Adenovirus

Abstract

The present invention relates to adenoviruses, in particular oncolytic adenoviruses, for use in the prevention or treatment of cancers, including interstitial-containing tumors and ovarian cancers. In particular, the present invention relates to adenoviruses having a deletion in the E3 region as compared to a wild-type adenovirus.

Description

[0001] This invention relates to adenoviruses, particularly oncolytic adenoviruses, for the prevention or treatment of cancers, including stromal tumors and ovarian cancer. In particular, this invention relates to adenoviruses with a deletion in the E3 region compared to wild-type adenoviruses.

[0002] Cancer originates from the epithelial tissue lining the exterior or luminal surfaces of organs, including the lungs, gastrointestinal tract, and reproductive organs. Cancer accounts for approximately 90% of all cancer cases worldwide and is the leading cause of the vast majority of cancer deaths.

[0003] A consistent theme in cancer is that cancer cells are often diagnosed as palpable lesions due to the presence and / or presence of extracellular material, fibroblasts, and immune cells around and / or within cancer cell islands. All cells and material in a tumor that are not described as cancerous (or malignant) are generally referred to collectively as the “stroma,” more specifically, the “tumor stroma.” Some cells of the immune system are typically included under the term stroma (usually macrophages, fibroblasts), while other cells (usually lymphocytes) are described as infiltrating or passing through the stroma.

[0004] All cancerous tissues that are visible to the naked eye and palpable contain a certain amount of stroma; this provides the necessary structure, nutrition, and environment for the survival, proliferation, and even spread of cancer cells.

[0005] The proportion of stroma can vary between 5% and 80%, depending in part on the underlying indication. However, it is obvious to anyone familiar with tumor structure that the greatest variation in stroma content for any given biopsy is due to the location of the biopsy, as some areas of the tumor visually appear to have more stroma than others.

[0006] The surface of stromal cells exhibits features recognizable from normal tissues and organs. They remodel the ECM, recruit blood vessels, and provide structure. However, unlike normal tissues, the function of these cells is dysregulated and often disordered. Excessive stromal mass leads to high pressure, poor blood flow, and hypoxic areas. Consequently, cancer cells experience temporal and spatial variability in their nutrient and oxygen supply. As the tumor grows, significant cell death and necrosis occur due to cyclical changes in nutrient levels. Fluctuations in nutrient and oxygen pressure appear to be undesirable conditions for tumor growth; however, it is increasingly recognized that this drives the clonal evolution of more resistant cells.

[0007] Over the years, the role of interstitial compartments in protecting cancer cells from drug or immune attacks has become increasingly recognized. Cancer-associated fibroblasts (CAFs) are particularly associated with excluding T cells from tumors or suppressing any T cells that infiltrate by expressing TGFβ and other cytokines.

[0008] The presence of CAFs and their effects on immune cells are often considered the root cause of poor responses to immunotherapy. However, the development of novel immunotherapies for cancer patients is hampered by the lack of animal models containing sufficient numbers of mesenchymal cells or the correct structure. When CAFs are artificially introduced into animal models, the efficacy of cancer vaccines and checkpoint inhibitor therapies is reduced or completely lost.

[0009] Despite the lack of models, the development of drugs specifically targeting the mesenchyme has garnered increasing attention. These drugs either target mesenchymal cells directly or the microenvironmental conditions created by the mesenchyme. However, mesenchymal-targeting strategies typically face two fundamental challenges. First, selectivity can be extremely challenging because the cells that make up the mesenchyme are essentially "normal": they are not malignant and are often found in other parts of the body. The second conceptual problem is the need for combination therapy, as mesenchymal-targeting therapies alone are unlikely to be entirely effective. Targeting both the mesenchyme and cancer cells may be necessary for treating refractory cancers if they do not respond to either approach alone. The basic principle of combination therapy is very strong, but developing drugs as a combination remains very difficult, especially for drugs that are ineffective or potentially ineffective as single agents.

[0010] The ideal intervention for treating cancer should target cancer cells and stromal cell populations simultaneously and selectively.

[0011] Oncolytic viruses are an emerging therapeutic approach because they can kill a variety of tumor cell types, including differentiated cancer cells and cancer-initiating cells or stem cells. They can be highly selective, while also utilizing "cancer markers" such as immunity or cell cycle dysregulation to proliferate and lyse cells.

[0012] The main drawback of oncolytic viruses is that, to date, they have not been specifically designed or developed to combat mesenchymal cells. This is because modern oncolytic viruses are engineered to be active only in malignant cells (by definition).

[0013] In human tumors, the stroma divides and surrounds various regions of cancer cells, posing a physical (and indeed conceptual) barrier to oncolytic therapy. Therefore, treatments that are successful in the laboratory rarely translate effectively into clinical application. In cases of clinical success in treating solid tumors, oncolytic viruses have been repeatedly injected to bypass 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, they can be targeted at very small or early-stage cancers with relatively low interstitial content (e.g., Packiam VT, et al. “An open label, single-arm, phase II multicenter study of the safety and efficacy of CG0070 oncolyticvector regimen in patients with BCG-unresponsive non-muscle-invasive bladder cancer: Interim results.” Urol Oncol. 2018 Oct;36(10):440-447. doi: 10.1016 / j.urolonc.2017.07.005. Epub 2017 Jul 26. PMID: 28755959).

[0014] To address the stromal tumor problem, some oncolytic viruses have been engineered to express biologics, including bispecific T-cell adjuvants (BiTEs), to selectively kill stromal cells. The concept here is to allow the oncolytic virus to treat cancer cells while the expressed BiTEs target stromal cells, thereby achieving complete tumor lysis.

[0015] Unfortunately, this concept remains insufficient due to the structure and arrangement of the tumor stroma. In human tumors, blood-supplying capillaries emerge through the stroma. Therefore, drugs, including oncolytic viruses, must penetrate multiple layers of stromal cells to reach cancer cells. This is not a problem for small molecule drugs due to diffusion, but oncolytic viruses are typically too large to migrate between cells. Once inside a human tumor, oncolytic viruses first come into contact with stromal cells, thus preventing replication or expression of transgenes, such as BiTE.

[0016] This deficiency is not apparent in animal models because animal models typically lack the stroma; or, even if the stroma is present in animal models, its structure is different, with capillaries appearing directly in cancer cells.

[0017] For oncolytic agents to be effective in treating human diseases, they must be active immediately upon entering the tumor microenvironment. This is because oncolytic viruses utilize three hallmark features of cancer to kill cancer cells, and these same features also apply to cancer cells with stromal inflammatory bowel disease (CAF). These include metabolic disorders, anti-apoptosis, and immune dysfunction. Therefore, developing an oncolytic agent that utilizes these shared characteristics of both cancer cells and stromal cells, making it active against both, is highly anticipated.

[0018] Ovarian cancer is one of the most common and deadliest cancers in women. Often, the disease is diagnosed at an advanced stage, and may have already spread to other parts of the body. Despite the availability of various treatments, the best survival rate for patients with stage 3 and above remains only 25%. Most ovarian cancer patients are diagnosed with the most severe type: high-grade serous ovarian cancer (HGSOC), which accounts for 70-80% of deaths. Current treatment options involve repeated platinum-based therapy until unavoidable resistance and recurrence occur. Second-line treatments include paclitaxel and doxorubicin, but their efficacy is limited. Only a small percentage of patients with BRCA1 mutations (13-15%) qualify for the recently approved PARP inhibitors. Recently, Mirvetuximab received accelerated approval for patients with high folate receptor levels. While these newer drugs are effective in the intermediate term, cancer recurrence is expected in most patients.

[0019] The potential role of oncolytic viruses in ovarian cancer has been explored, based on their ability to stimulate immune responses while killing heterogeneous cell populations, including cancer stem cells. Numerous clinical trials have demonstrated the safety and feasibility of delivering oncolytic viruses to ovarian cancer patients. However, overall efficacy has been limited. In fact, stromal fibroblasts (CAFs) inhibit the effects of oncolytic therapy through antiviral signaling (Arwert EN, et al. “STING and IRF3 in stromal fibroblasts enables ensing of genomic stress in cancer cells to undermine oncolytic viral therapy”. Nat Cell Biol. 2020 Jul;22(7):758-766. doi: 10.1038 / s41556-020-0527-7. Epub 2020 Jun 1. Erratum in: Nat Cell Biol. 2020 Jun 18;: PMID:32483388; PMCID: PMC7611090).

[0020] Given the important role that CAF plays in advancing ovarian cancer and providing resistance mechanisms to chemotherapy, immunotherapy, and oncolytic therapy, there is an urgent need to develop new therapies that can simultaneously combat CAF and cancer cells.

[0021] Cancer-associated fibroblasts (CAFs) play a crucial role in determining prognosis and treatment response by suppressing immune cells, preventing the infiltration of effective T cells, forming a dense barrier that hinders the spread of treatment, and promoting resistance to standard therapies. Furthermore, CAFs can promote and nourish tumors, supporting tumor growth and metastasis. Despite their importance in determining patient response to treatment, existing preclinical models, including more sophisticated in vivo PDX models, do not represent this key component of the human tumor microenvironment (TME). In vivo use of traditional cancer cell lines and xenograft tumor models fails to reproduce the complexity and heterogeneity of human cancers. They do not accurately capture the stroma, immunosuppression, metabolic dysregulation, cytokines, and stress proteins (such as TNF-α and TGF-β) present in the patient's TME. Developing therapies on realistic model systems (e.g., freshly resected patient biopsies, multicellular, nutrient-deprived, hypoxic tumor microenvironments) is crucial for identifying durable drugs that maintain efficacy even under such challenging and stressful conditions.

