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 some embodiments, the invention relates to adenoviruses which have a chimeric L2-L3 region compared to wild-type adenoviruses. In other embodiments, the invention relates to adenoviruses which have a deletion in the E3 region compared to wild-type adenoviruses. In other embodiments, the invention relates to adenoviruses having a mutation in the coding sequences of the DNA pol I polypeptide and / or the pTP (pre-Terminal Protein) polypeptide. In yet other embodiments, the invention relates to adenoviruses having the aforementioned chimeric L2-L3 region, deletion and mutation(s). In yet other embodiments, the invention relates to an adenovirus comprising the nucleotide sequence as shown in SEQ ID NO: 58 or a variant thereof having at least 93% sequence identity thereto and having oncolytic activity.

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 some embodiments, the invention relates to adenoviruses which have a chimeric L2-L3 region compared to wild-type adenoviruses. In other embodiments, the invention relates to adenoviruses which have a deletion in the E3 region compared to wild-type adenoviruses. In other embodiments, the invention relates to adenoviruses having a mutation in the coding sequences of the DNA pol I polypeptide and / or the pTP (pre-Terminal Protein) polypeptide. In yet other embodiments, the invention relates to adenoviruses having the aforementioned chimeric L2-L3 region, deletion and mutation(s). In yet other embodiments, the invention relates to an adenovirus comprising the nucleotide sequence as shown in SEQ ID NO: 58 or a variant thereof having at least 93% sequence identity thereto and having oncolytic activity.

[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. 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. 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. 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.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] An ideal intervention to treat carcinomas would simultaneously and selectively target both cancer cells and stromal populations.

[0009] 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.

[0010] 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).

[0011] 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 multi-center study of the safety and efficacy of CG0070 oncolytic vector regimen in patients with BCG-unresponsive non-muscle-invasive bladder cancer:

[0012] Interim results.” Urol Oncol. 2018 Oct;36(10):440-447. doi:

[0013] 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] From an initial mixed pool of Group B, C, D, F, and G adenoviruses, 23 rounds of bioselection (including mutational steps) were performed to enrich for oncolytic adenovirus candidates with the best combination of tumour lysis, spread, blood stability and immune stimulation. From the final pool, 60 adenoviruses were selected for sequencing and their genomes were analysed.

[0024] Genome analyses of the selected adenoviruses revealed that a number of features of their genomes were common to the majority of or all of the candidate adenoviruses. In particular, the candidate adenovirus genomes were all found to be from Group B adenoviruses, with the genomes all being chimeric: the 5’ end of the genome was from an Ad3 serotype virus; and the 3’ end of the genome was from an Ad7 serotype virus. The chimeric junction point was found to be within the L2-L3 region in all of the bioselected adenoviruses.

[0025] One structural result of this chimerism is that, in the adenoviruses of the invention, the penton base protein (which is encoded by a L2 gene) is from an Ad3 serotype and the hexon protein (which is encoded by a L3 gene) is from an Ad7 serotype. The penton base and hexon proteins both form parts of the adenoviral capsid. The third major adenoviral capsid component, the fibre (which is encoded by a L5 gene), is from an Ad7 serotype.

[0026] The penton base protein is present at each vertex of the adenovirus capsid and for many adenoviruses it serves as the attachment site for cell surface integrins avp3 and avp5; the latter integrins serve as receptors for adenovirus internalization into host cells. Consequently, the serotype of the penton base protein affects the cell tropism of the adenovirus.

[0027] The Applicants have now found that this feature confers, inter alia, an enhanced ability for systemic delivery and to spread through solid tumours by establishing productive infections in neighbouring cells.

[0028] Without being bound by theory, it is also possible that the chimeric nature of the adenoviruses of the invention may have been specifically selected for (during the bioselection process) due to an ability of these chimeric adenoviruses to overcome neutralising antibodies that bind to the surface proteins (e.g. penton base, hexon and fibre) of the adenoviruses. This makes the adenoviruses of the invention particularly useful as oncolytic adenoviruses and also as gene therapy vectors. CN103966174 discloses a recombinant Ad3 adenovirus comprising an Ad7 hexon protein expression cassette inserted into the E3 region of the adenoviral genome. The resultant capsids are chimeric, i.e. comprising Ad3 penton proteins, a mixture of Ad3 hexon proteins and Ad7 hexon proteins, and Ad3 fibre proteins. The recombinant adenovirus is stated to be for use as a bivalent vaccine, i.e. for stimulating immunity to both Ad3 and Ad7, to prevent both human adenovirus Ad3 and Ad7 infections (e.g. acute respiratory diseases). This is in contrast to the adenoviruses of the invention which are for use as oncolytic viruses and which are bioselected to overcome neutralising antibodies.

[0029] Furthermore, the bioselected adenovirus genomes were all found to comprise a deletion in their E3 region compared to wild-type Group B adenoviruses.

[0030] 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.

[0031] 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.

[0032] The Applicants have now found that this feature confers, inter alia, an enhanced capacity of adenoviruses to demonstrate oncolytic activity in tumours containing stroma, including ovarian cancer cells.

[0033] In particular, 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.

[0034] 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.

[0035] 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.

[0036] 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 10.4K 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.

[0037] Genome analyses of the bioselected adenoviruses also revealed that the genomes of the candidate adenoviruses had a missense mutation in the E2B DNA pol I gene. The mutation was a single nucleotide change ggc gac, which results in a G34D mutation in the amino acid sequence of the E2B DNA pol I polypeptide.

[0038] In the wild-type adenovirus genome, the coding sequences for the E2B DNA pol I gene and the E2B pre-Terminal Protein (pTP) overlap. The above-mentioned mutation falls within the coding sequences of both the DNA pol I polypeptide and the pTP polypeptide and hence the pTP gene was also mutated. With regard to the pTP polypeptide sequence, the mutation is a gcg acg mutation; this results in an A623T amino acid mutation in the amino acid sequence of the E2B pTP polypeptide.

[0039] The Applicants have now found that this feature confers, inter alia, an enhanced capacity of adenoviruses to demonstrate oncolytic activity in tumours containing stroma, including ovarian cancer cells.

[0040] In particular, on examination, one or both of these mutations were found to confer an advantage on the adenoviruses in terms of their levels of replication and production of infectious virus progeny. In the context of oncolytic adenoviruses, this advantage manifests itself in the ability of the adenovirus to achieve sustained oncolytic activity at the tumour sites. In the context of transgene encoding or ‘armed’ oncolytic adenoviruses, e.g. ones which encode a transgene, this advantage manifests itself in the ability of the adenovirus to produce the transgene, and the corresponding polypeptide product, to a higher abundance.

[0041] Adenoviruses comprising the nucleotide sequence as shown in SEQ ID NO: 58 were found to have particularly advantageous oncolytic properties. In particular, they have enhanced spreading ability, allowing them to infect neighbouring cells and to establish new productive infections. Their cancer cell-killing activity is not neutralised by whole blood or blood serum. Furthermore, such adenoviruses are capable of activating inflammatory T cell responses, thus promoting the targeting and killing of cancer cells by T cells.

[0042] It is therefore an object of the invention to provide adenoviruses having a chimeric genome, wherein the 5’ end of the genome is from an Ad3 serotype virus and the 3’ end of the genome is from an Ad7 serotype virus, particularly oncolytic adenoviruses, which have enhanced ability to infect neighbouring cells and to establish new productive infections, particularly in tumour cells.

[0043] It is also an object of the invention to provide adenoviruses which comprise a deletion in their E3 region compared to wild-type Group B adenoviruses, and 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.

[0044] It is also an object of the invention to provide oncolytic adenoviruses having mutations in the E2B DNA pol I gene and / or E2B pTP gene and which are capable of infecting and lysing cancer cells, particularly ovarian cancer cells and cancer-associated fibroblasts (CAFs). It is yet another object of the invention to provide adenoviruses having mutations in the E2B DNA pol I gene and / or E2B pTP gene and which are capable of replicating to greater levels and producing more infectious virus progeny compared to corresponding control adenoviruses.

[0045] A yet further object of the invention is to provide adenoviruses having the aforementioned chimeric genome, deletion and mutation(s); and adenoviruses of SEQ ID NO: 58 and variants thereof.

[0046] 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, including tumours containing stroma and ovarian cancer. It is another object of the invention to provide compositions comprising the adenoviruses of the invention for use as gene therapy vectors.

[0047] In a first aspect, the invention provides a chimeric adenovirus, wherein the genome of the chimeric adenovirus comprises:

[0048] (a) an Ad3 penton gene; and

[0049] (b) an Ad7 hexon gene; for use in the prevention or treatment of cancer, preferably ovarian cancer.

[0050] In one embodiment, the invention provides a method of treating cancer, preferably ovarian cancer, the method comprising administering an effective amount of a chimeric adenovirus, wherein the genome of the chimeric adenovirus comprises: (a) an Ad3 penton gene; and

[0051] (b) an Ad7 hexon gene; to a patient in need thereof.

[0052] In yet another embodiment, the invention provides the use of a chimeric adenovirus, wherein the genome of the chimeric adenovirus comprises:

[0053] (a) an Ad3 penton gene; and

[0054] (b) an Ad7 hexon gene; in the manufacture of a medicament for the prevention or treatment of cancer, preferably ovarian cancer.

[0055] In some embodiments, the genome of the chimeric adenovirus additionally comprises:

[0056] (c) an Ad7 fibre gene.

[0057] The invention also provides a chimeric adenovirus, wherein the genome of the chimeric adenovirus comprises:

[0058] (a) an Ad3 penton gene;

[0059] (b) an Ad7 hexon gene; and

[0060] (c) an Ad7 fibre gene.

[0061] The invention also provides an adenoviral gene therapy vector, wherein the genome of the adenoviral gene therapy vector comprises:

[0062] (a) an Ad3 penton gene;

[0063] (b) an Ad7 hexon gene; and

[0064] (c) a transgene.

[0065] The invention also provides a pharmaceutical composition comprising a chimeric adenovirus of the invention, optionally together with one or more pharmaceutically- acceptable carriers, excipients or diluents.

[0066] The invention also provides a pharmaceutical combination comprising: (A) a first pharmaceutical composition comprising a chimeric adenovirus of the invention; and

[0067] (B) a second pharmaceutical composition comprising a chemotherapeutic or immunotherapeutic agent, wherein the pharmaceutical combination is in the form of a combined preparation for simultaneous, separate or sequential use, preferably for the treatment of cancer.

[0068] Also provided is a chimeric adenovirus of the invention or an adenoviral gene therapy vector of the invention for use in therapy or for use as a medicament.

[0069] Also provided is the use of a chimeric adenovirus of the invention as a vector for protein production, wherein the chimeric adenovirus comprises a transgene encoding the protein to be produced.

[0070] In a second aspect, the invention provides an oncolytic adenovirus having a genome comprising an E3 region:

[0071] (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.

[0072] 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. 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:

[0073] (a) a Group B adenovirus E3 12.1 K ORF;

[0074] (b) a 3-truncated Group B adenovirus E3 16.1 K ORF;

[0075] (c) a 5’-truncated Group B adenovirus E3 10.3K ORF;

[0076] (d) a Group B adenovirus E3 14.9K ORF; and

[0077] (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.

[0078] 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:

[0079] (a) a Group B adenovirus E3 12.1 K ORF;

[0080] (b) a Group B adenovirus E3 16.1 K ORF;

[0081] (c) a 3-truncated Group B adenovirus E3 19.3K ORF;

[0082] (d) a 5’-truncated Group B adenovirus E3 14.9K ORF; and

[0083] (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.

[0084] 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. 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.

[0085] 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.

[0086] 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).

[0087] In a third aspect, the invention provides an adenovirus, wherein the genome of the adenovirus comprises an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the amino acid in the DNA pol I polypeptide sequence corresponding to amino acid 34 in SEQ ID NO: 49 is Asp or Glu, preferably Asp.

[0088] In another embodiment, the invention provides an adenovirus, wherein the genome of the adenovirus comprises an E2B pTP gene encoding a pre-Terminal Protein, wherein the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 51 is selected from the group consisting of Thr, Asn, Cys, Gin and Ser, preferably Thr. In some embodiments, if the adenovirus is an Ad1 adenovirus, then the amino acid is not Asn; if the adenovirus is an Ad41 adenovirus, then the amino acid is not Gin; and / or if the adenovirus is an Ad4 or 4a adenovirus, then the amino acid is not Thr.

[0089] In another embodiment, the invention provides a pharmaceutical composition comprising an adenovirus of the invention, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.

[0090] Also provided is an adenovirus or a pharmaceutical composition of the invention for use in therapy or for use as a medicament, particularly for use in treating cancer (preferably ovarian cancer).

[0091] Also provided is a method of treating cancer (preferably ovarian cancer) in a subject, the method comprising administering an effective amount of an adenovirus or a pharmaceutical composition of the invention to a subject in need thereof.

[0092] Also provided is the use of an oncolytic adenovirus of the invention in the manufacture of a medicament for treating cancer (preferably ovarian cancer).

[0093] Also provided are the use of a conditionally-replicating adenovirus of the invention as a vector for protein production, wherein the adenovirus comprises a transgene encoding the protein to be produced; and the use of a conditionally-replicating adenovirus of the invention as a helper virus for virus production.

[0094] The invention also provides mutant Group B or human E2B DNA pol I and E2B pTP genes as defined herein, and mutant Group B or human E2B DNA pol I and E2B pTP polypeptides, as defined herein.

[0095] In a fourth aspect, the invention provides an adenovirus, wherein the genome of the adenovirus comprises one, two or all of (A), (B) and (C): (A) (a) an Ad3 penton gene; and

[0096] (b) an Ad7 hexon gene; and

[0097] (c) optionally an Ad7 fibre gene;

[0098] (B) an E3 region:

[0099] (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

[0100] (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; and

[0101] (C) an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the amino acid in the DNA pol I polypeptide sequence corresponding to amino acid 34 in SEQ ID NO: 49 is Asp or Glu, preferably Asp; or an E2B pTP gene encoding a pre-Terminal Protein, wherein the amino acid in the preTerminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 51 is selected from the group consisting of Thr, Asn, Cys, Gin and Ser, preferably Thr.

[0102] In some embodiments, with regard to the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 51 , if the adenovirus is an Ad1 adenovirus, then the amino acid is not Asn; if the adenovirus is an Ad41 adenovirus, then the amino acid is not Gin; and / or if the adenovirus is an Ad4 or 4a adenovirus, then the amino acid is not Thr.

[0103] In some embodiments, the invention provides an adenovirus, wherein the genome of the adenovirus comprises (A) and (B). In some embodiments, the invention provides an adenovirus, wherein the genome of the adenovirus comprises (A) and (C).

[0104] In some embodiments, the invention provides an adenovirus, wherein the genome of the adenovirus comprises (B) and (C).

[0105] In some embodiments, the invention provides an adenovirus, wherein the genome of the adenovirus comprises (A), (B) and (C).

[0106] In a fifth aspect, the invention provides an adenovirus comprising the nucleotide sequence as shown in SEQ ID NO: 58 and an adenovirus comprising a nucleotide sequence having at least 93% (preferably at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to SEQ ID NO: 58 and which has oncolytic activity. In some preferred embodiments, the nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 58 and the adenovirus has oncolytic activity. In some other preferred embodiments, the nucleotide sequence has at least 99% sequence identity to SEQ ID NO: 58 and the adenovirus has oncolytic activity.

[0107] Tables of Sequences

[0108] Table 1 : Table of nucleotide and amino acid sequences of L2-L3 region

[0109] Table 2: Table of nucleotide and amino acid sequences of E3 regions

[0110] Table 3: Table of sequences of DNA pol I and pTP genes and polypeptides, and complete virus sequence.

[0111] In all aspects, the invention provides adenoviruses, particularly oncolytic 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.

[0112] 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.

[0113] 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.

[0114] The serotypes which fall within each of these adenovirus species include but are not limited to those which are given below:

[0115] 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, Ad 19, 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.

[0116] The references herein to different Ad serotypes include all different strains or variants of those serotypes.

[0117] 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. The chimeric oncolytic adenovirus of the invention has a genome. 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.

[0118] 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.

[0119] 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.

