Adenovirus

Abstract

The present invention relates to adenoviruses, in particular oncolytic adenoviruses, for use in the prevention or treatment of cancer, including interstitial-containing tumors and ovarian cancer. More specifically, the present invention relates to adenoviruses having a chimeric L2-L3 region as compared to a wild-type adenovirus.

Description

[0001] The present invention relates to adenoviruses, in particular oncolytic adenoviruses, for the prevention or treatment of cancer, including tumors containing stroma and ovarian cancer. In particular, the present invention relates to adenoviruses having a chimeric L2-L3 region compared to wild-type adenoviruses.

[0002] Carcinomas are malignant tumors that arise from epithelial tissues, which line the outer or luminal surfaces of organs, including the lung, gastrointestinal tract, and reproductive tissues. Carcinomas account for approximately 90% of all cancer cases worldwide and are the leading cause of most cancer deaths.

[0003] A common feature of carcinomas is that they are typically diagnosed as a palpable lesion due to the presence of extracellular stroma, fibroblasts, and immune cells surrounding and / or between islands of cancer cells. All cells and material in a tumor that are not defined as cancer cells (or malignant cells) are generally referred to as the “stroma,” or more specifically, the “tumor stroma.” Certain cells in the immune system, such as macrophages, fibroblasts, are often included in the stroma category, while others, such as lymphocytes, are described as infiltrating or traveling through it.

[0004] All macroscopically visible and palpable carcinomas contain some degree of stroma; the stroma provides the necessary structural support, nutrient supply, and suitable environment for the survival, proliferation, and even spread of cancer cells.

[0005] The proportion of stroma can vary between 5-80%, depending in part on the underlying lesion type. However, anyone familiar with the structure of tumors understands that the greatest variation in stroma content for a particular biopsy sample arises from the biopsy location, as certain regions of a tumor appear to have more stroma visually than others.

[0006] Stromal cells present features on the surface that resemble normal tissues and organs. They are responsible for remodeling the extracellular matrix, inducing angiogenesis, and providing structural support. However, unlike normal tissues, the behavior of these cells is out of control and often chaotic. Excessive stroma leads to increased tissue pressure, poor blood flow, and triggers local hypoxia. Thus, cancer cells face temporal and spatial differences in nutrient and oxygen supply. As the tumor grows, large numbers of cells die and necrotize due to periodic fluctuations in local nutrient levels. The fluctuating shortages of nutrients and oxygen appear to be detrimental to tumor growth, but increasing research suggests that this environment instead drives stronger cellular clonal evolution, which confers resistance to therapy.

[0007] In recent years, the role of the stromal tissue in protecting cancer cells from drug or immune attack has gained increasing attention. In particular, cancer-associated fibroblasts (CAFs), which have been shown to prevent T cell entry into tumor tissue or inhibit the activity of infiltrating T cells by expressing cytokines such as transforming growth factor beta (TGFp).

[0008] The presence of CAFs and their influence on immune cells are often considered to be the root cause of poor responses to immunotherapy. However, the development of new immunotherapies for cancer patients has been hampered by the lack of animal models containing sufficient stromal cells or the correct tissue architecture. Once CAFs are artificially introduced into animal models, the effectiveness of cancer vaccines and immune checkpoint inhibitor therapies is significantly reduced or even completely lost.

[0009] Despite the lack of models, the development of drugs targeting the stroma is increasingly gaining attention. Such drugs either act directly on stromal cells themselves or on the microenvironmental conditions created by the stroma. However, stromal targeting strategies often face two fundamental challenges. First, selectivity is extremely challenging because the cells that make up the stroma are essentially "normal" and not malignant and are also found in other parts of the body; second, the conceptual difficulty is that a combination therapy must be used because targeting the stroma alone is almost never sufficient. For refractory cancers, simultaneous targeting of the stroma and cancer cells can be necessary if a single targeting approach is ineffective. While the theoretical basis for combination therapy is well established, the development of combination drugs still poses a significant challenge, especially for drugs that are ineffective or can be ineffective as monotherapies.

[0010] An ideal cancer treatment intervention should be able to simultaneously and selectively target both cancer cells and stromal cell populations.

[0011] Oncolytic viruses, as an emerging therapeutic modality, are able to kill multiple types of tumor cells, including differentiated cancer cells and cancer cell initiating cells or stem cells. They are able to exploit "hallmark features" of cancer (such as immune or cell cycle dysregulation) to a high degree of selectivity, proliferate and lyse cells within the tumor.

[0012] The main disadvantage of oncolytic viruses is that, to date, no oncolytic virus has been explicitly designed or developed to target stromal cells. This is because contemporary oncolytic viruses have only been constructed to be active in malignant cells (by definition).

[0013] In human tumors, stromal cells divide and surround individual regions of cancer cells, which sets a physical barrier (conceptually, at least) to oncolytic virus therapy. As a result, successful therapeutic regimens in the laboratory rarely translate effectively to the clinic. In cases where clinical efficacy has been achieved against solid cancers, multiple injections of oncolytic viruses are often used to break through the stromal barrier (e.g., Khuri FR, et al. “A controlled trial of intra-tumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer”. Nat Med. 2000 Aug;6(8):879-85. doi: 10.1038 / 78638. PMID: 10932224). Alternatively, very small or early stage cancers with relatively low stromal content can be targeted (e.g., Packiam VT, et al. “An open label, single-arm, phase II multicenter study of the safety and efficacy of CG0070 oncolytic vector regimen in patients with BCG-unresponsive non-muscle-invasive bladder cancer: Interim results.” Urol Oncol. 2018 Oct;36(10):440-447. doi: 10.1016 / j.urolonc.2017.07.005. Epub 2017 Jul 26. PMID: 28755959).

[0014] To address stromal cell problems, some oncolytic viruses have been engineered to express biologies, including bispecific T-cell engager molecules (BiTEs), to selectively kill stromal cells. The design concept is that the oncolytic virus is responsible for clearing cancer cells, while the BiTE expressed by it targets stromal cells, thus enabling complete lysis of the tumor.

[0015] Unfortunately, this strategy remains fundamentally insufficient due to the structural and spatial arrangement characteristics of the tumor stroma. In human tumors, the capillaries supplying cancer cells emerge from the stroma, so drugs, including oncolytic viruses, must traverse multiple layers of stromal cells to reach the cancer cells. For small molecule drugs, diffusion makes this process relatively easy; however, oncolytic viruses are usually too large to easily penetrate the intercellular spaces. When oncolytic viruses enter human tumors, they are first exposed to stromal cells, where they cannot replicate or express transgenic products such as BiTE.

[0016] This defect is not apparent in animal models because these models typically lack the stroma; even when the stroma is present, its structure is different—capillaries emerge directly from cancer cells.

[0017] For oncolytic agents to be effective in treating human diseases, they must be activated immediately upon entering the tumor microenvironment. This mechanism is possible because the three hallmark features of cancer that oncolytic viruses rely on to kill cancer cells also apply to cancer cells (CAFs). These features include metabolic dysregulation, anti-apoptosis, and immune dysfunction. Therefore, the ideal strategy is to develop an oncolytic drug that can simultaneously utilize the shared features of both cancer cells and stromal cells, making it active against both types of cells.

[0018] Ovarian cancer is one of the most common and deadliest cancers in women. It is often diagnosed at an advanced stage, by which time the cancer cells have spread to other parts of the body. Despite the availability of various treatments, the best survival rate for patients with stage III and higher ovarian cancer remains only 25%. Most ovarian cancer patients are diagnosed at the most advanced stage—high-grade serous ovarian cancer (HGSOC), which accounts for 70-80% of ovarian cancer deaths. Current treatment options involve repeated platinum-based therapy until resistance and recurrence become inevitable. Second-line treatments include paclitaxel and doxorubicin, but their efficacy is limited. Only a small percentage of patients with BRCA1 mutations (13-15%) can use newly approved PARP inhibitors. Recently, mirvetuximab received accelerated approval for the treatment of patients with high folate receptor expression. Although these new drugs show some efficacy in the intermediate stages, most patients are expected to experience cancer recurrence.

[0019] The potential role of oncolytic viruses in the treatment of ovarian cancer has been explored. The mechanism is that oncolytic viruses can kill heterogeneous cell populations, including cancer stem cells, while stimulating the body to produce an immune response. Multiple clinical trials have confirmed that the delivery of oncolytic viruses to ovarian cancer patients is safe and feasible, but the overall efficacy is still limited. In fact, it is CAFs in the stroma that weaken the effect of oncolytic therapy through antiviral signaling pathways (Arwert EN, et al. "STING and IRF3 instromal fibroblasts enable sensing of genomic stress in cancer cells 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 crucial role of CAFs in promoting ovarian cancer progression and providing resistance mechanisms to chemotherapy, immunotherapy, and oncolytic virus therapy, there is an urgent need to develop novel therapeutic strategies that can simultaneously target CAFs and cancer cells.

[0021] Cancer-associated cells (CAFs) play a crucial role in determining patient prognosis and treatment response. They achieve these effects by suppressing immune cells, hindering effector T cell infiltration, forming dense barriers to prevent the spread of therapeutic drugs, and inducing resistance to standard-of-care (SOC) drugs. Furthermore, CAFs can promote tumor growth and provide nutritional support, aiding in tumor proliferation and metastasis. Despite the critical impact of CAFs on patient treatment response, existing preclinical models, including more complex in vivo PDX models, fail to represent this key component of the human tumor microenvironment (TME). Traditional cancer cell lines and in vivo xenograft tumor models cannot replicate the complexity and heterogeneity of human cancers, and cannot accurately capture the stromal components, immunosuppressive mechanisms, metabolic disorders, cytokines, and stress proteins (such as TNF-α and TGF-β) present in the patient's tumor microenvironment. Therefore, developing therapies based on realistic simulation systems (such as freshly excised patient biopsy tissue, multicellular, nutrient-deprived, and hypoxic tumor microenvironments) is crucial for identifying drugs that remain effective under these barriers and stress conditions.

[0022] The applicant observed that the tumor microenvironment is drastically different from the environment in which viruses naturally evolve: viruses typically reside in airway cells with a sustainable supply of nutrients and an excess of oxygen. The tumor microenvironment differs significantly from nutrient-rich cell culture systems or rapidly growing animal model environments. Therefore, wild-type viruses, or viruses screened through simple animal models, are often unsuitable for treating human diseases. It can be inferred that any virus optimized for the tumor environment is likely to differ significantly from wild-type or engineered viruses. To survive in the tumor environment, viruses must regulate their demand for various resources, otherwise they risk disruption of their replication cycle. Mutations (even subtle ones) enable this regulation because the viral mechanism—the synthesis of RNA, DNA, and proteins—is highly "gear-like." When a virus replicates and produces hundreds of thousands of progeny cells, even small changes in a single virus can have a significant impact.

[0023] The applicant has now identified a characteristic of the adenovirus that includes conferring enhanced systemic administration capability and the ability to spread in solid tumors by establishing effective infection in adjacent cells.

[0024] From an initial pool of B, C, D, F, and G adenoviruses, 23 rounds of biological screening (including mutation steps) were conducted to enrich oncolytic adenovirus candidate strains exhibiting optimal tumor lysis, spread, hematopoietic stability, and immunostimulatory activity. Finally, 60 adenovirus strains were selected from the pool for sequencing and genomic analysis.

[0025] Genome analysis of the selected adenoviruses revealed several genomic features common to most or all candidate adenoviruses. Specifically, all candidate adenovirus genomes were derived from type B adenovirus and were chimeric: the 5' end of the genome originated from Ad3 serotype virus, and the 3' end from Ad7 serotype virus. In all the adenoviruses screened, the chimeric junction was located within the L2-L3 region.