[0022] The applicant demonstrates insight by recognizing that the tumor microenvironment is fundamentally different from the environment in which viruses naturally evolve: viruses typically reside in oxygen-rich cells that are constantly supplied with nutrients. The tumor microenvironment also differs significantly from cell cultures or rapidly growing animal models, which are rich in nutrients. Therefore, wild-type viruses or viruses discovered using simple animal models are generally 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 demands for various resources, or risk the termination of their replication cycle. Mutations—even subtle ones—can achieve this effect because viral activity—the production of RNA, DNA, and proteins—is highly "precise," so even a small change in a single virus can become significant when it replicates to form 100,000 offspring.

[0023] The applicant has now identified a feature that endows the adenovirus with the ability to enhance its oncolytic activity in stromal tumors, including ovarian cancer cells.

[0024] From the initial pools of adenoviruses in 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 tumor lysis, spread, hematopoietic stability, and immunostimulation. From the final pool, 60 adenoviruses were selected for sequencing, and their genomes were analyzed.

[0025] Genome analysis of selected adenoviruses revealed that the genomes of most or all the selected adenoviruses shared some common characteristics. Specifically, the genomes of the selected adenoviruses all originated from group B adenoviruses, and compared to wild-type group B adenoviruses, these genomes all exhibited deletions in the E3 region.

[0026] The wild-type E3 region of group B adenovirus genomes typically contains nine open reading frames (ORFs). In all bioselective viruses, at least five ORFs (19.3K, 20K, 20.6K, 7.7K, and 10.3K) were found to be completely or partially deleted; and two other ORFs (16.1K and 14.9K) were partially deleted in some bioselective adenoviruses.

[0027] As mentioned above, the tumor microenvironment in cancer patients is filled with stress proteins (such as TNF-α), which, when combined with early viral infection, can lead to the rapid death of the first infected tumor cells. This rapid death of tumor cells prevents effective viral replication, thus limiting viral spread and causing premature viral clearance.

[0028] This study demonstrates the value of preserving all or part of the E3 14.9K ORF in blocking NF-κB signaling. The results show that this prevents premature adenovirus clearance, allowing the virus more time to begin infecting cells.

[0029] Furthermore, this study demonstrates that preserving the E3 14.7K ORF prevents early apoptosis in infected cells. This has been found to provide more time for viral replication within the cell, thereby increasing the infectious titer of the adenovirus of this invention.

[0030] This paper also demonstrates that large transgenes can be inserted into the E3 deletion site of the bioselective adenovirus of this invention without significantly reducing its efficacy against cancer cells. The loss of certain E3 ORFs also attenuates viral virulence in normal cells compared to the parent wild-type strain.

[0031] Therefore, the present invention provides an oncolytic adenovirus having one or more of the above-mentioned features, which can be used for the prevention or treatment of cancer.

[0032] US Patent 2002 / 106746 A1 discloses a recombinant adenovirus vector derived from the Ad5 adenovirus genome, wherein at least a portion of the E3 region is deleted or non-functional, but the adenovirus vector retains the E3 sequence encoding a functional 14.7K protein, a functional 14.5K protein, and / or a functional 10.4K protein. The corresponding proteins in group B (e.g., Ad3 or Ad7) adenoviruses would be 14.7K, 14.9K, and 10.3K proteins, respectively. The Enadenotucirev genome (ColoAd; Kuhn et al. PLoS One 2008; 3(6): e2409) contains a deletion of the E3 region, which includes the complete or partial deletion of the 14.9K and 14.7K ORFs.

[0033] One object of the present invention is to provide an adenovirus that can lyse a variety of different tumor cells and cancer-associated fibroblasts (CAFs) without lysing normal cells. The adenovirus is capable of spreading, is stable in the blood, and can induce immunogenic cell death and stimulate immune responses, including immune responses from T cells and dendritic cells, after tumor cell lysis.

[0034] Another object of the present invention is to provide compositions comprising the adenovirus of the present invention and their use in the prevention or treatment of cancer.

[0035] In one embodiment, the present invention provides an oncolytic adenovirus having a genome comprising an E3 region:

[0036] (a) wherein 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) 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.

[0037] In some embodiments, the E3 region additionally includes a functional (optionally 3'-truncated) 16.1K ORF from the E3 region of group B adenovirus or its corresponding ORF from the E3 region of non-group B adenovirus. In some embodiments, the E3 region additionally includes a 16.1K ORF and a functional (optionally 3'-truncated) 19.3K ORF from the E3 region of group B adenovirus, or its corresponding ORF from the E3 region of non-group B adenovirus. In one embodiment, the E3 region also includes a 14.9K ORF from the E3 region of group B adenovirus or its corresponding ORF from the E3 region of non-group B adenovirus.

[0038] In a further embodiment, the present invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region comprises or is composed of the following:

[0039] (a) Group B adenovirus E3 12.1K ORF;

[0040] (b) 3-truncated group B adenovirus E3 16.1K ORF;

[0041] (c) 5'-truncated group B adenovirus E3 10.3K ORF;

[0042] (d) Group B adenovirus E3 14.9K ORF; and,

[0043] (e) Group B adenovirus E3 14.7K ORF,

[0044] Or it may originate from the corresponding ORF of the E3 region of a non-group B adenovirus.

[0045] The ORFs are connected sequentially in the 5'-3' order described above, wherein the E3 region may optionally contain one or more transgenes located within or adjacent to one or more of the ORFs.

[0046] In a further embodiment, the present invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region comprises or is composed of the following:

[0047] (a) Group B adenovirus E3 12.1K ORF;

[0048] (b) Group B adenovirus E3 16.1K ORF;

[0049] (c) 3'-truncated group B adenovirus E3 19.3K ORF;

[0050] (d) 5'-truncated group B adenovirus E3 14.9K ORF; and,

[0051] (e) Group B adenovirus E3 14.7K ORF,

[0052] Or it may originate from the corresponding ORF of the E3 region of a non-group B adenovirus.

[0053] The transverse links are connected in the 5'-3' sequence described above, wherein the E3 region may optionally contain one or more transgenes located within or adjacent to one or more of the ORFs.

[0054] In a further embodiment, the present invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region encodes a fusion protein comprising a C-terminus of a C-terminus of a group B adenovirus E3 16.1K protein fused to the N-terminus of an N-terminus of an N-terminus of an N-terminus of a group B adenovirus E3 10.3K protein, or the fusion protein comprising a corresponding terminus of a corresponding protein from a non-group B adenovirus E3 region.

[0055] In a further embodiment, the present invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region encodes a fusion protein comprising a fusion of the C-terminus of a C-terminated group B adenovirus E3 19.3K protein and the N-terminus of an N-terminated group B adenovirus E3 14.9K protein, or a fusion protein comprising the corresponding terminus of a corresponding protein from a non-group B adenovirus E3 region.

[0056] In a further embodiment, the present invention provides a pharmaceutical composition comprising an oncolytic adenovirus as described in any of the preceding claims, optionally together with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0057] In a further embodiment, the present invention provides an oncolytic adenovirus of the present invention for treatment or as a pharmaceutical composition of the present invention. In a further embodiment, the present invention provides an oncolytic adenovirus of the present invention for treating cancer (preferably ovarian cancer) or a pharmaceutical composition of the present invention. In a further embodiment, the present invention provides a method of treating a subject with cancer (preferably ovarian cancer), the method comprising administering an 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 pharmaceutical for treating cancer (preferably ovarian cancer).

[0058] Table 1: Nucleotide and amino acid sequence listing

[0059]

[0060] This invention provides an adenovirus, preferably for the treatment of cancer, and more preferably for the treatment of ovarian cancer or stromal tumors. As used herein, "adenovirus" (also referred to as "Ad") means a virus belonging to the Adenoviridae family, including any of the five currently known genera: mammalian adenovirus, avian adenovirus, atadenovirus, and siadenovirus. Fish adenovirus (Ichtadenovirus) Preferably, the adenovirus is derived from mammalian adenoviruses; this includes all human serotypes. In one embodiment, the adenovirus is a human adenovirus.

[0061] Currently, more than 60 antigenic types or "serotypes" of human adenoviruses have been described, and these serotypes have been classified into seven species, namely Ad species AG (e.g., as described in Wold et al. Current gene therapy vol. 13, 6 (2013): 421-33), based on their physical, chemical, and biological characteristics. Therefore, the adenovirus species used in this article refer to the currently known adenovirus AG group, as well as any adenoviruses discovered in the future.

[0062] Therefore, in one embodiment, the adenovirus species is selected from the group consisting of AdA, AdB, AdC, AdD, AdE, AdF, and AdG. In some embodiments, the human adenovirus species is selected from the group consisting of AdB, AdC, AdD, AdE, AdF, and AdG.

[0063] The serotypes belonging to each of these adenovirus species include, but are not limited to, the following: 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. AdG includes Ad52. The different adenovirus serotypes mentioned in this document include all different strains or variants of these serotypes.

[0064] 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 serotype Ad3 or Ad7, or an Ad3 / Ad7 chimera.

[0065] In some implementations, the adenovirus is a human adenovirus. All adenovirus genomes studied to date share the same overall structure, meaning that genes encoding specific functions are located in the same positions within the adenovirus genome (referred to herein as structural elements). Each end of the adenovirus genome has a short sequence called an inverted terminal repeat (ITR), which is necessary for viral replication. In some implementations, the adenovirus is neither chimpanzee adenovirus nor AdC7 adenovirus.

[0066] 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 generate five late mRNA families (L1-L5). The proteins encoded by the early genes are primarily involved in 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, but is typically 34-36 kilobases. For example, the adenovirus Ad5 genome is typically 35,938 base pairs, with 103-base-pair terminal repeats at each end and a GC content of 58%.