[0120] Our understanding of adenovirus genetics, transcription and translation is primarily derived from virological studies using adenovirus species C Ad5. The sequential process of gene transcription from the adenovirus genome reflects the protein requirements of the virus at each stage of the replication process. The transcription from the adenovirus genome is therefore divided into early and late events dependent on the timing of initiation of transcription from each viral promoter. The first proteins produced from the virus genome are the E1A proteins. The E1A transcription unit produces multiple mRNA molecules through alternative splicing which in turn produce multiple proteins ranging from 6-36 kDa. The E1A proteins have two major roles within an infected cell. Firstly, they induce the cell to enter the S phase of the cell cycle to allow the efficient replication of the viral genome. Secondly, they induce transcription of the other early promoters within the viral genome through transactivation. These promoters control the production of the E1 B, E2, E3 and E4 proteins. E1A expression is immediately followed by VA RNA and E1 B and E3 protein production. These proteins and RNA molecules help to prevent the development of an anti-viral response. These early events in viral replication help to shape the intracellular environment to allow the replication of the viral genome before packaging. Later transcription events involve the production of structural proteins and proteins essential for cell lysis which are derived primarily from a single promoter (the major late promoter) which transcribes the late regions 1 -5. Ad5 induced cell lysis is dependent on the E3-11 ,6K protein (also termed the adenovirus death protein) which despite its labelling as an early gene is only produced late in infection and from the major late promoter.

[0121] 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.

[0122] 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).

[0123] 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).

[0124] The adenovirus of the invention comprises a plurality of adenoviral early genes (e.g. Figure 1A).

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] The adenovirus of the invention comprises a plurality of adenoviral late genes (Figure 1 A). 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).

[0132] 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. - T1 -

[0133] The L2 series of transcripts encode for the penton base, pVII, 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.

[0134] 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.

[0135] The L4 series of transcripts encode the 100K, 33K, 22K and pVIII 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.

[0136] 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.

[0137] 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.

[0138] In one embodiment, the adenovirus is replication-capable, replication-competent or conditionally replicating. These viruses may be oncolytic, virus vaccines, vectors for protein production.

[0139] “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.

[0140] “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.

[0141] “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.

[0142] "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 adenoviruses may be oncolytic in nature, or used as a vector for gene, vaccine or protein delivery.

[0143] In some embodiments, the adenovirus genome has at least 70% (preferably at least 80%, 85%, 90%, 95% or 99%; most preferably at least 99%) nucleotide sequence identity to the wild-type Ad3 genome sequence as given in the full Ad3 genome sequence (e.g. as given in Genbank Sequence ID: DQ086466.1). In some embodiments, the adenovirus genome has at least 70% (preferably at least 80%, 85%, 90%, 95% or 99%; most preferably at least 99%) nucleotide sequence identity to the wild-type Ad7 genome sequence as given in the full Ad7 genome sequence (e.g. as given in Genbank Sequence ID AY594255.1).

[0144] In some particularly-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.

[0145] Viral infection may be measured by an infectivity assay such as a plaque assay, Median Tissue Culture infectious Dose (TCID50) assay, or by using an anti-hexon antibody in an immuno-cytochemical (ICC) staining assay. The oncolytic adenoviruses of the invention may be cytolytic. Lysis may be measured by cell death or cell viability assays including MTS, MTT assays, PrestoBlue™, live / dead staining and flow cytometry. The oncolytic virus infection causes death and lysis of the cancer or stroma cells, preferably with release of newly-generated virus particles.

[0146] 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. In some embodiments, the adenovirus of the invention encodes one or more transgenes. Adenoviruses of the invention may be used, inter alia, as oncolytic vectors, conditionally-replicating vectors or replication-defective vectors. Adenoviruses of the invention may be used as gene therapy vectors. Such vectors may comprise transgenes in order to produce recombinant nucleic acids or polypeptides at the desired site, such as a tumour site.

[0147] In gene therapy vectors, the transgene may encode a therapeutic polypeptide or it may be a gene to correct a genetic defect. In cancer gene therapy vectors, the gene may encode a polypeptide (e.g. p53) which induces cell death.

[0148] 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). Preferred examples of transgenes include ones which encode EpCaAM-Lite, EpCAM-BiTE, CTLA4, CCL19, CCL5, CXCL11 or CXCL12.

[0149] 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.

[0150] A deletion in the E3 region creates more space for transgenes to be placed in other locations in the adenoviral genome. It also removes ORFs which help the virus to hide from the immune system and prevent host clearance of the virus. As a result, deletions in the E3 region attenuate virus activity in normal cells, with a functioning immune response, but not in immune dysfunctional tumour cells, thus increasing the therapeutic index of the oncolytic virus.

[0151] The adenovirus must have a genome within the packaging limit of the virus. For example, it must not carry a transgene of such a size that the total length of the genome surpasses the length which is able to be packaged into the protein capsid. 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.

[0152] The invention relates to a number of different aspects, as described herein, both individually and in combination. In particular, the invention provides adenoviruses having the features of the first and second aspects; first and third aspects; second and third aspects; and first, second and third aspects.

[0153] First aspect of the invention

[0154] In a first aspect, the invention provides an adenovirus, wherein the genome of the adenovirus comprises:

[0155] (A) (a) an Ad3 penton gene; and

[0156] (b) an Ad7 hexon gene; and

[0157] (c) optionally an Ad7 fibre gene.

[0158] The wild-type adenoviral L2 region comprises adenoviral penton, pVI I, V and pX genes encoding adenoviral penton polypeptides, and the pVI I, V and pX core polypeptides, respectively.

[0159] The wild-type adenoviral L3 region comprises adenoviral pVI, hexon and protease genes encoding adenoviral pVI, hexon and protease polypeptides, respectively.

[0160] The references herein to adenoviral genes (e.g. penton, hexon, fibre, etc.) relate to the ORFs or the coding sequences of these genes. In some embodiments, the L2 region comprises a penton gene which encodes an Ad3 penton polypeptide, and the L3 region comprises a hexon gene which encodes an Ad7 hexon polypeptide.

[0161] In some embodiments, the L3 region comprises a VI gene which encodes an Ad3 VI polypeptide; and the L3 region comprises a hexon gene which encodes an Ad7 hexon polypeptide.

[0162] In some embodiments, the L2 region comprises penton, pVI I , V and pX genes which encode Ad3 penton, Ad3 pVI I , Ad3 V and Ad3 pX polypeptides, respectively; the L3 region comprises a VI gene which encodes an Ad3 VI polypeptide; and the L3 region comprises hexon gene which encodes an Ad7 hexon polypeptide.

[0163] In some embodiments, the L2 region comprises a V gene which encodes an Ad3 V polypeptide; and the L2 region comprises a pX gene which encodes an Ad7 pX polypeptide.

[0164] In some embodiments, the L2 region comprises penton, pVII and V genes which encode Ad3 penton, Ad3 pVII and Ad3 V polypeptides, respectively; the L2 region comprises a pX gene which encodes an Ad7 pX polypeptide; and the L3 region comprises VI and hexon genes which encode Ad7 VI and Ad7 hexon polypeptides, respectively.

[0165] In some embodiments, the L2 region comprises a pX gene which encodes an Ad3 pX polypeptide; and the L2 region comprises a VI gene which encodes an Ad7 VI polypeptide.

[0166] In some embodiments, the L2 region comprises penton, pVII, V and pX genes which encode Ad3 penton, Ad3 pVII, Ad3 V and Ad3 pX polypeptides, respectively; and the L3 region comprises VI and hexon genes which encode Ad7 VI and Ad7 hexon polypeptides, respectively. In a particularly-preferred embodiment, the L2 region comprises penton, pVI I, V and pX genes which encode Ad3 penton, Ad3 pVI I , Ad3 V and Ad3 pX polypeptides, respectively; and the L3 region comprises VI and hexon genes which encode Ad3 VI and Ad7 hexon polypeptides, respectively.

[0167] In all embodiments, the L3 region may additionally comprise a protease gene encoding an Ad7 protease. In all embodiments, the L1 region preferably comprises L1 genes all of which encode Ad3 polypeptides. In all embodiments, the L4 region preferably comprises L4 genes all of which encode Ad7 polypeptides.

[0168] Preferably, the L2 region comprises an Ad3 penton gene, i.e. preferably one of the L2 polypeptides is an Ad3 penton polypeptide.

[0169] The terms “penton” and “penton base” are used herein interchangeably; both refer to penton polypeptides.

[0170] As used herein, the term "Ad3 penton gene" refers to a gene comprising or consisting of:

[0171] (a) the nucleotide sequence as given in SEQ ID NO: 3;

[0172] (b) a nucleotide sequence which has at least 95% or 99% nucleotide sequence identity to SEQ ID NO: 3, and which encodes an Ad3 penton polypeptide;

[0173] (c) a nucleotide sequence which has at least 99% nucleotide sequence identity to SEQ ID NO: 3, and which encodes an adenovirus (preferably Ad3) penton polypeptide; or

[0174] (d) a nucleotide sequence which encodes an Ad3 penton polypeptide.

[0175] As used herein, the term "Ad3 penton polypeptide" preferably includes, but is not limited to:

[0176] (a) a polypeptide whose amino acid sequence is given in SEQ ID NO: 5;

[0177] (b) a polypeptide having at least 99.3%, 99.5% or 99.7% (preferably at least 99.7%) sequence identity to SEQ ID NO: 5, the polypeptide encoding an adenovirus penton polypeptide (preferably an Ad3 penton polypeptide); or (c) a polypeptide having at least 95%, 99% or 99.5% (preferably at least 99.5%) sequence identity to SEQ ID NO: 5, wherein the amino acids in the penton polypeptide at the positions which correspond to positions 11 , 158, 178 and 326 in SEQ ID NO: 5 are V, T, I and D, respectively, the polypeptide encoding an adenovirus penton polypeptide (preferably an Ad3 penton polypeptide).

[0178] Adenovirus penton polypeptides are well characterised (Medina-Kauwe, Then Deliv. 2013 Feb; 4(2): 267-277). The penton base forms a homo-pentamer that non- covalently attaches to each vertex of the adenovirus capsid and binds to the N-terminal tail domain of the fibre polypeptide. The penton base homo-pentamer contributes to the formation of the adenoviral capsid's characteristic icosahedral shape and is essential for the structural stability of the adenovirus capsid. The penton polypeptides are therefore necessary for the efficient transduction of target cells.

[0179] The efficiency of transduction of target cells (e.g. HEK293 cells) by an adenovirus comprising penton polypeptides of the invention can be tested by exposing the target cells to the adenovirus and subsequently measuring the percentage of cells showing expression of a gene (e.g. E1 A gene or a GFP reporter gene) encoded by the genome packaged within the adenovirus capsid, relative to the results obtained with an adenovirus comprising a wild-type penton polypeptide (e.g. one of SEQ ID NO: 5) when tested under the same conditions.

[0180] The transduction assay may be performed for example by measuring the percentage of HEK293 cells expressing a GFP reporter gene encoded in the viral genome by flow cytometry as performed in Gueret et al. (Gueret V, Negrete-Virgen JA, Lyddiatt A, Al- Rubeai M. “Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells”. Cytotechnology. 2002 Jan;38(1 -3):87-97). In such assays, the two adenoviruses to be compared should have the same capsid and same genomes apart from different penton polypeptides and penton genes. Penton polypeptides of the invention include those wherein adenoviruses comprising such penton polypeptides have a transduction efficiency of at least 0.001 % or 0.01 %, preferably at least 0.1 %, and most preferably at least 1 % of the transduction efficiency obtainable under the same assay conditions using an adenovirus comprising a wild-type penton polypeptide of SEQ ID NO: 5.

[0181] Preferably, the L2 region comprises an Ad3 pVII gene, i.e. preferably one of the L2 polypeptides is an Ad3 pVII polypeptide.

[0182] As used herein, the term "Ad3 pVII gene" refers to a gene comprising or consisting of:

[0183] (a) the nucleotide sequence as given in SEQ ID NO: 7;

[0184] (b) a nucleotide sequence which has at least 95% or 99% nucleotide sequence identity to SEQ ID NO: 7, and which encodes an Ad3 pVII polypeptide;

[0185] (c) a nucleotide sequence which has at least 99% nucleotide sequence identity to SEQ ID NO: 7, and which encodes an adenovirus (preferably Ad3) pVII polypeptide; or

[0186] (d) a nucleotide sequence which encodes an Ad3 pVII polypeptide.

[0187] As used herein, the term "Ad3 pVII polypeptide" preferably includes, but is not limited to:

[0188] (a) a polypeptide whose amino acid sequence is given in SEQ ID NO: 9; or

[0189] (b) a polypeptide having at least 99% sequence identity to SEQ ID NO: 9, the polypeptide encoding an adenovirus pVII polypeptide; or

[0190] (c) a polypeptide having at least 95%, 99% or 99.5% (preferably at least 99.5%) sequence identity to SEQ ID NO: 9, and wherein the amino acids in the pVII polypeptide at the positions which correspond to positions 27 and 112 in SEQ ID NO: 9 are K and P, respectively, the polypeptide encoding an adenovirus pVII polypeptide (preferably an Ad3 pVII polypeptide).

[0191] Preferably, the L2 region comprises an Ad3 V gene, i.e. preferably one of the L2 polypeptides is an Ad3 V polypeptide.

[0192] As used herein, the term "Ad3 V gene" refers to a gene comprising or consisting of: (a) the nucleotide sequence as given in SEQ ID NO: 11 ;

[0193] (b) a nucleotide sequence which has at least 95% or 99% nucleotide sequence identity to SEQ ID NO: 11 , and which encodes an Ad3 V polypeptide; or

[0194] (c) a nucleotide sequence which has at least 99.5% nucleotide sequence identity to SEQ ID NO: 11 , and which encodes an Ad3 V polypeptide; or

[0195] (d) a nucleotide sequence which encodes an Ad3 V polypeptide.

[0196] As used herein, the term "Ad3 V polypeptide" preferably includes, but is not limited to:

[0197] (a) a polypeptide whose amino acid sequence is given in SEQ ID NO: 13; or

[0198] (b) a polypeptide having at least 99.6% sequence identity to SEQ ID NO: 13, the polypeptide encoding an Ad3 V polypeptide; or

[0199] (c) a polypeptide having at least 90%, 95% or 99% (preferably at least 99%) sequence identity to SEQ ID NO: 13, and wherein the amino acids in the V polypeptide at the positions which correspond to positions 45 and 234 in SEQ ID NO: 14 are D and are absent, respectively, the polypeptide encoding an adenovirus V polypeptide (preferably an Ad3 V polypeptide).

[0200] In some embodiments, the L2 region comprises an Ad3 pX gene, i.e. in some embodiments, one of the L2 polypeptides is an Ad3 pX polypeptide.

[0201] As used herein, the term "Ad3 pX gene" refers to a gene comprising or consisting of:

[0202] (a) the nucleotide sequence as sequence given in SEQ ID NO: 15;

[0203] (b) a nucleotide sequence which has at least 95% nucleotide sequence identity to SEQ ID NO: 15, and which encodes an Ad3 pX polypeptide;

[0204] (c) a nucleotide sequence which has at least 99% nucleotide sequence identity to SEQ ID NO: 15, and which encodes an adenovirus (preferably Ad3) pX polypeptide; or

[0205] (d) a nucleotide sequence which encodes an Ad3 pX polypeptide.

[0206] As used herein, the term "Ad3 pX polypeptide" preferably includes, but is not limited to:

[0207] (a) a polypeptide whose amino acid sequence is given in SEQ ID NO: 17; or (b) a polypeptide having at least 90%, 95% or 98.5% (preferably at least 98.5%) sequence identity to SEQ ID NO: 17, and wherein the amino acid in the pX polypeptide at the position which corresponds to position 43 in SEQ ID NO: 17 is G, the polypeptide encoding an adenovirus pX polypeptide (preferably an Ad3 pX polypeptide).

[0208] In some embodiments, the L2 region comprises an Ad7 pX gene, i.e. in some embodiments, one of the L2 polypeptides is an Ad7 pX polypeptide.

[0209] As used herein, the term "Ad7 pX gene" refers to a gene comprising or consisting of:

[0210] (a) the nucleotide sequence as sequence given in SEQ ID NO: 16;

[0211] (b) a nucleotide sequence which has at least 95% nucleotide sequence identity to SEQ ID NO: 16, and which encodes an Ad7 pX polypeptide;

[0212] (c) a nucleotide sequence which has at least 99% nucleotide sequence identity to SEQ ID NO: 16, and which encodes an adenovirus (preferably Ad7) pX polypeptide; or

[0213] (d) a nucleotide sequence which encodes an Ad7 pX polypeptide.

[0214] As used herein, the term "Ad7 pX polypeptide" preferably includes, but is not limited to:

[0215] (a) a polypeptide whose amino acid sequence is given in SEQ ID NO: 18; or

[0216] (b) a polypeptide having at least 90%, 95% or 98.5% (preferably at least 98.5%) sequence identity to SEQ ID NO: 18, and wherein the amino acid in the pX polypeptide at the position which corresponds to position 43 in SEQ ID NO: 18 is D, the polypeptide encoding an adenovirus pX polypeptide (preferably an Ad7 pX polypeptide).

[0217] The genome of the chimeric adenovirus comprises an L3 region, which comprises one or more L3 genes encoding one or more L3 polypeptides. Preferably, the L3 region is an Ad7 L3 region, optionally with the exception of the adenovirus pVI gene (which may be Ad3).