[0026] The structural result of this chimerism is that in the adenovirus involved in this invention, the pentagonal basal protein (encoded by the L2 gene) originates from the Ad3 serotype, while the hexagonal protein (encoded by the L3 gene) originates from the Ad7 serotype. These two proteins together constitute the components of the adenovirus capsid. The third major component of the adenovirus capsid—the spike protein (encoded by the L5 gene)—also originates from the Ad7 serotype.

[0027] Pentagonal basal proteins are located at each apex of the adenovirus capsid. For most adenoviruses, they serve as attachment sites for cell surface integrins αvβ3 and αvβ5, with αvβ5 being a key receptor for adenovirus internalization into host cells. Therefore, the serotype of pentagonal basal proteins directly influences the cell tropism of adenoviruses.

[0028] Not limited by theory, the chimeric characteristics of the adenoviruses of this invention may also be specifically screened out during biological selection processes, because these chimeric adenoviruses have the ability to overcome neutralizing antibodies, which can bind to the surface proteins of adenoviruses (such as pentameric basal proteins, hexameric proteins, and spike proteins). This characteristic makes the adenoviruses of this invention not only highly valuable as oncolytic adenoviruses, but also suitable as gene therapy vectors.

[0029] CN103966174 discloses a recombinant Ad3 adenovirus that inserts an Ad7 hexagonal protein expression cassette into the E3 region of the adenovirus genome. The resulting capsid is chimeric, containing a mixture of Ad3 pentagonal protein, Ad3 hexagonal protein, and Ad7 hexagonal protein, as well as an Ad3 spike protein. This recombinant adenovirus is described as a bivalent vaccine, preventing human Ad3 and Ad7 adenovirus infections (such as acute respiratory diseases) by eliciting an immune response to Ad3 and Ad7. This contrasts with the adenovirus described in this invention, which is used as an oncolytic virus and has been biologically screened to possess the ability to overcome neutralizing antibodies.

[0030] One object of the present invention is to provide adenoviruses, particularly oncolytic adenoviruses, which have enhanced ability to infect adjacent cells and establish new effective infections, especially in tumor cells. Another object of the present invention is to provide compositions comprising the oncolytic adenoviruses of the present invention, and their use in the prevention or treatment of cancer, preferably ovarian cancer. Yet another object of the present invention is to provide compositions comprising the adenoviruses of the present invention for use as gene therapy vectors.

[0031] In some embodiments, the present invention provides a chimeric adenovirus, wherein the genome of the chimeric adenovirus comprises:

[0032] (a) Ad3 pentagenesis gene; and

[0033] (b) Ad7 hexane gene;

[0034] It is used to prevent or treat cancer, with ovarian cancer being the preferred candidate.

[0035] In some embodiments, the present invention provides a method for treating cancer, preferably ovarian cancer, the method comprising administering an effective amount of chimeric adenovirus to a patient in need, wherein the genome of the chimeric adenovirus comprises:

[0036] (a) Ad3 pentagenesis gene; and

[0037] (b) Ad7 hexane gene.

[0038] In another embodiment, the present invention provides the use of a chimeric adenovirus in the preparation of a medicament for the prevention or treatment of cancer, preferably ovarian cancer, wherein the genome of said chimeric adenovirus comprises:

[0039] (a) Ad3 pentagenesis gene; and

[0040] (b) Ad7 hexane gene.

[0041] In some embodiments, the genome of the chimeric adenovirus further includes: (c) the Ad7 spike protein gene.

[0042] The present invention also provides a chimeric adenovirus, wherein the genome of the chimeric adenovirus comprises:

[0043] (a) Ad3 pentacol gene;

[0044] (b) Ad7 hexane gene; and

[0045] (c) Ad7 spike protein gene.

[0046] The present invention also provides an adenovirus gene therapy vector, wherein the genome of the adenovirus gene therapy vector comprises:

[0047] (a) Ad3 pentacol gene;

[0048] (b) Ad7 hexane gene; and

[0049] (c) Genetically modified organisms.

[0050] The present invention also provides a pharmaceutical composition comprising the chimeric adenovirus of the present invention, optionally in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0051] The present invention also provides a pharmaceutical combination comprising:

[0052] (A) A first pharmaceutical composition comprising the chimeric adenovirus of the present invention; and

[0053] (B) A second pharmaceutical composition comprising a chemotherapy drug or an immunotherapy drug.

[0054] The drug combination is in the form of a combination formulation for simultaneous, separate or sequential use, preferably for the treatment of cancer.

[0055] The invention also provides a chimeric adenovirus or an adenovirus gene therapy vector for treatment or as a medicine.

[0056] The invention also provides the use of the chimeric adenovirus as a protein production vector, wherein the chimeric adenovirus contains a transgene encoding the protein to be produced.

[0057] Table 1. Nucleotide and amino acid sequence list

[0058]

[0059]

[0060] The present invention provides adenoviruses, particularly oncolytic adenoviruses, preferably for the treatment of cancer, more preferably for ovarian cancer or stromal tumors.

[0061] As used herein, "adenovirus (Ad)" refers to viruses belonging to the family Adenoviridae, encompassing the five currently known genera: Mastadenovirus, Aviadenovirus, Atadenovirus, Siadenovirus, and Ichtadenovirus. Preferably, the adenovirus originates from the genus Mastadenovirus, which includes all human serotypes. In some embodiments, the adenovirus is a human adenovirus.

[0062] More than 60 antigenic or "serotypes" of human adenoviruses have been identified to date. These serotypes are classified into seven species, namely adenoviruses AG, based on their physical, chemical, and biological characteristics (e.g., as described in Wold et al. Current genetherapy vol. 13, 6 (2013): 421-33). Therefore, the adenovirus species referred to in this article include both currently known AG species and potential new species that may be discovered in the future.

[0063] In some embodiments, the adenovirus species is selected from subspecies AdA, AdB, AdC, AdD, AdE, AdF, and AdG. In some embodiments, the human adenovirus is selected from subspecies AdB, AdC, AdD, AdE, AdF, and AdG.

[0064] The serotypes belonging to these adenovirus subspecies include, but are not limited to, the following:

[0065] AdA includes Ad12, Ad18, Ad31, and Ad61. AdB includes Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad50, Ad55, Ad66, Ad68, and Ad79. AdC includes Ad1, Ad2, Ad5, Ad6, and Ad57. AdD includes Ad8, Ad9, Ad10, Ad13, Ad15, Ad17, Ad19, Ad20, Ad22, Ad23, Ad24, Ad25, Ad26, Ad27, Ad28, Ad29, Ad30, Ad32, Ad33, Ad36, Ad37, Ad38, Ad39, Ad42, Ad43, Ad44, Ad45, Ad46, Ad47, Ad48, Ad49, Ad51, Ad53, Ad54, and Ad56. Ad58, Ad59, Ad60, Ad62, Ad63, Ad64, Ad65, Ad67, Ad69, Ad70, Ad71, Ad73, Ad74, and Ad75. AdE includes Ad4. AdF includes Ad40 and Ad41, and AdG includes Ad52.

[0066] The different adenovirus serotypes mentioned in this article all include all different strains or variants of that serotype.

[0067] Preferably, the adenovirus is a type B adenovirus. Type B1 adenoviruses include Ad3, Ad7, Ad16, Ad21, Ad50, Ad66, and Ad68; Type B2 adenoviruses include Ad11, Ad14, Ad34, Ad35, Ad55, and Ad79. Preferably, the adenovirus is a type B1 adenovirus; most preferably, the adenovirus is Ad3 serotype, Ad7 serotype, or an Ad3 / Ad7 chimeric adenovirus.

[0068] The chimeric oncolytic adenovirus of this invention has a genome. In some embodiments, the adenovirus is a human adenovirus. To date, all studied human adenovirus genomes have the same overall structure, i.e., genes encoding specific functions are located in the same positions within the adenovirus genome (referred to in this invention as "structural elements"). At each end of the adenovirus genome is a short sequence called an inverted terminal repeat (ITR), which is essential for viral replication. In some embodiments, the adenovirus is neither a chimpanzee adenovirus nor an AdC7 adenovirus.

[0069] The adenovirus genome contains five early transcription units (E1A, E1B, E2, E3, and E4), three delayed early units (IX, IVa2, and E2 late), and one late unit (major late), which is processed to produce five sets of late mRNAs (L1-L5). The proteins encoded by the early genes are primarily involved in viral replication and regulating the host cell's response to infection, while the late genes encode viral structural proteins. Early genes are prefixed with the letter "E," and late genes with the letter "L." The length of the adenovirus DNA genome varies depending on the serotype, typically ranging from 34 to 36 kb. For example, the Ad5 genome typically contains 35,938 bp, with a 103 bp terminal repeat at each end and a GC content of 58%.

[0070] Our understanding of adenovirus genetics, transcription, and translation primarily stems from virological studies of the C subtype adenovirus Ad5. The transcriptional sequence of the adenovirus genome reflects the protein requirements of the virus at each stage of replication. Therefore, based on the transcription initiation time of each viral promoter, adenovirus genome transcription can be divided into early and late events. The first protein produced by the viral genome is the E1A protein. The E1A transcription unit generates various mRNAs through alternative splicing, which in turn generate proteins with molecular weights of 6-36 kDa. The E1A protein has two main functions in infected cells: first, it induces cells to enter the S phase of the cell cycle, ensuring efficient viral genome replication; second, it induces the transcription of other early promoters within the viral genome through transactivation. These promoters regulate the production of E1B, E2, E3, and E4 proteins. Following E1A expression, VA RNA, E1B protein, and E3 protein are subsequently produced; these proteins and RNA molecules help suppress the generation of antiviral responses. Early events in viral replication create conditions for viral genome replication before packaging by remodeling the intracellular environment. Late transcriptional events involve the synthesis of structural proteins and key proteins for cell lysis, which are primarily derived from a single promoter (the major late promoter) that transcribes late regions 1-5. Ad5-induced cell lysis depends on the E3-11.6K protein (also known as the adenovirus death protein), which, although labeled as an early gene, is produced only in the later stages of infection and is driven by the major late promoter.

[0071] Adenovirus genes are divided into early transcripts (E1-4) and late transcripts (L1-5), which can produce various protein isotypes through a series of splicing events. The early region is divided into E1, E2, E3, and E4. E1 is crucial for enabling cells to enter cell cycle phases conducive to viral replication, inhibiting apoptosis, and promoting cell division. The E2 region is mainly responsible for DNA genome replication, including DNA-binding proteins (E2A), pTP, and DNA polymerase (E2B). E3 includes genes involved in regulating the host immune response. E4 includes a series of genes involved in regulating cellular pathways, such as non-homologous end joining (NHEJ) and genes that bind to E1B-55K to mediate p53 degradation.

[0072] All adenovirus late genes are transcribed from the same promoter (major late promoter) and share the same 5' mRNA end, which contains three exons that together form a triplet leader sequence. Late genes are expressed through a series of splicing events, producing about 13 proteins that either form part of the viral particle (such as hexagonal proteins and spike proteins) or participate in the assembly of the viral particle (such as the 100K protein).

[0073] Adenoviral vectors are vectors based on or derived from the genome of viruses in the Adenoviridae family. These vectors can be classified as functional replicating, replication-deficient, and / or genetically engineered, capable of delivering genetic material (such as adenoviral genes or exogenous genes) into eukaryotic cells for expression. Adenoviral vectors can achieve the insertion of 8-30 kb transgenes by deleting early E1, E3, and / or E4 genes. The function of the deleted early genes can be provided by the transfected engineered cell lines. The most commonly used adenoviral vector is based on adenovirus type 5 (Ad5).

[0074] The adenovirus of the present invention contains multiple early adenovirus genes (e.g. Figure 1 ).

[0075] The E1A protein is the translation product of the adenovirus genome after the initial transcription in the E1A region of the cell nucleus. This initial transcription is driven by a strong constitutive enhancer element within the E1A promoter, producing a large amount of E1A mRNA. The E1A protein is one of two proteins in the adenovirus genome that can induce transformation; the E1B protein can also induce cell cycle progression. Both the E1A and E1B genes are essential for viral replication.

