Adenovirus

Abstract

The present invention relates to adenoviruses, in particular oncolytic adenoviruses, for use in the prevention or treatment of cancer, including interstitial-containing tumors and ovarian cancers. In some embodiments, the present invention relates to adenoviruses having a chimeric L2-L3 region as compared to a wild-type adenovirus. In other embodiments, the invention relates to adenoviruses having a deletion in the E3 region as compared to a wild-type adenovirus. In other embodiments, the present invention relates to adenoviruses having mutations in the coding sequence of a DNA polymerase I polypeptide and / or a pTP (proterminal protein) polypeptide. In other embodiments, the present invention relates to adenoviruses having the aforementioned chimeric L2-L3 region, deletions, and mutations at the same time. In other embodiments, the present invention relates to an adenovirus comprising the nucleotide sequence as shown in SEQ ID NO: 58, or a variant having at least 93% sequence identity thereto and having oncolytic activity.

Description

[0001] This invention relates to adenoviruses, particularly oncolytic adenoviruses, for the prevention or treatment of cancer, including stromal tumors and ovarian cancer. In some embodiments, this invention relates to adenoviruses having a chimeric L2-L3 region compared to wild-type adenoviruses. In other embodiments, this invention relates to adenoviruses with a deletion in the E3 region compared to wild-type adenoviruses. In other embodiments, this invention relates to adenoviruses with mutations in the coding sequences of DNA polymerase I polypeptide and / or pTP (pre-terminal protein) polypeptide. In other embodiments, this invention relates to adenoviruses simultaneously possessing the aforementioned chimeric L2-L3 region, deletion, and mutation. In other embodiments, this invention relates to an adenovirus comprising the nucleotide sequence shown in SEQ ID NO: 58, or a variant having at least 93% sequence identity with it and possessing oncolytic activity.

[0002] Cancer is a malignant tumor originating from epithelial tissues that cover the outer or inner surfaces of organs, including the lungs, gastrointestinal tract, and reproductive tissues. Cancer accounts for approximately 90% of all cancer cases worldwide and is the leading cause of cancer death. A common characteristic of cancers is that they are often diagnosed as palpable lesions due to the presence and / or presence of extracellular matrix, fibroblasts, and immune cells around and / or within cancer cell islands. All cells and material in a tumor that are not defined as cancer cells (or malignant cells) are generally referred to as the "stroma," or more specifically, the "tumor stroma." Some cells in the immune system (such as macrophages and fibroblasts) are often classified as part of the stroma, while others (such as lymphocytes) are described as infiltrating or permeating it. All visible and palpable cancers contain some degree of stroma; the stroma provides the necessary structural support, nutrient supply, and suitable environment for the survival, proliferation, and even spread of cancer cells.

[0003] The proportion of stroma can vary between 5% and 80%, depending to some extent on the type of underlying lesion. However, anyone familiar with tumor structure understands that the greatest variation in stroma content for a given biopsy sample stems from the biopsy location, as certain areas of the tumor visually appear to have more stroma than others.

[0004] Stromal cells superficially resemble normal tissues and organs. They are responsible for remodeling the extracellular matrix, inducing angiogenesis, and providing structural support. However, unlike normal tissues, these cells behave uncontrolled and often chaotically. Excessive stroma leads to increased tissue pressure, impaired blood flow, and local hypoxia. Therefore, cancer cells face spatiotemporal variability in nutrient and oxygen supply. As tumors grow, significant cell death and necrosis occur due to the cyclical fluctuations in local nutrient levels. While this fluctuating shortage of nutrients and oxygen may seem detrimental to tumor growth, increasing research suggests that this environment actually drives stronger clonal evolution, leading to drug resistance.

[0005] In recent years, the role of mesenchymal tissue in protecting cancer cells from drug or immune attacks has received increasing attention. In particular, cancer-associated fibroblasts (CAFs) have been shown to prevent T cells from entering tumor tissue or inhibit the activity of already infiltrated T cells by expressing cytokines such as transforming growth factor β (TGFβ).

[0006] The presence of cellular fibroblasts (CAFs) and their effects on immune cells are often considered the root cause of poor responses to immunotherapy. However, the development of new immunotherapies for cancer patients has been hampered by the lack of animal models containing sufficient amounts of mesenchymal cells or the correct tissue structure. Once CAFs are artificially introduced into animal models, the efficacy of cancer vaccines and immune checkpoint inhibitor therapies is significantly reduced or even completely lost.

[0007] Despite a lack of supporting models, the development of drugs targeting the mesenchyme has garnered increasing attention. These drugs either act directly on the mesenchymal cells themselves or target the microenvironment created by the mesenchyme. However, mesenchymal-targeting strategies typically face two fundamental challenges. First, selectivity is extremely challenging because the cells that make up the stroma are essentially "normal"—they are not malignant and are equally common in other parts of the body. Second, a conceptual challenge lies in the necessity of combination therapy, as targeting the mesenchyme alone is almost impossible to achieve complete efficacy. For refractory cancers, if single-targeting approaches are ineffective, simultaneous targeting of both the mesenchyme and cancer cells may become necessary. While the theoretical basis for combination therapy is strong, developing combination drugs remains extremely difficult, especially for those drugs that are ineffective or potentially unresponsive to monotherapy.

[0008] Ideal cancer treatment interventions should be able to target cancer cells and stromal cell populations simultaneously and selectively.

[0009] Oncolytic viruses, as an emerging therapeutic approach, can kill various types of tumor cells, including differentiated cancer cells and cancer cell initiation cells or stem cells. They can be highly selective, while utilizing "cancer markers" such as immunity or cell cycle dysregulation to proliferate and lyse cells.

[0010] The main drawback of oncolytic viruses is that, to date, no oncolytic virus has been specifically designed or developed for targeting mesenchymal cells. This is because contemporary oncolytic viruses are only constructed to be active in malignant cells (by definition).

[0011] In human tumors, stromal cells divide and surround various areas of cancer cells, creating a physical barrier (or rather, conceptual barrier) for oncolytic virus therapy. Therefore, successful laboratory treatments rarely translate effectively into clinical applications. In cases where clinical efficacy has been achieved against solid tumors, multiple injections of oncolytic viruses are typically used to overcome the interstitial barrier (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. PMID: 10932224). Alternatively, very small or early-stage cancers with relatively low interstitial content can be targeted (e.g., Packiam VT, et al. “An open label, single-arm, phase II multicenter study of the safety and efficacy of CG0070oncolytic vector regimen in patients with BCG-unresponsive non-muscle-invasive bladder cancer: Interim results.” Urol Oncol. 2018 Oct;36(10):440-447. doi: 10.1016 / j.urolonc.2017.07.005. Epub 2017 Jul 26. PMID: 28755959).

[0012] To address the problem of stromal cells, some oncolytic viruses have been modified to express biological agents, including bispecific T-cell connectives (BiTEs), to selectively kill stromal cells. The design concept is that the oncolytic virus is responsible for clearing cancer cells, while the BiTEs it expresses target stromal cells, thereby achieving complete tumor lysis.

[0013] Unfortunately, this strategy remains fundamentally insufficient due to the structural and spatial arrangement characteristics of the tumor stroma. In human tumors, the capillaries supplying cancer cells emerge from the stroma, so drugs, including oncolytic viruses, must traverse multiple layers of stromal cells to reach the cancer cells. For small molecule drugs, diffusion makes this process relatively easy; however, oncolytic viruses are usually too large to easily penetrate the intercellular spaces. When oncolytic viruses enter human tumors, they are first exposed to stromal cells, where they cannot replicate or express transgenic products such as BiTE.

[0014] This deficiency is not apparent in animal models because these models typically lack the stroma; even when the stroma is present, its structure differs, with capillaries appearing directly within cancer cells.

[0015] For oncolytic agents to be effective in treating human diseases, they must be activated immediately upon entering the tumor microenvironment. This mechanism is possible because the three hallmark features of cancer that oncolytic viruses rely on to kill cancer cells also apply to cancer cells in cancer cells (CCF). These features include metabolic dysregulation, anti-apoptosis, and immune dysfunction. Therefore, the ideal strategy is to develop an oncolytic drug that can simultaneously utilize the shared features of both cancer cells and stromal cells, making it active against both types of cells.

[0016] Ovarian cancer is one of the most common and deadliest cancers in women. It is often diagnosed at an advanced stage, by which time the cancer cells have spread to other parts of the body. Despite the availability of various treatments, the best survival rate for stage III and higher ovarian cancer remains only 25%. Most ovarian cancer patients are diagnosed at the most advanced type—high-grade serous ovarian cancer (HGSOC), which accounts for 70-80% of deaths. Current treatment options involve repeated platinum-based therapy until resistance and recurrence become inevitable. Second-line treatments include paclitaxel and doxorubicin, but their efficacy is limited. Only a small percentage of patients with BRCA1 mutations (13-15%) can use newly approved PARP inhibitors. Recently, mirvetuximab received accelerated approval for the treatment of patients with high folate receptor expression. While these newer drugs offer some efficacy in the intermediate stages, most patients are still expected to experience cancer recurrence.

[0017] The potential role of oncolytic viruses in the treatment of ovarian cancer has been explored. The mechanism is that oncolytic viruses can kill heterogeneous cell populations, including cancer stem cells, while stimulating the body to produce an immune response. Multiple clinical trials have confirmed that the delivery of oncolytic viruses to ovarian cancer patients is safe and feasible, but the overall efficacy is still limited. In fact, it is the CAF in the stroma that weakens the effect of oncolytic therapy through antiviral signaling pathways (Arwert EN, et al. “STING and IRF3 instromal fibroblasts enable sensing of genomic stress in cancer cells toundermine 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).

[0018] Given the crucial role of CAF in promoting ovarian cancer progression and providing resistance mechanisms to chemotherapy, immunotherapy, and oncolytic virus therapy, there is an urgent need to develop new therapies that can simultaneously target CAF and cancer cells.

[0019] Cancer-associated cells (CAFs) play a crucial role in determining patient prognosis and treatment response. They exert these effects by suppressing immune cells, hindering effector T cell infiltration, forming dense barriers to prevent the spread of therapeutic drugs, and inducing resistance to standard-of-care (SOC) drugs. Furthermore, CAFs can promote tumor growth and provide nutritional support, aiding in tumor proliferation and metastasis. Despite the critical impact of CAFs on patient treatment response, existing preclinical models, including more complex in vivo PDX models, fail to represent this key component of the human tumor microenvironment (TME). Traditional cancer cell lines and in vivo xenograft tumor models cannot replicate the complexity and heterogeneity of human cancers, and cannot accurately capture the stromal components, immunosuppressive mechanisms, metabolic disorders, cytokines, and stress proteins (such as TNF-α and TGF-β) present in the patient's tumor microenvironment. Therefore, developing therapies based on realistic simulation systems (such as freshly excised patient biopsy tissue, multicellular, nutrient-deprived, and hypoxic tumor microenvironments) is crucial for identifying drugs that remain effective under these barriers and stress conditions.

[0020] The applicant observed that the tumor microenvironment is drastically different from the environment in which viruses naturally evolve: viruses typically reside in airway cells with a sustainable supply of nutrients and an excess of oxygen. The tumor microenvironment differs significantly from nutrient-rich cell culture systems or rapidly growing animal model environments. Therefore, wild-type viruses, or viruses screened through simple animal models, are often unsuitable for treating human diseases. It follows that any virus optimized for the tumor environment is likely to differ significantly from wild-type or engineered viruses. To survive in the tumor environment, viruses must regulate their demand for various resources, otherwise they risk disrupting their replication cycle. Mutations (even subtle ones) enable this regulation because the viral mechanism—the synthesis of RNA, DNA, and proteins—is highly "gear-like." Therefore, even a small change in a single virus can become significant when the virus replicates to form 100,000 offspring.

[0021] From the initial mixed library of adenoviruses (groups B, C, D, F, and G), 23 rounds of biological screening (including mutation steps) were conducted to enrich oncolytic adenovirus candidate strains that exhibited optimal tumor lysis, spread, hematopoietic stability, and immunostimulatory activity. Finally, 60 adenovirus strains were selected from the library for sequencing and genomic analysis.

[0022] Genome analysis of the selected adenoviruses showed that many of their genome features were common to most or all candidate adenoviruses.

[0023] Specifically, all candidate adenovirus genomes were derived from group B adenoviruses and were chimeric: the 5' end of the genome came from Ad3 serotype virus, and the 3' end from Ad7 serotype virus. In all the adenoviruses screened, the chimeric junction was located within the L2-L3 region.

[0024] One structural consequence of this chimerism is that in the adenovirus involved in this invention, the pentagonal basal protein (encoded by the L2 gene) originates from the Ad3 serotype, while the hexagonal protein (encoded by the L3 gene) originates from the Ad7 serotype. These two proteins together constitute the components of the adenovirus capsid. The third major component of the adenovirus capsid—the spike protein (encoded by the L5 gene)—also originates from the Ad7 serotype.

[0025] Pentagonal basal proteins are located at each apex of the adenovirus capsid. For most adenoviruses, they serve as attachment sites for cell surface integrins αvβ3 and αvβ5, with αvβ5 being a key receptor for adenovirus internalization into host cells. Therefore, the serotype of pentagonal basal proteins directly influences the cell tropism of adenoviruses.

[0026] The applicant has now discovered that this feature also endows adenoviruses with a stronger systemic delivery capability and enables them to spread in solid tumors by establishing effective infection in neighboring cells.

[0027] Not limited by theory, the chimeric characteristics of the adenoviruses of this invention may also be specifically screened out during biological selection processes, because these chimeric adenoviruses have the ability to overcome neutralizing antibodies, which can bind to the surface proteins of adenoviruses (such as pentameric basal proteins, hexameric proteins, and spike proteins). This characteristic makes the adenoviruses of this invention not only highly valuable as oncolytic adenoviruses, but also suitable as gene therapy vectors.

[0028] CN103966174 discloses a recombinant Ad3 adenovirus that inserts an Ad7 hexagonal protein expression cassette into the E3 region of the adenovirus genome. The resulting capsid is chimeric, containing a mixture of Ad3 pentagonal protein, Ad3 hexagonal protein, and Ad7 hexagonal protein, as well as an Ad3 spike protein. This recombinant adenovirus is described as a bivalent vaccine, preventing human Ad3 and Ad7 adenovirus infections (such as acute respiratory diseases) by eliciting an immune response to Ad3 and Ad7. This contrasts with the adenovirus described in this invention, which is used as an oncolytic virus and has been biologically screened to possess the ability to overcome neutralizing antibodies.

[0029] Furthermore, compared with wild-type group B adenoviruses, all biologically selected adenovirus genomes were found to have deletions in the E3 region.

[0030] The wild-type E3 region of the adenovirus genome in group B typically contains 9 ORFs.

[0031] Among all the viruses obtained through bioscreening, at least five open reading frames (ORFs) (19.3K, 20K, 20.6K, 7.7K, and 10.3K) were found to be completely or partially deleted; in addition, two other open reading frames (16.1K and 14.9K) were partially deleted in some of the bioscreened adenoviruses.

[0032] As mentioned earlier, the tumor microenvironment of cancer patients is filled with stress proteins (such as TNF-α). These proteins bind to early viral infections, potentially causing the rapid death of the first infected tumor cells. This rapid death of tumor cells hinders efficient viral replication, limits viral transmission, and ultimately leads to the premature clearance of the virus.

[0033] The applicant has now discovered that this feature particularly enhances the ability of adenoviruses to exhibit oncolytic activity in tumors containing stroma, including ovarian cancer cells.

[0034] Specifically, this study demonstrates that preserving either a complete or partial E3 14.9K ORF can block NF-κB signaling. The study found that this feature prevents premature clearance of adenovirus, thus allowing more time for adenovirus to initiate cell infection.

[0035] Furthermore, this study demonstrates that preserving the E3 14.7K ORF can prevent early apoptosis in infected cells. This mechanism was found to provide more time for viral replication within cells, thereby increasing the infection titer of the adenovirus of this invention.

[0036] This invention also demonstrates that large transgenic structures can be inserted into the E3 deletion site of the biologically screened adenovirus of this invention without significantly reducing its killing efficacy against cancer cells. Compared to the parental wild-type strain, the partial deletion of the E3 ORF also weakens viral activity in normal cells.

[0037] US 2002 / 106746 A1 discloses a recombinant adenovirus vector derived from the Ad5 adenovirus genome, in which at least a portion of the E3 region is deleted or lost function, but the adenovirus vector still retains the E3 sequence encoding functional 14.7K, 14.5K, and / or 10.4K proteins. The corresponding proteins in group B adenoviruses (such as Ad3 or Ad7) are 14.7K, 14.9K, and 10.3K proteins, respectively. The genome of Enadenotucirev (ColoAd; Kuhn et al. PLoS One 2008; 3(6): e2409) contains deletions in the E3 region, including complete or partial deletions of the 14.9K and 14.7K ORFs.

[0038] Genome analysis of adenoviruses obtained through biological screening revealed a missense mutation in the E2B DNA polymerase I (DNA pol I) gene. This mutation is a single nucleotide substitution (ggc-gac), resulting in a G34D mutation in the amino acid sequence of the E2B DNA polymerase I polypeptide.

[0039] In the wild-type adenovirus genome, the coding sequence of the E2B DNA polymerase I gene overlaps with that of the E2B front-terminal protein (pTP). The aforementioned mutation is located precisely within the coding sequences of both the DNA polymerase I polypeptide and the pTP polypeptide, thus causing a mutation in the pTP gene as well. Specifically, the mutation in the pTP polypeptide sequence is gcg→acg, leading to an A623T mutation in the amino acid sequence of the E2B pTP polypeptide.

[0040] The applicant has now discovered that this feature particularly enhances the ability of adenoviruses to exhibit oncolytic activity in tumors containing stroma, including ovarian cancer cells.

[0041] Specifically, research has found that one or both of these mutations can confer advantages on adenoviruses in terms of viral replication levels and the production of infectious progeny viruses. For oncolytic adenoviruses, this advantage manifests as the adenovirus's ability to maintain sustained oncolytic activity at the tumor site. For transgenic or "armed" oncolytic adenoviruses (such as transgenic adenoviruses), this advantage is manifested in the virus's ability to express the transgene and its corresponding polypeptide products at a higher abundance.

[0042] Adenoviruses containing the nucleotide sequence shown in SEQ ID NO:58 have been found to possess excellent oncolytic properties. Specifically, these adenoviruses exhibit enhanced dispersibility, enabling them to infect neighboring cells and establish new, effective infections. Their cancer cell-killing activity is not neutralized by whole blood or serum, and they can activate inflammatory T cell responses, thereby promoting T cell targeting and killing of cancer cells.

[0043] Therefore, the object of the present invention is to provide an adenovirus with a chimeric genome, wherein the 5' end of the genome is derived from Ad3 serotype virus and the 3' end is derived from Ad7 serotype virus. Such adenoviruses (especially oncolytic adenoviruses) have enhanced ability to infect neighboring cells and establish new effective infections, especially in tumor cells.

[0044] Another objective of this invention is to provide an adenovirus that, compared to wild-type B adenoviruses, has a missing E3 region and can lyse a variety of different tumor cells and CAFs, but has no lysing effect on normal cells; at the same time, this type of adenovirus has the ability to spread, blood stability, and can induce immunogenic cell death and stimulate immune responses including T cells and dendritic cells when tumor cells are lysed.

[0045] Another object of the present invention is to provide an oncolytic adenovirus having mutations in its E2B DNA polymerase I gene and / or E2B front-terminal protein (pTP) gene, and being able to infect and lyse cancer cells, particularly ovarian cancer cells and CAF; furthermore, another object of the present invention is to provide an adenovirus with mutations in the E2B DNA polymerase I gene and / or E2B pTP gene, which, compared with the corresponding control adenovirus, can achieve a higher level of replication and produce more progeny viruses with infectious activity.

[0046] Another object of the present invention is to provide an adenovirus having the above-described chimeric genome, deletions and mutations; and an adenovirus and its variants as shown in SEQ ID NO:58.

[0047] Another object of the present invention is to provide a composition comprising the adenovirus, and its use in the prevention or treatment of cancer (including stromal tumors and ovarian cancer); another object of the present invention is to provide a composition comprising the adenovirus for use as a gene therapy vector.

[0048] In a first aspect, the present invention provides a chimeric adenovirus, wherein the genome of the chimeric adenovirus comprises:

[0049] (a) Ad3 pentagenesis gene; and

[0050] (b) Ad7 hexane gene;

[0051] It is used to prevent or treat cancer, with ovarian cancer being the preferred candidate.

[0052] In some embodiments, the present invention provides a method for treating cancer, preferably ovarian cancer, the method comprising administering an effective amount of chimeric adenovirus to a patient in need, wherein the genome of the chimeric adenovirus comprises:

[0053] (a) Ad3 pentagenesis gene; and

[0054] (b) Ad7 hexane gene.

[0055] In another embodiment, the present invention provides the use of a chimeric adenovirus in the preparation of a medicament for the prevention or treatment of cancer, preferably ovarian cancer, wherein the genome of said chimeric adenovirus comprises:

[0056] (a) Ad3 pentagenesis gene; and

[0057] (b) Ad7 hexane gene.

[0058] In some embodiments, the genome of the chimeric adenovirus further includes: (c) the Ad7 spike protein gene.

[0059] The present invention also provides a chimeric adenovirus, wherein the genome of the chimeric adenovirus comprises:

[0060] (a) Ad3 pentacol gene;

[0061] (b) Ad7 hexane gene; and

[0062] (c) Ad7 spike protein gene.

[0063] The present invention also provides an adenovirus gene therapy vector, wherein the genome of the adenovirus gene therapy vector comprises:

[0064] (a) Ad3 pentacol gene;

[0065] (b) Ad7 hexane gene; and

[0066] (c) Genetically modified organisms.

[0067] The present invention also provides a pharmaceutical composition comprising the chimeric adenovirus of the present invention, optionally in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0068] The present invention also provides a pharmaceutical combination comprising:

[0069] (A) A first pharmaceutical composition comprising the chimeric adenovirus of the present invention; and

[0070] (B) A second pharmaceutical composition comprising a chemotherapy drug or an immunotherapy drug.

[0071] The drug combination is in the form of a combination formulation for simultaneous, separate or sequential use, preferably for the treatment of cancer.

[0072] The invention also provides a chimeric adenovirus or an adenovirus gene therapy vector for treatment or as a medicine.

[0073] The invention also provides the use of the chimeric adenovirus as a protein production vector, wherein the chimeric adenovirus contains a transgene encoding the protein to be produced.

[0074] In a second aspect, the present invention provides an oncolytic adenovirus whose genome includes an E3 region:

[0075] (a) The E3 region contains 12.1K and 14.7K ORFs from the E3 region of group B adenovirus, or corresponding ORFs from the E3 region of non-group B adenovirus; and

[0076] (b) The E3 region does not contain a functional 20K, 20.6K, 7.7K or 10.3KORF from the E3 region of group B adenovirus, or a corresponding ORF from a non-group B adenovirus E3 region.

[0077] In some embodiments, the E3 region further includes a functional (optionally 3' truncated) 16.1K ORF from the E3 region of group B adenovirus, or a corresponding ORF from a non-group B adenovirus E3 region. In some embodiments, the E3 region further includes a 16.1K ORF and a functional (optionally 3' truncated) 19.3K ORF from the E3 region of group B adenovirus, or a corresponding ORF from a non-group B adenovirus E3 region. In some embodiments, the E3 region further includes a 14.9K ORF from the E3 region of group B adenovirus, or a corresponding ORF from a non-group B adenovirus E3 region.

[0078] In a further embodiment, the present invention provides an oncolytic adenovirus whose genome includes an E3 region, wherein the E3 region comprises or is composed of the following:

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

[0080] (b) 3' truncated group B adenovirus E3 16.1K ORF;

[0081] (c) 5' truncated group B adenovirus E3 10.3K ORF;

[0082] (d) Group B adenovirus E3 14.9K ORF; and

[0083] (e) Group B adenovirus E3 14.7K ORF,

[0084] Or the corresponding ORF from the E3 region of a non-group B adenovirus.

[0085] The E3 regions are connected sequentially in the order of 5'-3' described above, and the E3 regions may optionally contain one or more transgenes located inside or adjacent to one or more of the ORFs.

[0086] In a further embodiment, the present invention provides an oncolytic adenovirus whose genome includes an E3 region, wherein the E3 region comprises or is composed of the following:

[0087] (a) Group B adenovirus E3 12.1K ORF;

[0088] (b) Group B adenovirus E3 16.1K ORF;

[0089] (c) 3' truncated group B adenovirus E3 19.3K ORF;

[0090] (d) 5' truncated group B adenovirus E3 14.9K ORF; and

[0091] (e) Group B adenovirus E3 14.7K ORF,

[0092] Or the corresponding ORF from the E3 region of a non-group B adenovirus.

[0093] The E3 regions are connected sequentially in the order of 5'-3' described above, and the E3 regions may optionally contain one or more transgenes located inside or adjacent to one or more of the ORFs.