[0218] In some embodiments, the L3 region comprises an Ad3 pVI gene, i.e. in some embodiments, one of the L3 polypeptides is an Ad3 pVI polypeptide.

[0219] As used herein, the term "Ad3 pVI gene" refers to a gene comprising or consisting of: (a) the nucleotide sequence as sequence given in SEQ ID NO: 19;

[0220] (b) a nucleotide sequence which has at least 95% nucleotide sequence identity to SEQ ID NO: 19, and which encodes an Ad3 pVI polypeptide;

[0221] (c) a nucleotide sequence which has at least 99% nucleotide sequence identity to SEQ ID NO: 19, and which encodes an adenovirus (preferably Ad3) pVI polypeptide; or

[0222] (d) a nucleotide sequence which encodes an Ad3 pVI polypeptide.

[0223] As used herein, the term "Ad3 pVI polypeptide" preferably includes, but is not limited to:

[0224] (a) a polypeptide whose amino acid sequence is given in SEQ ID NO: 21 ; or

[0225] (b) a polypeptide having at least 90%, 95% or 98% (preferably at least 98%) sequence identity to SEQ ID NO: 21 , and wherein the amino acid in the pVI polypeptide at the position which corresponds to position 196 in SEQ ID NO: 21 is P, the polypeptide encoding an adenovirus pVI polypeptide (preferably an Ad3 pVI polypeptide).

[0226] In some embodiments, the L3 region comprises an Ad7 pVI gene, i.e. in some embodiments, one of the L3 polypeptides is an Ad7 pVI polypeptide.

[0227] As used herein, the term "Ad7 pVI gene" refers to a gene comprising or consisting of:

[0228] (a) the nucleotide sequence as sequence given in SEQ ID NO: 20;

[0229] (b) a nucleotide sequence which has at least 95% nucleotide sequence identity to SEQ ID NO: 20, and which encodes an Ad7 pVI polypeptide;

[0230] (c) a nucleotide sequence which has at least 99% nucleotide sequence identity to SEQ ID NO: 20, and which encodes an adenovirus (preferably Ad7) pVI polypeptide; or

[0231] (d) a nucleotide sequence which encodes an Ad7 pVI polypeptide.

[0232] As used herein, the term "Ad7 pVI polypeptide" preferably includes, but is not limited to:

[0233] (a) a polypeptide whose amino acid sequence is given in SEQ ID NO: 22; or

[0234] (b) a polypeptide having at least 90%, 95% or 98% (preferably at least 98%) sequence identity to SEQ ID NO: 22, and wherein the amino acid in the pVI polypeptide at the position which corresponds to position 196 in SEQ ID NO: 21 is L, the polypeptide encoding an adenovirus pVI polypeptide (preferably an Ad7 pVI polypeptide). In some embodiments, the L3 region comprises an Ad7 hexon gene, i.e. in some embodiments, one of the L3 polypeptides is an Ad7 hexon polypeptide.

[0235] As used herein, the term "Ad7 hexon gene" refers to a gene comprising or consisting of:

[0236] (a) the nucleotide sequence as sequence given in SEQ ID NO: 24;

[0237] (b) a nucleotide sequence which has at least 95% (or at least 99%) nucleotide sequence identity to SEQ ID NO: 24, and which encodes an Ad7 hexon polypeptide;

[0238] (c) a nucleotide sequence which has at least 97% (or at least 99%) nucleotide sequence identity to SEQ ID NO: 24, and which encodes an adenovirus (preferably Ad7) hexon polypeptide; or

[0239] (d) a nucleotide sequence which encodes an Ad7 hexon polypeptide.

[0240] As used herein, the term "Ad7 hexon polypeptide" preferably includes, but is not limited to:

[0241] (a) a polypeptide whose amino acid sequence is given in SEQ ID NO: 26; or

[0242] (b) a polypeptide having at least 97%, 98% or 99% (preferably at least 99%) sequence identity to SEQ ID NO: 26, and which encodes an adenovirus (preferably Ad7) hexon polypeptide.

[0243] In some embodiments, the genome of the adenovirus of the invention does not comprise an Ad7 hexon gene inserted into the E1 or E3 region.

[0244] Adenovirus hexon polypeptides are well characterised (Medina-Kauwe, Then Deliv. 2013 Feb; 4(2): 267-277). Hexon polypeptides are capable of forming a plurality (e.g. 240) of homotrimers that form the 20 facets of the icosahedral adenovirus capsid which serves to encapsulate the genome of the adenovirus and its associated proteins. The hexon trimers are essential for maintaining the capsid's structural integrity and stability and therefore are necessary for the efficient transduction of target cells.

[0245] The efficiency of transduction of target cells (e.g. HEK293 cells) by an adenovirus comprising variant hexon polypeptides of the invention can be tested by exposing the target cells to the adenovirus and subsequently measuring the percentage of cells showing expression of a gene (e.g. E1 A gene or a GFP reporter gene) encoded by the genome packaged within the adenovirus capsid, relative to the results obtained with an adenovirus comprising a wild-type hexon polypeptide (e.g. one of SEQ ID NO: 25) when tested under the same conditions.

[0246] The transduction assay may be performed for example by measuring the percentage of HEK293 cells expressing a GFP reporter gene encoded in the adenoviral genome by flow cytometry as performed in Gueret et al. (Gueret V, Negrete-Virgen JA, Lyddiatt A, Al-Rubeai M. “Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells”. Cytotechnology. 2002 Jan;38(1-3):87-97). In such assays, the two adenoviruses to be compared should have the same capsid and same genomes apart from different hexon polypeptides and hexon genes.

[0247] Hexon polypeptides of the invention include those wherein adenoviruses comprising such hexon polypeptides have a transduction efficiency of at least 0.001 % or 0.01 %, preferably at least 0.1 %, and most preferably at least 1 % of the transduction efficiency obtainable under the same assay conditions using an adenovirus comprising a wild-type hexon polypeptide of SEQ ID NO: 25.

[0248] In some embodiments, the L3 region comprises an adenovirus protease gene, i.e. in some embodiments, one of the L3 polypeptides is an adenovirus protease polypeptide. Preferably, the adenovirus protease gene is an Ad7 protease gene, encoding an Ad7 protease.

[0249] The genome of the chimeric adenovirus comprises an L4 region, which comprises one or more L4 genes encoding one or more L4 polypeptides. Preferably, the L4 region is an Ad7 L4 region.

[0250] The genome of the chimeric adenovirus comprises an E3 region. This E3 region may be a complete or substantially complete E3 region (i.e. one without any deletions compared to a wild-type adenoviral, preferably Ad3 or Ad7, E3 region). In other embodiments, the genome of the chimeric adenovirus comprises an E3 region having one or more deletions (compared to a wild-type adenoviral, preferably Ad3 or Ad7, E3 region).

[0251] The genome of the chimeric adenovirus additionally comprises an L5 region which comprises one or more L5 genes encoding one or more L5 polypeptides. Preferably, the L5 region is an Ad7 L5 region.

[0252] In some embodiments, one of the L5 genes is an adenovirus fibre gene which encodes a fibre polypeptide, i.e. in some embodiments, one of the L5 polypeptides is a fibre polypeptide. Preferably, the fibre gene is an Ad3 fibre gene or an Ad7 fibre gene, encoding an Ad3 or an Ad7 fibre polypeptide, respectively, more preferably an Ad7 fibre gene encoding an Ad7 fibre polypeptide.

[0253] As used herein, the term "Ad3 fibre gene" refers to a gene comprising or consisting of:

[0254] (a) the nucleotide sequence as sequence given in SEQ ID NO: 31 ;

[0255] (b) a nucleotide sequence which has at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) nucleotide sequence identity to SEQ ID NO: 31 , and which encodes an adenovirus (preferably Ad3) fibre polypeptide; or

[0256] (c) a nucleotide sequence which encodes an Ad3 fibre polypeptide.

[0257] As used herein, the term "Ad3 fibre polypeptide" preferably includes, but is not limited to:

[0258] (a) a polypeptide whose amino acid sequence is given in SEQ ID NO: 33; or

[0259] (b) a polypeptide having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) sequence identity to SEQ ID NO: 33, and which encodes an adenovirus (preferably Ad3) fibre polypeptide.

[0260] As used herein, the term "Ad7 fibre gene" refers to a gene comprising or consisting of: (a) the nucleotide sequence as sequence given in SEQ ID NO: 32; (b) a nucleotide sequence which has at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) nucleotide sequence identity to SEQ ID NO: 32, and which encodes an adenovirus (preferably Ad7) fibre polypeptide; or

[0261] (c) a nucleotide sequence which encodes an Ad7 fibre polypeptide.

[0262] As used herein, the term "Ad7 fibre polypeptide" preferably includes, but is not limited to:

[0263] (a) a polypeptide whose amino acid sequence is given in SEQ ID NO: 34; or

[0264] (b) a polypeptide having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) sequence identity to SEQ ID NO: 34, and which encodes an adenovirus (preferably Ad7) fibre polypeptide.

[0265] In some embodiments of the invention, the genome of the chimeric adenovirus does not comprise an Ad3 fibre gene. In some embodiments of the invention, the genome of the chimeric adenovirus does not comprise an L5 region which comprises an Ad3 fibre gene.

[0266] Adenovirus fibre polypeptides are well characterised (Medina-Kauwe, Ther. Deliv. 2013 Feb; 4(2): 267-277). The fibre comprises a homotrimer of polypeptides containing an amino (N-terminal) tail domain, which is capable of interacting with the penton base in the capsid, and a carboxy (C-terminal) globular knob. These two domains are separated by a shaft of differing length depending on serotype, wherein the shaft comprises a repeated sequence capable of forming a triple p-spiral in the fibre homotrimer. The fibre polypeptide plays an essential role in cell attachment and entry and therefore necessary for the efficient transduction of target cells.

[0267] The efficiency of transduction of target cells (e.g. HEK293 cells) by an adenovirus comprising fibre polypeptides of the invention can be tested by exposing the target cells to the adenovirus and subsequently measuring the percentage of cells showing expression of a gene (e.g. E1 A gene or a GFP reporter gene) encoded by the genome packaged within the adenovirus capsid, relative to the results obtained with an adenovirus comprising a wild-type fibre polypeptide (e.g. one of SEQ ID NO: 33) when tested under the same conditions.

[0268] The transduction assay may be performed for example by measuring the percentage of HEK293 cells expressing a GFP reporter gene encoded in the viral genome by flow cytometry as performed in Gueret et al. (Gueret V, Negrete-Virgen JA, Lyddiatt A, Al- Rubeai M. “Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells. Cytotechnology. 2002 Jan;38(1-3):87-97). In such assays, the two adenoviruses to be compared should have the same capsid and same genomes apart from different fibre polypeptides and fibre genes.

[0269] Fibre polypeptides of the invention include those wherein adenoviruses comprising such fibre polypeptides have a transduction efficiency of at least 0.001 % or 0.01 %, preferably at least 0.1 %, and most preferably at least 1 % of the transduction efficiency obtainable under the same assay conditions using an adenovirus comprising a wild-type fibre polypeptide of SEQ ID NO: 33.

[0270] Second aspect of the invention

[0271] In a second aspect, the invention provides an adenovirus, wherein the genome of the adenovirus comprises:

[0272] (B) an E3 region:

[0273] (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

[0274] (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.

[0275] The adenoviruses of this aspect of the invention comprise an E3 region. The E3 region is usually situated between the L4 and L5 genes (see Figure 1A). 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.

[0276] 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.

[0277] 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.

[0278] The nucleotide sequence of the Ad7 E3 region is given herein as SEQ ID NO: 35. As used herein, references to a Group B adenovirus E3 region relate preferably to a nucleotide sequence as given in SEQ ID NO: 35 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto.

[0279] 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.

[0280] The nucleotide sequence of the Ad7 12.1 K ORF is given herein as SEQ ID NO: 36.

[0281] 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: 36 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.

[0282] The nucleotide sequence of the Ad7 16.1 K ORF is given herein as SEQ ID NO: 37. 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: 37 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.

[0283] 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: 38.

[0284] 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: 38 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.

[0285] The nucleotide sequence of the Ad7 20K ORF is given herein as SEQ ID NO: 39.

[0286] As used herein, references to a Group B adenovirus E3 20K ORF relate preferably to a nucleotide sequence as given in SEQ ID NO:395 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto.

[0287] Preferably, the variant encodes a polypeptide having the same function as the Group B adenovirus E3 20K ORF.

[0288] The nucleotide sequence of the Ad7 20.6K ORF is given herein as SEQ ID NO: 40. 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: 40 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. The nucleotide sequence of the Ad7 7.7K ORF is given herein as SEQ ID NO: 41 . 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: 41 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto.

[0289] Preferably, the variant encodes a polypeptide having the same function as the Group B adenovirus E3 7.7K ORF.

[0290] 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: 42. 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: 42 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.

[0291] 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: 43. 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: 43 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.

[0292] 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: 44. 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: 44 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. 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.

[0293] 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.

[0294] 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 9 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.

[0295] 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.

[0296] 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.

[0297] 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. In some embodiments, the invention provides an oncolytic adenovirus, having a genome comprising an E3 region, wherein the E3 region comprises or consists of:

[0298] (a) a Group B adenovirus E3 12.1 K ORF;

[0299] (b) a 3-truncated Group B adenovirus E3 16.1 K ORF;

[0300] (c) a 5’-truncated Group B adenovirus E3 10.3K ORF;

[0301] (d) a Group B adenovirus E3 14.9K ORF; and

[0302] (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.

[0303] In other embodiments, the invention provides an oncolytic adenovirus, having a genome comprising an E3 region, wherein the E3 region comprises or consists of:

[0304] (a) a Group B adenovirus E3 12.1 K ORF;

[0305] (b) a Group B adenovirus E3 16.1 K ORF;

[0306] (c) a 3-truncated Group B adenovirus E3 19.3K ORF;

[0307] (d) a 5’-truncated Group B adenovirus 14.9K ORF; and

[0308] (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.

[0309] 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. 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: 37. The 3’-truncated Group B adenovirus (preferably Ad7) 16.1 K ORF may still retain some functional activity.

[0310] 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: 38. The 3’-truncated Group B adenovirus (preferably Ad7) 19.3K ORF may still retain some functional activity.

[0311] 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: 42.

[0312] 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: 43. 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.

[0313] 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.

[0314] 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: 35, 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: 35, or at the corresponding nucleotide in the nucleotide sequence of a non-Group B adenovirus E3 region.

[0315] 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: 35, 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: 35, or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region.

[0316] (The above nucleotide numberings refer to the nucleotides of the ends which are retained in the E3 region following the deletion.)

[0317] 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.)

[0318] The full genome of the Ad7 genome is available from Genbank (AY594255.1), the sequence of which is incorporated herein by reference.

[0319] In a particularly-preferred embodiment, the E3 region of the adenovirus has the nucleotide sequence as given in SEQ ID NO: 45, or a variant thereof having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto, preferably wherein the variant encodes:

[0320] (a) a Group B adenovirus E3 12.1 K ORF;

[0321] (b) a 3-truncated Group B adenovirus E3 16.1 K ORF;

[0322] (c) a 5’-truncated Group B adenovirus E3 10.3K ORF;

[0323] (d) a Group B adenovirus E3 14.9K ORF; and

[0324] (e) a Group B adenovirus E3 14.7K ORF.

[0325] In a particularly-preferred embodiment, the E3 region of the adenovirus has the nucleotide sequence as given in SEQ ID NO: 47, or a variant thereof having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity thereto, preferably wherein the variant encodes:

[0326] (a) a Group B adenovirus E3 12.1 K ORF;

[0327] (b) a Group B adenovirus E3 16.1 K ORF; (c) a 3-truncated Group B adenovirus E3 19.3K ORF;

[0328] (d) a 5’-truncated Group B adenovirus 14.9K ORF; and

[0329] (e) a Group B adenovirus E3 14.7K ORF.

[0330] Third aspect of the invention

[0331] In a third aspect, the invention provides an adenovirus, wherein the genome of the adenovirus comprises:

[0332] (C) an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the amino acid in the DNA pol I polypeptide sequence corresponding to amino acid 34 in SEQ ID NO: 49 is Asp or Glu, preferably Asp; or an E2B pTP gene encoding a pre-Terminal Protein, wherein the amino acid in the preTerminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 51 is selected from the group consisting of Thr, Asn, Cys, Gin and Ser, preferably Thr.

[0333] In some embodiments, if the adenovirus is an Ad1 adenovirus, then the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 51 is not Asn. In some embodiments, if the adenovirus is an Ad41 adenovirus, then the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 51 is not Gin. In some embodiments, if the adenovirus is an Ad4 or 4a adenovirus, then the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 51 is not Thr.

