 (expresses mGM-CSF but not
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`GALV R—); or 50ul 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
`
`compared to the virus expressing GM-CSF alone.
`
`Figure 16 shows the anti-tumor effects 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
`
`mGM—CSF and GALV—R— compared to virus 16 (expresses GALV and mGM—CSF).
`
`Brief Description of the Seguenee Listing
`
`SEQ ID NO: 1 is the nucleotide sequence of mouse GM-CSF.
`
`SEQ ID \10: 2 is the nucleotide sequence of a codon optimized version of mouse
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`GM-CSF.
`
`SEQ ID NO: 3 is the nucleotide sequence of human GM-CSF.
`
`SEQ ID NO: 4 is the nucleotide sequence of a codon optimized version of human
`
`GM—CSF.
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`
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`20
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`25
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`30
`
`SEQ ID NO: 5 is the amino acid sequence of mouse GM—CSF.
`
`SEQ ID NO: 6 is the amino acid sequence of human GM—CSF.
`
`SEQ ID NO: 7 is the nucleotide sequence of GALV-R-.
`
`SEQ ID \IO: 8 is the nucleotide sequence ofa codon optimized version of GALV-
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`R- (the first three nucleotides are optional)
`
`SEQ ID NO: 9 is the amino acid sequence of GALV-R-.
`
`SEQ ID NO: 10 is the nucleotide sequence of a codon optimized version of a
`
`human membrane bound version of CD40L.
`
`SEQ ID NO: 11 is the amino acid sequence of a human membrane
`
`bound version of CD40L.
`
`SEQ ID NO: 12 is the nucleotide sequence of a codon optimized version of a
`
`multimeric secreted version of human CD40L.
`
`SEQ ID NO: 13 is the amino acid sequence ofa multimeric secreted version of
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`human CD40L.
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`SEQ ID NO: 14 is the nucleotide sequence of a codon optimized version of a
`
`multimeric secreted version of mouse CD40L.
`
`SEQ ID NO: 15 is the amino acid sequence of a multimeric secreted version of
`
`5
`
`mouse CD40L.
`
`SEQ ID NO: 16 is a codon optimized version of the nucleotide sequence ofwild-
`
`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-
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`10
`
`type mouse CD40L.
`
`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
`
`4-1BBL.
`
`SEQ ID N O: 21 is the nucleotide sequence of a codon optimized version of human
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`15
`
`4-1BBL.
`
`SEQ ID NO: 22 is the nucleotide sequence of a codon optimized version of
`
`secreted mouse 4-1BBL.
`
`SEQ ID NO: 23 is the nucleotide sequence of a codon optimized version of human
`
`secreted 4—1BBL.
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`20
`
`SEQ ID NO: 24 is the nucleotide sequence of a codon optimized version of murine
`
`GITRL.
`
`SEQ ID NO: 25 is the nucleotide sequence of a codon optimizcd version of human
`
`GITRL.
`
`SEQ ID NO: 26 is the nucleotide sequence of a codon optimized version of
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`25
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`secreted murine GITRL.
`
`SEQ ID NO: 27 is the nucleotide sequence of a codon optimized version of
`
`secreted human GIT RL.
`
`SEQ ID NO: 28 is the nucleotide sequence of a codon optimized version of murine
`
`OX40L.
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`30
`
`SEQ ID NO: 29 is the nucleotide sequence of a codon optimizcd version of human
`
`OX40L.
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`SEQ ID NO: 30 is the nucleotide sequence of a codon optimized version of
`
`secreted murine OX40L.
`
`SEQ ID NO: 31 is the nucleotide sequence of a codon optimized version of
`
`secreted human OX40L.
`
`SEQ ID N O: 32 is the nucleotide sequence of a codon optimized version of murine
`
`ICOSL.
`
`SEQ ID NO: 33 is the nucleotide sequence of a codon optimized version of human
`
`ICOSL.
`
`SEQ. ID NO: 34 is the nucleotide sequence of a murine seFv CTLA~4 antibody.
`
`10
`
`The first six and last eight nucleotides are restriction sites added for Cloning purposes.
`
`SEQ ID NO: 35 is the nueieetide sequence of a murine seFv CTLA—4 antibody.
`
`The first six and last eight nucleotides are restriction sites added fer cloning purpeses.
`
`SEQ ID NO: 36 is the nuclcotidc sequence of the CMV promotcr.
`
`SEQ ID NO: 37 is the nucleotide sequence of the RSV promoter.
`
`SEQ ID \10: 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.
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`20
`
`SEQ ID NO: 43 is the nucleotide sequence of the MoMuLV LTR promoter.
`
`SEQ ID NO: 44 is the nucleotide sequence of the EFla promoter.
`
`SEQ ID NO: 45 is the nuclcotidc sequence of HGH polyA.
`
`Detailed Description of the Invention
`
`25
`
`()fléfffliiiiit‘? 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 if it
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`30
`
`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 ot‘the iirveittien may be wild type
`
`(i.e. unaltered from the parental virus species), er with gene disruptions er gene additions.
`
`Wl'iieh et‘tnese is the case will depend on the virus species to be used, Preferably the virus
`
`is a species eflierpes virus, more preferably a strain 0f HSV, ineiuding strains (if ilSVl
`
`and HSVZ, and is it‘lGSl preferably a strain 53f t'i-SV l .
`
`in particularly preferred embedintents
`
`the virus of the in rention is based on a clinical isetate of the virus species t0 be used. The
`
`elinieal isetate may have been selected on the basis of it having particular advantageous
`
`10
`
`properties 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 bc 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