Modified adenoviruses

Abstract

To provide a modified oncolytic adenovirus of serotype Ad5; a pharmaceutical composition comprising the adenovirus; and a method of treating cancer using the adenovirus.SOLUTION: A modified adenovirus comprises at least one point mutation(s) in the hexon hypervariable region 7 (HVR7 mutation) to prevent virus binding with coagulation factor 10 (FX); at least one point mutation(s) in the fiber knob region AB loop (KO1 mutation) to prevent virus binding with the coxsackie and adenovirus receptor (CAR); and at least one point mutation(s) in the penton integrin binding motif Arg-Gly-Asp (RGD) to prevent virus binding with ανβ3 / ανβ5 integrin.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a modified oncolytic adenovirus of serotype Ad5; and a method of treating cancer using the adenovirus, wherein said The modified adenovirus contains a hexon hyperpolymer that prevents viral binding to coagulation factor 10 (FX). At least one point mutation in variable region 7 (HVR7 mutation); Coxsackievirus Fiber knob region AB loop prevents viral binding to the adenovirus receptor (CAR) at least one point mutation in the αVβ3 / αVβ5 inte The penton integrin binding motif Arg-Gly-A prevents virus binding to integrin Contains at least one point mutation in sp(RGD). [Background technology]

[0002] Cancer virotherapy is a promising treatment for herpes simplex type 1 as the first oncolytic immunotherapy for advanced melanoma This is an emerging field driven by the PES-based talimogene-laherparepvec (T-VEC). Oncolytic adenoviruses are often used in various vaccine approaches against infectious diseases. These are highly immunogenic viruses used in vaccines. Importantly, they stimulate an immune response. Furthermore, the adenovirus has an extraordinary ability to simultaneously enhance the expression of oncolytic The presence and immunogenic cell death it induces is related to TH1-type immune modulators such as IFNg. By inducing expression of these receptors, we aim to develop a more sensitive approach to generate clinically relevant antitumor immunity. However, tumor lysis may shape the hostile tumor microenvironment toward a virulent state. The immunogenicity of hydrolyzed adenoviruses is a double-edged sword; antiviral immunity is often too suppressed is much weaker than that caused by self-antigens expressed by tumors. Neutralize the immune response.

[0003] Adenovirus 5 (Ad5) is a vector that can be genetically engineered and can tolerate large transgenes. To date, numerous cancer and gene therapy clinical trials (clinicaltrials.go) have been published. v, 2016). However, this serotype However, Ad5 has several suboptimal characteristics that prevent its widespread clinical use. It is a virus that has a seroprevalence approaching 100% in certain populations and produces neutralizing antibodies (nAbs) in all Rapidly inactivates the therapeutic vector delivered to the body. Other suboptimal features include viral hexameric expression Spleen and Extensive off-target sequestration in the liver, mediated by human coagulation factor 10 (FX) In vitro, Ad5 binds to the viral fiber protein It is ubiquitously expressed within tight junctions on polarized epithelial cells and is downregulated in advanced cancers. The virus is a virulent pathogen that causes inflammatory bowel disease in host cells through interactions between the Cussackievirus and adenovirus receptor (CAR). Ad5 then enters the cytoplasm, mediated by the viral penton base protein. The cells are transported to the host via αvβ3 / 5 integrin through clathrin-mediated endocytosis. Internalized into cells.

[0004] Immunotherapy and stimulating a patient's own immune system to target and attack cancer are gaining popularity Recognition of the role of oncolytic viruses in cancer therapy has increased over the past decade. This has changed dramatically since the turn of the century. At the beginning of the century, oncolytic viruses were used to destroy tumor cells through oncolysis. are recognized as active agents in cancer treatment acting solely through their inherent ability to dissolve In recent years, there has been interest in their use as cancer vaccines, and they have been shown to activate the immune system. Their ability to release tumor antigens from cancer cells upon oncolysis to induce tumor cell death may be a promising therapeutic target for cancer. This is recognized as an important feature in designing novel immunotherapies. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, although viral vectors are becoming increasingly attractive in clinical settings, The clinical efficacy of serotype-Ad5- is limited by poor tumor specificity, extensive off-target delivery, and These are hampered by inactivation by immune and innate immunity, necessitating innovative engineering strategies. [Means for solving the problem]

[0006] Described herein are modified adenoviral vectors designed to overcome the above-mentioned shortcomings. Disclose.

[0007] According to a first aspect of the present invention, a) I4 in hexon hypervariable region 7, which prevents virus binding to coagulation factor 10 (FX) 21G, T423N, E424S, E450Q or L426Y point mutations (HVR7 at least one of the following: b) Prevents viral binding to the Coxsackievirus-Adenovirus Receptor (CAR) The S408E or P409A point mutation in the AB loop of the fiber knob region (KO1 at least one of the following mutations: c) α V β 3 / α V β 5Penton integrin, which prevents virus binding to integrins The D342E or D342A point mutation in the RGD binding motif At least one of the mutations A modified Ad5 serotype adenovirus comprising:

[0008] The modified Ad5 serotype adenovirus of the present invention is capable of infecting off-target tissues. The ability of the body to actually infect the liver and spleen is severely impaired and is actually prevented / inhibited. This is advantageous because it also compromises the ability to infect a broad range of cells. Adenoviruses are compromised in terms of the tissues they can infect.

