Adenoviruses

Abstract

The present invention relates to adenoviruses, particularly oncolytic adenoviruses, for use in the prevention or treatment of cancer, including tumours containing stroma and ovarian cancer. In particular, the invention relates to adenoviruses which have a deletion in the E3 region compared to wild-type adenoviruses.

Description

[0001] ADENOVIRUSES

[0002] The present invention relates to adenoviruses, particularly oncolytic adenoviruses, for use in the prevention or treatment of cancer, including tumours containing stroma and ovarian cancer. In particular, the invention relates to adenoviruses which have a deletion in the E3 region compared to wild-type adenoviruses.

[0003] Carcinomas are cancers derived from epithelial tissue that line external or luminal surfaces of organs including the lung gastrointestinal tract and reproductive tissues. Carcinomas represent about 90% of all cancer cases globally and are the cause of the vast majority of cancer deaths.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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. An ideal intervention to treat carcinomas would simultaneously and selectively target both cancer cells and stromal populations.

[0011] 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.

[0012] 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).

[0013] 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).

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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 bioselected adenoviruses. In particular, the bioselected adenovirus genomes were all found to be from Group B adenoviruses, with the genomes all comprising a deletion in their E3 region compared to wild-type Group B adenoviruses.

[0026] The wild-type E3 region of Group B adenovirus genomes normally comprises 9 ORFs. In all of the bioselected viruses, at least 5 of these ORFs (19.3K, 20K, 20.6K, 7.7K and 10.3K) were found to be fully or partially deleted; and a further two ORFs (16.1 K and 14.9K) were partially deleted in some of the bioselected adenoviruses. As described above, the cancer patients’ tumour microenvironment is rife with stress proteins (e.g. TNF-alpha), which in combination with early virus infection may result in rapid death of the first infected tumour cells. Rapid death of tumour cells prevents effective replication of the virus to high yields, limiting virus propagation and resulting in premature clearance of the virus.

[0027] It is shown herein that the retention of the full or partial E3 14.9K ORF has value in blocking NF-KB signalling. This has been found to prevent premature clearance of the adenoviruses, thus allowing longer time for the adenoviruses to initiate infection of cells.

[0028] Additionally, it is shown herein that the retention of the E3 14.7K ORF provides protection against early apoptosis of infected cells. This have been found to allow further time for viral replication within cells thus increasing the infectious titre of adenoviruses of the invention.

[0029] It is also shown herein that large transgenes can be inserted into the E3 deletion site of the bioselected adenoviruses of the invention without significant loss of efficacy against cancer cells. Loss of some E3 ORFs also attenuates the virus in normal cells compared to the parental wildtype strains.

[0030] The invention therefore provides oncolytic adenoviruses having one or more of the above features and uses thereof in the prevention or treatment of cancer.

[0031] US 2002 / 106746 A1 discloses a recombinant adenoviral vector derived from an Ad5 adenovirus genome in which at least a part of the E3 region is deleted or is nonfunctional, but wherein said adenoviral vector retains E3 sequences encoding a functional 14.7K protein, a functional 14.5K protein, and / or a functional 10AK protein. The corresponding proteins in a Group B (e.g. Ad3 or Ad7) adenovirus would be the 14.7 K, 14.9K and 10.3K proteins, respectively. The genome of Enadenotucirev (ColoAd; Kuhn et al. PLoS One 2008; 3(6): e2409) comprises deletions in the E3 region which include full or partial deletions of the 14.9K and 14.7K ORFs. It is an object of the invention to provide adenoviruses which are capable of lysing a range of different tumour cells and cancer-associated fibroblasts (CAFs) but not normal cells, which are capable of spreading, which are stable in blood and which are capable of inducing immunogenic cell death and stimulating immune responses, including from T cell and dendritic cells, upon tumour cell lysis.

[0032] It is another object of the invention to provide compositions comprising the adenoviruses of the invention and uses thereof in the prevention or treatment of cancer.

[0033] In one embodiment, the invention provides an oncolytic adenovirus having a genome comprising an E3 region:

[0034] (a) wherein the E3 region comprises 12.1 K and 14.7K ORFs from a Group B adenovirus E3 region or ORFs corresponding thereto from non-Group B adenovirus E3 regions; and (b) wherein the E3 region does not comprise functional 20K, 20.6K, 7.7K or 10.3K ORFs from a Group B adenovirus E3 region or ORFs corresponding thereto from non- Group B adenovirus E3 regions.

[0035] In some embodiments, the E3 region additionally comprises a functional (optionally 3’- truncated) 16.1 K ORF from a Group B adenovirus E3 region or an ORF corresponding thereto from a non-Group B adenovirus E3 region. In some embodiments, the E3 region additionally comprises a 16.1 K ORF and a functional (optionally 3’-truncated) 19.3K ORF from a Group B adenovirus E3 region or ORFs corresponding thereto from a non- Group B adenovirus E3 region. In some embodiments, the E3 region additionally comprises a 14.9K ORF from a Group B adenovirus E3 region or an ORF corresponding thereto from a non-Group B adenovirus E3 region.

[0036] In a further embodiment, the invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region comprises or consists of:

[0037] (a) a Group B adenovirus E3 12.1 K ORF;

[0038] (b) a 3-truncated Group B adenovirus E3 16.1 K ORF;

[0039] (c) a 5’-truncated Group B adenovirus E3 10.3K ORF;

[0040] (d) a Group B adenovirus E3 14.9K ORF; and (e) a Group B adenovirus E3 14.7K ORF, or ORFs corresponding thereto from non-Group B adenovirus E3 regions, joined contiguously in the above 5‘-3‘ order, wherein the E3 region may optionally comprise one or more transgenes located within or adjacent to one or more of the said ORFs.

[0041] In a further embodiment, the invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region comprises or consists of:

[0042] (a) a Group B adenovirus E3 12.1 K ORF;

[0043] (b) a Group B adenovirus E3 16.1 K ORF;

[0044] (c) a 3-truncated Group B adenovirus E3 19.3K ORF;

[0045] (d) a 5’-truncated Group B adenovirus E3 14.9K ORF; and

[0046] (e) a Group B adenovirus E3 14.7K ORF, or ORFs corresponding thereto from non-Group B adenovirus E3 regions, joined contiguously in the above 5‘-3‘ order, wherein the E3 region may optionally comprise one or more transgenes located within or adjacent to one or more of the said ORFs.

[0047] In a further embodiment, the invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region encodes a fusion protein which comprises the C-terminal end of a C-terminal-truncated Group B adenovirus E3 16.1 K protein fused to the N-terminal end of an N-terminal truncated Group B adenovirus E3 10.3K protein, or a fusion protein comprising the corresponding ends of the proteins corresponding thereto from non-Group B adenovirus E3 regions.