[0067] Our understanding of adenovirus genetics, transcription, and translation primarily stems from virological studies using the adenovirus species CAD5. The transcriptional sequence of the adenovirus genome reflects the virus's protein requirements at each stage of replication. Therefore, transcription of the adenovirus genome is categorized into early and late events based on the transcriptional initiation time of each viral promoter. The first protein produced from the viral genome is the E1A protein. E1A transcription units generate multiple mRNA molecules through alternative splicing, leading to a variety of proteins ranging from 6 to 36 kDa. E1A proteins play two main roles in infected cells. First, they induce cells to enter the S phase of the cell cycle, enabling efficient viral genome replication. Second, they induce the transcription of other early promoters within the viral genome through transactivation. These promoters control the production of E1B, E2, E3, and E4 proteins. E1A expression follows immediately after the production of VA RNA and E1B and E3 proteins. These proteins and RNA molecules help prevent the generation of antiviral responses. These early events in viral replication help shape the intracellular environment to allow the viral genome to replicate before packaging. Subsequent transcriptional events involve the production of structural proteins and proteins essential 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 by the major late promoter in the later stages of infection.

[0068] Adenovirus genes are divided into early (E1-4) and late (L1-5) transcripts, with various protein isoforms generated by a series of splicing events. The early transcripts are further divided into E1, E2, E3, and E4. E1 is crucial for transitioning cells to cell cycle stages favorable for viral replication; it inhibits apoptosis and promotes cell division. The E2 region is primarily responsible for DNA genome replication and contains DNA-binding protein (E2A), pTP, and DNA polymerase (E2B). E3 contains genes involved in regulating the host immune response, and E4 contains a series of genes involved in regulating cellular pathways (such as non-homologous end joining (NHEJ)) and forming a complex with E1B-55K to mediate p53 degradation.

[0069] All adenovirus late genes are transcribed from the same promoter (the major late promoter) and share the same 5' mRNA end, which contains three exons that together form a three-part leader sequence. Late genes are expressed through a series of splicing events, resulting in the expression of approximately 13 proteins that either form part of the viral particle (e.g., hexagonal proteins and spike proteins) or participate in its assembly (e.g., the 100K protein).

[0070] Adenoviral vectors are vectors based on or derived from the genome of viruses in the Adenoviridae family. Adenoviral vectors are replicating, non-replicating, and / or genetically engineered adenoviruses that transport genetic material (e.g., adenoviral genes or transgenes) into eukaryotic cells for expression. Adenoviral vectors may carry deletions of early E1, E3, and / or E4 genes, allowing for the insertion of 8–30 kb transgenes. The function of the deleted early genes can be trans-complemented using engineered cell lines. The most commonly used adenoviral vector is based on adenovirus type 5 (Ad5).

[0071] The adenovirus of this invention contains multiple early adenovirus genes (Figure 1).

[0072] E1A proteins are the translation products of the first gene transcription event in the E1A region of the adenoviral genome within the cell nucleus. Initial transcription is driven by a strong constitutive enhancer element within the E1A promoter, allowing for the production of large amounts of E1A mRNA. They are one of two groups of proteins in the adenoviral genome capable of inducing transformation; E1B proteins also induce cell cycle progression. Both E1A and E1B genes are crucial for viral replication.

[0073] The E2 gene is divided into two regions in the adenovirus genome: E2A and E2B, both of which are essential for viral replication. E2B contains the DNA polymerase gene, which is necessary for the amplification of viral genomic DNA. This region also contains the terminal protein (pTP), which acts as a primer to initiate viral genome replication. The terminal protein is covalently linked to the ends of the viral genomic DNA. The E2A region contains the DNA-binding protein required for DNA replication. All E2 genes are essential for viral replication.

[0074] 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 essential 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.

[0075] E3 genes are primarily involved in regulating the cellular and host immune responses to viral infection. However, since most viruses used in biotechnological applications are cultured in vitro, many viral genes, if not all, can be removed without reducing 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 necessary for efficient replication and viral production.

[0076] The E4 region of the adenovirus genome is similar to the E1 region and is primarily involved in the production of proteins that help the virus control and regulate cells to ensure efficient viral replication and production. This region includes six open reading frames (ORFs), which help prevent non-homologous end joining and apoptosis, as well as many other discrete functions. The relative importance of each E4 transcriptional unit for viral replication varies; some are essential, while others can be deleted or modified with little effect on viral growth kinetics and yield.

[0077] The adenovirus described in this invention preferably contains sufficient early adenovirus genes so that the adenovirus can replicate its viral genome within the cell nucleus where it resides.

[0078] The adenovirus of this invention contains multiple adenovirus late genes ( Figure 1 ).

[0079] Late viral genes are divided into five major transcription families, named L1-L5. These transcriptions primarily encode proteins involved in viral assembly and viral structural proteins. During viral replication, they can account for 30-40% of the cellular protein content (Garnier, 1994; Ginsberg, 1984).

[0080] The L1 series of genes transcribes and 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.

[0081] The L2 series of transcriptions encodes the pentazocine base, pVII, V, and pX proteins. These constitute the structural parts of the viral capsid and are essential for the proper assembly of the viral particle. The pentazocine bases contain the RGD motif, which is crucial for the attachment of various adenoviruses to the cell surface during infection. The L2 gene is necessary for successful viral assembly, but not for genomic DNA replication.

[0082] The L3 series of transcriptional proteins encodes pVI, hexagonal proteins, and proteases. Hexagonal proteins are major components of the viral capsid and exhibit antigenic diversity across serotypes. The proteases are involved in cell entry and viral capsid maturation. The L3 gene is essential for successful viral assembly but not for genomic DNA replication.

[0083] The L4 series of transcriptions encodes 100K, 33K, 22K, and pVII proteins. These proteins are involved in a range of functions. The 100K protein is involved in assisting with viral hexagon assembly and nuclear importation, and may also play a role in converting cellular mRNA translation into cap-independent translation. The 22K protein is involved in viral envelope formation. The L4 gene is essential for successful viral assembly, but not for genomic DNA replication. However, the 100K protein may help redirect cellular protein translation to transcription containing a three-part leader sequence (TPL).

[0084] L5 encodes the spike protein. The spike protein is a viral structural protein involved in viral attachment to the cell surface and mediating viral infection. The production of the spike protein far exceeds the amount required for viral particle formation. The L5 gene is essential for successful viral assembly, but not for genomic DNA replication.

[0085] The genome of the adenovirus of this invention contains multiple early and late adenovirus genes sufficient for 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 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.

[0086] In one implementation, the adenovirus is a replicative, reproducible, or conditionally replicative virus. These viruses can be oncolytic viruses, viral vaccines, protein production vectors, or helper viruses for virus production.

[0087] As used herein, "replicating" refers to adenoviruses capable of replicating their genome within a host cell. In one implementation, "replicating" encompasses both replicable and conditionally replicating viruses.

[0088] In this invention, "replicable" means that the adenovirus possesses all the mechanisms required for replication in vitro and in vivo cells, i.e., without the assistance of packaging cell lines.

[0089] As used in this article, "conditional replication," "replication selectivity," or "selective replication" refers to the ability of oncolytic adenoviruses to replicate in cancer cells by utilizing elements specific to or upregulated in cancer cells, such as defective cellular mechanisms like p53 mutations, thereby exhibiting a degree of selectivity for healthy / normal cells.

[0090] "Conditional replication" also refers to the ability of adenoviruses to infect and replicate under specific conditions. Such conditions may include placing the virus under the control of a tumor-specific promoter (e.g., the PSA promoter) to drive viral replication in cells, or placing the virus under the control of an inhibitory element (e.g., a tetracycline repressor (TetR) binding site or a microRNA binding site) to limit viral replication under specific conditions. Conditional replication may have oncolytic properties or may be used as a vector for gene delivery, vaccines, protein production, or viral production (e.g., as a helper virus in AAV production).

[0091] In some embodiments, the adenovirus genome has at least 70% (preferably at least 80%, 85%, 90%, 95%, or 99%) nucleotide sequence identity with the wild-type Ad3 genome sequence given in the complete Ad3 genome sequence (e.g., given in Genbank sequence ID DQ086466.1). In some embodiments, the adenovirus genome has at least 70% (preferably at least 80%, 85%, 90%, 95%, or 99%) nucleotide sequence identity with the wild-type Ad7 genome sequence given in the complete Ad7 genome sequence (e.g., given in Genbank sequence ID AY594255.1).

[0092] In some preferred embodiments, the adenovirus of the present invention is oncolytic. As used herein, the term "oncolytic" refers to the ability of an adenovirus to infect, replicate, and lyse cancer cells. Preferably, the oncolytic adenovirus of the present invention preferentially infects and / or preferentially lyses 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.

[0093] Viral infection can be detected by infectivity assays such as plaque assays and median tissue culture infectious dose (TCID). 50 It can be measured by assay or by using anti-hexamethylenetetramine antibodies in immunocytochemical (ICC) staining assay.

[0094] The oncolytic adenovirus of this invention can be cytolytic. Lysis can be measured by cell death or cell viability assays, including MTS, MTT assay, PrestoBlue™, live / dead staining, and flow cytometry.

[0095] Oncolytic virus infection leads to the death and lysis of cancer cells or mesenchymal cells, preferably releasing newly generated viral particles.

[0096] Viral production can be measured using a variety of methods, including the infectivity assays described above. Other methods exist for measuring the physical count of viral particles: these include UV absorbance measurement (OD260), dynamic light scattering, and HPLC quantification. Other methods still allow for the measurement of viral yield by quantifying DNA and interpolating from a standard curve with known DNA content. These detection methods include quantitative real-time PCR and Pico Green dye-based detection methods. Purified virus can also be quantified by measuring total protein content in a dioctanine acid (BCA) assay.