[0076] The E2 gene in the adenovirus genome is divided into two regions: E2A and E2B, both essential for viral replication. E2B contains the DNA polymerase gene, which is fundamentally required for the amplification of viral genomic DNA. This region also contains the pTP protein, which acts as a primer to initiate viral genome replication. The pTP protein is covalently linked to the ends of the viral genomic DNA. The E2A region contains a DNA-binding protein, which is also essential for DNA replication. All E2 genes are fundamentally necessary for viral replication.

[0077] For Ad5, pTP assembles with DNA polymerase, DNA-binding proteins, host NFI, and OCT1 at the viral origin of replication to form the initiation complex. pTP is an approximately 80 kDa protein that functions as an initiation primer. The DNA polymerase possesses 5'–3' polymerase activity and a 3'–5' exonuclease domain, which is crucial for its intrinsic proofreading capabilities. In the final stage of replication, pTP is cleaved into TP by viral proteases, producing progeny DNA, which is then packaged into viral particles.

[0078] E3 genes are primarily involved in regulating the cellular and host immune responses to viral infection. However, since most viruses in biotechnological applications are cultured in vitro, most of these viral genes (if not all) can be removed without affecting viral replication efficiency. Most E3 genes are not essential for viral replication in cell lines. However, some genes, such as adenovirus death protein (ADP), are essential for efficient replication and viral production.

[0079] The E4 region of the adenovirus genome is similar to the E1 region, primarily involved in the production of proteins that help the virus control and regulate cells, ensuring efficient viral replication and production. This region contains six ORFs, which assist in preventing non-homologous end joining and apoptosis, and have several other functions. The importance of different E4 transcripts to viral replication varies: some transcripts are indispensable, while others, even if deleted or modified, have little effect on viral growth kinetics and production.

[0080] The adenovirus of the present invention preferably contains sufficient early adenovirus genes to ensure that the virus can replicate its genome within the host cell nucleus.

[0081] The adenovirus of this invention contains multiple adenovirus late genes ( Figure 1 Late viral genes are divided into five major transcription families, named L1-L5. These transcripts primarily encode proteins involved in viral assembly and viral structural proteins. During viral replication, these proteins can account for 30-40% of the cellular protein content (Garnier, 1994; Ginsberg, 1984).

[0082] The L1 series of transcripts encodes 13.6K, 52K, and PIIIa proteins. These proteins are all involved in viral assembly and particle production. The L1 gene is essential for successful viral assembly but not for genomic DNA replication.

[0083] The L2 series transcripts encode the pentazocine basement membrane, pVII, V, and pX proteins. These proteins constitute the structural parts of the viral capsid and are essential for the proper assembly of viral particles. The pentazocine basement membrane contains the RGD motif, which is important for attachment to the cell surface during infection with various adenoviruses. The L2 genes are required for successful viral assembly but are not essential for genomic DNA replication.

[0084] The L3 series transcripts encode pVI, hexagonal proteins, and protease proteins. Hexagonal proteins are major components of the viral capsid and exhibit antigenic diversity across different serotypes. Protease proteins are involved in cell entry and viral capsid maturation. The L3 gene is essential for successful viral assembly but not for genomic DNA replication.

[0085] The L4 series of transcripts encodes 100K, 33K, 22K, and pVIII proteins. These proteins are involved in multiple functions. The 100K protein is involved in assisting viral hexapod assembly and nuclear importation, and may also play a role in shifting cellular mRNA translation to cap-independent translation. The 22K protein is involved in viral capsid formation. The L4 gene is essential for successful viral assembly but not for genomic DNA replication. However, the 100K protein may help shift cellular protein translation to transcripts containing a triplet leader sequence (TPL).

[0086] L5 encodes the spike protein gene. The spike protein is a viral structural protein involved in attaching to the cell surface and mediating viral infection. The production of spike protein far exceeds what is needed to form viral particles. The L5 gene is essential for successful viral assembly but not for genomic DNA replication.

[0087] The genome of the adenovirus of this invention contains multiple early and late adenovirus genes, sufficient to support adenovirus replication. Preferably, the adenovirus of this invention contains at least the early genes E1, E2, and E4, and the late genes L1-L5. E3 is not essential for replication in cell lines. However, some genes in these regions can be modified, mutated, or even deleted without inhibiting viral replication in cell lines.

[0088] In one embodiment, the adenovirus is capable of replication, has replicative activity, or is conditionally replicating. These viruses may be oncolytic viruses, viral vaccines, or protein production vectors.

[0089] As used herein, the term "replicating" refers to an adenovirus capable of replicating its genome within a host cell. In some embodiments, "replicating" includes viruses with replication activity and those with conditional replication capability.

[0090] In the context of this invention, "replicating" refers to an adenovirus that possesses all the mechanisms necessary for replication in vitro and in vivo in cells, i.e., without the aid of a packaging cell line.

[0091] The terms "conditional replication," "selective replication," or "replication selectivity" used in this article specifically refer to an oncolytic adenovirus that can replicate in cancer cells. It can replicate within cancer cells by utilizing elements specific to or upregulated in cancer cells (such as defective cellular mechanisms, like p53 mutations), thereby exhibiting a certain selectivity for healthy / normal cells.

[0092] "Conditional replication" also refers to the ability of adenoviruses to infect and replicate under specific conditions. These conditions may include viral regulation by tumor-specific promoters to drive intracellular viral replication (such as the PSA promoter), or viral regulation by repressive factors (such as tetracycline repressor (TetR) binding sites or microRNA binding sites) to restrict viral replication under specific conditions. Conditionally replicating adenoviruses can be oncolytic or used as vectors for gene, vaccine, or protein delivery.

[0093] In some embodiments, the adenovirus genome shares at least 70% (preferably at least 80%, 85%, 90%, 95%, or 99%) nucleotide sequence identity with the wild-type Ad3 genome sequence (e.g., Genbank Sequence ID: DQ086466.1). In some embodiments, the adenovirus genome shares at least 70% (preferably at least 80%, 85%, 90%, 95%, or 99%) nucleotide sequence identity with the wild-type Ad7 genome sequence (e.g., Genbank Sequence ID AY594255.1).

[0094] In some preferred embodiments, the adenovirus of the present invention is oncolytic. As used herein, "oncolytic" refers to the adenovirus's ability to infect, replicate, and lyse cancer cells. Preferably, the oncolytic adenovirus of the present invention is more likely to infect and / or preferentially lyse cancer cells compared to non-cancer cells. In some embodiments, the oncolytic adenovirus of the present invention is also capable of infecting and / or lysing stromal cells, particularly cellular fibroblasts (CAFs).

[0095] Viral infection can be measured using infectivity assays, such as plaque assays, median tissue culture infectious dose (TCID50) assays, or immunocytochemical (ICC) staining using anti-hexamethylenetetramine antibodies. The oncolytic adenovirus of this invention can have cell-lysing activity. Lysis can be measured using cell death or cell viability assays, including MTS, MTT assays, and PrestoBlue. TM Live / dead staining and flow cytometry. Oncolytic virus infection causes the death and lysis of cancer cells or mesenchymal cells, preferably accompanied by the release of newly generated viral particles.

[0096] Virus production can be determined by a variety of methods, including the infectivity assays described above. Other methods can be used to measure the physical count of viral particles, including ultraviolet absorption measurement (OD260), dynamic light scattering, and HPLC quantification. Furthermore, virus production can be measured by DNA quantification and interpolation based on a standard curve of known DNA content; these assays include real-time quantitative PCR and Pico Green dye-based assays. Purified virus can also be quantified by determining total protein content using the dioctanine acid (BCA) method.

[0097] In some embodiments, the adenovirus of the present invention encodes one or more transgenes. The adenovirus of the present invention is particularly useful as an oncolytic vector, a conditionally replicative vector, or a replication-deficient vector, and can also be used as a gene therapy vector. Such vectors may contain transgenes to produce recombinant nucleic acids or polypeptides at a target site (such as a tumor site).

[0098] In gene therapy vectors, the transgene may encode a therapeutic polypeptide, or may be a gene for correcting genetic defects. In cancer gene therapy vectors, the gene may encode a polypeptide that induces cell death (e.g., p53).

[0099] Examples of transgenics include transgenics encoding anticancer agents, immunostimulants, or imaging agents (e.g., antibodies, bispecific adaptors, checkpoint inhibitors, cytokines, chemokines, and enzymes including extracellular matrix degrading enzymes and anti-angiogenic inhibitors). Preferred examples of transgenics include transgenics encoding EpCAM-Lite, EpCAM-BiTE, CTLA4, CCL19, CCL5, CXCL11, or CXCL12.

[0100] The transgene can be located in, for example, the E1 or E3 region of an adenovirus, or in the E1 / E3 deletion region, or it can be inserted near or inside the L3 or L5 region.

[0101] The deletion of the E3 region creates more space for transgene insertion at other locations in the adenovirus genome, while removing the ORF that helps the virus evade immune system recognition and hinders host clearance. Therefore, the deletion of the E3 region weakens viral activity in normal cells with a functional immune response, but does not weaken its activity in immune-dysfunctional tumor cells, thereby increasing the therapeutic index of oncolytic viruses.

[0102] The genome of an adenovirus must be within the viral packaging limits. For example, the size of the transgene it carries must not make the total length of the genome exceed the length that can be packaged into the protein capsid.

[0103] 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, such as a non-natural adenovirus whose genome contains continuous gene sequences that are not normally found in the genome of a wild-type adenovirus.

[0104] The L2 region of wild-type adenovirus contains the adenovirus pentagon gene encoding the adenovirus pentagon polypeptide, as well as the adenovirus pVII, V, and pX genes encoding the pVII, V, and pX core polypeptides, respectively.

[0105] The L3 region of wild-type adenovirus contains adenovirus pVI, hexagonal, and protease genes that encode adenovirus pVI, hexagonal, and protease polypeptides, respectively.

[0106] The references to adenovirus genes (such as penta-neighbone, hexa-neighbone, spike protein, etc.) in this article refer to the ORF or coding sequences of these genes.

[0107] In some embodiments, the L2 region contains a pentagonal gene encoding an Ad3 pentagonal polypeptide, and the L3 region contains a hexagonal gene encoding an Ad7 hexagonal polypeptide.

[0108] In some embodiments, the L3 region contains a VI gene encoding an Ad3 VI polypeptide; and the L3 region contains a hexagon gene encoding an Ad7 hexagon polypeptide.

[0109] In some embodiments, the L2 region contains pentagonal, pVII, V, and pX genes encoding Ad3 pentagonal, Ad3 pVII, Ad3 V, and Ad3 pX peptides, respectively; the L3 region contains the VI gene encoding the Ad3 VI peptide; and the L3 region contains the hexagonal gene encoding the Ad7 hexagonal peptide.

[0110] In some embodiments, the L2 region contains the V gene encoding the Ad3 V polypeptide; and the L2 region contains the pX gene encoding the Ad7 pX polypeptide.

[0111] In some embodiments, the L2 region contains the pentagon, pVII, and V genes encoding Ad3 pentagon, Ad3 pVII, and Ad3 V polypeptides, respectively; the L2 region contains the pX gene encoding the Ad7 pX polypeptide; and the L3 region contains the VI and hexagon genes encoding the Ad7 VI and Ad7 hexagon polypeptides, respectively.

[0112] In some embodiments, the L2 region contains a pX gene encoding an Ad3 pX polypeptide; and the L2 region contains a VI gene encoding an Ad7 VI polypeptide.

[0113] In some embodiments, the L2 region contains the pentagon, pVII, V and pX genes encoding the Ad3 pentagon, Ad3 pVII, Ad3 V and Ad3 pX peptides, respectively; and the L3 region contains the VI and hexagon genes encoding the Ad7 VI and Ad7 hexagon peptides, respectively.