[0048] In a further embodiment, the invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region encodes a fusion protein which comprises the C-terminal end of a C-terminal truncated Group B adenovirus E3 19.3K protein fused to the N-terminal end of an N-terminal truncated Group B adenovirus E3 14.9K protein, or a fusion protein comprising the corresponding ends of the proteins corresponding thereto from non-Group B adenovirus E3 regions. In a further embodiment, the invention provides a pharmaceutical composition comprising an oncolytic adenovirus as claimed in any one of the preceding claims, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.

[0049] In a further embodiment, the invention provides an oncolytic adenovirus of the invention or a pharmaceutical composition of the invention for use in therapy or for use as a medicament. In a further embodiment, the invention provides an oncolytic adenovirus of the invention or a pharmaceutical composition of the invention for use in treating cancer (preferably ovarian cancer). In a further 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 or a pharmaceutical composition of the invention to a subject in need thereof. In a further 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).

[0050] Table 1 : Table of nucleotide and amino acid sequences

[0051] The invention provides adenoviruses, preferably for use in treatment of cancer, preferably ovarian cancer or stroma-containing tumours. 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.

[0052] 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.

[0053] 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.

[0054] The serotypes which fall within each of these adenovirus species include but are not limited to the following: 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. The references herein to different Ad serotypes include all different strains or variants of those serotypes.

[0055] 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 , Ad 14, 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.

[0056] 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.

[0057] 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.

[0058] 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. 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 E1 A 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.

[0059] 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.

[0060] 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).

[0061] 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).

[0062] The adenovirus of the invention comprises a plurality of adenoviral early genes (Figure 1 ).

[0063] 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.

[0064] 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.

[0065] 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

[0066] 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.

[0067] 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.

[0068] 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. The adenovirus of the invention comprises a plurality of adenoviral late genes (Figure 1 ).

[0069] 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).

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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. 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.

[0074] 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.

[0075] 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.

[0076] “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.

[0077] “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.

[0078] “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. "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).

[0079] In some embodiments, the adenovirus genome has at least 70% (preferably at least 80%, 85%, 90%, 95% or 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%) 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).

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] In some embodiments, the adenovirus of the invention is oncolytic and it encodes one or more transgenes. Adenoviruses of the invention may be used as oncolytic 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).

[0085] 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.

[0086] The 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. The adenovirus must have a genome size which is 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. The deletion in the E3 region creates more space for transgenes to be placed in other locations in the adenoviral genome.

[0087] 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.

[0088] The adenoviruses of the invention comprise an E3 region. The E3 region is usually situated between the L4 and L5 genes (see Figure 1). The E3 region comprises a plurality of ORFs (Open Reading Frames). The E3 region of wild-type Group B adenoviruses comprise the following nine ORFs: 12.1 K, 16.1 K, 19.3K, 20K, 20.6K, 7.7K, 10.3K, 14.9K and 14.7K.

[0089] Most published research on the functions of the proteins encoded by the ORFs in the E3 region of adenoviruses relates to the Group C adenovirus, Ad5. In Ad5, the E3 ORFs encode proteins which modulate the host immune system.

[0090] The E3 region is frequently entirely deleted from adenovirus vectors; such a deletion has a minimal effect on the virus phenotype under standard and optimised cell culture conditions. Hence, for adenovirus vectors which are used under such conditions, it could be argued that the E3 region has no essential functions. However, in the context of more relevant models and in actual real world infections, at least some of the E3 ORFs appear to have essential roles in host immune evasion.

[0091] The nucleotide sequence of the Ad7 E3 region is given herein as SEQ ID NO: 1.

[0092] As used herein, references to a Group B adenovirus E3 region relate preferably to a nucleotide sequence as given in SEQ ID NO: 1 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto. Preferably, the variant encodes the following nine ORFs: 12.1 K, 16.1 K, 19.3K, 20K, 20.6K, 7.7K, 10.3K, 14.9K and 14.7K.

[0093] The nucleotide sequence of the Ad7 12.1 K ORF is given herein as SEQ ID NO: 2.

[0094] As used herein, references to a Group B adenovirus E3 12.1 K ORF relate preferably to a nucleotide sequence as given in SEQ ID NO: 2 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto. Preferably, the variant encodes a polypeptide having the same function as the Group B adenovirus E3 12.1 K ORF.

[0095] The nucleotide sequence of the Ad7 16.1 K ORF is given herein as SEQ ID NO: 3.

[0096] As used herein, references to a Group B adenovirus E3 16.1 K ORF relate preferably to a nucleotide sequence as given in SEQ ID NO: 3 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto. Preferably, the variant encodes a polypeptide having the same function as the Group B adenovirus E3 16.1 K ORF.

[0097] The Group B adenovirus E3 19.3K ORF encodes a polypeptide which blocks MHC class-l-restricted antigen presentation; this diminishes cell killing by cytotoxic T cells. The nucleotide sequence of the Ad7 19.3K ORF is given herein as SEQ ID NO: 4.

[0098] As used herein, references to a Group B adenovirus E3 19.3K ORF relate preferably to a nucleotide sequence as given in SEQ ID NO: 4 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto. Preferably, the variant encodes a polypeptide which blocks MHC class-l- restricted antigen presentation.

[0099] The nucleotide sequence of the Ad7 20K ORF is given herein as SEQ ID NO: 5.

[0100] As used herein, references to a Group B adenovirus E3 20K ORF relate preferably to a nucleotide sequence as given in SEQ ID NO: 5 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto. Preferably, the variant encodes a polypeptide having the same function as the Group B adenovirus E3 20K ORF.

[0101] The nucleotide sequence of the Ad7 20.6K ORF is given herein as SEQ ID NO: 6.

[0102] As used herein, references to a Group B adenovirus E3 20.6K ORF relate preferably to a nucleotide sequence as given in SEQ ID NO: 6 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto. Preferably, the variant encodes a polypeptide having the same function as the Group B adenovirus E3 20.6K ORF.

[0103] The nucleotide sequence of the Ad7 7.7K ORF is given herein as SEQ ID NO: 7. As used herein, references to a Group B adenovirus E3 7.7K ORF relate preferably to a nucleotide sequence as given in SEQ ID NO: 7 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto. Preferably, the variant encodes a polypeptide having the same function as the Group B adenovirus E3 7.7K ORF.

[0104] The Group B adenovirus E3 10.3K ORF encodes RIDa; this inhibits immune-induced cell death. The nucleotide sequence of the Ad7 10.3K ORF is given herein as SEQ ID NO: 8. As used herein, references to a Group B adenovirus E3 10.3K ORF relate preferably to a nucleotide sequence as given in SEQ ID NO: 8 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto. Preferably, the variant encodes a RIDa polypeptide.