[0097] In some embodiments, the adenovirus of the present invention is oncolytic and encodes one or more transgenes. The adenovirus of the present invention can be used as an oncolytic vector containing transgenes to generate recombinant nucleic acids and peptides at the tumor site. Examples of transgenes include those encoding anticancer, immunostimulant, or imaging agents (e.g., antibodies, bispecific conjugates, checkpoint inhibitors, cytokines, chemokines, and enzymes (including extracellular matrix degrading enzymes) and angiogenesis inhibitors).

[0098] For example, the transgene can be located within the E1 or E3 region of the adenovirus, or within the region where E1 / E3 has been deleted. It can also be inserted near or inside the L3 or L5 region.

[0099] The deletion of the E3 region creates more space for transgenes to be placed in other locations on the adenovirus genome. It also removes open reading frames (ORFs) that help the virus evade the immune system and prevent the host from clearing the virus. Therefore, the deletion of the E3 region weakens the activity of the virus in normal cells with a normal immune response, but does not weaken the activity in immune-dysfunctional tumor cells, thereby increasing the therapeutic index of oncolytic viruses.

[0100] The size of an adenovirus genome must be within the limits of viral packaging capacity. For example, it must not carry transgenes of such size that the total length of the genome exceeds the length that can be packaged into the protein capsid. The absence of the E3 region creates more space for transgenes in other locations on the adenovirus genome.

[0101] 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.

[0102] The adenovirus of this invention comprises an E3 region. The E3 region is typically located between the L4 and L5 genes (see Figure 1). The E3 region contains multiple ORFs (open reading frames). 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.

[0103] Most published studies on the function of proteins encoded by the E3 region ORF of adenoviruses involve group C adenovirus Ad5. In Ad5, the E3 ORF encodes proteins that regulate the host immune system.

[0104] The E3 region is often completely deleted from adenoviral vectors; under standard and optimized cell culture conditions, this deletion has little impact on viral phenotype. Therefore, for adenoviral vectors used under these conditions, the E3 region can be considered to lack essential function. However, in more relevant models and actual real-world infections, at least some E3 ORFs appear to play an important role in host immune evasion.

[0105] This article provides the nucleotide sequence of the Ad7 E3 region, denoted as SEQ ID NO: 1.

[0106] As used herein, reference to the E3 region of group B adenovirus preferably involves the nucleotide sequence given in SEQ ID NO: 1 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. 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.

[0107] This article provides the nucleotide sequence of Ad7 12.1K ORF, denoted as SEQ ID NO: 2.

[0108] As used herein, reference to group B adenovirus E3 12.1K ORF preferably refers to the nucleotide sequence as given in SEQ ID NO: 2 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a polypeptide having the same function as group B adenovirus E3 12.1K ORF.

[0109] This article provides the nucleotide sequence of Ad7 16.1K ORF, denoted as SEQ ID NO: 3.

[0110] As used herein, reference to group B adenovirus E3 16.1K ORF preferably refers to the nucleotide sequence as given in SEQ ID NO: 3 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a polypeptide having the same function as group B adenovirus E3 16.1K ORF.

[0111] Group B adenovirus E3 19.3K ORF encodes a polypeptide that blocks the presentation of MHC class I restriction antigens; this reduces cytotoxic T cell killing. The nucleotide sequence of Ad7 19.3K ORF is provided in this article, designated SEQ ID NO: 4.

[0112] As used herein, reference to group B adenovirus E3 19.3K ORF preferably relates to the nucleotide sequence given in SEQ ID NO: 4 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.

[0113] This article provides the nucleotide sequence of Ad7 20K ORF, denoted as SEQ ID NO: 5.

[0114] As used herein, references to group B adenovirus E3 20K ORF preferably refer to the nucleotide sequence given in SEQ ID NO: 5 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it.

[0115] Preferably, the variant encodes a polypeptide with the same function as group B adenovirus E3 20K ORF.

[0116] This article provides the nucleotide sequence of Ad7 20.6K ORF, denoted as SEQ ID NO: 6.

[0117] As used herein, reference to group B adenovirus E3 20.6K ORF preferably refers to the nucleotide sequence as given in SEQ ID NO: 6 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, the variant encodes a polypeptide having the same function as group B adenovirus E3 20.6K ORF.

[0118] This document provides the nucleotide sequence of Ad7 7.7K ORF, designated SEQ ID NO: 7. As used herein, reference to group B adenovirus E3 7.7K ORF preferably refers to the nucleotide sequence given in SEQ ID NO: 7 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.

[0119] Group B adenovirus E3 10.3k ORF encodes RIDα; this inhibits immune-induced cell death. The nucleotide sequence of Ad7 10.3k ORF is provided herein, designated SEQ ID NO: 8. As used herein, reference to group B adenovirus E3 10.3k ORF preferably refers to the nucleotide sequence given in SEQ ID NO: 8 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, this variant encodes the RIDα polypeptide.

[0120] Group B adenovirus E3 14.9K ORF encodes RIDβ; this inhibits immune-induced cell death. The nucleotide sequence of Ad7 14.9K ORF is provided herein, designated SEQ ID NO: 9. As used herein, reference to group B adenovirus E3 14.9K ORF preferably refers to the nucleotide sequence given in SEQ ID NO: 9 or a variant nucleotide sequence having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity with it. Preferably, this variant encodes the RIDβ polypeptide.

[0121] Group B adenovirus E3 14.7K ORF encodes a TNF-mediated inhibitor of apoptosis. The nucleotide sequence of Ad7 14.7K ORF, designated SEQ ID NO: B10, is provided herein. As used herein, reference to group B adenovirus E3 14.7K ORF preferably refers to the nucleotide sequence given in SEQ ID NO: 10 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.

[0122] 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 comprises one or more specific E3 region ORFs present in wild-type adenovirus.

[0123] In some embodiments, the present invention provides an oncolytic adenovirus having a genome comprising an E3 region: (a) wherein the E3 region comprises 12.1K and 14.7K ORFs from the E3 region of group B adenoviruses, or corresponding ORFs from the E3 region of non-group B adenoviruses; 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 adenoviruses, or corresponding ORFs from the E3 region of non-group B adenoviruses.

[0124] By comparing the sequence of the gene described in this article with the E3 region of non-group B adenoviruses (e.g., using BLAST), it is easy to find the gene or ORF that "corresponds" to the gene in the E3 region of the group B adenovirus described in this article. Figure 3 The E3 region from group B adenovirus and the corresponding E3 region from representative viruses from groups A and C through G are shown.

[0125] For example, the following nucleotide sequences of the E3 region of non-group B adenoviruses can be found in: Ad26 - GenBank EF153474.1; Ad6 - GenBank OP871032.1; Ad49 - GenBank DQ393829.1; and Ad10 - GenBank JN226746.1.

[0126] 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 an ORF will render the ORF nonfunctional.

[0127] Therefore, in some embodiments, the E3 region does not contain more than 50%, 60%, 70%, 80%, or 90% (preferably not more than 90%) of the 20K, 20.6K, 7.7K, or 10.3K ORFs from the E3 region of group B adenoviruses or their corresponding ORFs from the E3 region of non-group B adenoviruses. In some embodiments, the term "does not contain a functional…ORF" means that the E3 region does not contain that ORF.

[0128] In some embodiments, the present invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region comprises or is composed of the following:

[0129] (a) Group B adenovirus E3 12.1K ORF;

[0130] (b) 3-truncated group B adenovirus E3 16.1 K ORF;

[0131] (c) 5'-truncated group B adenovirus E3 10.3 K ORF;

[0132] (d) Group B adenovirus E3 14.9K ORF; and,

[0133] (e) Group B adenovirus E3 14.7K ORF,

[0134] Alternatively, the corresponding ORF from the E3 region of a non-group B adenovirus may be continuously linked in the 5'-3' sequence described above, wherein the E3 region may optionally contain one or more transgenes located within or adjacent to one or more of the ORFs.

[0135] In other embodiments, the present invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region comprises or is composed of the following:

[0136] (a) Group B adenovirus E3 12.1K ORF;

[0137] (b) Group B adenovirus E3 16.1K ORF;

[0138] (c) 3'-truncated group B adenovirus E3 19.3 K ORF;

[0139] (d) 5'-truncated group B adenovirus 14.9K ORF; and,

[0140] (e) Group B adenovirus E3 14.7K ORF,

[0141] Alternatively, the corresponding ORF from the E3 region of a non-group B adenovirus may be continuously linked in the 5'-3' sequence described above, wherein the E3 region may optionally contain one or more transgenes located within or adjacent to one or more of the ORFs.

[0142] As used herein, the term "continuous linkage" means that the specified ORFs are linked together without any inserted nucleotides or any inserted nucleotides of significant length. In other words, the E3 region consists essentially only of the specified ORFs. However, in some embodiments, the E3 region may optionally contain one or more transgenes located within or near one or more of the stated ORFs. In such embodiments, the term "continuous linkage" means that the specified ORFs are linked together except for the presence of one or more transgenes.

[0143] In some embodiments, the E3 region contains a 3'-truncated group B adenovirus (preferably Ad7) 16.1K ORF. As used herein, 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 when measured from the 3' end). Preferably, the truncation is due to the deletion of nucleotides 356 to 441 in SEQ ID NO: 3. The 3'-truncated group B adenovirus (preferably Ad7) 16.1K ORF may still retain some functional activity.

[0144] In some embodiments, the E3 region contains a 3'-truncated group B adenovirus (preferably Ad7) 19.3 K ORF. As used herein, the term "3'-truncated" means that the 3' end of the 19.3 K ORF has been deleted. For example, at least 10%, 20%, 30%, 40%, or 50% of the 3' end of the 19.3 K ORF has been deleted (preferably when measured from the 3' end). Preferably, the truncation is due to the deletion of nucleotides 402 to 519 (the end of the 19.3 K ORF) in SEQ ID NO: 4. The 3'-truncated group B adenovirus (preferably Ad7) 19.3 K ORF may still retain some functional activity.