[0009] Adenoviruses have an icosahedral nucleocapsid that contains a double-stranded DNA genome. They are medium-sized (90-100 nm) non-enveloped (without an outer lipid bilayer) proteins. In humans, there are seven species of adenovirus (human adenovirus A to G),57 There are four commonly recognized human adenovirus serotypes (Ad-1 to 57): A-12, 1 8, 31;B-3, 7, 11, 14, 16, 21, 34, 35, 50, 55;C-1, 2 , 5, 6, 57; D-8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 32, 33, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49, 51, 53, 54, 56;E-4;F-4 0, 41; and G-52. Thus, any reference herein to Ad5 is to Adenovirus 1 (Adenovirus 1), Adenovirus 2 (Adenovirus 2), Adenovirus 3 (Adenovirus 3), Adenovirus 4 (Adenovirus 4), Adenovirus 5 (Adenovirus 5), Adenovirus 6 (Adenovirus 6), Adenovirus 7 (Adenovirus 7), Adenovirus 8 (Adenovirus 9), Adenovirus 10 (Ad This refers to adenovirus serotype 5, which belongs to the C subclass of the virus.

[0010] As known to those skilled in the art, adenovirus virions are composed of viral DNA-protein complexes. It consists of a non-enveloped icosahedral capsid surrounding a cellular core complex. It is the most abundant structural protein, with 240 copies of trimeric molecules per capsid. Twelve copies of the hexon trimer form each of the 20 triangular facets of the capsid. The penton base and fiber protein complex seals the apex of the capsid, and Promotes viral attachment (fiber) and internalization (penton base).

[0011] The hexon protein contains nine hypervariable regions (HVRs) that are essential for the differentiation of various adenoviruses. These HVRs are highly conserved among the R. serovars. The HVRs are located in two different loops that form the DE1 loop, HVR1-6 in the DE2 loop, and HVR7 in the DE3 loop. ~9 is located within the FG1 loop.

[0012] Thus, reference herein to at least one mutation in HVR7 is Specifically, the at least one HVR7 mutation is Prevents interaction with coagulation factor X, thereby preventing off-targeting of the modified adenovirus to the liver. Limiting get sequestration and improving targeting to target cancer cells.

[0013] In a preferred embodiment, the at least one HVR7 mutation is I421G, T4 23N, E424S, E450Q or L426Y. Most preferably, the FXA-binding domain comprises at least one amino acid substitution mutation that prevents FX interaction with the FXA-binding domain. The at least one HVR7 mutation may additionally or alternatively be I421G, T42 Contains at least one of the following point mutations: 3N, E424S and L426Y.

[0014] As known to those skilled in the art, adenoviral vectors have a wide distribution of vectors. Group C adenoviruses such as Ad5 have a natural tropism that affects the entry of the virus into cells. , viral fiber proteins and the coxsackievirus-adenovirus receptor (CA R) interactions, which are thought to involve high affinity binding of the virus to cells. Therefore, adenoviral vectors can be used to target specific tissues or cell types for the purpose of anti-cancer therapy. Targeting to a target gene requires modification of the vector's normal tropism to improve specificity .

[0015] Adenovirus infection is caused by a special protein on the surface of the virus, the adenovirus The fiber protein, specifically the adenovirus fiber domain called the carboxy-terminal knob domain, It begins with the recognition of host cell receptors by the globular carboxy-terminal domain of the fiber protein. Thus, references herein to the knob of the adenovirus fiber protein , a reference to the globular carboxy-terminal domain of the adenovirus fiber protein.

[0016] Thus, at least one reference to the KO1 mutation is in the fiber knob region AB loop. Specifically, the term "KO1 mutation" refers to at least one of the above-mentioned KO1 mutations. In a preferred embodiment, the at least one mutation prevents viral binding to the CAR. One KO1 mutation selected from one or more of the following groups: S408E or P409A Most preferably, the at least one point mutation prevents CAR binding to the target site. Mutations include S408E and P409A point mutations.

[0017] The adenovirus penton base contains five Arg-Gly-Asp sequences and is integrated Phosphoryl alpha v beta 3 and alpha v beta 5 (α V β 3 / α V β 5 ) and It facilitates viral infection by enabling viral internalization. By reducing the number of splenic targeting sites, off-site targeting to the spleen is reduced, thereby allowing tumor-specific targeting. and, when used in the context of anticancer therapy, inhibits otherwise harmful immune responses of the host. The release of inflammatory cytokines that lead to the response was found to be attenuated.

[0018] Thus, reference to at least one RGD mutation is V β 3 / α V β 5 Intel The penton integrin binding motif Arg-Gly-, which prevents virus binding to integrin It refers to at least one mutation of Asp (RGD mutation). In one embodiment, the at least one RGD mutation is RGE or RGA, respectively. At least one selected from one or more of the group including D342E or D342A Most preferably, the point mutation is D342E resulting in RGE. It is.