[0114] In a particularly preferred embodiment, the L2 region contains the pentagon, pVII, V and pX genes encoding the Ad3 pentagon, Ad3 pVII, Ad3 V and Ad3 pX polypeptides, respectively; and the L3 region contains the VI and hexagon genes encoding the Ad3 VI and Ad7 hexagon polypeptides, respectively.

[0115] In all embodiments, the L3 region may additionally contain a protease gene encoding the Ad7 protease. In all embodiments, the L1 region preferably contains the L1 gene encoding the entire Ad3 polypeptide. In all embodiments, the L4 region preferably contains the L4 gene encoding the entire Ad7 polypeptide.

[0116] Preferably, the L2 region contains the Ad3 pentagon gene, that is, preferably, one of the L2 polypeptides is the Ad3 pentagon polypeptide.

[0117] The terms “penton” and “penton base” used in this article are used interchangeably; both refer to penton polypeptides.

[0118] The term "Ad3 pentagonal gene" as used in this article refers to a gene that contains or consists of the following:

[0119] (a) The nucleotide sequence shown in SEQ ID NO: 3;

[0120] (b) A nucleotide sequence having at least 95% or 99% nucleotide sequence identity with SEQ ID NO: 3 and encoding an Ad3 pentagonal polypeptide;

[0121] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 3 and encoding an adenovirus (preferably Ad3) pentagonal polypeptide; or

[0122] (d) Nucleotide sequence encoding the Ad3 pentagonal polypeptide.

[0123] The term “Ad3 pentam polypeptide” as used herein preferably includes, but is not limited to:

[0124] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 5;

[0125] (b) A polypeptide having at least 99.3%, 99.5%, or 99.7% (preferably at least 99.7%) sequence identity with SEQ ID NO: 5 and encoding an adenovirus pentagonal polypeptide (preferably an Ad3 pentagonal polypeptide); or

[0126] (c) Having at least 95%, 99% or 99.5% (preferably at least 99.5%) sequence identity with SEQ ID NO: 5, wherein the pentagonal polypeptide contains a polypeptide whose amino acids at positions 11, 158, 178 and 326 of SEQ ID NO: 5 are V, T, I and D, respectively, and the polypeptide encodes an adenovirus pentagonal polypeptide (preferably an Ad3 pentagonal polypeptide).

[0127] Adenoviral pentagonal peptides have been well characterized (Medina-Kauwe, Ther. Deliv. 2013 Feb; 4(2): 267-277). The pentagonal base forms a homopentamer, non-covalently attached to each vertices of the adenoviral capsid and binding to the N-terminal tail domain of the spike peptide. The pentagonal base homopentamer contributes to the formation of the characteristic icosahedral shape of the adenoviral capsid and is crucial for the structural stability of the adenoviral capsid. Therefore, pentagonal peptides are essential for efficient transfection of target cells.

[0128] The transfection efficiency of adenovirus containing the pentagonal polypeptide of the present invention on target cells (e.g., HEK293 cells) can be tested by exposing target cells to the adenovirus and then measuring the percentage of cells expressing genes encoded by the genome packaged in the adenovirus capsid (e.g., the E1A gene or the GFP reporter gene), and comparing the results with those obtained by testing adenovirus containing wild-type pentagonal polypeptide (e.g., SEQ ID NO: 5) under the same conditions.

[0129] Transfection assays can be performed, for example, by measuring the percentage of HEK293 cells expressing the GFP reporter gene encoded by the viral genome using flow cytometry, as described by Gueret et al. (Gueret V, Negrete-Virgen JA, Lyddiatt A, Al-Rubeai M. "Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells". Cytotechnology. 2002 Jan;38(1-3):87-97). In such assays, the two adenoviruses being compared should have the same capsid and the same genome, differing only in the pentagonal polypeptide and pentagonal gene.

[0130] The pentagonal polypeptide of the present invention comprises: an adenovirus containing such a pentagonal polypeptide, wherein the transfection efficiency is at least 0.001% or 0.01% of the transfection efficiency obtainable using an adenovirus containing the wild-type pentagonal polypeptide of SEQ ID NO: 5 under the same assay conditions, preferably at least 0.1%, and most preferably at least 1%.

[0131] Preferably, the L2 region contains the Ad3 pVII gene, that is, preferably, one of the L2 polypeptides is the Ad3 pVII polypeptide.

[0132] The term "Ad3 pVII gene" as used in this article refers to a gene that contains or consists of the following:

[0133] (a) The nucleotide sequence shown in SEQ ID NO: 7;

[0134] (b) A nucleotide sequence having at least 95% or 99% nucleotide sequence identity with SEQ ID NO: 7 and encoding the Ad3 pVII polypeptide;

[0135] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 7 and encoding an adenovirus (preferably Ad3) pVII polypeptide; or

[0136] (d) Nucleotide sequence encoding the Ad3 pVII polypeptide.

[0137] The term “Ad3 pVII polypeptide” as used in this article preferably includes, but is not limited to:

[0138] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 9; or

[0139] (b) A polypeptide having at least 99% sequence identity with SEQ ID NO: 9 and encoding an adenovirus pVII polypeptide; or

[0140] (c) Having at least 95%, 99% or 99.5% (preferably at least 99.5%) sequence identity with SEQ ID NO: 9, and wherein the pVII polypeptide contains a polypeptide whose amino acids at positions 27 and 112 of SEQ ID NO: 9 are K and P, respectively, and the polypeptide encodes an adenovirus pVII polypeptide (preferably Ad3 pVII polypeptide).

[0141] Preferably, the L2 region contains the Ad3V gene, that is, preferably, one of the L2 polypeptides is the Ad3V polypeptide.

[0142] The term "Ad3 V gene" as used in this article refers to a gene that contains or consists of the following:

[0143] (a) The nucleotide sequence shown in SEQ ID NO: 11;

[0144] (b) A nucleotide sequence having at least 95% or 99% nucleotide sequence identity with SEQ ID NO: 11 and encoding an Ad3V polypeptide; or

[0145] (c) A nucleotide sequence having at least 99.5% nucleotide sequence identity with SEQ ID NO: 11 and encoding an Ad3V polypeptide; or

[0146] (d) Nucleotide sequence encoding the Ad3 V polypeptide.

[0147] The term "Ad3 V peptide" as used in this article is preferably including, but not limited to:

[0148] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 13; or

[0149] (b) A polypeptide having at least 99.6% sequence identity with SEQ ID NO: 13 and encoding an Ad3 V polypeptide; or

[0150] (c) Having at least 90%, 95% or 99% (preferably at least 99%) sequence identity with SEQ ID NO: 13, wherein the V polypeptide contains amino acids D and missing at positions 45 and 234 of SEQ ID NO: 14, respectively, and the polypeptide encodes an adenovirus V polypeptide (preferably Ad3 V polypeptide).

[0151] In some embodiments, the L2 region contains the Ad3 pX gene, that is, in some embodiments, one of the L2 polypeptides is the Ad3 pX polypeptide.

[0152] The term "Ad3 pX gene" as used in this article refers to a gene that contains or consists of the following:

[0153] (a) The nucleotide sequence shown in SEQ ID NO: 15;

[0154] (b) A nucleotide sequence having at least 95% nucleotide sequence identity with SEQ ID NO: 15 and encoding the Ad3 pX polypeptide;

[0155] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 15 and encoding an adenovirus (preferably Ad3) pX polypeptide; or

[0156] (d) Nucleotide sequence encoding the Ad3 pX polypeptide.

[0157] The term "Ad3 pX peptide" as used in this article is preferably including, but not limited to:

[0158] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 17; or

[0159] (b) Having at least 90%, 95% or 98.5% (preferably at least 98.5%) sequence identity with SEQ ID NO: 17, and wherein the pX polypeptide contains a polypeptide whose amino acid at position 43 of SEQ ID NO: 17 is G, the polypeptide encoding an adenovirus pX polypeptide (preferably Ad3 pX polypeptide).

[0160] In some embodiments, the L2 region contains the Ad7 pX gene, that is, in some embodiments, one of the L2 polypeptides is the Ad7 pX polypeptide.

[0161] The term "Ad7 pX gene" as used in this article refers to a gene that contains or consists of the following:

[0162] (a) The nucleotide sequence shown in SEQ ID NO: 16;

[0163] (b) A nucleotide sequence having at least 95% nucleotide sequence identity with SEQ ID NO: 16 and encoding the Ad7 pX polypeptide;

[0164] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 16 and encoding an adenovirus (preferably Ad7) pX polypeptide; or

[0165] (d) Nucleotide sequence encoding the Ad7 pX polypeptide.

[0166] The term "Ad7 pX peptide" as used in this article is preferably including, but not limited to:

[0167] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 18; or

[0168] (b) Having at least 90%, 95% or 98.5% (preferably at least 98.5%) sequence identity with SEQ ID NO: 18, and wherein the pX polypeptide contains a polypeptide whose amino acid corresponding to position 43 in SEQ ID NO: 18 is D, the polypeptide encoding an adenovirus pX polypeptide (preferably Ad7 pX polypeptide).

[0169] The genome of the chimeric adenovirus includes an L3 region containing one or more L3 genes encoding one or more L3 polypeptides. Preferably, the L3 region is the Ad7 L3 region, except for the adenovirus pVI gene (which may be Ad3).

[0170] In some embodiments, the L3 region contains the Ad3 pVI gene, that is, in some embodiments, one of the L3 polypeptides is the Ad3 pVI polypeptide.

[0171] The term "Ad3 pVI gene" as used in this article refers to a gene that contains or consists of the following:

[0172] (a) The nucleotide sequence shown in SEQ ID NO: 19;

[0173] (b) A nucleotide sequence having at least 95% nucleotide sequence identity with SEQ ID NO: 19 and encoding an Ad3 pVI polypeptide;

[0174] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 19 and encoding an adenovirus (preferably Ad3) pVI polypeptide; or

[0175] (d) Nucleotide sequence encoding the Ad3 pVI polypeptide.

[0176] The term “Ad3 pVI peptide” as used in this article preferably includes, but is not limited to:

[0177] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 21; or

[0178] (b) Having at least 90%, 95% or 98% (preferably at least 98%) sequence identity with SEQ ID NO: 21, and wherein the pVI polypeptide has an amino acid P corresponding to position 196 in SEQ ID NO: 21, the polypeptide encoding an adenovirus pVI polypeptide (preferably Ad3 pVI polypeptide).

[0179] In some embodiments, the L3 region contains the Ad7 pVI gene, that is, in some embodiments, one of the L3 polypeptides is the Ad7 pVI polypeptide.

[0180] The term "Ad7 pVI gene" as used in this article refers to a gene that contains or consists of the following:

[0181] (a) The nucleotide sequence shown in SEQ ID NO: 20;

[0182] (b) A nucleotide sequence having at least 95% nucleotide sequence identity with SEQ ID NO: 20 and encoding an Ad7 pVI polypeptide;

[0183] (c) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 20 and encoding an adenovirus (preferably Ad7) pVI polypeptide; or

[0184] (d) Nucleotide sequence encoding the Ad7 pVI polypeptide.

[0185] The term "Ad7 pVI peptide" as used in this article preferably includes, but is not limited to:

[0186] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 22; or

[0187] (b) Having at least 90%, 95% or 98% (preferably at least 98%) sequence identity with SEQ ID NO: 22, and wherein the pVI polypeptide contains a polypeptide whose amino acid at position 196 of SEQ ID NO: 21 is L, the polypeptide encoding an adenovirus pVI polypeptide (preferably Ad7 pVI polypeptide).

[0188] In some embodiments, the L3 region contains the Ad7 hexagon gene, that is, in some embodiments, one of the L3 polypeptides is the Ad7 hexagon polypeptide.