[0145] In some embodiments, the E3 region contains a 5'-truncated group B adenovirus (preferably Ad7) 10.3 K ORF. As used herein, the term "5'-truncated" means that the 5' end of the 10.3 K ORF has been deleted. For example, at least 10%, 20%, 30%, 40%, or 50% of the 3' end of the 10.3 K ORF has been deleted (preferably when measured from the 5' end). Preferably, the truncation is due to the deletion of nucleotides 1 to 236 in SEQ ID NO: 8.

[0146] In some embodiments, the E3 region contains a 5'-truncated group B adenovirus (preferably Ad7) 14.9 K ORF. As used herein, the term "5'-truncated" means that the 5' end of the 14.9 K ORF has been deleted. For example, at least 10%, 20%, 30%, 40%, or 50% of the 3' end of the 14.9 K ORF has been deleted (preferably when measured from the 5' end). Preferably, the truncation is due to the deletion of nucleotides 1 to 380 in SEQ ID NO: 9 (i.e., from the beginning of the 14.9 K ORF to the last nucleotide deleted in the B deletion).

[0147] In another embodiment, the present invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region encodes a fusion protein comprising a C-terminus of a C-terminus of a group B adenovirus E3 16.1K protein fused to the N-terminus of an N-terminus of an N-terminus of a group B adenovirus E3 10.3K protein, or the fusion protein comprising a corresponding terminus of a corresponding protein from a non-group B adenovirus E3 region.

[0148] In other embodiments, the present invention provides an oncolytic adenovirus having a genome comprising an E3 region, wherein the E3 region encodes a fusion protein comprising a C-terminus of a C-terminus of a group B adenovirus E3 19.3K protein fused to the N-terminus of an N-terminus of an N-terminus of an N-terminus of a group B adenovirus E3 14.9K protein, or the fusion protein comprising a corresponding terminus of a corresponding protein from a non-group B adenovirus E3 region.

[0149] In some preferred embodiments, the adenovirus of the present invention comprises an E3 region, wherein the E3 region has a deletion compared to the corresponding region of a wild-type group B adenovirus: (a) wherein the start point of the deletion is located at nucleotide 629 in the group B adenovirus E3 region nucleotide sequence as shown in SEQ ID NO: 1, or at the corresponding nucleotide in a non-group B adenovirus E3 region nucleotide sequence; and (b) wherein the end point of the deletion is located at nucleotide 2,892 in the group B adenovirus E3 region nucleotide sequence as shown in SEQ ID NO: 1, or at the corresponding nucleotide in a non-group B adenovirus E3 region nucleotide sequence.

[0150] In some other preferred embodiments, the adenovirus of the present invention comprises an E3 region, wherein the E3 region has a deletion compared to the corresponding region of a 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 as shown in SEQ ID NO: 1, or at the corresponding nucleotide in a 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 as shown in SEQ ID NO: 1, or at the corresponding nucleotide in a non-group B adenovirus E3 region nucleotide sequence.

[0151] (The nucleotide numbers mentioned above refer to the nucleotides at the ends of the E3 region that remain after deletion.)

[0152] In other preferred embodiments, the adenovirus of the present invention comprises an E3 region, wherein the E3 region has a deletion compared to wild-type group B adenovirus, wherein the deletion corresponds to: (a) Ad7 genomic nucleotides 28,011-30,274; or (b) Ad7 genomic nucleotides 28,482-30,665. (These nucleotide numbers refer to the terminal nucleotides retained in the E3 region after deletion.)

[0153] The complete genome of Ad7 is available from Genbank (AY594255.1), and its sequence is incorporated herein by reference.

[0154] In a particularly preferred embodiment, the E3 region of the adenovirus has a nucleotide sequence as given in SEQ ID NO: 11, or a variant thereof having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity, preferably wherein said variant encodes:

[0155] (a) Group B adenovirus E3 12.1K ORF;

[0156] (b) 3-truncated group B adenovirus E3 16.1K ORF;

[0157] (c) 5'-truncated group B adenovirus E3 10.3 K ORF;

[0158] (d) Group B adenovirus E3 14.9K ORF; and,

[0159] (e) Group B adenovirus E3 14.7K ORF.

[0160] In a particularly preferred embodiment, the E3 region of the adenovirus has a nucleotide sequence as given in SEQ ID NO: 13, or a variant thereof having at least 90% (preferably at least 95% or 99%) nucleotide sequence identity, preferably wherein said variant encodes:

[0161] (a) Group B adenovirus E3 12.1K ORF;

[0162] (b) Group B adenovirus E3 16.1K ORF;

[0163] (c) 3'-truncated group B adenovirus E3 10.3 K ORF;

[0164] (d) 5'-truncated group B adenovirus 14.9 K ORF; and,

[0165] (e) Group B adenovirus E3 14.7K ORF.

[0166] Numerous established algorithms exist for aligning two amino acid or nucleic acid sequences. Typically, one sequence serves as a reference sequence, which the test sequence is compared to. Sequence comparison algorithms calculate the percentage of sequence identity between the test sequence and the reference sequence, based on specified program parameters. The alignment of amino acid or nucleic acid sequences used for comparison can be performed, for example, by computer-implemented algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA) or Clustal Omega, BLASTn, and BLASTp algorithms.

[0167] The standard protein-protein BLAST (blastp) can be used to find similar sequences in protein databases. Like other BLAST programs, blastp aims to find locally similar regions. When sequence similarity spans the entire sequence, blastp will also report a global alignment, which is preferred for protein identification purposes. Preferably, standard or default alignment parameters are used. In some cases, the "low complexity filter" can be removed.

[0168] For nucleotide sequence comparisons, MEGABLAST, discontiguous-megablast, and blastn can be used to achieve this goal. Preferably, standard or default alignment parameters are used. MEGABLAST is specifically designed to efficiently find long alignments between very similar sequences. Discontiguous MEGABLAST can be used to discover nucleotide sequences that are similar to but not identical to the nucleic acids of this invention.

[0169] The BLAST nucleotide algorithm finds similar sequences by breaking down a query into short subsequences called words. The program first identifies exact matches (word hits) with the query word. Then, the BLAST program expands these word hits in multiple steps to generate the final gap alignment. In some implementations, the BLAST nucleotide search can be performed using the BLASTN program with a score of 100 and a word length of 12.

[0170] One of the important parameters controlling the search sensitivity of BLAST is the 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 outperforms MEGABLAST in finding alignments with relevant nucleotide sequences from other organisms. In blastn, the word length is adjustable and can be reduced from the default value to a minimum of 7 to improve search sensitivity.

[0171] More sensitive searches can be achieved by using the newly introduced discontinuous 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). Discontinuous megablasts do not require exact word matches as seeds for alignment expansion, but instead use discontinuous words within a longer window of the template. In the encoding mode, the third base wobble is considered by focusing on finding matches at the first and second codon positions while ignoring mismatches at the third position. Searching in discontinuous MEGABLAST using the same word length is more sensitive and efficient than using standard blastn using the same word length. The parameters specific to discontinuous megablasts are: word length: 11 or 12; standard: 16, 18 or 21; standard type: encoded (0), non-encoded (1) or both (2).

[0172] In some implementations, the BLASTP 2.5.0+ algorithm (e.g., an algorithm available from NCBI) can be used with default parameters.

[0173] In other implementations, a BLAST global alignment procedure (e.g., provided by NCBI) can be used, which uses Needleman-Wunsch alignment of two protein sequences with a gap cost of 11 for presence and 1 for extension.

[0174] As used herein, the term “sequence identity” can be replaced by “sequence similarity” in the context of amino acid sequences. The term “similarity” allows for the conserved substitution of amino acid residues with similar physicochemical properties at a given alignment length. The similarity percentage can be determined using any reasonable similarity scoring matrix.

[0175] The present invention also provides a pharmaceutical composition comprising the adenovirus of the present invention, optionally used with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0176] As used herein, the term “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents.

[0177] 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 such as mannitol, sorbitol, and sodium chloride.

[0178] 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.

[0179] In one embodiment, the pharmaceutical composition is a liquid parenteral preparation of the adenovirus of the present invention, for example for infusion or injection.

[0180] As used herein, the term "parenteral preparation" refers to a pharmaceutical composition designed for delivery without passing through the gastrointestinal tract. Typical routes of parenteral administration include injection, implantation, or infusion. In one embodiment, the pharmaceutical composition is provided in a form intended for bolus delivery.

[0181] In one implementation, the parenteral preparation is in the form of an injectable. Injection includes intravenous injection, subcutaneous injection, intratumoral injection, or intramuscular injection. As used herein, injection refers to the administration of liquid into the body via a syringe.

[0182] In one implementation, 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 driver, or equivalent device.

[0183] In one implementation, the parenteral preparation is administered via intravenous infusion.

[0184] In another embodiment, the pharmaceutical composition is provided as a formulation for topical application, including inhalation.

[0185] Suitable inhalable formulations include inhalable powders, metered aerosols containing propellant gases, or inhalable solutions without propellant gases. Inhalable powders according to this disclosure typically contain viruses and physiologically acceptable excipients as described herein.

[0186] 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 used appropriately, with lactose or glucose used, particularly but not limited to their hydrated forms.

[0187] The particles used for deposition in the lungs need to be smaller than 10 micrometers in size, for example, 1 to 9 micrometers, or 0.1 to 5 micrometers, particularly 1 to 5 micrometers. The size of the virus-carrying particle is of utmost importance, and therefore, in one embodiment, the virus according to the invention can be adsorbed or absorbed onto particles, such as lactose particles of a given size.

[0188] 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.

[0189] Inhalable aerosols containing propellant gases may also contain other components, such as solubilizers, stabilizers, surfactants, antioxidants, lubricants, and means for adjusting pH. All of these components are known in the art.