[0094] In a further embodiment, the present invention provides an oncolytic adenovirus whose genome includes an E3 region, the E3 region encoding a fusion protein, the fusion protein being a fusion protein formed by fusing the C-terminus of a C-terminated group B adenovirus E3 16.1K protein with the N-terminus of an N-terminated group B adenovirus E3 10.3K protein, or a fusion protein formed by fusing the corresponding end of a protein from a non-group B adenovirus E3 region.

[0095] In a further embodiment, the present invention provides an oncolytic adenovirus whose genome includes an E3 region, the E3 region encoding a fusion protein, the fusion protein being a fusion protein formed by fusing the C-terminus of a C-terminated group B adenovirus E3 19.3K protein with the N-terminus of an N-terminated group B adenovirus E3 14.9K protein, or a fusion protein formed by fusing the corresponding ends of a protein from a non-group B adenovirus E3 region.

[0096] In a further embodiment, the present invention provides a pharmaceutical composition comprising an adenovirus as described in any of the preceding claims, optionally further comprising one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0097] In a further embodiment, the present invention provides the oncolytic adenovirus of the present invention or the pharmaceutical composition of the present invention for treatment or use as a medicine. In a further embodiment, the present invention provides the oncolytic adenovirus of the present invention or the pharmaceutical composition of the present invention for treating cancer (preferably ovarian cancer). In a further embodiment, the present invention provides a method of treating a subject with cancer (preferably ovarian cancer), the method comprising administering a therapeutically effective amount of the oncolytic adenovirus of the present invention or the pharmaceutical composition of the present invention to a subject in need. In a further embodiment, the present invention provides the use of the oncolytic adenovirus of the present invention in the preparation of a medicine for treating cancer (preferably ovarian cancer).

[0098] In a third aspect, the present invention provides an adenovirus, wherein the genome of the adenovirus comprises an E2B DNA polymerase I gene encoding a DNA polymerase I polypeptide, wherein the amino acid corresponding to the 34th amino acid in SEQ ID NO: 49 of the DNA polymerase I polypeptide sequence is Asp or Glu, preferably Asp.

[0099] In another embodiment, the present invention provides an adenovirus, wherein the genome of the adenovirus comprises an E2B pTP gene encoding a terminal protein, wherein the amino acid corresponding to amino acid position 623 of SEQ ID NO: 51 in the terminal protein sequence is selected from Thr, Asn, Cys, Gln, and Ser, preferably Thr. In some embodiments, if the adenovirus is an Ad1 adenovirus, the amino acid is not Asn; if the adenovirus is an Ad41 adenovirus, the amino acid is not Gln; and / or if the adenovirus is an Ad4 or 4a adenovirus, the amino acid is not Thr.

[0100] In another embodiment, the present invention provides a pharmaceutical composition comprising the adenovirus of the present invention, optionally in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0101] The adenovirus of the present invention or the pharmaceutical composition of the present invention are also provided for treatment or use as a medicine, particularly for the treatment of cancer (preferably ovarian cancer).

[0102] A method for treating a subject with cancer (preferably ovarian cancer) is also provided, the method comprising administering a therapeutically effective amount of the adenovirus of the present invention or the pharmaceutical composition of the present invention to the subject in need.

[0103] The use of the oncolytic adenovirus of the present invention in the preparation of a medicament for treating cancer (preferably ovarian cancer) is also provided.

[0104] The invention also provides the use of a conditionally replicating adenovirus as a protein production vector, wherein the adenovirus contains a transgene encoding a protein to be produced; and the use of a conditionally replicating adenovirus as a virus production aid.

[0105] The present invention also provides mutant group B or human E2B DNA polymerase I and E2B pTP genes as defined herein, as well as mutant group B or human E2B DNA polymerase I and E2B pTP peptides as defined herein.

[0106] In a fourth aspect, the present invention provides an adenovirus, wherein the genome of the adenovirus comprises one, two, or all of (A), (B), and (C):

[0107] (A)(a)Ad3 pentacol gene; and

[0108] (b) Ad7 hexane gene; and

[0109] (c) Optionally, the Ad7 spike protein gene;

[0110] (B) Area E3:

[0111] (a) The E3 region contains 12.1K and 14.7K ORFs from the E3 region of group B adenovirus, or corresponding ORFs from the E3 region of non-group B adenovirus; and

[0112] (b) The E3 region does not contain a functional 20K, 20.6K, 7.7K or 10.3KORF from the E3 region of group B adenovirus, or a corresponding ORF from a non-group B adenovirus E3 region;

[0113] as well as

[0114] (C) The E2B DNA polymerase I gene encoding the DNA polymerase I polypeptide.

[0115] Wherein, the amino acid corresponding to the 34th amino acid in SEQ ID NO: 49 of the DNA polymerase I polypeptide sequence is Asp or Glu, preferably Asp; or

[0116] The E2B pTP gene encodes the terminal protein.

[0117] The amino acid corresponding to amino acid 623 in SEQ ID NO: 51 in the preterminal protein sequence is selected from Thr, Asn, Cys, Gln and Ser, preferably Thr.

[0118] In some embodiments, regarding the amino acid corresponding to amino acid 623 in SEQ ID NO: 51 in the terminal protein sequence, 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 Gln; and / or if the adenovirus is an Ad4 or 4a adenovirus, then the amino acid is not Thr.

[0119] In some embodiments, the present invention provides an adenovirus, wherein the genome of the adenovirus comprises (A) and (B).

[0120] In some embodiments, the present invention provides an adenovirus, wherein the genome of the adenovirus comprises (A) and (C).

[0121] In some embodiments, the present invention provides an adenovirus, wherein the genome of the adenovirus comprises (B) and (C).

[0122] In some embodiments, the present invention provides an adenovirus, wherein the genome of the adenovirus comprises (A), (B) and (C).

[0123] Fifthly, the present invention provides an adenovirus comprising the nucleotide sequence 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 with SEQ ID NO: 58 and having oncolytic activity. In some preferred embodiments, the nucleotide sequence has at least 95% sequence identity with SEQ ID NO: 58 and the adenovirus has oncolytic activity. In other preferred embodiments, the nucleotide sequence has at least 99% sequence identity with SEQ ID NO: 58 and the adenovirus has oncolytic activity.

[0124] sequence list

[0125] Table 1. Nucleotide and amino acid sequence listing of the L2-L3 region

[0126]

[0127]

[0128] Table 2. Nucleotide and amino acid sequence listing of the E3 region

[0129]

[0130] Table 3. DNA polymerase I and pTP gene and polypeptide sequences, and complete viral sequence.

[0131]

[0132] In all respects, the present invention provides adenoviruses, particularly oncolytic adenoviruses, preferably for the treatment of cancer, more preferably for ovarian cancer or stromal-containing tumors.

[0133] As used herein, "adenovirus (Ad)" refers to viruses belonging to the family Adenoviridae, encompassing the five currently known genera: Mastadenovirus, Aviadenovirus, Atadenovirus, Siadenovirus, and Ichtadenovirus. Preferably, the adenovirus originates from the genus Mastadenovirus, which includes all human serotypes. In some embodiments, the adenovirus is a human adenovirus.

[0134] More than 60 antigenic or "serotypes" of human adenoviruses have been identified to date. These serotypes are classified into seven species, namely adenoviruses AG, based on their physical, chemical, and biological characteristics (e.g., as described in Wold et al. Current genetherapy vol. 13, 6 (2013): 421-33). Therefore, the adenovirus species referred to in this article include both currently known AG species and potential new species that may be discovered in the future.

[0135] In some embodiments, the adenovirus species is selected from the AdA, AdB, AdC, AdD, AdE, AdF, and AdG groups. In some embodiments, the human adenovirus is selected from the AdB, AdC, AdD, AdE, AdF, and AdG groups.

[0136] The serotypes belonging to these adenovirus subspecies include, but are not limited to, the following:

[0137] AdA includes Ad12, Ad18, Ad31, and Ad61. AdB includes Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad50, Ad55, Ad66, Ad68, and Ad79. AdC includes Ad1, Ad2, Ad5, Ad6, and Ad57. AdD includes Ad8, Ad9, Ad10, Ad13, Ad15, Ad17, Ad19, Ad20, Ad22, Ad23, Ad24, Ad25, Ad26, Ad27, Ad28, Ad29, Ad30, Ad32, Ad33, Ad36, Ad37, Ad38, Ad39, Ad42, Ad43, Ad44, Ad45, Ad46, Ad47, Ad48, Ad49, Ad51, Ad53, Ad54, and Ad56. Ad58, Ad59, Ad60, Ad62, Ad63, Ad64, Ad65, Ad67, Ad69, Ad70, Ad71, Ad73, Ad74, and Ad75. AdE includes Ad4. AdF includes Ad40 and Ad41, and AdG includes Ad52.

[0138] The different adenovirus serotypes mentioned in this article all include all different strains or variants of that serotype.

[0139] Preferably, the adenovirus is a group B adenovirus. Group B1 adenoviruses include Ad3, Ad7, Ad16, Ad21, Ad50, Ad66, and Ad68; group B2 adenoviruses include Ad11, Ad14, Ad34, Ad35, Ad55, and Ad79. Preferably, the adenovirus is a group B1 adenovirus; most preferably, the adenovirus is Ad3 serotype, Ad7 serotype, or Ad3 / Ad7 chimeric adenovirus.

[0140] The chimeric oncolytic adenovirus of this invention has a genome. In some embodiments, the adenovirus is a human adenovirus. To date, all studied human adenovirus genomes have the same overall structure, i.e., genes encoding specific functions are located in the same positions within the adenovirus genome (referred to in this invention as "structural elements"). At each end of the adenovirus genome is a short sequence called an inverted terminal repeat (ITR), which is essential for viral replication. In some embodiments, the adenovirus is neither a chimpanzee adenovirus nor an AdC7 adenovirus.

[0141] The adenovirus genome contains five early transcription units (E1A, E1B, E2, E3, and E4), three delayed early units (IX, IVa2, and E2 late), and one late unit (major late), which is processed to produce five sets of late mRNAs (L1-L5). The proteins encoded by the early genes are primarily involved in viral replication and regulating the host cell's response to infection, while the late genes encode viral structural proteins. Early genes are prefixed with the letter "E," and late genes with the letter "L." The length of the adenovirus DNA genome varies depending on the serotype, typically ranging from 34 to 36 kb. For example, the Ad5 genome typically contains 35,938 bp, with a 103 bp terminal repeat at each end and a GC content of 58%.

[0142] Our understanding of adenovirus genetics, transcription, and translation primarily stems from virological studies of the C subtype adenovirus Ad5. The transcriptional sequence of the adenovirus genome reflects the protein requirements of the virus at each stage of replication. Therefore, based on the transcription initiation time of each viral promoter, adenovirus genome transcription can be divided into early and late events. The first protein produced by the viral genome is the E1A protein. The E1A transcription unit generates various mRNAs through alternative splicing, which in turn generate proteins with molecular weights of 6-36 kDa. The E1A protein has two main functions in infected cells: first, it induces cells to enter the S phase of the cell cycle, ensuring efficient viral genome replication; second, it induces the transcription of other early promoters within the viral genome through transactivation. These promoters regulate the production of E1B, E2, E3, and E4 proteins. Following E1A expression, VA RNA, E1B protein, and E3 protein are subsequently produced; these proteins and RNA molecules help suppress the generation of antiviral responses. Early events in viral replication create conditions for viral genome replication before packaging by remodeling the intracellular environment. Late transcriptional events involve the synthesis of structural proteins and key proteins for cell lysis, which are primarily derived from a single promoter (the major late promoter) that transcribes late regions 1-5. Ad5-induced cell lysis depends on the E3-11.6K protein (also known as the adenovirus death protein), which, although labeled as an early gene, is produced only in the later stages of infection and is driven by the major late promoter.

[0143] Adenovirus genes are divided into early transcripts (E1-4) and late transcripts (L1-5), which can produce various protein isotypes through a series of splicing events. The early region is divided into E1, E2, E3, and E4. E1 is crucial for enabling cells to enter cell cycle phases conducive to viral replication, inhibiting apoptosis, and promoting cell division. The E2 region is mainly responsible for DNA genome replication, including DNA-binding proteins (E2A), pTP, and DNA polymerase (E2B). E3 includes genes involved in regulating the host immune response. E4 includes a series of genes involved in regulating cellular pathways, such as non-homologous end joining (NHEJ) and genes that bind to E1B-55K to mediate p53 degradation.

[0144] All adenovirus late genes are transcribed from the same promoter (major late promoter) and share the same 5' mRNA end, which contains three exons that together form a triplet leader sequence. Late genes are expressed through a series of splicing events, producing about 13 proteins that either form part of the viral particle (such as hexagonal proteins and spike proteins) or participate in the assembly of the viral particle (such as the 100K protein).

[0145] Adenoviral vectors are vectors based on or derived from the genome of viruses in the Adenoviridae family. These vectors can be classified as functional replicating, replication-deficient, and / or genetically engineered, capable of delivering genetic material (such as adenoviral genes or exogenous genes) into eukaryotic cells for expression. Adenoviral vectors can achieve the insertion of 8-30 kb transgenes by deleting early E1, E3, and / or E4 genes. The function of the deleted early genes can be provided by the transfected engineered cell lines. The most commonly used adenoviral vector is based on adenovirus type 5 (Ad5).

[0146] The adenovirus of the present invention contains multiple early adenovirus genes (e.g. Figure 1 A).

[0147] The E1A protein is the translational product of the adenovirus genome after the initial transcription in the E1A region of the cell nucleus. This initial transcription is driven by a strong constitutive enhancer element within the E1A promoter, producing a large amount of E1A mRNA. The E1A protein is one of two proteins in the adenovirus genome that can induce transformation; the E1B protein can also induce cell cycle progression. Both the E1A and E1B genes are essential for viral replication.

[0148] The E2 gene in the adenovirus genome is divided into two regions: E2A and E2B, both essential for viral replication. E2B contains the DNA polymerase gene, which is fundamentally required for the amplification of viral genomic DNA. This region also contains the pTP protein, which acts as a primer to initiate viral genome replication. The pTP protein is covalently linked to the ends of the viral genomic DNA. The E2A region contains a DNA-binding protein, which is also essential for DNA replication. All E2 genes are fundamentally necessary for viral replication.

[0149] For Ad5, pTP assembles with DNA polymerase, DNA-binding proteins, host NFI, and OCT1 at the viral origin of replication to form the initiation complex. pTP is an approximately 80 kDa protein that functions as an initiation primer. The DNA polymerase possesses 5'–3' polymerase activity and a 3'–5' exonuclease domain, which is crucial for its intrinsic proofreading capabilities. In the final stage of replication, pTP is cleaved into TP by viral proteases, producing progeny DNA, which is then packaged into viral particles.

[0150] E3 genes are primarily involved in regulating the cellular and host immune responses to viral infection. However, since most viruses in biotechnological applications are cultured in vitro, most of these viral genes (if not all) can be removed without affecting viral replication efficiency. Most E3 genes are not essential for viral replication in cell lines. However, some genes, such as adenovirus death protein (ADP), are essential for efficient replication and viral production.

[0151] The E4 region of the adenovirus genome is similar to the E1 region, primarily involved in the production of proteins that help the virus control and regulate cells to ensure efficient viral replication and production. This region contains six ORFs, which assist in preventing non-homologous end joining and apoptosis, and have several other functions. The importance of different E4 transcripts to viral replication varies: some transcripts are indispensable, while others, even if deleted or modified, have little effect on viral growth kinetics and production.

[0152] The adenovirus of the present invention preferably contains sufficient early adenovirus genes to ensure that the virus can replicate its genome within the host cell nucleus.

[0153] The adenovirus of this invention contains multiple adenovirus late genes ( Figure 1 A). Late viral genes are divided into five major transcriptional families, named L1-L5. These transcripts primarily encode proteins involved in viral assembly and viral structural proteins. During viral replication, these proteins can account for 30-40% of the cellular protein content (Garnier, 1994; Ginsberg, 1984).

[0154] The L1 series of transcripts encodes 13.6K, 52K, and PIIIa proteins. These proteins are all involved in viral assembly and particle production. The L1 gene is essential for successful viral assembly but not for genomic DNA replication.

[0155] The L2 series transcripts encode the pentazocine basement membrane, pVII, V, and pX proteins. These proteins constitute the structural parts of the viral capsid and are essential for the proper assembly of viral particles. The pentazocine basement membrane contains the RGD motif, which is important for attachment to the cell surface during infection with various adenoviruses. The L2 genes are required for successful viral assembly but are not essential for genomic DNA replication.

[0156] The L3 series transcripts encode pVI, hexagonal proteins, and protease proteins. Hexagonal proteins are major components of the viral capsid and exhibit antigenic diversity across different serotypes. Protease proteins are involved in cell entry and viral capsid maturation. The L3 gene is essential for successful viral assembly but not for genomic DNA replication.

[0157] The L4 series of transcripts encodes 100K, 33K, 22K, and pVIII proteins. These proteins are involved in multiple functions. The 100K protein is involved in assisting viral hexapod assembly and nuclear importation, and may also play a role in shifting cellular mRNA translation to cap-independent translation. The 22K protein is involved in viral capsid formation. The L4 gene is essential for successful viral assembly but not for genomic DNA replication. However, the 100K protein may help shift cellular protein translation to transcripts containing a triplet leader sequence (TPL).

[0158] L5 encodes the spike protein gene. The spike protein is a viral structural protein involved in attaching to the cell surface and mediating viral infection. The production of spike protein far exceeds what is needed to form viral particles. The L5 gene is essential for successful viral assembly but not for genomic DNA replication.

[0159] The genome of the adenovirus of this invention contains multiple early and late adenovirus genes, sufficient to support adenovirus replication. Preferably, the adenovirus of this invention contains at least the early genes E1, E2, and E4, and the late genes L1-L5. E3 is not essential for replication in cell lines. However, some genes in these regions can be modified, mutated, or even deleted without inhibiting viral replication in cell lines.

[0160] In one implementation, the adenovirus is capable of replication, is reproducible, or conditionally replicates. These viruses may be oncolytic viruses, viral vaccines, or protein production vectors.

[0161] As used herein, the term "replicating" refers to an adenovirus capable of replicating its genome within a host cell. In some implementations, "replicating" includes viruses that are capable of replication or conditionally replicating.

[0162] In the context of this invention, "replicable" means an adenovirus that possesses all the mechanisms necessary for replication in vitro and in vivo in cells, i.e., without the aid of a packaging cell line.

[0163] The terms "conditional replication," "selective replication," or "replication selectivity" used in this article specifically refer to an oncolytic adenovirus that can replicate in cancer cells. It can replicate within cancer cells by utilizing elements specific to or upregulated in cancer cells (such as defective cellular mechanisms, like p53 mutations), thereby exhibiting a certain selectivity for healthy / normal cells.

[0164] "Conditional replication" also refers to the ability of adenoviruses to infect and replicate under specific conditions. These conditions may include viral regulation by tumor-specific promoters to drive intracellular viral replication (such as the PSA promoter), or viral regulation by repressive factors (such as tetracycline repressor (TetR) binding sites or microRNA binding sites) to restrict viral replication under specific conditions. Conditionally replicating adenoviruses can be oncolytic or used as vectors for gene, vaccine, or protein delivery.

[0165] In some embodiments, the adenovirus genome shares at least 70% (preferably at least 80%, 85%, 90%, 95%, or 99%; most preferably at least 99%) nucleotide sequence identity with the wild-type Ad3 genome sequence (e.g., Genbank Sequence ID: DQ086466.1). In some embodiments, the adenovirus genome shares at least 70% (preferably at least 80%, 85%, 90%, 95%, or 99%; most preferably at least 99%) nucleotide sequence identity with the wild-type Ad7 genome sequence (e.g., Genbank Sequence ID AY594255.1).

[0166] In some preferred embodiments, the adenovirus of the present invention is oncolytic. As used herein, "oncolytic" refers to the adenovirus's ability to infect, replicate, and lyse cancer cells. Preferably, the oncolytic adenovirus of the present invention is more likely to infect and / or preferentially lyse cancer cells compared to non-cancer cells. In some embodiments, the oncolytic adenovirus of the present invention is also capable of infecting and / or lysing stromal cells, particularly CAF.

[0167] Viral infection can be measured using infectivity assays, such as plaque assays and the median infectious dose (TCID) of tissue culture. 50 The oncolytic adenovirus of the present invention can be measured by an assay, or by immunocytochemical (ICC) staining using anti-hexamethylenetetramine antibodies. The oncolytic adenovirus of the present invention can have cell lysis activity. Lysis can be measured by cell death or cell viability assays, including MTS, MTT assay, and PrestoBlue. TM Live / dead staining and flow cytometry. Oncolytic virus infection causes the death and lysis of cancer cells or mesenchymal cells, preferably accompanied by the release of newly generated viral particles.

[0168] Virus production can be determined by a variety of methods, including the infectivity assays described above. Other methods can be used to measure the physical count of viral particles, including ultraviolet absorption measurement (OD260), dynamic light scattering, and HPLC quantification. Furthermore, virus production can be measured by DNA quantification and interpolation based on a standard curve of known DNA content; these assays include real-time quantitative PCR and Pico Green dye-based assays. Purified virus can also be quantified by determining total protein content using the dioctanine acid (BCA) method.

[0169] In some embodiments, the adenovirus of the present invention encodes one or more transgenes. The adenovirus of the present invention is particularly useful as an oncolytic vector, a conditionally replicative vector, or a replication-deficient vector, and can also be used as a gene therapy vector. Such vectors may contain transgenes to produce recombinant nucleic acids or polypeptides at a target site (such as a tumor site).

[0170] In gene therapy vectors, the transgene may encode a therapeutic polypeptide, or may be a gene for correcting genetic defects. In cancer gene therapy vectors, the gene may encode a polypeptide that induces cell death (e.g., p53).

[0171] Examples of transgenics include transgenics encoding anticancer agents, immunostimulants, or imaging agents (e.g., antibodies, bispecific adaptors, checkpoint inhibitors, cytokines, chemokines, and enzymes including extracellular matrix degrading enzymes and anti-angiogenic inhibitors). Preferred examples of transgenics include transgenics encoding EpCAM-Lite, EpCAM-BiTE, CTLA4, CCL19, CCL5, CXCL11, or CXCL12.

[0172] The transgene can be located in, for example, the E1 or E3 region of an adenovirus, or in the E1 / E3 deletion region, or it can be inserted near or inside the L3 or L5 region.

[0173] The deletion of the E3 region creates more space for transgene insertion at other locations in the adenovirus genome, while removing the ORF that helps the virus evade immune system recognition and hinders host clearance. Therefore, the deletion of the E3 region weakens viral activity in normal cells with a functional immune response, but does not weaken its activity in immune-dysfunctional tumor cells, thereby increasing the therapeutic index of oncolytic viruses.

[0174] The genome of an adenovirus must be within the viral packaging limits. For example, the size of the transgene it carries must not make the total length of the genome exceed the length that can be packaged into the protein capsid.

[0175] The adenovirus described in this invention may be referred to as a "recombinant adenovirus".

[0176] The adenovirus described in this invention may be referred to as a "recombinant" adenovirus. As used herein, the term "recombinant adenovirus" refers to a non-natural adenovirus that differs from a wild-type adenovirus by at least one nucleotide, for example, a non-natural adenovirus whose genome has a continuous gene sequence that is not simultaneously present in the genome of a wild-type adenovirus.

[0177] This invention relates to multiple aspects, as described herein, and can be implemented individually or in combination. Specifically, the invention provides adenoviruses having first and second aspects; adenoviruses having first and third aspects; adenoviruses having second and third aspects; and adenoviruses having first, second, and third aspects.

[0178] First aspect of the present invention

[0179] In a first aspect, the present invention provides an adenovirus, wherein the genome of the adenovirus comprises:

[0180] (A) (a) Ad3 pentagenesis gene; and

[0181] (b) Ad7 hexane gene; and

[0182] (c) Optionally, the Ad7 spike protein gene.

[0183] The L2 region of wild-type adenovirus contains the adenovirus pentagon gene encoding the adenovirus pentagon polypeptide, and the adenovirus pVII, V and pX genes encoding the pVII, V and pX core polypeptides, respectively.

[0184] The L3 region of wild-type adenovirus contains adenovirus pVI, hexagonal, and protease genes that encode adenovirus pVI, hexagonal, and protease polypeptides, respectively.

[0185] The references to adenovirus genes (such as penta-neighbone, hexa-neighbone, spike protein, etc.) in this article refer to the ORF or coding sequences of these genes.

[0186] In some embodiments, the L2 region contains a pentagonal gene encoding an Ad3 pentagonal polypeptide, and the L3 region contains a hexagonal gene encoding an Ad7 hexagonal polypeptide.

[0187] In some embodiments, the L3 region contains a VI gene encoding an Ad3 VI polypeptide; and the L3 region contains a hexagon gene encoding an Ad7 hexagon polypeptide.

