Mutated adenoviruses with improved replication and their use for treating cancer

Abstract

The present invention relates to adenoviruses, particularly oncolytic adenoviruses, and pharmaceutical compositions comprising adenoviruses, for use in the treatment of cancer, including tumours containing stroma and ovarian cancer. In particular, the invention relates to adenoviruses having a mutation in the coding sequences of the DNA pol I polypeptide and / or the pTP (pre-Terminal Protein) polypeptide.

Description

[0001] MUTATED ADENOVIRUSES WITH IMPROVED REPLICATION AND THEIR USE FOR TREATING CANCER

[0002] The present invention relates to adenoviruses, particularly oncolytic adenoviruses, and pharmaceutical compositions comprising adenoviruses, for use in the treatment of cancer, including tumours containing stroma and ovarian cancer. In particular, the invention relates to adenoviruses having a mutation in the coding sequences of the DNA pol I polypeptide and / or the pTP (pre-Terminal Protein) polypeptide.

[0003] Carcinomas are cancers derived from epithelial tissue that line external or luminal surfaces of organs including the lung gastrointestinal tract and reproductive tissues.

[0004] Carcinomas represent about 90% of all cancer cases globally and are the cause of the vast majority of cancer deaths.

[0005] A consistent theme of carcinomas is that they are often diagnosed as palpable lesions due to the presence of extracellular material, fibroblasts and immune cells surrounding and / or between islands of cancer cells. All the cells and materials of a tumour that are not described as a cancer cell (or a malignant cell) are often collectively referred to as “the stroma” or more specifically “tumour stroma”. Some cells of the immune system are often included under the term stroma (typically macrophages, fibroblasts), whereas others (typically lymphocytes) are described as infiltrating it or transiting through it.

[0006] All carcinomas of a visible and palpable size have some degree of stroma; this provides the essential structures, nutrients and environment for carcinoma cells to survive, proliferate and even spread.

[0007] The proportion of stroma can vary between 5-80%, depending to some extent on the underlying indication. However, it would be apparent to anyone familiar with tumour structures that the greatest variance in stromal content for any given biopsy is due to the location of the biopsy since some regions of a tumour visually appear to have more stroma than others.

[0008] Stromal cells superficially present with recognisable characteristics from normal tissue and organs. They remodel the ECM, recruit blood vessels and provide structure. However, unlike normal tissues, the actions of these cells are dysregulated and often chaotic. Too much stroma can lead to high pressure, poor blood flow and regions of hypoxia. As a result, cancer cells experience temporal and spatial disparity in nutrients and oxygen. As tumours grow, a great deal of cellular death and necrosis occurs due to regions cycling in and out of nutrient levels. Fluctuating episodes of nutrient and oxygen stress may feel like less than ideal conditions for tumours to thrive; however, it is increasingly appreciated that this drives clonal evolution of stronger cells with resistance to therapy.

[0009] The role of the stromal compartment in protecting cancer cells from exposure to drugs or immune attack has been increasingly recognised over the years. Fibroblasts associated with cancer (cancer associated fibroblasts, CAFs) in particular have been associated with excluding T-cells from the tumour or suppressing any T-cells that do infiltrate by expressing TGFp amongst other cytokines.

[0010] The presence of CAFs and their impact on immune cells have often been cited as the underlying cause of poor responses to immunotherapy. However, the development of new immunotherapies to treat cancer patients has been hampered by the lack of animal models that contain sufficient quantities of stromal cells or the correct architecture. When CAFs are artificially introduced into animal models, cancer vaccines and checkpoint inhibitor therapy is less effective or completely fails.

[0011] Despite the lack of models, the development of drugs that specifically target the stroma has increasingly received attention. These drugs target either the stromal cells directly or the micro-environmental conditions that the stroma creates. However, stromal- targeting strategies generally have two underlying challenges. Firstly, selectivity can be very challenging because cells that make up the stroma are essentially ‘normal’: they are not malignant and often found elsewhere in the body. The second conceptual problem is the need for combination therapy since it is unlikely that a stromal targeting approach (alone) is going to be completely effective. Targeting both the stroma and the cancer cells is likely to be necessary to treat an intransigent carcinoma if it is unresponsive to either approach alone. The rationale for combination therapies is very strong but it remains very difficult to develop drugs as a combination especially for pharmaceuticals that are or likely to be ineffective as single agents.

[0012] An ideal intervention to treat carcinomas would simultaneously and selectively target both cancer cells and stromal populations.

[0013] Oncolytic viruses are an emerging treatment modality because they can kill a wide range of tumour cell types including both differentiated cancer cells and cancer-initiating or stem cells. They can be highly selective while taking advantage of the ‘hallmarks of cancer’ such as immune or cell-cycle dysregulation to proliferate and lyse cells.

[0014] The main disadvantage of oncolytic viruses is they have not to date been explicitly designed or developed to deal with stromal cells. This is because contemporary oncolytic viruses are constructed to only have activity in malignant cells (by definition).

[0015] In human tumours, where the stroma divides and surrounds individual regions of cancer cells, this presents a physical (and indeed a conceptual) barrier to oncolytic therapy. Consequently, treatments which have been successful in the laboratory rarely translate effectively to the clinics. Where there has been clinical success in treating solid carcinomas, oncolytic viruses have been injected multiple times to bypass stromal barriers (e.g. Khuri FR, et al. “A controlled trial of intra-tumoral ONYX-015, a selectively- replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer”. Nat Med. 2000 Aug;6(8):879-85. doi: 10.1038 / 78638. PM ID: 10932224). Alternatively, very small or early stage carcinomas with relatively low levels of stroma have been targeted (e.g. Packiam VT, et al. “An open label, single-arm, phase II multicenter study of the safety and efficacy of CG0070 oncolytic vector regimen in patients with BCG-unresponsive non-muscle-invasive bladder cancer: Interim results.” Urol Oncol. 2018 Oct;36(10):440-447. doi: 10.1016 / j.urolonc.2017.07.005. Epub 2017 Jul 26. PMID: 28755959).

[0016] To address the stromal problem, some oncolytic viruses have been adapted to express biologies, including bi-specific T-cell engagers (BiTEs), to selectively kill stromal cells. The concept here is to allow the oncolytic virus to deal with the cancer cells while the expressed BiTE targets stromal cells to effect complete tumour lysis. Unfortunately, this concept remains insufficient due to the architecture and arrangement of tumour stroma. In human tumours, the feeding capillaries emerge through the stroma. Drugs, including oncolytic viruses, must therefore cross multiple layers of stromal cells in order to reach the cancer cells. For small molecule drugs this is less of an issue due to diffusion, but oncolytic viruses are generally too large to transit between cells. An oncolytic virus entering a human tumour would be exposed to stromal cells first and be unable to replicate or express transgenes such as BiTEs.

[0017] This deficiency is not noticeable in animal models where stroma is usually absent, or when present in animal models, it has a different architecture where the capillaries emerge directly amongst cancer cells.

[0018] In order for an oncolytic agent to be effective at treating human disease it must be active as soon as it enters the tumour micro-environment. This is made possible because three of the hallmarks of cancer that oncolytic viruses exploit to kill cancer cells, can also apply to CAFs. These include metabolic dysregulation, resistance to apoptosis and immune dysfunction. It would be desirable, therefore, to develop an oncolytic agent to exploit these shared features of cancer and stromal cells in order to be active in both.

[0019] Ovarian cancer is one of the most frequently fatal forms of neoplasm affecting women. It is typically diagnosed late, with the disease already having spread to other sites. The best survival rate for stage 3+ remains at 25% despite all currently available drugs. The majority of ovarian cancer patients are diagnosed with the most serious form of the disease: high grade serous ovarian cancer (HGSOC) which accounts for 70-80% of fatalities. Current treatment plans include repeated platinum-based therapy until inevitable resistance and relapse. Second line therapies include paclitaxel and doxorubicin with only marginal benefit. A small proportion of patients (13-15%) carrying the BRCA1 mutation patients are eligible for recently approved PARP inhibitors. Most recently Mirvetuximab received accelerated approval in patients with high folate receptor levels. Although these new drugs are effective in the medium term, the cancer is expected to return in most patients. A potential role for oncolytic viruses in ovarian cancer has been explored on the basis that they stimulate immune responses while killing heterogeneous cell populations including cancer stem cells. A number of clinical trials have demonstrated safety and feasibility of delivering oncolytic viruses to ovarian cancer in patients. However, the overall efficacy has been limited. The fact is that stromal CAFs frustrate the impact of oncolytic therapies through antiviral signalling (Arwert EN, et al. ’’STING and IRF3 in stromal fibroblasts enable sensing of genomic stress in cancer cells to undermine oncolytic viral therapy”. Nat Cell Biol. 2020 Jul;22(7):758-766. doi: 10.1038 / s41556-020- 0527-7. Epub 2020 Jun 1. Erratum in: Nat Cell Biol. 2020 Jun 18;: PMID: 32483388; PMCID: PMC7611090).

[0020] Given the prominent role of CAFs in advancing ovarian cancer and providing resistance mechanisms for chemotherapy, immunotherapy and oncolytic approaches, there is an urgent need to develop new treatments that can tackle CAFs and cancer cells simultaneously.

[0021] CAFs play a critical role in determining prognosis and response to therapies via their ability to suppress immune cells, block effective T cells from infiltrating, forming dense barriers to the spread of therapies and promoting resistance to SOC drugs. In addition, CAFs promote and feed the tumour, supporting it to grow and metastasis. Despite their importance in determining patients' response to therapy, existing preclinical models, including the more sophisticated in vivo PDX models, do not represent this critical component of the human TME. Use of traditional cancer cell lines and xenograft tumour models in vivo do not recapitulate the complexity and heterogeneity of human carcinomas. They do not accurately capture the stroma, the immunosuppression, dysregulated metabolism, cytokines and stress proteins (e.g. TNF-alpha, TGF-beta) present in the patient TME. Development of therapies on realistic model systems (e.g. freshly resected patient biopsies, multicellular, nutrient deprived, hypoxic tumour microenvironments) is critical to identifying efficacious drugs that are durable in the presence of such barriers and stressed conditions. The Applicant has had the insight to recognise that the tumour microenvironment is completely different from the environment in which viruses have naturally evolved: viruses are often found in hyper-oxygenated airway cells which are continually supplied with nutrients. The tumour microenvironment is also completely different from cell culture or fast-growing animal models where nutrients are in abundance. Consequently, wild-type viruses or viruses discovered using simple animal models are often not suitable for treating human disease. By extension, any virus which has been optimised for the tumour environment is likely to be significantly different from wild-type viruses or engineered viruses. In order to thrive in the tumour environment, a virus will have to regulate its demand for various resources or risk an aborted replication cycle. Mutations (even subtle ones) can achieve this because the actions of viruses - making RNA, DNA, and proteins - are highly ‘geared’ so even a small change in a single virus may become significant when the virus reproduces to form 100,000 progeny.