[0190] 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 to 5% by weight, 0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% by weight, 0.5 to 2% by weight, or 0.5 to 1% by weight of active ingredient.

[0191] Alternatively, local administration to the lungs can also be achieved by administering a liquid solution or suspension, for example using a device such as a nebulizer, such as a nebulizer connected to a compressor (e.g., the Pari LC-JetPlus nebulizer connected to a Pari Master® compressor, manufactured by Pari Respiratory Equipment, Inc., Richmond, Va).

[0192] The atomizable formulations according to this disclosure can be provided, for example, as single-dose units packaged in foil sleeves (e.g., sealed plastic containers or vials). Each vial contains a volume of one unit dose, such as 2 mL of solvent / solution buffer.

[0193] The pharmaceutical compositions of the present invention are generally sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other parenteral preparations suitable for human administration, and can be formulated as pre-filled devices such as syringes or vials, particularly for single-dose applications.

[0194] Subjects treated with the adenovirus of this invention may additionally use one or more other chemotherapeutic 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 or immunotherapeutic agents may, for example, include one or more of the following: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, checkpoint inhibitors, antibodies, and other antitumor agents.

[0195] Specific examples of chemotherapy agents include doxorubicin, 5-fluorouracil (5-FU), taxane derivatives (such as paclitaxel and docetaxel), capecitabine, irinotecan, abraxen, and platinum (such as cisplatin, carboplatin, and oxaliplatin).

[0196] Chemotherapy agents can be those that do not interfere with adenovirus activity (e.g., beneficial properties or characteristics 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).

[0197] Typically, this drug combination will be provided as two components:

[0198] (A) The first pharmaceutical composition of the adenovirus of the present invention; and,

[0199] (B) A second pharmaceutical composition comprising a chemotherapeutic agent or an immunotherapeutic agent.

[0200] Therefore, the pharmaceutical combinations of the present invention can be in the form of combination formulations for simultaneous, individual, or sequential use, and are preferably used 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, individually, or sequentially.

[0201] The term "combination formulation" includes both fixed and non-fixed combinations.

[0202] The term "fixed composition" means that the active ingredients (e.g., components (A) and (B)) are in the form of a single entity or dosage unit. In other words, the active ingredients are present in a single composition or formulation.

[0203] The term "non-fixed combination" refers to active ingredients (e.g., components (A) and (B)) existing in different entities or doses (e.g., as separate compositions or formulations), such as as a part of a kit. Independent components (A) and (B) (in their respective compositions or formulations) can then be administered simultaneously, individually, or sequentially at the same time point or at different time points.

[0204] 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.

[0205] 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).

[0206] 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.

[0207] In another embodiment, the present invention provides an oncolytic adenovirus of the present invention for treatment or use as a medicine. In another embodiment, the present invention provides an oncolytic adenovirus of the present invention for treating cancer (preferably ovarian cancer).

[0208] In another embodiment, the present invention provides a method for treating a subject with cancer (preferably ovarian cancer), the method comprising administering an effective amount of the oncolytic adenovirus of the present invention to the subject in need.

[0209] 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).

[0210] The present invention also provides the use of the oncolytic adenovirus of the present invention in the treatment of cancer (preferably ovarian cancer); and the oncolytic adenovirus of the present invention when used in the treatment of cancer (preferably ovarian cancer).

[0211] As used in this article, the term "treating cancer" includes killing cancer cells and / or preventing or inhibiting the spread of cancer cells.

[0212] The therapeutic index (TI, also known as the therapeutic ratio) is a quantitative measure of the relative safety of a drug. It is a comparison of the amount of therapeutic agent that causes a therapeutic effect to the amount that causes toxicity. Related terms, therapeutic window and safety window, refer to the optimized dosage range between efficacy and toxicity, achieving maximum therapeutic benefit without causing unacceptable side effects or toxicity.

[0213] For humans in clinical trials, the treatment index is defined as TD. 50 / ED 50 (of which TD) 50 This is the toxic dose for 50% of subjects; and ED 50 This is the minimum effective dose for 50% of the population.

[0214] The subjects are humans. Subjects can be human males or females (biologically). For example, humans can be 0-10 years old, 10-20 years old, 20-30 years old, 30-40 years old, 40-50 years old, 50-60 years old, 60-70 years old, 70-80 years old, 80-90 years old, 90-100 years old, or over 100 years old. Humans can have a specific disease or condition, such as cancer, preferably ovarian cancer, or be at risk of a specific disease or condition. In some preferred embodiments, the subject is someone who has or has previously had cancer, preferably ovarian cancer.

[0215] As used herein, the term "interstitial tissue" refers to the cells and tissues that support and structure organs, glands, or other tissues in the body. The interstitial tissue 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 interstitial tissue also participates in the body's immune response and the growth and spread of cancer cells. In some implementations, cancer is a tumor.

[0216] 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 weight of the tumor). In some embodiments, a stromal tumor contains 20-80% stromal cells. The stromal cells may contain CAF. The cancer or tumor can be a cancer or tumor containing CAF.

[0217] 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 a stromal-containing cancer.

[0218] In some embodiments, the adenovirus of the present invention can be used as a pretreatment for treatment, such as surgery (neoadjuvant therapy), to shrink tumors, treat metastases, and / or prevent metastasis or further metastasis. In other embodiments, the adenovirus of the present invention can be used after treatment, such as after surgery (adjuvant therapy), to treat metastases and / or prevent metastasis or further metastasis.

[0219] The pharmaceutical compositions of the present invention can be administered via one or more routes using one or more of a variety of methods known in the art. (Components (A) and (B) may be administered via the same route or different routes.) As those skilled in the art will understand, the route and / or pattern of administration will vary depending on the desired outcome.

[0220] Preferred routes of administration for the pharmaceutical compositions of the present invention include intravenous, intratumoral, intraperitoneal, intrapleural, intravesical, intradermal, or other parenteral administration routes, such as by injection or infusion.

[0221] As used herein, the phrase “parenteral administration” refers to a mode of administration other than enteral and local administration, usually by injection, and including but not limited to intravenous, intratumoral, intraperitoneal, intrapleural, intrabladder, intramuscular, intraarterial, intrasheath, intracystic, intraorbital, intracardiac, intradermal, intratracheal, subcutaneous, subepidermal, intra-articular, sub-bursary, subarachnoid, intracavitary, intra-arachnoid, injection and infusion.

[0222] Alternatively, the pharmaceutical compositions of the present invention can be administered via non-parenteral routes, such as local, epidermal or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual or local administration.

[0223] In one embodiment, the formulation is administered intravenously (iv). This route is particularly effective for the delivery of oncolytic viruses because it allows rapid access to most organs and tissues and is especially suitable for treating metastatic tumors, such as established metastatic tumors, particularly those located in highly vascularized areas such as the liver and lungs.

[0224] In one implementation, the formulation is administered intraperitoneally (ip). This route is particularly effective for delivering oncolytic viruses to cancer types primarily located in the peritoneal cavity, such as ovarian, colorectal, and gastric cancer. 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.

[0225] In one embodiment, the formulation is administered intratumorally (it). This route is particularly effective for delivering oncolytic viruses to local cancer types that can be accessed via injection needles. This is because it allows for rapid access to the tumor and minimizes off-target effects.

[0226] 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.

[0227] In one embodiment, each dose of the pharmaceutical composition of the present invention may contain 1x10 9 Up to 1x10 14 Each viral particle. Preferably, each dose of the pharmaceutical composition of the present invention contains 1 × 10⁻⁶ viral particles. 11 Up to 1 × 10 13 A virus particle.

[0228] 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 not be given consecutively over several months.

[0229] Preferably, the method steps are performed in a specified order.

[0230] The adenovirus of the present invention can be readily prepared using techniques known in the art, including introducing one or more nucleotide mutations into a known adenovirus genome using standard cloning techniques (e.g., restriction enzymes, site-directed mutagenesis, or Gibson assembly) or CRISPR-based techniques (e.g., CRISPR Cas9 / guide RNA).

[0231] All publicly available information in each reference mentioned in this article is incorporated herein by reference in its entirety. Attached Figure Description

[0232] Figure 1 A simplified diagram illustrating the locations of early (E) and late (L) genes in the adenovirus genome. Arrows represent viral genes, and their direction indicates the coding direction. ITR = inverted terminal repeat.

[0233] Figure 2A 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 variant viruses (Ov26_E3_Δ14.7K, Ov26_E3_Δ14.9K and Ov26_E3_ΔX1). Black bars represent retained ORFs.

[0234] Figure 3 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.

[0235] Figure 4 Samples from patients with high-grade serous ovarian cancer were infected with increasing concentrations of Ov26. Six days after infection, the viability of cancer cells (A) and cancer-associated fibroblasts (CAFs) (B) in the samples was determined by multiparameter flow cytometry. FAP = fibroblast activation protein, a cell surface marker present on CAFs. CA125 = cancer antigen 125, a cell surface marker present on cancer cells. VG / cell = viral genome per cell.

[0236] Figure 5. Cell viability of various cell lines and primary cells in the presence of Ov26. Cells were infected with escalating doses of Ov26, and viability was measured by MTS assay 5 days post-infection. Data are expressed as viability relative to uninfected controls. VG / cell = viral genome per cell.

[0237] Figure 6 Activity of Ov26 and wild-type parental viruses in cancer cells and normal cells. (A) Viral genome replication was measured by qPCR at 7 days post-infection and expressed as viral genome (VG) per cell. (B) Infectious progeny viruses were quantified by ICC assay and expressed as infection-forming units (IFU) / cell. For each virus, data were normalized to levels observed in A549 cells.

[0238] Figure 7 TNFα levels in ascites samples from ovarian cancer patients, serum from 250 healthy donors, and A549 cancer cell culture medium.