[0334] The invention particularly relates to pharmaceutical compositions comprising such adenoviruses, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.

[0335] The E2B DNA pol I gene encodes the adenovirus’ DNA polymerase I (DNA pol I). DNA polymerase I is required for the amplification of the adenovirus’ genomic DNA. The nucleotide and corresponding amino acid sequences of the wild-type Ad3 E2B DNA pol I gene and polypeptide are given herein as SEQ ID NOs: 48 and 49, respectively.

[0336] As used herein, the term “E2B DNA pol I gene” refers to a gene comprising or consisting of:

[0337] (i) the nucleotide sequence as given in SEQ ID NO: 48;

[0338] (ii) a nucleotide sequence which has at least 80%, 85%, 90%, 95% or 99% (preferably at least 99%) nucleotide sequence identity to SEQ ID NO: 48; and which preferably encodes a DNA polymerase I; or

[0339] (iii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 49.

[0340] As used herein, the term “E2B DNA pol I” refers to a polypeptide comprising or consisting of:

[0341] (i) the amino acid sequence as given in SEQ ID NO: 49; or

[0342] (ii) a variant of (i) having at least 80%, 85%, 90%, 95% or 99% (preferably at least 99%) amino acid sequence identity or sequence similarity to (i), and which preferably has DNA polymerase I activity.

[0343] The E2B pTP gene encodes the adenovirus pre-Terminal Protein (pTP). This polypeptide is also known as the Terminal Protein Precursor (TPP) or Terminal Precursor Protein (TPP). The pTP is required for the initiation of virus genome replication.

[0344] The nucleotide and corresponding amino acid sequences of the wild-type Ad3 E2B pTP gene and polypeptide are given herein as SEQ ID NOs: 50 and 51 , respectively.

[0345] As used herein, the term “E2B pTP gene” refers to a gene comprising or consisting of:

[0346] (i) the nucleotide sequence as given in SEQ ID NO: 50;

[0347] (ii) a nucleotide sequence which has at least 80%, 85%, 90%, 95% or 99% (preferably at least 99%) nucleotide sequence identity to SEQ ID NO: 50; and which preferably encodes a pre-Terminal Protein; or (iii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 51 .

[0348] As used herein, the term “E2B pre-Terminal Protein” refers to a polypeptide comprising or consisting of:

[0349] (i) the amino acid sequence as given in SEQ ID NO: 51 ; or

[0350] (ii) a variant of (i) having at least 80%, 85%, 90%, 95% or 99% (preferably at least 99%) amino acid sequence identity or sequence similarity to (i), and which preferably encodes a pre-Terminal Protein.

[0351] The inventors have found that adenoviruses comprising the G34D mutation in the E2B DNA pol I polypeptide have enhanced adenovirus DNA replication and enhanced oncolytic activity compared to control adenoviruses without this mutation.

[0352] Aspartic acid (Asp, D) and glutamic acid (Glu, E) are both negatively charged amino acids. In one embodiment, therefore, the invention provides an adenovirus, wherein the genome of the adenovirus comprises an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the amino acid in the DNA pol I polypeptide sequence corresponding to amino acid 34 in SEQ ID NO: 49 is Asp or Glu, preferably Asp. For example, the codon at the position in the genome of the adenovirus corresponding to nucleotide positions 100-102 in SEQ ID NO: 48 is gac, gau, gaa or gag.

[0353] In the wild-type adenoviral genome, the coding sequences of the E2B DNA pol I gene and the E2B pTP gene overlap, in different reading frames. One consequence of this is that some mutations in the 5’-end of the E2B DNA pol gene lead to mutations in the 3’- end of the E2B pTP gene, and also in the corresponding polypeptides.

[0354] For example, the G34D mutation in the E2B DNA polypeptide (e.g. mutation of the codon ggc -^gac) results in the mutation A623T (e.g. codon gcg acg) in the E2B pTP polypeptide.

[0355] The corresponding mutations in the E2B pTP polypeptide which result from the mutations G34D and G34E are shown in the table below: Table 4: Effect of G34D and G34E mutations in E2B DNA pol I polypeptide on E2B pTP polypeptide sequence.

[0356] As shown in the above table, the mutations G34D and G34E in E2B DNA pol I polypeptide result in Thr, Met, Lys or Arg mutations at position 623 in the E2B pTP polypeptide.

[0357] In a further preferred embodiment, therefore, the invention provides an adenovirus of the invention wherein the genome of the adenovirus additionally comprises an E2B pTP gene encoding a pre-Terminal Protein, wherein the coding region of the E2B DNA pol I gene overlaps with the coding region of the E2B pTP gene, and wherein the amino acid in the terminal protein precursor sequence at the position corresponding to amino acid 623 in SEQ ID NO: 51 is selected from the group consisting of Thr, Met, Lys and Arg, preferably Thr. Preferably, the amino acid in the E2B pre-Terminal Protein polypeptide at the position corresponding to amino acid 623 in SEQ ID NO: 51 is not a nonsense (i.e. stop) mutation.

[0358] The inventors have found that adenoviruses comprising the A623T mutation in the E2B pTP polypeptide have enhanced adenovirus DNA replication and production of infectious virus progeny compared to control adenoviruses without this mutation. Thr (T), Asn (N), Cys (C), Gin (Q) and Ser (S) are all polar or neutral amino acids. In yet another embodiment, therefore, the invention provides an adenovirus, wherein the genome of the adenovirus comprises an E2B pTP gene encoding a pre-Terminal Protein, wherein the amino acid in the pre-Terminal Protein sequence at the position corresponding to amino acid 623 in SEQ ID NO: 51 is selected from the group consisting of Thr, Asn, Cys, Gin and Ser, preferably Thr. In some embodiments, if the adenovirus is an Ad1 adenovirus, then the amino acid is not Asn. In some embodiments, if the adenovirus is an Ad41 adenovirus, then the amino acid is not Gin. In some embodiments, if the adenovirus is an Ad41 adenovirus, then the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 51 is not Gin.

[0359] For example, the codon at the position in the genome of the adenovirus corresponding to nucleotide positions 5,326-5,328 in SEQ ID NO: 50 encodes:

[0360] (i) Thr e.g. acu, acc, aca, acg

[0361] (ii) Asn e.g. aau, aac,

[0362] (iii) Cys e.g. ugu, ugc

[0363] (iv) Gin e.g. caa, cag

[0364] (v) Ser e.g. agu, age, ucu, ucc, uca, ucg

[0365] As noted above, in the wild-type adenoviral genome, the coding sequences of the E2B DNA pol I gene and the E2B pTP gene overlap, in different reading frames. One consequence of this is that some mutations in the 3’-end of the E2B pTP gene lead to mutations in the 5’- end of the E2B DNA pol I gene, and also in the corresponding polypeptides. For example, the A623T mutation in the E2B pTP polypeptide (e.g. codon gcg acg) results in the mutation G34D in the E2B DNA polypeptide (e.g. mutation of the codon ggc -^gac).

[0366] The corresponding mutations in the E2B DNA pol I polypeptide which result from mutations to polar or neutral amino acids at position 623 in the E2B pTP polypeptide are shown in the table below: Table 5: Effect of polar or neutral amino acid mutations at position 623 of E2B pTP polypeptide on E2B DNA pol I polypeptide sequence. ln a further preferred embodiment, therefore, the invention provides an adenovirus of the invention, wherein the genome of the adenovirus additionally comprises an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the coding region of the E2B pTP gene overlaps with the coding region of the E2B DNA pol I gene, and wherein the amino acids in the DNA pol I polypeptide sequence at the positions corresponding to amino acids 34-35 in SEQ ID NO: 49 are selected from the group consisting of Asp-Ser, Asp-Pro, Asp-Thr, Asp-Ala, Glu-Ser, Glu-Pro, Vai-Ser, Val-Pro, Ala-Thr, Ala-Ala, Val- Thr and Val-Ala.

[0367] With regard to the above list (i.e. Asp-Ser, Asp-Pro, etc.), the first amino acid listed in each pair is the one at the position corresponding to amino acid 34 in SEQ ID NO: 49, and the second amino acid is the one at the position corresponding to amino acid 35 in SEQ ID NO: 49. Preferably, neither of the amino acids in the E2B DNA pol I polypeptide at positions corresponding to amino acids 34-35 in SEQ ID NO: 49 are nonsense (i.e. stop) mutations.

[0368] In yet further embodiments, the invention provides a mutant E2B DNA pol I gene, wherein the nucleotide in the mutant E2B DNA pol I gene at the position corresponding to nucleotide 101 in SEQ ID NO: 48 is A. Preferably, the nucleotide sequence of the mutant E2B DNA pol I gene also has at least 80%, 85%, 90%, 95% or 99% (preferably at least 99%) nucleotide sequence identity to SEQ ID NO: 48; and preferably it encodes a polypeptide having E2B DNA pol I activity.

[0369] In yet further embodiments, the invention provides a mutant E2B pTP gene, wherein the nucleotide in the mutant E2B pTP gene at the position corresponding to nucleotide 5,326 in SEQ ID NO: 50 is a G. Preferably, the nucleotide sequence of the mutant E2B pTP gene also has at least 80%, 85%, 90%, 95% or 99% (preferably at least 99%) nucleotide sequence identity to SEQ ID NO: 50; and preferably it encodes a polypeptide having E2B terminal precursor protein activity.

[0370] The genes of the invention are preferably isolated or purified. As used herein, the term "isolated gene" means that the nucleic acid molecule is not contiguous with other genes or nucleotide sequences with which it is normally associated in an adenovirus. For example, an isolated nucleic acid of the invention comprising a DNA pol I gene of interest will not carry said DNA pol I gene contiguously with a directly neighbouring nucleotide sequence, e.g. a nucleic acid encoding its directly neighbouring gene(s), in an adenovirus genome. The references to a nucleic acid comprising the DNA pol I gene (and, mutatis mutandis, the pTP gene) should be construed accordingly. Thus, the isolated gene is not a wild-type gene of an adenovirus.

[0371] In yet further embodiments, the invention provides a mutant E2B DNA pol I polypeptide, wherein the amino acid in the mutant E2B DNA pol I polypeptide at the position corresponding to amino acid 34 in SEQ ID NO: 49 is D or E, preferably D. Preferably, the amino acid sequence of the mutant E2B DNA pol I polypeptide also has at least 80%, 85%, 90%, 95% or 99% (preferably at least 99%) amino acid sequence identity or similarity to SEQ ID NO: 49; and preferably the polypeptide has E2B DNA pol I activity.

[0372] In yet further embodiments, the invention provides a mutant E2B pTP polypeptide, wherein the amino acid in the mutant E2B pTP polypeptide at the position corresponding to amino acid 623 in SEQ ID NO: 49 is T. Preferably, the amino acid sequence of the mutant E2B pTP polypeptide has at least 80%, 85%, 90%, 95% or 99% (preferably at least 99%) amino acid sequence identity or similarity to SEQ ID NO: 51 ; and preferably the polypeptide has E2B pTP activity.

[0373] Fourth aspect of the invention

[0374] In a fourth aspect, the invention provides an adenovirus, wherein the genome of the adenovirus comprises one, two or all of (A), (B) and (C): (A) an Ad3 penton gene, wherein the Ad3 penton gene comprises or consists of:

[0375] (a) the nucleotide sequence as given in SEQ ID NO: 3;

[0376] (b) a nucleotide sequence which has at least 99% nucleotide sequence identity to SEQ ID NO: 3, and which encodes an adenovirus penton polypeptide; or

[0377] (c) a nucleotide sequence which encodes an Ad3 penton polypeptide, wherein the Ad3 penton polypeptide is:

[0378] (i) a polypeptide whose amino acid sequence is given in SEQ ID NO: 5;

[0379] (ii) a polypeptide having at least 99.3%, 99.5% or 99.7% sequence identity to SEQ ID NO: 5, the polypeptide encoding an adenovirus penton polypeptide; or

[0380] (iii) a polypeptide having at least 95%, 99% or 99.5% sequence identity to SEQ ID NO: 5, wherein the amino acids in the penton polypeptide at the positions which correspond to positions 11 , 158, 178 and 326 in SEQ ID NO: 5 are V, T, I and D, respectively, the polypeptide encoding an adenovirus penton polypeptide; and an Ad7 hexon gene, wherein the Ad7 hexon gene comprises or consists of:

[0381] (a) the nucleotide sequence as sequence given in SEQ ID NO: 24;

[0382] (b) a nucleotide sequence which has at least 97% or at least 99% nucleotide sequence identity to SEQ ID NO: 24, and which encodes an adenovirus hexon polypeptide; or

[0383] (c) a nucleotide sequence which encodes an Ad7 hexon polypeptide, wherein the Ad7 hexon polypeptide is:

[0384] (i) a polypeptide whose amino acid sequence is given in SEQ ID NO: 26; or

[0385] (ii) a polypeptide having at least 97%, 98% or 99% sequence identity to SEQ ID NO: 26, and which encodes an adenovirus hexon polypeptide. andoptionally an Ad7 fibre gene;

[0386] (B) 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

[0387] (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; and

[0388] (C) an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the amino acid in the DNA pol I polypeptide sequence corresponding to amino acid 34 in SEQ ID NO: 49 is Asp or Glu, preferably Asp; or an E2B pTP gene encoding a pre-Terminal Protein, wherein the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 51 is selected from the group consisting of Thr, Asn, Cys, Gin and Ser, preferably Thr.

[0389] The adenoviruses of the fourth aspect of the invention may comprise any combination of:

[0390] (A) any of the features of the adenovirus of the first aspect of the invention;

[0391] (B) any of the features of the adenovirus of the second aspect of the invention; and

[0392] (C) any of the features of the adenovirus of the third aspect of the invention.

[0393] In particular, in a fourth aspect of the invention, the genome of the adenovirus may comprise:

[0394] (A) an L2 region and an L3 region, wherein:

[0395] (a) the L2 region comprises:

[0396] (i) an Ad3 penton gene as defined above;

[0397] (ii) an Ad3 pVIII gene;

[0398] (iii) an Ad3 V gene; and

[0399] (iv) an Ad3 or Ad7 pX gene; and / or

[0400] (b) the L3 region comprises: (i) an Ad3 or Ad7 pVI gene;

[0401] (ii) an Ad7 hexon gene as defined above; and

[0402] (iii) an Ad7 protease gene;

[0403] (B) an E3 region which comprises or consists of:

[0404] (a) a Group B adenovirus E3 12.1 K ORF;

[0405] (b) a 3-truncated Group B adenovirus E3 16.1 K ORF;

[0406] (c) a 5’-truncated Group B adenovirus E3 10.3K ORF;

[0407] (d) a Group B adenovirus E3 14.9K ORF; and

[0408] (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; and

[0409] (C) an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the coding region of the E2B pTP gene overlaps with the coding region of the E2B DNA pol I gene, and wherein the amino acids in the DNA pol I polypeptide at the positions corresponding to amino acids 34-35 in SEQ ID NO: 49 are selected from the group consisting of Asp-Ser, Asp-Pro, Asp-Thr, Asp-Ala, Glu-Ser, Glu-Pro, Val- Ser, Val-Pro, Ala-Thr, Ala-Ala, Val-Thr and Val-Ala.

[0410] In particular, in another fourth aspect of the invention, the genome of the adenovirus may comprise:

[0411] (A) an L2 region and an L3 region, wherein:

[0412] (a) the L2 region comprises:

[0413] (i) an Ad3 penton gene as defined above;

[0414] (ii) an Ad3 pVIII gene; (iii) an Ad3 V gene; and

[0415] (iv) an Ad3 pX gene; and / or

[0416] (b) the L3 region comprises:

[0417] (i) an Ad3 pVI gene;

[0418] (ii) an Ad7 hexon gene as defined above; and

[0419] (iii) an Ad7 protease gene; and the L5 region comprises an Ad7 fibre gene;

[0420] (B) an E3 region which has a deletion compared to the wild-type Group B adenovirus, wherein the deletion corresponds to:

[0421] (a) Ad7 genome nucleotides 28,011 - 30,274; or

[0422] (b) Ad7 genome nucleotides 28,482 - 30,665; and

[0423] (C) an E2B DNA pol I gene which comprises or consists of:

[0424] (i) the nucleotide sequence as given in SEQ ID NO: 48; or

[0425] (ii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 49; or an E2B pTP gene which comprises or consists of:

[0426] (i) the nucleotide sequence as given in SEQ ID NO: 50; or

[0427] (ii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 51 .