[0022] The Applicants have now identified a feature which confers, inter alia, an enhanced capacity of adenoviruses to demonstrate oncolytic activity in tumours containing stroma, including ovarian cancer cells.

[0023] From an initial mixed pool of Group B, C, D, F, and G adenoviruses, 23 rounds of bioselection (including mutational steps) were performed to enrich for oncolytic adenovirus candidates with the best combination of tumour lysis, spread, blood stability and immune stimulation. From the final pool, 60 adenoviruses were selected for sequencing and their genomes were analysed.

[0024] Genome analyses of the selected adenoviruses revealed that a number of features of their genomes were common to the majority of or all of the candidate adenoviruses, including the presence of a missense mutation in the E2B DNA pol I gene. The mutation was a single nucleotide change ggc gac, which results in a G34D mutation in the amino acid sequence of the E2B DNA pol I polypeptide.

[0025] In the wild-type adenovirus genome, the coding sequences for the E2B DNA pol I gene and the E2B pre-Terminal Protein (pTP) overlap. The above-mentioned mutation falls within the coding sequences of both the DNA pol I polypeptide and the pTP polypeptide and hence the pTP gene was also mutated. With regard to the pTP polypeptide sequence, the mutation is a gcg acg mutation; this results in an A623T amino acid mutation in the amino acid sequence of the E2B pTP polypeptide.

[0026] On examination, one or both of these mutations were found to confer an advantage on the adenoviruses in terms of their levels of replication and production of infectious virus progeny. In the context of oncolytic adenoviruses, this advantage manifests itself in the ability of the adenovirus to achieve sustained oncolytic activity at the tumour sites. In the context of transgene encoding or ‘armed’ oncolytic adenoviruses, e.g. ones which encode a transgene, this advantage manifests itself in the ability of the adenovirus to produce the transgene, and the corresponding polypeptide product, to a higher abundance.

[0027] It is an object of the invention therefore to provide adenoviruses and pharmaceutical compositions comprising adenoviruses, the adenoviruses having mutations in the E2B DNA pol I gene and / or E2B pTP gene and which are capable of infecting and lysing cancer cells, particularly ovarian cancer cells and cancer-associated fibroblasts (CAFs). It is another object of the invention to provide methods of treating cancers, particularly ovarian cancers and stroma-containing cancers, using the oncolytic adenoviruses of the invention. It is yet another object of the invention to provide adenoviruses having mutations in the E2B DNA pol I gene and / or E2B pTP gene and which are capable of replicating to greater levels and producing more infectious virus progeny compared to corresponding control adenoviruses.

[0028] In one embodiment, the invention provides a pharmaceutical composition comprising an adenovirus, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents, wherein the genome of the adenovirus comprises an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the amino acid in the DNA pol I polypeptide sequence corresponding to amino acid 34 in SEQ ID NO: 2 is Asp or Glu, preferably Asp. In another embodiment, the invention provides a pharmaceutical composition comprising an adenovirus, optionally together with one or more pharmaceutically- acceptable carriers, excipients or diluents , wherein the genome of the adenovirus comprises an E2B pTP gene encoding a pre-Terminal Protein, wherein the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 4 is selected from the group consisting of Thr, Asn, Cys, Gin and Ser, preferably Thr. In some embodiments, if the adenovirus is an Ad1 adenovirus, then the amino acid is not Asn; if the adenovirus is an Ad41 adenovirus, then the amino acid is not Gin; and if the adenovirus is an Ad4 or 4a adenovirus, then the amino acid is not Thr.

[0029] In another embodiment, the invention provides a pharmaceutical composition comprising an adenovirus of the invention, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.

[0030] Also provided is an adenovirus or a pharmaceutical composition of the invention for use in therapy or for use as a medicament, particularly for use in treating cancer (preferably ovarian cancer).

[0031] Also provided is a method of treating cancer (preferably ovarian cancer) in a subject, the method comprising administering an effective amount of an adenovirus or a pharmaceutical composition of the invention to a subject in need thereof.

[0032] Also provided is the use of an oncolytic adenovirus of the invention in the manufacture of a medicament for treating cancer (preferably ovarian cancer).

[0033] Also provided are the use of a conditionally-replicating adenovirus of the invention as a vector for protein production, wherein the adenovirus comprises a transgene encoding the protein to be produced; and the use of a conditionally-replicating adenovirus of the invention as a helper virus for virus production.

[0034] The invention also provides mutant Group B adenovirus E2B DNA pol I and E2B pTP genes as defined herein, and mutant Group B adenovirus E2B DNA pol I and E2B pTP polypeptides, as defined herein.

[0035] Table 1 : Table of Sequences

[0036] The invention provides adenoviruses, preferably for use in treatment of cancer, preferably ovarian cancer or stroma-containing tumours.

[0037] As used herein, “adenovirus” (also abbreviated herein to “Ad”) refers to those viruses belonging to the family Adenoviridae, included in any one of the currently known five genera: Mastadenovirus, Aviadenovirus, Atadenovirus, Siadenovirus and Ichtadenovirus. Preferably, the adenoviruses are from the genus Mastadenovirus', this includes all human serotypes. In one embodiment, the adenoviruses are human adenoviruses.

[0038] At present more than 60 antigenic types or “serotypes” of human adenoviruses have been described and these serotypes have been classified into seven species, i.e. Ad species A-G, on the basis of their physical, chemical and biological properties (as described, for example, in Wold et al. Current gene therapy vol. 13, 6 (2013): 421 -33). Thus adenoviral species as used herein refers to those currently-known Ad groups A-G, as well as any identified in the future.

[0039] In one embodiment, therefore, the adenovirus species are species of human adenoviruses selected from the group consisting of AdA, AdB, AdC, AdD, AdE, AdF and AdG. In some embodiments, the species of human adenoviruses are selected from the group consisting of AdB, AdC, AdD, AdE, AdF and AdG.

[0040] The serotypes which fall within each of these adenovirus species include but are not limited to those which are given below:

[0041] AdA comprises Ad12, Ad18 and Ad31 and Ad61. AdB comprises Ad3, Ad7, Ad11 , Ad14, Ad16, Ad21 , Ad34, Ad35, Ad50 and Ad55, Ad66, Ad68 and Ad79. AdC comprises Ad1 , Ad2, Ad5, Ad6 and Ad57. AdD comprises Ad8, Ad9, Ad10, Ad13, Ad15, Ad 17, Ad19, Ad20, Ad22, Ad23, Ad24, Ad25, Ad26, Ad27, Ad28, Ad29, Ad30, Ad32, Ad33, Ad36, Ad37, Ad38, Ad39, Ad42, Ad43, Ad44, Ad45, Ad46, Ad47, Ad48, Ad49, Ad51 , Ad53, Ad54 and Ad56. Ad58, Ad59, Ad60, Ad62, Ad63, Ad64, Ad65, Ad67, Ad69, Ad70, Ad71 , Ad73, Ad74 and Ad75. AdE comprises Ad4. AdF comprises Ad40 and Ad41 . AdG comprises Ad52.

[0042] The references herein to different Ad serotypes include all different strains or variants of those serotypes.

[0043] Preferably, the adenovirus is a Group B adenovirus. Group B1 adenoviruses include Ad3, Ad7, Ad16, Ad21 , Ad50, Ad66 and Ad68. Group B2 adenoviruses include Ad11 , Ad14, Ad34, Ad35, Ad55 and Ad79. Preferably, the adenovirus is a Group B1 adenovirus. Most preferably, the adenovirus is an Ad3 or Ad7 serotype, or an Ad3 / Ad7 chimera.

[0044] In some embodiments, the adenovirus is a human adenovirus. All human adenovirus genomes examined to date have the same general organisation, i.e. the genes encoding specific functions are located at the same position in the adenovirus genome (referred to herein as structural elements). Each end of the adenovirus genome has a short sequence known as the inverted terminal repeat (or ITR), which is required for viral replication. In some embodiments, the adenovirus is not a chimpanzee adenovirus or is not an AdC7 adenovirus. The adenovirus genome contains five early transcription units (E1A, E1 B, E2, E3, and E4), three delayed early units (IX, IVa2 and E2 late) and one late unit (major late) that is processed to generate five families of late mRNAs (L1 -L5). Proteins encoded by the early genes are primarily involved in replication and modulation of the host cell response to infection, whereas the late genes encode viral structural proteins. Early genes are prefixed by the letter E and the late genes are prefixed by the letter L.

[0045] The DNA genome of adenovirus varies in length dependent on serotype but is typically 34-36 kilobases in length. For example, the adenovirus Ad5 genome is usually 35938 base pairs with a 103 base pair terminal repeat at each end and a 58% GC content.

