PCTIGBZO17/050038
`
`Concept House
`Cardiff Road
`
`Newport
`South Wales
`
`NP 10 8QQ
`
`I, the undersigned, being an officer duly authorised in accordance with Section 74(1) and (4)
`of the Deregulation & Contracting Out Act 1994, to sign and issue certificates on behalf of
`the Comptroller—General, hereby certify that annexed hereto is a true copy of the documents
`as stored electronically on the Patents Electronic Case file System in connection with patent
`application
`GB1600381.6
`filed on 8th January 2016
`
`The Patents Electronic Case-file System is compliant with British Standard BS10008 —
`Evidential weight and legal admissibility of information stored electronically and 1S015801 —
`Electronic imaging — information stored electronically, recommendations for trustworthiness
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`Signed
`
`Cb“
`
`Dated
`
`12th January 2017
`
`UK Intellectual Property Office is an operating name of the Patent Office
`
`

`

`. *
`
` _
`Intellectual
`
`Property.
`OffiCe
`
`1200178714
`
`08/01/2016 0.00 NONE
`
`Patents Form 1
`Patents Act 1977 (Rule 12)
`
`Request for grant of a patent
`
`.
`.
`Application number
`1. Your reference
`
`GB 16003816
`
`2.
`
`Full name, address and postcode of the applicant or of
`each applicant
`
`Concept House
`CardiffRoad
`Newport
`South Wales
`NP10 SQQ
`
`N406715GB
`
`Replimune Limited
`The Magdalen Centre Oxford Science Park
`Robert Robinson Avenue
`Oxford OX4 4GA
`
`Patents ADP number (ifyou know it)
`1 1627528001
`
`3. Title of the invention
`VIRUS STRAIN
`
`United Kingdom
`
`4. Name of your agent (ifyou have one)
`“Address for service" to which all correspondence should
`be sent. This may be in the European Economic area or
`Channel Islands (see warning note below)
`(including the postcode)
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`Patents ADP number (ifyou know it)
`5. Priority declaration: Are you claiming priority from one or
`more earlier-filed patent applications? If so, please give
`details ofthe application(s)
`
`J A Kemp
`J A Kemp
`14 South Square
`Gray's Inn
`London WC1 R 5JJ
`Greater London
`
`United Kingdom
`
`10645901001
`
`Country
`
`Application number
`
`Date of filing
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`PDAS Access Code
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`6. Divisionals etc: Is this application a divisional application,
`or being made following resolution of an entitlement
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`lnventorship: (Inventors must be individuals not
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`app'mat'on
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`(REV DEC07)
`Intellectual Property Office is an operating name ofthe Patent Office
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`Patents Form 1(e)
`https://www.gov.uk/ipo
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`Patents Form 1
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`Continuation sheets of this form
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`Description:
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`Claim(s):
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`Abstract:
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`If you are n_ot filing a description, please give details of
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`11. l/We request the grant of a patent on the basis of this application.
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`Any other documents (please specify): Sequence Listing
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`Signature: Subject: Pamela Tuxworth 23470; Issuer:
`European Patent Office, European Patent
`
`Office CA GZ
`
`08 Jan 2016
`
`Date:
`
`12. Name, e-mail address, telephone, fax and/or mobile
`number, if any, of a contact point forthe applicant
`
`TUXWORTH, Ms. Pamela Mary
`Email: mail@jakemp.com
`Telephone: +44 20 3077 8600
`Fax: +44 20 7430 1000
`
`Warning
`After an application for a patent has been filed, the Comptroller will consider whether publication or communication of the
`invention should be prohibited or restricted under section 22 of the Patents Act 1977. You will be informed if it is necessary to
`prohibit or restrict your invention in this way. Furthermore, if you are resident in the United Kingdom and your application contains
`information which relates to military technology, or would be prejudicial to national security or the safety of the public, section 23 of
`the Patents Act 1977 prohibits you from applying for a patent abroad without first getting written permission from the Office unless
`an application has been filed at least 6 weeks beforehand in the United Kingdom for a patent for the same invention and either no
`direction prohibiting publication or communication has been given, or any such direction has been revoked. Until such time or until
`the revocation of any direction, for any such application the address for service referred to at part 4 above must be in the United
`Kingdom.