[0105] The Group B adenovirus E3 14.9K ORF encodes RIDp; this inhibits immune-induced cell death. The nucleotide sequence of the Ad7 14.9K ORF is given herein as SEQ ID NO: 9. As used herein, references to a Group B adenovirus E3 14.9K ORF relate preferably to a nucleotide sequence as given in SEQ ID NO: 9 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto. Preferably, the variant encodes a RIDp polypeptide. The Group B adenovirus E3 14.7K ORF encodes an inhibitor of TNF-mediated apoptosis. The nucleotide sequence of the Ad7 14.7K ORF is given herein as SEQ ID NO: B10. As used herein, references to a Group B adenovirus E3 14.7K ORF relate preferably to a nucleotide sequence as given in SEQ ID NO: 10 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto. Preferably, the variant encodes an inhibitor of TNF-mediated apoptosis.

[0106] The adenoviruses of the invention have a deletion in the E3 region compared to the corresponding region of a wild-type adenovirus, wherein the deletion comprises one or more specified E3 region ORFs which are present in the wild-type adenovirus.

[0107] In some embodiments, the invention provides an oncolytic adenovirus, having a genome comprising an E3 region: (a) wherein the E3 region comprises 12.1 K and 14.7K ORFs from a Group B adenovirus E3 region or ORFs corresponding thereto from non-Group B adenovirus E3 regions; and (b) wherein the E3 region does not comprise functional 20K, 20.6K, 7.7K or 10.3K ORFs from a Group B adenovirus E3 region or ORFs corresponding thereto from non-Group B adenovirus E3 regions.

[0108] Genes or ORFs which “correspond to” the Group B adenovirus E3 region genes referred to herein may readily be found by sequence comparisons (e.g. using BLAST) between the genes referred to herein and non-Group B adenovirus E3 regions. Figure 3 shows the E3 regions from Group B adenoviruses and the corresponding E3 regions from representative viruses from each of Groups A and C to G.

[0109] For example, the nucleotide sequences of the following non-Group B adenovirus E3 regions may be found at: Ad26 - GenBank EF 153474.1 ; Ad6 - GenBank OP871032.1 ; Ad49: GenBank DQ393829.1 ; and Ad10 - GenBank JN226746.1.

[0110] As used herein, the term “functional ... ORF” refers to the ability of the ORF to encode a mRNA or protein which fully or substantially performs its usual function. In some embodiments, the deletion of at least 10%, 20%, 30%, 40% or 50% or more of the ORF will render the ORF non-functional. Hence in some embodiments, the E3 region does not comprise more than 50%, 60%, 70%, 80% or 90% (preferably not comprise more than 90%) of a 20K, 20.6K, 7.7K or 10.3K ORF from a Group B adenovirus E3 region or of an ORF corresponding thereto from a non-Group B adenovirus E3 region. In some embodiment, the term “does not comprise a functional ... ORF” means that the E3 region does not comprise that ORF.

[0111] In some embodiments, the invention provides an oncolytic adenovirus, having a genome comprising an E3 region, wherein the E3 region comprises or consists of:

[0112] (a) a Group B adenovirus E3 12.1 K ORF;

[0113] (b) a 3-truncated Group B adenovirus E3 16.1 K ORF;

[0114] (c) a 5’-truncated Group B adenovirus E3 10.3K ORF;

[0115] (d) a Group B adenovirus E3 14.9K ORF; and

[0116] (e) a Group B adenovirus E3 14.7K ORF, or ORFs corresponding thereto from non-Group B adenovirus E3 regions, joined contiguously in the above 5‘-3‘ order, wherein the E3 region may optionally comprise one or more transgenes located within or adjacent to one or more of the said ORFs.

[0117] In other embodiments, the invention provides an oncolytic adenovirus, having a genome comprising an E3 region, wherein the E3 region comprises or consists of:

[0118] (a) a Group B adenovirus E3 12.1 K ORF;

[0119] (b) a Group B adenovirus E3 16.1 K ORF;

[0120] (c) a 3-truncated Group B adenovirus E3 19.3K ORF;

[0121] (d) a 5’-truncated Group B adenovirus 14.9K ORF; and

[0122] (e) a Group B adenovirus E3 14.7K ORF, or ORFs corresponding thereto from non-Group B adenovirus E3 regions, joined contiguously in the above 5‘-3‘ order, wherein the E3 region may optionally comprise one or more transgenes located within or adjacent to one or more of the said ORFs. As used herein, the term “joined contiguously” refers to the joining of the specified ORFs to each other without intervening nucleotides or without any significant lengths of intervening nucleotides. In other words, the E3 region consists essentially of only the specified ORFs. In some embodiments, however, the E3 region may optionally comprise one or more transgenes located within or adjacent to one or more of the said ORFs. In such embodiments, the term “joined contiguously” refers to the joining of the specified ORFs to each other except for the presence of the one or more transgenes.

[0123] In some embodiments, the E3 region comprises a 3’-truncated Group B adenovirus (preferably Ad7) 16.1 K ORF. As used in this context, the term “3’-truncated” means that the 3’-end of the 16.1 K ORF has been deleted. For example, at least 10%, 20%, 30%, 40% or 50% of the 3’ end of the 16.1 K ORF has been deleted (preferably when measured from the 3’-end). Preferably, the truncation is due to a deletion of nucleotides 356 to 441 in SEQ ID NO: 3. The 3’-truncated Group B adenovirus (preferably Ad7) 16.1 K ORF may still retain some functional activity.

[0124] In some embodiments, the E3 region comprises a 3’-truncated Group B adenovirus (preferably Ad7) 19.3K ORF. As used in this context, the term “3’-truncated” means that the 3’-end of the 19.3K ORF has been deleted. For example, at least 10%, 20%, 30%, 40% or 50% of the 3’ end of the 19.3K ORF has been deleted (preferably when measured from the 3’-end). Preferably, the truncation is due to a deletion of nucleotides 402 to 519 (the end of the 19.3K ORF) in SEQ ID NO: 4. The 3’-truncated Group B adenovirus (preferably Ad7) 19.3K ORF may still retain some functional activity.

[0125] In some embodiments, the E3 region comprises a 5’-truncated Group B adenovirus (preferably Ad7) 10.3K ORF. As used in this context, the term “5’-truncated” means that the 5’-end of the 10.3K ORF has been deleted. For example, at least 10%, 20%, 30%, 40% or 50% of the 3’ end of the 10.3K ORF has been deleted (preferably when measured from the 5’-end). Preferably, the truncation is due to a deletion of nucleotides 1 to 236 in SEQ ID NO: 8. In some embodiments, the E3 region comprises a 5’-truncated Group B adenovirus (preferably Ad7) 14.9K ORF. As used in this context, the term “5’-truncated” means that the 5’-end of the 14.9K ORF has been deleted. For example, at least 10%, 20%, 30%, 40% or 50% of the 3’ end of the 14.9K ORF has been deleted (preferably when measured from the 5’-end). Preferably, the truncation is due to a deletion of nucleotides 1 to 380 (i.e. the start of the 14.9K ORF to the last nucleotide deleted in the B deletion) in SEQ ID NO: 9.