[0046] Our understanding of adenovirus genetics, transcription and translation is primarily derived from virological studies using adenovirus species C Ad5. The sequential process of gene transcription from the adenovirus genome reflects the protein requirements of the virus at each stage of the replication process. The transcription from the adenovirus genome is therefore divided into early and late events dependent on the timing of initiation of transcription from each viral promoter. The first proteins produced from the virus genome are the E1A proteins. The E1A transcription unit produces multiple mRNA molecules through alternative splicing which in turn produce multiple proteins ranging from 6-36 kDa. The E1A proteins have two major roles within an infected cell. Firstly, they induce the cell to enter the S phase of the cell cycle to allow the efficient replication of the viral genome. Secondly, they induce transcription of the other early promoters within the viral genome through transactivation. These promoters control the production of the E1 B, E2, E3 and E4 proteins. E1A expression is immediately followed by VA RNA and E1 B and E3 protein production. These proteins and RNA molecules help to prevent the development of an anti-viral response. These early events in viral replication help to shape the intracellular environment to allow the replication of the viral genome before packaging. Later transcription events involve the production of structural proteins and proteins essential for cell lysis which are derived primarily from a single promoter (the major late promoter) which transcribes the late regions 1 -5. Ad5 induced cell lysis is dependent on the E3-11 ,6K protein (also termed the adenovirus death protein) which despite its labelling as an early gene is only produced late in infection and from the major late promoter. Adenovirus genes are divided into early (E1 -4) and late (L1 -5) transcripts, with multiple protein isoforms driven from a range of splicing events. The early regions are divided into E1 , E2, E3 and E4. E1 is essential for transitioning the cell into a phase of the cell cycle that is conducive to virus replication and inhibiting apoptosis and promoting cell division. The E2 region is largely responsible for the replication of the DNA genome, containing the DNA binding protein (E2A), the pre-terminal protein (pTP) and the DNA polymerase (E2B). E3 contains genes involved in immune regulation of host responses and E4 contains a range of genes involved in regulating cell pathways such as non- homologous end joining (NHEJ) and complexing with E1 B-55K to mediate p53 degradation.

[0047] The adenovirus late genes are all transcribed from the same promoter, the Major Late Promoter and all share the same 5’ mRNA terminus which contains three exons that collectively form the tri-partite leader sequence. The late genes are expressed by a series of splice events that allow the expression of approximately 13 proteins that either form a part of the virus particle (e.g. Hexon and Fibre) or involved in its assembly (e.g. 100K protein).

[0048] Adenoviral vectors are vectors which are based on or derived from the genome of a virus of the family Adenoviridae. Adenoviral vectors are vectors which are based on or derived from the genome of a virus of the family Adenoviridae. Adenoviral vectors are functional replication-capable, replication-incompetent and / or genetically-engineered adenoviruses which transport genetic material (e.g. adenovirus genes or a transgene) into a eukaryotic cell, where it is subsequently expressed. Adenoviral vectors may carry deletions of E1 , E3 and / or E4 early genes, thus allowing the insertion of 8-30 kb transgenes. The functions of the deleted early genes may be supplied by engineered cell lines in trans. The most commonly-used adenoviral vector is based on adenovirus type 5 (Ad5).

[0049] The adenovirus of the invention comprises a plurality of adenoviral early genes (Figure 1 ). The E1 A proteins are the translation products of the first gene transcription events from the adenovirus genome within the nucleus at the E1A region. This initial transcription is driven by a strong constitutively active enhancer element within the E1 A promoter and allows significant quantities of E1 A mRNA to be produced. They are one of two sets of proteins in the adenovirus genome which are capable of inducing transformation with E1 B proteins also able to induce cell cycle progression. E1 A and E1 B genes are essential for virus replication.

[0050] The E2 genes are divided into two sections in the adenovirus genome: the E2A and the E2B regions, and both are required for virus replication. E2B contains the DNA polymerase gene which is fundamentally required for the amplification of the virus genomic DNA. Similarly, this region also contains the pre-terminal protein (pTP) which functions as a primer for the initiation of virus genome replication. The terminal protein is covalently attached to the end of the virus genomic DNA. The E2A region contains a DNA binding protein that is required for DNA replication. All E2 genes are fundamentally required for virus replication.

[0051] For Ad5, the pTP assembles on the viral origin of replication in an initiation complex with DNA polymerase, DNA binding protein, host NFI and OCT1. pTP is ~80-kDa protein that functions as the primer for initiation. The DNA polymerase is associated with 5-3' polymerase activity, and a 3-5' exonuclease domain that is necessary for its intrinsic proofreading ability. In the final stages of replication, pTP is cleaved by a viral protease to TP, resulting in progeny DNA, which is subsequently packaged in virions

[0052] The E3 genes are primarily involved in regulating cell and host immune responses to virus infection, however, as the majority of viruses used for biotechnology applications are in vitro many, if not all, of these virus genes can be removed without reducing virus replication efficiency. The majority of the E3 genes are not essential for virus replication in cell lines. However, some genes such as the Adenovirus Death Protein (ADP) are required for efficient replication and virus production. The E4 region of the adenovirus genome is similar to the E1 region in that it is primarily involved in producing proteins that help the virus control and regulate the cell to ensure efficient virus replication and production. The region includes 6 open reading frames (ORFs) that are able to aid in preventing non-homologous end joining and apoptosis amongst a number of other discrete functions. The relative importance of each E4 transcript to virus replication is variable with some being essential whilst others can be deleted or modified with little to no effect of virus growth kinetics and production.

[0053] The adenovirus of the invention preferably comprises sufficient adenoviral early genes in order for the adenovirus to be capable of replicating the viral genome in the nucleus of a cell in which it is placed.

[0054] The adenovirus of the invention comprises a plurality of adenoviral late genes (Figure 1 ).

[0055] The virus late genes are divided into five main transcript families named L1-L5. These transcripts primarily encode proteins that are involved in virus assembly and the structural proteins of the virus. During virus replication they can represent as much as 30-40% of the cells protein content (Gamier, 1994; Ginsberg, 1984).

[0056] The L1 series of transcripts encode for the 13.6K, 52K, and Pllla proteins. These proteins are all involved in virus assembly and particle production. L1 genes are required for successful virus assembly but not genomic DNA replication.

[0057] The L2 series of transcripts encode for the penton base, pVI I, V, pX proteins. These form structural parts of the virus capsid and are required for the particle to assemble correctly. Penton base contains an RGD motif that is important for attachment of several adenoviruses to the cell surface during infection. L2 genes are required for successful virus assembly but not genomic DNA replication.

[0058] The L3 series of transcripts encode for the pVI, hexon, and protease proteins. The hexon protein is a major component of the virus capsid and is antigenically diverse between serotypes. The protease protein is involved in cellular entry and the virus capsid maturation. L3 genes are required for successful virus assembly but not genomic DNA replication.

[0059] The L4 series of transcripts encode the 100K, 33K, 22K and pVII proteins. These proteins are involved in a range of functions. 100K protein is involved in both aiding virus hexon assembly and nuclear import but may also play a role in shifting cell mRNA translation to cap-independent translation. The 22K protein is involved in virus encapsidation. L4 genes are required for successful virus assembly but not genomic DNA replication. However, the 100K protein may aid in shifting cellular protein translation towards those transcripts that contain a tripartite leader (TPL) sequence.

[0060] L5 encodes the Fibre gene. Fibre is a virus structural protein involved in attachment to cell surfaces and in mediating virus cellular infection. The Fibre protein is produced in significant excess of its requirement for virus particle formation. L5 genes are required for successful virus assembly but not genomic DNA replication.

[0061] The genome of the adenovirus of the invention comprises a plurality of adenoviral early and late genes which are sufficient for replication of the adenovirus. Preferably, the adenovirus of the invention comprises at least the E1 , E2 and E4 early genes and the L1-L5 late genes. E3 is dispensable for replication in cell lines. However, some genes within each of these regions can be modified, mutated or even deleted without inhibiting virus replication in cell lines.

[0062] In one embodiment, the adenovirus is replication-capable, replication-competent or conditionally replicating. These viruses may be oncolytic, virus vaccines, vectors for protein production or helper viruses for virus manufacture.

[0063] “Replication-capable” as employed herein refers to an adenovirus that can replicate its genome in a host cell. In one embodiment, “replication-capable” encompasses replication competent and conditionally replicating viruses. “Replication-competent” in the context of the present invention refers to an adenovirus that possesses all the necessary machinery to replicate in cells in vitro and in vivo, i.e. without the assistance of a packaging cell line.

[0064] “Conditionally-replicating”, “Replication-selective” or “selective replication” as employed herein is intended to mean an oncolytic adenovirus that is able to replicate in cancer cells employing an element which is specific to said cancer cells or upregulated therein, for example defective cellular machinery, such as a p53 mutation, thereby allowing a degree of selectivity over healthy / normal cells.

[0065] "Conditionally replicating" also refers to the ability of the adenovirus to infect and replicate under specific conditions. Such conditions may include having the virus under the control of a tumour specific promoter to drive virus replication in cells (e.g. PSA promoter), or having the virus under the control of a repressive element (e.g. tetracycline repressor (TetR) binding site or a microRNA binding site) to limit virus replication under specific conditions. Conditionally replicating may be oncolytic in nature, or used as a vector for gene delivery, vaccine, protein or virus production (e.g. as a helper for AAV production).

[0066] In some embodiments, the adenovirus genome has at least 70% (preferably at least 80%, 85%, 90%, 95% or 99%, most preferably at least 99%) nucleotide sequence identity to the wild-type Ad3 genome sequence as given in the full Ad3 genome sequence (e.g. as given in Genbank Sequence ID: DQ086466.1). In some embodiments, the adenovirus genome has at least 70% (preferably at least 80%, 85%, 90%, 95% or 99%, most preferably at least 99%) nucleotide sequence identity to the wild-type Ad7 genome sequence as given in the full Ad7 genome sequence (e.g. as given in Genbank Sequence ID AY594255.1).

[0067] In some preferred embodiments, the adenovirus of the invention is oncolytic. As used herein, the term “oncolytic” refers to the ability of the adenovirus to infect, replicate and to lyse cancer cells. Preferably, the oncolytic adenoviruses of the invention preferentially infect and / or preferentially lyse cancer cells compared to non-cancer cells. In some embodiments, the oncolytic adenoviruses of the invention are also capable of infecting and / or lysing stroma cells, particularly CAFs.

[0068] Viral infection may be measured by an infectivity assay such as a plaque assay, Median Tissue Culture infectious Dose (TCID50) assay, or by using an anti-hexon antibody in an immuno-cytochemical (ICC) staining assay. The oncolytic adenoviruses of the invention may be cytolytic. Lysis may be measured by cell death or cell viability assays including MTS, MTT assays, PrestoBlue™ , live / dead staining and flow cytometry. The oncolytic virus infection causes death and lysis of the cancer or stroma cells, preferably with release of newly-generated virus particles.