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`Although you may have an address for service in the Channel Islands, any agent instructed to act for you must reside or have a
`place of business in the European Economic Area or Isle of Man.
`
`(REV DEC07)
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`Patents Form 1(e)
`
`

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`w
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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
`
`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.
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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. toll-like receptors
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`and cGAS/ STING signalling resulting in the activation of interferon responses and
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`inflammation which are also immunogenic signals to the host. These immune responses
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`may result in the immunogenic benefit to cancer patients such that immune responses to
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`tumor antigens provide a systemic overall benefit resulting in the treatment of tumors
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`which have not been infected with the virus, including micro-metastatic disease, and
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`providing vaccination 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 increase the immunogenicity of antigens released following
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`virus replication and cell death, to increase the general immune activation status of the
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`tumor, or to increase the direct oncolytic properties (i.e. cytotoxic effects) of the virus.
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`It has been demonstrated that a number of viruses including herpes simplex virus
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`(HSV) have utility in the oncolytic treatment of cancer. HSV for use in the oncolytic
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`treatment of cancer must be disabled such that it is no longer pathogenic, but can still enter
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`into and kill tumor cells. A number of disabling mutations to HSV, including disruption of
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`the genes encoding ICP34.5, ICP6, and/or thymidine kinase, have been identified which do
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`not prevent the virus from replicating in culture or in tumor tissue in vivo, but which
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`prevent significant replication in normal tissue. HSVs in which only the ICP34.5 genes
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`have been disrupted replicate in many tumor cell types in vitro, and replicate selectively in
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`tumor tissue, but not in surrounding tissue, in mouse tumor models. Clinical trials of
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`ICP34.5 deleted, or ICP34.5 and ICP6 deleted, HSV have also shown safety and selective
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`replication in tumor tissue in man.
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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. The trial data demonstrated that tumor responses could be
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`seen in injected tumors, and to a lesser extent in uninj ected tumors. Responses tended to
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`be highly durable (months-years), and a survival benefit appeared to be achieved in
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`responding patients. Each of these indicated engagement of the immune system in the
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`treatment of cancer in addition to the direct oncolytic effect. However, this and other data
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`with oncolytic viruses generally showed that not all tumors respond to treatment and not all
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`patients achieve a survival advantage. Thus, improvements to the art of oncolytic therapy
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`and oncolytic immunotherapy are clearly needed. These may serve to increase the direct
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`oncolytic effects of therapy, the anti-tumor immune stimulating effects of the therapy, or
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`both of these effects together.
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`Recently it has been shown that oncolytic immunotherapy can result in additive or
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`synergistic therapeutic effects in conjunction with immune checkpoint blockade (ie.
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`inhibition or ‘antagonism’ of immune checkpoint pathways). Checkpoint blockade is
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`intended to block host immune inhibitory mechanisms which usually serve to prevent the
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`occurrence of auto-immunity. However, in cancer patients these mechanisms can also
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`serve to inhibit or block the potentially beneficial effects of any immune responses induced
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`to tumors. Alternatively, immune responses may not be fully potentiated due to a lack of
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`activation or lack of full activation of immune potentiating pathways. Therefore, drugs
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`which alleviate these blocks or stimulate immune potentiating pathways (ie. which
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`activate, or are ‘agonists’ of these immune potentiating pathways) are attractive for testing
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`and developing cancer treatments. Targets for such approved or experimental drugs
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`include CTLA~4, PIN, 93—1,..1, LAG«3, Trix/Ls, VISTA, CSPlR, mo, CEACAME, GITR,
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`4-1 “BB, KER, SLAIWFI 0X40, CD40, EGGS or CD47.