[0019] In an even more preferred embodiment, said adenovirus is directed against tumor cells, particularly those of certain types. Further modified to include at least one cancer targeting modification that selectively targets tumor cells. As will be appreciated by those skilled in the art, altering the natural tropism of an adenovirus can be and also incorporating at least one targeted modification. Through the introduction of mutations / sequences, the modified viruses exhibit improved tumor specificity and reduced off-cell proliferation. Examples of cancer targeting modifications / sequences include: , known to those skilled in the art, for example, but not limited to, binding to aminopeptidase N NGR (containing) peptides, especially those that bind to the HI loop of the adenovirus fiber protein Adenovirus (Ad) with NGR; YSA ( The peptide, which contains EphA2, is particularly effective in transducing EphA2-positive cancer cells. Adenoviruses carrying YSA in chimeric fibers do not result in transduction of 2-negative cancer cells growth factor antibodies, particularly chemical conjugation of their targeting moieties, e.g. bFGF, EGFR, For example, antibodies against adenoviruses using avidin / biotin conjugates (e.g., Sec Cimab, Herceptin, Avastin, etc.); and once attached to the outside of the virus (Typically chemically linked, e.g., via hyaluronidase) to degrade the outer cell membrane and matrix-degrading enzymes that allow viruses to penetrate the tumor microenvironment more efficiently. There is a source.

[0020] In a preferred embodiment, the cancer targeting modification comprises the insertion or expression of an αvβ6 integrin binding peptide (identified using conventional techniques such as sequence binding and homology techniques), such as the A20 peptide sequence NAVPNLRGDLQVLAQKVART (SEQ ID NO:1), into or by the virus, ideally into or at the viral fiber knob HI loop (this modified virus is hereinafter referred to as Ad5.3D.A20). A20 is originally derived from the foot and mouth disease virus (FMDV) capsid protein VP1 and has high affinity for αvβ6 integrin. αvβ6 integrin is expressed in one third of ovarian cancers and various other epithelial cancers, and is undetectable in healthy adult tissues. Thus, as will be appreciated by those skilled in the art, through the expression and incorporation of this sequence in the modified virus, the modified virus can selectively target αvβ6 integrin overexpressing cancers such as, but not limited to, ovarian cancer, pancreatic cancer, oesophageal cancer, lung cancer, cervical cancer, head and neck cancer, oral cancer, cancer of the larynx, skin cancer, breast cancer, kidney cancer and colorectal cancer.

[0021] Those skilled in the art will also recognize homologs, orthologs, or functional derivatives of the listed mutations. It will be understood that such finds use in the context of the present invention. Mutations including additions, deletions, substitutions, etc. of certain amino acids are encompassed by the present invention. It may be possible to replace a given amino acid with another amino acid of a similar "type". Replacing an amino acid with another hydrophobic amino acid can be done using programs such as CLUSTAL. This can be accomplished by comparing amino acid sequences using a program. The program compares amino acid sequences and inserts spaces into either sequence if necessary. For optimal alignment, the amino acid sequence is It is possible to calculate identity or similarity (identity means conservation of amino acid type) Programs such as BLASTx align the longest similar sequences and give a value of Therefore, we assign several similarities, each with a different score. It is possible to obtain a comparison in which regions of interest are found. Both types of analysis are contemplated in the present invention. can be.

[0022] In an even more preferred embodiment, the modified adenoviral vector (Ad5.3D. A20) is a) I4 in hexon hypervariable region 7, which prevents virus binding to coagulation factor 10 (FX) 21G, T423N, E424S, E450Q or L426Y point mutations (HVR7 at least one of the following: b) Prevents viral binding to the Coxsackievirus-Adenovirus Receptor (CAR) The S408E or P409A point mutation in the AB loop of the fiber knob region (KO1 at least one of the following: c) α V β 3 / α V β 5 Penton integrin, which prevents virus binding to integrins The D342E or D342A point mutation in the RGD binding motif at least one of the mutations; and d) A20 peptide sequence NAVPNLRGDL in the viral fiber knob HI loop Insertion or expression of QVLAQKVART (SEQ ID NO: 1) Includes.

[0023] More preferably, the modified adenoviral vector (Ad5.3D.A20) comprises: a) I4 in hexon hypervariable region 7, which prevents virus binding to coagulation factor 10 (FX) 21G, T423N, E424S and L426Y point mutations (HVR7 mutation); b) Prevents viral binding to the Coxsackievirus-Adenovirus Receptor (CAR) The S408E and P409A point mutations in the AB loop of the fiber knob region (KO1 mutations); c) Preventing viral binding to αVβ3 / αVβ5 integrins (RGE mutations) The D342E punctuation of the penton integrin binding motif Arg-Gly-Asp results in natural mutations; and d) A20 peptide sequence NAVPNLRGDL in the viral fiber knob HI loop Insertion or expression of QVLAQKVART (SEQ ID NO: 1) Includes.

[0024] In the above viral vector of the present invention, the inventors have determined that the major capsid protein: The natural tropism of Ad5 was abolished by mutating the hexon, fiber, and penton. The tumor was successfully excised. Residues responsible for binding to the receptor (CAR) and αvβ3 / 5 integrin, respectively hexon hypervariable region 7 (HVR7 mutation), fiber knob A, including substitution mutations in B-loop (KO1 mutation) and the penton integrin binding motif Arg-Gly- Triple de-targeted by Asp modification (RGD mutation) An Ad5-based vector backbone was generated (Ad5.3D).