[0126] In yet other embodiments, the invention provides an oncolytic adenovirus, having a genome comprising an E3 region, wherein the E3 region encodes a fusion protein which comprises the C-terminal end of a C-terminal-truncated Group B adenovirus E3 16.1 K protein fused to the N-terminal end of an N-terminal truncated Group B adenovirus E3 10.3K protein, or a fusion protein comprising the corresponding ends of the proteins corresponding thereto from non-Group B adenovirus E3 regions.

[0127] In yet other embodiments, the invention provides an oncolytic adenovirus, having a genome comprising an E3 region, wherein the E3 region encodes a fusion protein which comprises the C-terminal end of a C-terminal truncated Group B adenovirus E3 19.3K protein fused to the N-terminal end of an N-terminal truncated Group B adenovirus E3 14.9K protein, or a fusion protein comprising the corresponding ends of the proteins corresponding thereto from non-Group B adenovirus E3 regions.

[0128] In some preferred embodiments, the adenovirus of the invention comprises an E3 region, wherein the E3 region has a deletion in the E3 region compared to the corresponding region of a wild-type Group B adenovirus: (a) wherein the start of the deletion is located at nucleotide 629 in the Group B adenovirus E3 region nucleotide sequence as given in SEQ ID NO: 1 , or at the corresponding nucleotide in the nucleotide sequence of a non-Group B adenovirus E3 region; and (b) wherein the end of the deletion is located at nucleotide 2,892 in the Group B adenovirus E3 region nucleotide sequence as given in SEQ ID NO: 1 , or at the corresponding nucleotide in the nucleotide sequence of a non-Group B adenovirus E3 region. - T1 -

[0129] In some other preferred embodiments, the adenovirus of the invention comprises an E3 region, wherein the E3 region has a deletion in the E3 region compared to the corresponding region of a wild-type Group B adenovirus: (a) wherein the start of the deletion is located at position 1 ,099 in the Group B adenovirus E3 region nucleotide sequence as given in SEQ ID NO: 1 , or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region; and (b) wherein the end of the deletion is located at nucleotide 3,283 in the Group B adenovirus E3 region nucleotide sequence as given in SEQ ID NO: 1 , or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region.

[0130] (The above nucleotide numberings refer to the nucleotides of the ends which are retained in the E3 region following the deletion.)

[0131] In other preferred embodiments, the adenovirus of the invention comprises an E3 region, wherein the E3 region has a deletion compared to the wild-type Group B adenovirus, wherein the deletion corresponds to: (a) Ad7 genome nucleotides 28,011 - 30,274; or (b) Ad7 genome nucleotides 28,482 - 30,665. (These nucleotide numberings refer to the nucleotides of the ends which are retained in the E3 region following the deletion.)

[0132] The full genome of the Ad7 genome is available from Genbank (AY594255.1), the sequence of which is incorporated herein by reference.

[0133] In a particularly-preferred embodiment, the E3 region of the adenovirus has the nucleotide sequence as given in SEQ ID NO: 11 , or a variant thereof having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto, preferably wherein the variant encodes:

[0134] (a) a Group B adenovirus E3 12.1 K ORF;

[0135] (b) a 3-truncated Group B adenovirus E3 16.1 K ORF;

[0136] (c) a 5’-truncated Group B adenovirus E3 10.3K ORF;

[0137] (d) a Group B adenovirus E3 14.9K ORF; and

[0138] (e) a Group B adenovirus E3 14.7K ORF. In a particularly-preferred embodiment, the E3 region of the adenovirus has the nucleotide sequence as given in SEQ ID NO: 13, or a variant thereof having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto, preferably wherein the variant encodes:

[0139] (a) a Group B adenovirus E3 12.1 K ORF;

[0140] (b) a Group B adenovirus E3 16.1 K ORF;

[0141] (c) a 3-truncated Group B adenovirus E3 19.3K ORF;

[0142] (d) a 5’-truncated Group B adenovirus 14.9K ORF; and

[0143] (e) a Group B adenovirus E3 14.7K ORF.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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).

[0150] 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 .

[0151] 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.

[0152] 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.

[0153] 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.

[0154] Example 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.

[0155] 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.

[0156] In one embodiment, the pharmaceutical composition is a liquid parenteral formulation, for example for infusion or injection, of an adenovirus of the invention. 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.

[0157] 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.

[0158] 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.

[0159] In one embodiment, the parenteral formulation is in the form of an infusion for intra venous administration.

[0160] In another embodiment, the pharmaceutical composition is provided as a formulation for topical administration, including inhalation.

[0161] 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.

[0162] 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. 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.). 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.

[0167] 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.

[0168] 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.

[0169] 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).

[0170] 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).

[0171] Generally, such a pharmaceutical combination will be provided as two components:

[0172] (A) a first pharmaceutical composition of the adenovirus of the invention; and

[0173] (B) a second pharmaceutical composition comprising a chemotherapeutic or immunotherapeutic agent. 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. Similarly, in the methods of the invention, the first and second pharmaceutical compositions may be administered to a patient simultaneously, separately or sequentially.

[0174] The term “combined preparation” includes both fixed combinations and non-fixed combinations.

[0175] 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.

[0176] The term “non-fixed combination” means that the active ingredients (e.g. components

[0177] (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

[0178] (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.

[0179] 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. 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.

[0180] 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. 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).

[0181] 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.

[0182] 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).

[0183] 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.

[0184] In another embodiment, the invention provides use of an oncolytic adenovirus of the invention in the manufacture of a medicament for treating cancer (preferably ovarian cancer).

[0185] 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).

[0186] As used herein, the term “treating cancer” includes killing cancer cells and / or preventing or inhibiting the spread of cancer cells.

[0187] 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.

[0188] For humans in clinical trials, the Therapeutic Index is defined as TD50 / ED50 (where TD50is the toxic dose in 50% of subjects; and ED50is the minimum effective dose for 50% of the population).

[0189] 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.

[0190] 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.

[0191] 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.

[0192] The stromal cells may comprise CAFs. The cancer or tumour may be one which comprises CAFs.

[0193] 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. 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. 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.

[0194] 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 (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.

[0195] Preferred routes of administration for pharmaceutical composition of the present invention include intravenous, intra-tumoural, intraperitoneal, intrapleural, intravesical, intramuscular, intradermal, or other parenteral routes of administration, for example by injection or infusion.