[0189] The term "Ad7 hexane gene" as used in this article refers to a gene that contains or consists of the following:

[0190] (a) The nucleotide sequence shown in SEQ ID NO: 24;

[0191] (b) A nucleotide sequence having at least 95% (or at least 99%) nucleotide sequence identity with SEQ ID NO: 24 and encoding an Ad7 hexagonal polypeptide;

[0192] (c) A nucleotide sequence having at least 97% (or at least 99%) nucleotide sequence identity with SEQ ID NO: 24 and encoding an adenovirus (preferably Ad7) hexagonal polypeptide; or

[0193] (d) Nucleotide sequence encoding Ad7 hexagonal polypeptide.

[0194] The term "Ad7 hexagonal polypeptide" as used herein preferably includes, but is not limited to:

[0195] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 26; or

[0196] (b) A polypeptide having at least 97%, 98% or 99% (preferably at least 99%) sequence identity with SEQ ID NO: 26 and encoding an adenovirus (preferably Ad7) hexagonal polypeptide.

[0197] In some embodiments, the genome of the adenovirus of the present invention does not contain the Ad7 hexane gene inserted into the E1 or E3 region.

[0198] Adenoviral hexagonal peptides have been well characterized (Medina-Kauwe, Ther. Deliv. 2013 Feb; 4(2): 267-277). These hexagonal peptides can form multiple (e.g., 240) homotrimers that constitute the 20 faces of the icosahedral adenoviral capsid, encapsulating the adenoviral genome and related proteins. The hexagonal trimers are crucial for maintaining the structural integrity and stability of the capsid, and are therefore essential for efficient transfection of target cells.

[0199] The transfection efficiency of adenoviruses containing hexagonal peptide variants of the present invention on target cells (e.g., HEK293 cells) can be tested by exposing target cells to the adenovirus and then measuring the percentage of cells expressing genes encoded by the genome packaged within the adenovirus capsid (e.g., the E1A gene or the GFP reporter gene), and comparing the results with those obtained by testing adenoviruses containing wild-type hexagonal peptides (e.g., one of SEQ ID NO: 25) under the same conditions.

[0200] Transfection assays can be performed, for example, by measuring the percentage of HEK293 cells expressing the GFP reporter gene encoded by the adenoviral genome using flow cytometry, as described by Gueret et al. (Gueret V, Negrete-Virgen JA, Lyddiatt A, Al-Rubeai M. "Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells". Cytotechnology. 2002 Jan;38(1-3):87-97). In such assays, the two adenoviruses being compared should have the same capsid and the same genome, differing only in the hexagonal polypeptide and hexagonal gene.

[0201] The hexagonal polypeptide of the present invention comprises: an adenovirus containing such hexagonal polypeptide, wherein the transfection efficiency is at least 0.001% or 0.01% of the transfection efficiency obtainable using an adenovirus containing the wild-type hexagonal polypeptide of SEQ ID NO: 25 under the same assay conditions, preferably at least 0.1%, and most preferably at least 1%.

[0202] In some embodiments, the L3 region contains an adenoviral protease gene; that is, in some embodiments, one of the L3 polypeptides is an adenoviral protease polypeptide. Preferably, the adenoviral protease gene is an Ad7 protease gene, encoding the Ad7 protease.

[0203] The genome of the chimeric adenovirus includes an L4 region containing one or more L4 genes encoding one or more L4 polypeptides. Preferably, the L4 region is the Ad7 L4 region.

[0204] The genome of the chimeric adenovirus contains an E3 region. This E3 region can be a complete or substantially complete E3 region (i.e., a region without any missing parts compared to the E3 region of wild-type adenovirus (preferably Ad3 or Ad7)).

[0205] In other embodiments, the genome of the chimeric adenovirus includes one or more missing E3 regions (compared to the E3 regions of wild-type adenovirus (preferably Ad3 or Ad7)).

[0206] The genome of the chimeric adenovirus further includes an L5 region, which contains one or more L5 genes encoding one or more L5 polypeptides. Preferably, the L5 region is the Ad7 L5 region.

[0207] In some embodiments, one of the L5 genes is an adenoviral spike protein gene encoding a spike polypeptide; that is, in some embodiments, one of the L5 polypeptides is a spike polypeptide. Preferably, the spike protein gene is an Ad3 spike protein gene or an Ad7 spike protein gene, encoding an Ad3 or Ad7 spike polypeptide, respectively; more preferably, it is an Ad7 spike protein gene encoding an Ad7 spike polypeptide.

[0208] The term "Ad3 spike protein gene" as used in this article refers to a gene that contains or consists of the following:

[0209] (a) The nucleotide sequence shown in SEQ ID NO: 31;

[0210] (b) A nucleotide sequence having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) nucleotide sequence identity with SEQ ID NO: 31 and encoding an adenovirus (preferably Ad3) spike polypeptide; or

[0211] (c) Nucleotide sequence encoding Ad3 spike polypeptide.

[0212] The term "Ad3 spike polypeptide" as used in this article is preferably including, but not limited to:

[0213] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 33; or

[0214] (b) A polypeptide having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) sequence identity with SEQ ID NO: 33 and encoding an adenovirus (preferably Ad3) spike polypeptide.

[0215] The term "Ad7 spike protein gene" as used in this article refers to a gene that contains or consists of the following:

[0216] (a) The nucleotide sequence shown in SEQ ID NO: 32;

[0217] (b) A nucleotide sequence having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) nucleotide sequence identity with SEQ ID NO: 32 and encoding an adenovirus (preferably Ad7) spike polypeptide; or

[0218] (c) Nucleotide sequence encoding Ad7 spike polypeptide.

[0219] The term "Ad7 spike polypeptide" as used in this article preferably includes, but is not limited to:

[0220] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 34; or

[0221] (b) A polypeptide having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) sequence identity with SEQ ID NO: 34 and encoding an adenovirus (preferably Ad7) spike polypeptide.

[0222] In some embodiments of the present invention, the genome of the chimeric adenovirus does not contain the Ad3 spike protein gene. In some embodiments of the present invention, the genome of the chimeric adenovirus does not contain the L5 region containing the Ad3 spike protein gene.

[0223] Adenoviral spike peptides have been well characterized (Medina-Kauwe, Ther. Deliv. 2013 Feb; 4(2): 267-277). The spike consists of a polypeptide homotrimer containing an amino (N-terminal) tail domain and a carboxyl (C-terminal) globular head. The tail domain is capable of interacting with the pentavalent base in the capsid. These two domains are separated by an axial portion of varying length depending on the serotype, which contains repeating sequences capable of forming triple β-helices within the spike homotrimer. The spike peptide plays a crucial role in cell attachment and entry, and is therefore essential for efficient transfection of target cells.

[0224] The transfection efficiency of adenovirus containing the spike peptide of the present invention on target cells (e.g., HEK293 cells) can be tested by exposing target cells to the adenovirus and then measuring the percentage of cells expressing genes encoded by the genome packaged in the adenovirus capsid (e.g., the E1A gene or the GFP reporter gene), and comparing the results with those obtained by testing adenovirus containing wild-type spike peptide (e.g., one of SEQ ID NO: 33) under the same conditions.

[0225] Transfection assays can be performed, for example, by measuring the percentage of HEK293 cells expressing the GFP reporter gene encoded by the viral genome using flow cytometry, as described by Gueret et al. (Gueret V, Negrete-Virgen JA, Lyddiatt A, Al-Rubeai M. "Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells". Cytotechnology. 2002 Jan;38(1-3):87-97). In such assays, the two adenoviruses being compared should have the same capsid and the same genome, differing only in the spike polypeptide and spike protein genes.

[0226] The spike peptide of the present invention comprises: an adenovirus containing such a spike peptide, wherein the transfection efficiency is at least 0.001% or 0.01% of the transfection efficiency obtainable using an adenovirus containing the wild-type spike peptide of SEQ ID NO: 33 under the same assay conditions, preferably at least 0.1%, and most preferably at least 1%.

[0227] The gene of the present invention is preferably isolated or purified. As used herein, the term "isolated gene" means that the nucleic acid molecule is not linked to other genes or nucleotide sequences that are typically associated with it in adenoviruses. Therefore, the isolated gene is not a wild-type gene of an adenovirus.

[0228] The polypeptides of the present invention are preferably isolated. As used herein, the term "isolated polypeptide" means that the polypeptide is not in a mixture with other polypeptides that are normally mixed with in adenoviruses.

[0229] Many mature algorithms exist for aligning two amino acid or nucleic acid sequences. Typically, one sequence is used as a reference sequence, and the test sequence is compared to it. Sequence comparison algorithms calculate the percentage sequence identity of the test sequence relative to the reference sequence based on specified program parameters. The alignment of amino acid or nucleic acid sequences used for comparison can be performed using computer-implemented algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA) or Clustal Omega, BLASTn, and BLASTp algorithms.

[0230] The standard protein-protein BLAST (blastp) can be used to find similar sequences in protein databases. Similar to other BLAST programs, blastp aims to find locally similar regions. When sequence similarity spans the entire sequence, blastp also reports a global alignment, which is the preferred result for protein identification purposes. It is preferable to use standard or default alignment parameters. In some cases, the "low complexity filter" can be turned off.

[0231] For nucleotide sequence comparison, MEGABLAST, non-contiguous MEGABLAST, and blastn can be used to achieve this goal. Standard or default alignment parameters are preferred. MEGABLAST is specifically designed for efficiently finding long alignments between highly similar sequences. Non-contiguous MEGABLAST can be used to find nucleotide sequences that are similar to but not identical to the nucleic acids of this invention.

[0232] The BLAST nucleotide algorithm searches for similar sequences by breaking down the query sequence into short subsequences called "words". The program first identifies sequences that exactly match the query word (word hits). Then, the BLAST program expands these word hits in multiple steps to generate a final alignment including gaps. In some implementations, the BLAST nucleotide search can be performed using the BLASTN program with parameters set to score=100 and wordlength=12.

[0233] One of the important parameters for controlling the search sensitivity of BLAST is word length. The most important reason why blastn is more sensitive than MEGABLAST is that it uses a shorter default word length (11). Therefore, blastn is superior to MEGABLAST in finding alignments with other biologically relevant nucleotide sequences. The word length in blastn is adjustable and can be reduced from the default value to a minimum of 7 to improve search sensitivity.

[0234] More sensitive searches can be achieved by using the newly introduced non-contiguous megablast page (www.ncbi.nlm.nih.gov / Web / Newsltr / FallWinter02 / blastlab.html). This page uses an algorithm similar to that reported by Ma et al. (Bioinformatics. 2002 Mar; 18(3): 440-5). Instead of requiring exact word matches as seeds for alignment expansion, non-contiguous megablasts use non-contiguous words within a longer template window. In encoded mode, third base wobbles are considered by focusing on finding matches at the first and second positions of the codon while ignoring mismatches at the third position. Searching in non-contiguous MEGABLAST using the same word length is more sensitive and efficient than using standard blastn with the same word length. Parameters specific to non-contiguous megablasts include: word length: 11 or 12; template: 16, 18, or 21; template type: encoded (0), non-encoded (1), or both (2).

[0235] In some implementations, the BLASTP 2.5.0+ algorithm (e.g., the version provided by NCBI) with default parameters can be used. In other implementations, the BLAST global alignment program (e.g., the version provided by NCBI) can be used to align two protein sequences using the Needleman-Wunsch algorithm, with gap costs set as: presence penalty of 11 and extension penalty of 1.

[0236] In this paper, the term "sequence identity" can be replaced with "sequence similarity" in the context of amino acid sequences. The term "similarity" allows for conservative substitutions with amino acid residues having similar physicochemical properties within a given alignment length. The similarity percentage can be determined using any reasonable similarity scoring matrix.

[0237] The present invention also provides a pharmaceutical composition comprising the adenovirus of the present invention, optionally in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0238] As used herein, the term "pharmaceutically acceptable carrier" includes all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Examples of suitable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Examples of suitable isotonic agents include sugars, polyols (e.g., mannitol, sorbitol), and sodium chloride.

[0239] 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.