[0025] The Ad5.3D vector was generated using homologous recombination technology and is a compromised adenovirus. The complementation cell lines were engineered to rescue the HIV-1 virus and provide high virus titers. A combination of tropic ablative mutations enhances the expression of αvβ3 / 5 integrin, CA R- and FX-mediated cell entry was completely blocked. They reported that a heterologous αvβ6 integrin-binding peptide (A20, NAVPNLRGDLQVL AQKVART (SEQ ID NO: 1) was inserted into the HI loop of the viral fiber knob. By incorporating αvβ6, the αvβ6 antigen preferentially targets and infects certain cancer tumor cells. The adenovirus was further modified by the incorporation of additional targeting sequences for the purpose of targeting the adenovirus (Ad 5.3D.A20). Using 296-β6 cells engineered to overexpress β6 , Ad5.3D.A20 adenovirus was propagated.

[0026] The αvβ6 integrin is expressed in one-third of ovarian cancers and a variety of other epithelial cancers. It is not detectable in healthy adult tissues. Ad5.3D.A20 is highly neutralized in the presence of ascites fluid. Even so, αvβ6+ ovarian cancer cell lines and primary clinical ascites-derived EOC ex vivo cultures Efficiently transduced Ad5.3D.A20 after systemic delivery in non-tumor-bearing mice. The in vivo biodistribution profile was significantly altered compared to Ad5, and all off-target Luciferase expression was reduced in the organs, and viral genome load was reduced by 7 logs in the liver. Furthermore, the antitumor activity of oncolytic Ad5.3D.A20 (OAd5.3D.A20) after intraperitoneal delivery Tumor efficacy was assessed in immunodeficient mice bearing intraperitoneal SKOV3(-β6)EOC xenografts. Oncolytic treatment with OAd5.3D.A20 significantly reduced overall tumor lysis compared to Ad5 treatment. Improved survival rate.

[0027] Thus, Ad5.3D modified adenoviruses can be used to target additional cancer-specific mutations Through the incorporation of It is a viral vector that provides the platform.

[0028] In an even more preferred embodiment, the adenovirus is an adenovirus that is capable of transporting, without limitation, a therapeutic agent. The method further comprises the steps of: Thus, this embodiment exerts a therapeutic effect on targeted cancer cells. Examples of agents include those that directly stimulate the immune response. , e.g., GM-CSF, IL-12; agents that indirectly stimulate the immune system, e.g., CTLA -4, immune checkpoint inhibitors that inhibit corepressors such as PD-L1, PD1, or Lag3 Antibodies (or antibody fragments) encoding cross-linking inhibitors; bispecific T cell binding Bispecific Natural Killer Cell-associated (BiTE) antibody constructs; Bispecific Natural Killer Cell-associated (BiKE) antibody constructs agents that sensitize tumors to cell-based immunotherapy, such as anti-CD19 25Antibody-encoding agents that deplete regulatory T cells in the tumor microenvironment; e.g., Natri Cortex is a serogroup that encodes the nitrate / iodide symporter (NIS) or somatostatin receptor type 2 (SSTR2). These drugs contain agents that sensitize tumors to radiation therapy or for imaging. Alternatively, the transgene may be, for example, a Reduced Expression sion in Immortalized Cells (REIC / DKK3) Enzymes that sensitize cancer cells via the conversion of a non-toxic prodrug to a toxic drug For example, genes encoding cytosine deaminase, nitroreductase, and thymidine kinase By using the nucleotide sequence of ... Other transgenes known in the art and useful in the treatment of cancer may be used in the practice of the present invention. can be done.

[0029] In an even more preferred embodiment, the adenovirus is pRB-deficient for viral replication. To restrict the expression of β-terminal β-terminal endonuclease (β-terminal ... It has been further modified to include the A gene.

[0030] Even more preferably, the adenovirus is modified with E3 / 1 to enhance oncolytic potential. A single adenine base addition (T1 mutation) at position 445 within the endoplasmic reticulum (ER) retention domain of 9K It has been further modified to include

[0031] According to a second aspect of the present invention, there is provided a modified form as defined herein for use as a medicament. Adenoviruses are provided.

[0032] According to a third aspect of the present invention there is provided a modified erythrocyte colony-stimulating agent as defined herein for use in the treatment of cancer. Mutant adenoviruses are provided.

[0033] According to a fourth aspect of the present invention, there is provided a method for the preparation of a medicament for treating cancer. In accordance with the present invention, there is provided a modified adenovirus as defined herein.