[0069] Viral production may be measured by numerous methods including the infectivity assays mentioned above. Other methods exist to measure the physical count of virus particles: these include UV absorbance measurements (OD260), dynamic light scattering, and HPLC quantification. Further methods still are able to measure virus production by quantification of DNA and interpolation from a standard curve of known DNA content. These assays include quantitative real time PCR and the Pico Green dye based assay. Purified virus can also be quantified by measuring total protein content in a bicinchoninic acid assay (BCA) assay.

[0070] In some embodiments, the adenovirus of the invention is oncolytic or conditionally replicating and it encodes one or more transgenes. Adenoviruses of the invention may be used as oncolytic vectors or conditionally replicating vectors comprising transgenes in order to produce recombinant nucleic acids and polypeptides at the tumour site. Examples of transgenes include those which encode anti-cancer, immune stimulatory or imaging agent(s) (e.g. antibodies, bispecific engagers, checkpoint inhibitors, cytokines, chemokines, and enzymes including extracellular matrix degrading enzymes, and angiogenesis inhibitors). The transgene(s) may be located, for example, within the adenovirus in the E1 or E3 region, or an E1 / E3-deleted region. It may also be inserted adjacent to or into the L3 or L5 region. A deletion in the E3 region creates more space for transgenes to be placed in other locations in the adenoviral genome. It also removes ORFs which help the virus to hide from the immune system and prevent host clearance of the virus. As a result, deletions in the E3 region attenuate virus activity in normal cells, with a functioning immune response, but not in immune dysfunctional tumour cells, thus increasing the therapeutic index of the oncolytic virus.

[0071] The adenovirus must have a genome within the packaging limit of the virus. For example, it must not carry a transgene of such a size that the total length of the genome surpasses the length which is able to be packaged into the protein capsid.

[0072] The adenoviruses of the invention may be referred to as “recombinant” adenoviruses”. As used herein, the term “recombinant adenovirus” refers to non-natural adenoviruses which have at least one nucleotide difference from wild-type adenoviruses, e.g. a nonnatural adenovirus whose genome has contiguous gene sequences that would not otherwise be found together in the genome of a wild-type adenovirus.

[0073] The E2B DNA pol I gene encodes the adenovirus’ DNA polymerase I (DNA pol I). DNA polymerase I is required for the amplification of the adenovirus’ genomic DNA.

[0074] The nucleotide and corresponding amino acid sequences of the wild-type Ad3 E2B DNA pol I gene and polypeptide are given herein as SEQ ID NOs: 1 and 2, respectively. As used herein, the term “E2B DNA pol I gene” refers to a gene comprising or consisting of:

[0075] (i) the nucleotide sequence as given in SEQ ID NO: 1 ;

[0076] (ii) a nucleotide sequence which has at least 80%, 85%, 90%, 95% or 99% (most preferably at least 99%) nucleotide sequence identity to SEQ ID NO: 1 ; and which preferably encodes a DNA polymerase I; or

[0077] (iii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 2.

[0078] As used herein, the term “E2B DNA pol I” refers to a polypeptide comprising or consisting of: (i) the amino acid sequence as given in SEQ ID NO: 2; or

[0079] (ii) a variant of (i) having at least 80%, 85%, 90%, 95% or 99% (most preferably at least 99%) amino acid sequence identity or sequence similarity to (i), and which preferably has DNA polymerase I activity.

[0080] The E2B pTP gene encodes the adenovirus pre-Terminal Protein (pTP). This polypeptide is also known as the Terminal Protein Precursor (TPP) or Terminal Precursor Protein (TPP). The pTP is required for the initiation of virus genome replication.

[0081] The nucleotide and corresponding amino acid sequences of the wild-type Ad3 E2B pTP gene and polypeptide are given herein as SEQ ID NOs: 3 and 4, respectively.

[0082] As used herein, the term “E2B pTP gene” refers to a gene comprising or consisting of:

[0083] (i) the nucleotide sequence as given in SEQ ID NO: 3;

[0084] (ii) a nucleotide sequence which has at least 80%, 85%, 90%, 95% or 99% (most preferably at least 99%) nucleotide sequence identity to SEQ ID NO: 3; and which preferably encodes a pre-Terminal Protein; or

[0085] (iii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 4.

[0086] As used herein, the term “E2B pre-Terminal Protein” refers to a polypeptide comprising or consisting of:

[0087] (i) the amino acid sequence as given in SEQ ID NO: 4; or

[0088] (ii) a variant of (i) having at least 80%, 85%, 90%, 95% or 99% (most preferably at least 99%) amino acid sequence identity or sequence similarity to (i), and which preferably encodes a pre-Terminal Protein.

[0089] The inventors have found that adenoviruses comprising the G34D mutation in the E2B DNA pol I polypeptide have enhanced adenovirus DNA replication and enhanced oncolytic activity compared to control adenoviruses without this mutation. Aspartic acid (Asp, D) and glutamic acid (Glu, E) are both negatively charged amino acids. In one embodiment, therefore, the invention provides an adenovirus, wherein the genome of the adenovirus comprises an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the amino acid in the DNA pol I polypeptide sequence corresponding to amino acid 34 in SEQ ID NO: 2 is Asp or Glu, preferably Asp. For example, the codon at the position in the genome of the adenovirus corresponding to nucleotide positions 100-102 in SEQ ID NO: 1 is gac, gau, gaa or gag.

[0090] In the wild-type adenoviral genome, the coding sequences of the E2B DNA pol I gene and the E2B pTP gene overlap, in different reading frames. One consequence of this is that some mutations in the 5’-end of the E2B DNA pol gene lead to mutations in the 3’- end of the E2B pTP gene, and also in the corresponding polypeptides.

[0091] For example, the G34D mutation in the E2B DNA polypeptide (e.g. mutation of the codon ggc -^gac) results in the mutation A623T (e.g. codon gcg acg) in the E2B pTP polypeptide.

[0092] The corresponding mutations in the E2B pTP polypeptide which result from the mutations G34D and G34E are shown in the table below:

[0093] Table 2: Effect of G34D and G34E mutations in E2B DNA pol I polypeptide on E2B pTP polypeptide sequence.

[0094] As shown in the above table, the mutations G34D and G34E in E2B DNA pol I polypeptide result in Thr, Met, Lys or Arg mutations at position 623 in the E2B pTP polypeptide.

[0095] In a further preferred embodiment, therefore, the invention provides an adenovirus of the invention wherein the genome of the adenovirus additionally comprises an E2B pTP gene encoding a pre-Terminal Protein, wherein the coding region of the E2B DNA pol I gene overlaps with the coding region of the E2B pTP gene, and wherein the amino acid in the terminal protein precursor sequence at the position corresponding to amino acid 623 in SEQ ID NO: 4 is selected from the group consisting of Thr, Met, Lys and Arg, preferably Thr. Preferably, the amino acid in the E2B pre-Terminal Protein polypeptide at the position corresponding to amino acid 623 in SEQ ID NO: 4 is not a nonsense (i.e. stop) mutation.

[0096] The inventors have found that adenoviruses comprising the A623T mutation in the E2B pTP polypeptide have enhanced adenovirus DNA replication and production of infectious virus progeny compared to control adenoviruses without this mutation.

[0097] Thr (T), Asn (N), Cys (C), Gin (Q) and Ser (S) are all polar or neutral amino acids. In yet another embodiment, therefore, the invention provides an adenovirus, wherein the genome of the adenovirus comprises an E2B pTP gene encoding a pre-Terminal Protein, wherein the amino acid in the pre-Terminal Protein sequence at the position corresponding to amino acid 623 in SEQ ID NO: 4 is selected from the group consisting of Thr, Asn, Cys, Gin and Ser, preferably Thr. If the adenovirus is an Ad1 adenovirus, then the amino acid is not Asn. If the adenovirus is an Ad41 adenovirus, then the amino acid is not Gin.

[0098] For example, the codon at the position in the genome of the adenovirus corresponding to nucleotide positions 5,326-5,328 in SEQ ID NO: 3 encodes: (i) Thr e.g. acu, acc, aca, acg

[0099] (ii) Asn e.g. aau, aac,

[0100] (iii) Cys e.g. ugu, ugc

[0101] (iv) Gin e.g. caa, cag

[0102] (v) Ser e.g. agu, age, ucu, ucc, uca, ucg

[0103] As noted above, in the wild-type adenoviral genome, the coding sequences of the E2B DNA pol I gene and the E2B pTP gene overlap, in different reading frames. One consequence of this is that some mutations in the 3’-end of the E2B pTP gene lead to mutations in the 5’- end of the E2B DNA pol I gene, and also in the corresponding polypeptides. For example, the A623T mutation in the E2B pTP polypeptide (e.g. codon gcg acg) results in the mutation G34D in the E2B DNA polypeptide (e.g. mutation of the codon ggc -^gac).

[0104] The corresponding mutations in the E2B DNA pol I polypeptide which result from mutations to polar or neutral amino acids at position 623 in the E2B pTP polypeptide are shown in the table below:

[0105] Table 3: Effect of polar or neutral amino acid mutations at position 623 of E2B pTP polypeptide on E2B DNA pol I polypeptide sequence. In a further preferred embodiment, therefore, the invention provides an adenovirus of the invention, wherein the genome of the adenovirus additionally comprises an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the coding region of the E2B pTP gene overlaps with the coding region of the E2B DNA pol I gene, and wherein the amino acids in the DNA pol I polypeptide sequence at the positions corresponding to amino acids 34-35 in SEQ ID NO: 2 are selected from the group consisting of Asp-Ser, Asp-Pro, Asp-Thr, Asp-Ala, Glu-Ser, Glu-Pro, Vai-Ser, Val-Pro, Ala-Thr, Ala-Ala, Val- Thr and Val-Ala.