[0428] Fifth aspect of the invention

[0429] In yet another embodiment, the invention provides an adenovirus comprising the nucleotide sequence as shown in SEQ ID NO: 58 or a variant thereof having at least 93% (preferably at least 94%, 95%, 96%, 97%, 98%, 99% or 99.5%) sequence identity thereto and having oncolytic activity (preferably against ovarian cancer cells, e.g. A549 cancer cells). More preferably, the variant thereof has at least 95% or at least 99% sequence identity to SEQ ID NO: 58 and the adenovirus has oncolytic activity (preferably against ovarian cancer cells, e.g. A549 cancer cells). In some embodiments, the adenovirus comprises the nucleotide sequence as shown in SEQ ID NO: 58.

[0430] In other embodiments, the invention provides an adenovirus or adenoviral vector whose genome has 100% nucleotide sequence identity with nucleotides 1-479, 3,410- 27,380 and 28,869-33,044 of SEQ ID NO: 58 (i.e. the whole of the genome of SEQ ID NO: 58 excluding the E1 and E3 regions, which may be deleted). The genome of this adenovirus or adenoviral vector may additionally comprise a transgene. Preferably, the transgene is located between nucleotides 480-3,409 or 27,381 -28,868 of SEQ ID NO: 58.

[0431] In other embodiments, the invention provides an adenovirus or adenoviral vector whose genome has 100% nucleotide sequence identity with a continuous or discontinuous region which spans at least 85%, 90%, 95% or 99% (preferably at least 99%) of SEQ ID NO: 58. The discontinuous region may, for example, comprise 2, 3 or 4 regions, preferably 2 regions.

[0432] An adenovirus in accordance with the fifth aspect of the invention has a genome comprising all of (A), (B) and (C):

[0433] (A) (a) an Ad3 penton gene; and

[0434] (b) an Ad7 hexon gene; and

[0435] (c) optionally an Ad7 fibre gene;

[0436] (B) an E3 region:

[0437] (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; and

[0438] (C) an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the amino acid in the DNA pol I polypeptide sequence corresponding to amino acid 34 in SEQ ID NO: 49 is Asp or Glu, preferably Asp; or an E2B pTP gene encoding a pre-Terminal Protein, wherein the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 51 is selected from the group consisting of Thr, Asn, Cys, Gin and Ser, preferably Thr.

[0439] The genes of the invention are preferably isolated or purified. As used herein, the term "isolated gene" means that the nucleic acid molecule is not contiguous with other genes or nucleotide sequences with which it is normally associated in an adenovirus. Thus, the isolated gene is not a wild-type gene of an adenovirus.

[0440] The polypeptides of the invention are preferably isolated. As used herein, the term "isolated polypeptide" means that the polypeptide is not in a mixture with other polypeptides with which it is normally associated in an adenovirus.

[0441] 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. 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.

[0442] 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.

[0443] 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.

[0444] 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.

[0445] 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).

[0446] 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 .

[0447] 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.

[0448] 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.

[0449] 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.

[0450] Examples of suitable carriers include water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Examples of suitable isotonic agents include sugars, poly-alcohols such as mannitol, sorbitol and sodium chloride.

[0451] 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.

[0452] In one embodiment, the pharmaceutical composition is a liquid parenteral formulation, for example for infusion or injection, of an adenovirus of the invention.

[0453] 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.

[0454] 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.

[0455] 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.

[0456] In one embodiment, the parenteral formulation is in the form of an infusion for intra venous administration.

[0457] In another embodiment, the pharmaceutical composition is provided as a formulation for topical administration, including inhalation. Suitable inhalable preparations include inhalable powders, metering aerosols containing propellant gases or inhalable solutions free from propellant gases. Inhalable powders according to the disclosure will generally contain a virus as described herein with a physiologically acceptable excipient. These inhalable powders may include monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo- and polysaccharides (e.g. dextranes), poly-alcohols (e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures of these with one another. Mono- or disaccharides are suitably used, the use of lactose or glucose, particularly but not exclusively in the form of their hydrates.

[0458] 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.

[0459] 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.

[0460] 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.

[0461] 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.

[0462] 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.).

[0463] 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.

[0464] 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.

[0465] 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.

[0466] 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). 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).

[0467] Generally, such a pharmaceutical combination will be provided as two components:

[0468] (A) a first pharmaceutical composition of the adenovirus of the invention; and

[0469] (B) a second pharmaceutical composition comprising a chemotherapeutic or immunotherapeutic agent.

[0470] 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.

[0471] The term “combined preparation” includes both fixed combinations and non-fixed combinations. The term “fixed combination” means that the active ingredients (e.g. components (A) and (B)) are in the form of a single entity or dosage unit. In other words, the active ingredients are present in a single composition or formulation. The term “nonfixed combination” means that the active ingredients (e.g. components (A) and (B)) are present in different entities or dosages (e.g. as separate compositions or formulations), for example as a kit of parts. The independent components (A) and (B) (in their desired compositions or formulations) can then be administered simultaneously, separately or sequentially, at the same time point or at different time points.

[0472] 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.

[0473] 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.

[0474] 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.

[0475] 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).

[0476] 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.

[0477] In another embodiment, the invention provides an adenovirus of the invention for use in therapy or for use as a medicament.

[0478] In another embodiment, the invention provides an oncolytic adenovirus of the invention for use in treating cancer (preferably ovarian cancer). In another embodiment, the invention provides a method of treating cancer (preferably ovarian cancer) in a subject, the method comprising administering an effective amount of an oncolytic adenovirus of the invention to a subject in need thereof.

[0479] In another embodiment, the invention provides the use of an oncolytic adenovirus of the invention in the manufacture of a medicament for treating cancer (preferably ovarian cancer). The invention also provides the use of an oncolytic adenovirus of the invention for the treatment of cancer (preferably ovarian cancer); and an oncolytic adenovirus of the invention when used to treat cancer (preferably ovarian cancer). As used herein, the term “treating cancer” includes killing cancer cells and / or preventing or inhibiting the spread of cancer cells.

[0480] 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.

[0481] 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).

[0482] 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.

[0483] 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.

[0484] 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.

[0485] The stromal cells may comprise CAFs. The cancer or tumour may be one which comprises CAFs.

[0486] 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.

[0487] 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.

[0488] 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.

[0489] A pharmaceutical composition of the present invention may be administered via one or more routes using one or more of a variety of methods known in the art. Components or compositions (A) and (B) may be administered by the same route or by different routes. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results.

[0490] 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.

[0491] 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.

[0492] 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.

[0493] 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.

[0494] In one embodiment the formulation is for intra-peritoneal (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.

[0495] In one embodiment the formulation is for intra-tumoural (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.

[0496] 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.

[0497] In one embodiment, the pharmaceutical composition of the invention may comprise 1x1010to 1x1014viral particles per dose. Preferably, the pharmaceutical composition of the invention comprises 1x1011to 1x1013viral particles per dose. In one embodiment, the pharmaceutical composition of the invention may be administered over 1-8 cycles, with each cycle comprising one or multiple administrations over a 1 month period. Cycles may not be given in consecutive months.

[0498] Preferably, the method steps are carried out in the order specified.

[0499] 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.

[0500] In another embodiment of the first aspect (A) or third aspect (C) of the invention, there is provided the use of a conditionally-replicating adenovirus of the invention as a vector for protein production, wherein the adenovirus comprises a transgene encoding the protein to be produced. In yet another embodiment of the first aspect (A) or third aspect (C) of the invention, there is provided the use of a conditionally-replicating adenovirus of the invention as a helper virus for virus production.

[0501] The disclosure of each reference set forth herein is specifically incorporated herein by reference in its entirety.

[0502] BRIEF DESCRIPTION OF THE FIGURES

[0503] Figure 1 : A) 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. B) Schematic representation of the open reading frames encoded within the L2 and L3 regions.

[0504] Figure 2: Sequence comparisons of the Ad3 v Ad7 penton (Figure 2A; SEQ ID NOs: 5- 6) and hexon (Figure 2B; SEQ ID NOs: 25-26) amino acid sequences.

[0505] Figure 3: A high grade serous ovarian cancer patient sample was infected with increasing concentrations of one of the bio-selected adenoviruses, Ov26. Six days postinfection, the viability of cancer cells (A) and cancer associated fibroblasts (CAFs) (B) within the sample was determined by multiparametric flow cytometry. FAP= fibroblast activation protein, a cell surface marker present on CAFs. CA125 = cancer antigen 125, a cell surface marker present on cancer cells. VG / cell = viral genomes per cell.

[0506] Figure 4: 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.

[0507] Figure 5: Oncolytic activity of Ov26 compared to wild-type parental viruses.

[0508] (A) Viral genome replication was measured by qPCR 7 days post-infection and is expressed as viral genomes per cell. (B) Infectious progeny virus was quantified by ICC assay and is expressed as Infection Forming Units (I FU) / cell . For each virus, data is normalised to the level observed in A549 cells. Parental A = wild-type Ad3; Parental B = wild-type Ad7.

[0509] Figure 6: A549 cancer cells were seeded on sterile coverslips and infected with 100 VG / cell of either Ov26 or Ov26_L2Ad7. The coverslip was transferred onto a coculture of A549 cancer cells and cancer associated fibroblasts (CAFs). The cells were immediately overlaid with agarose and DMEM. The cells were then incubated for 2 weeks after which point MTT reagent was added to stain live cells and reveal the extent of virus spread. A) Representative image of a spreading assay; the dashed circle indicates the position of the coverslip. The unstained area delineated by a continuous line shows the extent of viral spread. B) Quantification of spread in multiple spreading assays in which MRC5 fibroblast cells were co cultured with A549 cells. C) Quantification of spread in multiple spreading assays in which the CAFs used to coculture with the A549 cells were isolated from ascites samples donated by ovarian cancer patients. A total of 4 separate samples were used in independent experiments. Each point represents spread in a single assay, error bars represent the standard deviation of the mean. Spread was calculated using imaged software; significance was assessed by T-test, *** P < 0.001 ** P < 0.01 , * P< 0.05.

[0510] Figure 7. A dilution series of each virus was performed and was incubated for 30 minutes in DMEM medium or in DMEM supplemented with pooled serum from 300 healthy human donors to a final concentration of 15%. A549 cells were infected with the serially diluted virus for 4 hours, after which time infection media on cells was replaced with DMEM supplemented with 2% FBS. 7 days post-infection cell viability was measured by MTS viability assay. Cell viability is plotted relative to uninfected cells. Non-linear regression was performed in order to calculate the IC50 value, from which a fold change in IC50 upon addition of serum was determined.

[0511] Figure 8. 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.

[0512] Figure 9. 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.

[0513] Figure 10. 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.

[0514] Figure 11. 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.

[0515] Figure 12. Quantification of TNFo 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 TNFo in combination with 12.5 pM of cyclohexamide (CHX) in order to initiate TNFo-induced apoptosis. 16 hours posttreatment with TNFo 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 TNFo 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.

[0516] Figure 13. (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).

[0517] Figure 14. 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.

[0518] Figure 15. 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.

[0519] Figure 16. Introduction of the E3 deletion enables oncolytic adenovirus CRC74 to accommodate exogenous DNA at either of two distinct arming sites

[0520] 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.

[0521] Figure 17. Ov26 and Ov26_DNAPol_WT differ in a single substitution mutation: Ov26 has the mutation; Ov26_DNAPol_WT has the wild-type sequence.

[0522] A) Schematic diagram of the genome of Ov26. The position of the difference between Ov26 and Ov26_WT_DNAPol within the virus genome is indicated by the vertical black arrow. Unfilled horizontal arrows show the positions and orientations of the surrounding viral genes. B) Nucleotide and amino acid sequences (SEQ ID NOs: 52-57, in appropriate 5’-3’ and N-C orientation) of the region containing the mutation. On the right, the mutation in Ov26 is shown in bold. The left displays the sequence of Ov26_DNAPol_WT. This single mutation in Ov26 gives rise to an amino acid change in both the DNA Pol and pTP genes since they have overlapping open reading frames.

[0523] Figure 18. OV26 displays enhanced viral genome replication relative to OV26_DNAPol_WT in different cancer cell lines. Viral genomes (VG) were quantified by qPCR 48 hours post-infection of (A) Ad-293, (B) Hela, (C) Panel and (D) A549 cells at a dose of 100 viral genomes / cell. The mean of three biological replicates is plotted; significance was tested by unpaired T-test.

[0524] Figure 19. Viral replication in patient-derived cancer samples and ovarian cancer cell line OvCAR3. Primary samples of cells from patient derived ascites fluid and ovarian cancer cell line OVCAR3 cells were infected at a dose of 100 virus genomes / cell. 6 days after infection viral genomes were measured by qPCR. Data is expressed as a fold change over the input virus dose. Significance was assessed by multiple T tests, ** P < 0.01.

[0525] Figure 20. A549 cells were infected with 100 viral genomes per cell of Ov26 or Ov26_DNAPol_WT. At 3 days post-infection wells were harvested. Samples were freeze-thawed to achieve cell lysis, and then infectious virus particles were quantified by immunocytochemistry (ICC) assay using an antibody against the viral hexon protein. Significance was assessed by T test *** P < 0.001 .

[0526] Figure 21. Genome replication in normal cell lines 6 days post-infection. Data is normalised to the fold change measured for Ov26 in each cell line.

[0527] NHDF = Normal human dermal fibroblasts

[0528] HPF = Human Pulmonary Fibroblasts

[0529] HCF = Human Cardiac Fibroblast

[0530] HUF = Human Uterine Fibroblasts HAoAF = Human Aortic Adventitial Fibroblasts

[0531] Figure 22. Analysis of Ov26 genome over 30 passages in vitro. Ov26 was used to infect Ad-293 cells. Upon formation of widespread cytopathic effect, material was harvested and used to infect a subsequent passage of Ad-293 cells. After 30 passages of virus infections, material was harvested and DNA was extracted. DNA was also extracted from the input virus; DNA was sequenced using whole genome Illumina® sequencing. A graphical representation of sequence alignment and read coverage for input virus (left) and virus after 30 infection cycles (right) is shown and reveals no mutations.

[0532] Figure 23. Quantification of Luciferase expression from A549 cells infected with either Ov26_SA-Fluc or Ov26_DNAPol_WT_SA-Fluc. Cells were infected at a multiplicity of infection of 0.6 infectious units per cell. 48 hours post infection luciferase expression was measured by lysis of cells and addition of luciferin, luminescence was measured on a plate reader.

[0533] Figure 24. A549 cancer cells were seeded on sterile coverslips and infected with 100 VG / cell of either Ov26, Ad3 or Ad7. The coverslip was transferred onto a monolayer of OVCAR-3 ovarian cancer cells. The cells were immediately overlaid with agarose and DMEM. The cells were then incubated for 2 weeks after which point MTT reagent was added to stain live cells and reveal the extent of virus spread. A) Representative images of a spreading assay; the white circle indicates the position of the coverslip and the dashed dark line delineates the unstained spread area. B) Quantification of spread in duplicate or triplicate spreading assays. Spread was calculated using imaged software; significance was assessed by one-way ANOVA, ** P < 0.01.

[0534] Figure 25. Ov26 is neutralised to a lesser degree than its parental viruses by whole blood and healthy donor serum. The clinically relevant dose (8x1012vp / patient) is shown here. Each virus was incubated with whole blood or serum for 30 minutes before being used to infect human cancer cells at a dose equivalent to the clinically relevant dose of 8x1012vp / patient. After an infection period of 2 hours blood or serum was washed from cells using cell growth medium. Viability was measured 6 days post-infection using PrestoBlue viability reagent. Data shows mean ± the standard deviation (SD).

[0535] Figure 26. Tumour cells (A549) were left untreated (mock) or infected with 1 vppc of Ov26 (A, B and C), Ad3 or Ad7 (C). After 4 days immature dendritic cells (iDCs) were added to cultures, and after a further 2 days, autologous T cells were also added as indicated (DCs and T cells were present in all conditions in C). Immune cells were derived from one of two donors as indicated. After 72 hours of incubation IFNy (A and C) and IL2 expression (B) was quantified. Data was collected in triplicate wells and is presented as the mean + / - SD.

[0536] EXAMPLES

[0537] 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.

[0538] Example 1 : Bioselection for oncolytic adenoviruses and sequence analysis

[0539] 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. The initial mixed pool of Group B, C, D, F, and G adenoviruses were obtained either from commercial sources (American Type Culture Collection or Public Health England) or from a UK Collaboration Network. From the final pool, 60 adenoviruses were selected for sequencing and their genomes were analysed.

[0540] Genome analyses of the selected adenoviruses revealed that the L2-L3 regions of all 60 bioselected adenoviruses were Ad3 / Ad7 chimeric.

[0541] Table 6: Details of chimeric breakpoints in the nucleotide and amino acid sequences of bioselected adenoviruses.

[0542] Nucleotide sequences Amino acid sequences

[0543] The fact that all 60 of the bioselected adenoviruses had chimeric Ad3 / Ad7 L2-L3 regions is evidence of the advantages conferred on the adenoviruses by these regions in terms of their ability to lyse ovarian cancer cells, and their spread, blood stability and immune stimulation.