[0188] In some embodiments, the L2 region contains pentagonal, pVII, V, and pX genes encoding Ad3 pentagonal, Ad3 pVII, Ad3 V, and Ad3 pX peptides, respectively; the L3 region contains the VI gene encoding the Ad3 VI peptide; and the L3 region contains the hexagonal gene encoding the Ad7 hexagonal peptide.

[0189] In some embodiments, the L2 region contains the V gene encoding the Ad3 V polypeptide; and the L2 region contains the pX gene encoding the Ad7 pX polypeptide.

[0190] In some embodiments, the L2 region contains the pentagon, pVII, and V genes encoding Ad3 pentagon, Ad3 pVII, and Ad3 V polypeptides, respectively; the L2 region contains the pX gene encoding the Ad7 pX polypeptide; and the L3 region contains the VI and hexagon genes encoding the Ad7 VI and Ad7 hexagon polypeptides, respectively.

[0191] In some embodiments, the L2 region contains a pX gene encoding an Ad3 pX polypeptide; and the L2 region contains a VI gene encoding an Ad7 VI polypeptide.

[0192] In some embodiments, the L2 region contains the pentagon, pVII, V and pX genes encoding the Ad3 pentagon, Ad3 pVII, Ad3 V and Ad3 pX peptides, respectively; and the L3 region contains the VI and hexagon genes encoding the Ad7 VI and Ad7 hexagon peptides, respectively.

[0193] In a particularly preferred embodiment, the L2 region contains the pentagon, pVII, V and pX genes encoding the Ad3 pentagon, Ad3 pVII, Ad3 V and Ad3 pX polypeptides, respectively; and the L3 region contains the VI and hexagon genes encoding the Ad3 VI and Ad7 hexagon polypeptides, respectively.

[0194] In all embodiments, the L3 region may additionally contain a protease gene encoding the Ad7 protease. In all embodiments, the L1 region preferably contains the L1 gene encoding the entire Ad3 polypeptide. In all embodiments, the L4 region preferably contains the L4 gene encoding the entire Ad7 polypeptide.

[0195] Preferably, the L2 region contains the Ad3 pentagon gene, that is, preferably, one of the L2 polypeptides is the Ad3 pentagon polypeptide.

[0196] The terms “penton” and “penton base” used in this article are used interchangeably; both refer to penton polypeptides.

[0197] The term "Ad3 pentagonal gene" as used in this article refers to a gene that contains or consists of the following:

[0198] (a) The nucleotide sequence shown in SEQ ID NO: 3;

[0199] (b) A nucleotide sequence having at least 95% or 99% nucleotide sequence identity with SEQ ID NO: 3 and encoding an Ad3 pentagonal polypeptide;

[0200] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 3 and encoding an adenovirus (preferably Ad3) pentagonal polypeptide; or

[0201] (d) Nucleotide sequence encoding the Ad3 pentagonal polypeptide.

[0202] The term “Ad3 pentam polypeptide” as used herein preferably includes, but is not limited to:

[0203] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 5;

[0204] (b) A polypeptide having at least 99.3%, 99.5%, or 99.7% (preferably at least 99.7%) sequence identity with SEQ ID NO: 5 and encoding an adenovirus pentagonal polypeptide (preferably an Ad3 pentagonal polypeptide); or

[0205] (c) Having at least 95%, 99% or 99.5% (preferably at least 99.5%) sequence identity with SEQ ID NO: 5, wherein the pentagonal polypeptide contains polypeptides corresponding to amino acids V, T, I and D at positions 11, 158, 178 and 326 of SEQ ID NO: 5, respectively, which encode an adenovirus pentagonal polypeptide (preferably an Ad3 pentagonal polypeptide).

[0206] Adenoviral pentagonal peptides have been well characterized (Medina-Kauwe, Ther. Deliv. 2013 Feb; 4(2): 267–277). The pentagonal base forms a homopentamer, non-covalently attached to each vertices of the adenoviral capsid and binding to the N-terminal tail domain of the spike peptide. The pentagonal base homopentamer contributes to the formation of the characteristic icosahedral shape of the adenoviral capsid and is crucial for the structural stability of the adenoviral capsid. Therefore, pentagonal peptides are essential for efficient transfection of target cells.

[0207] The transfection efficiency of adenovirus containing the pentagonal polypeptide of the present invention on target cells (e.g., HEK293 cells) can be tested by exposing target cells to the adenovirus and then measuring the percentage of cells expressing genes encoded by the genome packaged in the adenovirus capsid (e.g., the E1A gene or the GFP reporter gene), and comparing the results with those obtained by testing adenovirus containing wild-type pentagonal polypeptide (e.g., SEQ ID NO: 5) under the same conditions.

[0208] Transfection assays can be performed, for example, by measuring the percentage of HEK293 cells expressing the GFP reporter gene encoded by the viral genome using flow cytometry, as described by 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 being compared should have the same capsid and the same genome, differing only in the pentagonal polypeptide and pentagonal gene.

[0209] The pentagonal polypeptide of the present invention comprises: an adenovirus containing such a pentagonal polypeptide, wherein the transfection efficiency is at least 0.001% or 0.01% of the transfection efficiency obtainable using an adenovirus containing the wild-type pentagonal polypeptide of SEQ ID NO: 5 under the same assay conditions, preferably at least 0.1%, and most preferably at least 1%.

[0210] Preferably, the L2 region contains the Ad3 pVII gene, that is, preferably, one of the L2 polypeptides is the Ad3 pVII polypeptide.

[0211] As used in this article, the term "Ad3 pVII gene" refers to a gene that contains or consists of the following:

[0212] (a) The nucleotide sequence shown in SEQ ID NO: 7;

[0213] (b) A nucleotide sequence having at least 95% or 99% nucleotide sequence identity with SEQ ID NO: 7 and encoding the Ad3 pVII polypeptide;

[0214] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 7 and encoding an adenovirus (preferably Ad3) pVII polypeptide; or

[0215] (d) Nucleotide sequence encoding the Ad3 pVII polypeptide.

[0216] The term “Ad3 pVII polypeptide” as used in this article preferably includes, but is not limited to:

[0217] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 9; or

[0218] (b) A polypeptide having at least 99% sequence identity with SEQ ID NO: 9 and encoding an adenovirus pVII polypeptide; or

[0219] (c) Having at least 95%, 99% or 99.5% (preferably at least 99.5%) sequence identity with SEQ ID NO: 9, and wherein the pVII polypeptide contains a polypeptide whose amino acids at positions 27 and 112 of SEQ ID NO: 9 are K and P, respectively, and the polypeptide encodes an adenovirus pVII polypeptide (preferably Ad3 pVII polypeptide).

[0220] Preferably, the L2 region contains the Ad3 V gene, that is, preferably, one of the L2 polypeptides is the Ad3 V polypeptide.

[0221] The term "Ad3 V gene" as used in this article refers to a gene that contains or consists of the following:

[0222] (a) The nucleotide sequence shown in SEQ ID NO: 11;

[0223] (b) A nucleotide sequence having at least 95% or 99% nucleotide sequence identity with SEQ ID NO: 11 and encoding an Ad3V polypeptide; or

[0224] (c) A nucleotide sequence having at least 99.5% nucleotide sequence identity with SEQ ID NO: 11 and encoding an Ad3V polypeptide; or

[0225] (d) Nucleotide sequence encoding the Ad3 V polypeptide.

[0226] The term "Ad3 V peptide" as used in this article is preferably including, but not limited to:

[0227] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 13; or

[0228] (b) A polypeptide having at least 99.6% sequence identity with SEQ ID NO: 13 and encoding an Ad3 V polypeptide; or

[0229] (c) Having at least 90%, 95% or 99% (preferably at least 99%) sequence identity with SEQ ID NO: 13, wherein the V polypeptide contains amino acids D and missing at positions 45 and 234 of SEQ ID NO: 14, respectively, and the polypeptide encodes an adenovirus V polypeptide (preferably Ad3 V polypeptide).

[0230] In some embodiments, the L2 region contains the Ad3 pX gene, that is, in some embodiments, one of the L2 polypeptides is the Ad3 pX polypeptide.

[0231] The term "Ad3 pX gene" as used in this article refers to a gene that contains or consists of the following:

[0232] (a) The nucleotide sequence shown in SEQ ID NO: 15;

[0233] (b) A nucleotide sequence having at least 95% nucleotide sequence identity with SEQ ID NO: 15 and encoding the Ad3 pX polypeptide;

[0234] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 15 and encoding an adenovirus (preferably Ad3) pX polypeptide; or

[0235] (d) Nucleotide sequence encoding the Ad3 pX polypeptide.

[0236] The term "Ad3 pX peptide" as used in this article is preferably including, but not limited to:

[0237] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 17; or

[0238] (b) Having at least 90%, 95% or 98.5% (preferably at least 98.5%) sequence identity with SEQ ID NO: 17, and wherein the pX polypeptide contains a polypeptide whose amino acid at position 43 of SEQ ID NO: 17 is G, the polypeptide encoding an adenovirus pX polypeptide (preferably Ad3 pX polypeptide).

[0239] In some embodiments, the L2 region contains the Ad7 pX gene, that is, in some embodiments, one of the L2 polypeptides is the Ad7 pX polypeptide.

[0240] The term "Ad7 pX gene" as used in this article refers to a gene that contains or consists of the following:

[0241] (a) The nucleotide sequence shown in SEQ ID NO: 16;

[0242] (b) A nucleotide sequence having at least 95% nucleotide sequence identity with SEQ ID NO: 16 and encoding the Ad7 pX polypeptide;

[0243] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 16 and encoding an adenovirus (preferably Ad7) pX polypeptide; or

[0244] (d) Nucleotide sequence encoding the Ad7 pX polypeptide.

[0245] The term "Ad7 pX peptide" as used in this article is preferably including, but not limited to:

[0246] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 18; or

[0247] (b) Having at least 90%, 95% or 98.5% (preferably at least 98.5%) sequence identity with SEQ ID NO: 18, and wherein the pX polypeptide contains a polypeptide whose amino acid corresponding to position 43 of SEQ ID NO: 18 is D, the polypeptide encoding an adenovirus pX polypeptide (preferably Ad7 pX polypeptide).

[0248] The genome of the chimeric adenovirus includes an L3 region containing one or more L3 genes encoding one or more L3 polypeptides. Preferably, the L3 region is the Ad7 L3 region, except for the adenovirus pVI gene (which may be Ad3).

[0249] In some embodiments, the L3 region contains the Ad3 pVI gene, that is, in some embodiments, one of the L3 polypeptides is the Ad3 pVI polypeptide.

[0250] The term "Ad3 pVI gene" as used in this article refers to a gene that contains or consists of the following:

[0251] (a) The nucleotide sequence shown in SEQ ID NO: 19;

[0252] (b) A nucleotide sequence having at least 95% nucleotide sequence identity with SEQ ID NO: 19 and encoding an Ad3 pVI polypeptide;

[0253] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 19 and encoding an adenovirus (preferably Ad3) pVI polypeptide; or

[0254] (d) Nucleotide sequence encoding the Ad3 pVI polypeptide.

[0255] The term “Ad3 pVI peptide” as used in this article preferably includes, but is not limited to:

[0256] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 21; or

[0257] (b) Having at least 90%, 95% or 98% (preferably at least 98%) sequence identity with SEQ ID NO: 21, and wherein the pVI polypeptide has a peptide corresponding to amino acid P at position 196 in SEQ ID NO: 21, the polypeptide encoding an adenovirus pVI polypeptide (preferably Ad3 pVI polypeptide).

[0258] In some embodiments, the L3 region contains the Ad7 pVI gene, that is, in some embodiments, one of the L3 polypeptides is the Ad7 pVI polypeptide.

[0259] The term "Ad7 pVI gene" as used in this article refers to a gene that contains or consists of the following:

[0260] (a) The nucleotide sequence shown in SEQ ID NO: 20;

[0261] (b) A nucleotide sequence having at least 95% nucleotide sequence identity with SEQ ID NO: 20 and encoding the Ad7 pVI polypeptide;

[0262] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 20 and encoding an adenovirus (preferably Ad7) pVI polypeptide; or

[0263] (d) Nucleotide sequence encoding the Ad7 pVI polypeptide.

[0264] The term "Ad7 pVI peptide" as used in this article preferably includes, but is not limited to:

[0265] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 22; or

[0266] (b) Having at least 90%, 95% or 98% (preferably at least 98%) sequence identity with SEQ ID NO: 22, and wherein the pVI polypeptide contains a polypeptide whose amino acid at position 196 of SEQ ID NO: 21 is L, the polypeptide encoding an adenovirus pVI polypeptide (preferably Ad7 pVI polypeptide).

[0267] In some embodiments, the L3 region contains the Ad7 hexagon gene, that is, in some embodiments, one of the L3 polypeptides is the Ad7 hexagon polypeptide.

[0268] The term "Ad7 hexane gene" as used in this article refers to a gene that contains or consists of the following:

[0269] (a) The nucleotide sequence shown in SEQ ID NO: 24;

[0270] (b) A nucleotide sequence having at least 95% (or at least 99%) nucleotide sequence identity with SEQ ID NO: 24 and encoding an Ad7 hexagonal polypeptide;

[0271] (c) A nucleotide sequence having at least 97% (or at least 99%) nucleotide sequence identity with SEQ ID NO: 24 and encoding an adenovirus (preferably Ad7) hexagonal polypeptide; or

[0272] (d) Nucleotide sequence encoding the Ad7 hexagonal polypeptide.

[0273] The term "Ad7 hexagonal polypeptide" as used herein preferably includes, but is not limited to:

[0274] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 26; or

[0275] (b) A polypeptide having at least 97%, 98% or 99% (preferably at least 99%) sequence identity with SEQ ID NO: 26 and encoding an adenovirus (preferably Ad7) hexagonal polypeptide.

[0276] In some embodiments, the genome of the adenovirus of the present invention does not contain the Ad7 hexane gene inserted into the E1 or E3 region.

[0277] Adenoviral hexagonal peptides have been well characterized (Medina-Kauwe, Ther. Deliv. 2013 Feb; 4(2): 267–277). These hexagonal peptides can form multiple (e.g., 240) homotrimers that constitute the 20 faces of the icosahedral adenoviral capsid, encapsulating the adenoviral genome and related proteins. The hexagonal trimers are crucial for maintaining the structural integrity and stability of the capsid, and are therefore essential for efficient transfection of target cells.

[0278] The transfection efficiency of adenoviruses containing hexagonal peptide variants of the present invention on target cells (e.g., HEK293 cells) can be tested by exposing target cells to the adenovirus and then measuring the percentage of cells expressing genes encoded by the genome packaged within the adenovirus capsid (e.g., the E1A gene or the GFP reporter gene), and comparing the results with those obtained by testing adenoviruses containing wild-type hexagonal peptides (e.g., one of SEQ ID NO: 25) under the same conditions.

[0279] Transfection assays can be performed, for example, by measuring the percentage of HEK293 cells expressing the GFP reporter gene encoded by the adenoviral genome using flow cytometry, as described by 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 being compared should have the same capsid and the same genome, differing only in the hexagonal polypeptide and hexagonal gene.

[0280] The hexagonal polypeptide of the present invention comprises: an adenovirus containing such hexagonal polypeptide, wherein the transfection efficiency is at least 0.001% or 0.01% of the transfection efficiency obtainable using an adenovirus containing the wild-type hexagonal polypeptide of SEQ ID NO: 25 under the same assay conditions, preferably at least 0.1%, and most preferably at least 1%.

[0281] In some embodiments, the L3 region contains an adenoviral protease gene; that is, in some embodiments, one of the L3 polypeptides is an adenoviral protease polypeptide. Preferably, the adenoviral protease gene is an Ad7 protease gene, encoding the Ad7 protease.

[0282] The genome of the chimeric adenovirus includes an L4 region containing one or more L4 genes encoding one or more L4 polypeptides. Preferably, the L4 region is the Ad7 L4 region.

[0283] The genome of the chimeric adenovirus contains an E3 region. This E3 region can be a complete or substantially complete E3 region (i.e., a region without any missing parts compared to the E3 region of wild-type adenovirus (preferably Ad3 or Ad7)).

[0284] In other embodiments, the genome of the chimeric adenovirus includes one or more missing E3 regions (compared to the E3 regions of wild-type adenovirus (preferably Ad3 or Ad7)).

[0285] The genome of the chimeric adenovirus further includes an L5 region, which contains one or more L5 genes encoding one or more L5 polypeptides. Preferably, the L5 region is the Ad7 L5 region.

[0286] In some embodiments, one of the L5 genes is an adenoviral spike protein gene encoding a spike polypeptide; that is, in some embodiments, one of the L5 polypeptides is a spike polypeptide. Preferably, the spike protein gene is an Ad3 spike protein gene or an Ad7 spike protein gene, encoding an Ad3 or Ad7 spike polypeptide, respectively; more preferably, it is an Ad7 spike protein gene encoding an Ad7 spike polypeptide.

[0287] The term "Ad3 spike protein gene" as used in this article refers to a gene that contains or consists of the following:

[0288] (a) The nucleotide sequence shown in SEQ ID NO: 31;

[0289] (b) A nucleotide sequence having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) nucleotide sequence identity with SEQ ID NO: 31 and encoding an adenovirus (preferably Ad3) spike polypeptide; or

[0290] (c) Nucleotide sequence encoding Ad3 spike polypeptide.

[0291] The term "Ad3 spike polypeptide" as used in this article preferably includes, but is not limited to:

[0292] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 33; or

[0293] (b) A polypeptide having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) sequence identity with SEQ ID NO: 33 and encoding an adenovirus (preferably Ad3) spike polypeptide.

[0294] The term "Ad7 spike protein gene" as used in this article refers to a gene that contains or consists of the following:

[0295] (a) The nucleotide sequence shown in SEQ ID NO: 32;

[0296] (b) A nucleotide sequence having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) nucleotide sequence identity with SEQ ID NO: 32 and encoding an adenovirus (preferably Ad7) spike polypeptide; or

[0297] (c) Nucleotide sequence encoding Ad7 spike polypeptide.

[0298] The term "Ad7 spike polypeptide" as used in this article preferably includes, but is not limited to:

[0299] (a) A polypeptide with the amino acid sequence shown in SEQ ID NO: 34; or

[0300] (b) A polypeptide having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) sequence identity with SEQ ID NO: 34 and encoding an adenovirus (preferably Ad7) spike polypeptide.

[0301] In some embodiments of the present invention, the genome of the chimeric adenovirus does not contain the Ad3 spike protein gene. In some embodiments of the present invention, the genome of the chimeric adenovirus does not contain the L5 region containing the Ad3 spike protein gene.

[0302] Adenoviral spike peptides have been well characterized (Medina-Kauwe, Ther. Deliv. 2013 Feb; 4(2): 267–277). The spike consists of a polypeptide homotrimer comprising an amino (N-terminal) tail domain and a carboxyl (C-terminal) globular head. The tail domain is capable of interacting with the pentavalent base in the capsid. These two domains are separated by an axial portion of varying length depending on the serotype, which contains repeating sequences capable of forming triple β-helices within the spike homotrimer. The spike peptide plays a crucial role in cell attachment and entry, and is therefore essential for efficient transfection of target cells.

[0303] The transfection efficiency of adenovirus containing the spike peptide of the present invention on target cells (e.g., HEK293 cells) can be tested by exposing target cells to the adenovirus and then measuring the percentage of cells expressing genes encoded by the genome packaged in the adenovirus capsid (e.g., the E1A gene or the GFP reporter gene), and comparing the results with those obtained by testing adenovirus containing wild-type spike peptide (e.g., one of SEQ ID NO: 33) under the same conditions.

[0304] Transfection assays can be performed, for example, by measuring the percentage of HEK293 cells expressing the GFP reporter gene encoded by the viral genome using flow cytometry, as described by 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 being compared should have the same capsid and the same genome, differing only in the spike polypeptide and spike protein genes.

[0305] The spike peptide of the present invention comprises: an adenovirus containing such a spike peptide, wherein the transfection efficiency is at least 0.001% or 0.01% of the transfection efficiency obtainable using an adenovirus containing the wild-type spike peptide of SEQ ID NO: 33 under the same assay conditions, preferably at least 0.1%, and most preferably at least 1%.

[0306] Second aspect of the invention

[0307] In a second aspect, the present invention provides an adenovirus, wherein the genome of the adenovirus comprises:

[0308] (B) E3 area:

[0309] (a) The E3 region contains 12.1K and 14.7K ORFs from the E3 region of group B adenovirus, or corresponding ORFs from the E3 region of non-group B adenovirus; and

[0310] (b) The E3 region therein does not contain a functional 20K, 20.6K, 7.7K or 10.3K ORF from a group B adenovirus E3 region, or a corresponding ORF from a non-group B adenovirus E3 region.

[0311] The adenovirus of this invention comprises an E3 region. The E3 region is typically located between the L4 and L5 genes (see...). Figure 1 A). The E3 region contains multiple ORFs. The E3 region of wild-type group B adenovirus contains the following nine ORFs: 12.1K, 16.1K, 19.3K, 20K, 20.6K, 7.7K, 10.3K, 14.9K, and 14.7K.

[0312] Most published studies on the function of proteins encoded by the E3 region ORF of adenoviruses are related to group C adenovirus Ad5. In Ad5, the E3 ORF encodes proteins that regulate the host immune system.

[0313] The E3 region is often completely deleted in adenoviral vectors; under standard and optimized cell culture conditions, this deletion has minimal impact on viral phenotype. Therefore, for adenoviral vectors used under such conditions, the E3 region can be considered non-essential. However, in more relevant models and in real-world infections, at least a portion of the E3 ORF appears to play an important role in host immune evasion.

[0314] The nucleotide sequence of the Ad7 E3 region in this article is SEQ ID NO: 35.

[0315] In this document, references to the E3 region of group B adenovirus preferably refer to the nucleotide sequence shown in SEQ ID NO: 35, or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it.

[0316] Preferably, the variant encodes the following nine ORFs: 12.1K, 16.1K, 19.3K, 20K, 20.6K, 7.7K, 10.3K, 14.9K, and 14.7K.

[0317] The nucleotide sequence of Ad7 12.1K ORF in this article is SEQ ID NO: 36.

[0318] In this document, references to group B adenovirus E3 12.1K ORF preferably refer to the nucleotide sequence shown in SEQ ID NO: 36, or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a polypeptide having the same function as group B adenovirus E3 12.1K ORF.

[0319] The nucleotide sequence of Ad7 16.1K ORF in this article is SEQ ID NO: 37.

[0320] In this document, references to group B adenovirus E3 16.1K ORF preferably refer to the nucleotide sequence shown in SEQ ID NO: 37, or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a polypeptide having the same function as group B adenovirus E3 16.1K ORF.

[0321] Group B adenovirus E3 19.3K ORF encodes a polypeptide that blocks the presentation of MHC class I restricted antigens; this reduces the killing effect of cytotoxic T cells on cells.

[0322] The nucleotide sequence of Ad7 19.3K ORF in this article is SEQ ID NO: 38.

[0323] In this document, references to group B adenovirus E3 19.3K ORF preferably refer to the nucleotide sequence shown in SEQ ID NO: 38, or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a polypeptide that blocks the presentation of MHC class I restricted antigens.

[0324] The nucleotide sequence of Ad7 20K ORF in this article is SEQ ID NO: 39.

[0325] In this document, references to group B adenovirus E3 20K ORF preferably refer to the nucleotide sequence shown in SEQ ID NO: 39, or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a polypeptide having the same function as group B adenovirus E3 20K ORF.

[0326] The nucleotide sequence of Ad7 20.6K ORF in this article is SEQ ID NO: 40.

[0327] In this document, references to group B adenovirus E3 20.6K ORF preferably refer to the nucleotide sequence shown in SEQ ID NO: 40, or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a polypeptide having the same function as group B adenovirus E3 20.6K ORF.

[0328] The nucleotide sequence of Ad7 7.7K ORF in this article is SEQ ID NO: 41.

[0329] In this document, references to group B adenovirus E3 7.7K ORF preferably refer to the nucleotide sequence shown in SEQ ID NO: 41, or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a polypeptide having the same function as group B adenovirus E3 7.7K ORF.

[0330] Group B adenovirus E3 10.3K ORF encodes RIDα; this inhibits immune-induced cell death.

[0331] The nucleotide sequence of Ad7 10.3K ORF in this article is SEQ ID NO: 42.

[0332] In this document, references to group B adenovirus E3 10.3K ORF preferably refer to the nucleotide sequence shown in SEQ ID NO: 42, or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a RIDα polypeptide.

[0333] Group B adenovirus E3 14.9K ORF encodes RIDβ; this inhibits immune-induced cell death.

[0334] The nucleotide sequence of Ad7 14.9K ORF in this article is SEQ ID NO: 43.

[0335] In this document, references to group B adenovirus E3 14.9K ORF preferably refer to the nucleotide sequence shown in SEQ ID NO: 43, or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a RIDβ polypeptide.

[0336] Group B adenovirus E3 14.7K ORF encodes a TNF-mediated inhibitor of apoptosis.

[0337] The nucleotide sequence of Ad7 14.7K ORF in this article is SEQ ID NO: 44.