[0106] With regard to the above list (i.e. Asp-Ser, Asp-Pro, etc.), the first amino acid listed in each pair is the one at the position corresponding to amino acid 34 in SEQ ID NO: 2, and the second amino acid is the one at the position corresponding to amino acid 35 in SEQ ID NO: 2. Preferably, neither of the amino acids in the E2B DNA pol I polypeptide at positions corresponding to amino acids 34-35 in SEQ ID NO: 2 are nonsense (i.e. stop) mutations.

[0107] In yet further embodiments, the invention provides a mutant E2B DNA pol I gene, wherein the nucleotide in the mutant E2B DNA pol I gene at the position corresponding to nucleotide 101 in SEQ ID NO: 1 is A. Preferably, the nucleotide sequence of the mutant E2B DNA pol I gene also has at least 80%, 85%, 90%, 95% or 99% nucleotide sequence identity to SEQ ID NO: 1 ; and preferably it encodes a polypeptide having E2B DNA pol I activity.

[0108] In yet further embodiments, the invention provides a mutant E2B pTP gene, wherein the nucleotide in the mutant E2B pTP gene at the position corresponding to nucleotide 5,326 in SEQ ID NO: 3 is a G. Preferably, the nucleotide sequence of the mutant E2B pTP gene also has at least 80%, 85%, 90%, 95% or 99% (most preferably at least 99%) nucleotide sequence identity to SEQ ID NO: 3; and preferably it encodes a polypeptide having E2B terminal precursor protein activity.

[0109] The genes of the invention are preferably isolated or purified. As used herein, the term "isolated gene" means that the nucleic acid molecule is not contiguous with other genes or nucleotide sequences with which it is normally associated in an adenovirus. For example, an isolated nucleic acid of the invention comprising a DNA pol I gene of interest will not carry said DNA pol I gene contiguously with a directly neighbouring nucleotide sequence, e.g. a nucleic acid encoding its directly neighbouring gene(s), in an adenovirus genome. The references to a nucleic acid comprising the DNA pol I gene (and, mutatis mutandis, the pTP gene) should be construed accordingly. Thus, the isolated gene is not a wild-type gene of an adenovirus.

[0110] In yet further embodiments, the invention provides a mutant E2B DNA pol I polypeptide, wherein the amino acid in the mutant E2B DNA pol I polypeptide at the position corresponding to amino acid 34 in SEQ ID NO: 2 is D or E, preferably D. Preferably, the amino acid sequence of the mutant E2B DNA pol I polypeptide also has at least 80%, 85%, 90%, 95% or 99% (most preferably at least 99%) amino acid sequence identity or similarity to SEQ ID NO: 2; and preferably the polypeptide has E2B DNA pol I activity.

[0111] In yet further embodiments, the invention provides a mutant E2B pTP polypeptide, wherein the amino acid in the mutant E2B pTP polypeptide at the position corresponding to amino acid 623 in SEQ ID NO: 2 is T. Preferably, the amino acid sequence of the mutant E2B pTP polypeptide has at least 80%, 85%, 90%, 95% or 99% (most preferably at least 99%) amino acid sequence identity or similarity to SEQ ID NO: 4; and preferably the polypeptide has E2B pTP activity.

[0112] The polypeptides of the invention are preferably isolated. As used herein, the term "isolated polypeptide" means that the polypeptide is not in a mixture with other polypeptides with which it is normally associated in an adenovirus.

[0113] There are many established algorithms available to align two amino acid or nucleic acid sequences. Typically, one sequence acts as a reference sequence, to which test sequences may be compared. The sequence comparison algorithm calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alignment of amino acid or nucleic acid sequences for comparison may be conducted, for example, by computer- implemented algorithms (e.g. GAP, BESTFIT, FASTA or TFASTA), or Clustal Omega, BLASTn and BLASTp algorithms.

[0114] Standard protein-protein BLAST (blastp) may be used for finding similar sequences in protein databases. Like other BLAST programs, blastp is designed to find local regions of similarity. When sequence similarity spans the whole sequence, blastp will also report a global alignment, which is the preferred result for protein identification purposes. Preferably the standard or default alignment parameters are used. In some instances, the "low complexity filter" may be taken off.

[0115] With regard to nucleotide sequence comparisons, MEGABLAST, discontiguous- megablast, and blastn may be used to accomplish this goal. Preferably the standard or default alignment parameters are used. MEGABLAST is specifically designed to efficiently find long alignments between very similar sequences. Discontiguous MEGABLAST may be used to find nucleotide sequences which are similar, but not identical, to the nucleic acids of the invention.

[0116] The BLAST nucleotide algorithm finds similar sequences by breaking the query into short subsequences called words. The program identifies the exact matches to the query words first (word hits). The BLAST program then extends these word hits in multiple steps to generate the final gapped alignments. In some embodiments, the BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12.

[0117] One of the important parameters governing the sensitivity of BLAST searches is the word size. The most important reason that blastn is more sensitive than MEGABLAST is that it uses a shorter default word size (11). Because of this, blastn is better than MEGABLAST at finding alignments to related nucleotide sequences from other organisms. The word size is adjustable in blastn and can be reduced from the default value to a minimum of 7 to increase search sensitivity. - T1 -

[0118] A more sensitive search can be achieved by using the newly-introduced discontiguous megablast page (www.ncbi.nlm.nih.gov / Web / Newsltr / FallWinterO2 / blastlab.html). This page uses an algorithm which is similar to that reported by Ma et al. (Bioinformatics. 2002 Mar; 18(3): 440-5). Rather than requiring exact word matches as seeds for alignment extension, discontiguous megablast uses non-contiguous word within a longer window of template. In coding mode, the third base wobbling is taken into consideration by focusing on finding matches at the first and second codon positions while ignoring the mismatches in the third position. Searching in discontiguous MEGABLAST using the same word size is more sensitive and efficient than standard blastn using the same word size. Parameters unique for discontiguous megablast are: word size: 11 or 12; template: 16, 18, or 21 ; template type: coding (0), non-coding (1), or both (2).

[0119] In some embodiments, the BLASTP 2.5.0+ algorithm may be used (such as that available from the NCBI) using the default parameters. In other embodiments, a BLAST Global Alignment program may be used (such as that available from the NCBI) using a Needleman-Wunsch alignment of two protein sequences with the gap costs: Existence 11 and Extension 1 .

[0120] As used herein, the term “sequence identity” in the context of amino acid sequences may alternatively be replaced by “sequence similarity”. The term “similarity” allows conservative substitutions of amino acid residues having similar physicochemical properties over a defined length of a given alignment. The percentage of similarity is determinable with any reasonable similarity-scoring matrix.

[0121] The invention also provides a pharmaceutical composition comprising an adenovirus of the invention, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.

[0122] As used herein, the term “pharmaceutically-acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically-compatible. Examples of suitable carriers include water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Examples of suitable isotonic agents include sugars, poly-alcohols such as mannitol, sorbitol and sodium chloride.

[0123] Excipients are well known in the art and include buffers (e.g. citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g. serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres. Diluents include sterile water.

[0124] In one embodiment, the pharmaceutical composition is a liquid parenteral formulation, for example for infusion or injection, of an adenovirus of the invention.

[0125] As used herein, the term “parenteral formulation” refers to a pharmaceutical composition designed not to be delivered through the Gl tract. Typical parenteral delivery routes include injection, implantation or infusion. In one embodiment, the pharmaceutical composition is provided in a form for bolus delivery.

[0126] In one embodiment, the parenteral formulation is in the form of an injection. Injection includes intravenous, subcutaneous, intra-tumoral or intramuscular injection. Injection as employed herein means the insertion of liquid into the body via a syringe.

[0127] In one embodiment, the parenteral formulation is in the form of an infusion. As used herein, the term “infusion” refers to the administration of fluids at a slower rate by drip, infusion pump, syringe driver or equivalent device.

[0128] In one embodiment, the parenteral formulation is in the form of an infusion for intra venous administration. In another embodiment, the pharmaceutical composition is provided as a formulation for topical administration, including inhalation. Suitable inhalable preparations include inhalable powders, metering aerosols containing propellant gases or inhalable solutions free from propellant gases. Inhalable powders according to the disclosure will generally contain a virus as described herein with a physiologically acceptable excipient. These inhalable powders may include monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo- and polysaccharides (e.g. dextranes), poly-alcohols (e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures of these with one another. Mono- or disaccharides are suitably used, the use of lactose or glucose, particularly but not exclusively in the form of their hydrates.

[0129] Particles for deposition in the lung require a particle size less than 10 microns, such as 1 -9 microns for example from 0.1 to 5 microns, in particular from 1 to 5 microns. The particle size of the carrying the virus is of primary importance and thus in one embodiment the virus according to the present invention may be adsorbed or absorbed onto a particle, such as a lactose particle of the given size.

[0130] The propellant gases which can be used to prepare the inhalable aerosols are known in the art. Suitable propellant gases are selected from among hydrocarbons such as n- propane, n-butane or isobutane and halo-hydrocarbons such as chlorinated and / or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. The above-mentioned propellant gases may be used on their own or in mixtures thereof. Particularly suitable propellant gases are halogenated alkane derivatives selected from among TG 11 , TG 12, TG 134a and TG227. Of the abovementioned halogenated hydrocarbons, TG134a (1 ,1 ,1 ,2-tetrafluoroethane) and TG227 (1 ,1 ,1 ,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable.

[0131] The propellant gas-containing inhalable aerosols may also contain other ingredients, such as cosolvents, stabilisers, surface-active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All these ingredients are known in the art. The propellant gas-containing inhalable aerosols according to the invention may contain up to 5 % by weight of active substance. Aerosols according to the invention contain, for example, 0.002 to 5 % by weight, 0.01 to 3 % by weight, 0.015 to 2 % by weight, 0.1 to 2 % by weight, 0.5 to 2 % by weight or 0.5 to 1 % by weight of active ingredient.

[0132] Alternatively topical administrations to the lung may also be by administration of a liquid solution or suspension formulation, for example employing a device such as a nebulizer, for example, a nebulizer connected to a compressor (e.g., the Pari LC-Jet Plus nebulizer connected to a Pari Master(R) compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.).