[0544] Amino acid sequence comparisons of the Ad3 v Ad7 penton and hexon polypeptides are given in Figures 2A and 2B, respectively.

[0545] One of the chimeric adenoviruses, i.e. Ov26, was chosen for further study.

[0546] Example 2: Ov26 kills cancer cells and CAFs from an ex vivo ovarian cancer sample A high grade serous ovarian cancer (HGSOC) patient sample was infected with increasing concentrations of Ov26. Six days post-infection, the viability of cancer cells and cancer associated fibroblasts (CAFs) within the sample was determined by multiparametric flow cytometry. The results are shown in Figure 3. These results show that the viability of the cancer cells and the CAFs decreased with increasing concentrations of Ov26.

[0547] Example 3: Ov26 kills a range of ovarian patient cancer subtypes including treatment-naive and platinum-resistant cells

[0548] Ovarian patient samples (n=24) were treated with a clinically-relevant viral dose of Ov26 (equivalent to 1e13 vp / 5L / patient) and the viability of cancer cells and cancer associated fibroblasts (CAFs) was assessed. More specifically, cell viability was assessed after 6 days using flow cytometry. Cells were stained for viability using Live / Dead Near I R, EpCAM / CA125 marker to identify cancer cells and fibroblast activation protein (FAP+) to identify cancer associated fibroblasts. Each condition was measured in triplicate and presented as the mean viability relative to an uninfected control. HGSOC = High grade serous ovarian cancer. LGSOC = Low grade serous ovarian cancer. The results are shown in Table 7 below:

[0549]

[0550] The above table demonstrates that Ov26 effectively kills cancer cells and cancer associated fibroblasts (CAFs) across a range of different ovarian cancer patient subtypes.

[0551] Example 4: Use of a panel of cell lines to assess oncolytic activity of Ov26

[0552] The oncolytic activity of Ov26 was assessed in a number of cancer cell lines and cancer associated fibroblasts including the following:

[0553] Ad-293 Human embryonic kidney cell line A549 Lung cancer cell line

[0554] Hela Cervical cancer cell line

[0555] MDA-MB-231 Breast cancer cell line

[0556] PANC1 Pancreatic cell line

[0557] PSN1 Pancreatic cell line

[0558] HCT116 Colorectal cancer cell line

[0559] HT29 Colorectal cancer cell line

[0560] SKOV-3 Ovarian cell line

[0561] Huh-7 Hepatoma cell line

[0562] MRC5 Fibroblast cell line

[0563] OE-21 Oesophageal cell line

[0564] OVCAR-3 Ovarian cell line

[0565] OVSAHO Ovarian carcinoma cell line

[0566] The results are shown in Figure 4. Ov26 demonstrated efficacy against all cancer cell lines tested.

[0567] The IC50 values of Ov26 are given in Table 8 below.

[0568] Table 8. IC50 values of Ov26 in various cancer cell lines

[0569] Example 5: Oncolytic activity of Ov26 compared to wild-type parental viruses

[0570] Various parameters of Ov26 virus infection were measured 72 hours after infection of lung cancer cells (A549s), of non-cancerous normal primary human hepatocytes and of normal human dermal fibroblasts (NHDFs). The results are shown in Figure 5.

[0571] Production of viral genomes (A) and infectious virus particles (B) was lower in normal cells relative to A549 cancer cells during Ov26 infection as compared to infections with wild type parental viruses. The results demonstrate the ability of Ov26 to preferentially infect the lung cancer cells compared to normal hepatocytes and normal fibroblasts.

[0572] Example 6: Production of adenoviruses with chimeric L2-L3 regions

[0573] Homologous recombination was used to replace the L2 region in Ov26 with a selectable cassette. Insertion of the selectable cassette was verified by Sanger sequencing. The cassette was then removed by restriction digestion and an alternative L2 sequence was inserted by Gibson assembly. In this way, a new adenovirus, Ov26_L2Ad7, was produced having both L2 and L3 regions from an Ad7 adenovirus (apart from an Ad3 pVI polypeptide).

[0574] Example 7: Spreading assays on cocultured A549 cancer cells and fibroblasts

[0575] After infecting and lysing a target cell, the ability of progeny virus particles to infect neighbouring cells and establish new productive infections is key to the persistence of the virus and its spread throughout the tumour. Consequently, the ability of Ov26 to spread through a layer of cancer cells and cocultured with either MRC5 fibroblasts or patient derived cancer associated fibroblasts was compared to Ov26_L2Ad7. The results shown in Figure 6 demonstrate that Ov26 has significantly enhanced spreading ability compared to Ov26_L2Ad7 in both cases.

[0576] Example 8: Serum neutralisation assays on A549 cancer cells.

[0577] The ability of an oncolytic virus to evade pre-existing immunity such as neutralising antibodies in the bloodstream is important if it is to be successful in reaching the tumour following intravenous delivery. The humoral immune response to adenovirus results in production of antibodies against the three major capsid proteins: hexon, penton and fibre. However, to what extent antibodies against each capsid protein are produced and which of these are most efficient in neutralising the virus is not fully resolved

[0578] The ability of Ov26 to establish an infection and kill A549 cancer cells in the presence and absence of serum was compared to the wild-type parental viruses Ad3 and Ad7.

[0579] The results shown in Figure 7 demonstrate that the propensity of Ov26 to evade neutralisation by serum is similar to that of Ad7, whilst it is substantially greater than that of Ad3. These findings suggest that Ov26 is able to incorporate the penton protein from Ad3 without suffering from a dramatic rise in neutralisation, likely due to the fact that it has a fibre protein from an alternative parental virus, namely Ad7. This underlines the importance of the chimeric capsid arrangement found in Ov26 in which the penton and fibre genes are derived from different parental viruses. Example 9: Bioselected adenoviruses with E3 deletions

[0580] Genome analyses of the selected adenoviruses from Example 1 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

[0581] Figure 8. Ov26 and Ov91 are two of the bioselected adenoviruses which have Deletion (A). Ov20 has Deletion (B).

[0582] Table 9: Details of the two E3 region deletions

[0583] Deletion A: sequence between nucleotides 629 - 2,892 is deleted from SEQ ID NO: 35.

[0584] Deletion B: sequence between nucleotides 954 - 3,283 is deleted from SEQ ID NO: 35. Table 10 Details of the corresponding ORFs in other adenovirus Groups are given in Figure 9.

[0585] 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.

[0586] Example 10: Quantification of TNFa levels in patient derived samples.

[0587] 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 10 show that levels of TNFa were highest in patient-derived samples, and that no TNFa was detected in healthy serum or in the cancer cell line culture. This suggests that the effects of TNFa on virotherapies can only be effectively evaluated in models such as patient-derived tumour samples where TNFa 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.

[0588] Example 11 : Comparison of Ov26, Ov26_E3_A14.7K, Ov26_E3_A14.9K and Ov26_E3_AX1

[0589] 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.

[0590] 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.

[0591] 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 11).

[0592] The results show that Ov26 is able to block induction of NF-KB signalling to a greater extent than is Ov26_E3_A14.9K.

[0593] 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 12). 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. Example 12: Comparison of growth kinetics between Ov91 candidate and “armed” Ov candidate

[0594] 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.

[0595] 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 13 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.

[0596] Example 13: Quantification of infectious virus production in the presence of TNFa The E3 14.7K protein was shown to be important for the blockade of TNFa 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 TNFa treatment was assessed. The results shown in Figure 14 demonstrate that treatment of cells with TNFa 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. Example 14: Transferability of the E3 deletion to other oncolytic viruses for improved selectivity towards cancer and cancer associated cells

[0597] 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 8, Theolytics A) was introduced into an alternative oncolytic adenovirus, CRC74, generating the E3-deleted variant CRC74_AE3.

[0598] 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 15 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.

[0599] Example 15: Effect of the E3 deletion on the ability of the virus to accommodate exogenous transgenes without affecting virus activity

[0600] 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. 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 16A 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.

[0601] Meanwhile, the results in Figure 16B 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 16 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.

[0602] Example 16: Selection of adenoviruses with missense mutation

[0603] Genome analyses of the selected adenoviruses from Example 1 revealed that a large number of the selected adenoviruses had a missense mutation in the E2B DNA pol I gene. The mutation was the single nucleotide change ggc gac in the adenovirus genome. This nucleotide change results in a G34D mutation in the E2B DNA pol I polypeptide and an A623T mutation in the E2B TPP polypeptide.

[0604] One of the selected adenoviruses which had the mutation, i.e. Ov26, was chosen for further study.

[0605] Given that both D (aspartic acid) and E (glutamic acid) are both negatively-charged amino acids of similar structure, similar results would be expected from a G34E mutation. Given that T (threonine), N (asparagine), C (cysteine), Q (glutamine) and S (serine) are all polar / neutral amino acids of similar structure, similar results would be expected from A623N, A623C, A623Q and A623S mutations.

[0606] Example 17: Generation of control adenovirus without mutation

[0607] Sequence data from adenovirus Ov26 (which has the ggc gac mutation) is represented in Figure 17, compared to that of a control adenovirus (Ov26_DNAPol_WT) which has the same genome sequence as Ov26 except for the latter single nucleotide mutation.

[0608] Example 18: The growth kinetics of Ov26 and Ov26_DNAPol_WT in cancer cell lines

[0609] The growth kinetics of Ov26 and Ov26_DNAPol_WT over 48 hours in four different cell lines is shown in Figure 18. Viral genomes accumulated to higher levels in Ad-293 (A), HeLa (B), Panc-1 (C) and A549 (D) cells, as measured by qPCR 48 hours postinfection.

[0610] The data shows that Ov26 is capable of replicating to high levels post-infection than Ov26_DNAPol_WT.

[0611] Example 19: Genome replication in ascites samples and ovarian cancer cell line Replication of Ov26 and Ov26_DNAPol_WT was assessed in patient-derived cancer samples and the ovarian cancer cell line OvCAR3.

[0612] Primary samples of cells from patient-derived ascites fluid, and ovarian cancer cell line OVCAR3 cells were infected at a dose of 100 virus genomes / cell, 6 days after infection viral genomes were measured by qPCR. The results are shown in Figure 19 and show that Ov26 is able to replicate to a greater extent than Ov26_DNAPol_WT in ascites samples leading to an accumulation of a greater number of genomes by day 6 post infection.

[0613] Example 20: Production of infectious virus particles in A549 cells

[0614] A549 cells were infected with 100 viral genomes per cell of Ov26 or Ov26_DNAPol_WT virus. Infectious virus particles were then measured at various time points post-infection

[0615] Figure 20 shows that infection with Ov26 leads to the production of a greater number of infectious virus particles compared to Ov26_DNAPol_WT. Example 21 : Genome replication in normal cell lines 6 days post infection

[0616] Genome replication of Ov26 and Ov26_DNAPol_WT in various normal cell lines was quantified by qPCR 3 days post-infection of cells with 100 viral genomes per cell. The results are shown in Figure 21 , which show that the mutation present in the DNA pol I gene of Ov26 does not confer enhanced viral replication in normal cells, and that this mutation serves to increase the therapeutic index of the virus.

[0617] Example 22: Mutations in the E2B DNA pol I and pTP genes do not impair the virus proof-reading or result in mutation of the virus genome

[0618] The Ov26 genome was analysed over 30 passages in vitro. Ad-293 cells were infected with Ov26 and harvested when signs of CPE were observed. The harvested material was then used to infect the following passage. DNA was extracted from the input virus, as well as passage 10, 20, and 30, and libraries were produced for whole genome Illumina® sequencing.

[0619] The results for the input virus versus the virus after 30 passages are shown in Figure 22. These results show that no mutations were detected by Illumina® sequencing in the viral genome after 30 passages in cell culture, indicating that the E2B mutation in Ov26 does not impair the fidelity of the virus polymerase.

[0620] Example 23: Production of adenovirus with fluorescent reporter transgene

[0621] Viral genomes of Ov26 and Ov26_DNAPol_WT were vectorised into Bacterial Artificial Chromosomes (BACs) and a recombineering approach was used to insert a firefly luciferase reporter gene driven by an SA promoter (SAFIuc) into the viral E3 region. The BACs, now encoding viral genomes harbouring reporter transgenes, were transfected into Ad-293 cells and replicating virus was recovered, amplified and titred by immunocytochemistry assay. The virus was then used to infect A549 cells at a dose of 0.6 infectious units per cell. Luciferase expression was measured at 48 hours post-infection.

[0622] The data (Figure 23) shows that Ov26 is capable of producing greater levels of transgene expression than Ov26_DNAPol_WT. Example 24: Spreading assays on OVACR-3 Ovarian cancer cells

[0623] After infecting and lysing a target cell, the ability of progeny virus particles to infect neighbouring cells and establish new productive infections is key to the persistence of the virus and its spread throughout the tumour. Consequently, the ability of Ov26 to spread through a layer of cancer cells was compared to its parental viruses Ad3 and Ad7. The results shown in Figure 24 demonstrate that Ov26 has significantly enhanced spreading ability compared to Ad3 and Ad7.

[0624] Example 25: Neutralisation assays using whole blood and healthy donor serum For an oncolytic virus to be successfully administered intravenously, it must be able to overcome neutralisation by components of the blood in order to reach and infect its target cancer cells. Consequently, the ability of Ov26 and parental viruses Ad3 and Ad7 to infect and kill human cancer (A549) cells after exposure to whole blood or serum was compared. The results in Figure 25 show that Ov26 retains its ability to kill cancer cells after exposure to whole blood or serum whilst its parental viruses Ad3 and ad7 are effectively neutralised.

[0625] Example 26: Ov26 induces IL-2 and IFNy expression only when both antigen- presenting cells (DC cells) and T cells are present in co-culture with lysed tumour cells, and induces IFNy activation to a greater extent than its parental viruses Ad3 and Ad7

[0626] One of the mechanisms of action for cancer cell killing of an oncolytic virus is activation of immune cells to subsequently target and kill cancer cells. Therefore, the ability of Ov26 to activate an inflammatory T cell response against infected A549 cancer cells was assessed. The results in Figure 26A and 26B show that only when both antigen presenting DC cells and T cells are both present is an inflammatory response, characterised by IFNy and IL2 upregulation, mounted. This data indicates that pathogen and danger associated molecular patterns (PAMPS / DAMPS) released from cancer cells lysed by Ov26 are specifically presented to the T cells by the antigen presenting cell, thereby stimulating the immune response. Using the same assay set up, IFNy upregulation was compared between conditions where the A549 cancer cells were infected with Ov26 or with either parental virus Ad3 or Ad7. The results in Figure 26C show that Ov26 infection induced a greater IFNy response from the T-cells than that induced by either parental virus Ad3 or Ad7.

[0627] The Sequence Listing filed with this patent application is fully incorporated herein as part of the description.

[0628] FIRST ASPECT OF THE INVENTION

[0629] Preferred Embodiments of the first aspect of the invention include the following:

[0630] 1 . A chimeric oncolytic adenovirus for use in the prevention or treatment of cancer, wherein the genome of the chimeric oncolytic adenovirus comprises:

[0631] (A) an Ad3 penton gene, wherein the Ad3 penton gene comprises or consists of:

[0632] (a) the nucleotide sequence as given in SEQ ID NO: 3;

[0633] (b) a nucleotide sequence which has at least 99% nucleotide sequence identity to SEQ ID NO: 3, and which encodes an adenovirus penton polypeptide; or

[0634] (c) a nucleotide sequence which encodes an Ad3 penton polypeptide, wherein the Ad3 penton polypeptide is:

[0635] (i) a polypeptide whose amino acid sequence is given in SEQ ID NO: 5;

[0636] (ii) a polypeptide having at least 99.3%, 99.5% or 99.7% sequence identity to SEQ ID NO: 5, the polypeptide encoding an adenovirus penton polypeptide; or

[0637] (iii) a polypeptide having at least 95%, 99% or 99.5% sequence identity to SEQ ID NO: 5, wherein the amino acids in the penton polypeptide at the positions which correspond to positions 11 , 158, 178 and 326 in SEQ ID NO: 5 are V, T, I and D, respectively, the polypeptide encoding an adenovirus penton polypeptide; and

[0638] (B) an Ad7 hexon gene, wherein the Ad7 hexon gene comprises or consists of:

[0639] (a) the nucleotide sequence as sequence given in SEQ ID NO: 24; (b) a nucleotide sequence which has at least 97% or at least 99% nucleotide sequence identity to SEQ ID NO: 24, and which encodes an adenovirus hexon polypeptide; or

[0640] (c) a nucleotide sequence which encodes an Ad7 hexon polypeptide, wherein the Ad7 hexon polypeptide is:

[0641] (i) a polypeptide whose amino acid sequence is given in SEQ ID NO: 26; or

[0642] (ii) a polypeptide having at least 97%, 98% or 99% sequence identity to SEQ ID NO: 26, and which encodes an adenovirus hexon polypeptide.