[0338] In this document, references to group B adenovirus E3 14.7K ORF preferably refer to the nucleotide sequence shown in SEQ ID NO: 44, or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a TNF-mediated inhibitor of apoptosis.

[0339] Compared to the corresponding region of wild-type adenovirus, the adenovirus of the present invention has a deletion in the E3 region, wherein the deletion includes one or more specific E3 region ORFs present in wild-type adenovirus.

[0340] In some embodiments, the present invention provides an oncolytic adenovirus whose genome includes an E3 region: (a) wherein the E3 region includes 12.1K and 14.7K ORFs from the E3 region of group B adenovirus, or corresponding ORFs from the E3 region of non-group B adenovirus; and (b) wherein the E3 region does not contain functional 20K, 20.6K, 7.7K, or 10.3K ORFs from the E3 region of group B adenovirus, or corresponding ORFs from the E3 region of non-group B adenovirus.

[0341] By comparing the sequences of the genes mentioned in this article with the E3 region of non-group B adenoviruses (e.g., using BLAST), it is easy to find genes or ORFs that "correspond" to the genes in the E3 region of group B adenoviruses mentioned in this article. Figure 9 The E3 region of group B adenovirus and the corresponding E3 regions of representative viruses from groups A, C to G are shown.

[0342] For example, the following nucleotide sequences of the E3 region of non-group B adenoviruses can be found at the following locations: Ad26 - GenBank EF153474.1; Ad6 - GenBank OP871032.1; Ad49: GenBank DQ393829.1; Ad10 - GenBank JN226746.1.

[0343] As used herein, the term "functional...ORF" refers to the ability of an ORF to encode an mRNA or protein that performs its normal function fully or substantially. In some implementations, the absence of at least 10%, 20%, 30%, 40%, or 50% or more of the ORF will result in the ORF losing its function.

[0344] Therefore, in some embodiments, the E3 region contains no more than 20K, 20.6K, 7.7K, or 10.3K ORFs from the E3 region of group B adenovirus, or 50%, 60%, 70%, 80%, or 90% (preferably no more than 90%) of the corresponding ORFs from the E3 region of non-group B adenovirus. In some embodiments, the term "does not contain a functional...ORF" means that the E3 region does not contain that ORF.

[0345] In some embodiments, the present invention provides an oncolytic adenovirus whose genome includes an E3 region, wherein the E3 region comprises or is composed of the following:

[0346] (a) Group B adenovirus E3 12.1K ORF;

[0347] (b) 3' truncated group B adenovirus E3 16.1K ORF;

[0348] (c) 5' truncated group B adenovirus E3 10.3K ORF;

[0349] (d) Group B adenovirus E3 14.9K ORF; and

[0350] (e) Group B adenovirus E3 14.7K ORF,

[0351] Or the corresponding ORF from the E3 region of a non-group B adenovirus.

[0352] Connected sequentially in the 5'-3' order described above, wherein the E3 region may optionally contain one or more transgenes located inside or adjacent to the one or more ORFs.

[0353] In other embodiments, the present invention provides an oncolytic adenovirus whose genome includes an E3 region, wherein the E3 region comprises or is composed of the following:

[0354] (a) Group B adenovirus E3 12.1K ORF;

[0355] (b) Group B adenovirus E3 16.1K ORF;

[0356] (c) 3' truncated group B adenovirus E3 19.3K ORF;

[0357] (d) 5' truncated group B adenovirus E3 14.9K ORF; and

[0358] (e) Group B adenovirus E3 14.7K ORF,

[0359] Or the corresponding ORF from the E3 region of a non-group B adenovirus.

[0360] The E3 region is connected in the order of 5'→3' as described above, and may optionally contain one or more transgenes located inside or adjacent to the one or more ORFs.

[0361] As used herein, the term "continuous linkage" means that the specific ORFs are linked together without any nucleotides in between or without any spacer nucleotides of significant length. In other words, the E3 region consists essentially of only the specific ORF. However, in some embodiments, the E3 region may optionally contain one or more transgenes located within or adjacent to the one or more ORFs. In such embodiments, the term "continuous linkage" means that the specific ORFs are linked together, except where one or more transgenes are present.

[0362] In some embodiments, the E3 region comprises a 3'-truncated group B adenovirus (preferably Ad7) 16.1K ORF. In this context, the term "3'-truncated" means that the 3' end of the 16.1K ORF has been deleted. For example, at least 10%, 20%, 30%, 40%, or 50% of the 3' end of the 16.1K ORF has been deleted (preferably measured from the 3' end). Preferably, the truncation is due to the deletion of nucleotides 356 to 441 in SEQ ID NO: 37. The 3'-truncated group B adenovirus (preferably Ad7) 16.1K ORF may still retain some functional activity.

[0363] In some embodiments, the E3 region comprises a 3'-truncated group B adenovirus (preferably Ad7) 19.3K ORF. 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 measured from the 3' end). Preferably, the truncation is due to the deletion of nucleotides 402 to 519 (the ends of the 19.3K ORF) in SEQ ID NO: 38. This 3'-truncated group B adenovirus (preferably Ad7) 19.3K ORF may still retain some functional activity.

[0364] In some embodiments, the E3 region comprises a 5'-truncated group B adenovirus (preferably Ad7) 10.3K ORF. 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 5' end of the 10.3K ORF has been deleted (preferably measured from the 5' end). Preferably, the truncation is due to the deletion of nucleotides 1 to 236 in SEQ ID NO: 42.

[0365] In some embodiments, the E3 region comprises a 5'-truncated 14.9K ORF of group B adenovirus (preferably Ad7). 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 5' end of the 14.9K ORF has been deleted (preferably measured from the 5' end). Preferably, the truncation is due to the deletion of nucleotides 1 to 380 in SEQ ID NO: 43 (i.e., from the beginning of the 14.9K ORF to the last deleted nucleotide in the B deletion).

[0366] In other embodiments, the present invention provides an oncolytic adenovirus whose genome includes an E3 region, wherein the E3 region encodes a fusion protein comprising the fusion of the C-terminus of a C-terminated group B adenovirus E3 16.1K protein with the N-terminus of an N-terminated group B adenovirus E3 10.3K protein, or a fusion protein comprising the fusion of the corresponding terminus of a corresponding protein from a non-group B adenovirus E3 region.

[0367] In other embodiments, the present invention provides an oncolytic adenovirus whose genome includes an E3 region, wherein the E3 region encodes a fusion protein comprising the fusion of the C-terminus of a C-terminal truncated group B adenovirus E3 19.3K protein with the N-terminus of an N-terminal truncated group B adenovirus E3 14.9K protein, or a fusion protein comprising the fusion of the corresponding terminus of a corresponding protein from a non-group B adenovirus E3 region.

[0368] In some preferred embodiments, the adenovirus of the present invention comprises an E3 region, wherein the E3 region is missing compared to the corresponding region of a wild-type group B adenovirus: (a) the start point of the deletion is located at nucleotide 629 of the group B adenovirus E3 region nucleotide sequence shown in SEQ ID NO: 35, or at the corresponding nucleotide in the non-group B adenovirus E3 region nucleotide sequence; and (b) the end point of the deletion is located at nucleotide 2,892 of the group B adenovirus E3 region nucleotide sequence shown in SEQ ID NO: 35, or at the corresponding nucleotide in the non-group B adenovirus E3 region nucleotide sequence.

[0369] In other preferred embodiments, the adenovirus of the present invention comprises an E3 region, wherein the E3 region is deleted compared to the corresponding region of wild-type group B adenovirus: (a) wherein the start point of the deletion is located at nucleotide 1,099 of the group B adenovirus E3 region nucleotide sequence shown in SEQ ID NO: 35, or at the corresponding nucleotide in the non-group B adenovirus E3 region nucleotide sequence; and (b) wherein the end point of the deletion is located at nucleotide 3,283 of the group B adenovirus E3 region nucleotide sequence shown in SEQ ID NO: 35, or at the corresponding nucleotide in the non-group B adenovirus E3 region nucleotide sequence. (The nucleotide numbers above refer to the nucleotides retained at the ends of the E3 region after deletion.)

[0370] In other preferred embodiments, the adenovirus of the present invention includes an E3 region, wherein the E3 region is deleted compared to wild-type group B adenovirus, wherein the deletion corresponds to: (a) Ad7 genomic nucleotides 28011-30274; or (b) Ad7 genomic nucleotides 28482-30665. (These nucleotide numbers refer to the nucleotides retained at the ends of the E3 region after deletion.)

[0371] The complete sequence of the Ad7 genome is available from Genbank (AY594255.1), and its sequence is incorporated herein by reference.

[0372] In a particularly preferred embodiment, the E3 region of the adenovirus has a nucleotide sequence as shown in SEQ ID NO: 45, or a variant having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it, preferably wherein the variant encodes:

[0373] (a) Group B adenovirus E3 12.1K ORF;

[0374] (b) 3' truncated group B adenovirus E3 16.1K ORF;

[0375] (c) 5' truncated group B adenovirus E3 10.3K ORF;

[0376] (d) Group B adenovirus E3 14.9K ORF; and

[0377] (e) Group B adenovirus E3 14.7K ORF.

[0378] In a particularly preferred embodiment, the E3 region of the adenovirus has a nucleotide sequence as shown in SEQ ID NO: 47, or a variant having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it, preferably wherein the variant encodes:

[0379] (a) Group B adenovirus E3 12.1K ORF;

[0380] (b) Group B adenovirus E3 16.1K ORF;

[0381] (c) 3' truncated group B adenovirus E3 19.3K ORF;

[0382] (d) 5' truncated group B adenovirus E3 14.9K ORF; and

[0383] (e) Group B adenovirus E3 14.7K ORF.

[0384] Third aspect of the invention

[0385] In a third aspect, the present invention provides an adenovirus, wherein the genome of the adenovirus comprises:

[0386] (C) The E2B DNA polymerase I gene encoding a DNA polymerase I polypeptide, wherein the amino acid corresponding to the 34th amino acid in SEQ ID NO: 49 in the DNA polymerase I polypeptide sequence is Asp or Glu, preferably Asp;

[0387] or

[0388] The E2B pTP gene encoding the terminal protein, wherein the amino acid corresponding to amino acid 623 in SEQ ID NO:51 in the terminal protein sequence is selected from the group consisting of Thr, Asn, Cys, Gln and Ser, preferably Thr.

[0389] In some embodiments, if the adenovirus is an Ad1 adenovirus, the amino acid corresponding to amino acid position 623 of SEQ ID NO: 51 in the terminal protein sequence is not Asn. In some embodiments, if the adenovirus is an Ad41 adenovirus, the amino acid corresponding to amino acid position 623 of SEQ ID NO: 51 in the terminal protein sequence is not Gln. In some embodiments, if the adenovirus is an Ad4 or 4a adenovirus, the amino acid corresponding to amino acid position 623 of SEQ ID NO: 51 in the terminal protein sequence is not Thr.

[0390] The present invention particularly relates to pharmaceutical compositions comprising such adenoviruses, optionally in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0391] The E2B DNA polymerase I gene encodes adenovirus DNA polymerase I. DNA polymerase I is essential for adenovirus genomic DNA amplification.

[0392] The nucleotide and corresponding amino acid sequences of the wild-type Ad3 E2B DNA polymerase I gene and its polypeptide in this article are listed as SEQ ID NO: 48 and 49, respectively.

[0393] The term "E2B DNA polymerase I gene" as used in this article refers to a gene that contains or consists of the following:

[0394] (i) The nucleotide sequence shown in SEQ ID NO: 48;

[0395] (ii) Having at least 80%, 85%, 90%, 95%, or 99% (preferably at least 99%) nucleotide sequence identity with SEQ ID NO: 48, and preferably encoding a nucleotide sequence of DNA polymerase I; or

[0396] (iii) The nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 49.

[0397] The term "E2B DNA polymerase I" as used in this article refers to a polypeptide that contains or is composed of the following:

[0398] (i) The amino acid sequence shown in SEQ ID NO: 49; or

[0399] (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, and preferably having DNA polymerase I activity.

[0400] The E2B pTP gene encodes an adenoviral proterminus protein. This polypeptide is also known as a proterminus protein. pTP is essential for the initiation of viral genome replication.

[0401] The nucleotide and corresponding amino acid sequences of the wild-type Ad3 E2B pTP gene and its polypeptide in this article are listed as SEQ ID NO: 50 and 51, respectively.

[0402] The term "E2B pTP gene" as used in this article refers to a gene that contains or consists of the following:

[0403] (i) The nucleotide sequence shown in SEQ ID NO: 50;

[0404] (ii) Having at least 80%, 85%, 90%, 95%, or 99% (preferably at least 99%) nucleotide sequence identity with SEQ ID NO: 50, and preferably encoding a nucleotide sequence of a terminal protein; or

[0405] (iii) The nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 51.

[0406] The term "E2B terminal protein" as used in this article refers to a polypeptide that contains or is composed of the following:

[0407] (i) The amino acid sequence shown in SEQ ID NO: 51; or

[0408] (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, and preferably encoding a terminal protein.

[0409] The inventors discovered that, compared with control adenoviruses that do not carry the mutation, adenoviruses containing the G34D mutation in the E2B DNA polymerase I polypeptide have enhanced adenoviral DNA replication ability and enhanced oncolytic activity.

[0410] Aspartic acid (Asp, D) and glutamic acid (Glu, E) are both negatively charged amino acids. Therefore, in one embodiment, the present invention provides an adenovirus, wherein the genome of said adenovirus contains an E2B DNA polymerase I gene encoding a DNA polymerase I polypeptide, wherein the amino acid corresponding to amino acid position 34 in SEQ ID NO: 49 of said DNA polymerase I polypeptide sequence is Asp or Glu, preferably Asp. For example, the codons corresponding to nucleotides 100-102 in SEQ ID NO: 48 in the adenovirus genome are gac, gau, gaa, or gag.

[0411] In the wild-type adenovirus genome, the coding sequences of the E2B DNA polymerase I gene and the E2B pTP gene overlap in different reading frames. One consequence of this is that some mutations at the 5' end of the E2B DNA polymerase gene can lead to mutations at the 3' end of the E2B pTP gene and the corresponding polypeptide.

[0412] For example, the G34D mutation in the E2B DNA polymerase polypeptide (e.g., the mutation of the codon ggc→gac) leads to the A623T mutation in the E2BpTP polypeptide (e.g., the codon gcg→acg).

[0413] The table below shows the corresponding mutations in the E2B pTP peptide caused by G34D and G34E mutations in the E2B DNA polymerase I peptide:

[0414] Table 4: Effects of G34D and G34E mutations in E2B DNA polymerase I peptide on the E2B pTP peptide sequence

[0415]

[0416] As shown in the table above, mutations in G34D and G34E in the E2B DNA polymerase I polypeptide result in a mutation at amino acid position 623 of the E2B pTP polypeptide to Thr, Met, Lys, or Arg.

[0417] Therefore, in another preferred embodiment, the present invention provides an adenovirus, wherein the genome of the adenovirus further comprises an E2B pTP gene encoding a terminal protein, wherein the coding region of the E2B DNA polymerase I gene overlaps with the coding region of the E2B pTP gene, and wherein the amino acid corresponding to amino acid 623 in SEQ ID NO: 51 in the terminal protein precursor sequence is selected from the group consisting of Thr, Met, Lys, and Arg, preferably Thr. Preferably, the amino acid corresponding to amino acid 623 in SEQ ID NO: 51 in the E2B terminal protein polypeptide is not a nonsense (i.e., termination) mutation.

[0418] The inventors discovered that, compared with control adenoviruses that do not carry the mutation, adenoviruses containing the A623T mutation in the E2B pTP peptide have enhanced adenovirus DNA replication capacity and infectious viral progeny yield.

[0419] Thr (T), Asn (N), Cys (C), Gln (Q), and Ser (S) are all polar or neutral amino acids. Therefore, in yet another embodiment, the present invention provides an adenovirus whose genome includes an E2B pTP gene encoding a terminal protein, wherein the amino acid corresponding to amino acid position 623 of SEQ ID NO: 51 in the terminal protein sequence is selected from the group consisting of Thr, Asn, Cys, Gln, 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 Gln. In some embodiments, if the adenovirus is an Ad41 adenovirus, then the amino acid corresponding to amino acid position 623 of SEQ ID NO: 51 in the terminal protein sequence is not Gln.

[0420] For example, the codons in the adenovirus genome corresponding to nucleotides 5326-5328 of SEQ ID NO: 50 encode:

[0421] (i)Thr For example, acu, acc, aca, acg

[0422] (ii)Asn For example, aau, aac,

[0423] (iii) Cys, for example, ugu, ugc

[0424] (iv)Gln For example, caa, cag

[0425] (v) Ser For example, agu, agc, ucu, ucc, uca, ucg

[0426] As previously mentioned, in the wild-type adenovirus genome, the coding sequences of the E2B DNA polymerase I gene and the E2B pTP gene overlap in different reading frames. One consequence of this is that some mutations at the 3' end of the E2B pTP gene can lead to mutations at the 5' end of the E2B DNA polymerase I gene and the corresponding polypeptide. For example, the A623T mutation in the E2B pTP polypeptide (e.g., codon gcg→acg) leads to the G34D mutation in the E2B DNA polymerase polypeptide (e.g., codon ggc→gac).

[0427] The table below shows the corresponding mutations in the E2B DNA polymerase I polypeptide caused by the mutation of amino acid 623 of the E2B pTP polypeptide to a polar or neutral amino acid:

[0428] Table 5: Effects of polar or neutral amino acid mutation at position 623 of the E2B pTP peptide on the E2B DNA polymerase I peptide sequence.

[0429]

[0430]

[0431] Therefore, in another preferred embodiment, the present invention provides an adenovirus, wherein the genome of the adenovirus further comprises an E2B DNA polymerase I gene encoding a DNA polymerase I polypeptide, wherein the coding region of the E2B pTP gene overlaps with the coding region of the E2B DNA polymerase I gene, and wherein the amino acid corresponding to amino acids 34-35 in SEQ ID NO: 49 in the DNA polymerase I polypeptide sequence is 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.

[0432] Regarding the above list (i.e., Asp-Ser, Asp-Pro, etc.), the first amino acid listed in each pair corresponds to the 34th amino acid in SEQ ID NO: 49, and the second amino acid corresponds to the 35th amino acid in SEQ ID NO: 49. Preferably, the amino acids in the E2B DNA polymerase I polypeptide corresponding to positions 34-35 in SEQ ID NO: 49 are not nonsense (i.e., termination) mutations.

[0433] In a further embodiment, the present invention provides a mutant E2B DNA polymerase I gene, wherein the nucleotide corresponding to nucleotide 101 in SEQ ID NO: 48 is A. Preferably, the nucleotide sequence of the mutant E2B DNA polymerase I gene also has at least 80%, 85%, 90%, 95% or 99% (preferably at least 99%) nucleotide sequence identity with SEQ ID NO: 48; and preferably it encodes a polypeptide having E2B DNA polymerase I activity.

[0434] In a further embodiment, the present invention provides a mutant E2B pTP gene, wherein the nucleotide corresponding to nucleotide 5,326 of SEQ ID NO: 50 in the mutant E2B pTP gene is 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 with SEQ ID NO: 50; and preferably it encodes a polypeptide having E2B terminal protein precursor activity.

[0435] The genes of the present invention are preferably isolated or purified. The term "isolated gene" as used herein means that the nucleic acid molecule is not linked to other genes or nucleotide sequences typically associated with it in adenoviruses. For example, the isolated nucleic acid containing the target DNA polymerase I gene of the present invention will not cause the DNA polymerase I gene to be continuously linked to its directly adjacent nucleotide sequences (e.g., nucleic acids encoding its directly adjacent genes) in the adenovirus genome. References to nucleic acids containing the DNA polymerase I gene (and, similarly, the pTP gene) should be interpreted accordingly. Therefore, the isolated gene is not a wild-type gene of adenovirus.

[0436] In a further embodiment, the present invention provides a mutant E2B DNA polymerase I polypeptide, wherein the amino acid corresponding to the 34th amino acid in SEQ ID NO: 49 is D or E, preferably D. Preferably, the amino acid sequence of the mutant E2B DNA polymerase 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 polymerase I activity.

[0437] In a further embodiment, the present invention provides a mutant E2B pTP polypeptide, wherein the amino acid corresponding to amino acid position 623 of SEQ ID NO: 51 in the mutant E2B pTP polypeptide 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.

[0438] Fourth aspect of the invention

[0439] In a fourth aspect, the present invention provides an adenovirus, wherein the genome of the adenovirus comprises one, two, or all of (A), (B), and (C):

[0440] (A) Ad3 pentagonal genes, wherein the Ad3 pentagonal genes comprise or consist of the following:

[0441] (a) The nucleotide sequence shown in SEQ ID NO: 3;

[0442] (b) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 3 and encoding an adenovirus pentagonal polypeptide; or

[0443] (c) The nucleotide sequence encoding the Ad3 pentagonal polypeptide, wherein the Ad3 pentagonal polypeptide is:

[0444] (i) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 5;

[0445] (ii) A polypeptide having at least 99.3%, 99.5%, or 99.7% sequence identity with SEQ ID NO: 5 and encoding an adenovirus pentagonal polypeptide; or

[0446] (iii) Having at least 95%, 99% or 99.5% sequence identity with SEQ ID NO: 5, wherein the pentagonal polypeptide contains polypeptides corresponding to amino acids V, T, I and D at positions 11, 158, 178 and 326 of SEQ ID NO: 5, respectively, and the polypeptide encodes an adenovirus pentagonal polypeptide;

[0447] as well as

[0448] Ad7 hexagonal genes, wherein the Ad7 hexagonal genes comprise or consist of the following:

[0449] (a) The nucleotide sequence shown in SEQ ID NO: 24;

[0450] (b) A nucleotide sequence having at least 97% or at least 99% nucleotide sequence identity with SEQ ID NO: 24 and encoding an adenovirus hexapod polypeptide; or

[0451] (c) The nucleotide sequence encoding the Ad7 hexagonal polypeptide, wherein the Ad7 hexagonal polypeptide is:

[0452] (i) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 26; or

[0453] (ii) A polypeptide having at least 97%, 98% or 99% sequence identity with SEQ ID NO: 26 and encoding an adenovirus hexapod polypeptide;

[0454] And optionally, it also includes the Ad7 spike protein gene;

[0455] (B) E3 area:

[0456] (a) The E3 region contains 12.1K and 14.7K ORFs from the E3 region of group B adenovirus, or corresponding ORFs from the E3 region of non-group B adenovirus; and

[0457] (b) The E3 region therein does not contain a functional 20K, 20.6K, 7.7K or 10.3K ORF from a group B adenovirus E3 region, or a corresponding ORF from a non-group B adenovirus E3 region;

[0458] as well as

[0459] (C) The E2B DNA polymerase I gene encoding the DNA polymerase I polypeptide.

[0460] The amino acid corresponding to the 34th amino acid in SEQ ID NO: 49 of the DNA polymerase I polypeptide sequence is Asp or Glu, preferably Asp;

[0461] or

[0462] The E2B pTP gene encodes the terminal protein.

[0463] The amino acid corresponding to amino acid 623 in SEQ ID NO: 51 in the preterminal protein sequence is selected from the group consisting of Thr, Asn, Cys, Gln and Ser, preferably Thr.

[0464] The adenovirus of the fourth aspect of this invention may comprise any combination of the following features:

[0465] (A) Any feature of the adenovirus in the first aspect of the present invention;

[0466] (B) Any features of the adenovirus in the second aspect of the invention; and

[0467] (C) Any feature of the adenovirus in the third aspect of the present invention.

[0468] Specifically, in one embodiment of the fourth aspect, the genome of the adenovirus may include:

[0469] (A) Regions L2 and L3, where:

[0470] (a) The L2 region includes:

[0471] (i) The Ad3 pentagonal gene as defined above;

[0472] (ii) Ad3 pVIII gene;

[0473] (iii) Ad3V gene; and

[0474] (iv) Ad3 or Ad7 pX gene; and / or

[0475] (b) The L3 region includes:

[0476] (i) Ad3 or Ad7 pVI gene;

[0477] (ii) the Ad7 hexane gene as defined above; and

[0478] (iii) Ad7 protease gene;

[0479] (B) Region E3, which contains or consists of the following:

[0480] (a) Group B adenovirus E3 12.1K ORF;

[0481] (b) 3' truncated group B adenovirus E3 16.1K ORF;

[0482] (c) 5' truncated group B adenovirus E3 10.3K ORF;

[0483] (d) Group B adenovirus E3 14.9K ORF; and

[0484] (e) Group B adenovirus E3 14.7K ORF,

[0485] Or the corresponding ORF from the E3 region of a non-group B adenovirus.

[0486] The E3 region is connected in the order of 5'→3' as described above, and may optionally contain one or more transgenes located inside or adjacent to the one or more ORFs.

[0487] as well as

[0488] (C) The E2B DNA polymerase I gene encoding the DNA polymerase I polypeptide.