[0239] Figure 8A549 cells stably transduced with the NF-KB-induced promoter-controlled secretory alkaline phosphatase (SEAP) gene 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 TNF-α to induce NF-KB signaling. Sixteen hours after TNF-α treatment, the cells were transduced via QuantiBlue. TM Quantitative SEAP expression was determined.

[0240] Figure 9 Quantification 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 5' 217 bp deletion, the 16.1K ORF was intact, and the 19.1K ORF was present but had a 362 bp 3' deletion (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.

[0241] Figure 10(A) A549 cancer cells were seeded onto sterile coverslips and infected with Ov91 at 500 vg / cell or with Ov91 prepared 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; viability 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).

[0242] Figure 11 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.

[0243] Figure 12. A) A549 cancer cells or normal human primary cells were infected with CRC74 or CRC74_ΔE3 containing 10 viral genomes per cell. Total infectious viral particles were quantified by ICC assay after 4 days. The number of infectious units per cell was calculated and expressed as a percentage of IFU produced during A549 infection. B) 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 were infected with increased doses of CRC74 or CRC74_ΔE3. Cell viability was assessed by MTS assay after 7 days. Viability is expressed as a percentage of viable cells relative to untreated controls.

[0244] Figure 13 The introduction of the E3 deletion enables the oncolytic adenovirus CRC74 to accommodate exogenous DNA at either of the two different equivalence sites.

[0245] The DNA expression cassette encodes ligation sites 1 and 2 of CRC74 (A) and CRC74_ΔE3 (B) in the viral genome. A549 cancer cells were infected with ligated and unligated 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 associated unligated control virus. Example

[0246] Unless otherwise stated, the invention is further illustrated by the following examples, wherein parts and percentages are by weight, and degrees are in degrees Celsius. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Based on the foregoing discussion and these examples, those skilled in the art can determine the essential features of the invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from its spirit and scope. Therefore, various modifications to the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. These modifications are also intended to fall within the scope of the appended claims.

[0247] Example 1: Bioscreening and Sequence Analysis of Oncolytic Adenoviruses

[0248] From the initial pools of adenoviruses in 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 tumor lysis, spread, hematopoietic stability, and immunostimulation for ovarian cancer cells. From the final pool, 60 adenoviruses were selected for sequencing and their genomes were analyzed. The initial pools of adenoviruses in groups B, C, D, F, and G were obtained from commercial sources (US Type Culture Collection or Public Health England) or from UK collaborative networks.

[0249] Genome analysis of the selected adenoviruses revealed that the E3 region of all 60 biologically screened adenoviruses exhibited one of two distinct deletions. Further details of the two deletions are given in the table below; they are also... Figure 2 As shown in the figure, Ov26 and Ov91 are two adenoviruses with deletion (A) for biological screening. Ov20 has deletion (B).

[0250] Table 2: Detailed information on the two missing E3 areas

[0251]

[0252] Deletion A: Delete the sequence between nucleotides 629 and 2,892 from SEQ ID NO:1.

[0253] Deletion B: Delete nucleotides between 954 and 3,283 from SEQ ID NO:1.

[0254] Table 3

[0255]

[0256] For details on the corresponding open reading frames (ORFs) in other adenovirus groups, see [link to relevant documentation]. Figure 3 .

[0257] The fact that all 60 biologically screened adenoviruses possessed one of the two aforementioned deficiencies demonstrates that these deficiencies endow adenoviruses with advantages, namely their ability to lyse ovarian cancer cells, as well as their spread, blood stability, and immune stimulation.

[0258] Example 2: Ov26 kills cancer cells and cancer-associated fibroblasts (CAF) from in vitro ovarian cancer samples.

[0259] Samples from patients with high-grade serous ovarian cancer (HGSOC) were infected with increasing concentrations of Ov26 (one of the adenoviruses with deletion (A) for biological screening). Six days after infection, the viability of cancer cells (A) and cancer-associated fibroblasts (CAF) in the samples was determined by multiparameter flow cytometry.

[0260] The results show Figure 4 These results indicate that the activity of cancer cells and CAFs decreases with increasing Ov26 concentration.

[0261] Example 3: Ov26 kills a range of ovarian cancer subtypes, including untreated and platinum-resistant cells.

[0262] Ovarian patient samples (n=24) were treated with a clinically relevant viral dose of Ov26 (equivalent to 1e13 vp / 5L / patient), and the viability of cancer cells and cancer-associated fibroblasts (CAF) was assessed.

[0263] More specifically, cell viability was assessed by flow cytometry after 6 days. Cell viability was stained using live / dead near-infrared spectroscopy, EpCAM / CA125 markers to identify cancer cells, and fibroblast activation protein (FAP+) to identify cancer-associated fibroblasts. Each condition was measured three times and presented as mean viability relative to uninfected controls. HGSOC = high-grade serous ovarian cancer. LGSOC = low-grade serous ovarian cancer.

[0264] The results are shown in Table 4 below:

[0265] Table 4 :

[0266]

[0267] The table above demonstrates that Ov26 effectively kills cancer cells and cancer-associated fibroblasts (CAFs) in a range of different ovarian cancer subtypes.

[0268] Example 4: Evaluation of the oncolytic activity of Ov26 using a cell line group

[0269] The oncolytic activity of Ov26 was evaluated in many cancer cell lines and cancer-associated fibroblasts, including the following:

[0270] 293A Human Embryonic Kidney Cell Line

[0271] A549 lung cancer cell line

[0272] Hela cervical cancer cell line

[0273] MDA-MB-231 breast cancer cell line

[0274] PANC1 pancreatic cell line

[0275] PSN1 pancreatic cell line

[0276] HCT116 colorectal cancer cell line

[0277] HT29 colorectal cancer cell line

[0278] SKOV-3 ovarian cell line

[0279] Huh-7 liver cancer cell line

[0280] MRC5 fibroblast cell line

[0281] OE-21 esophageal cell line

[0282] OVCAR-3 ovarian cell line

[0283] OVSAHO ovarian cancer cell line

[0284] The results are shown in Figure 5. Ov26 demonstrated efficacy against all tested cancer cell lines.

[0285] The IC50 values ​​for Ov26 are given in the table below.

[0286] Table 5. IC50 values ​​of Ov26 in various cancer cell lines

[0287]

[0288] Example 5: Oncolytic activity of Ov26 compared to wild-type parent virus

[0289] Parameters of Ov26 virus infection were measured 72 hours after infection of lung cancer cells (A549s), non-cancerous normal primary human hepatocytes, and normal human skin fibroblasts (NHDF).

[0290] like Figure 6 As shown, during Ov26 infection, compared to parental virus infection, normal cells produced lower levels of viral genome (A) and infectious viral particles (B) relative to A549 cancer cells. These results demonstrate Ov26's ability to preferentially infect lung cancer cells compared to normal hepatocytes and normal fibroblasts.

[0291] Example 6: Quantification of TNFα levels in patient-derived samples.

[0292] Tumor necrosis factor-alpha (TNFα) is an inflammatory cytokine that plays a role in a variety of cellular signaling events, many of which ultimately lead to cell necrosis or apoptosis. TNFα signaling can enhance resistance to infection by inducing apoptosis in infected cells, and therefore may have important implications for oncolytic virus therapy. TNFα levels were measured in ascites fluid samples from ovarian cancer patients, in serum from 250 conjoined healthy donors, and in culture medium from A549 cancer cells. Quantification was performed by ELISA. Figure 7The results showed that TNFα levels were highest in patient-derived samples, and TNFα was not detected in healthy serum or cancer cell line cultures. This indicates that the effects of TNFα on viral therapy can only be effectively assessed in models, such as patient-derived tumor samples, where TNFα or other related cytokines, chemokines, and stress factors (such as IFN) are upregulated or at physiologically relevant concentrations, while these effects cannot be reflected under normal cell culture conditions.

[0293] Example 7: Comparison of Ov26, Ov26_E3_Δ14.7K, Ov26_E3_Δ14.9K, and Ov26_E3_ΔX1

[0294] The E3 region of adenovirus encodes proteins that 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 localize to the plasma membrane. The RID complex promotes the survival of infected cells by inhibiting apoptosis typically induced by receptors containing death domains 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; the role of RIDβ in NF-κB signaling remains unclear. The 14.7K ORF of the E3 region protects infected cells from TNFα-induced apoptosis.

[0295] Ov26 variants with deletions at E3 ORF 14.9K (RIDβ) or 14.7K were generated and named OV26_E3_Δ14.9K and OV26_E3_Δ14.7K, respectively. A variant named Ov26_E3_ΔX1 was also generated, with a complete deletion of 14.9K ORF, a 5'217 bp deletion of 14.7K ORF, an intact 16.1K ORF, and a 19.1K ORF present but with a 362 bp 3' deletion. These deletion constructs were generated by vectorizing the viral genome into a bacterial artificial chromosome (BAC). The E3 region of Ov26 was then replaced with a selectable cassette flanked by unique restriction sites using a homologous recombination-based approach. The cassettes were then removed by restriction digestion, and synthetic DNA encoding the modified E3 region was inserted via Gibson assembly. The constructs were validated, and the virus was recovered by transfecting them into Ad-293 cells.

[0296] The ability of these adenoviruses to induce NF-KB signaling was determined using cell lines encoding the secretory alkaline phosphatase (SEAP) gene under the control of the NF-KB inducible promoter. Figure 8 ).

[0297] The results showed that Ov26 could block the induction of NF-KB signaling to a greater extent than Ov26_E3_Δ14.9K.