[0034] Most preferably, the cancer referred to herein is any one or more of the following cancers: Including: nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, cancer of the connective tissue, melanoma, lung cancer , intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, Oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, brown cancer Chromocytoma, Prolactinoma, T-cell leukemia / lymphoma, Neuroma, von Hippel-Lyssoma Dow disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, urinary Tube cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal tumor, bone cancer, osteochondroma , chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, fibro Sarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, and gallbladder cancer Head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, Prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer cer), skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma , pancreatic cancer, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer , stomach cancer, thymic cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal Cancer of the vulva, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostimuli Tachinoma, gum cancer, heart cancer, lip cancer, meningeal cancer, mouth cancer, Transverse cancer, palate cancer, parotid cancer, peritoneal cancer, pharynx cancer, pleural cancer, salivary Liquid gland cancer, tongue cancer and tonsillar cancer.

[0035] Compounds for use in medicine are generally provided in pharmaceutical or veterinary compositions, thus Thus, according to a further fifth aspect of the present invention, there is provided an adenovirus as defined herein, and and a pharma- ceutical composition comprising a pharma- ceutical agent, an adjuvant, a diluent or an excipient. will be done.

[0036] Suitable pharmaceutical excipients are well known to those skilled in the art. The pharmaceutical compositions can be administered by any suitable route, e.g. , formulated for oral, buccal, nasal or bronchial (inhalation), transdermal or parenteral administration. and may be prepared by any of the methods well known in the art of pharmacy.

[0037] The composition can be prepared by associating an adenovirus as defined above with a carrier. Generally, the formulation comprises the adenovirus in a liquid carrier or a finely divided solid carrier or by uniformly and intimately bringing into association both the raw materials and the finished product, and then, if necessary, shaping the product. The present invention relates to a method for preparing a pharmaceutical composition comprising administering to a subject an adenovirus as defined above. in combination or association with a pharma- ceutical or veterinarily acceptable carrier or vehicle. The present invention extends to a method comprising the step of:

[0038] According to yet a further aspect of the present invention, there is provided a method of treating cancer comprising administering to said patient an effective amount of Administering a modified adenovirus or pharmaceutical composition as defined in claim 1 to a patient in need thereof. A method is provided that includes the steps.

[0039] An "effective amount" of an adenovirus or a composition comprising the adenovirus as used herein References to effective doses are to amounts sufficient to achieve a desired biological effect, such as cancer cell death. The dosage will depend on the recipient's age, sex, health status, and weight, as well as type of concomitant treatment, if any. It will be understood that the frequency of treatment will depend on the nature of the effect desired. The amount of efficacy will be determined by the person administering the treatment.

[0040] In the following claims and the preceding description of the invention, the following claims and the preceding claims, whether expressly stated or implied, Unless the context otherwise requires, the word "comprises" shall be used The word "comprises" or "comprising" g)" in the inclusive sense, i.e., to specify the presence of the stated feature, It is not used to exclude the presence or addition of further features in various embodiments of the invention. do.

[0041] All references cited herein, including any patents or patent applications, are hereby incorporated by reference. No admission is made that any reference constitutes prior art. It is not recognized that any of the prior art forms part of the general knowledge in the art. I can't.

[0042] Preferred features of each aspect of the invention are as described in connection with any of the other aspects. It is possible.

[0043] Other features of the present invention will become apparent from the following examples. Generally speaking, the present invention Any novel feature disclosed in this specification (including the accompanying claims and drawings) is Accordingly, the present invention is not limited to the specific embodiments, implementations, and / or aspects thereof. Any features, integers, properties, compounds or chemical moieties stated in connection with any embodiment or example are not to be construed as being inconsistent with the present disclosure. Unless otherwise stated, the present invention is applicable to any other aspect, embodiment or example described herein. It should be understood as follows.

[0044] Moreover, unless expressly stated otherwise, features disclosed herein may be used in combination with other features having the same or similar purpose. can be replaced by alternative features that fulfill the same function.

[0045] Throughout the description and claims of this specification, unless the context otherwise requires, the singular In particular, where the indefinite article is used, the specification shall not be construed as limiting the scope of the invention unless the context requires otherwise. Unless otherwise specified, plural as well as singular is to be understood as being intended.

[0046] Embodiments of the present invention will now be described, by way of example only, with reference to the following. [Brief description of the drawings]