[0489] The coding region of the E2B pTP gene overlaps with the coding region of the E2B DNA polymerase I gene, and the amino acid corresponding to amino acids 34-35 in SEQ ID NO: 49 in the DNA polymerase I polypeptide is 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.

[0490] Specifically, in another embodiment of the fourth aspect, the genome of the adenovirus may include:

[0491] (A) Regions L2 and L3, where:

[0492] (a) The L2 region includes:

[0493] (i) The Ad3 pentagonal gene as defined above;

[0494] (ii) Ad3 pVIII gene;

[0495] (iii) Ad3V gene; and

[0496] (iv) Ad3 pX gene; and / or

[0497] (b) The L3 region includes:

[0498] (i) Ad3 pVI gene;

[0499] (ii) the Ad7 hexane gene as defined above; and

[0500] (iii) Ad7 protease gene; and

[0501] The L5 region contains the Ad7 spike protein gene;

[0502] (B) The E3 region, which is deleted compared to wild-type group B adenovirus, wherein the deletion corresponds to:

[0503] (a) Ad7 genomic nucleotides 28011-30274; or

[0504] (b) Ad7 genomic nucleotides 28482-30665;

[0505] as well as

[0506] (C) The E2B DNA polymerase I gene, which contains or is composed of the following:

[0507] (i) The nucleotide sequence shown in SEQ ID NO: 48; or

[0508] (ii) The nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 49;

[0509] or

[0510] The E2B pTP gene contains or is composed of the following:

[0511] (i) The nucleotide sequence shown in SEQ ID NO: 50; or

[0512] (ii) The nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 51.

[0513] Fifth aspect of the invention

[0514] In another embodiment, the present invention provides an adenovirus comprising the nucleotide sequence 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 and having oncolytic activity (preferably against ovarian cancer cells, such as A549 cancer cells). More preferably, the variant has at least 95% or at least 99% sequence identity with SEQ ID NO: 58, and the adenovirus has oncolytic activity (preferably against ovarian cancer cells, such as A549 cancer cells).

[0515] In some embodiments, the adenovirus comprises a nucleotide sequence as shown in SEQ ID NO: 58.

[0516] In other embodiments, the present invention provides an adenovirus or adenovirus 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 genome of SEQ ID NO: 58, excluding potentially missing E1 and E3 regions). The genome of this adenovirus or adenovirus vector may additionally contain a transgene. Preferably, the transgene is located between nucleotides 480-3,409 or 27,381-28,868 of SEQ ID NO: 58.

[0517] In other embodiments, the present invention provides an adenovirus or adenovirus vector whose genome has 100% nucleotide sequence identity with at least 85%, 90%, 95%, or 99% (preferably at least 99%) of a continuous or discontinuous region of SEQ ID NO: 58. The discontinuous region may, for example, comprise 2, 3, or 4 regions, preferably 2 regions.

[0518] An adenovirus conforming to the fifth aspect of this invention has a genome containing all the features of (A), (B), and (C):

[0519] (A)

[0520] (a) Ad3 pentagenesis gene; and

[0521] (b) Ad7 hexane gene; and

[0522] (c) Optionally, the Ad7 spike protein gene;

[0523] (B) E3 area:

[0524] (a) The E3 region contains 12.1K and 14.7K ORFs from the E3 region of group B adenovirus, or corresponding ORFs from the E3 region of non-group B adenovirus; and

[0525] (b) The E3 region therein does not contain a functional 20K, 20.6K, 7.7K or 10.3K ORF from a group B adenovirus E3 region, or a corresponding ORF from a non-group B adenovirus E3 region;

[0526] as well as

[0527] (C) The E2B DNA polymerase I gene encoding the DNA polymerase I polypeptide.

[0528] The amino acid corresponding to the 34th amino acid in SEQ ID NO: 49 of the DNA polymerase I polypeptide sequence is Asp or Glu, preferably Asp;

[0529] or

[0530] The E2B pTP gene encodes the terminal protein.

[0531] The amino acid corresponding to amino acid 623 in SEQ ID NO: 51 in the preterminal protein sequence is selected from the group consisting of Thr, Asn, Cys, Gln and Ser, preferably Thr.

[0532] The gene of the present invention is preferably isolated or purified. As used herein, the term "isolated gene" means that the nucleic acid molecule is not linked to other genes or nucleotide sequences that are typically associated with it in adenoviruses. Therefore, the isolated gene is not a wild-type gene of an adenovirus.

[0533] The polypeptides of the present invention are preferably isolated. As used herein, the term "isolated polypeptide" means that the polypeptide is not in a mixture with other polypeptides that are normally mixed with in adenoviruses.

[0534] Many mature algorithms exist for aligning two amino acid or nucleic acid sequences. Typically, one sequence is used as a reference sequence, and the test sequence is compared to it. Sequence comparison algorithms calculate the percentage sequence identity of the test sequence relative to the reference sequence based on specified program parameters. The alignment of amino acid or nucleic acid sequences used for comparison can be performed using computer-implemented algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA) or Clustal Omega, BLASTn, and BLASTp algorithms.

[0535] The standard protein-protein BLAST (blastp) can be used to find similar sequences in protein databases. Similar to other BLAST programs, blastp aims to find locally similar regions. When sequence similarity spans the entire sequence, blastp also reports a global alignment, which is the preferred result for protein identification purposes. It is preferable to use standard or default alignment parameters. In some cases, the "low complexity filter" can be turned off.

[0536] For nucleotide sequence comparison, MEGABLAST, non-contiguous MEGABLAST, and blastn can be used to achieve this goal. Standard or default alignment parameters are preferred. MEGABLAST is specifically designed for efficiently finding long alignments between highly similar sequences. Non-contiguous MEGABLAST can be used to find nucleotide sequences that are similar to but not identical to the nucleic acids of this invention.

[0537] The BLAST nucleotide algorithm searches for similar sequences by breaking down the query sequence into short subsequences called words. The program first identifies sequences that exactly match the query word (word hits). Then, the BLAST program expands these word hits in multiple steps to generate a final alignment including gaps. In some implementations, the BLAST nucleotide search can be performed using the BLASTN program with parameters set to score=100 and wordlength=12.

[0538] One of the important parameters for controlling the search sensitivity of BLAST is word length. The most important reason why blastn is more sensitive than MEGABLAST is that it uses a shorter default word length (11). Therefore, blastn is superior to MEGABLAST in finding alignments with other biologically relevant nucleotide sequences. The word length in blastn is adjustable and can be reduced from the default value to a minimum of 7 to improve search sensitivity.

[0539] More sensitive searches can be achieved by using the newly introduced non-contiguous megablast page (www.ncbi.nlm.nih.gov / Web / Newsltr / FallWinter02 / blastlab.html). This page uses an algorithm similar to that reported by Ma et al. (Bioinformatics. 2002 Mar; 18(3): 440-5). Instead of requiring exact word matches as seeds for alignment expansion, non-contiguous megablasts use non-contiguous words within a longer template window. In encoded mode, third base wobbles are considered by focusing on finding matches at the first and second positions of the codon while ignoring mismatches at the third position. Searching in non-contiguous MEGABLAST using the same word length is more sensitive and efficient than using standard blastn with the same word length. Parameters specific to non-contiguous megablasts include: word length: 11 or 12; template: 16, 18, or 21; template type: encoded (0), non-encoded (1), or both (2).

[0540] In some implementations, the BLASTP 2.5.0+ algorithm (e.g., the version provided by NCBI) with default parameters can be used. In other implementations, the BLAST global alignment program (e.g., the version provided by NCBI) can be used to align two protein sequences using the Needleman-Wunsch algorithm, with gap costs set as: presence penalty of 11 and extension penalty of 1.

[0541] In this paper, the term "sequence identity" can be replaced with "sequence similarity" in the context of amino acid sequences. The term "similarity" allows for conservative substitutions with amino acid residues having similar physicochemical properties within a given alignment length. The similarity percentage can be determined using any reasonable similarity scoring matrix.

[0542] The present invention also provides a pharmaceutical composition comprising the adenovirus of the present invention, optionally in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0543] As used herein, the term "pharmaceutically acceptable carrier" includes all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Examples of suitable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Examples of suitable isotonic agents include sugars, polyols (e.g., mannitol, sorbitol), and sodium chloride.

[0544] 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 may be encapsulated in liposomes or biodegradable microspheres. Diluents include sterile water.

[0545] In one embodiment, the pharmaceutical composition is a liquid parenteral preparation of the adenovirus of the present invention, for example for infusion or injection.

[0546] As used herein, the term "parenteral preparation" refers to a pharmaceutical composition delivered outside the gastrointestinal tract. Typical parenteral delivery routes include injection, implantation, or infusion. In one embodiment, the pharmaceutical composition is provided in a form suitable for bolus administration.

[0547] In one embodiment, the parenteral preparation is in the form of an injectable. Injection includes intravenous, subcutaneous, intratumoral, or intramuscular injection. As used herein, "injection" means the administration of a liquid into the body via a syringe.

[0548] In one embodiment, the parenteral preparation is in the form of infusion. As used herein, the term "infusion" refers to the administration of fluid at a slower rate via drip, infusion pump, syringe pump, or equivalent device.

[0549] In one embodiment, the parenteral preparation is an infusion form for intravenous administration.

[0550] In another embodiment, the pharmaceutical composition is provided in a formulation for topical administration, including inhalation. Suitable inhalable formulations include inhalable powders, metered aerosols containing propellant gases, or inhalable solutions without propellant gases. Inhalable powders according to this disclosure generally contain the virus described herein and physiologically acceptable excipients. These inhalable powders may include monosaccharides (e.g., glucose or arabinose), disaccharides (e.g., lactose, sucrose, maltose), oligosaccharides and polysaccharides (e.g., dextran), polyols (e.g., sorbitol, mannitol, xylitol), salts (e.g., sodium chloride, calcium carbonate), or mixtures thereof. Monosaccharides or disaccharides are suitable for use, with lactose or glucose used, especially but not limited to their hydrated forms.

[0551] Particles deposited in the lungs need to have a diameter of less than 10 micrometers, for example, 1-9 micrometers, such as from 0.1 to 5 micrometers, particularly from 1 to 5 micrometers. The particle size of the virus-carrying particles is critical, so in one embodiment, the virus of the present invention can be adsorbed or absorbed onto particles, such as lactose particles of a given size.

[0552] Propellant gases suitable for preparing inhalable aerosols are well known in the art. Suitable propellant gases are selected from hydrocarbons such as n-propane, n-butane, or isobutane, and chlorinated and / or fluorinated derivatives of halogenated hydrocarbons such as methane, ethane, propane, butane, cyclopropane, or cyclobutane. These propellant gases can be used alone or in mixtures thereof. Particularly suitable propellant gases are halogenated alkane derivatives selected from TG11, TG12, TG134a, and TG227. Among these halogenated hydrocarbons, TG134a (1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable.

[0553] Inhalable aerosols containing propellant gases may also contain other components such as solubilizers, stabilizers, surfactants, antioxidants, lubricants, and pH adjusters. All of these components are known in the art.

[0554] The inhalable aerosol containing propellant gas according to the present invention may contain up to 5% by weight of active material. The aerosol according to the present invention contains, for example, 0.002-5% by weight, 0.01-3% by weight, 0.015-2% by weight, 0.1-2% by weight, 0.5-2% by weight, or 0.5-1% by weight of active ingredient.

[0555] Alternatively, local administration to the lungs can be achieved by administering a liquid solution or suspension, for example using a device such as a nebulizer, specifically a nebulizer connected to a compressor (e.g., the Pari Respiratory Equipment, Inc., Richmond, Va., connected to the Pari Master). (Pari LC-Jet Plus atomizer for compressor).

[0556] The atomizable formulations according to this disclosure can be provided, for example, in single-dose units (e.g., sealed plastic containers or vials) packaged in aluminum foil bags. Each vial contains one unit dose, with a volume of, for example, 2 mL, of solvent / solution buffer.

[0557] The pharmaceutical compositions of the present invention are generally sterile and stable under the conditions of manufacture and storage. The compositions can be formulated into solutions, microemulsions, liposomes, or other parenteral preparations suitable for human administration, and can be formulated into pre-filled devices, such as syringes or vials, particularly for single doses.

[0558] Subjects treated with the adenovirus of this invention may also receive additional treatment with one or more other chemotherapeutic agents or immunotherapeutic agents, namely specific antitumor chemotherapeutic agents, or drugs that selectively destroy malignant cells or tissues or induce an antitumor immune response. Such other chemotherapeutic agents or immunotherapeutic agents may include, for example, one or more of the following: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, checkpoint inhibitors, antibodies, and other antitumor agents.

[0559] Specific examples of chemotherapy agents include doxorubicin, 5-fluorouracil (5-FU), taxane derivatives (such as paclitaxel and docetaxel), capecitabine, irinotecan, albumin-bound paclitaxel, and platinum-based drugs (such as cisplatin, carboplatin, and oxaliplatin).

[0560] The chemotherapeutic agent may be an agent that does not interfere with adenovirus activity (e.g., does not affect the beneficial properties of the virus, such as oncolytic activity and / or the ability of the virus to replicate in cancer cells, such as viral replication in vivo).

[0561] Typically, such drug combinations are provided as two components:

[0562] (A) The first pharmaceutical composition of the adenovirus of the present invention; and

[0563] (B) A second pharmaceutical composition comprising a chemotherapeutic agent or an immunotherapeutic agent.

[0564] Therefore, the pharmaceutical combinations of the present invention can exist in combined formulations for simultaneous, single, or sequential administration, preferably for the treatment of cancer. Similarly, in the method of the present invention, the first and second pharmaceutical compositions can be administered to the patient simultaneously, single, or sequentially.

[0565] The term "combination formulation" includes fixed and non-fixed combinations. The term "fixed combination" means that the active ingredients (e.g., components (A) and (B)) are present in a single entity or dosage unit. In other words, the active ingredient is present in a single composition or formulation. The term "non-fixed 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), such as as kit components. Then, the independent components (A) and (B) (in their desired composition or formulation form) can be administered simultaneously, individually, or sequentially at the same time point or at different time points.

[0566] In cases of simultaneous administration, components (A) and (B) are administered to the subject at the same time, but not necessarily together. Components (A) and (B) may be present in a single composition or they may be present in different compositions. Components (A) and (B) may be administered at the same or different sites (inside or on the body of the subject). Components (A) and (B) may be administered via the same or different routes.

[0567] When applied sequentially, the delay in applying the second component should not result in the loss of the synergistic benefits derived from using the combination. Components (A) and (B) may each be applied once or multiple times. Components (A) and (B) may be applied in any order, such as applying component (A) first, then component (B); or applying component (B) first, then component (A).

[0568] The present invention also provides a kit comprising: (A) a first pharmaceutical composition of the adenovirus of the present invention; and (B) a second pharmaceutical composition comprising a chemotherapeutic agent, optionally accompanied by instructions for use.

[0569] In another embodiment, the present invention provides the adenovirus of the present invention for use as a treatment or as a medicine.

[0570] In another embodiment, the present invention provides an oncolytic adenovirus of the present invention for treating cancer (preferably ovarian cancer). In another embodiment, the present invention provides a method for treating a subject with cancer (preferably ovarian cancer), the method comprising administering a therapeutically effective amount of the oncolytic adenovirus of the present invention to a subject in need.

[0571] In another embodiment, the present invention provides the use of the oncolytic adenovirus of the present invention in the preparation of a medicament for treating cancer (preferably ovarian cancer). The present invention also provides the use of the oncolytic adenovirus of the present invention for treating cancer (preferably ovarian cancer); and the use of the oncolytic adenovirus of the present invention in treating cancer (preferably ovarian cancer).

[0572] The term “treating cancer” as used in this article includes killing cancer cells and / or preventing or inhibiting the spread of cancer cells.

[0573] The therapeutic index (TI, also known as the treatment ratio) is a quantitative measure of the relative safety of a drug. It is a comparison between the therapeutic dose that produces a therapeutic effect and the dose that causes toxicity. Related terms, therapeutic window and safety window, refer to the optimized dose range between efficacy and toxicity, achieving maximum therapeutic benefit with unacceptable side effects or toxicity.

[0574] For humans in clinical trials, the treatment index is defined as TD. 50 / ED 50 (of which TD) 50 This is the dose that causes toxicity in 50% of the subjects; ED 50 This is the dose that produces the minimum effective effect in 50% of the population.

[0575] The subject is a human being. The subject can be biologically male or female. The human being can be, for example, aged 0-10 years, 10-20 years, 20-30 years, 30-40 years, 40-50 years, 50-60 years, 60-70 years, 70-80 years, 80-90 years, 90-100 years, or over 100 years old. The human being can be an individual currently suffering from or at risk of suffering from a specific disease or condition (e.g., cancer, preferably ovarian cancer). In some preferred embodiments, the subject is an individual currently suffering from or who has previously suffered from cancer (preferably ovarian cancer).

[0576] As used in this article, the term "interstitium" refers to the cells and tissues that support and supply the structure of organs, glands, or other tissues in the body. The interstitium is primarily composed of connective tissue, blood vessels, lymphatic vessels, and nerves. It provides nutrients to tissues or organs and removes waste and excess fluid. The interstitium may also participate in the body's immune response as well as the growth and spread of cancer cells.

[0577] In some implementations, the cancer is a tumor.

[0578] In some preferred embodiments, the cancer is a stromal tumor. As used herein, the term "stromal tumor" includes a tumor containing at least 1%, preferably at least 5%, 10%, 20%, or 50% stromal cells (by tumor weight). In some embodiments, the stromal tumor contains 20-80% stromal cells. The stromal cells may contain CAFs. The cancer or tumor may be a type containing CAFs.

[0579] In some preferred embodiments, the cancer is a carcinoma of epithelial origin. In some preferred embodiments, the cancer is ovarian cancer, colorectal cancer, lung cancer, liver cancer, multiple myeloma, esophageal cancer, breast cancer, or pancreatic cancer. Most preferably, the cancer is ovarian cancer or stromal carcinoma.

[0580] In some embodiments, the adenovirus of the present invention can be used as a pretreatment for a therapy (e.g., surgery (neoadjuvant therapy)) to shrink tumors, treat metastases and / or prevent metastasis or further metastasis.

[0581] In other embodiments, the adenovirus of the present invention can be used post-treatment (e.g., post-operative (adjuvant therapy)) to treat metastatic lesions and / or prevent metastasis or further metastasis.

[0582] The pharmaceutical compositions of the present invention can be administered via one or more routes, using one or more methods known in the art. Components or compositions (A) and (B) can be administered via the same route or different routes. As will be understood by those skilled in the art, the route and / or manner of administration will vary depending on the expected results.

[0583] Preferred routes of administration for the pharmaceutical compositions of the present invention include intravenous, intratumoral, intraperitoneal, intrapleural, intravesical, intradermal, or other parenteral routes, such as by injection or infusion.

[0584] The phrase "parenteral administration" as used in this article refers to administration methods other than enteral and local administration, usually by injection, including but not limited to intravenous, intratumoral, intraperitoneal, intrapleural, intrabladder, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, tracheal, subcutaneous, subcutaneous tissue, intra-articular, subcapsular, and subarachnoid injections and infusions.

[0585] Alternatively, the pharmaceutical compositions of the present invention can be administered via non-parenteral routes, such as local, epidermal, or mucosal routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration.

[0586] In one embodiment, the formulation is administered intravenously (iv). This route is particularly effective for delivering oncolytic viruses because it allows rapid entry into most organs and tissues, and is especially useful for treating metastatic lesions, such as established metastases, particularly those located in highly vascularized areas such as the liver and lungs.

[0587] In one embodiment, the formulation is administered intraperitoneally (ip). This route is particularly effective for delivering oncolytic viruses to cancer types primarily confined to the peritoneum, such as ovarian, colorectal, and gastric cancers. This is because it allows rapid entry into most relevant organs and tissues to treat primary tumors and metastases without blood dilution and minimizes off-target effects.

[0588] In one embodiment, the formulation is used for intratumoral (it) administration. This route is particularly effective for delivering oncolytic viruses to localized cancer types accessible through an injection needle, as it allows for rapid entry into the tumor and minimizes off-target effects.

[0589] The pharmaceutical compositions of the present invention may comprise a therapeutically effective dose of the adenovirus of the present invention. The term "therapeuticly effective dose" refers to the amount of adenovirus suitable for achieving the desired therapeutic effect when used in a suitable treatment regimen, such as improving symptoms or condition of a disease, particularly without causing dose-limiting side effects. A dose may be considered a therapeutic dose for treating cancer or metastases when the number of viral particles is sufficient to cause a slowing or cessation of tumor or metastatic growth, or the discovery of a reduction in tumor or metastatic size, and / or an increase in patient lifespan. A suitable therapeutic dose is generally a balance between therapeutic efficacy and tolerable toxicity, for example, when the side effects and toxicity are tolerable considering the benefits achieved through treatment.

[0590] In one embodiment, the pharmaceutical composition of the present invention may contain 1×1010 Up to 1×10 14 Virus particles / agent. Preferably, the pharmaceutical composition of the present invention comprises 1×10 11 Up to 1×10 13 Virus particles / dosage. In one embodiment, the pharmaceutical composition of the present invention can be administered over 1-8 cycles, each cycle comprising one or more administrations within a 1-month period. Cycles may be administered in non-consecutive months.

[0591] Preferably, the method steps are performed in a specified order.

[0592] The adenovirus of the present invention can be readily produced using techniques well known in the art, including introducing one or more nucleotide mutations into a known adenovirus genome using standard cloning techniques (e.g., restriction endonucleases, site-directed mutagenesis, or Gibson assembly) or CRISPR-based techniques (e.g., CRISPR Cas9 / guide RNA).

[0593] In another embodiment of the first aspect (A) or the third aspect (C) of the present invention, the use of the conditionally replicating adenovirus of the present invention as a protein production vector is provided, wherein the adenovirus contains a transgene encoding a protein to be produced.

[0594] In yet another embodiment of the first aspect (A) or the third aspect (C) of the present invention, the conditionally replicating adenovirus of the present invention is provided for use as a virus production aid virus.

[0595] The publicly available information of each reference listed in this article is incorporated into this article in its entirety through citation. Attached Figure Description

[0596] Figure 1 A) Simplified diagram of the locations of early (E) and late (L) genes in the adenovirus genome. Arrows indicate viral genes, and their direction indicates the coding direction. ITR = Inverted Terminal Repeat. B) Schematic diagram of the open reading frames encoded in the L2 and L3 regions.

[0597] Figure 2: Five neighbors of Ad3 and Ad7 ( Figure 2A (SEQ ID NO: 5-6) and hexagonal ( Figure 2B Sequence alignment of amino acid sequences (SEQ ID NO: 25–26).

[0598] Figure 3A sample from a patient with high-grade serous ovarian cancer was infected with different concentrations of adenovirus Ov26, which was obtained through biological screening. On day 6 post-infection, the survival rates of cancer cells (A) and cancer-associated fibroblasts (CAFs) (B) in the sample were determined by multiparameter flow cytometry. FAP = fibroblast activation protein, a cell surface marker present on the surface of CAFs. CA125 = cancer antigen 125, a cell surface marker present on the surface of cancer cells. VG / cell = viral genome number per cell.

[0599] Figure 4: Cell viability of various cell lines and primary cells in the presence of Ov26. Cells were infected with escalating doses of Ov26, and cell viability was determined by MTS assay on day 5 post-infection. Data are expressed as viability relative to uninfected controls. VG / cell = viral genome number per cell.

[0600] Figure 5 Comparison of oncolytic activity between Ov26 and wild-type parental viruses. (A) Viral genome replication was determined by qPCR on day 7 post-infection, expressed as the number of viral genomes per cell. (B) Infectious progeny viruses were quantified by ICC assay, expressed as infection-forming units (IFU) per cell. For each virus, data were normalized to levels observed in A549 cells. Parental A = wild-type Ad3; Parental B = wild-type Ad7.

[0601] Figure 6 A549 cancer cells were seeded onto sterile coverslips and infected with Ov26 or Ov26_L2Ad7 cells at a dose of 100 VG / cell. The coverslips were then transferred to a co-culture system consisting of A549 cancer cells and cancer-associated fibroblasts (CAFs). Cells were immediately covered with agarose and DMEM medium. After two weeks of culture, MTT assay was added to stain surviving cells and visualize the extent of viral spread. A) Representative images of the spread assay; dashed circles indicate coverslip locations. Unstained areas marked with solid lines indicate the extent of viral spread. B) Quantitative analysis of viral spread in multiple spread assays under MRC5 fibroblast and A549 cell co-culture conditions. C) Quantitative analysis of viral spread in multiple spread assays under A549 cell co-culture conditions with CAFs isolated from ascites samples of ovarian cancer patients. Four independent samples were used for independent experiments. Each data point represents the spread result in a single experiment, and the error bar represents the standard deviation of the mean. The diffusion range was calculated using ImageJ software; statistical significance was assessed using a t-test, with *** P < 0.001, ** P < 0.01, and * P < 0.05.