[0643] 2. A method of treating cancer in a patient, the method comprising administering an effective amount of a chimeric oncolytic adenovirus, wherein the genome of the chimeric oncolytic adenovirus comprises:

[0644] (A) an Ad3 penton gene as defined in Embodiment 1 ; and

[0645] (B) an Ad7 hexon gene as defined in Embodiment 1 ; to a patient in need thereof.

[0646] 3. Use of a chimeric oncolytic adenovirus, wherein the genome of the chimeric oncolytic adenovirus comprises:

[0647] (A) an Ad3 penton gene as defined in Embodiment 1 ; and

[0648] (B) an Ad7 hexon gene as defined in Embodiment 1 ; in the manufacture of a medicament for the prevention or treatment of cancer.

[0649] 4. A chimeric oncolytic adenovirus, method or use as defined in any one of the preceding Embodiments, wherein the genome of the chimeric oncolytic adenovirus comprises an L2 region and an L3 region, and wherein:

[0650] (a) the Ad3 penton gene is located within the L2 region; and / or

[0651] (b) the Ad7 hexon gene is located within the L3 region.

[0652] 5. A chimeric oncolytic adenovirus, method or use as defined in any one of the preceding Embodiments, wherein the genome of the chimeric oncolytic adenovirus comprises an L2 region and an L3 region, and wherein: (a) the L2 region comprises:

[0653] (i) an Ad3 penton gene as defined in Embodiment 1 ;

[0654] (ii) an Ad3 pVIII gene;

[0655] (iii) an Ad3 V gene; and

[0656] (iv) and an Ad3 or Ad7 pX gene; and / or

[0657] (b) the L3 region comprises:

[0658] (i) an Ad3 or Ad7 pVI gene;

[0659] (ii) an Ad7 hexon gene as defined in Embodiment 1 ; and

[0660] (iii) an Ad7 protease gene.

[0661] 6. A chimeric oncolytic adenovirus, method or use as defined in any one of the preceding Embodiments, wherein the genome of the chimeric oncolytic adenovirus additionally comprises an Ad7 fibre gene, wherein the Ad7 fibre gene comprises or consists of:

[0662] (a) the nucleotide sequence as sequence given in SEQ ID NO: 32;

[0663] (b) a nucleotide sequence which has at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) nucleotide sequence identity to SEQ ID NO: 32, and which encodes an adenovirus fibre polypeptide; or

[0664] (c) a nucleotide sequence which encodes an Ad7 fibre polypeptide, wherein the Ad7 fibre polypeptide is:

[0665] (i) a polypeptide whose amino acid sequence is given in SEQ ID NO: 34; or

[0666] (ii) a polypeptide having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) sequence identity to SEQ ID NO: 34, and which encodes an adenovirus fibre polypeptide; preferably wherein the genome of the chimeric oncolytic adenovirus comprises an L5 region and the Ad7 fibre gene is located within the L5 region.

[0667] 7. A chimeric oncolytic adenovirus, method or use as defined in any one of the preceding Embodiments, wherein the genome of the chimeric oncolytic adenovirus comprises a transgene, preferably wherein the transgene is located in or adjacent to the E1 region, E3 region, L3 region, L5 region or in an E1 / E3-deleted region of the genome of the chimeric oncolytic adenovirus.

[0668] 8. A chimeric oncolytic adenovirus, method or use as defined in Embodiment 7, wherein the transgene encodes an antibody, bispecific engager, checkpoint inhibitor, cytokine, chemokine, an enzyme or an angiogenesis inhibitor.

[0669] 9. A chimeric oncolytic adenovirus, method or use as defined in any one of the preceding Embodiments, wherein the cancer is selected from the group consisting of ovarian cancer, colorectal cancer, lung cancer, hepatoma, multiple myeloma, oesophageal cancer, breast cancer and pancreatic cancer, preferably ovarian cancer or a stroma-containing carcinoma.

[0670] 10. A chimeric adenovirus, wherein the genome of the chimeric adenovirus comprises:

[0671] (A) an Ad3 penton gene as defined in Embodiment 1 ;

[0672] (B) an Ad7 hexon gene as defined in Embodiment 1 ; and

[0673] (C) an Ad7 fibre gene as defined in Embodiment 6.

[0674] 11. A chimeric adenovirus as defined in Embodiment 10, wherein the genome of the chimeric adenovirus comprises an L2 region and an L3 region, and wherein:

[0675] (a) the Ad3 penton gene is located within the L2 region; and / or

[0676] (b) the Ad7 hexon gene is located within the L3 region.

[0677] 12. A chimeric adenovirus as defined in Embodiment 10 or Embodiment 11 , wherein the genome of the chimeric adenovirus comprises an L2 region and an L3 region, and wherein:

[0678] (a) the L2 region comprises:

[0679] (i) an Ad3 penton gene as defined in Embodiment 1 ;

[0680] (ii) an Ad3 pVIII gene;

[0681] (iii) an Ad3 V gene; and

[0682] (iv) and an Ad3 or Ad7 pX gene; and / or (b) the L3 region comprises:

[0683] (i) an Ad3 or Ad7 pVI gene;

[0684] (ii) an Ad7 hexon gene as defined in Embodiment 1 ; and

[0685] (iii) an Ad7 protease gene.

[0686] 13. A chimeric adenovirus as defined in any one of Embodiments 10 to 12, wherein the genome of the chimeric adenovirus comprises an L5 region and the Ad7 fibre gene is located within the L5 region.

[0687] 14. A chimeric adenovirus as defined in any one of Embodiments 10 to 13, wherein the genome of the chimeric adenovirus comprises a transgene, preferably wherein the transgene is located in or adjacent to the E1 region, E3 region, L3 region, L5 region or in an E1 / E3-deleted region of the genome of the chimeric adenovirus.

[0688] 15. A chimeric adenovirus, as defined in Embodiment 14, wherein the transgene encodes an antibody, bispecific engager, checkpoint inhibitor, cytokine, chemokine, an enzyme or an angiogenesis inhibitor.

[0689] 16. A chimeric adenovirus comprising an adenoviral capsid, wherein the adenoviral capsid comprises:

[0690] (A) an Ad3 penton polypeptide as defined in claim 1 ;

[0691] (B) an Ad7 hexon polypeptide as defined in claim 1 ; and

[0692] (C) an Ad7 fibre polypeptide as defined in claim 6.

[0693] 17. A chimeric oncolytic adenovirus, chimeric adenovirus, method or use as claimed in any one of the preceding Embodiments, wherein the adenovirus is a Group B adenovirus or a human adenovirus or an Ad3 / A37 chimeric adenovirus.

[0694] 18. An adenoviral gene therapy vector, wherein the genome of the adenoviral gene therapy vector comprises:

[0695] (a) an Ad3 penton gene as defined in Embodiment 1 ;

[0696] (b) an Ad7 hexon gene as defined in Embodiment 1 ; and (c) a transgene.

[0697] 19. An adenoviral gene therapy vector as defined in Embodiment 17, wherein the genome of the adenoviral gene therapy vector comprises an L2 region and an L3 region, and wherein:

[0698] (a) the Ad3 penton gene is located within the L2 region; and / or

[0699] (b) the Ad7 hexon gene is located within the L3 region.

[0700] 20. An adenoviral gene therapy vector as defined in Embodiment 18 or Embodiment

[0701] 19, wherein the genome of the adenoviral gene therapy vector comprises an L2 region and an L3 region, and wherein:

[0702] (a) the L2 region comprises:

[0703] (i) an Ad3 penton gene as defined in Embodiment 1 ;

[0704] (ii) an Ad3 pVIII gene;

[0705] (iii) an Ad3 V gene; and

[0706] (iv) and an Ad3 or Ad7 pX gene; and / or

[0707] (b) the L3 region comprises:

[0708] (i) an Ad3 or Ad7 pVI gene;

[0709] (ii) an Ad7 hexon gene as defined in Embodiment 1 ; and

[0710] (iii) an Ad7 protease gene.

[0711] 21. An adenoviral gene therapy vector as defined in any one of Embodiments 18 to

[0712] 20, wherein the genome of the adenoviral gene therapy vector additionally comprises an Ad7 fibre gene as defined in Embodiment 6; preferably wherein the genome of the adenoviral gene therapy vector comprises an L5 region and the Ad7 fibre gene is located within the L5 region.

[0713] 22. An adenoviral gene therapy vector as defined in any one of Embodiments 18 to

[0714] 21 , wherein the transgene is located in or adjacent to the E1 region, E3 region, L3 region, L5 region or in an E1 / E3-deleted region of the genome of the adenoviral gene therapy vector. 23. An adenoviral gene therapy vector as defined in Embodiment 22, wherein the transgene encodes an antibody, bispecific engager, checkpoint inhibitor, cytokine, chemokine, an enzyme or an angiogenesis inhibitor.

[0715] 24. An adenoviral gene therapy vector as claimed in any one of claims 18-23, wherein the adenoviral gene therapy vector is obtained from or derived from a Group B or a human adenovirus or an Ad3 / Ad7 chimeric adenovirus.

[0716] 25. A pharmaceutical composition comprising a chimeric adenovirus as defined in any one of Embodiments 10 to 17 or an adenoviral gene therapy vector as defined in any one of Embodiments 18 to 24, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.

[0717] 26. A pharmaceutical combination comprising:

[0718] (A) a first pharmaceutical composition comprising a chimeric adenovirus as defined in any one of Embodiments 10 to 17 or an adenoviral gene therapy vector as defined in any one of Embodiments 18 to 24; and

[0719] (B) a second pharmaceutical composition comprising a chemotherapeutic or immunotherapeutic agent, wherein the pharmaceutical combination is in the form of a combined preparation for simultaneous, separate or sequential use, preferably for the treatment of cancer.

[0720] 27. A chimeric adenovirus as defined in any one of Embodiments 10 to 17 or an adenoviral gene therapy vector as defined in any one of Embodiments 18 to 24 for use in therapy or for use as a medicament.

[0721] 28. Use of a chimeric adenovirus as defined in any one of Embodiments 10 to 17 as a vector for protein production, wherein the chimeric adenovirus comprises a transgene encoding the protein to be produced. SECOND ASPECT OF THE INVENTION

[0722] Preferred Embodiments of the second aspect of the invention include the following:

[0723] 1 . An oncolytic adenovirus, having a genome comprising an E3 region:

[0724] (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

[0725] (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.

[0726] 2. An oncolytic adenovirus as defined in Embodiment 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.

[0727] 3. An oncolytic adenovirus as defined in Embodiment 1 or Embodiment 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.

[0728] 4. An oncolytic adenovirus as defined in any one of the preceding Embodiments, 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.

[0729] 5. An oncolytic adenovirus as defined in Embodiment 1 , wherein the E3 region comprises or consists of:

[0730] (a) a Group B adenovirus E3 12.1 K ORF;

[0731] (b) a 3-truncated Group B adenovirus E3 16.1 K ORF;

[0732] (c) a 5’-truncated Group B adenovirus E3 10.3K ORF;

[0733] (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.

[0734] 6. An oncolytic adenovirus as defined in Embodiment 1 , wherein the E3 region comprises or consists of:

[0735] (a) a Group B adenovirus E3 12.1 K ORF;

[0736] (b) a Group B adenovirus E3 16.1 K ORF;

[0737] (c) a 3-truncated Group B adenovirus E3 19.3K ORF;

[0738] (d) a 5’-truncated Group B adenovirus E3 14.9K ORF; and

[0739] (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.

[0740] 7. An oncolytic adenovirus as defined in Embodiment 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.

[0741] 8. An oncolytic adenovirus, as defined in Embodiment 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 defined in Embodiment 1 , wherein the E3 region has a deletion in the E3 region compared to the corresponding region of a wild-type Group B adenovirus:

[0742] (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: 35, or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region; and

[0743] (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: 35, or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region.

[0744] 10. An oncolytic adenovirus as defined in Embodiment 1 , wherein the E3 region has a deletion in the E3 region compared to the corresponding region of a wild-type Group B adenovirus:

[0745] (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: 35, or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region; and

[0746] (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: 35, or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region.

[0747] 11. An oncolytic adenovirus as defined in Embodiment 1 , wherein the E3 region has a deletion compared to the wild-type Group B adenovirus, wherein the deletion corresponds to:

[0748] (a) Ad7 genome nucleotides 28,011 - 30,274; or

[0749] (b) Ad7 genome nucleotides 28,482 - 30,665. - I l l -

[0750] 12. An oncolytic adenovirus as claimed in any one of the preceding Embodiments, wherein the adenovirus is a Group B adenovirus, preferably an Ad3 or Ad7 serotype.

[0751] 13. An oncolytic adenovirus as defined in any one of the preceding Embodiments, wherein the adenovirus additionally comprises a transgene, preferably a transgene which encodes an antibody, bispecific engager, checkpoint inhibitor, cytokine, chemokine or enzyme.

[0752] 14. An oncolytic adenovirus as defined in Embodiment 13, wherein the transgene is located in or adjacent to the E1 region, E3 region, L3 region or L5 region of the adenovirus.

[0753] 15. A pharmaceutical composition comprising an oncolytic adenovirus as defined in any one of the preceding Embodiments, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.

[0754] 16. An oncolytic adenovirus as defined in any one of Embodiments 1 to 14 or a pharmaceutical composition as defined in Embodiment 15 for use in therapy or for use as a medicament.

[0755] 17. An oncolytic adenovirus as defined in any one of Embodiments 1 to 14 or a pharmaceutical composition as defined in Embodiment 15 for use in treating cancer (preferably ovarian cancer).

[0756] 18. A method of treating cancer (preferably ovarian cancer) in a subject, the method comprising administering an effective amount of an oncolytic adenovirus as defined in any one of Embodiments 1 to 14 or a pharmaceutical composition as defined in Embodiment 15 to a subject in need thereof.

[0757] 19. Use of an oncolytic adenovirus as defined in any one of Embodiments 1 to 14 in the manufacture of a medicament for treating cancer (preferably ovarian cancer). 20. An oncolytic adenovirus for use as defined in Embodiment 17, a method as defined in Embodiment 18 or a use as defined in Embodiment 19, wherein the cancer is a stroma-containing tumour.

[0758] 21 . An oncolytic adenovirus for use as defined in Embodiment 17, a method as defined in Embodiment 18 or a use as defined in Embodiment 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.

[0759] THIRD ASPECT OF THE INVENTION

[0760] Preferred Embodiments of the third aspect of the invention include the following:

[0761] 1 . A pharmaceutical composition comprising an adenovirus, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents, wherein the genome of the adenovirus comprises an E2B DNA pol I gene encoding a DNA pol I polypeptide, and wherein the amino acid in the DNA pol I polypeptide sequence corresponding to amino acid 34 in SEQ ID NO: 2 is Asp or Glu, preferably Asp.

[0762] 2. A pharmaceutical composition as claimed in Embodiment 1 , wherein the genome of the adenovirus additionally comprises an E2B pTP gene encoding a pre-Terminal Protein, wherein the coding region of the E2B DNA pol I gene overlaps with the coding region of the E2B pTP gene, and wherein the amino acid in the pTP sequence at the position corresponding to amino acid 623 in SEQ ID NO: 4 is selected from the group consisting of Thr, Met, Lys and Arg, preferably Thr.

[0763] 3. A pharmaceutical composition as claimed in Embodiment 1 , wherein the nucleotide in the E2B DNA pol I gene at the position corresponding to nucleotide 5,326 in SEQ ID NO: 1 is A. 4. A pharmaceutical composition comprising an adenovirus, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents , wherein the genome of the adenovirus comprises an E2B pTP gene encoding a pre-Terminal Protein, and wherein the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 4 is selected from the group consisting of Thr, Asn, Cys, Gin and Ser, preferably Thr.

[0764] 5. A pharmaceutical composition as claimed in Embodiment 4, wherein if the adenovirus is an Ad1 adenovirus, then the amino acid is not Asn, if the adenovirus is an Ad41 adenovirus, then the amino acid is not Gin; or if the adenovirus is an Ad4 or 4a adenovirus, then the amino acid is not Thr.

[0765] 6. A pharmaceutical composition as claimed in Embodiment 4 or Embodiment 5, wherein the genome of the adenovirus additionally comprises an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the coding region of the E2B pTP gene overlaps with the coding region of the E2B DNA pol I gene, and wherein the amino acids in the DNA pol I polypeptide at the positions corresponding to amino acids 34-35 in SEQ ID NO: 2 are selected from the group consisting of Asp-Ser, Asp-Pro, Asp-Thr, Asp-Ala, Glu-Ser, Glu-Pro, Vai-Ser, Val-Pro, Ala-Thr, Ala-Ala, Val-Thr and Val-Ala.