[0196] 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, intra-tumoural, intraperitoneal, intrapleural, intravesical, intramuscular, intraarterial, intrathecal, intracapsular, intra-orbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, injection and infusion.

[0197] 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. 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] In one embodiment, the pharmaceutical composition of the invention may comprise 1x109to 1x1014viral particles per dose. Preferably, the pharmaceutical composition of the invention comprises 1x1011to 1x1013viral particles per dose.

[0202] 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.

[0203] Preferably, the method steps are carried out in the order specified.

[0204] 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.

[0205] The disclosure of each reference set forth herein is specifically incorporated herein by reference in its entirety.

[0206] BRIEF DESCRIPTION OF THE FIGURES

[0207] 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.

[0208] Figure 2. Schematic representation of the open reading frames (ORFs) encoded in the E3 region of Group B adenoviruses, and the positions in relation to these ORFs of the two deletions discovered in the viruses of the invention isolated following bioselection, as well as the variant viruses generated (Ov26_E3_A14.7K, Ov26_E3_A14.9K and Ov26_E3_AX1). The black bars represent the ORFs which are retained.

[0209] Figure 3. Schematic representation showing a comparison of the E3 region structure between different adenovirus groups in relation to that of Group B adenoviruses. Arrows represent ORFs and the size of the protein encoded by each ORF is shown within the arrow. White arrows are used where the ORF has a homolog in Group B viruses. Black arrows indicate that there is no ORF with a homologous sequence in Group B adenoviruses. White arrows indicate that homologous sequences in Group B adenoviruses exist.

[0210] Figure 4. A high grade serous ovarian cancer patient sample was infected with increasing concentrations of Ov26. Six days post-infection, 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.

[0211] Figure 5. Cell viability of a variety of different cell lines and primary cells in the presence of Ov26. Cells were infected with increasing doses of Ov26 and viability was measured 5 days post-infection by MTS assay. Data is expressed as viability relative to the uninfected control. VG / cell = viral genomes per cell.

[0212] Figure 6. Activity of Ov26 and wild-type parental viruses in cancer cells and normal cells. (A) Viral genome replication was measured by qPCR 7 days post-infection and is expressed as viral genomes (VG) 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.

[0213] Figure 7. TNFo levels from samples of ascites fluid drained from ovarian cancer patients, in serum from 250 pooled healthy donors, and in the medium from a cell culture of A549 cancer cells.

[0214] Figure 8. A549 cells stably transduced with a secreted alkaline phosphatase (SEAP) gene under the control of an NF-KB induced promoter were infected with 50 viral genomes (VG) per cell of Ov26, or Ov26 variants in which the E3 ORFs 14.7K or 14.9K had been deleted, named Ov26_E3_A14.7k and Ov26_E3_A14.9k, respectively. After 24 hours, cells were treated with increasing concentrations of TNFa in order to induce NF-KB signalling. 16 hours post-TNFo treatment SEAP expression was quantified by QuantiBlue™ assay.

[0215] Figure 9. Quantification of TNFa induced apoptosis in virally infected H2199 cells. (A) Cells were infected with 50 viral genomes (VG) per cell of Ov26 or an Ov26 variant in which the E3 ORF 14.7K had been deleted (Ov26_E3_A14.7k). 24 hours after infection, cells were treated with increasing concentrations of TNFa in combination with 12.5 pM of cyclohexamide (CHX) in order to initiate TNFa-induced apoptosis. 16 hours posttreatment with TNFa and CHX, induction of apoptosis was quantified by Caspase-Gio 3 / 7 luminescent assay. The mean luminescent signal of three biological replicates is plotted as a percentage of signal in the untreated mock with error bars indicating the standard deviation (SD). (B) Cells were infected at a dose of 2.5 IFU / cell with Ov26, Ov26_E3_A14.7k or an Ov26 variant in which 14.9K is entirely deleted, the 14.7K ORF has a 5’ 217 bp deletion, the 16.1 K ORF is intact, and the 19.1 K ORF is present but has a 362 bp 3’ deletion (Ov26_E3_AX1). 24 hours after infection, cells were treated with 10 ng / mL of TNFa in combination with 12.5 pM of cyclohexamide (CHX). 16 hours posttreatment induction of apoptosis was quantified by Caspase-Gio 3 / 7 luminescent assay. Relative fluorescent units (RLU) of three individual biological replicates are plotted with the median. Significance of differences between Ov26 infected cells and variant infected cells were assessed by one-way ANOVA. *** P < 0.001 ** P < 0.01 , * P< 0.05.

[0216] Figure 10. (A) A549 cancer cells were seeded on sterile coverslips and infected with 500 VG / cell of either Ov91 or Ov91 armed with a therapeutic agent (Tag) encoded within its E3 region Tag91 (Ov91_Tag26) and Tag57 (Ov91_Tag57). Tag26 is 1853 bp in length and Tag57 is 2207 bp in length. The coverslip was transferred to a layer of uninfected A549 cells and immediately overlaid with agarose and DMEM. The cells were incubated for 2 weeks after which point MTT reagent was added to stain live cells and reveal the extent of virus spread. (B) A549 cells were infected with a range of virus doses of Ov91 or Ov91_Tag26; 5 days after infection viability was assessed by MTS assay. (C) A549 cells were infected with 100 VG / cell of Ov91 or Ov91_Tag44. Cell pellets and supernatants were collected at various time points post-infection and viral genomes were quantified by qPCR. The mean of three values is plotted with error bars indicating the standard deviation. (Tag44 is Green Fluorescent Protein; Tag26 is NCAM BiTE; and Tag57 is VEGF TRAP).

[0217] Figure 11. H1299 cancer cells were infected with each virus Ov26, Ov26_E3_ 14.7K or Ov26_E3_AX1 at an MOI of 2.5 IFU / cell. 24 hours post infection cells were treated with 10 ng / mL TNFa and 12.5 ug / mL CHX or vehicle control (PBS). 72 hours post infection cells and supernatants were harvested and were subjected to 3 rounds of freeze thawing to achieve cell lysis. Infectious virus content of samples was quantified by ICC assay. All conditions were performed in triplicate. The number of infectious particles produced in TNFa treated conditions was expressed as a percentage of the number of particles produced in untreated conditions and then data were normalised to the Ov26. Therefore data shows the extent to which TNFa treatment reduces infectious virus particle production of the two variant viruses relative to the reduction seen for Ov26. Significance of differences was measured by one-way ANOVA. *** P < 0.001 ** P < 0.01 , * P< 0.05.