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`For these approaches to be successful, pre-existing immune responses to tumors are
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`needed, so that a pre-existing immune response can be potentiated or a block to an anti-
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`tumor immune response can be relieved. The presence of an inflamed tumor micro-
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`environment, which is indicative of such an ongoing response, is also needed. Pre-existing
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`immune responses to tumor neo-antigens appear to be particularly important for the
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`activity of checkpoint blockade and related drugs. Only some patients may have an
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`ongoing immune response to tumor antigens including neoantigens and/or an inflamed
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`tumor microenvironment, both of which are required for the activity of these drugs.
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`Therefore, oncolytic agents which can induce immune responses to tumor antigens,
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`including neoantigens, and/or which can induce an inflamed tumor microenvironment are
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`attractive for use in combination with immune checkpoint blockade and immune
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`potentiating drugs. This likely also explains the promising combined anti-tumor effects of
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`oncolytic agents and immune checkpoint blockade in mice and humans that have so far
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`been observed.
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`The indoleamine 2,3-dioxygenase (IDO) pathway contributes to tumor-induced
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`tolerance by creating a tolerogenic environment in the tumor and the tumor-draining lymph
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`nodes, both by direct suppression of T cells and enhancement of local regulatory T cell
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`(Treg)—mediated immunosuppression. IDO catalyses the rate-limiting step of tryptophan
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`degradation along the kynurenine pathway, and both the reduction in local tryptophan
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`concentration and the production of immunomodulatory tryptophan metabolites contribute
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`to the immunosuppressive effects of IDO. IDO is chronically activated in many cancer
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`patients with IDO activation correlating with more extensive disease. It can also function
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`as an antagonist to other activators of antitumor immunity. Therefore, inhibitors of the
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`[DO pathway are being developed as anticancer agents, particularly in combination with
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`checkpoint blockade agents such as those which target CTLA-4, PD-l or PDL-l. IDO
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`inhibitors may also be synergistic with oncolytic immunotherapy, including together with
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`drugs targeting other immune checkpoint or immune co-stimulatory pathways.
`
`Summary of the Invention
`
`The invention provides improved oncolytic viruses. The improved direct oncolytic
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`effects provided by the viruses of the invention will also lead to improved systemic anti-
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`tumor immune effects. Enhanced replication in and killing of tumor cells will result in
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`enhanced tumor antigen release and enhanced systemic immune responses to the released
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`antigens. The expression levels of any genes inserted to augment the direct oncolytic
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`effects and/or immune stimulation will also be increased.
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`Virus species naturally exist in a range of variants (strains) within the natural
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`population which may differ by a small or larger number of nucleotides while still
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`retaining the antigenic characteristics and sufficient sequence identity to still be recognized
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`as the same species. These strains, due to their differing sequences, may exhibit a range of
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`differing properties, including properties which have been selected for by natural selection
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`in their natural host or hosts (for example the ability to infect or replicate in the target cell
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`types of the virus in question, spread between these cells, or to evade the host innate or
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`adaptive immune system, or to spread between infected individuals of the host species) and
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`properties which have not been specifically selected for (e. g. the ability to replicate in and
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`kill or spread between cell types which are not the natural targets of the virus in question,
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`including tumor or other non-target cell types or tissues). The inventors have recognised
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`that sampling a range of viral strains of a particular viral species which are present in the
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`natural host population (in the case of viruses infecting humans, here termed ‘clinical
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`isolates’) and comparing these to each other to select for the strain with the best properties
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`for the intended purpose for which it is to be used (e. g. infection and killing of tumor cells)
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`can be used to identify a virus with optimal properties for that purpose. A virus identified
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`by this approach is likely to have more optimal properties for the intended purpose than a
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`‘prototype’ or ‘laboratory’ virus strain or a clinical strain which has not been selected for
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`the required property or properties from a broad group of viral strains. This is because the
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`full biological complexity in the natural population, particularly with respect to the
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`particular desirable property or properties, is unlikely to have been sampled through taking
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`a narrow approach to screening for the desired property or properties, bearing in mind the
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`degree of sequence variation present in natural virus populations. In particular, these may
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`vary in sequence within an infected host (as is often the case with RNA or retroviral
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`populations where so-called quasi-species are often present), between individual infected
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`hosts, or between different geographically separated viral populations.