[0047] [Figure 1] Generated vectors. (A) Viral titers and predicted tropism of Ad5 and triple detargeted avβ6 integrin retargeting vector, Ad5.3D.A20. (B) Vector map of oncolytic Ad5.3D.A20. (C) Comparative predictive 3D modeling of adenovirus serotype 5 (Ad5) fiber knob and modified Ad5.3D.A20 fiber knob with A20 peptide (NAVPNLRGDLQVLAQKVART; SEQ ID NO: 1) insertion in the HI loop (green). CAR, coxsackievirus-adenovirus receptor; FX, coagulation factor 10; HVR7, FX-binding mutation in hexon hypervariable region 7; KO1, CAR-binding mutation in fiber knob AB loop; Luc, luciferase transgene; vp, viral particle. [Diagram 2]Excision of natural receptor tropism. (A) Binding of replication-deficient Ad5 and Ad5.3D.A20 vectors to the Coxsackievirus-Adenovirus Receptor (CAR). The ratio of viral transgene expression from Ad5.3D.A20 compared to Ad5 is shown above the bars. (B) Binding of replication-deficient Ad5 and HVR7 mutant Ad5 variants to coagulation factor 10 (FX0 was assessed in a luciferase assay by infecting cells in the presence of human FX with (+) or without (-) the anticoagulant X-bp for 3 h at 37°C). HVR7, FX binding mutation. Statistical significance: ns, p?0.05; **, p<0.01. [Diagram 3] In vitro evaluation of αvβ6 integrin targeting. (A) Transduction efficiency of replication-deficient wild-type (Ad5) and triple-detargeted, integrin-retargeted (Ad5.3D.A20) vectors in αvβ6+BT-20 breast cancer cells. (B) Transduction efficiency of replication-deficient wild-type (Ad5) and triple-detargeted, integrin-retargeted (Ad5.3D.A20) vectors in αvβ6+ primary epithelial ovarian cancer (EOC) cells from patient 004. (C) Luciferase expression by oncolytic vector (T1 / Δ24) in infected avβ6-low / CAR+SKOV3 and avβ6-high / CAR+SKOV3-β6 cells (in-house SKOV3 cells with retroviral expression of avβ6). (D) Competitive inhibition of αvβ6 integrin-mediated cell entry. The highest 10% αvβ6 expressing SKOV3-β6 cells were FACS sorted, subcultured and infected. IgG, normal mouse IgG control; 10D5, anti-αvβ6 function blocking antibody. Ratios of viral transgene expression are shown above the bars. Statistical significance ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 4]Effect of malignant ovarian ascites on vector transduction ex vivo. (A) Quantification of anti-Ad5 antibodies in 20 clinical ovarian ascites (OAS) samples and control serum (solid black line) from healthy male volunteers by ELISA. Horizontal lines indicate 50% and 100% binding of anti-Ad5 ab in control serum. (B) Antigen specificity of anti-Ad5 antibodies in ascites and serum by Western blot. (C) Vector transduction efficiency of replication-deficient (Ad5) and Ad5.3D.A20 vectors in the absence and presence of various dilutions of ascites from ovarian cancer patient 004 in BT-20 cells. (D) Vector transduction efficiency of replication-deficient (Ad5) and Ad5.3D.A20 vectors in the absence and presence of various dilutions of ascites from ovarian cancer patient 004 in primary ex vivo cultures of epithelial ovarian cancer cells from patient 004. Cells were preincubated and infected with increasing concentrations of ascites. [Diagram 5] Biodistribution of replication-deficient vectors 72 hours after systemic delivery. (A) Biodistribution study schedule. (B) In vivo imaging of the biodistribution of replication-deficient (Ad5) and triple-detargeted Ad5.3D.A20 viruses 3 days after intravenous injection. (C) Quantification of total luminescent signal from panel B in the whole body. (D) Quantification of total luminescent signal from panel B in the liver, 335. (E) Quantification of total luminescent signal from panel B in the spleen. (F) Quantification of total luminescent signal from panel B in the lung. (G) Quantification of total luminescent signal from panel B in the ovary. (H) Quantification of total luminescent signal from panel B in the heart. ip, intraperitoneal; IVIS, in vivo imaging system; pi, post-infection; vp, viral particles. Error bars represent standard error of the mean; n=5 / group; ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 6]Viral genome copy numbers in off-target organs after systemic delivery. (A) Adenoviral genome copy numbers from tissues excised in FIG. 5: liver by qPCR for the hexon gene after systemic vector delivery. (B) Adenoviral genome copy numbers from tissues excised in FIG. 5: spleen by qPCR for the hexon gene after systemic vector delivery. (C) Adenoviral genome copy numbers from tissues excised in FIG. 5: lung by qPCR for the hexon gene after systemic vector delivery. (D) Adenoviral genome copy numbers from tissues excised in FIG. 5: ovary by qPCR for the hexon gene after systemic vector delivery. (E) Adenoviral genome copy numbers from tissues excised in FIG. 5: heart by qPCR for the hexon gene after systemic vector delivery. Data were normalized and analyzed by one-way ANOVA and Sidak's multiple comparison post-hoc test in GraphPad Prism. Error bars represent standard error of the mean; n=5 / group; *, p<0.05; **, p<0.01; ***, p<0.001; p<0.0001; ns, no statistically significant difference. Numbers below the graphs indicate fold reduction in the Ad5.3D.A20 group compared to the Ad5 group. [Figure 7]Oncolytic efficacy study: intraperitoneal delivery of oncolytic vectors in ovarian cancer xenograft models. (A) Study schedule. Intraperitoneal xenografts of human ovarian cancer cells (SKOV3 and SKOV3-6) were implanted in immunodeficient mice (n=5 / group) and then treated with three doses of intravenous oncolytic Ad5 or triple-detargeted, integrin-retargeted Ad5.3D.A20 on days 14, 16, and 18. (B) Luminescence heatmap images measured at 349 48 hours after the first treatment (day 16). (C) Quantification of whole-body luminescence measured at 349 48 hours after the first treatment (day 16). (D) Luminescence heatmap images measured at 349 7 days after the first treatment (day 21). (E) Quantification of whole-body luminescence measured at 349 7 days after the first treatment (day 21). (F) Overall survival of animals inoculated with SKOV3 (αvβ6-low / CAR+) cells and then treated with virus as above, shown as Kaplan-Meier survival curves, until the final study endpoint of 101 days. (G) Overall survival of animals inoculated with SKOV3-β6 (αvβ6-high / CAR+) cells and then treated with virus as above, shown as Kaplan-Meier survival curves, until the final study endpoint of 101 days. ip, intraperitoneal; IVIS, in vivo imaging system; vp, viral particles *, p<0.05; **, p<0.01; ***, p<0.001. IVIS, in vivo imaging system. [Figure 8]Biodistribution study: Heatmap images of ex vivo luminescence intensity. (A) Liver, spleen, lungs, ovaries and heart collected immediately after death from animals inoculated intravenously with PBS (control). (B) Liver, spleen, lungs, ovaries and heart collected immediately after death from animals inoculated intravenously