[0133] Nebulisable formulation according to the present disclosure may be provided, for example, as single dose units (e.g., sealed plastic containers or vials) packed in foil envelopes. Each vial contains a unit dose in a volume, e.g., 2 mL, of solvent / solution buffer.

[0134] The pharmaceutical composition of the invention will typically be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other parenteral formulation suitable for administration to a human and may be formulated as a pre-filled device such as a syringe or vial, particular as a single dose.

[0135] Subjects who are treated with the adenovirus of the invention may additionally be treated with one or more other chemotherapeutic or immunotherapeutic agents, i.e. specific anti-neoplastic chemical agents, or drugs that are selectively destructive to malignant cells or tissues or raise an anti-tumour immune response. Such other chemotherapeutic or immunotherapeutic agents may, for example, include one or more of the following: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, checkpoint inhibitors, antibodies and other anti-tumour agents. Examples of specific chemotherapeutic agents include doxorubicin, 5-fluorouracil (5- FU), a taxane derivative (e.g. paclitaxel, docetaxel), capecitabine, irinotecan, abraxane and a platin (e.g. cisplatin, carboplatin and oxaliplatin).

[0136] The chemotherapeutic agent may be one which does not interfere with adenovirus activity (e.g. a beneficial property or characteristic of the virus, for example the oncolytic activity and or the ability of the virus to replicate in cancer cells, such as viral replication in vivo).

[0137] Generally, such a pharmaceutical combination will be provided as two components:

[0138] (A) a first pharmaceutical composition of the adenovirus of the invention; and

[0139] (B) a second pharmaceutical composition comprising a chemotherapeutic or immunotherapeutic agent.

[0140] The pharmaceutical combination of the invention may therefore be in the form of a combined preparation for simultaneous, separate or sequential use, preferably for the treatment of cancer.

[0141] Similarly, in the methods of the invention, the first and second pharmaceutical compositions may be administered to a patient simultaneously, separately or sequentially.

[0142] The term “combined preparation” includes both fixed combinations and non-fixed combinations. The term “fixed combination” means that the active ingredients (e.g. components (A) and (B)) are in the form of a single entity or dosage unit. In other words, the active ingredients are present in a single composition or formulation. The term “nonfixed combination” means that the active ingredients (e.g. components (A) and (B)) are present in different entities or dosages (e.g. as separate compositions or formulations), for example as a kit of parts. The independent components (A) and (B) (in their desired compositions or formulations) can then be administered simultaneously, separately or sequentially, at the same time point or at different time points. Where the administration is simultaneous, components (A) and (B) are administered to the subject at the same time, but not necessarily together. Components (A) and (B) may be present in a single composition or they may be present in different compositions.

[0143] Components (A) and (B) may be administered at the same site or at different sites (in or on the subject). Components (A) and (B) may be administered by the same route or different routes.

[0144] Where the administration is sequential, the delay in administering the second component should not be such as to lose the benefit of the synergistic effect arising from use of the combination.

[0145] Components (A) and (B) may each be administered once or at a plurality of times. Components (A) and (B) may be administered in any order, e.g. component (A) first and then component (B); or component (B) first and then component (A).

[0146] The invention also provides a kit comprising: (A) a first pharmaceutical composition of the adenovirus of the invention; and (B) a second pharmaceutical composition comprising a chemotherapeutic agent, optionally together with instructions for use.

[0147] In another embodiment, the invention provides an oncolytic adenovirus of the invention for use in therapy or for use as a medicament. In another embodiment, the invention provides an oncolytic adenovirus of the invention for use in treating cancer (preferably ovarian cancer). In another embodiment, the invention provides a method of treating cancer (preferably ovarian cancer) in a subject, the method comprising administering an effective amount of an oncolytic adenovirus of the invention to a subject in need thereof. In another embodiment, the invention provides the use of an oncolytic adenovirus of the invention in the manufacture of a medicament for treating cancer (preferably ovarian cancer). The invention also provides the use of an oncolytic adenovirus of the invention for the treatment of cancer (preferably ovarian cancer); and an oncolytic adenovirus of the invention when used to treat cancer (preferably ovarian cancer). As used herein, the term “treating cancer” includes killing cancer cells and / or preventing or inhibiting the spread of cancer cells.

[0148] The Therapeutic Index (Tl, also referred to as the Therapeutic Ratio) is a quantitative measurement of the relative safety of a drug. It is a comparison of the amount of a therapeutic agent that causes the therapeutic effect to the amount that causes toxicity. The related terms Therapeutic Window and Safety Window refer to a range of doses optimized between efficacy and toxicity, achieving the greatest therapeutic benefit without resulting in unacceptable side-effects or toxicity.

[0149] For humans in clinical trials, the Therapeutic Index is defined as TD50 / ED50 (where TD50 is the toxic dose in 50% of subjects; and ED50is the minimum effective dose for 50% of the population).

[0150] The subject is a human. The subject may be human male or female (biologically). The human may, for example, be 0-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80- 90, 90-100 or above 100 years old. The human may be one who is suffering from or at risk from a particular disease or disorder, e.g. cancer, preferably ovarian cancer. In some preferred embodiments, the subject is one who is suffering from or has previously suffered from cancer, preferably ovarian cancer.

[0151] As used herein, the term “stroma” refers to cells and tissues that support and give structure to organs, glands, or other tissues in the body. The stroma is mostly made up of connective tissue, blood vessels, lymphatic vessels and nerves. It provides nutrients to the tissue or organ and removes waste and extra fluid. The stroma may also be involved in the body’s immune response and in the growth and spread of cancer cells. In some embodiments, the cancer is a tumour.

[0152] In some preferred embodiments, the cancer is a stroma-containing tumour. As used herein, the term “stroma-containing tumour” includes tumours which comprise at least 1 %, preferably at least 5%, 10%, 20% or 50% stromal cells (by weight of the tumour). In some embodiments, the stroma-containing tumour comprises 20-80% stromal cells. The stromal cells may comprise CAFs. The cancer or tumour may be one which comprises CAFs.

[0153] In some preferred embodiments, the cancer is a carcinoma. In some preferred embodiments, the cancer is ovarian cancer, colorectal cancer, lung cancer, hepatoma, multiple myeloma, oesophageal cancer, breast cancer or pancreatic cancer. Most preferably, the cancer is ovarian cancer or a stroma-containing carcinoma.

[0154] In some embodiments, the adenovirus of the present invention may be used as a pretreatment to a therapy, such as a surgery (neo-adjuvant therapy), to shrink the tumour, to treat metastasis and / or prevent metastasis or further metastasis.

[0155] In other embodiments, the adenovirus of the invention may be used after the therapy, such as after a surgery (adjuvant therapy), to treat metastasis and / or prevent metastasis or further metastasis.

[0156] A pharmaceutical composition of the present invention may be administered via one or more routes using one or more of a variety of methods known in the art. Components or compositions (A) and (B) may be administered by the same route or by different routes. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results.

[0157] Preferred routes of administration for pharmaceutical composition of the present invention include intravenous, intratumoural, intraperitoneal, intrapleural, intravesical, intramuscular, intradermal, or other parenteral routes of administration, for example by injection or infusion.

[0158] The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intratumoural, intraperitoneal, intrapleural, intravesical, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, injection and infusion. Alternatively, pharmaceutical composition of the present invention may be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0159] In one embodiment the formulation is for intravenous (i.v.) administration. This route is particularly effective for delivery of oncolytic viruses because it allows rapid access to the majority of the organs and tissues, and is particularly useful for the treatment of metastases, for example established metastases, especially those located in highly- vascularised regions such as the liver and lungs.

[0160] In one embodiment the formulation is for intraperitoneal (i.p.) administration. This route is particularly effective for delivery of oncolytic viruses to cancer types predominantly localised to peritoneum (e.g. ovarian, colorectal, stomach cancers). This is because it allows rapid access to the majority of the relevant organs and tissues, to treat the primary tumour and metastasis, without dilution by the blood stream and minimises off- target effects.

[0161] In one embodiment the formulation is for intratumoural (i.t.) administration. This route is particularly effective for delivery of oncolytic viruses to localised cancer types, accessible by injection needle. This is because it allows rapid access to the tumour and minimises off-target effects.

[0162] The pharmaceutical composition of the invention may comprise a therapeutically- effective dose of the adenovirus of the invention. The term “therapeutically-effective dose” refers to the amount of adenovirus that is suitable for achieving the intended therapeutic effect when employed in a suitable treatment regimen, for example ameliorates symptoms or conditions of a disease, in particular without eliciting dose limiting side effects. A dose may be considered a therapeutic dose in the treatment of cancer or metastases when the number of viral particles may be sufficient to result in the following: tumour or metastatic growth is slowed or stopped, or the tumour or metastasis is found to shrink in size, and / or the life span of the patient is extended. Suitable therapeutic doses are generally a balance between therapeutic effect and tolerable toxicity, for example where the side-effect and toxicity are tolerable given the benefit achieved by the therapy.

[0163] In one embodiment, the pharmaceutical composition of the invention may comprise 1x1010to 1x1014viral particles per dose. Preferably, the pharmaceutical composition of the invention comprises 1x1011to 1x1013viral particles per dose. In one embodiment, the pharmaceutical composition of the invention may be administered over 1-8 cycles, with each cycle comprising one or multiple administrations over a 1 month period. Cycles may not be given in consecutive months.

[0164] Preferably, the method steps are carried out in the order specified.

[0165] The adenoviruses of the invention may readily be produced using techniques which are well known in the art, including using standard cloning techniques (e.g. restriction enzyme, site-directed mutagenesis or Gibson assembly) or CRISPR-based technologies (e.g. CRISPR Cas9 / guideRNA) to introduce one or more nucleotide mutations into a known adenoviral genome.

[0166] In another embodiment of the invention, there is provided the use of a conditionally- replicating adenovirus of the invention as a vector for protein production, wherein the adenovirus comprises a transgene encoding the protein to be produced.

[0167] In yet another embodiment of the invention, there is provided the use of a conditionally- replicating adenovirus of the invention as a helper virus for virus production.