[0240] In one embodiment, the pharmaceutical composition is a liquid parenteral preparation of the adenovirus of the present invention, for example for infusion or injection.

[0241] As used herein, the term "parenteral preparation" refers to a pharmaceutical composition delivered outside the gastrointestinal tract. Typical parenteral delivery routes include injection, implantation, or infusion. In one embodiment, the pharmaceutical composition is provided in a form suitable for bolus administration.

[0242] In one embodiment, the parenteral preparation is in the form of an injectable. Injection includes intravenous, subcutaneous, intratumoral, or intramuscular injection. As used herein, "injection" means the administration of a liquid into the body via a syringe.

[0243] In one embodiment, the parenteral preparation is in the form of infusion. As used herein, the term "infusion" refers to the administration of fluid at a slower rate via drip, infusion pump, syringe pump, or equivalent device.

[0244] In one embodiment, the parenteral preparation is an infusion form for intravenous administration.

[0245] In another embodiment, the pharmaceutical composition is provided in a formulation for topical administration, including inhalation. Suitable inhalable formulations include inhalable powders, metered aerosols containing propellant gases, or inhalable solutions without propellant gases. Inhalable powders according to this disclosure generally contain the virus described herein and physiologically acceptable excipients. These inhalable powders may include monosaccharides (e.g., glucose or arabinose), disaccharides (e.g., lactose, sucrose, maltose), oligosaccharides and polysaccharides (e.g., dextran), polyols (e.g., sorbitol, mannitol, xylitol), salts (e.g., sodium chloride, calcium carbonate), or mixtures thereof. Monosaccharides or disaccharides are suitable for use, with lactose or glucose used, especially but not limited to their hydrated forms.

[0246] Particles deposited in the lungs need to have a diameter of less than 10 micrometers, for example, 1-9 micrometers, for example, from 0.1 to 5 micrometers, particularly from 1 to 5 micrometers. The particle size of the virus-carrying particles is crucial, therefore, in one embodiment, the virus of the present invention can be adsorbed or absorbed onto particles having the given diameters described above (e.g., lactose particles).

[0247] Propellant gases suitable for preparing inhalable aerosols are known in the art. Suitable propellant gases are selected from hydrocarbons (such as n-propane, n-butane, or isobutane) and halogenated hydrocarbons (such as chlorinated and / or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane, or cyclobutane). The aforementioned propellant gases can be used alone or in mixtures thereof. Particularly suitable propellant gases are halogenated alkane derivatives selected from TG 11, TG 12, TG 134a, and TG227. Among the aforementioned halogenated hydrocarbons, TG134a (1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable.

[0248] Inhalable aerosols containing propellant gases may also contain other components such as cosolvents, stabilizers, surfactants, antioxidants, lubricants, and pH adjusters. All of these components are known in the art.

[0249] The inhalable aerosol containing a propellant gas according to the present invention may contain up to 5% by weight of an active ingredient. The aerosol according to the present invention comprises, 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 an active ingredient.

[0250] Alternatively, local administration to the lungs can be achieved by administering a liquid solution or suspension, for example using a device such as a nebulizer, such as a nebulizer connected to a compressor (e.g., the Pari LC-Jet Plus nebulizer connected to a Pari Master® compressor, manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.).

[0251] The atomizable formulations according to this disclosure can be provided, for example, in single-dose units (e.g., sealed plastic containers or vials) packaged in aluminum foil bags. Each vial contains one unit dose, with a volume of, for example, 2 mL, of solvent / solution buffer.

[0252] The pharmaceutical compositions of the present invention are generally sterile and stable under the conditions of manufacture and storage. The compositions can be formulated into solutions, microemulsions, liposomes, or other parenteral preparations suitable for human administration, and can be formulated into pre-filled devices, such as syringes or vials, particularly for single doses.

[0253] Subjects treated with the adenovirus of this invention may also receive additional treatment with one or more other chemotherapeutic agents or immunotherapeutic agents, namely specific antitumor chemotherapeutic agents, or drugs that selectively destroy malignant cells or tissues or induce an antitumor immune response. Such other chemotherapeutic agents or immunotherapeutic agents may include, for example, one or more of the following: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, checkpoint inhibitors, antibodies, and other antitumor agents.

[0254] Specific examples of chemotherapy agents include doxorubicin, 5-fluorouracil (5-FU), taxane derivatives (such as paclitaxel and docetaxel), capecitabine, irinotecan, albumin-bound paclitaxel, and platinum-based drugs (such as cisplatin, carboplatin, and oxaliplatin).

[0255] The chemotherapeutic agent may be an agent that does not interfere with adenovirus activity (e.g., does not affect the beneficial properties of the virus, such as oncolytic activity and / or the ability of the virus to replicate in cancer cells, such as viral replication in vivo).

[0256] Typically, such drug combinations are provided as two components:

[0257] (A) The first pharmaceutical composition of the adenovirus of the present invention; and

[0258] (B) A second pharmaceutical composition comprising a chemotherapeutic agent or an immunotherapeutic agent.

[0259] Therefore, the pharmaceutical combinations of the present invention can exist in combined formulations for simultaneous, separate, or sequential use, preferably for the treatment of cancer. Similarly, in the method of the present invention, the first and second pharmaceutical compositions can be administered to the patient simultaneously, separately, or sequentially.

[0260] The term "combination formulation" includes fixed-formulation and non-fixed-formulation. The term "fixed-formulation" means that the active ingredients (e.g., components (A) and (B)) are present in a single entity or dosage unit. In other words, the active ingredient is present in a single composition or formulation. The term "non-fixed-formulation" means that the active ingredients (e.g., components (A) and (B)) are present in different entities or dosages (e.g., as separate compositions or formulations), such as as kit components. Then, the independent components (A) and (B) (in their desired composition or formulation form) can be administered simultaneously, separately, or sequentially, at the same or different time points.

[0261] In cases of simultaneous administration, components (A) and (B) are given to the subject at the same time, but not necessarily together. Components (A) and (B) may be present in a single composition or in different compositions. Components (A) and (B) may be administered at the same or different sites (inside or on the body surface of the subject). Components (A) and (B) may be administered via the same or different routes.

[0262] In the case of sequential dosing, delaying the administration of the second component should not result in the loss of the synergistic effect produced by using the combination.

[0263] Both components (A) and (B) can be administered once or multiple times.

[0264] Components (A) and (B) can be administered in any order, such as component (A) before component (B); or component (B) before component (A).

[0265] 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 together with instructions for use.

[0266] In another embodiment, the present invention provides the oncolytic adenovirus of the present invention for use as a treatment or as a medicine.

[0267] In another embodiment, the present invention provides an oncolytic adenovirus of the present invention for treating cancer (preferably ovarian cancer). In another embodiment, the present invention provides a method for treating a subject with cancer (preferably ovarian cancer), the method comprising administering a therapeutically effective amount of the oncolytic adenovirus of the present invention to a subject in need.

[0268] In another embodiment, the present invention provides the use of the oncolytic adenovirus of the present invention in the preparation of a medicament for treating cancer (preferably ovarian cancer). The present invention also provides the use of the oncolytic adenovirus of the present invention for treating cancer (preferably ovarian cancer); and the application of the oncolytic adenovirus of the present invention in treating cancer (preferably ovarian cancer).

[0269] The term “treating cancer” as used in this article includes killing cancer cells and / or preventing or inhibiting the spread of cancer cells.

[0270] The therapeutic index (TI, also known as the treatment ratio) is a quantitative measure of the relative safety of a drug. It is a comparison between the therapeutic dose that produces a therapeutic effect and the dose that causes toxicity. Related terms, therapeutic window and safety window, refer to the optimized dose range between efficacy and toxicity, achieving maximum therapeutic benefit with unacceptable side effects or toxicity.

[0271] For humans in clinical trials, the treatment index is defined as TD50 / ED50 (where TD50 is the dose that causes toxicity in 50% of subjects; ED50 is the dose that produces the minimum effective effect in 50% of the population).

[0272] The subject is a human being. The subject can be biologically male or female. The human being can be, for example, aged 0-10 years, 10-20 years, 20-30 years, 30-40 years, 40-50 years, 50-60 years, 60-70 years, 70-80 years, 80-90 years, 90-100 years, or over 100 years old. The human being can be an individual currently suffering from or at risk of suffering from a specific disease or condition (e.g., cancer, preferably ovarian cancer). In some preferred embodiments, the subject is an individual currently suffering from or who has previously suffered from cancer (preferably ovarian cancer).

[0273] As used in this article, the term "interstitium" refers to the cells and tissues that support and supply the structure of organs, glands, or other tissues in the body. The interstitium is primarily composed of connective tissue, blood vessels, lymphatic vessels, and nerves. It provides nutrients to tissues or organs and removes waste and excess fluid. The interstitium may also participate in the body's immune response as well as the growth and spread of cancer cells.

[0274] In some implementations, the cancer is a tumor.

[0275] In some preferred embodiments, the cancer is a stromal tumor. As used herein, the term "stromal tumor" includes a tumor containing at least 1%, preferably at least 5%, 10%, 20%, or 50% stromal cells (by tumor weight). In some embodiments, the stromal tumor contains 20-80% stromal cells. The stromal cells may contain CAFs (cellular stromal cells). The cancer or tumor may be a type containing CAFs.

[0276] In some preferred embodiments, the cancer is carcinoma. In some preferred embodiments, the cancer is ovarian cancer, colorectal cancer, lung cancer, liver cancer, multiple myeloma, esophageal cancer, breast cancer, or pancreatic cancer. Most preferably, the cancer is ovarian cancer or stromal carcinoma.

[0277] In some embodiments, the adenovirus of the present invention can be used as a pretreatment for a therapy (e.g., surgery (neoadjuvant therapy)) to shrink tumors, treat metastases and / or prevent metastasis or further metastasis.

[0278] In other embodiments, the adenovirus of the present invention can be used post-treatment (e.g., post-operative (adjuvant therapy)) to treat metastatic lesions and / or prevent metastasis or further metastasis.

[0279] The pharmaceutical compositions of the present invention can be administered via one or more routes, using one or more methods known in the art. Components or compositions (A) and (B) can be administered via the same route or different routes. As will be understood by those skilled in the art, the route and / or manner of administration will vary depending on the expected results.

[0280] Preferred routes of administration for the pharmaceutical compositions of the present invention include intravenous, intratumoral, intraperitoneal, intrapleural, intravesical, intradermal, or other parenteral routes, such as by injection or infusion.

[0281] The phrase "parenteral administration" as used in this article refers to administration methods other than enteral and local administration, usually by injection, including but not limited to intravenous, intratumoral, intraperitoneal, intrapleural, intrabladder, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, tracheal, subcutaneous, subcutaneous tissue, intra-articular, subcapsular, and subarachnoid injections and infusions.

[0282] Alternatively, the pharmaceutical compositions of the present invention can be administered via non-parenteral routes, such as local, epidermal, or mucosal routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration.

[0283] In one embodiment, the formulation is administered intravenously (iv). This route is particularly effective for delivering oncolytic viruses because it allows rapid entry into most organs and tissues, and is especially useful for treating metastatic lesions, such as established metastases, particularly those located in highly vascularized areas such as the liver and lungs.

[0284] In one embodiment, the formulation is administered intraperitoneally (ip). This route is particularly effective for delivering oncolytic viruses to cancer types primarily confined to the peritoneum, such as ovarian, colorectal, and gastric cancers. This is because it allows rapid entry into most relevant organs and tissues to treat primary tumors and metastases without blood dilution and minimizes off-target effects.

[0285] In one embodiment, the formulation is used for intratumoral (it) administration. This route is particularly effective for delivering oncolytic viruses to localized cancer types accessible through an injection needle, as it allows for rapid entry into the tumor and minimizes off-target effects.