[0218] Figure 12. A) A549 cancer cells or normal primary human cells were infected with 10 viral genomes per cell of either virus CRC74 or CRC74_AE3. 4 days later total infectious virus particles were quantified by ICC assay. The number of infectious units per cell was calculated and expressed as a percentage of the IFU produced during A549 infections. B) HCT116 cancer cells, cancer associated fibroblast cell lines vCAF and MRC5, and primary CAFs isolated from ovarian cancer patient liquid biopsies ovCAF 1 and 2, were infected with increasing doses of CRC74 or CRC74_AE3. 7 days later cell viability was assessed by MTS assay. Viability is expressed as the percentage of viable cells relative to the untreated control.

[0219] Figure 13. Introduction of the E3 deletion enables oncolytic adenovirus CRC74 to accommodate exogenous DNA at either of two distinct arming sites

[0220] DNA expression cassettes were encoded within the viral genome at arming sites 1 and 2 in CRC74 (A) and in CRC74_AE3 (B). Armed viruses and unarmed control viruses were used to infect A549 cancer cells at equivalent doses. At 72 and 96 hours post infection cell viability was assessed by MTS assay. Killing activity of the viruses is expressed as the percentage of killing displayed by the relevant unarmed control virus.

[0221] EXAMPLES

[0222] 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.

[0223] Example 1 : Bioselection for oncolytic adenoviruses and sequence analysis

[0224] 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 ovarian cancer cells, spread, blood stability and immune stimulation. From the final pool, 60 adenoviruses were selected for sequencing and their genomes were analysed. 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.

[0225] Genome analyses of the selected adenoviruses revealed that the E3 regions of all 60 bioselected adenoviruses had one of two different deletions. Further details of the two deletions are given in the tables below; they are also illustrated in Figure 2. Ov26 and Ov91 are two of the bioselected adenoviruses which have Deletion (A). Ov20 has Deletion (B). Table 2: Details of the two E3 region deletions

[0226] Deletion A: sequence between nucleotides 629 - 2,892 is deleted from SEQ ID NO:1. Deletion B: sequence between nucleotides 954 - 3,283 is deleted from SEQ ID NO:1 .

[0227] Table 3 Details of the corresponding ORFs in other adenovirus Groups are given in Figure 3.

[0228] The fact that all 60 of the bioselected adenoviruses had one of the two above-defined deletions is evidence of the advantages conferred on the adenoviruses by these deletions in terms of their ability to lyse ovarian cancer cells, and their spread, blood stability and immune stimulation.

[0229] Example 2: Ov26 kills cancer cells and CAFs from an ex vivo ovarian cancer sample

[0230] A high grade serous ovarian cancer (HGSOC) patient sample was infected with increasing concentrations of Ov26 (one of the bioselected adenoviruses which has Deletion (A)). Six days post-infection, the viability of cancer cells and cancer associated fibroblasts (CAFs) within the sample was determined by multiparametric flow cytometry.

[0231] The results are shown in Figure 4. These results show that the viability of the cancer cells and the CAFs decreased with increasing concentrations of Ov26.

[0232] Example 3: Ov26 kills a range of ovarian patient cancer subtypes including treatment-naive and platinum-resistant cells

[0233] 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.

[0234] More specifically, cell viability was assessed after 6 days using flow cytometry. Cells were stained for viability using Live / Dead Near IR, 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.

[0235] The results are shown in the Table 4 below: Table 4:

[0236] The above table demonstrates that Ov26 effectively kills cancer cells and cancer associated fibroblasts (CAFs) across a range of different ovarian cancer patient subtypes.

[0237] Example 4: Use of a panel of cell lines to assess oncolytic activity of Ov26

[0238] The oncolytic activity of Ov26 was assessed in a number of cancer cell lines and cancer associated fibroblasts including the following: 293A Human embryonic kidney cell line

[0239] A549 Lung cancer cell line

[0240] Hela Cervical cancer cell line

[0241] MDA-MB-231 Breast cancer cell line

[0242] PANC1 Pancreatic cell line

[0243] PSN1 Pancreatic cell line

[0244] HCT116 Colorectal cancer cell line

[0245] HT29 Colorectal cancer cell line

[0246] SKOV-3 Ovarian cell line

[0247] Huh-7 Hepatoma cell line

[0248] MRC5 Fibroblast cell line

[0249] OE-21 Oesophageal cell line

[0250] OVCAR-3 Ovarian cell line

[0251] OVSAHO Ovarian carcinoma cell line

[0252] The results are shown in Figure 5. Ov26 demonstrated efficacy against all cancer cell lines tested against.

[0253] The IC50 values of Ov26 are given in the table below.

[0254] Table 5. IC50 values of Ov26 in various cancer cell lines

[0255] Example 5: Oncolytic activity of Ov26 compared to wild-type parental viruses

[0256] Parameters of Ov26 virus infection were measured 72 hours after infection of lung cancer cells (A549s), of non-cancerous normal primary human hepatocytes and normal human dermal fibroblasts (NHDFs).

[0257] As shown in Figure 6, production of viral genomes (A) and infectious virus particles (B) is lower in normal cells relative to A549 cancer cells during Ov26 infection as compared to infections with parental viruses. The results demonstrate the ability of Ov26 to preferentially infect the lung cancer cells compared to normal hepatocytes and normal fibroblasts.

[0258] Example 6: Quantification of TNFa levels in patient derived samples.

[0259] Tumour necrosis factor alpha (TNFa) is an inflammatory cytokine and plays a role in various cell signalling events many of which ultimately lead to necrosis or apoptosis of cells. TNFa signalling can promote resistance to infection by inducing apoptosis in infected cells and thus may have important consequences for oncolytic virotherapies. TNFa levels were measured in samples of ascites fluid drained from ovarian cancer patients, in serum from 250 pooled healthy donors, and in the medium from a cell culture of A549 cancer cells. Quantification was carried out by ELISA assay. The results in Figure 7 show that levels of TNFo were highest in patient-derived samples, and that no TNFo was detected in healthy serum or in the cancer cell line culture. This suggests that the effects of TNFo on virotherapies can only be effectively evaluated in models such as patient-derived tumour samples where TNFo or other relevant cytokines, chemokines, stress factors (e.g. IFN) are upregulated or at physiologically-relevant concentrations and these effects cannot be appreciated under normal cell culture conditions.

[0260] Example 7: Comparison of Ov26, Ov26_E3_A14.7K, Ov26_E3_A14.9K and Ov26_E3_AX1

[0261] The adenovirus E3 region encodes proteins that protect virus-infected cells from elimination by the host immune system. These proteins include those encoded by the 10.3K ORF (RIDa) and the 14.9K ORF (RIDp) which form a complex and localise to the plasma membrane. The RID complex promotes survival of infected cells by inhibition of apoptosis normally initiated through death domain-containing receptors of the tumour necrosis factor receptor (TNFR) superfamily (McNees et al., J. Virol. 2002 Oct; 76(19): 9716-9723). Whilst RIDa is known to down-regulate TNFa-induced NF-KB signalling; the role of RIDp in NF-KB signalling is not understood. E3 ORF 14.7K protects infected cells against TNFa- induced apoptosis.