[0168] The disclosure of each reference set forth herein is specifically incorporated herein by reference in its entirety. BRIEF DESCRIPTION OF THE FIGURES

[0169] Figure 1 : A simplified diagram of the positions of the early (E) and late (L) genes in the adenovirus genome. Arrows represent viral genes, the directions of which represent the coding direction. ITR = Inverted Terminal Repeat.

[0170] Figure 2: A high grade serous ovarian cancer patient sample was infected with increasing concentrations of one of the bioselected adenoviruses, Ov26. Six days postinfection, the viability of cancer cells (A) and cancer associated fibroblasts (CAFs) (B) within the sample was determined by multiparametric flow cytometry. FAP= fibroblast activation protein, a cell surface marker present on CAFs. CA125 = cancer antigen 125, a cell surface marker present on cancer cells. VG / cell = viral genomes per cell

[0171] Figure 3: Cell viability of a variety of different cell lines in the presence of Ov26. Cells were infected with increasing doses of Ov26 and viability was measured 5 days postinfection by MTS assay. Data is expressed as viability relative to the uninfected control. VG / cell = viral genomes per cell.

[0172] Figure 4: Oncolytic activity of Ov26 compared to wild-type parental viruses.

[0173] (A) Viral genome replication was measured by qPCR 7 days post-infection and is expressed as viral genomes per cell. (B) Infectious progeny virus was quantified by ICC assay and is expressed as Infection Forming Units (I FU) / cell . For each virus, data is normalised to the level observed in A549 cells.

[0174] Figure 5: Ov26 and Ov26_DNAPol_WT differ in a single substitution mutation: Ov26 has the mutation; Ov26_DNAPol_WT has the wild-type sequence.

[0175] A) Schematic diagram of the genome of Ov26. The position of the difference between Ov26 and Ov26_WT_DNAPol within the virus genome is indicated by the vertical black arrow. Unfilled horizontal arrows show the positions and orientations of the surrounding viral genes.

[0176] B) Nucleotide sequence of the region containing the mutation. On the right, the mutation in Ov26 is shown in bold. The left displays the sequence of Ov26_DNAPol_WT. This single mutation in Ov26 gives rise to an amino acid change in both the DNA Pol and pTP genes since they have overlapping open reading frames. (SEQ ID NOs: 5-10, when read in the appropriate 5’-3’ and N-C orientation.)

[0177] Figure 6: OV26 displays enhanced viral genome replication relative to OV26_DNAPol_WT in different cancer cell lines. Viral genomes (VG) were quantified by qPCR 48 hours post-infection of (A) Ad-293, (B) Hela, (C) Panel and (D) A549 cells at a dose of 100 viral genomes / cell. The mean of three biological replicates is plotted; significance was tested by unpaired T-test.

[0178] Figure 7: Viral replication in patient-derived cancer samples and ovarian cancer cell line OvCAR3. Primary samples of cells from patient derived ascites fluid and ovarian cancer cell line OVCAR3 cells were infected at a dose of 100 virus genomes / cell. 6 days after infection viral genomes were measured by qPCR. Data is expressed as a fold change over the input virus dose. Significance was assessed by multiple T tests, ** P < 0.01.

[0179] Figure 8: A549 cells were infected with 100 viral genomes per cell of Ov26 or Ov26_DNAPol_WT. At 3 days post-infection wells were harvested. Samples were freeze-thawed to achieve cell lysis, and then infectious virus particles were quantified by immunocytochemistry (ICC) assay using an antibody against the viral hexon protein. Significance was assessed by T test *** P < 0.001 .

[0180] Figure 9: Genome replication in normal cell lines 6 days post-infection. Data is normalised to the fold change measured for Ov26 in each cell line.

[0181] NHDF = Normal human dermal fibroblasts

[0182] HPF = Human Pulmonary Fibroblasts

[0183] HCF = Human Cardiac Fibroblast

[0184] HUF = Human Uterine Fibroblasts

[0185] HAoAF = Human Aortic Adventitial Fibroblasts

[0186] Figure 10: Analysis of OV26 genome over 30 passages in vitro. Ov26 was used to infect Ad-293 cells. Upon formation of widespread cytopathic effect, material was harvested and used to infect a subsequent passage of Ad-293 cells. After 30 passages of virus infections, material was harvested and DNA was extracted. DNA was also extracted from the input virus; DNA was sequenced using whole genome Illumina® sequencing. A graphical representation of sequence alignment and read coverage for input virus (left) and virus after 30 infection cycles (right) is shown and reveals no mutations.

[0187] Figure 11 : Quantification of Luciferase expression from A549 cells infected with either Ov26_SA-Fluc or Ov26_DNAPol_WT_SA-Fluc. Cells were infected at a multiplicity of infection of 0.6 infectious units per cell. 48 hours post infection luciferase expression was measured by lysis of cells and addition of luciferin, luminescence was measured on a plate reader.

[0188] EXAMPLES

[0189] The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0190] Example 1 : Bioselection for oncolytic adenoviruses

[0191] From an initial mixed pool of Group B, C, D, F, and G adenoviruses, 23 rounds of bioselection (including mutational steps) were performed to enrich for oncolytic adenovirus candidates with the best combination of tumour lysis of patient derived ovarian cancer cells, spread, blood stability and immune stimulation. The initial mixed pool of Group B, C, D, F, and G adenoviruses were obtained either from commercial sources (American Type Culture Collection or Public Health England) or from a UK Collaboration Network. From the final pool, 60 adenoviruses were isolated, selected for sequencing and their genomes were analysed. Genome analyses of the selected adenoviruses revealed that a large number of the selected adenoviruses had a missense mutation in the E2B DNA pol I gene. The mutation was the single nucleotide change ggc gac in the adenovirus genome. This nucleotide change results in a G34D mutation in the E2B DNA pol I polypeptide and an A623T mutation in the E2B TPP polypeptide.

[0192] One of the selected adenoviruses which had the mutation, i.e. Ov26, was chosen for further study.

[0193] Given that both D (aspartic acid) and E (glutamic acid) are both negatively-charged amino acids of similar structure, similar results would be expected from a G34E mutation. Given that T (threonine), N (asparagine), C (cysteine), Q (glutamine) and S (serine) are all polar / neutral amino acids of similar structure, similar results would be expected from A623N, A623C, A623Q and A623S mutations.

[0194] Example 2: Ov26 kills cancer cells and CAFs from an ex vivo ovarian cancer sample

[0195] A high grade serous ovarian cancer (HGSOC) patient sample was infected with increasing concentrations of Ov26. Six days post-infection, the viability of cancer cells and cancer associated fibroblasts (CAFs) within the sample was determined by multiparametric flow cytometry.

[0196] The results are shown in Figure 2. These results show that the viability of the cancer cells and the CAFs decreased with increasing concentrations of Ov26.

[0197] Example 3: Ov26 kills a range of ovarian patient cancer subtypes including treatment-naive and platinum-resistant cells

[0198] Ovarian patient samples (n=24) were treated with a clinically-relevant viral dose of Ov26 (equivalent to 1e13 vp / 5L / patient) and the viability of cancer cells and cancer associated fibroblasts (CAFs) was assessed. More specifically, cell viability was assessed after 6 days using flow cytometry. Cells were stained for viability using Live / Dead Near I R , EpCAM / CA125 marker to identify cancer cells and fibroblast activation protein (FAP+) to identify cancer associated fibroblasts. Each condition was measured in triplicate and presented as the mean viability relative to an uninfected control. HGSOC = High grade serous ovarian cancer. LGSOC = Low grade serous ovarian cancer.

[0199] The results are shown in Table 4 below:

[0200] The above table demonstrates that Ov26 effectively kills cancer cells and cancer associated fibroblasts (CAFs) across a range of different ovarian cancer patient subtypes.

[0201] Example 4: Use of a panel of cell lines to assess oncolytic activity of Ov26

[0202] The oncolytic activity of Ov26 was assessed in a number of cancer cell lines and cancer associated fibroblasts including the following:

[0203] Ad-293 Human embryonic kidney cell line

[0204] A549 Lung cancer cell line

[0205] Hela Cervical cancer cell line

[0206] MDA-MB-231 Breast cancer cell line

[0207] PANC1 Pancreatic cell line

[0208] PSN1 Pancreatic cell line

[0209] HCT116 Colorectal cancer cell line

[0210] HT29 Colorectal cancer cell line

[0211] SKOV-3 Ovarian cell line

[0212] Huh-7 Hepatoma cell line

[0213] MRC5 Fibroblast cell line

[0214] OE-21 Oesophageal cell line

[0215] OVCAR-3 Ovarian cell line

[0216] OVSAHO Ovarian carcinoma cell line

[0217] The results are shown in Figure 3. Ov26 demonstrated efficacy against all cancer cell lines tested against.

[0218] The IC50 values of Ov26 are given in Table 5 below. Table 5. IC50 values of Ov26 in various cancer cell lines

[0219] Example 5: Oncolytic activity of Ov26 compared to wild-type parental viruses Various parameters of Ov26 virus infection were measured 72 hours after infection of lung cancer cells (A549s), of non-cancerous normal primary human hepatocytes and of normal human dermal fibroblasts (NHDFs). The results are shown in Figure 4.

[0220] Production of viral genomes (A) and infectious virus particles (B) was lower in normal cells relative to A549 cancer cells during Ov26 infection as compared to infections with wild type parental viruses. The results demonstrate the ability of Ov26 to preferentially infect the lung cancer cells compared to normal hepatocytes and normal fibroblasts.

[0221] Example 6: Generation of control adenovirus without mutation

[0222] Sequence data from adenovirus Ov26 (which has the ggc gac mutation) is represented in Figure 5, compared to that of a control adenovirus (Ov26_DNAPol_WT) which has the same genome sequence as Ov26 except for the latter single nucleotide mutation.

[0223] Example 7: The growth kinetics of Ov26 and Ov26_DNAPol_WT in cancer cell lines

[0224] The growth kinetics of Ov26 and Ov26_DNAPol_WT over 48 hours in four different cell lines is shown in Figure 6. Viral genomes accumulated to higher levels in Ad-293 (A), HeLa (B), Panc-1 (C) and A549 (D) cells, as measured by qPCR 48 hours postinfection. The data shows that Ov26 is capable of replicating to high levels postinfection than Ov26_DNAPol_WT.