[0602] Figure 7Each virus was serially diluted and incubated for 30 minutes in DMEM medium or in DMEM medium supplemented with a mixed serum from 300 healthy human donors (final concentration 15%). A549 cells were then infected with the serially diluted virus for 4 hours, after which the infection medium was replaced with DMEM supplemented with 15% FBS. Cell viability was determined by the MTS viability assay on day 7 post-infection. Cell viability is expressed as a proportion of uninfected cells. The IC50 value was calculated using nonlinear regression to determine the fold change in IC50 after serum addition.

[0603] Figure 8 : A schematic diagram of the open reading frames (ORFs) encoded by the E3 region of group B adenovirus, and two deletions associated with these ORFs found in the viruses of this invention isolated after biological selection, and the resulting mutant viruses (Ov26_E3_Δ14.7K, Ov26_E3_Δ14.9K and Ov26_E3_ΔX1). Black bars represent retained ORFs.

[0604] Figure 9 The diagram illustrates a comparison of the E3 region structure between different adenovirus groups and that of group B adenovirus. Arrows represent ORFs, and the size of the protein encoded by each ORF is shown within the arrow. White arrows are used when an ORF has homologous sequences in group B virus. Black arrows indicate the absence of ORFs with homologous sequences in group B adenovirus. White arrows indicate the presence of homologous sequences in group B adenovirus.

[0605] Figure 10 TNFα levels were detected in ascites samples from ovarian cancer patients, mixed serum from 250 healthy donors, and culture supernatant from A549 cancer cells.

[0606] Figure 11 A549 cells stably transduced with the secretory alkaline phosphatase (SEAP) gene under NF-KB promoter induction were infected with 50 Ov26 viral genomes (VGs) per cell, or Ov26 variants in which the E3 ORF 14.7 K or 14.9 K had been deleted, named Ov26_E3_Δ14.7k and Ov26_E3_Δ14.9k, respectively. After 24 hours, cells were treated with escalating concentrations of TNFα to induce NF-KB signaling. Sixteen hours after TNFα treatment, the cells were transduced via QuantiBlue. TM Quantitative SEAP expression was determined.

[0607] Figure 12Quantification of TNFα-induced apoptosis in virus-infected H2199 cells. (A) Cells were infected with Ov26 or an Ov26 variant (OV26_E3_Δ14.7k) containing 50 viral genomes (VGs) per cell. Twenty-four hours post-infection, cells were treated with escalating concentrations of TNFα in combination with 12.5 μM cyclohexylamide (CHX) to initiate TNFα-induced apoptosis. Sixteen hours post-treatment with TNFα and CHX, the induction of apoptosis was quantified by Caspase-Glo 3 / 7 chemiluminescence assay. The average chemiluminescence signal from three biological replicates was plotted as a percentage of the signal in the untreated simulant, with error bars representing standard deviation (SD). (B) Cells were infected with Ov26, Ov26_E3_Δ14.7k, or the Ov26 variant at a dose of 2.5 IFU / cell, where 14.9K was completely deleted, the 14.7K ORF had a 217 bp deletion at the 5' end, the 16.1K ORF was intact, and the 19.1K ORF was present but had a 362 bp deletion at the 3' end (Ov26_E_3_ΔX1). 24 hours post-infection, cells were treated with a combination of 10 ng / mL TNFα and 12.5 μM cyclohexylamide (CHX). Apoptosis induction 16 hours post-treatment was quantified by caspase-Glo 3 / 7 chemiluminescence assay. Relative fluorescence units (RLU) of three individual biological replicates were plotted against the median. The significance of the difference between Ov26-infected cells and variant-infected cells was assessed by one-way ANOVA. ***P<0.001**P<0.01, *P<0.05.

[0608] Figure 13 (A) A549 cancer cells were seeded onto sterile coverslips and infected with Ov91 at 500 vg / cell or with Ov91 armed with a therapeutic agent (Tag) encoded in its E3 region Tag91 (Ov91_Tag26) and Tag57 (Ov91_Tag57). Tag26 was 1853 bp in length, and Tag57 was 2207 bp in length. The coverslips were transferred to uninfected A549 cell layers and immediately covered with agarose and DMEM. Cells were cultured for 2 weeks, after which MTT assay was added to stain live cells and reveal the extent of viral spread. (B) A549 cells were infected with a series of viral doses of Ov91 or Ov91_Tag26; survival was assessed by MTT assay 5 days post-infection. (C) A549 cells were infected with Ov91 or Ov91_Tag44 at 100 vg / cell. Cell pellets and supernatants were collected at different time points post-infection, and viral genome was quantified by qPCR. The average of the three values ​​was plotted using error bars indicating standard deviation. (Tag44 is green fluorescent protein; Tag26 is NCAM BiTE; and Tag57 is VEGF TRAP).

[0609] Figure 14 H1299 cancer cells were infected with each of the following viruses: Ov26, Ov26_E3_Δ14.7K, or Ov26_E3_ΔX1, at an MOI of 2.5 IFU / cell. Twenty-four hours post-infection, cells were treated with 10 ng / ml TNFα and 12.5 ug / mL CHX or a mediator control (PBS). Seventy-two hours post-infection, cells and supernatant were harvested and subjected to three freeze-thaw cycles to achieve cell lysis. The infectious viral load was determined by ICC. All conditions were performed in triplicate. The number of infectious particles produced under TNFα treatment was expressed as a percentage of the number of particles produced under untreated conditions, and the data were then normalized to Ov26. Therefore, the data showed that TNFα treatment reduced the degree of infectious viral particle production of both virus variants relative to the reduction observed with Ov26. Significance of differences was measured by one-way ANOVA. ***P<0.001, **P<0.01, *P<0.05.

[0610] Figure 15 A) Infect A549 cancer cells or normal human primary cells with CRC74 or CRC74_ΔE3 virus at 10 viral genomes per cell. After 4 days, quantify total infectious viral particles by ICC assay. Calculate the number of infectious units per cell and express it as a percentage of IFU generated during A549 infection. B) Infect HCT116 cancer cells, cancer-associated fibroblast lines vCAF and MRC5, and primary CAFs isolated from ovCAF 1 and 2 via liquid biopsy from ovarian cancer patients with increased doses of CRC74 or CRC74_ΔE3. After 7 days, assess cell viability by MTS assay. Viability is expressed as a percentage of live cells relative to untreated controls.

[0611] Figure 16 The introduction of E3 deletion enables the oncolytic adenovirus CRC74 to accommodate exogenous DNA at either of two distinct arming sites. The DNA expression cassette encodes arming sites 1 and 2 of CRC74 (A) and CRC74_ΔE3 (B) in the viral genome. A549 cancer cells were infected with armed and unarmed control viruses at equal doses. Cell viability was assessed by MTS assay at 72 and 96 hours post-infection. Viral killing activity is expressed as the percentage of cell death shown by the corresponding unarmed control virus.

[0612] Figure 17Ov26 and Ov26_DNAPol_WT differ in a single substitution mutation: Ov26 has the mutation; Ov26_DNAPol_WT has the wild-type sequence. A) Schematic diagram of the Ov26 genome. The location of the difference between Ov26 and Ov26_WT_DNAPol within the viral genome is indicated by vertical black arrows. Unfilled horizontal arrows show the location and orientation of surrounding viral genes. B) Nucleotide and amino acid sequences containing the mutated region (SEQ ID NO: 52-57, in appropriate 5'-3' and NC orientations). The mutation in Ov26 is shown in bold on the right. The sequence of Ov26_DNAPol_WT is shown on the left. This single-point mutation in Ov26 results in amino acid changes in both genes due to the overlapping open reading frames of the DNA polymerase and pTP genes.

[0613] Figure 18 Compared to OV26_DNAPol_WT, OV26 showed enhanced viral genome replication in different cancer cell lines. Viral genome (VG) was quantified by qPCR 48 hours after infection of (A)Ad-293, (B)HeLa, (C)Panc1, and (D)A549 cells at a dose of 100 viral genomes / cell. The mean of three biological replicates was plotted; significance was tested by an unpaired t-test.

[0614] Figure 19 Viral replication in patient-derived cancer samples and the ovarian cancer cell line OvCAR3. Primary cell samples from patient-derived ascites and the ovarian cancer cell line OvCAR3 were infected with a dose of 100 viral genomes / cell. Viral genomes were measured by qPCR 6 days post-infection. Data are expressed as fold changes in the input viral dose. Significance was assessed by multiple t-tests, **P < 0.01.

[0615] Figure 20 A549 cells were infected with Ov26 or Ov26_DNAPol_WT containing 100 viral genomes per cell. Samples were collected from wells on day 3 post-infection. Cells were lysed by freeze-thaw cycles, and infectious viral particles were quantified using immunocytochemistry (ICC) with an antibody against the antiviral hexagonal protein. Significance was assessed using a t-test; ***P < 0.001.

[0616] Figure 21 Genome replication in normal cell lines 6 days post-infection. Data were normalized to the fold change of Ov26 measured in each cell line.

[0617] NHDF = Normal human dermal fibroblasts

[0618] HPF = Human lung fibroblasts

[0619] HCF = Human cardiac fibroblasts

[0620] HUF = Human uterine fibroblasts

[0621] HAoAF = Human aortic adventitia fibroblasts

[0622] Figure 22 Analysis of the Ov26 genome after 30 in vitro passages. Ad-293 cells were infected with Ov26. After extensive cytopathic effects, materials were collected for infecting the next generation of Ad-293 cells. After 30 viral passages, materials were collected and DNA extracted. DNA from the initial virus was also extracted; Illumina was used for analysis. Whole-genome sequencing was performed on the DNA. The diagram shows sequence alignment and read coverage of the initial virus (left) and the virus after 30 infection cycles (right), with no mutations detected.

[0623] Figure 23 Quantitative analysis of luciferase expression in A549 cells infected with Ov26_SA-Fluc or Ov26_DNAPol_WT_SA-Fluc. Cells were infected at a multiplicity of infection of 0.6 units per cell. Luciferase expression was measured by lysing cells and adding luciferin 48 hours post-infection, and luminescence was measured on a microplate reader.

[0624] Figure 24 A549 cancer cells were seeded onto sterile coverslips and infected with 100 vG of Ov26, Ad3, or Ad7 per cell. The coverslips were then transferred to a monolayer of OVCAR-3 ovarian cancer cells. Cells were immediately covered with agarose and DMEM. After 2 weeks of incubation, MTT assay was used to stain viable cells to visualize the extent of viral spread. A) Representative images of the spread assay; white circles indicate coverslip positions, and black dashed lines delineate unstained spread areas. B) Quantitative spread extent in repeated or triplicate spread assays. Spread extent was calculated using ImageJ software; significance was assessed by one-way ANOVA, **P < 0.01.

[0625] Figure 25 Whole blood and healthy donor serum showed weaker neutralizing activity against Ov26 than their parent virus. Clinically relevant doses (8 × 10⁻⁶) are shown here. 12 (vp / patient). Each virus was incubated with whole blood or serum for 30 minutes, followed by a clinically relevant dose of 8 × 10⁻⁶. 12 The VP / patient dose was used to infect human cancer cells. After a 2-hour infection period, blood or serum was washed away with cell growth medium. Survival rates were measured using the PrestoBlue survival reagent on day 6 post-infection. Data are presented as mean ± standard deviation (SD).

[0626] Figure 26 Tumor cells (A549) were either untreated (simulated) or infected with 1 vppc of Ov26 (A, B, and C), Ad3, or Ad7 (C). Immature dendritic cells (iDCs) were added to the culture after 4 days, and autologous T cells were added as needed after another 2 days (DCs and T cells were present in all conditions in C). Immune cells were derived from one of the two donors shown. IFNγ (A and C) and IL2 expression (B) were quantified after 72 hours of incubation. Data were collected from three replicate wells and are presented as mean ± standard deviation. Detailed Implementation

[0627] The present invention is further illustrated by the following embodiments. Unless otherwise stated, all components and percentages are by weight, and temperatures are in degrees Celsius. It should be understood that while these embodiments demonstrate preferred embodiments of the invention, they are merely illustrative. Based on the foregoing discussion and these embodiments, those skilled in the art can identify the core features of the invention and can make various changes and modifications to adapt it to different uses and conditions without departing from the concept and scope of the invention. Therefore, in addition to the contents described herein, those skilled in the art can readily derive other modifications to the invention from the foregoing description. Such modifications are also intended to fall within the scope of protection of the appended claims.

[0628] Example 1: Bioscreening and Sequence Analysis of Oncolytic Adenovirus

[0629] From an initial mixed viral library containing adenoviruses of groups B, C, D, F, and G, 23 rounds of biological screening (including mutation steps) were performed to enrich oncolytic adenovirus candidates with the optimal combination of properties in terms of ovarian cancer cell tumor lysis, dissemination, hematopoietic stability, and immunostimulation. The initial mixed viral library containing adenoviruses of groups B, C, D, F, and G was either from commercial sources (US Type Culture Collection or Public Health England) or from a UK collaborative network. Sixty adenoviruses were selected from the final viral library for sequencing, and their genomes were analyzed.

[0630] Genomic analysis of the selected adenoviruses showed that the L2-L3 regions of all 60 adenoviruses obtained through biological screening were Ad3 / Ad7 chimeric structures.

[0631] Table 6. Detailed information on chimeric breakpoints in the nucleotide and amino acid sequences of adenoviruses obtained through biological screening.

[0632] nucleotide sequence

[0633]

[0634] amino acid sequence

[0635]

[0636] The fact that all 60 adenovirus strains obtained through biological screening possessed chimeric Ad3 / Ad7 L2-L3 regions demonstrates the role of these regions in conferring advantages to adenoviruses, including their ability to lyse ovarian cancer cells, as well as their transmissibility, hematopoietic stability, and immune stimulation.

[0637] The amino acid sequences of the pentagonal and hexaagonal polypeptides of Ad3 and Ad7 are compared as shown in the figures below. Figure 2A and Figure 2B .

[0638] One of the chimeric adenoviruses, Ov26, was selected for further research.

[0639] Example 2: Ov26 kills cancer cells and CAF derived from ex vivo ovarian cancer samples.

[0640] A sample from a patient with high-grade serous ovarian cancer (HGSOC) was infected with gradually increasing concentrations of Ov26. On day 6 post-infection, the survival rates of cancer cells and cancer-associated fibroblasts (CAFs) in the sample were determined by multiparameter flow cytometry.

[0641] The results are as follows Figure 3 As shown in the figure, the results indicated that the survival rates of both cancer cells and CAF decreased with increasing Ov26 concentration.

[0642] Example 3: Ov26 kills cancer cells of various ovarian cancer subtypes, including untreated cells and platinum-resistant cells.

[0643] A clinically relevant viral dose of Ov26 (equivalent to 1×10⁻⁶) was used. 13 Samples from ovarian cancer patients (n=24) were treated with VP / 5 L / patients, and the survival of cancer cells and cancer-associated fibroblasts (CAFs) was assessed. More specifically, cell viability was assessed by flow cytometry 6 days after treatment. Cells were viable stained with Live / Dead Near IR, cancer cells were identified using the EpCAM / CA125 marker, and cancer-associated fibroblasts were identified using fibroblast activation protein (FAP+). Each condition was performed in triplicate, and the results are presented as mean survival relative to uninfected controls. HGSOC = High-grade serous ovarian cancer. LGSOC = Low-grade serous ovarian cancer.

[0644] The results are shown in Table 7 below.

[0645]

[0646] The table above shows that Ov26 can effectively kill cancer cells and CAF in patients with a variety of different ovarian cancer subtypes.

[0647] Example 4: Evaluation of the oncolytic activity of Ov26 using a cell line group

[0648] The oncolytic activity of Ov26 was evaluated in a variety of cancer cell lines and cancer-associated fibroblasts, including the following cell types:

[0649] Ad-293: Human embryonic kidney cell line

[0650] A549: Lung Cancer Cell Line

[0651] HeLa: Cervical Cancer Cell Line

[0652] MDA-MB-231: Breast Cancer Cell Line

[0653] PANC-1: Pancreatic cancer cell line

[0654] PSN-1: Pancreatic cancer cell line

[0655] HCT116: Colorectal cancer cell line

[0656] HT-29: Colorectal cancer cell line

[0657] SKOV-3: Ovarian Cancer Cell Line

[0658] Huh-7: Liver cancer cell line

[0659] MRC-5: Fibroblast line

[0660] OE-21: Esophageal cancer cell line

[0661] OVCAR-3: Ovarian Cancer Cell Line

[0662] OVSAHO: Ovarian Cancer Cell Line

[0663] The results are shown in Figure 4. Ov26 showed efficacy against all tested cancer cell lines.

[0664] The IC50 values ​​for Ov26 are listed in Table 8 below.

[0665] Table 8: IC50 values ​​of Ov26 in different cancer cell lines

[0666]

[0667] Example 5: Oncolytic activity of Ov26 compared to wild-type parent virus

[0668] Several parameters of Ov26 virus infection were measured 72 hours after infecting lung cancer cells (A549), non-cancerous normal primary human hepatocytes, and normal human skin fibroblasts (NHDF). Results are as follows: Figure 5 As shown.

[0669] During Ov26 infection, compared with wild-type parental virus infection, normal cells produced fewer viral genomes (A) and fewer infectious viral particles (B) than A549 cancer cells. These results indicate that Ov26 has the ability to preferentially infect lung cancer cells compared with normal hepatocytes and normal fibroblasts.

[0670] Example 6: Preparation of adenovirus with chimeric L2-L3 regions

[0671] Homologous recombination was used to replace the L2 region in Ov26 with a selectable cassette. The insertion of the selectable cassette was verified by Sanger sequencing. Subsequently, the cassette was removed by restriction endonuclease digestion, and another L2 sequence was inserted via Gibson assembly.

[0672] In this way, a novel adenovirus, Ov26_L2Ad7, was prepared, whose L2 and L3 regions were derived from Ad7 adenovirus (except for an Ad3-derived pVI polypeptide).

[0673] Example 7: Diffusion experiment in co-cultured A549 cancer cells and fibroblasts

[0674] The ability of progeny viral particles to infect neighboring cells and establish new toxigenic infections after infecting and lysing target cells is crucial for viral persistence and its spread within tumors. Therefore, the ability of Ov26 to spread in cancer cell layers (co-cultured with MRC5 fibroblasts or patient-derived cancer-associated fibroblasts) was compared with that of Ov26_L2Ad7. Figure 6 The results show that, in both cases, Ov26 has a significantly stronger diffusion capacity than Ov26_L2Ad7.

[0675] Example 8: Serum neutralization experiment in A549 cancer cells

[0676] For oncolytic viruses to successfully reach tumors after intravenous administration, their ability to evade existing immune responses in the bloodstream, such as neutralizing antibodies, is crucial. The humoral immune response to adenoviruses produces antibodies against three major capsid proteins: hexagonal, pentaagonal, and spike proteins. However, the extent to which antibodies are produced against each capsid protein and which antibodies are most effective at neutralizing the virus are not yet fully understood.

[0677] The ability of Ov26 to establish infection and kill A549 cancer cells under serum- and serum-free conditions was compared and compared with wild-type parent viruses Ad3 and Ad7.

[0678] Figure 7 The results indicate that Ov26's tendency to evade serum neutralization is similar to that of Ad7, but significantly higher than that of Ad3. These findings suggest that Ov26 can integrate pentagonal proteins derived from Ad3 without leading to a significant increase in neutralization levels, possibly because its fibrin originates from another parent virus, Ad7. This further underscores the importance of the chimeric capsid structure in Ov26, where the pentagonal and spike protein genes originate from different parent viruses.

[0679] Example 9: E3 region deletion type adenovirus obtained through biological screening

[0680] Genome analysis of the adenoviruses screened in Example 1 revealed that all 60 adenovirus strains obtained through biological screening exhibited one of two deletion types in their E3 region. Details of these two deletions are shown in the table below, and related information is also available in [the table below]. Figure 8 The diagram below illustrates this. Ov26 and Ov91 are two bioselective adenoviruses with deletion (A). Ov20 has deletion (B).

[0681] Table 9: Detailed information on the two types of E3 region missing data

[0682]

[0683] Deletion A: The sequence between nucleotides 629-2892 in SEQ ID NO: 35 has been deleted.

[0684] Deletion B: The sequence between nucleotides 954-3283 in SEQ ID NO: 35 has been deleted.

[0685] Table 10

[0686]

[0687] For detailed information on the corresponding ORFs for other adenovirus types, see [link to relevant documentation]. Figure 9 .

[0688] All 60 adenovirus strains screened carried one of the two deletion mutations mentioned above. This fact proves that these deletion mutations bring advantages to adenoviruses, specifically in their ability to lyse ovarian cancer cells, spread, blood stability, and immune stimulation.

[0689] Example 10: Quantitative analysis of TNFα levels in patient samples

[0690] Tumor necrosis factor-alpha (TNFα) is an inflammatory cytokine involved in various cellular signaling processes, most of which ultimately lead to cell necrosis or apoptosis. TNFα signaling can enhance anti-infection capabilities by inducing apoptosis in infected cells, and therefore may have a significant impact on the efficacy of oncolytic virus therapy.

[0691] This study used ELISA to quantitatively detect TNFα levels in ascites samples from ovarian cancer patients, mixed serum samples from 250 healthy donors, and culture supernatant from A549 cancer cell lines. Figure 10 The results showed that TNFα levels were highest in patient-derived samples, while TNFα was not detected in healthy serum or cancer cell line cultures. This result indicates that the effect of TNFα on viral therapy can only be effectively assessed in models such as patient-derived tumor samples, because in these models, the expression of TNFα or other related cytokines, chemokines, and stress factors (such as IFN) is upregulated or at physiologically relevant concentrations; under conventional cell culture conditions, these effects cannot be fully reflected.

[0692] Example 11: Comparison of Ov26, Ov26_E3_Δ14.7K, Ov26_E3_Δ14.9K, and Ov26_E3_ΔX1

[0693] Proteins encoded by the E3 region of adenovirus protect virus-infected cells from clearance by the host immune system. These proteins include those encoded by the 10.3K ORF (RIDα) and 14.9K ORF (RIDβ), which form a complex and are localized to the cell membrane. The RID complex promotes the survival of infected cells by inhibiting apoptosis, which is typically initiated by the death domain receptor of the tumor necrosis factor receptor (TNFR) superfamily (McNees et al., J. Virol. 2002 Oct; 76(19): 9716–9723). RIDα is known to downregulate TNFα-induced NF-κB signaling; however, the role of RIDβ in NF-κB signaling remains unclear. The 14.7K E3 ORF protects infected cells from TNFα-induced apoptosis.

[0694] We constructed Ov26 mutants with deletions of the E3 ORF at 14.9K (RIDβ) or 14.7K, named Ov26_E3_Δ14.9K and Ov26_E3_Δ14.7K, respectively. We also constructed a mutant named Ov26_E3_ΔX1, in which the 14.9K ORF was completely deleted, the 14.7K ORF had a 217 bp deletion at the 5' end, the 16.1K ORF was intact, and the 19.1K ORF was present but had a 362 bp deletion at the 3' end. These deletion constructs were generated by cloning the viral genome into a bacterial artificial chromosome (BAC). Subsequently, homologous recombination was used to replace the E3 region of Ov26 with a selectable cassette flanked by specific restriction enzyme sites. The selectable cassette was then removed by restriction enzyme digestion, and synthetic DNA encoding the modified E3 region was inserted using Gibson assembly. After validation of the constructs, the virus was recovered by transfection into Ad-293 cells.

[0695] The ability of these adenoviruses to induce the NF-KB signaling pathway was examined using cell lines expressing the secretory alkaline phosphatase (SEAP) gene controlled by the NF-KB inducible promoter. Figure 11 ).

[0696] The results showed that Ov26 had a stronger ability to block NF-KB signal transduction induction than Ov26_E3_Δ14.9K.

[0697] The ability of Ov26, Ov26_E3_Δ14.7K, and Ov26_E3_ΔX1 to protect infected cells from TNFα-induced apoptosis was determined by detecting the quantitative level of caspase activation in infected cells after TNFα treatment. Figure 12 The results showed that cells infected with Ov26 did not exhibit any caspase activation response after TNFα treatment, thus avoiding TNFα-induced apoptosis. However, cells infected with Ov26_E3_Δ14.9K or Ov26_E3_ΔX1 showed significant caspase activation after TNFα treatment.

[0698] Example 12: Comparison of the proliferation kinetics of candidate virus Ov91 and "armed" Ov candidate virus

[0699] Ov91 is an adenovirus obtained through biological selection. Its E3 region contains a deletion type A, making its E3 region sequence completely identical to Ov26. By cloning the Ov91 genome into a bacterial artificial chromosome (BAC), an Ov91 variant simultaneously encoding therapeutic transgenes was constructed. Subsequently, homologous recombination technology was used to replace the E3 region of Ov91 with a selectable cassette flanked by specific restriction enzyme sites. This selectable cassette was then removed by restriction enzyme digestion, and synthetic DNA fragments encoding different therapeutic transgenes were embedded into the E3 region of Ov91 using Gibson assembly technology. After validation of the construct, the virus was recovered by transfecting Ad-293 cells.