[0262] Ov26 variants in which the E3 ORFs 14.9K (RIDp) or 14.7K were deleted were produced and named Ov26_E3_A14.9K and Ov26_E3_A14.7K, respectively. Also produced was a variant named Ov26_E3_AX1 in which the 14.9K ORF was entirely deleted, the 14.7K ORF has a 5’ 217 bp deletion, the 16.1 K ORF is intact, and the 19.1 K ORF is present but has a 362 bp 3’ deletion. These deletion constructs were generated by vectorising the viral genomes into bacterial artificial chromosomes (BACs). A homologous recombination based approach was then used to replace the E3 region of Ov26 with a selectable cassette flanked by unique restriction sites. The cassette was then removed by restriction digestion and synthetic DNA encoding the modified E3 regions was inserted by Gibson assembly. Constructs were validated and virus was recovered by their transfection into Ad-293 cells.

[0263] The ability of these adenoviruses to induce NF-KB signalling was assayed using a cell line encoding a secreted alkaline phosphatase (SEAP) gene under the control of an NF- KB induced promoter (Figure 8).

[0264] The results show that Ov26 is able to block induction of NF-KB signalling to a greater extent than is Ov26_E3_A14.9K.

[0265] The ability of the Ov26,Ov26_E3_A14.7K and Ov26_E3_AX1 to protect infected cells from apoptosis induced by TNFa was measured by quantifying caspase activation in infected cells after exposure to TNFa (Figure 9). The results show that cells infected with Ov26 do not respond to TNFa treatment with any caspase activation and thus are protected from TNFa-induced apoptosis. Cells infected with Ov26_E3_A14.9K or Ov26_E3_AX1 , however, show a significant activation of caspase following TNFa treatment.

[0266] Example 8: Comparison of growth kinetics between Ov91 candidate and “armed” Ov candidate

[0267] Ov91 is one of the bioselected adenovirus which has an E3 region comprising deletion A and its E3 region is therefore the same as that of Ov26. Ov91 variants also encoding therapeutic transgenes were generated by vectorising the genome of Ov91 into a bacterial artificial chromosome (BAC). A homologous recombination based approach was then used to replace the E3 region of Ov91with a selectable cassette flanked by unique restriction sites. The cassette was then removed by restriction digestion and synthetic DNA encoding different therapeutic transgenes embedded within the Ov91 E3 region was inserted by Gibson assembly. Constructs were validated and virus was recovered by their transfection into Ad-293 cells.

[0268] The ability of the virus to spread from an infected cell to neighbouring cells upon cell lysis, cancer cell killing and virus growth were all quantified. The results given in Figure 10 show that the E3 deletion A found in both Ov91 and Ov26 is such that it is able to accommodate the insertion of large transgenes of up to but not limited to 2,207 bp representing 6.7% of the length of its genome without significant loss of efficacy against cancer cells.

[0269] Example 9

[0270] The E3 14.7K protein was shown to be important for the blockade of TNFo induced NF- KB signalling, and Ov26 was better able resist cellular apoptosis than virus variants Ov26_E3_A14.7K and Ov26_E3_AX1 (Example 7). One way in which the resistance to apoptosis would offer a clear advantage to an oncolytic virus would be if this delay in cell death offered a greater opportunity for the virus to replicate and produce a greater number of progeny virus particles. Consequently the quantity of infectious units (IFU) produced by each of the viruses Ov26, Ov26_E3_A14.7K and Ov26_E3_AX during infection of cancer cells in the presence and absence of TNFo treatment was assessed. The results shown in Figure 11 demonstrate that treatment of cells with TNFo reduces the number of IFU produced by the variant viruses Ov26_E3_A14.7K and Ov26_E3_AX to a greater extent than for Ov26.

[0271] Example 10

[0272] Ov26 has been shown to display enhanced selectivity towards cancer cells over normal cells relative to its parental viruses; this is likely to be due to the attenuating effect of the deletion within its E3 region. To assess whether this deletion arrangement also enhances selectivity in the context of other oncolytic viruses, the same E3 deletion (shown in Figure 2, Theolytics A) was introduced into an alternative oncolytic adenovirus, CRC74, generating the E3-deleted variant CRC74_AE3.

[0273] To investigate the selectivity of CRC74 and the variant CRC74_AE3, infectious virus particle production following infection of A549 cancer cells and of 3 different normal primary cell types was quantified. The results in Figure 12. A show that CRC74 infection of a subset of normal primary cells results in the production of infectious virus particles; however, infection of these cells with the CRC74_AE3 variant produced significantly less infectious virus. However, the E3 deletion introduced into CRC74 did not substantially affect the killing of cancer cells or cancer associated fibroblasts, as demonstrated in Figure 12B. Therefore, taken together, the data displayed in Figure 12 shows that the introduction of the E3 deletion into CRC74 improves the selectivity of the oncolytic virus and demonstrates that this particular E3 arrangement enhances selectivity in different oncolytic adenoviruses.

[0274] Example 11

[0275] Ov26 is able to accommodate the insertion of transgene expression cassettes into its genome without affecting its ability to replicate and spread. In order to assess whether the E3 deletion is an important feature that allows for the inclusion of transgene sequences into the viral genome, the same E3 deletion was introduced into an alternative oncolytic adenovirus, CRC74, generating the E3 deleted variant CRC74_AE3.

[0276] To assess the ability of the two viruses, CRC74 and CRC74_AE3, to accommodate exogenous DNA within their genomes, cancer cell-killing activity was quantified after encoded expression cassettes were inserted at one of two sites within the viral genome. The results in Figure 13.A show that whilst arming CRC74 is possible, killing activity is significantly delayed, and 100% activity relative to the unarmed CRC74 virus was not achieved by 96 hours.

[0277] Meanwhile, the results in Figure 13.B show that arming is much better tolerated by the CRC74_AE3 virus, with cell-killing activity comparable to the control virus at 96 hours post-infection. Therefore, taken together, the data displayed in Figure 13 shows that the introduction of the E3 deletion into CRC74 greatly improves the ability of the oncolytic virus to tolerate the insertion of expression cassettes within its genome, without impacting oncolytic activity.

[0278] The Sequence Listing filed with this patent application is fully incorporated herein as part of the description.