with Ad5.Luc vector. (C) Liver, spleen, lungs, ovaries and heart collected immediately after death from animals inoculated intravenously with Ad5.3D.A20 vector. Organs were soaked in D-luciferin and imaged with an IVIS imager. Tissue color indicates relative luminescence intensity emitted by the luciferase transgene; scale normalized to exclude background luminescence. (D) Fold reduction in total luminescence (photons / sec) compared to Ad5.Luc. The mean luminescence intensity of the Ad5.Luc group was divided by the mean luminescence of each organ of the Ad5.3D.A20 group. The mean value of the PBS control group was subtracted from all values. [Figure 9] Biodistribution study: Immunohistochemistry of formalin-fixed, paraffin-embedded liver sections. (A) Hematoxylin-eosin staining for visualization of cellular structures and mouse liver staining using rabbit IgG isotype control antibody (1 μg / mL), primary rabbit anti-CAR antibody (1:100) and primary rabbit anti-ITGB6 (αvβ6) antibody (1:10). (B) Staining of Ad5-infected hepatocytes from animals infected with Ad5 or Ad5.3D.A20 vectors using primary rabbit anti-Ad5 antibody (1 μg / mL). DAB was used as substrate and sections were counterstained with hematoxylin, mounted on coverslips and observed under a light microscope. [Figure 10] Pilot study: Tumor localization and uptake rate in NOD / SCID mice. 1x107 SKOV3-β6 cells / animal were implanted ip on day 0, and 2 mice were sacrificed at each time point on days 7, 14, 21, and 48 / 49 (final endpoint). Approximate tumor size was measured and ascites volume was quantified. [Figure 11]Oncolytic efficacy testing: endpoint tumor characterization. (A) Viral genome copy number (per 40ng of DNA) in postmortem tumors of OAd5 and OAd5.3D.A20 from SKOV3 and SKOV3-β6 cohorts by qPCR. (B) αvβ6 integrin (ITGB6) gene expression in postmortem tumors of OAd5 and OAd5.3D.A20 from SKOV3 and SKOV3-β6 cohorts by qPCR. The level of αvβ6 expression is shown in comparison to mouse number 1 from the negative control PBS group in the SKOV3 cohort by using human ACTB (β-actin) as an endogenous control. [Figure 12] Transduction activity of Ad5.3D.A20 and Ad5 expressing luciferase in pancreatic cancer cell lines. (A) Expression levels of αvβ6 and hCAR determined in ASPC-1 pancreatic cancer cell line. (B) Expression levels of αvβ6 and hCAR determined in BxPc pancreatic cancer cell line. (C) Expression levels of αvβ6 and hCAR determined in CFPAC pancreatic cancer cell line. (D) Expression levels of αvβ6 and hCAR determined in PANC10-05 pancreatic cancer cell line. (E) Expression levels of αvβ6 and hCAR determined in SW1990 pancreatic cancer cell line. (F) Expression levels of αvβ6 and hCAR determined in PANC0403 pancreatic cancer cell line. (G) Expression levels of αvβ6 and hCAR determined in SUIT-2 pancreatic cancer cell line. (H) Expression levels of αvβ6 and hCAR determined in MiPaCa2 pancreatic cancer cell line. (I) Expression levels of αvβ6 and hCAR determined in the PT45 pancreatic cancer cell line. Cells were infected with 5,000 vp / cell of a virus expressing luciferase, and transgene expression was quantified 48 h post-infection and normalized for total cellular protein. [Figure 13]Figure 1 shows the transduction activity of Ad5.3D.A20 and Ad5 expressing luciferase in esophageal cancer cell lines. Expression levels of αvβ6 and hCAR were determined in Kyse-30 esophageal cancer cells. Cells were infected with 5,000 vp / cell of virus expressing luciferase, and transgene expression was quantified 48 hours post-infection and normalized for total cellular protein. [Figure 14] Transduction activity of Ad5.3D.A20 and Ad5 expressing luciferase in breast cancer cell lines. (A) Expression levels of αvβ6 and hCAR determined in BT-20 breast cancer cells. (B) Expression levels of αvβ6 and hCAR determined in BT-474 breast cancer cells. (C) Expression levels of αvβ6 and hCAR determined in MDA-MB-361 breast cancer cells. (D) Expression levels of αvβ6 and hCAR determined in MDA-MB-231 breast cancer cells. Cells were infected with 5,000 vp / cell of virus expressing luciferase and transgene expression was quantified 48 hours post-infection and normalized for total cellular protein. [Figure 15] Transduction activity of Ad5.3D.A20 and Ad5 expressing luciferase in lung cancer cell lines. (A) Expression levels of αvβ6 and hCAR determined in A427 lung cancer cells. (B) Expression levels of αvβ6 and hCAR determined in A549 lung cancer cells. (C) Expression levels of αvβ6 and hCAR determined in NCI-H460 lung cancer cells. Cells were infected with 5,000 vp / cell of virus expressing luciferase and 48 hours post-infection, transgene expression was quantified and corrected for total cellular protein. [Figure 16]Figure 1 shows the oncolytic activity of replication-deficient and oncolytic (O) Ad5.3D.A20 and Ad5 in pancreatic and breast cancer cell lines. Pancreatic cancer cell lines Suit 2 (αvβ6high / hCARhigh), MiCaPa2 (αvβ6low / hCARhigh), PANC0403 (αvβ6vvhigh / hCARhig h) and PT45 (αvβ6neg / hCARhigh) and breast cancer cell lines BT-20 (αvβ6high / hCARneg) and MDA-MB-231 (αvβ6neg / hCARhigh) were plated in 96-well plates at a density of 20,000 cells / well. Cells were infected with 5,000 vp / cell and cell viability was quantified every 24 hours using a standard MTS cell viability assay. As expected, the replication-deficient vectors showed no deleterious effects on cell viability, whereas the cell killing (oncolytic) activity of OAd5.3D.A20 and OAd5 was directly related to the presence / absence of cellular αvβ6 and hCAR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] material and method Adenoviral vectors, cell lines and clinical ascites All the constructed vectors expressed luciferase (Luc) and were expressed in bacterial artificial chromosomes (BACs). C) were based on wild-type Ad5 genomes captured by Us i-Kerttula et al., 2016), all genetic modifications were performed using the AdZ homologous recombination method (St The virus was introduced into the BAC by the T-Rex 29 3 or HEK293-β6 cells (A20 modified virus). The oncolytic vector has an E1 / E3 gene deletion, whereas the oncolytic vector controls viral replication via pR 24 bases in the E1A gene to restrict it to B-deficient cells (Sherr, 1996) The paired deletion dl922-947(Δ24) (Fueyo et al., 2000) and oncolysis The endoplasmic reticulum (ER) retention domain of E3 / 19K for enhanced cytotoxicity (Gros et al., 2008). It has a single adenine base addition, the T1 mutation at position 445 in the FMDV genome. The heterologous A20 peptide sequence (NAVPNLRGDLQVLAQKVART; SEQ ID NO: 1) The gene was inserted into the fiber knob HI loop. High titer virus was generated essentially as previously described. As reported in Uusi-Kerttula et al., 2015, a et al., 2016), produced in T-REx-293 cells or HEK293-β6 cells. .