[0286] The pharmaceutical compositions of the present invention may comprise a therapeutically effective dose of the adenovirus of the present invention. The term "therapeutically effective dose" refers to the amount of adenovirus suitable for achieving the desired therapeutic effect, such as improving symptoms or condition of disease, when used in a suitable treatment regimen, particularly without causing dose-limiting side effects. In the treatment of cancer or metastases, a dose may be considered therapeutic when the number of viral particles is sufficient to result in: slowing or stopping tumor or metastatic growth, or finding a reduction in tumor or metastatic size, and / or increased patient lifespan. A suitable therapeutic dose is generally a balance between therapeutic efficacy and tolerable toxicity, for example, when side effects and toxicity are tolerable considering the benefits achieved by the therapy.

[0287] In one embodiment, the pharmaceutical composition of the present invention may contain 1×10¹ 0 Up to 1×10¹ 4 Virus particles / dose. Preferably, the pharmaceutical composition of the present invention comprises 1×10¹¹ to 1×10¹³ virus particles / dose. In one embodiment, the pharmaceutical composition of the present invention may be administered over 1-8 cycles, each cycle comprising one or more administrations within a 1-month period. The cycles may be administered in non-consecutive months.

[0288] Preferably, the method steps are performed in a specified order.

[0289] The adenovirus of the present invention can be readily produced using techniques well known in the art, including introducing one or more nucleotide mutations into a known adenovirus genome using standard cloning techniques (e.g., restriction endonucleases, site-directed mutagenesis, or Gibson assembly) or CRISPR-based techniques (e.g., CRISPR Cas9 / guide RNA).

[0290] In another embodiment of the invention, the use of the conditionally replicating adenovirus of the invention as a protein production vector is provided, wherein the adenovirus contains a transgene encoding a protein to be produced.

[0291] The publicly available information of each reference listed in this article is incorporated into this article in its entirety through citation. Attached Figure Description

[0292] Figure 1 A) Simplified diagram of the locations of early (E) and late (L) genes in the adenovirus genome. Arrows indicate viral genes, and their direction indicates the coding direction. ITR = Inverted Terminal Repeat. B) Schematic representation of the open reading frames encoded in the L2 and L3 regions.

[0293] Figure 2: Five neighboring cells of Ad3 and Ad7 ( Figure 2A(SEQ ID NOs: 5-6) and hexagonal ( Figure 2B Sequence alignment of amino acid sequences (SEQ ID NOs:25-26).

[0294] Figure 3 A sample from a patient with high-grade serous ovarian cancer was infected with different concentrations of adenovirus Ov26, which was obtained through biological screening. On day 6 post-infection, the survival rates of cancer cells (A) and cancer-associated fibroblasts (CAFs) (B) in the sample were determined by multiparameter flow cytometry. FAP = fibroblast activation protein, a cell surface marker present on the surface of CAFs. CA125 = cancer antigen 125, a cell surface marker present on the surface of cancer cells. VG / cell = viral genome number per cell.

[0295] Figure 4: Cell viability of various cell lines and primary cells in the presence of Ov26. Cells were infected with escalating doses of Ov26, and cell viability was determined by MTS assay on day 5 post-infection. Data are expressed as viability relative to uninfected controls. VG / cell = viral genome number per cell.

[0296] Figure 5 Comparison of oncolytic activity between Ov26 and wild-type parental viruses. (A) Viral genome replication was determined by qPCR on day 7 post-infection, expressed as the number of viral genomes per cell. (B) Infectious progeny viruses were quantified by ICC assay, expressed as infection-forming units (IFU) per cell. For each virus, data were normalized to levels observed in A549 cells. Parental A = wild-type Ad3; Parental B = wild-type Ad7.

[0297] Figure 6A549 cancer cells were seeded onto sterile coverslips and infected with Ov26 or Ov26_L2Ad7 cells at a dose of 100 VG / cell. The coverslips were then transferred to a co-culture system consisting of A549 cancer cells and cancer-associated fibroblasts (CAFs). Cells were immediately covered with agarose and DMEM medium. After two weeks of culture, MTT assay was added to stain surviving cells and visualize the extent of viral spread. A) Representative images of the spread assay; dashed circles indicate coverslip locations. Unstained areas marked with solid lines indicate the extent of viral spread. B) Quantitative analysis of viral spread in multiple spread assays under MRC5 fibroblast and A549 cell co-culture conditions. C) Quantitative analysis of viral spread in multiple spread assays under A549 cell co-culture conditions with CAFs isolated from ascites samples from ovarian cancer patients. Four independent samples were used for independent experiments. Each data point represents the spread result in a single experiment, and the error bar represents the standard deviation of the mean. The diffusion range was calculated using ImageJ software; statistical significance was assessed using a t-test, with *** P < 0.001, ** P < 0.01, and * P < 0.05.

[0298] Figure 7 Each virus was serially diluted and incubated for 30 minutes in DMEM medium or in DMEM medium supplemented with a mixed serum from 300 healthy human donors (final concentration 15%). A549 cells were then infected with the serially diluted virus for 4 hours, after which the infection medium was replaced with DMEM supplemented with 2% FBS. Cell viability was determined by the MTS viability assay on day 7 post-infection. Cell viability is expressed as a proportion of uninfected cells. The IC50 value was calculated using nonlinear regression to determine the fold change in IC50 after serum addition. Detailed Implementation

[0299] The present invention is further illustrated by the following embodiments, wherein, unless otherwise stated, parts and percentages are by weight, and temperature is in degrees Celsius. It should be understood that these embodiments, while indicating preferred embodiments of the invention, are given by way of illustration only. Based on the foregoing discussion and these embodiments, 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 different uses and conditions without departing from the spirit and scope of the invention. Therefore, various modifications to the invention will be readily apparent to those skilled in the art from the foregoing description, in addition to the embodiments shown and described herein. Such modifications are also intended to be included within the scope of the appended claims.

[0300] Example 1: Bioscreening and Sequence Analysis of Oncolytic Adenovirus

[0301] Starting from an initial pool of mixed viruses containing adenoviruses of groups B, C, D, F, and G, 23 rounds of biological screening (including mutation steps) were conducted to enrich oncolytic adenovirus candidates with the optimal combination of properties in terms of ovarian cancer cell tumor lysis, dissemination, hematopoietic stability, and immunostimulation. Sixty adenoviruses were selected from the final viral library for sequencing, and their genomes were analyzed. The initial pool of mixed viruses containing adenoviruses of groups B, C, D, F, and G were either from commercial sources (USC or Public Health England) or from a UK collaborative network.

[0302] Genomic analysis of the selected adenoviruses showed that the L2-L3 regions of all 60 adenoviruses obtained through biological screening were Ad3 / Ad7 chimeric structures.

[0303] Table 2. Detailed information on chimeric breakpoints in adenovirus nucleotide sequences obtained through biological screening.

[0304]

[0305] Table 3. Detailed information on chimeric breakpoints in the amino acid sequences of adenoviruses obtained through biological screening.

[0306]

[0307] The fact that all 60 adenovirus strains obtained through biological screening possessed chimeric Ad3 / Ad7 L2-L3 regions demonstrates the role of these regions in conferring advantages to adenoviruses, including their ability to lyse ovarian cancer cells, as well as their ability to spread, blood stability, and immune stimulation.

[0308] The amino acid sequences of the pentagonal and hexaagonal polypeptides of Ad3 and Ad7 are compared as shown in the figures below. Figure 2A and Figure 2B .

[0309] One of the chimeric adenoviruses, Ov26, was selected for further research.

[0310] Example 2: Ov26 kills cancer cells and CAFs derived from ex vivo ovarian cancer samples

[0311] A sample from a patient with high-grade serous ovarian cancer (HGSOC) was infected with gradually increasing concentrations of Ov26. On day 6 post-infection, the survival rates of cancer cells and cancer-associated fibroblasts (CAFs) in the sample were determined by multiparameter flow cytometry.

[0312] The results are as follows Figure 3 As shown in the figure, the results indicated that the survival rates of both cancer cells and CAFs decreased with increasing Ov26 concentration.

[0313] Example 3: Ov26 kills cancer cells of various ovarian cancer subtypes, including untreated cells and platinum-resistant cells.

[0314] A clinically relevant viral dose of Ov26 (equivalent to 1×10⁻⁶) was used. 13 Ovarian cancer patient samples (n = 24) were treated with VP / 5 L / patient, and the survival of cancer cells and cancer-associated fibroblasts (CAFs) was assessed. More specifically, cell viability was assessed by flow cytometry 6 days after treatment. Cells were viable stained with Live / Dead Near IR, cancer cells were identified using the EpCAM / CA125 marker, and cancer-associated fibroblasts were identified using fibroblast activation protein (FAP+). Each condition was performed in triplicate, and the results are presented as mean survival relative to uninfected controls. HGSOC = High-grade serous ovarian cancer. LGSOC = Low-grade serous ovarian cancer.

[0315] The results are shown in Table 4 below.

[0316] Table 4. Survival rates (%) of cancer cells and cancer-associated fibroblasts (CAFs) in samples from different stages / types.

[0317]

[0318]

[0319] The table above shows that Ov26 can effectively kill cancer cells and cancer-associated fibroblasts (CAFs) in a variety of different ovarian cancer patient subtypes.

[0320] Example 4: Evaluation of the oncolytic activity of Ov26 using a cell line group

[0321] The oncolytic activity of Ov26 was evaluated in a variety of cancer cell lines and cancer-associated fibroblasts, including the following cell types:

[0322] Ad-293: Human embryonic kidney cell line

[0323] A549: Lung Cancer Cell Line

[0324] HeLa: Cervical Cancer Cell Line

[0325] MDA-MB-231: Breast Cancer Cell Line

[0326] PANC-1: Pancreatic cancer cell line

[0327] PSN-1: Pancreatic cancer cell line

[0328] HCT116: Colorectal cancer cell line

[0329] HT-29: Colorectal cancer cell line

[0330] SKOV-3: Ovarian Cancer Cell Line

[0331] Huh-7: Liver cancer cell line

[0332] MRC-5: Fibroblast line

[0333] OE-21: Esophageal cancer cell line

[0334] OVCAR-3: Ovarian Cancer Cell Line

[0335] OVSAHO: Ovarian Cancer Cell Line

[0336] The results are shown in Figure 4. Ov26 showed efficacy against all tested cancer cell lines.

[0337] The IC50 values ​​for Ov26 are listed in Table 5 below.

[0338] Table 5: IC50 values ​​of Ov26 in different cancer cell lines

[0339]

[0340] Example 5: Oncolytic activity of Ov26 compared to wild-type parent virus

[0341] Several parameters of Ov26 virus infection were measured 72 hours after infecting lung cancer cells (A549), non-cancerous normal primary human hepatocytes, and normal human skin fibroblasts (NHDFs). Results are as follows: Figure 5 As shown.

[0342] During Ov26 infection, compared with wild-type parental virus infection, normal cells produced fewer viral genomes (A) and fewer infectious viral particles (B) than A549 cancer cells. These results indicate that Ov26 has the ability to preferentially infect lung cancer cells compared with normal hepatocytes and normal fibroblasts.

[0343] Example 7: Preparation of adenovirus with chimeric L2-L3 regions

[0344] Homologous recombination was used to replace the L2 region in Ov26 with a selectable cassette. The insertion of the selectable cassette was verified by Sanger sequencing. Subsequently, the cassette was removed by restriction endonuclease digestion, and another L2 sequence was inserted via Gibson assembly.

[0345] In this way, a novel adenovirus, Ov26_L2Ad7, was prepared, whose L2 and L3 regions were derived from Ad7 adenovirus (except for an Ad3-derived pVI polypeptide).

[0346] Example 8: Diffusion experiment in co-cultured A549 cancer cells and fibroblasts

[0347] The ability of progeny viral particles to infect neighboring cells and establish new toxigenic infections after infecting and lysing target cells is crucial for viral persistence and its spread within tumors. Therefore, the ability of Ov26 to spread in cancer cell layers (co-cultured with MRC5 fibroblasts or patient-derived cancer-associated fibroblasts) was compared with that of Ov26_L2Ad7. Figure 6 The results show that, in both cases, Ov26 has a significantly stronger diffusion capacity than Ov26_L2Ad7.