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`Viruses of the invention have therefore been selected by sampling a range of viral
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`strains present in the natural population of a particular viral species and testing these
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`against each other for the desired property or properties (e.g. the ability to infect and kill
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`tumor cells). The virus strain or strains with the best properties for the intended purpose
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`are used for fiirther development
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`Where the intended use is oncolytic viral therapy, taking such an approach provides
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`an improved starting point for development of an oncolytic agent, which requires further
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`manipulation of the advantageous virus strains. Such manipulation includes the deletion of
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`viral genes to provide, for example, tumor selectivity, and/or the insertion of exogenous
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`genes to improve oncolytic or immune potentiating properties further.
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`The viruses of the invention therefore include novel clinical isolates of a viral
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`species that have better anti-tumor effects than the other clinical isolates to which they
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`were compared and through which comparison they were identified.
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`In particular, the
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`clinical isolates of the invention kill tumor cell lines in vitro more quickly and/or at a lower
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`dose than these reference clinical isolates of the same virus type. Typically, a clinical
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`isolate of the invention will have been identified through comparison of >5 clinical isolates
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`of a viral species for the required property or properties, preferably through comparison of
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`>10 clinical isolates of the viral species, and more preferably through comparison of >20
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`clinical isolates of the viral species. A clinical isolate of the invention typically shows
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`better tumor cell killing activity than 3/5, 6/10 or 11/20ths, preferably better than 4/5, 8/10
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`or 17/20ths, more preferably better than 9/10 or 19/20ths of the viral strains tested.
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`Typically, a clinical isolate of the invention can kill two or more tumor cell lines in
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`vitro within 24 to 48 hours after infection at a multiplicity of infection (MOI) of 0.01 to
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`0.001 or less.
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`The clinical isolates of the invention may be modified to further enhance their anti-
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`tumor effects. The genome of a clinical isolate of the invention may be modified to delete
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`or alter expression of one or more viral genes, and/or the genome of the clinical isolate
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`may be modified to express one or more heterologous genes, such as genes encoding a
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`fusogenic protein and/or an immune stimulatory molecule or molecules.
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`Oncolytic viruses of the invention provide improved treatment of cancer through
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`improved direct oncolytic effects, viral replication and spread through tumors, which (i)
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`increases the amount of tumor antigens, including neoanti gens, which are released for the
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`induction of an anti-tumor immune response, and (ii) enhances the expression of the virus-
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`encoded immune stimulatory molecule(s). Expression of immune stimulatory molecule(s)
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`by the virus can further enhance and potenti ate the anti—tumor immune effect. Expression
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`of fiisogenic protein(s) by the virus can further enhance viral spread through tumors.
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`Anti-tumor efficacy of an oncolytic virus of the invention is achieved when the
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`virus is used as a single agent and also when the virus is used in combination with other
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`anti-cancer modalities, including chemotherapy, treatment with targeted agents, radiation,
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`immune checkpoint blockade (i.e. administration of one or more antagonist of an immune
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`co-inhibitory pathway) and/or immune potentiating drugs (egone or more agonists of an
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`immune co-stimulatory pathway). The improved direct oncolytic effects (i.e. virus
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`replication in, spread between, and direct killing of tumor cells) and improved systemic
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`anti-tumor immune effects of the viruses of the invention improve on the combined
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`benefits of oncolytic therapy and immune co-inhibitory pathway blockade and/or immune
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`co-stimulatory pathway activation.