[0225] Example 8: Genome replication in ascites samples and ovarian cancer cell line Replication of Ov26 and Ov26_DNAPol_WT was assessed in patient-derived cancer samples and the ovarian cancer cell line OvCAR3.

[0226] Primary samples of cells from patient-derived ascites fluid, and ovarian cancer cell line OVCAR3 cells were infected at a dose of 100 virus genomes / cell, 6 days after infection viral genomes were measured by qPCR. The results are shown in Figure 7 and show that Ov26 is able to replicate to a greater extent than Ov26_DNAPol_WT in ascites samples leading to an accumulation of a greater number of genomes by day 6 post infection.

[0227] Example 9: Production of infectious virus particles in A549 cells

[0228] A549 cells were infected with 100 viral genomes per cell of Ov26 or Ov26_DNAPol_WT virus. Infectious virus particles were then measured at various time points post-infection Figure 8 shows that infection with Ov26 leads to the production of a greater number of infectious virus particles compared to Ov26_DNAPol_WT.

[0229] Example 10: Genome replication in normal cell lines 6 days post infection

[0230] Genome replication of Ov26 and Ov26_DNAPol_WT in various normal cell lines was quantified by qPCR 3 days post-infection of cells with 100 viral genomes per cell. The results are shown in Figure 9, which show that the mutation present in the DNA pol I gene of Ov26 does not confer enhanced viral replication in normal cells, and that this mutation serves to increase the therapeutic index of the virus.

[0231] Example 11 : Mutations in the E2B DNA pol I and pTP genes do not impair the virus proof-reading or result in mutation of the virus genome

[0232] The Ov26 genome was analysed over 30 passages in vitro. Ad-293 cells were infected with Ov26, and harvested when signs of CPE were observed. The harvested material was then used to infect the following passage. DNA was extracted from the input virus, as well as passage 10, 20, and 30, and libraries were produced for whole genome Illumina® sequencing.

[0233] The results for the input virus versus the virus after 30 passages are shown in Figure 10. These results show that no mutations were detected by Illumina® sequencing in the viral genome after 30 passages in cell culture, indicating that the E2B mutation in Ov26 does not impair the fidelity of the virus polymerase.

[0234] Example 12: Production of adenovirus with fluorescent reporter transgene

[0235] Viral genomes of Ov26 and Ov26_DNAPol_WT were vectorised into Bacterial Artificial Chromosomes (BACs) and a recombineering approach was used to insert a firefly luciferase reporter gene driven by an SA promoter (SAFIuc) into the viral E3 region. The BACs, now encoding viral genomes harbouring reporter transgenes, were transfected into Ad-293 cells and replicating virus was recovered, amplified and titred by immunocytochemistry assay. The virus was then used to infect A549 cells at a dose of 0.6 infectious units per cell. Luciferase expression was measured at 48 hours post-infection. The data (Figure 11) shows that Ov26 is capable of producing greater levels of transgene expression than Ov26_DNAPol_WT. The Sequence Listing filed with this patent application is fully incorporated herein as part of the description.

Claims

CLAIMS1 . A pharmaceutical composition comprising an adenovirus, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents, wherein the genome of the adenovirus comprises an E2B DNA pol I gene encoding a DNA pol I polypeptide, and wherein the amino acid in the DNA pol I polypeptide sequence corresponding to amino acid 34 in SEQ ID NO: 2 is Asp or Glu, preferably Asp.

2. A pharmaceutical composition as claimed in claim 1 , wherein the genome of the adenovirus additionally comprises an E2B pTP gene encoding a pre-Terminal Protein, wherein the coding region of the E2B DNA pol I gene overlaps with the coding region of the E2B pTP gene, and wherein the amino acid in the pTP sequence at the position corresponding to amino acid 623 in SEQ ID NO: 4 is selected from the group consisting of Thr, Met, Lys and Arg, preferably Thr.

3. A pharmaceutical composition as claimed in claim 1 , wherein the nucleotide in the E2B DNA pol I gene at the position corresponding to nucleotide 5,326 in SEQ ID NO: 1 is A.

4. A pharmaceutical composition comprising an adenovirus, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents , wherein the genome of the adenovirus comprises an E2B pTP gene encoding a pre-Terminal Protein, and wherein the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 4 is selected from the group consisting of Thr, Asn, Cys, Gin and Ser, preferably Thr.

5. A pharmaceutical composition as claimed in claim 4, wherein if the adenovirus is an Ad1 adenovirus, then the amino acid is not Asn, if the adenovirus is an Ad41 adenovirus, then the amino acid is not Gin; or if the adenovirus is an Ad4 or 4a adenovirus, then the amino acid is not Thr.

6. A pharmaceutical composition as claimed in claim 4 or claim 5, wherein the genome of the adenovirus additionally comprises an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the coding region of the E2B pTP gene overlaps with the coding region of the E2B DNA pol I gene, and wherein the amino acids in the DNA pol I polypeptide at the positions corresponding to amino acids 34-35 in SEQ ID NO: 2 are selected from the group consisting of Asp-Ser, Asp-Pro, Asp-Thr, Asp-Ala, Glu-Ser, Glu-Pro, Vai-Ser, Val-Pro, Ala-Thr, Ala-Ala, Val-Thr and Val-Ala.

7. A pharmaceutical composition as claimed in claim 4 or claim 5, wherein the nucleotide in the E2B pTP gene at the position corresponding to nucleotide 5,326 in SEQ ID NO: 3 is A.

8. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the adenovirus is a conditionally-replicating adenovirus or an oncolytic adenovirus.

9. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the adenovirus is a Group B adenovirus or a human adenovirus, preferably a Group B1 adenovirus.

10. A pharmaceutical composition as claimed in claim 9, wherein the adenovirus is an Ad3 or Ad7 adenovirus or an Ad3 / Ad7 chimeric adenovirus.

11. A pharmaceutical composition as claimed in any one of claims 1 to 3 or 6 to 10, wherein the E2B DNA pol I gene comprises or consists of:(i) the nucleotide sequence as given in SEQ ID NO: 1 ;(ii) a nucleotide sequence which has at least 80%, 85%, 90%, 95% or 99% nucleotide sequence identity to SEQ ID NO: 1 , and which preferably encodes a DNA polymerase I; or(iii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 2.

12. A pharmaceutical composition as claimed in any one of claims 1 to 3 or 6 to 11 , wherein the E2B DNA pol I polypeptide comprises or consists of:(i) the amino acid sequence as given in SEQ ID NO: 2; or(ii) a variant of (i) having at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity or sequence similarity to (i), and which preferably has DNA polymerase I activity.

13. A pharmaceutical composition as claimed in any one of claims 2 to 12, wherein the E2B pTP gene comprises or consists of:(i) the nucleotide sequence as given in SEQ ID NO: 3;(ii) a nucleotide sequence which has at least 80%, 85%, 90%, 95% or 99% nucleotide sequence identity to SEQ ID NO: 3, and which preferably encodes a pre-Terminal Protein; or(iii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 4.

14. A pharmaceutical composition as claimed in any one of claims 2 to 13, wherein the E2B pre-Terminal Protein is a polypeptide comprising or consisting of:(i) the amino acid sequence as given in SEQ ID NO: 4; or(ii) a variant of (i) having at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity or sequence similarity to (i), and which preferably encodes a pre-Terminal Protein.

15. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the adenovirus comprises a transgene, preferably wherein the transgene is located in or adjacent to the E1 region, E3 region, L3 region, L5 region or in an E1 / E3- deleted region of the adenovirus.

16. A pharmaceutical combination comprising:(A) a first pharmaceutical composition as claimed in any one of claims 1 to 15; and(B) a second pharmaceutical composition comprising a chemotherapeutic or immunotherapeutic agent,wherein the pharmaceutical combination is in the form of a combined preparation for simultaneous, separate or sequential use, preferably for the treatment of cancer.

17. An adenovirus as defined in any one of claims 1 to 15 or a pharmaceutical composition as claimed in any one of claims 1 to 15 for use in therapy or for use as a medicament.

18. An adenovirus as defined in any one of claims 1 to 15 or a pharmaceutical composition as claimed in any one of claims 1 to 15 for use in treating cancer (preferably ovarian cancer).

19. A method of treating cancer (preferably ovarian cancer) in a subject, the method comprising administering an effective amount of an adenovirus as defined in any one of claims 1 to 15 or a pharmaceutical composition as claimed in any one of claims 1 to 15 to a subject in need thereof.

20. Use of an adenovirus as defined in any one of claims 1 to 15 in the manufacture of a medicament for treating cancer (preferably ovarian cancer).21 . An adenovirus for use as claimed in claim 18, a method as claimed in claim 19 or a use as claimed in claim 20, wherein the cancer is selected from the group consisting of ovarian cancer, colorectal cancer, lung cancer, hepatoma, multiple myeloma, oesophageal cancer, breast cancer and pancreatic cancer, preferably ovarian cancer or a stroma-containing carcinoma.

22. Use of a conditionally-replicating adenovirus as defined in any one of claims 1 to 15 as a vector for protein production, wherein the adenovirus comprises a transgene encoding the protein to be produced.

23. Use of a conditionally-replicating adenovirus as defined in any one of claims 1 to 15 as a helper virus for virus production.

24. A mutant Group B or human adenovirus E2B DNA pol I gene, wherein the nucleotide in the mutant E2B DNA pol I gene at the position corresponding to nucleotide 101 in SEQ ID NO: 1 is A.

25. A mutant Group B or human adenovirus E2B pTP gene, wherein the nucleotide in the mutant E2B pTP gene at the position corresponding to nucleotide 5,326 in SEQ ID NO: 3 is a G.

26. A mutant Group B or human adenovirus E2B DNA pol I polypeptide, wherein the amino acid in the mutant E2B DNA pol I polypeptide at the position corresponding to amino acid 34 in SEQ ID NO: 2 is D or E, preferably D.

27. A mutant Group B or human adenovirus E2B pTP polypeptide, wherein the amino acid in the mutant E2B pTP polypeptide at the position corresponding to amino acid 623 in SEQ ID NO: 2 is T.

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