[0700] The ability of the virus to spread from infected cells to neighboring cells after cell lysis, its ability to kill cancer cells, and its viral proliferation were all quantified.

[0701] Figure 13 The results showed that the E3 region deletion type A present in both Ov91 and Ov26 allows for the insertion of large transgenes up to 2,207 bp (approximately 6.7% of their genome length) without significantly reducing their killing efficacy against cancer cells.

[0702] Example 13: Quantitative analysis of infectious virus production in the presence of TNFα

[0703] Studies have shown that the E3 14.7K protein is crucial for blocking TNFα-induced NF-κB signaling, and the Ov26 strain is more resistant to apoptosis than the Ov26_E3_Δ14.7K and Ov26_E3_ΔX1 variants (Example 7). One of the significant advantages of anti-apoptotic properties for oncolytic viruses is that delayed cell death creates more favorable conditions for viral replication, resulting in the production of more progeny viral particles. Based on this, the number of infectious units (IFUs) produced when Ov26, Ov26_E3_Δ14.7K, and Ov26_E3_ΔX viruses infect cancer cells with or without TNFα treatment was evaluated. Figure 14 The results showed that TNFα treatment significantly reduced the number of IFUs produced by the mutant viruses Ov26_E3_Δ14.7K and Ov26_E3_ΔX, with a much higher degree of inhibition than that of the Ov26 virus.

[0704] Example 14: The transplantability of E3 deletion in other oncolytic viruses and its effect on enhancing the selectivity of cancer cells and cancer-related cells.

[0705] Studies have confirmed that Ov26 exhibits significantly better targeting selectivity for cancer cells than its parent strain; this is likely due to the attenuation effect resulting from the deletion of its E3 region. To assess whether this deletion structure can similarly enhance the targeting selectivity of other oncolytic viruses, the same E3 deletion (e.g., Figure 8 As shown, Theolytics A) introduced another oncolytic adenovirus, CRC74, to obtain the E3 deletion mutant CRC74_ΔE3.

[0706] To investigate the selectivity of CRC74 and its mutant CRC74_ΔE3, the infectious viral particle yield was quantitatively analyzed after infecting A549 cancer cells and three different normal primary cell types. Figure 12 Results showed that CRC74 infection of some normal primary cells produced infectious viral particles; however, when these cells were infected with the CRC74_ΔE3 variant, the production of infectious virus was significantly reduced. However, as... Figure 12 As shown in B, the E3 deletion introduced in CRC74 did not significantly affect its killing effect on cancer cells or cancer-associated fibroblasts. Therefore, Figure 15 The data shown indicate that introducing E3 deletion in CRC74 can improve the selectivity of oncolytic viruses, and confirm that this specific E3 structure can enhance the selectivity of different oncolytic adenoviruses.

[0707] Example 15: Effect of E3 deletion on the ability of a virus to accommodate exogenous transgenes without affecting viral activity

[0708] Ov26 can accommodate the insertion of transgene expression cassettes into its genome without affecting its replication and diffusion capabilities. To assess whether E3 deletion is an important feature that allows the integration of transgene sequences into the viral genome, the same E3 deletion was introduced into another oncolytic adenovirus, CRC74, resulting in the E3 deletion mutant CRC74_ΔE3.

[0709] To assess the ability of CRC74 and CRC74_ΔE3 viruses to contain exogenous DNA in their genomes, expression cassettes encoding genes were inserted into one of two sites in the genomes of the two viruses, and their cancer cell killing activity was quantitatively detected. Figure 16 Results showed that although CRC74 could be armed, its lethal activity was significantly delayed, and it did not reach 100% activity relative to the unarmed CRC74 virus even after 96 hours.

[0710] and Figure 16 The results indicate that the CRC74_ΔE3 virus exhibits stronger resistance to arming, and its lethal activity is comparable to that of the control virus after 96 hours of infection. In conclusion, Figure 16The data shown indicate that the introduction of the E3 gene deletion mutation into the CRC74 viral genome significantly enhances the tolerance of the oncolytic virus to expression cassette insertion without affecting its oncolytic activity.

[0711] Example 16: Screening of adenoviruses with missense mutations

[0712] Genome analysis of the adenoviruses screened in Example 1 revealed a missense mutation in the E2B DNA polymerase I gene in a large number of the screened adenoviruses. This mutation is a single nucleotide change in the adenovirus genome, ggc→gac. This nucleotide change leads to the G34D mutation in the E2B DNA polymerase I polypeptide and the A623T mutation in the E2B pTP polypeptide.

[0713] One of the adenoviruses carrying the mutation (i.e., Ov26) was selected for further research.

[0714] Given that D (aspartic acid) and E (glutamic acid) are both structurally similar negatively charged amino acids, the G34E mutation is expected to produce similar results. Similarly, given that T (threonine), N (asparagine), C (cysteine), Q (glutamine), and S (serine) are all structurally similar polar / neutral amino acids, the A623N, A623C, A623Q, and A623S mutations are also expected to produce similar results.

[0715] Example 17: Construction of a mutation-free control adenovirus

[0716] Figure 17 The sequence data of adenovirus Ov26 (carrying the ggc→gac mutation) is presented and compared with the sequence of the control adenovirus (Ov26_DNAPol_WT). Except for the aforementioned single nucleotide mutation, the genome sequence of the control adenovirus is identical to that of Ov26.

[0717] Example 18: Proliferation kinetics of Ov26 and Ov26_DNAPol_WT in cancer cell lines

[0718] Figure 18 The proliferation kinetics of Ov26 and Ov26_DNAPol_WT in four different cell lines over 48 hours were shown. qPCR detection at 48 hours post-infection revealed higher levels of viral genome accumulation in Ad-293(A), HeLa(B), Panc-1(C), and A549(D) cells.

[0719] Data show that Ov26 is able to replicate at a higher level after infection compared to Ov26_DNAPol_WT.

[0720] Example 19: Genome duplication in ascites samples and ovarian cancer cell lines

[0721] The replication of Ov26 and Ov26_DNAPol_WT was evaluated in patient-derived cancer samples and the ovarian cancer cell line OvCAR3.

[0722] Primary ascites cell samples and ovarian cancer cell line OVCAR3 were infected with a dose of 100 viral genomes / cell. The viral genome count was measured by qPCR on day 6 post-infection. Results are as follows... Figure 19 As shown, in ascites samples, Ov26 was able to replicate to a higher degree than Ov26_DNAPol_WT, resulting in a greater accumulation of the genome on day 6 post-infection.

[0723] Example 20: Production of infectious viral particles in A549 cells

[0724] A549 cells were infected with Ov26 or Ov26_DNAPol_WT virus at a dose of 100 viral genomes per cell. Infectious viral particles were then detected at different time points post-infection.

[0725] Figure 20 The results showed that Ov26 infection produces a greater number of infectious viral particles compared to Ov26_DNAPol_WT.

[0726] Example 21: Genome replication in normal cell lines 6 days post-infection

[0727] Three days after infecting cells with a dose of 100 viral genomes per cell, the genome replication of Ov26 and Ov26_DNAPol_WT in various normal cell lines was quantitatively detected by qPCR. Results are as follows: Figure 21 As shown, the mutation in the Ov26 DNA polymerase I gene did not enhance its replication ability in normal cells, but instead increased the therapeutic index of the virus.

[0728] Example 22: Mutations in the E2B DNA polymerase I and pTP genes do not impair the virus's proofreading ability, nor do they lead to viral genome mutations.

[0729] The Ov26 genome was analyzed in 30 in vitro passages. Ad-293 cells were infected with Ov26 and harvested when signs of CPE were observed. The harvested material was then used to infect the next generation. DNA was extracted from the initial virus and from the 10th, 20th, and 30th generations of the virus, and libraries were constructed for Illumina analysis. Whole genome sequencing.

[0730] Figure 22The results show the initial virus and the virus after 30 passages. These results indicate that after 30 passages of cell culture, Illumina... No mutations were detected in the viral genome during sequencing, indicating that the E2B mutation in Ov26 does not impair the fidelity of the viral polymerase.

[0731] Example 23: Preparation of adenovirus carrying fluorescent reporter gene transgene

[0732] The viral genomes of Ov26 and Ov26_DNAPol_WT were cloned into bacterial artificial chromosomes (BACs), and a firefly luciferase reporter gene (SAFluc) driven by the SA promoter was inserted into the E3 region of the virus using recombinant engineering techniques. BACs encoding the viral genome carrying the reporter gene transgene were transfected into Ad-293 cells. Replicating virus was recovered, amplified, and titers were determined by immunocytochemistry. A549 cells were then infected with the virus at a dose of 0.6 infection units per cell, and luciferase expression was measured 48 hours post-infection.

[0733] data( Figure 23 The results showed that the expression level of the Ov26 transgene was significantly higher than that of Ov26_DNAPol_WT.

[0734] Example 24: OVACR-3 Ovarian Cancer Cell Metastasis Experiment

[0735] After an oncolytic virus infects and lyses target cells, the ability of progeny viral particles to continue infecting neighboring cells and establishing new toxin-producing infections is crucial for viral persistence and spread within the tumor. Therefore, the diffusion capacity of Ov26 in cancer cell layers was compared with that of its parent viruses, Ad3 and Ad7. Figure 24 The results showed that Ov26 had a significantly better diffusion capacity than Ad3 and Ad7.

[0736] Example 25: Neutralization test using whole blood and healthy donor serum

[0737] For oncolytic viruses to be successfully administered intravenously, they must overcome the neutralizing effect of blood components in order to reach and infect their target cancer cells. Therefore, the ability of Ov26 to infect and kill human cancer cells (A549) after exposure to whole blood or serum was compared with that of its parent viruses Ad3 and Ad7. Figure 25 The results showed that Ov26 retained its ability to kill cancer cells after exposure to whole blood or serum, while its parent viruses Ad3 and Ad7 were effectively neutralized.

[0738] Example 26: Ov26 induces IL-2 and IFNγ expression only when antigen-presenting cells (DCs) and T cells are co-cultured with lysed tumor cells, and the degree of IFNγ activation induced is higher than that induced by its parent viruses Ad3 and Ad7.

[0739] One of the mechanisms by which oncolytic viruses kill cancer cells is by activating immune cells, thereby targeting and killing cancer cells. Therefore, the ability of Ov26 to activate inflammatory T cell responses against infected A549 cancer cells was evaluated. Figure 26 Results A and 26B showed that an inflammatory response characterized by upregulation of IFNγ and IL2 was only induced when antigen-presenting dendritic cells (DCs) and T cells were present simultaneously. This data suggests that pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) released by Ov26-lysed cancer cells are specifically presented to T cells by antigen-presenting cells, thereby stimulating an immune response. Using the same experimental setup, the upregulation of IFNγ in A549 cancer cells was compared when infected with Ov26 or the parental viruses Ad3 or Ad7, respectively. Figure 26 The results showed that the T cell IFNγ response induced by Ov26 infection was significantly stronger than that induced by the parental Ad3 or Ad7 viruses.

[0740] The sequence list filed with this patent application is incorporated herein as part of the specification.

[0741] First aspect of the present invention

[0742] A preferred embodiment of the first aspect of the present invention includes:

[0743] 1. A chimeric oncolytic adenovirus for the prevention or treatment of cancer, wherein the genome of the chimeric oncolytic adenovirus comprises:

[0744] (A) Ad3 pentagonal genes, wherein the Ad3 pentagonal genes comprise or consist of the following:

[0745] (a) The nucleotide sequence shown in SEQ ID NO: 3;

[0746] (b) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 3 and encoding an adenovirus pentagonal polypeptide; or

[0747] (c) The nucleotide sequence encoding the Ad3 pentagonal polypeptide, wherein the Ad3 pentagonal polypeptide is:

[0748] (i) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 5;

[0749] (ii) A polypeptide having at least 99.3%, 99.5%, or 99.7% sequence identity with SEQ ID NO: 5 and encoding an adenovirus pentagonal polypeptide; or

[0750] (iii) Having at least 95%, 99% or 99.5% sequence identity with SEQ ID NO: 5, wherein the pentagonal polypeptide contains polypeptides corresponding to amino acids V, T, I and D at positions 11, 158, 178 and 326 of SEQ ID NO: 5, respectively, and the polypeptide encodes an adenovirus pentagonal polypeptide;

[0751] as well as

[0752] (B) Ad7 hexagonal gene, wherein the Ad7 hexagonal gene comprises or consists of the following:

[0753] (a) The nucleotide sequence shown in SEQ ID NO: 24;

[0754] (b) A nucleotide sequence having at least 97% or at least 99% nucleotide sequence identity with SEQ ID NO: 24 and encoding an adenovirus hexapod polypeptide; or

[0755] (c) The nucleotide sequence encoding the Ad7 hexagonal polypeptide, wherein the Ad7 hexagonal polypeptide is:

[0756] (i) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 26; or

[0757] (ii) A polypeptide having at least 97%, 98% or 99% sequence identity with SEQ ID NO: 26 and encoding an adenovirus hexapod polypeptide.

[0758] 2. A method of treating a patient with cancer, the method comprising administering a therapeutically effective amount of a chimeric oncolytic adenovirus to a patient in need, wherein the genome of the chimeric oncolytic adenovirus comprises:

[0759] (A) The Ad3 quinant genes as defined in Implementation Scheme 1; and

[0760] (B) The Ad7 hexane gene as defined in Implementation Scheme 1.

[0761] 3. Use of chimeric oncolytic adenovirus in the preparation of medicaments for the prevention or treatment of cancer, wherein the genome of said chimeric oncolytic adenovirus comprises:

[0762] (A) The Ad3 quinant genes as defined in Implementation Scheme 1; and

[0763] (B) The Ad7 hexane gene as defined in Implementation Scheme 1.

[0764] 4. The chimeric oncolytic adenovirus, method, or use as defined in any of the foregoing embodiments, wherein the genome of the chimeric oncolytic adenovirus comprises an L2 region and an L3 region, and wherein:

[0765] (a) The Ad3 quinant gene is located within the L2 region; and / or

[0766] (b) The Ad7 hexane gene is located in the L3 region.

[0767] 5. The chimeric oncolytic adenovirus, method, or use as defined in any of the foregoing embodiments, wherein the genome of the chimeric oncolytic adenovirus comprises an L2 region and an L3 region, and wherein:

[0768] (a) The L2 region includes:

[0769] (i) The Ad3 quinant gene as defined in Implementation Scheme 1;

[0770] (ii) Ad3 pVIII gene;

[0771] (iii) Ad3V gene; and

[0772] (iv) Ad3 or Ad7 pX gene; and / or

[0773] (b) The L3 region includes:

[0774] (i) Ad3 or Ad7 pVI gene;

[0775] (ii) the Ad7 hexane gene as defined in Implementation Scheme 1; and

[0776] (iii) Ad7 protease gene.

[0777] 6. The chimeric oncolytic adenovirus, method, or use as defined in any of the foregoing embodiments, wherein the genome of the chimeric oncolytic adenovirus additionally includes an Ad7 spike protein gene, wherein the Ad7 spike protein gene comprises or is composed of the following:

[0778] (a) The nucleotide sequence shown in SEQ ID NO: 32;

[0779] (b) A nucleotide sequence having at least 60%, 70%, 80%, 90%, or 95% (preferably at least 95%) nucleotide sequence identity with SEQ ID NO: 32 and encoding an adenovirus spike polypeptide; or

[0780] (c) The nucleotide sequence encoding the Ad7 spike polypeptide, wherein the Ad7 spike polypeptide is:

[0781] (i) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 34; or

[0782] (ii) A polypeptide having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) sequence identity with SEQ ID NO: 34 and encoding an adenovirus spike polypeptide;

[0783] Preferably, the genome of the chimeric oncolytic adenovirus includes an L5 region, and the Ad7 spike protein gene is located within the L5 region.

[0784] 7. The chimeric oncolytic adenovirus, method, or use as defined in any of the foregoing embodiments, wherein the genome of the chimeric oncolytic adenovirus contains a transgene, preferably wherein the transgene is located in or near the E1, E3, L3, or L5 regions of the chimeric oncolytic adenovirus genome, or in the E1 / E3 deletion region.

[0785] 8. The chimeric oncolytic adenovirus, method, or use as defined in Embodiment 7, wherein the transgene encodes an antibody, bispecific adapter, checkpoint inhibitor, cytokine, chemokine, enzyme, or angiogenesis inhibitor.

[0786] 9. The chimeric oncolytic adenovirus, method, or use as defined in any of the foregoing embodiments, wherein the cancer is selected from the group consisting of ovarian cancer, colorectal cancer, lung cancer, liver cancer, multiple myeloma, esophageal cancer, breast cancer, and pancreatic cancer, preferably ovarian cancer or stromal cancer.

[0787] 10. A chimeric adenovirus, wherein the genome of said chimeric adenovirus comprises:

[0788] (A) The Ad3 quinant gene as defined in Implementation Scheme 1;

[0789] (B) The Ad7 hexane gene as defined in Implementation Scheme 1; and

[0790] (C) The Ad7 spike protein gene as defined in Implementation Scheme 6.

[0791] 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:

[0792] (a) The Ad3 quinant gene is located within the L2 region; and / or

[0793] (b) The Ad7 hexane gene is located in the L3 region.

[0794] 12. A chimeric adenovirus as defined in embodiment 10 or 11, wherein the genome of the chimeric adenovirus comprises an L2 region and an L3 region, and wherein:

[0795] (a) The L2 region includes:

[0796] (i) The Ad3 quinant gene as defined in Implementation Scheme 1;

[0797] (ii) Ad3 pVIII gene;

[0798] (iii) Ad3V gene; and

[0799] (iv) Ad3 or Ad7 pX gene; and / or

[0800] (b) The L3 region includes:

[0801] (i) Ad3 or Ad7 pVI gene;

[0802] (ii) the Ad7 hexane gene as defined in Implementation Scheme 1; and

[0803] (iii) Ad7 protease gene.

[0804] 13. A chimeric adenovirus as defined in any one of embodiments 10-12, wherein the genome of the chimeric adenovirus includes an L5 region and the Ad7 spike protein gene is located within the L5 region.

[0805] 14. A chimeric adenovirus as defined in any one of embodiments 10-13, wherein the genome of the chimeric adenovirus contains a transgene, preferably wherein the transgene is located in or near the E1, E3, L3, or L5 regions of the chimeric adenovirus genome, or in an E1 / E3 deletion region.

[0806] 15. A chimeric adenovirus as defined in embodiment 14, wherein the transgene encodes an antibody, a bispecific adapter, a checkpoint inhibitor, a cytokine, a chemokine, an enzyme, or an angiogenesis inhibitor.

[0807] 16. A chimeric adenovirus comprising an adenovirus capsid, wherein the adenovirus capsid comprises:

[0808] (A) The Ad3 pentagonal polypeptide as defined in claim 1;

[0809] (B) The Ad7 hexagonal polypeptide as defined in claim 1; and

[0810] (C) The Ad7 fibrous polypeptide as defined in claim 6.

[0811] 17. The chimeric oncolytic adenovirus, chimeric adenovirus, method, or use as described in any of the foregoing embodiments, wherein the adenovirus is a group B adenovirus, a human adenovirus, or an Ad3 / Ad7 chimeric adenovirus.

[0812] 18. An adenovirus gene therapy vector, wherein the genome of the adenovirus gene therapy vector comprises:

[0813] (a) The Ad3 pentagonal gene as defined in Implementation Scheme 1;

[0814] (b) The Ad7 hexane gene as defined in Implementation Scheme 1; and

[0815] (c) Genetically modified organisms.

[0816] 19. An adenovirus gene therapy vector as defined in embodiment 18, wherein the genome of the adenovirus gene therapy vector comprises an L2 region and an L3 region, and wherein:

[0817] (a) The Ad3 quinant gene is located within the L2 region; and / or

[0818] (b) The Ad7 hexane gene is located in the L3 region.

[0819] 20. An adenovirus gene therapy vector as defined in embodiment 18 or 19, wherein the genome of the adenovirus gene therapy vector comprises an L2 region and an L3 region, and wherein:

[0820] (a) The L2 region includes:

[0821] (i) The Ad3 quinant gene as defined in Implementation Scheme 1;

[0822] (ii) Ad3 pVIII gene;

[0823] (iii) Ad3V gene; and

[0824] (iv) Ad3 or Ad7 pX gene; and / or

[0825] (b) The L3 region includes:

[0826] (i) Ad3 or Ad7 pVI gene;

[0827] (ii) the Ad7 hexane gene as defined in Implementation Scheme 1; and

[0828] (iii) Ad7 protease gene.

[0829] 21. An adenovirus gene therapy vector as defined in any one of embodiments 18-20, wherein the genome of the adenovirus gene therapy vector additionally includes the Ad7 spike protein gene as defined in embodiment 6; preferably, wherein the genome of the adenovirus gene therapy vector includes an L5 region, and the Ad7 spike protein gene is located within the L5 region.

[0830] 22. An adenovirus gene therapy vector as defined in any of embodiments 18-21, wherein the transgene is located in or near the E1, E3, L3, or L5 regions of the genome of the adenovirus gene therapy vector, or in the E1 / E3 deletion region.

[0831] 23. An adenovirus gene therapy vector as defined in embodiment 22, wherein the transgene encodes an antibody, a bispecific adapter, a checkpoint inhibitor, a cytokine, a chemokine, an enzyme, or an angiogenesis inhibitor.

[0832] 24. The adenovirus gene therapy vector according to any one of claims 18-23, wherein the adenovirus gene therapy vector is obtained from or derived from group B adenovirus or human adenovirus or Ad3 / Ad7 chimeric adenovirus.

[0833] 25. A pharmaceutical composition comprising a chimeric adenovirus as defined in any one of embodiments 10-17 or an adenovirus gene therapy vector as defined in any one of embodiments 18-24, optionally in combination with one or more pharmaceutically acceptable vectors, excipients or diluents.

[0834] 26. A drug combination comprising:

[0835] (A) A first pharmaceutical composition comprising a chimeric adenovirus as defined in any one of embodiments 10-17 or an adenovirus gene therapy vector as defined in any one of embodiments 18-24; and

[0836] (B) A second pharmaceutical composition comprising a chemotherapeutic agent or an immunotherapeutic agent.

[0837] The drug combination is present in the form of a combination formulation and is administered simultaneously, alone or sequentially, preferably for the treatment of cancer.

[0838] 27. A chimeric adenovirus as defined in any of embodiments 10-17 or an adenovirus gene therapy vector as defined in any of embodiments 18-24, used for treatment or as a medicine.

[0839] 28. Use of a chimeric adenovirus as a protein production vector as defined in any one of embodiments 10-17, wherein the chimeric adenovirus contains a transgene encoding the protein to be produced.

[0840] Second aspect of the invention

[0841] A preferred embodiment of the second aspect of the present invention includes:

[0842] 1. An oncolytic adenovirus whose genome contains an E3 region:

[0843] (a) The E3 region contains 12.1K and 14.7K ORFs from the E3 region of group B adenovirus, or corresponding ORFs from the E3 region of non-group B adenovirus; and

[0844] (b) The E3 region therein does not contain a functional 20K, 20.6K, 7.7K or 10.3K ORF from a group B adenovirus E3 region, or a corresponding ORF from a non-group B adenovirus E3 region.

[0845] 2. The oncolytic adenovirus as defined in Implementation Scheme 1, wherein the E3 region additionally includes a functional (optionally 3' truncated) 16.1K ORF from the E3 region of a group B adenovirus, or a corresponding ORF from the E3 region of a non-group B adenovirus.

[0846] 3. The oncolytic adenovirus as defined in embodiment 1 or 2, wherein the E3 region additionally comprises a 16.1K ORF and a functional (optionally 3' truncated) 19.3K ORF from the E3 region of a group B adenovirus, or a corresponding ORF from the E3 region of a non-group B adenovirus.

[0847] 4. The oncolytic adenovirus as defined in any of the foregoing embodiments, wherein the E3 region additionally includes 14.9K ORF from the E3 region of group B adenovirus, or a corresponding ORF from the E3 region of a non-group B adenovirus.

[0848] 5. An oncolytic adenovirus as defined in Embodiment 1, wherein the E3 region comprises or is composed of the following:

[0849] (a) Group B adenovirus E3 12.1K ORF;

[0850] (b) 3' truncated group B adenovirus E3 16.1K ORF;

[0851] (c) 5' truncated group B adenovirus E3 10.3K ORF;

[0852] (d) Group B adenovirus E3 14.9K ORF; and

[0853] (e) Group B adenovirus E3 14.7K ORF,

[0854] Or the corresponding ORF from the E3 region of a non-group B adenovirus.

[0855] The E3 region is connected in the order of 5'→3' as described above, and may optionally contain one or more transgenes located inside or adjacent to the one or more ORFs.

[0856] 6. An oncolytic adenovirus as defined in Embodiment 1, wherein the E3 region comprises or is composed of the following:

[0857] (a) Group B adenovirus E3 12.1K ORF;

[0858] (b) Group B adenovirus E3 16.1K ORF;

[0859] (c) 3' truncated group B adenovirus E3 19.3K ORF;

[0860] (d) 5' truncated group B adenovirus E3 14.9K ORF; and

[0861] (e) Group B adenovirus E3 14.7K ORF,

[0862] Or the corresponding ORF from the E3 region of a non-group B adenovirus.