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`Accordingly, the present invention provides an oncolytic virus which is, or is
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`derived from, a clinical isolate which has been selected by comparing the abilities of a.
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`panel of three or more clinical isolates of the same viral species to kill tumor cells of two
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`or n'ioi'e tumor cell lines in Wire and selecting a clinical isolate which is capable ot‘killing
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`cells of two or more tumor cell lines more rapidly and,"or at a lower dose in Wire than one
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`or more ofthe other clinical isolates in the panel. The clinical isolate may be modified. A
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`modified clinical isolate may have mutations, such as deletions in the viral genome and/or
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`may express one or more l‘leterologeus genes.
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`The virus may he a strain of any virus species which may be used for the oncolytic
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`treatment of cancer, ii'iclutling strains a herpes virus, pox virus, adenovirus, retrovirus,
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`rhabdovirus, paramyxovirus or reovirus. The virus is preferably a herpes simplex vi 111s
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`(HSV), such as HSVl. The HSV typically does not express functional lCPEtl-S and/or
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`functional lCP47 and/or expresses the USl l gene as an immediate early gene.
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`The virus may comprise (i) a tusogenic protein—encoding gene; and/or (ii) an
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`immune stimulatory molecule or an immune stimulatory rnolecule—ei‘ieoding gene. The
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`Virus may encode more than one fusogenie protein and/or more than one ininiune
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`stimulatory molecule The fusogenic protein is preferably the glycoprotein from gibbon
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`ape leukemia virus (GALV) and has the R transniemhrane peptide mutated or removed
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`(GAQLV—Rw) The immune stimulatory molecule is preferably GMECSF an d/or an agoni st
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`ofan ii’nrnune co—stin’iulatory patl’iway in eluding GETRL, ltd—BBL 0&1?)le lCOSL or
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`CD401; or a modified version in each case thereof.
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`The invention also proyi des:
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`*
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`—
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`—
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`-
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`—
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`a pharmaceutical composition comprising a Virus of the invention and a
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`pharmaceutically acceptable carrier or diluent;
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`the Virus ofthe invention for use in a method of treating the human or animal hotly
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`by therapy;
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`the virus of the invention for use in a method of treating cancer, wherein the
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`method optionally comprises administering, a further anti-cancer agent;
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`a product of n'ianufacture comprising a Vitus of the iiwention in a steiile Vial,
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`anipoule or syringe;
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`a method of treating cancer; which comprises administering a therapeutically
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`effective amount of a Virus or a pharmaceutical composition ot‘the invention to a
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`patient in need thereol; wherein the method optionally comprises administering a
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`further anti—eancer agent;
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`—
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`use of a Viius of the invention in the manufacture of a medicament for use in a
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`method of treating cancer, wherein the method option ally comprises administering
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`a further anti—cancer agent;
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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 fuIther antagonist of an immune co-inhibitory
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`pathway, or agonist of an immune co-stimulatory pathway to a patient in need
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`thereof; and
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`—
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`a method of selecting an oncolytic Virus, the method comprising:
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`(i)
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`comparing the ahilities of a panel of three or more clinical isolates of the
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`same viral strain to kill tumor cells or" two or more tumor cell lines in w’r‘ro;
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`(ii)
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`scoring the abilities of each of the panel of viruses to kill tumor cells;
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`(iii)
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`selecting a Virus which has one of the best scores;
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`(iv)
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`option ally modifying the virus to inactivate one or more viral genes; and/or
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`(v)
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`optionally modit‘yin g the virus to express one or more immune stimulatory
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`molecule encoding genes and/or one or more firsogenie protein-encoding
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`genes.
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`The further anti-cancer agent may be an ai’itagonist of an inn'nnne co~inhibiteiy
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`pathway or an agonist of an immune constimuiatory pathway
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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 GALV-R- and a gene encoding GM-CSF inserted into the ICP34.5 gene
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`locus, and in which the ICP47 gene is deleted such that the US 11 gene is under the control
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`of the ICP47 immediate early promoter.