[0348] Example 9: Serum neutralization experiment in A549 cancer cells

[0349] For oncolytic viruses to successfully reach tumors after intravenous administration, their ability to evade existing immune responses in the bloodstream, such as neutralizing antibodies, is crucial. The humoral immune response to adenoviruses produces antibodies against three major capsid proteins: hexagonal, pentaagonal, and spike proteins. However, the extent to which antibodies are produced against each capsid protein and which antibodies are most effective at neutralizing the virus are not yet fully understood.

[0350] The ability of Ov26 to establish infection and kill A549 cancer cells under serum- and serum-free conditions was compared and compared with wild-type parent viruses Ad3 and Ad7.

[0351] Figure 7 The results indicate that Ov26's tendency to evade serum neutralization is similar to that of Ad7, but significantly higher than that of Ad3. These findings suggest that Ov26 can integrate pentagonal proteins derived from Ad3 without leading to a significant increase in neutralization levels, possibly because its fibrin originates from another parent virus, Ad7. This further underscores the importance of the chimeric capsid structure in Ov26, where the pentagonal and spike protein genes originate from different parent viruses.

[0352] The sequence list submitted with this patent application is incorporated herein by reference in its entirety.

Claims

1. A chimeric oncolytic adenovirus for the prevention or treatment of cancer, wherein, The genome of the chimeric oncolytic adenovirus includes: (A) Ad3 pentagonal genes, wherein the Ad3 pentagonal genes include or consist of the following: (a) A nucleotide sequence as shown in SEQ ID NO: 3; (b) A nucleotide sequence having at least 99% nucleotide sequence identity with SEQ ID NO: 3 and encoding an adenoviral polypeptide; or (c) The nucleotide sequence encoding the Ad3 pentagonal polypeptide, wherein the Ad3 pentagonal polypeptide is: (i) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 5; (ii) A polypeptide having at least 99.3%, 99.5%, or 99.7% sequence identity with SEQ ID NO: 5 and encoding an adenovirus pentagonal polypeptide; or, (iii) A polypeptide having at least 95%, 99% or 99.5% sequence identity with SEQ ID NO: 5, and having amino acids V, T, I and D at positions 11, 158, 178 and 326 of SEQ ID NO: 5, respectively, said polypeptide encoding an adenovirus pentavalent polypeptide; as well as, (B) Ad7 hexagonal gene, wherein the Ad7 hexagonal gene comprises or consists of the following: (a) A nucleotide sequence as shown in SEQ ID NO: 24; (b) A nucleotide sequence having at least 97% or at least 99% nucleotide sequence identity with SEQ ID NO: 24 and encoding an adenovirus hexapod polypeptide; or, (c) The nucleotide sequence encoding the Ad7 hexagonal polypeptide, wherein the Ad7 hexagonal polypeptide is: (i) A polypeptide with the amino acid sequence shown in SEQ ID NO: 26; or, (ii) A polypeptide having at least 97%, 98% or 99% sequence identity with SEQ ID NO: 26 and encoding an adenovirus hexapod polypeptide.

2. A method of treating a patient with cancer, the method comprising administering an effective amount of chimeric oncolytic adenovirus to the patient in need, wherein, The genome of the chimeric oncolytic adenovirus includes: (A) The Ad3 quinant gene as defined in claim 1; and, (B) The Ad7 hexane gene as defined in claim 1; And apply it to patients in need.

3. The use of a chimeric oncolytic adenovirus, wherein, The genome of the chimeric oncolytic adenovirus includes: (A) The Ad3 quinant gene as defined in claim 1; and, (B) The Ad7 hexane gene as defined in claim 1; Used to manufacture drugs for the prevention or treatment of cancer.

4. The chimeric oncolytic adenovirus, method, or use as described in any of the preceding claims, wherein, The genome of the chimeric oncolytic adenovirus includes an L2 region and an L3 region, wherein: (a) The Ad3 quinant gene is located within the L2 region; and / or, (b) The Ad7 hexane gene is located in the L3 region.

5. The chimeric oncolytic adenovirus, method, or use as described in any of the preceding claims, wherein, The genome of the chimeric oncolytic adenovirus includes an L2 region and an L3 region, wherein: (a) The L2 region includes: (i) the Ad3 quinant gene as defined in claim 1; (ii) Ad3 pVIII gene; (iii) The Ad3V gene; and, (iv) Ad3 or Ad7 pX gene; and / or, (b) The L3 region includes: (i) Ad3 or Ad7 pVI gene; (ii) the Ad7 hexane gene as defined in claim 1; and, (iii) Ad7 protease gene.

6. The chimeric oncolytic adenovirus, method, or use as described in any of the preceding claims, wherein, The genome of the chimeric oncolytic adenovirus further includes the Ad7 spike protein gene, wherein the Ad7 spike protein gene comprises or consists of the following: (a) A nucleotide sequence as shown in SEQ ID NO: 32; (b) A nucleotide sequence having at least 60%, 70%, 80%, 90%, or 95% (preferably at least 95%) nucleotide sequence identity with SEQ ID NO: 32 and encoding an adenovirus spike protein polypeptide; or, (c) The nucleotide sequence encoding the Ad7 spike protein polypeptide, wherein the Ad7 spike protein polypeptide is: (i) A polypeptide with the amino acid sequence shown in SEQ ID NO: 34; or, (ii) A polypeptide having at least 60%, 70%, 80%, 90% or 95% (preferably at least 95%) sequence identity with SEQ ID NO: 34 and encoding an adenovirus spike protein polypeptide; Preferably, the genome of the chimeric oncolytic adenovirus includes the L5 region, and the Ad7 spike protein gene is located within the L5 region.

7. The chimeric oncolytic adenovirus, method, or use as described in any of the preceding claims, wherein, The genome of the chimeric oncolytic adenovirus contains transgenes, preferably located in or near the E1, E3, L3, or L5 regions of the chimeric oncolytic adenovirus genome, or in regions where E1 / E3 is missing.

8. The chimeric oncolytic adenovirus, method, or use as described in claim 7, wherein, The transgene encodes an antibody, a bispecific linker molecule, a checkpoint inhibitor, a cytokine, a chemokine, an enzyme, or an angiogenesis inhibitor.

9. The chimeric oncolytic adenovirus, method, or use as described in any of the preceding claims, wherein, The cancer is selected from the group consisting of ovarian cancer, colorectal cancer, lung cancer, liver cancer, multiple myeloma, esophageal cancer, breast cancer, and pancreatic cancer; preferably ovarian cancer or cancer containing stromal tissue.

10. A chimeric adenovirus, wherein, The genome of the chimeric adenovirus includes: (A) The Ad3 quinant gene as defined in claim 1; (B) The Ad7 hexane gene as defined in claim 1; and, (C) The Ad7 spike protein gene as defined in claim 6.

11. The chimeric adenovirus of claim 10, wherein, The genome of the chimeric adenovirus includes an L2 region and an L3 region, wherein: (a) The Ad3 quinant gene is located within the L2 region; and / or (b) The Ad7 hexane gene is located in the L3 region.

12. The chimeric adenovirus as claimed in claim 10 or 11, wherein, The genome of the chimeric adenovirus includes an L2 region and an L3 region, wherein: (a) The L2 region includes: (i) the Ad3 quinant gene as defined in claim 1; (ii) Ad3 pVIII gene; (iii) Ad3V gene; and (iv) Ad3 or Ad7 pX gene; and / or (b) The L3 region includes: (i) Ad3 or Ad7 pVI gene; (ii) the Ad7 hexane gene as defined in claim 1; and (iii) Ad7 spike protein gene.

13. The chimeric adenovirus according to any one of claims 10-12, wherein, The genome of the chimeric adenovirus includes the L5 region, and the Ad7 spike protein gene is located within the L5 region.

14. The chimeric adenovirus according to any one of claims 10-13, wherein, The genome of the chimeric adenovirus contains transgenes; preferably, the transgenes are located in or adjacent to the E1, E3, L3, or L5 regions of the chimeric adenovirus genome, or in the E1 / E3 deletion region.

15. The chimeric adenovirus of claim 14, wherein, The transgene encodes an antibody, a bispecific linker molecule, a checkpoint inhibitor, a cytokine, a chemokine, an enzyme, or an angiogenesis inhibitor.

16. A chimeric adenovirus comprising an adenovirus capsid, wherein, The adenovirus capsid comprises: (A) The Ad3 pentagonal polypeptide as defined in claim 1; (B) The Ad7 hexagonal polypeptide as defined in claim 1; and, (C) The Ad7 spike protein polypeptide as defined in claim 6.

17. The chimeric oncolytic adenovirus, chimeric adenovirus, method, or use as described in any of the preceding claims, wherein, Adenoviruses are either group B adenoviruses or human adenoviruses.

18. An adenovirus gene therapy vector, wherein, The genome of the adenovirus gene therapy vector includes: (a) The Ad3 quinone gene as defined in claim 1; (b) the Ad7 hexane gene as defined in claim 1; and (c) Genetically modified organisms.

19. The adenovirus gene therapy vector as described in claim 18, wherein, The genome of the adenovirus gene therapy vector includes an L2 region and an L3 region, wherein: (a) The Ad3 quinant gene is located within the L2 region; and / or, (b) The Ad7 hexane gene is located in the L3 region.

20. The adenovirus gene therapy vector as described in claim 18 or 19, wherein, The genome of the adenovirus gene therapy vector includes an L2 region and an L3 region, wherein: (a) The L2 region includes: (i) the Ad3 quinant gene as defined in claim 1; (ii) Ad3 pVIII gene; (iii) The Ad3V gene; and, (iv) Ad3 or Ad7 pX gene; and / or, (b) The L3 region includes: (i) Ad3 or Ad7 pVI gene; (ii) the Ad7 hexane gene as defined in claim 1; and, (iii) Ad7 protease gene.

21. The adenovirus gene therapy vector according to any one of claims 18-20, wherein, The genome of the adenovirus gene therapy vector further includes the Ad7 spike protein gene as defined in claim 6; preferably, the genome of the adenovirus gene therapy vector includes an L5 region, and the Ad7 spike protein gene is located within the L5 region.

22. The adenovirus gene therapy vector according to any one of claims 18-21, wherein, The transgene is located in or near the E1, E3, L3, or L5 regions of the chimeric oncolytic adenovirus genome, or in the E1 / E3 deletion region.

23. The adenovirus gene therapy vector as described in claim 22, wherein, The transgenic encoding antibody, bispecific adaptor molecule, checkpoint inhibitor, cytokine, chemokine, enzyme or angiogenesis inhibitor.

24. The adenovirus gene therapy vector according to any one of claims 18-23, wherein, The adenovirus gene therapy vector is derived from or derived from group B adenovirus or human adenovirus.

25. A pharmaceutical composition comprising a chimeric adenovirus as described in any one of claims 10-17, or an adenovirus gene therapy vector as described in any one of claims 18-24, optionally further comprising one or more pharmaceutically acceptable vectors, excipients, or diluents.

26. A drug combination comprising: (A) A first pharmaceutical composition comprising a chimeric adenovirus as described in any one of claims 10-17, or an adenovirus gene therapy vector as described in any one of claims 18-24; and, (B) A second pharmaceutical composition comprising a chemotherapeutic agent or an immunotherapeutic agent; The drug combination is a combination preparation for simultaneous, separate, or sequential use, and is preferably used for the treatment of cancer.

27. The chimeric adenovirus as described in any one of claims 10-17, or the adenovirus gene therapy vector as described in any one of claims 18-24, for use in treatment or as a medicine.

28. Use of the chimeric adenovirus as described in any one of claims 10-17 as a protein production vector, wherein the chimeric adenovirus comprises a transgene encoding a protein to be produced.

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