[0298] The ability of Ov26, Ov26_E3_Δ14.7K, and Ov26_E3_ΔX1 to protect infected cells from TNFα-induced apoptosis was measured by quantifying caspase activation in infected cells after exposure to TNFα. Figure 9 The results showed that cells infected with Ov26 did not respond to TNFα treatment and any caspase activation, and were therefore protected from TNFα-induced apoptosis. However, cells infected with OV26_E3_Δ14.9K or OV26_E3_ΔX1 showed significant caspase activation after TNFα treatment.

[0299] Example 8: Comparison of growth kinetics between Ov91 candidate and “equipped” Ov candidate

[0300] Ov91 is one of the biologically selected adenoviruses with an E3 region containing a deleted A, and therefore its E3 region is identical to that of Ov26. Ov91 variants encoding therapeutic transgenes were generated by vectorizing the Ov91 genome into a bacterial artificial chromosome (BAC). The E3 region of Ov91 was then replaced with a selectable cassette flanked by unique restriction sites using a homologous recombination-based approach. The cassettes were then removed by restriction digestion, and synthetic DNA encoding the modified E3 region was inserted via Gibson assembly. The construct was validated, and the virus was recovered by transfecting it into Ad-293 cells.

[0301] The ability of viruses to spread from infected cells to neighboring cells after cell lysis, cancer cell killing, and viral growth was quantified. Figure 10 The results presented show that the E3 deletion A found in both Ov91 and Ov26 can accommodate the insertion of large transgenes of up to, but not limited to, 2,207 bp (representing 6.7% of their genome length) without significantly losing their efficacy against cancer cells.

[0302] Example 9:

[0303] Studies have shown that the E3 14.7K protein is crucial for blocking TNFα-induced NF-κB signaling, and that Ov26 is more resistant to apoptosis than viral variants Ov26_E3_Δ14.7K and Ov26_E3_ΔX1 (Example 7). One way in which resistance to apoptosis provides a significant advantage for oncolytic viruses is whether delayed cell death offers a greater opportunity for viral replication and the production of more progeny viral particles. Therefore, the number of infectious units (IFUs) produced by each of the viruses Ov26, Ov26_E3_Δ14.7K, and Ov26_E3_ΔX during cancer cell infection was assessed in the presence and absence of TNFα treatment. Figure 11 The results shown indicate that treatment of cells with TNFα reduced the number of IFUs produced by variant viruses Ov26_E3_Δ14.7K and Ov26_E3_ΔX to a greater extent than Ov26.

[0304] Example 10:

[0305] Compared to its parent virus, Ov26 has shown higher selectivity for cancer cells than for normal cells; this is likely due to the attenuation effect of deletions within its E3 region. To assess whether this deletion arrangement also enhances the selectivity of other oncolytic viruses, the same E3 deletion (e.g., Figure 2 As shown, Theolytics A) introduced an alternative oncolytic adenovirus CRC74, producing the E3-deficient variant CRC74_ΔE3.

[0306] To investigate the selectivity of CRC74 and its variant CRC74_ΔE3, the production of infectious viral particles following infection of A549 cancer cells and three different normal primary cell types was quantified. Figure 12.A shows that CRC74 infection of a subset of normal primary cells leads to the production of infectious viral particles; however, infection of these cells with the CRC74_ΔE3 variant significantly reduces the production of infectious viral particles. However, the introduction of E3 deletion in CRC74 does not substantially affect the killing of cancer cells or cancer-associated fibroblasts, such as… Figure 12B As shown in Figure 12, the data demonstrate that introducing E3 deletion into CRC74 improves the selectivity of oncolytic viruses and proves that this specific E3 arrangement enhances the selectivity of different oncolytic adenoviruses.

[0307] Example 11:

[0308] Ov26 is able to adapt to the insertion of transgene expression cassettes into its genome without affecting its replication and spread. To assess whether the E3 deletion is an important feature that allows transgene sequences to be included in the viral genome, the same E3 deletion was introduced into the alternative oncolytic adenovirus CRC74, generating the E3-deleted variant CRC74_ΔE3.

[0309] To assess the ability of the two viruses, CRC74 and CRC74_ΔE3, to contain exogenous DNA in their genomes, cancer cell killing activity was quantified after the encoded expression cassette was inserted into one of two sites within the viral genome. Figure 13 The results in .A showed that although CRC74 could be equipped, its killing activity was significantly delayed, and it did not reach 100% activity by 96 hours compared to the unequipped CRC74 virus.

[0310] at the same time, Figure 13 The results in .B showed that the CRC74_ΔE3 virus exhibited significantly better tolerance to the strain, with cytotoxic activity comparable to the control virus at 96 hours post-infection. Therefore, in summary, Figure 13 The data shown in the study indicate that introducing E3 deletion into CRC74 significantly improves the ability of oncolytic viruses to tolerate the insertion of expression cassettes into their genome without affecting oncolytic activity.

[0311] The sequence list filed with this patent application is incorporated herein as part of the specification.

Claims

1. An oncolytic adenovirus having a genome, said genome containing an E3 region: (a) wherein the E3 region comprises 12.1K and 14.7K open reading frames (ORFs) from the E3 region of group B adenovirus, or corresponding ORFs from the E3 region of non-group B adenovirus; and, (b) In which, 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 their corresponding ORFs from the E3 region of non-group B adenovirus.

2. The oncolytic adenovirus according to claim 1, wherein, The E3 region also includes functional (optionally 3'-truncated) E3 regions from group B adenoviruses. 16.1K ORF or its corresponding ORF from the E3 region of a non-group B adenovirus.

3. The oncolytic adenovirus according to claim 1 or claim 2, wherein, The E3 region further includes a 16.1K ORF from the E3 region of group B adenovirus and a functional (optionally 3'-truncated) 19.3K ORF from the E3 region of group B adenovirus, or its corresponding ORF from the E3 region of non-group B adenovirus.

4. The oncolytic adenovirus according to any one of the preceding claims, wherein, The E3 region also contains 14.9K ORF from the E3 region of group B adenovirus or its corresponding ORF from the E3 region of non-group B adenovirus.

5. The oncolytic adenovirus according to claim 1, 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 it may originate from the corresponding ORF of the E3 region of a non-group B adenovirus. The E3 regions are connected consecutively in the 5'-3' sequence described above, and the E3 regions may optionally contain one or more transgenes located within or adjacent to one or more of the ORFs.

6. The oncolytic adenovirus according to claim 1, wherein, The E3 region includes or consists of the following: (a) Group B adenovirus E3 12.1K ORF; (b) Group B adenovirus E3 16.1K ORF; (c) 3-truncated group B adenovirus E3 19.3 K ORF; (d) 5'-truncated group B adenovirus E3 14.9 K ORF; and, (e) Group B adenovirus E3 14.7K ORF, Or it may originate from the corresponding ORF of the E3 region of a non-group B adenovirus. The E3 regions are connected consecutively in the 5'-3' sequence described above, and the E3 regions may optionally contain one or more transgenes located within or adjacent to one or more of the ORFs.

7. The oncolytic adenovirus of claim 1, wherein the E3 region encodes a fusion protein, the fusion protein comprising a C-terminus of a C-terminus of a group B adenovirus E3 16.1K protein fused to the N-terminus of an N-terminus of an N-terminus of an N-terminus of an N-terminus of a group B adenovirus E3 10.3K protein, or the fusion protein comprising a corresponding terminus of a corresponding protein from a non-group B adenovirus E3 region.

8. The oncolytic adenovirus of claim 1, wherein the E3 region encodes a fusion protein, the fusion protein comprising a C-terminus of a C-terminus of a group B adenovirus E3 19.3K protein fused to the N-terminus of an N-terminus of an N-terminus of an N-terminus of a group B adenovirus E3 14.9K protein, or the fusion protein comprising a corresponding terminus of a corresponding protein from a non-group B adenovirus E3 region.

9. The oncolytic adenovirus of claim 1, wherein, compared to the corresponding region of wild-type group B adenovirus, the E3 region has a deletion: (a) wherein the deletion start point is located at nucleotide 629 in the group B adenovirus E3 region nucleotide sequence as shown in SEQ ID NO: 1, 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: 1, or at the corresponding nucleotide in the nucleotide sequence of a non-group B adenovirus E3 region.

10. The oncolytic adenovirus of claim 1, wherein, compared to the corresponding region of wild-type group B adenovirus, the E3 region has a deletion: (a) wherein the deletion start point is located at nucleotide 1099 in the group B adenovirus E3 region nucleotide sequence as shown in SEQ ID NO: 1, 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 3283 in the E3 region nucleotide sequence of group B adenovirus as shown in SEQ ID NO: 1, or at the corresponding nucleotide in the nucleotide sequence of a non-group B adenovirus E3 region.

11. The oncolytic adenovirus of claim 1, wherein the E3 region has a deletion 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.

12. The oncolytic adenovirus according to any one of the preceding claims, wherein the adenovirus is a group B adenovirus or a human adenovirus, preferably Ad3 or Ad7 serotype, or an Ad3 / Ad7 chimera.

13. The oncolytic adenovirus according to any one of the preceding claims, wherein the adenovirus further comprises a transgene, preferably a transgene encoding an antibody, a bispecific conjugate, a checkpoint inhibitor, a cytokine, a chemokine, or an enzyme.

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

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

16. The oncolytic adenovirus according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 15, for use as a treatment or as a medicine.

17. The oncolytic adenovirus according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 15, for the treatment of cancer (preferably ovarian cancer).

18. 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 oncolytic adenovirus as described in any one of claims 1 to 14 or a pharmaceutical composition as described in claim 15.

19. Use of the oncolytic adenovirus according to any one of claims 1 to 14 in the preparation of a medicament for treating cancer (preferably ovarian cancer).

20. The oncolytic adenovirus of claim 17, the method of claim 18, or the use of claim 19, wherein the cancer is a stromal tumor.

21. The oncolytic adenovirus of claim 17, the method of claim 18, or the use of claim 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 cancer.

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