[0766] 7. A pharmaceutical composition as claimed in Embodiment 4 or Embodiment 5, wherein the nucleotide in the E2B pTP gene at the position corresponding to nucleotide 5,326 in SEQ ID NO: 3 is A.

[0767] 8. A pharmaceutical composition as claimed in any one of the preceding Embodiments, wherein the adenovirus is a conditionally-replicating adenovirus or an oncolytic adenovirus.

[0768] 9. A pharmaceutical composition as claimed in any one of the preceding Embodiments, wherein the adenovirus is a Group B adenovirus or a human adenovirus, preferably a Group B1 adenovirus. 10. A pharmaceutical composition as claimed in Embodiment 9, wherein the adenovirus is an Ad3 or Ad7 adenovirus or an Ad3 / Ad7 chimeric adenovirus.

[0769] 11. A pharmaceutical composition as claimed in any one of Embodiment 1 to 3 or 6 to 10, wherein the E2B DNA pol I gene comprises or consists of:

[0770] (i) the nucleotide sequence as given in SEQ ID NO: 1 ;

[0771] (ii) a nucleotide sequence which has at least 80%, 85%, 90%, 95% or 99% nucleotide sequence identity to SEQ ID NO: 1 , and which preferably encodes a DNA polymerase I; or

[0772] (iii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 2.

[0773] 12. A pharmaceutical composition as claimed in any one of Embodiments 1 to 3 or 6 to 11 , wherein the E2B DNA pol I polypeptide comprises or consists of:

[0774] (i) the amino acid sequence as given in SEQ ID NO: 2; or

[0775] (ii) a variant of (i) having at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity or sequence similarity to (i), and which preferably has DNA polymerase I activity.

[0776] 13. A pharmaceutical composition as claimed in any one of Embodiments 2 to 12, wherein the E2B pTP gene comprises or consists of:

[0777] (i) the nucleotide sequence as given in SEQ ID NO: 3;

[0778] (ii) a nucleotide sequence which has at least 80%, 85%, 90%, 95% or 99% nucleotide sequence identity to SEQ ID NO: 3, and which preferably encodes a pre-Terminal Protein; or

[0779] (iii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 4.

[0780] 14. A pharmaceutical composition as claimed in any one of Embodiments 2 to 13, wherein the E2B pre-Terminal Protein is a polypeptide comprising or consisting of: (i) the amino acid sequence as given in SEQ ID NO: 4; or (ii) a variant of (i) having at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity or sequence similarity to (i), and which preferably encodes a pre-Terminal Protein.

[0781] 15. A pharmaceutical composition as claimed in any one of the preceding Embodiments, wherein the adenovirus comprises a transgene, preferably wherein the transgene is located in or adjacent to the E1 region, E3 region, L3 region, L5 region or in an E1 / E3-deleted region of the adenovirus.

[0782] 16. A pharmaceutical combination comprising:

[0783] (A) a first pharmaceutical composition as claimed in any one of Embodiments 1 to 15; and

[0784] (B) a second pharmaceutical composition comprising a chemotherapeutic or immunotherapeutic agent, wherein the pharmaceutical combination is in the form of a combined preparation for simultaneous, separate or sequential use, preferably for the treatment of cancer.

[0785] 17. An adenovirus as defined in any one of Embodiments 1 to 15 or a pharmaceutical composition as claimed in any one of Embodiments 1 to 15 for use in therapy or for use as a medicament.

[0786] 18. An adenovirus as defined in any one of Embodiments 1 to 15 or a pharmaceutical composition as claimed in any one of Embodiments 1 to 15 for use in treating cancer (preferably ovarian cancer).

[0787] 19. A method of treating cancer (preferably ovarian cancer) in a subject, the method comprising administering an effective amount of an adenovirus as defined in any one of Embodiments 1 to 15 or a pharmaceutical composition as claimed in any one of Embodiments 1 to 15 to a subject in need thereof. 20. Use of an adenovirus as defined in any one of Embodiments 1 to 15 in the manufacture of a medicament for treating cancer (preferably ovarian cancer).

[0788] 21 . An adenovirus for use as claimed in Embodiment 18, a method as claimed in Embodiment 19 or a use as claimed in Embodiment 20, wherein the cancer is selected from the group consisting of ovarian cancer, colorectal cancer, lung cancer, hepatoma, multiple myeloma, oesophageal cancer, breast cancer and pancreatic cancer, preferably ovarian cancer or a stroma-containing carcinoma.

[0789] 22. Use of a conditionally-replicating adenovirus as defined in any one of Embodiments 1 to 15 as a vector for protein production, wherein the adenovirus comprises a transgene encoding the protein to be produced.

[0790] 23. Use of a conditionally-replicating adenovirus as defined in any one of Embodiments 1 to 15 as a helper virus for virus production.

[0791] 24. A mutant Group B or human adenovirus E2B DNA pol I gene, wherein the nucleotide in the mutant E2B DNA pol I gene at the position corresponding to nucleotide 101 in SEQ ID NO: 1 is A.

[0792] 25. A mutant Group B or human adenovirus E2B pTP gene, wherein the nucleotide in the mutant E2B pTP gene at the position corresponding to nucleotide 5,326 in SEQ ID NO: 3 is a G.

[0793] 26. A mutant Group B or human adenovirus E2B DNA pol I polypeptide, wherein the amino acid in the mutant E2B DNA pol I polypeptide at the position corresponding to amino acid 34 in SEQ ID NO: 2 is D or E, preferably D.

[0794] 27. A mutant Group B or human adenovirus E2B pTP polypeptide, wherein the amino acid in the mutant E2B pTP polypeptide at the position corresponding to amino acid 623 in SEQ ID NO: 2 is T.

Claims

CLAIMS1 . An adenovirus, wherein the genome of the adenovirus comprises one, two or all of (A), (B) and (C):(A) an Ad3 penton gene, wherein the Ad3 penton gene comprises or consists of:(a) the nucleotide sequence as given in SEQ ID NO: 3;(b) a nucleotide sequence which has at least 99% nucleotide sequence identity to SEQ ID NO: 3, and which encodes an adenovirus penton polypeptide; or(c) a nucleotide sequence which encodes an Ad3 penton polypeptide, wherein the Ad3 penton polypeptide is:(i) a polypeptide whose amino acid sequence is given in SEQ ID NO: 5;(ii) a polypeptide having at least 99.3%, 99.5% or 99.7% sequence identity to SEQ ID NO: 5, the polypeptide encoding an adenovirus penton polypeptide; or(iii) a polypeptide having at least 95%, 99% or 99.5% sequence identity to SEQ ID NO: 5, wherein the amino acids in the penton polypeptide at the positions which correspond to positions 11 , 158, 178 and 326 in SEQ ID NO: 5 are V, T, I and D, respectively, the polypeptide encoding an adenovirus penton polypeptide; and an Ad7 hexon gene, wherein the Ad7 hexon gene comprises or consists of:(a) the nucleotide sequence as sequence given in SEQ ID NO: 24;(b) a nucleotide sequence which has at least 97% or at least 99% nucleotide sequence identity to SEQ ID NO: 24, and which encodes an adenovirus hexon polypeptide; or(c) a nucleotide sequence which encodes an Ad7 hexon polypeptide, wherein the Ad7 hexon polypeptide is:(i) a polypeptide whose amino acid sequence is given in SEQ ID NO: 26; or(ii) a polypeptide having at least 97%, 98% or 99% sequence identity to SEQ ID NO: 26, and which encodes an adenovirus hexon polypeptide. and optionally an Ad7 fibre gene;(B) 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; and(C) an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the amino acid in the DNA pol I polypeptide sequence corresponding to amino acid 34 in SEQ ID NO: 49 is Asp or Glu, preferably Asp; or an E2B pTP gene encoding a pre-Terminal Protein, wherein the amino acid in the pre-Terminal Protein sequence corresponding to amino acid 623 in SEQ ID NO: 51 is selected from the group consisting of Thr, Asn, Cys, Gin and Ser, preferably Thr,2. An adenovirus as claimed in claim 1 , wherein the genome of the adenovirus comprises (A), (B) or (C).

3. An adenovirus as claimed in claim 1 , wherein the genome of the adenovirus comprises (A) and (B); (A) and (C); or (B) and (C).

4. An adenovirus as claimed in claim 1 , wherein the genome of the adenovirus comprises (A), (B) and (C).

5. An adenovirus as claimed in any one of the preceding claims, wherein the genome of the adenovirus comprises an L2 region and an L3 region, and wherein:(a) the Ad3 penton gene is located within the L2 region; and / or(b) the Ad7 hexon gene is located within the L3 region.

6. An adenovirus as claimed in any one of the preceding claims, wherein the genome of the adenovirus comprises an L2 region and an L3 region, and wherein:(a) the L2 region comprises:(i) an Ad3 penton gene as defined in claim 1 ;(ii) an Ad3 pVIII gene;(iii) an Ad3 V gene; and(iv) and an Ad3 or Ad7 pX gene; and / or(b) the L3 region comprises:(i) an Ad3 or Ad7 pVI gene;(ii) an Ad7 hexon gene as defined in claim 1 ; and(iii) an Ad7 protease gene.

7. An adenovirus as claimed in any one of the preceding claims, wherein the genome of the adenovirus additionally comprises an Ad7 fibre gene, wherein the Ad7 fibre gene comprises or consists of:(a) the nucleotide sequence as sequence given in SEQ ID NO: 32;(b) a nucleotide sequence which has at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) nucleotide sequence identity to SEQ ID NO: 32, and which encodes an adenovirus fibre polypeptide; or(c) a nucleotide sequence which encodes an Ad7 fibre polypeptide, wherein the Ad7 fibre polypeptide is:(i) a polypeptide whose amino acid sequence is given in SEQ ID NO: 34; or(ii) a polypeptide having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) sequence identity to SEQ ID NO: 34, and which encodes an adenovirus fibre polypeptide; preferably wherein the genome of the adenovirus comprises an L5 region and the Ad7 fibre gene is located within the L5 region.

8. An adenovirus as claimed in any one of the preceding claims, wherein the E3 region additionally comprises a functional (optionally 3’-truncated) 16.1 K ORF from aGroup B adenovirus E3 region or an ORF corresponding thereto from a non-Group B adenovirus E3 region.

9. An adenovirus as claimed in any one of the preceding claims, 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.

10. An 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.

11. An adenovirus as claimed in any one of the preceding claims, 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.

12. An adenovirus as claimed in any one of the preceding claims, 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.

13. An adenovirus as claimed in any one of the preceding claims, 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.

14. An adenovirus as claimed in any one of the preceding claims, 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.

15. An adenovirus as claimed in any one of the preceding claims, wherein the E3 region has a deletion in the E3 region compared to the corresponding region of a wildtype 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: 35, 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: 35, or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region.

16. An adenovirus as claimed in any one of the preceding claims, wherein the E3 region has a deletion in the E3 region compared to the corresponding region of a wildtype 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: 35, 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: 35, or at the corresponding nucleotide(s) in the nucleotide sequence of a non-Group B adenovirus E3 region.

17. An adenovirus as claimed in any one of the preceding claims, 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.

18. An adenovirus as claimed in any one of the preceding claims, wherein the genome of the adenovirus comprises an E2B pTP gene encoding a pre-Terminal Protein, wherein the coding region of the E2B DNA pol I gene overlaps with the coding region of the E2B pTP gene, and wherein the amino acid in the pTP sequence at the position corresponding to amino acid 623 in SEQ ID NO: 51 is selected from the group consisting of Thr, Met, Lys and Arg, preferably Thr.

19. An adenovirus as claimed in any one of the preceding claims, wherein the nucleotide in the E2B DNA pol I gene at the position corresponding to nucleotide 5,326 in SEQ ID NO: 48 is A.

20. An adenovirus as claimed in any one of the preceding claims, wherein the genome of the adenovirus comprises an E2B DNA pol I gene encoding a DNA pol I polypeptide, wherein the coding region of the E2B pTP gene overlaps with the codingregion of the E2B DNA pol I gene, and wherein the amino acids in the DNA pol I polypeptide at the positions corresponding to amino acids 34-35 in SEQ ID NO: 49 are selected from the group consisting of Asp-Ser, Asp-Pro, Asp-Thr, Asp-Ala, Glu-Ser, Glu-Pro, Vai-Ser, Val-Pro, Ala-Thr, Ala-Ala, Val-Thr and Val-Ala.21 . An adenovirus as claimed in any one of the preceding claims, wherein the nucleotide in the E2B pTP gene at the position corresponding to nucleotide 5,326 in SEQ ID NO: 50 is A.

22. An adenovirus as claimed in any one of the preceding claims, wherein the adenovirus is a conditionally-replicating adenovirus or an oncolytic adenovirus.

23. An adenovirus as claimed in any one of the preceding claims, wherein the E2B DNA pol I gene comprises or consists of:(i) the nucleotide sequence as given in SEQ ID NO: 48;(ii) a nucleotide sequence which has at least 80%, 85%, 90%, 95% or 99% nucleotide sequence identity to SEQ ID NO: 48, and which preferably encodes a DNA polymerase I; or(iii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 49.

24. An adenovirus as claimed in any one of the preceding claims, wherein the E2B DNA pol I polypeptide comprises or consists of:(i) the amino acid sequence as given in SEQ ID NO: 49; or(ii) a variant of (i) having at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity or sequence similarity to (i), and which preferably has DNA polymerase I activity.

25. An adenovirus as claimed in any one of the preceding claims, wherein the E2B pTP gene comprises or consists of:(i) the nucleotide sequence as given in SEQ ID NO: 50;(ii) a nucleotide sequence which has at least 80%, 85%, 90%, 95% or 99% nucleotide sequence identity to SEQ ID NO: 50, and which preferably encodes a pre-Terminal Protein; or(iii) a nucleotide sequence which encodes the polypeptide of SEQ ID NO: 51.

26. An adenovirus as claimed in any one of the preceding claims, wherein the E2B pre-Terminal Protein is a polypeptide comprising or consisting of:(i) the amino acid sequence as given in SEQ ID NO: 51 ; or(ii) a variant of (i) having at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity or sequence similarity to (i), and which preferably encodes a pre-Terminal Protein.

27. An adenovirus as claimed in any one of the preceding claims, wherein the adenovirus is a Group B adenovirus or a human adenovirus, preferably a Group B1 adenovirus, an Ad3 or Ad7 adenovirus, or an Ad3 / Ad7 chimeric adenovirus.

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

29. An adenovirus as claimed in claim 28, wherein the transgene encodes an antibody, bispecific engager, checkpoint inhibitor, cytokine, chemokine, an enzyme or an angiogenesis inhibitor.

30. An adenovirus comprising the nucleotide sequence as shown in SEQ ID NO: 58 or a variant thereof having at least 93% (preferably at least 94%, 95%, 96%, 97%, 98%, or 99% or 99.5%) sequence identity thereto and having oncolytic activity.31 . An adenovirus or adenoviral vector whose genome has 100% nucleotide sequence identity with nucleotides 1-479, 3,410-27,380 and 28,869-33,044 of SEQ IDNO: 58; preferably wherein the genome of the adenovirus or adenoviral vector additionally comprises a transgene; more preferably, wherein the transgene is located between nucleotides 480-3,409 or 27,381 -28,868 of SEQ ID NO: 58.

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

33. A pharmaceutical combination comprising:(A) a first pharmaceutical composition comprising an adenovirus or adenoviral vector as claimed in any one of claims 1 to 31 ; and(B) a second pharmaceutical composition comprising a chemotherapeutic or immunotherapeutic agent, wherein the pharmaceutical combination is in the form of a combined preparation for simultaneous, separate or sequential use, preferably for the treatment of cancer.

34. An adenovirus or adenoviral vector as claimed in any one of claims 1 to 31 or a pharmaceutical composition as claimed in claim 32 for use in therapy or for use as a medicament.

35. An adenovirus or adenoviral vector as claimed in any one of claims 1 to 31 or a pharmaceutical composition as claimed in claim 32 for use in treating cancer (preferably ovarian cancer).

36. A method of treating cancer (preferably ovarian cancer) in a subject, the method comprising administering an effective amount of an adenovirus or adenoviral vector as claimed in any one of claims 1 to 31 or a pharmaceutical composition as claimed in claim 32 to a subject in need thereof.

37. Use of an adenovirus or adenoviral vector as claimed in any one of claims 1 to 31 in the manufacture of a medicament for treating cancer (preferably ovarian cancer).

38. An adenovirus for use as claimed in claim 35, a method as claimed in claim 36 or a use as claimed in claim 37, wherein the cancer is selected from the group consisting of ovarian cancer, colorectal cancer, lung cancer, hepatoma, multiple myeloma, oesophageal cancer, breast cancer and pancreatic cancer, preferably ovarian cancer or a stroma-containing carcinoma.

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