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`Figure 2 depicts the structure of an exemplary virus of the invention that comprises
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`a gene encoding GALV-R-, a gene encoding GM-CSF and a gene encoding CD4OL.
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`Brief Description of the Seguence Listing
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`SEQ ID NO: 1 is the nucleotide sequence of mouse GM-CSF.
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`SEQ ID NO: 2 is the nucleotide sequence of a codon optimized version of mouse
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`GM-CSF.
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`SEQ ID I\O: 3 is the nucleotide sequence of human GM-CSF.
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`SEQ ID I\O: 4 is the nucleotide sequence of a codon optimized version of human
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`GM-C SF.
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`SEQ ID I\O: 9 is the amino acid sequence of GALV-R-.
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`SEQ ID l\O: 10 is the nucleotide sequence of a codon optimized version of a
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`human/mouse hybrid membrane bound version of CD40L.
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`SEQ ID l\O:11 is the amino acid sequence of a human/mouse hybrid membrane
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`bound version of CD40L .
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`SEQ ID l\O: 5 is the amino acid sequence of mouse GM-CSF.
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`SEQ ID I\O: 6 is the amino acid sequence of human GM-CSF.
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`SEQ ID I\O: 7 is the nucleotide sequence of GALV-R-.
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`SEQ ID I\O: 8 is the nucleotide sequence of a codon optimized version of GALV-
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`SEQ ID NO: 12 is the nucleotide sequence of a codon optimized version of a
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`multimeric secreted version of human CD40L.
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`SEQ ID NO:13 is the amino acid sequence ofa multimeric secreted version of
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`human CD40L.
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`SEQ ID 1\O: 14 is the nucleotide sequence of a codon optimized version of a
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`multimeric secreted version of mouse CD4OL.
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`SEQ ID 1\O:15 is the amino acid sequence ofa multimeric secreted version of
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`mouse CD4OL.
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`SEQ ID I\O: 16 is the nucleotide sequence of wild-type human CD40L.
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`SEQ ID I\O: 17 is the amino acid sequence of Wild-type human CD40L.
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`SEQ ID I\O:18 is the nucleotide sequence of wild-type mouse CD4OL.
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`U‘I
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`10
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`SEQ ID 1\O: 19 is the amino acid sequence of wild-type mouse CD4OL.
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`Detailed Description of the Invention
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`15
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`()rzcozf)..'zic Virus
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`The virus of the invention is oncolytic. An oncolytic virus is a virus that infects
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`and replicates in tumor cells, such that the tumor cells are killed. Therefore, the virus of
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`the invention is replication competent. Preferably, the virus is selectively replication
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`competent in tumor tissue. A virus is selectively replication competent in tumor tissue if it
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`replicates more effectively in tumor tissue than in non-tumor tissue. The ability of a virus
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`to replicate in different tissue types can be determined using standard techniques in the art.
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`The virus of the invention may be any virus Which has these properties, including a
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`herpes virus, pox virus, adenovirus, retrovirus, rhabdovirus, paramyxovirus or reovirus, or
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`any species or strain Within these larger groups. Viruses of the invention may be Wild type
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`(ie. unaltered from the parental Virus species), or witl'i gene disruptions or gene additions.
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`Which of these is the case will depend on the virus species tn he used. Preferably the virus
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`is a. species of herpes virus, m ore preferably a strain ot‘HSV, including strains ofHSV l
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`and HSVQ, and is rnest preferably a strain 0f HSVi. The virus of the invention is based On
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`a clinical iselate Ofthe virus species to be used. The clinical isolate is selected 011 the basis
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`oi‘it having particular advantageous properties for the treatment of cancer. The virus of the
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`invention has surprisingly good anti-tumor effects compared to other strains of the same
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`virus isolated from other patients. The virus strains used for comparison to identify viruses
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`25
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`of the invention may be isolated from a patient or an otherwise healthy (i.e. other than
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`harboring the virus species to be tested) volunteer, preferably an otherwise healthy
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`volunteer, HSVl strains used to identify a virus of the invention are typically isolated
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`from cold sores ofindividuals harboring HSVl, typically by taking a swab using e.g.