[0863] The E3 region is connected in the order of 5'→3' as described above, and may optionally contain one or more transgenes located inside or adjacent to the one or more ORFs.

[0864] 7. The oncolytic adenovirus as defined in Embodiment 1, wherein the E3 region encodes a fusion protein comprising the fusion of the C-terminus of a C-terminus of a group B adenovirus E3 16.1K protein with the N-terminus of an N-terminus of a group B adenovirus E3 10.3K protein, or comprising the fusion of the corresponding terminus of a corresponding protein from a non-group B adenovirus E3 region.

[0865] 8. The oncolytic adenovirus as defined in Embodiment 1, wherein the E3 region encodes a fusion protein comprising the fusion of the C-terminus of a C-terminus of a group B adenovirus E3 19.3K protein with the N-terminus of an N-terminus of a group B adenovirus E3 14.9K protein, or comprising the fusion of the corresponding terminus of a corresponding protein from a non-group B adenovirus E3 region.

[0866] 9. An oncolytic adenovirus as defined in Implementation Scheme 1, wherein the E3 region is missing compared to the corresponding region of wild-type group B adenovirus:

[0867] (a) The deletion start point is located at nucleotide 629 in the E3 region nucleotide sequence of group B adenovirus shown in SEQ ID NO: 35, or at the corresponding nucleotide in a non-group B adenovirus E3 region nucleotide sequence; and

[0868] (b) The missing termination point is located at nucleotide 2,892 in the E3 region nucleotide sequence of group B adenovirus shown in SEQ ID NO: 35, or at the corresponding nucleotide in the E3 region nucleotide sequence of a non-group B adenovirus.

[0869] 10. An oncolytic adenovirus as defined in Implementation Scheme 1, wherein the E3 region is missing compared to the corresponding region of wild-type group B adenovirus:

[0870] (a) The deletion start point is located at nucleotide 1,099 of the group B adenovirus E3 region nucleotide sequence shown in SEQ ID NO: 35, or at the corresponding nucleotide in a non-group B adenovirus E3 region nucleotide sequence; and

[0871] (b) The missing termination point is located at nucleotide 3,283 in the E3 region nucleotide sequence of group B adenovirus shown in SEQ ID NO: 35, or at the corresponding nucleotide in the E3 region nucleotide sequence of a non-group B adenovirus.

[0872] 11. An oncolytic adenovirus as defined in Embodiment 1, wherein the E3 region is missing compared to wild-type group B adenovirus, wherein the missing region corresponds to:

[0873] (a) Ad7 genomic nucleotides 28,011–30,274; or

[0874] (b) Ad7 genomic nucleotides 28,482-30,665.

[0875] 12. The oncolytic adenovirus as described in any of the foregoing embodiments, wherein the adenovirus is a group B adenovirus, preferably Ad3 or Ad7 serotype.

[0876] 13. An oncolytic adenovirus as defined in any of the preceding embodiments, wherein the adenovirus additionally comprises a transgene, preferably encoding an antibody, a bispecific adapter, a checkpoint inhibitor, a cytokine, a chemokine, or an enzyme.

[0877] 14. An oncolytic adenovirus as defined in embodiment 13, wherein the transgene is located in or near the E1, E3, L3, or L5 region of the adenovirus.

[0878] 15. A pharmaceutical composition comprising an oncolytic adenovirus as defined in any of the preceding embodiments, optionally in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0879] 16. An oncolytic adenovirus as defined in any of embodiments 1-14 or a pharmaceutical composition as defined in embodiment 15, used for treatment or as a medicine.

[0880] 17. An oncolytic adenovirus as defined in any one of embodiments 1-14 or a pharmaceutical composition as defined in embodiment 15, for the treatment of cancer (preferably ovarian cancer).

[0881] 18. A method of treating a subject with cancer (preferably ovarian cancer), the method comprising administering to a subject in need a therapeutically effective amount of an oncolytic adenovirus as defined in any one of embodiments 1-14 or a pharmaceutical composition as defined in embodiment 15.

[0882] 19. Use of oncolytic adenovirus as defined in any one of embodiments 1-14 in the preparation of a medicament for treating cancer (preferably ovarian cancer).

[0883] 20. The oncolytic adenovirus as defined in embodiment 17, the method as defined in embodiment 18, or the use as defined in embodiment 19, wherein the cancer is a stromal tumor.

[0884] 21. The oncolytic adenovirus as defined in embodiment 17, the method as defined in embodiment 18, or the use as defined in embodiment 19, wherein the cancer is ovarian cancer, colorectal cancer, lung cancer, liver cancer, multiple myeloma, esophageal cancer, breast cancer, or pancreatic cancer, preferably wherein the cancer is ovarian cancer or stromal carcinoma.

[0885] Third aspect of the invention

[0886] A preferred embodiment of the third aspect of the present invention includes:

[0887] 1. A pharmaceutical composition comprising an adenovirus, optionally combined with one or more pharmaceutically acceptable carriers, excipients or diluents, wherein the genome of the adenovirus comprises an E2B DNA polymerase I gene encoding a DNA polymerase I polypeptide, and wherein the amino acid corresponding to amino acid 34 in SEQ ID NO: 2 of the DNA polymerase I polypeptide sequence is Asp or Glu, preferably Asp.

[0888] 2. The pharmaceutical composition of embodiment 1, wherein the genome of the adenovirus additionally includes an E2B pTP gene encoding a terminal protein, wherein the coding region of the E2B DNA polymerase I gene overlaps with the coding region of the E2B pTP gene, and wherein the amino acid corresponding to amino acid 623 in SEQ ID NO: 4 in the pTP sequence is selected from the group consisting of Thr, Met, Lys and Arg, preferably Thr.

[0889] 3. The pharmaceutical composition according to embodiment 1, wherein the nucleotide in the E2B DNA polymerase I gene corresponding to nucleotide 5,326 in SEQ ID NO: 1 is A.

[0890] 4. A pharmaceutical composition comprising an adenovirus, optionally combined with one or more pharmaceutically acceptable vectors, excipients, or diluents, wherein the genome of the adenovirus comprises an E2B pTP gene encoding a terminal protein, and wherein the amino acid corresponding to amino acid 623 in SEQ ID NO: 4 in the terminal protein sequence is selected from the group consisting of Thr, Asn, Cys, Gln, and Ser, preferably Thr.

[0891] 5. The pharmaceutical composition as described in embodiment 4,

[0892] If the adenovirus is Ad1 adenovirus, then the amino acid is not Asn; if the adenovirus is Ad41 adenovirus, then the amino acid is not Gln; or if the adenovirus is Ad4 or 4a adenovirus, then the amino acid is not Thr.

[0893] 6. The pharmaceutical composition of embodiment 4 or 5, wherein the genome of the adenovirus additionally comprises an E2B DNA polymerase I gene encoding a DNA polymerase I polypeptide, wherein the coding region of the E2B pTP gene overlaps with the coding region of the E2B DNA polymerase I gene, and wherein the amino acid corresponding to amino acids 34-35 in SEQ ID NO: 2 in the DNA polymerase I polypeptide is 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.

[0894] 7. The pharmaceutical composition according to embodiment 4 or 5, wherein the nucleotide in the E2B pTP gene corresponding to nucleotide 5,326 of SEQ ID NO: 3 is A.

[0895] 8. The pharmaceutical composition as described in any of the foregoing embodiments, wherein the adenovirus is a conditionally replicating adenovirus or an oncolytic adenovirus.

[0896] 9. The pharmaceutical composition as described in any of the foregoing embodiments, wherein the adenovirus is a group B adenovirus or a human adenovirus, preferably a type B1 adenovirus.

[0897] 10. The pharmaceutical composition of embodiment 9, wherein the adenovirus is an Ad3 or Ad7 adenovirus or an Ad3 / Ad7 chimeric adenovirus.

[0898] 11. The pharmaceutical composition according to any one of embodiments 1-3 or 6-10, wherein the E2B DNA polymerase I gene comprises or is composed of:

[0899] (i) The nucleotide sequence shown in SEQ ID NO: 1;

[0900] (ii) A nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% nucleotide sequence identity with SEQ ID NO: 1, and preferably encoding DNA polymerase I; or

[0901] (iii) The nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 2.

[0902] 12. The pharmaceutical composition according to any one of embodiments 1-3 or 6-11, wherein the E2B DNA polymerase I polypeptide comprises or is composed of:

[0903] (i) The amino acid sequence shown in SEQ ID NO: 2; or

[0904] (ii) A variant of (i) having at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity or sequence similarity, and preferably having DNA polymerase I activity.

[0905] 13. The pharmaceutical composition according to any one of embodiments 2-12, wherein the E2B pTP gene comprises or is composed of:

[0906] (i) The nucleotide sequence shown in SEQ ID NO: 3;

[0907] (ii) A nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% nucleotide sequence identity with SEQ ID NO: 3, and preferably encoding a terminal protein; or

[0908] (iii) The nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 4.

[0909] 14. The pharmaceutical composition of any one of embodiments 2 to 13, wherein the E2B terminal protein is a polypeptide comprising or composed of:

[0910] (i) The amino acid sequence shown in SEQ ID NO: 4; or

[0911] (ii) A variant of (i) having at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity or sequence similarity, and preferably encoding a terminal protein.

[0912] 15. The pharmaceutical composition according to any of the foregoing embodiments, wherein the adenovirus comprises a transgene, preferably wherein the transgene is located in or near the E1, E3, L3, or L5 regions of the adenovirus, or in the E1 / E3 deletion region.

[0913] 16. A drug combination comprising:

[0914] (A) The first pharmaceutical composition as described in any one of embodiments 1-15; and

[0915] (B) A second pharmaceutical composition comprising a chemotherapeutic agent or an immunotherapeutic agent.

[0916] The drug combination is present in the form of a combination formulation and is administered simultaneously, alone or sequentially, preferably for the treatment of cancer.

[0917] 17. An adenovirus as defined in any one of embodiments 1-15 or a pharmaceutical composition as described in any one of embodiments 1-15, for use as a treatment or as a medicine.

[0918] 18. An adenovirus as defined in any one of embodiments 1-15 or a pharmaceutical composition as described in any one of embodiments 1-15, for the treatment of cancer (preferably ovarian cancer).

[0919] 19. A method of treating a subject with cancer (preferably ovarian cancer), the method comprising administering to a subject in need a therapeutically effective amount of an adenovirus as defined in any one of embodiments 1-15 or a pharmaceutical composition as described in any one of embodiments 1-15.

[0920] 20. Use of adenovirus as defined in any one of embodiments 1-15 in the preparation of a medicament for treating cancer (preferably ovarian cancer).

[0921] 21. The adenovirus as defined in embodiment 18, the method as defined in embodiment 19, or the use as defined in embodiment 20, wherein the cancer is selected from the group consisting of ovarian cancer, colorectal cancer, lung cancer, liver cancer, multiple myeloma, esophageal cancer, breast cancer, and pancreatic cancer, preferably ovarian cancer or stromal cancer.

[0922] 22. Use of a conditionally replicating adenovirus as defined in any of embodiments 1-15 as a protein production vector, wherein the adenovirus contains a transgene encoding the protein to be produced.

[0923] 23. The use of a conditionally replicating adenovirus as defined in any of Implementation Schemes 1-15 as an auxiliary virus for virus production.

[0924] 24. A mutant group B or human adenovirus E2B DNA polymerase I gene, wherein the nucleotide corresponding to nucleotide 101 in SEQ ID NO: 1 in the mutant E2B DNA polymerase I gene is A.

[0925] 25. A mutant group B or human adenovirus E2B pTP gene, wherein the nucleotide corresponding to nucleotide 5,326 in SEQ ID NO: 3 in the mutant E2B pTP gene is G.

[0926] 26. A mutant group B or human adenovirus E2B DNA polymerase I polypeptide, wherein the amino acid corresponding to the 34th amino acid in SEQ ID NO: 2 is D or E, preferably D.

[0927] 27. A mutant group B or human adenovirus E2B pTP polypeptide, wherein the amino acid corresponding to amino acid position 623 in SEQ ID NO: 2 in the mutant E2B pTP polypeptide is T.

Claims

1. An adenovirus whose genome contains one, two, or all of (A), (B), and (C): (A) Ad3 pentagonal genes, among which, The Ad3 quinone gene comprises or consists of the following: (a) A nucleotide sequence as shown in SEQ ID NO: 3; (b) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 3 and encoding an adenovirus pentagonal polypeptide; or (c) The nucleotide sequence encoding the Ad3 pentagonal polypeptide, wherein the Ad3 pentagonal polypeptide is: (i) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 5; (ii) A polypeptide having at least 99.3%, 99.5%, or 99.7% sequence identity with SEQ ID NO: 5 and encoding an adenovirus pentagonal polypeptide; or, (iii) A polypeptide having at least 95%, 99% or 99.5% sequence identity with SEQ ID NO: 5, and having amino acids V, T, I and D at positions 11, 158, 178 and 326 of SEQ ID NO: 5, respectively, said polypeptide encoding an adenovirus pentavalent polypeptide; and, Ad7 hexagonal genes, wherein the Ad7 hexagonal genes comprise or consist of the following: (a) A nucleotide sequence as shown in SEQ ID NO: 24; (b) A nucleotide sequence having at least 97% or at least 99% nucleotide sequence identity with SEQ ID NO: 24 and encoding an adenovirus hexapod polypeptide; or, (c) The nucleotide sequence encoding the Ad7 hexagonal polypeptide, wherein the Ad7 hexagonal polypeptide is: (i) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 26; or, (ii) A polypeptide having at least 97%, 98% or 99% sequence identity with SEQ ID NO: 26 and encoding an adenovirus hexapod polypeptide; Optionally, it also includes the Ad7 spike protein gene; (B) Area E3: (a) The E3 region contains 12.1K and 14.7K ORFs from the E3 region of group B adenovirus, or corresponding ORFs from the E3 region of non-group B adenovirus; and (b) The E3 region does not contain a functional 20K, 20.6K, 7.7K, or 10.3K ORF from the E3 region of group B adenovirus, or a corresponding ORF from a non-group B adenovirus E3 region; as well as (C) The E2B DNA polymerase I gene encoding the DNA polymerase I polypeptide. Wherein, the amino acid corresponding to the 34th amino acid in SEQ ID NO: 49 of the DNA polymerase I polypeptide sequence is Asp or Glu, preferably Asp; or The E2B pTP gene encodes the terminal protein. The amino acid corresponding to amino acid 623 in SEQ ID NO: 51 in the preterminal protein sequence is selected from Thr, Asn, Cys, Gln and Ser, preferably Thr.

2. The adenovirus of claim 1, wherein the genome comprises (A), (B) or (C).

3. The adenovirus of claim 1, wherein the genome comprises (A) and (B); (A) and (C); or (B) and (C).

4. The adenovirus as claimed in claim 1, wherein its genome comprises (A), (B) and (C).

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

6. The adenovirus as claimed in any of the preceding claims, wherein the genome of the adenovirus comprises an L2 region and an L3 region, and: (a) The L2 region includes: (i) the Ad3 quinant gene as defined in claim 1; (ii) Ad3 pVIII gene; (iii) Ad3V gene; and (iv) Ad3 or Ad7 pX gene; and / or (b) The L3 region includes: (i) Ad3 or Ad7 pVI gene; (ii) the Ad7 hexane gene as defined in claim 1; and (iii) Ad7 protease gene.

7. The adenovirus of any of the preceding claims, wherein the genome of the adenovirus further comprises an Ad7 spike protein gene, wherein the Ad7 spike protein gene comprises or is composed of: (a) A nucleotide sequence as shown in SEQ ID NO: 32; (b) A nucleotide sequence having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) nucleotide sequence identity with SEQ ID NO: 32 and encoding an adenovirus spike polypeptide; or (c) A nucleotide sequence encoding an Ad7 spike protein polypeptide, wherein the Ad7 spike protein polypeptide is: (i) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 34; or (ii) A polypeptide having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) sequence identity with SEQ ID NO: 34 and encoding an adenovirus spike protein polypeptide; Preferably, the genome of the adenovirus includes an L5 region, and the Ad7 spike protein gene is located within the L5 region.

8. The adenovirus as claimed in any of the preceding claims, wherein the E3 region further comprises a functional (optionally 3' truncated) 16.1K ORF from the E3 region of a group B adenovirus, or a corresponding ORF from the E3 region of a non-group B adenovirus.

9. The adenovirus as claimed in any of the preceding claims, wherein the E3 region further comprises a 16.1K ORF and a functional (optionally 3' truncated) 19.3K ORF from the E3 region of a group B adenovirus, or a corresponding ORF from the E3 region of a non-group B adenovirus.

10. The adenovirus as claimed in any of the preceding claims, wherein the E3 region further comprises 14.9 KORF from the E3 region of group B adenovirus, or a corresponding ORF from the E3 region of a non-group B adenovirus.

11. The adenovirus as claimed in any of the preceding claims, wherein the E3 region comprises or is composed of: (a) Group B adenovirus E3 12.1K ORF; (b) 3' truncated group B adenovirus E3 16.1K ORF; (c) 5' truncated group B adenovirus E3 10.3K ORF; (d) Group B adenovirus E3 14.9K ORF; and (e) Group B adenovirus E3 14.7K ORF, Or the corresponding ORF from the E3 region of a non-group B adenovirus. The E3 regions are connected sequentially in the order of 5'-3' described above, and the E3 regions may optionally contain one or more transgenes located inside or adjacent to one or more of the ORFs.

12. The adenovirus as claimed in any of the preceding claims, wherein the E3 region comprises or is composed of: (a) Group B adenovirus E3 12.1K ORF; (b) Group B adenovirus E3 16.1K ORF; (c) 3' truncated group B adenovirus E3 19.3K ORF; (d) 5' truncated group B adenovirus E3 14.9K ORF; and (e) Group B adenovirus E3 14.7K ORF, Or the corresponding ORF from the E3 region of a non-group B adenovirus. The E3 regions are connected sequentially in the order of 5'-3' described above, and the E3 regions may optionally contain one or more transgenes located inside or adjacent to one or more of the ORFs.

13. The adenovirus as claimed in any of the preceding claims, wherein the E3 region encodes a fusion protein, the fusion protein being a fusion of the C-terminus of a C-terminated group B adenovirus E3 16.1K protein with the N-terminus of an N-terminated group B adenovirus E3 10.3K protein, or a fusion of the corresponding end of a protein from a non-group B adenovirus E3 region.

14. The adenovirus as claimed in any of the preceding claims, wherein the E3 region encodes a fusion protein, the fusion protein being a fusion of the C-terminus of a C-terminus truncated group B adenovirus E3 19.3K protein and the N-terminus of an N-terminus truncated group B adenovirus E3 14.9K protein, or a fusion of the corresponding end of a protein from a non-group B adenovirus E3 region.

15. The adenovirus as claimed in any of the preceding claims, wherein the E3 region is missing compared to the corresponding region of wild-type group B adenovirus: (a) The deletion start point is located at nucleotide 629 in the E3 region nucleotide sequence of group B adenovirus as shown in SEQ ID NO: 35, or at the corresponding nucleotide in a non-group B adenovirus E3 region nucleotide sequence; and (b) The missing termination point is located at nucleotide 2892 in the E3 region nucleotide sequence of group B adenovirus as shown in SEQ ID NO: 35, or at the corresponding nucleotide in the E3 region nucleotide sequence of a non-group B adenovirus.

16. The adenovirus as claimed in any of the preceding claims, wherein the E3 region is missing compared to the corresponding region of wild-type group B adenovirus: (a) The deletion start point is located at position 1099 of the group B adenovirus E3 region nucleotide sequence as shown in SEQ ID NO: 35, or at the corresponding nucleotide position in a non-group B adenovirus E3 region nucleotide sequence; and (b) The missing termination point is located at nucleotide 3283 in the E3 region nucleotide sequence of group B adenovirus as shown in SEQ ID NO: 35, or at the corresponding nucleotide in the E3 region nucleotide sequence of non-group B adenovirus.

17. The adenovirus as claimed in any of the preceding claims, wherein the E3 region is missing compared to wild-type group B adenovirus, the missing region corresponding to: (a) Ad7 genomic nucleotides 28011-30274; or (b) Ad7 genomic nucleotides 28482-30665.

18. The adenovirus as claimed in any of the preceding claims, wherein the genome of the adenovirus comprises an E2B pTP gene encoding a terminal protein, wherein the coding region of the E2B DNA polymerase I gene overlaps with the coding region of the E2B pTP gene, and the amino acid corresponding to amino acid 623 in SEQ ID NO: 51 in the pTP sequence is selected from Thr, Met, Lys, and Arg, preferably Thr.

19. The adenovirus as claimed in any of the preceding claims, wherein the nucleotide corresponding to position 5326 in SEQ ID NO: 48 of the E2B DNA polymerase I gene is A.

20. The adenovirus of any of the preceding claims, wherein the genome of the adenovirus comprises an E2B DNA polymerase I gene encoding a DNA polymerase I polypeptide, wherein the coding region of the E2B pTP gene overlaps with the coding region of the E2B DNA polymerase I gene, and wherein the amino acid corresponding to amino acids 34-35 in SEQ ID NO: 49 of the DNA polymerase I polypeptide is selected from 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.

21. The adenovirus as claimed in any of the preceding claims, wherein the nucleotide corresponding to position 5326 in SEQ ID NO: 50 of the E2B pTP gene is A.

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

23. The adenovirus as claimed in any of the preceding claims, wherein the E2B DNA polymerase I gene comprises or is composed of: (i) A nucleotide sequence as shown in SEQ ID NO: 48; (ii) A nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% nucleotide sequence identity with SEQ ID NO: 48 and preferably encoding DNA polymerase I; or (iii) The nucleotide sequence encoding the polypeptide shown in SEQ ID NO:

49.

24. The adenovirus as claimed in any of the preceding claims, wherein the E2B DNA polymerase I polypeptide comprises or is composed of: (i) The amino acid sequence as shown 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 with (i), and preferably having DNA polymerase I activity.

25. The adenovirus as claimed in any of the preceding claims, wherein the E2B pTP gene comprises or is composed of: (i) A nucleotide sequence as shown in SEQ ID NO: 50; (ii) A nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% nucleotide sequence identity with SEQ ID NO: 50 and preferably encoding a terminal protein; or (iii) The nucleotide sequence encoding the polypeptide shown in SEQ ID NO:

51.

26. The adenovirus as claimed in any of the preceding claims, wherein the E2B terminal protein comprises or is composed of: (i) The amino acid sequence as shown in SEQ ID NO: 51; (ii) A variant of (i) having at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity or sequence similarity with (i), and preferably encoding a terminal protein.

27. The adenovirus as claimed in any of the preceding claims, wherein the adenovirus is a group B adenovirus or a human adenovirus, preferably a type B1 adenovirus, an Ad3 or Ad7 serotype adenovirus, or an Ad3 / Ad7 chimeric adenovirus.

28. The adenovirus as claimed in any of the preceding claims, wherein the adenovirus comprises a transgene, preferably the transgene is located in or near the E1 region, E3 region, L3 region, L5 region of the adenovirus, or in the E1 / E3 deletion region of the adenovirus.

29. The adenovirus of claim 28, wherein the transgene encodes an antibody, a bispecific adapter, a checkpoint inhibitor, a cytokine, a chemokine, an enzyme, or an angiogenesis inhibitor.

30. An adenovirus comprising the nucleotide sequence 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 and having oncolytic activity.

31. An adenovirus or adenovirus vector whose genome has 100% nucleotide sequence identity with nucleotides 1-479, 3410-27380 and 28869-33044 of SEQ ID NO: 58; preferably, the genome of the adenovirus or adenovirus vector further comprises a transgene; more preferably, the transgene is located between nucleotides 480-3409 or 27381-28868 of SEQ ID NO:

58.

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

33. A drug combination comprising: (A) A first pharmaceutical composition comprising an adenovirus or adenovirus vector as described in any one of claims 1-31; and (B) A second pharmaceutical composition comprising a chemotherapeutic agent or an immunotherapeutic agent. The drug combination is in the form of a combination formulation for simultaneous, separate or sequential use, preferably for the treatment of cancer.

34. The adenovirus or adenovirus vector as described in any one of claims 1-31, or the pharmaceutical composition as described in claim 32, for treatment or use as a medicine.

35. The adenovirus or adenovirus vector as described in any one of claims 1-31, or the pharmaceutical composition as described in claim 32, for the treatment of cancer (preferably ovarian cancer).

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

37. Use of the adenovirus or adenovirus vector as described in any one of claims 1-31 in the preparation of a medicament for treating cancer (preferably ovarian cancer).

38. The adenovirus of claim 35, the method of claim 36, or the use of claim 37, wherein the cancer is selected from ovarian cancer, colorectal cancer, lung cancer, liver cancer, multiple myeloma, esophageal cancer, breast cancer, and pancreatic cancer, preferably ovarian cancer or cancer containing stromal tissue.

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