Claims

CLAIMS1 . An oncolytic adenovirus, having a genome comprising an E3 region:(a) wherein the E3 region comprises 12.1 K and 14.7K ORFs from a Group B adenovirus E3 region or ORFs corresponding thereto from non-Group B adenovirus E3 regions; and(b) wherein the E3 region does not comprise functional 20K, 20.6K, 7.7K or 10.3K ORFs from a Group B adenovirus E3 region or ORFs corresponding thereto from non- Group B adenovirus E3 regions.

2. An oncolytic adenovirus as claimed in claim 1 , wherein the E3 region additionally comprises a functional (optionally 3’-truncated) 16.1 K ORF from a Group B adenovirus E3 region or an ORF corresponding thereto from a non-Group B adenovirus E3 region.

3. An oncolytic adenovirus as claimed in claim 1 or claim 2, wherein the E3 region additionally comprises a 16.1 K ORF and a functional (optionally 3’-truncated) 19.3K ORF from a Group B adenovirus E3 region or ORFs corresponding thereto from a non- Group B adenovirus E3 region.

4. An oncolytic adenovirus as claimed in any one of the preceding claims, wherein the E3 region additionally comprises a 14.9K ORF from a Group B adenovirus E3 region or an ORF corresponding thereto from a non-Group B adenovirus E3 region.

5. An oncolytic adenovirus as claimed in claim 1 , wherein the E3 region comprises or consists of:(a) a Group B adenovirus E3 12.1 K ORF;(b) a 3-truncated Group B adenovirus E3 16.1 K ORF;(c) a 5’-truncated Group B adenovirus E3 10.3K ORF;(d) a Group B adenovirus E3 14.9K ORF; and(e) a Group B adenovirus E3 14.7K ORF, or ORFs corresponding thereto from non-Group B adenovirus E3 regions,joined contiguously in the above 5‘-3‘ order, wherein the E3 region may optionally comprise one or more transgenes located within or adjacent to one or more of the said ORFs.

6. An oncolytic adenovirus as claimed in claim 1 , wherein the E3 region comprises or consists of:(a) a Group B adenovirus E3 12.1 K ORF;(b) a Group B adenovirus E3 16.1 K ORF;(c) a 3-truncated Group B adenovirus E3 19.3K ORF;(d) a 5’-truncated Group B adenovirus E3 14.9K ORF; and(e) a Group B adenovirus E3 14.7K ORF, or ORFs corresponding thereto from non-Group B adenovirus E3 regions, joined contiguously in the above 5‘-3‘ order, wherein the E3 region may optionally comprise one or more transgenes located within or adjacent to one or more of the said ORFs.

7. An oncolytic adenovirus as claimed in claim 1 , wherein the E3 region encodes a fusion protein which comprises the C-terminal end of a C-terminal-truncated Group B adenovirus E3 16.1 K protein fused to the N-terminal end of an N-terminal truncated Group B adenovirus E3 10.3K protein, or a fusion protein comprising the corresponding ends of the proteins corresponding thereto from non-Group B adenovirus E3 regions.

8. An oncolytic adenovirus, as claimed in claim 1 , wherein the E3 region encodes a fusion protein which comprises the C-terminal end of a C-terminal truncated Group B adenovirus E3 19.3K protein fused to the N-terminal end of an N-terminal truncated Group B adenovirus E3 14.9K protein, or a fusion protein comprising the corresponding ends of the proteins corresponding thereto from non-Group B adenovirus E3 regions.

9. An oncolytic adenovirus as claimed in claim 1 , wherein the E3 region has a deletion in the E3 region compared to the corresponding region of a wild-type Group B adenovirus:(a) wherein the start of the deletion is located at nucleotide 629 in the Group B adenovirus E3 region nucleotide sequence as given in SEQ ID NO: 1 , or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region; and(b) wherein the end of the deletion is located at nucleotide 2,892 in the Group B adenovirus E3 region nucleotide sequence as given in SEQ ID NO: 1 , or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region.

10. An oncolytic adenovirus as claimed in claim 1 , wherein the E3 region has a deletion in the E3 region compared to the corresponding region of a wild-type Group B adenovirus:(a) wherein the start of the deletion is located at position 1 ,099 in the Group B adenovirus E3 region nucleotide sequence as given in SEQ ID NO: 1 , or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region; and(b) wherein the end of the deletion is located at nucleotide 3,283 in the Group B adenovirus E3 region nucleotide sequence as given in SEQ ID NO: 1 , or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region.

11. An oncolytic adenovirus as claimed in claim 1 , wherein the E3 region has a deletion compared to the wild-type Group B adenovirus, wherein the deletion corresponds to:(a) Ad7 genome nucleotides 28,011 - 30,274; or(b) Ad7 genome nucleotides 28,482 - 30,665.

12. An oncolytic adenovirus as claimed in any one of the preceding claims, wherein the adenovirus is a Group B adenovirus or a human adenovirus, preferably an Ad3 or Ad7 serotype, or an Ad3 / Ad7 chimera.

13. An oncolytic adenovirus as claimed in any one of the preceding claims, wherein the adenovirus additionally comprises a transgene, preferably a transgene which encodes an antibody, bispecific engager, checkpoint inhibitor, cytokine, chemokine or enzyme.

14. An oncolytic adenovirus as claimed in claim 13, wherein the transgene is located in or adjacent to the E1 region, E3 region, L3 region or L5 region of the adenovirus.

15. A pharmaceutical composition comprising an oncolytic adenovirus as claimed in any one of the preceding claims, optionally together with one or more pharmaceutically- acceptable carriers, excipients or diluents.

16. An oncolytic adenovirus as claimed in any one of claims 1 to 14 or a pharmaceutical composition as claimed in claim 15 for use in therapy or for use as a medicament.

17. An oncolytic adenovirus as claimed in any one of claims 1 to 14 or a pharmaceutical composition as claimed in claim 15 for use in treating cancer (preferably ovarian cancer).

18. A method of treating cancer (preferably ovarian cancer) in a subject, the method comprising administering an effective amount of an oncolytic adenovirus as claimed in any one of claims 1 to 14 or a pharmaceutical composition as claimed in claim 15 to a subject in need thereof.

19. Use of an oncolytic adenovirus as claimed in any one of claims 1 to 14 in the manufacture of a medicament for treating cancer (preferably ovarian cancer).

20. An oncolytic adenovirus for use as claimed in claim 17, a method as claimed in claim 18 or a use as claimed in claim 19, wherein the cancer is a stroma-containing tumour.21 . An oncolytic adenovirus for use as claimed in claim 17, a method as claimed in claim 18 or a use as claimed in claim 19, wherein the cancer is ovarian cancer, colorectal cancer, lung cancer, hepatoma, multiple myeloma, oesophageal cancer, breast cancer or pancreatic cancer, preferably wherein the cancer is ovarian cancer or a stroma-containing carcinoma.

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