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`Virocult (Sigma) brand swab/container containing transport media followed by transport to
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`the facility to be used for further testing.
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`After isolation of viruses to be compared from individuals, stocks of the viruses are
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`typically prepared, for example by growing the isolated viruses on BHK or vero cells.
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`This is done following no more than 3 cycles of freeze thaw between taking the sample and
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`it being grown on, for example, BHK or vero cells to prepare the virus stock for further
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`use. Preferably the virus sample has undergone 2 or less than 2 cycles of freeze thaw prior
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`to preparation of the stock for further use, more preferably one cycle of freeze thaw, most
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`preferably no cycles of freeze thaw. Lysates from the cell lines infected with the viruses
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`prepared in this way after isolation are compared, typically by testing for the ability of the
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`virus to kill tumor cell lines in vitro. Alternatively, the viral stocks may be stored under
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`suitable conditions, for example by freezing, prior to testing. Viruses of the invention have
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`surprisingly good anti-tumor effects compared to other strains of the same virus isolated
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`from other individuals, preferably when compared to those isolated from >5 individuals,
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`more preferably >10 other individuals, most preferably >20 other individuals.
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`The stocks of the clinical isolates identified as viruses of the invention (i.e. having
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`surprisingly good properties for the killing of tumor cells as compared to other viral strains
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`to which they were compared) may be stored under suitable conditions, before or after
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`modification, and used to generate further stocks as appropriate.
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`A clinical isolate is a strain of a virus species which has been isolated from its
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`natural host. The clinical isolate has preferably been isolated for the purposes of testing
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`and comparing the clinical isolate with other clinical isolates of that virus species for a
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`desired property, in the case of viruses of the invention that being the ability to kill human
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`tumor cells. Clinical isolates which may be used for comparison also include those from
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`clinical samples present in clinical repositories, i.e. previously collected for clinical
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`diagnostic or other purposes.
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`in either case the clinical isolates used for comparison and
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`identification of viruses of the invention will preferably have undergone minimal culture in
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`vim; prior to being tested for the desired property, preferably having only undergone
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`sufficient culture to enable generation of sufficient stocks for comparative testing purposes.
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`As such, the viruses used for comparison to identify viruses of the invention may also
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`include deposited strains, wherein the deposited strain has been isolated from a patient,
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`preferably an HSVl strain isolated from the cold sore of a patient.
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`The virus of the invention is an oncolytic virus which is, or is derived from, a
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`clinical isolate which has heen selected lily comparing the abilities ofa panel of three or
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`more clinical isolates of the same viral specl es to l<ill tumor cells of two or more tumor cell
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`lines in WW0 and selecting a clinical isolate which is capable of killing cells of two or more
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`tuntor cell lines more rapidly and/or at a lower Close in vitro than one or more of the other
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`clinical isolates in the panel Thus, the virus is a. clinical isolate that kills two or more
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`tumor cell lines more rapidly and/or at a lower dose in virm than one or more reference
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`clinical isolates ot‘the same species of virus.
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`'l‘ypically, the clinical isolate of the invention will kill two or more tumor cell lines
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`within 72 hours, preferably within 48 hours, more prelerahly within 24 hours, of infection
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`at multiplicities of infection (hi/ESE) of less than or equal to 0. l, preferably less than or
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`equal to an iVlOl of Gill. Preferably the clinical isolate will kill a broad range of human
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`tumor cell lines, such as 2, fl 4, S, 6, 7 or all ofthe following cell lines; HT29 (colorectal),
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`MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu (squamous cell carcinom