[0049] The SKOV3-β6 cell line was developed in-house. It is puromycin-selective and has a β6 gene insertion. The pBABE-β6 plasmid (no. 13596; Addgene) was transfected with Effecten e was used to transfect the 293Phoenix packaging cell line. After 2 h, the retrovirus was harvested, filtered, and used to infect SKOV3 cells. αvβ6 integrin expressing cells were selected in the presence of 5 μg / mL puromycin Permission to collect and culture primary EOC cells from ascites was granted by the Wales Center for Biomaterials. All patients were granted a 100% CI of 0.01 to 0.05% prior to collection. All subjects gave written informed consent. Ascites clinical samples were collected from the Venipuncture Centre, Cardiff. From a patient undergoing treatment for advanced ovarian cancer at the Indre Cancer Centre Data were collected and anonymized as previously described (Uusi-Kerttula et al., 201 5, Uusi-Kerttula et al., 2016), cells were treated and subcultured.

[0050] In vitro assays Cell surface receptor expression was assessed using anti-αvβ6 clone 10D5 and anti-CAR antibody clone Rm cB, followed by secondary F(ab')2 goat α-mouse IgG(H+L) IgG Alex aFluor647 was used as previously described (Uusi-Kerttula (2016) and evaluated by flow cytometry. Anti-A in ovarian ascites and serum The presence of d5 antibodies was assayed using an ELISA method essentially as previously described (Stallwood et al., 2013). 000). Antigen specificity of the antibodies was evaluated by Western blot.

[0051] In vitro cell transduction efficiency was assessed using a multimode plate reader essentially as previously described. As described in (Uusi-Kerttula et al., 2015, Uusi-Kerttula et al., 2015). ula et al., 2016), assessed by luciferase reporter gene assay and expressed as relative light units. The reversible translation units (RLU) were normalized to the total protein concentration (RLU / mg) in each well. To evaluate the effect of FX on the rate of IFN-γ transfection, the transduction medium was supplemented with 10 μg / mL of human FX. The vector tropism of the cell receptor was confirmed by using anti-αvβ6 antibody (10 μg / mL; clone 10 D5, Millipore) or normal anti-mouse control IgG (10 μg / mL; San ta Cruz) as previously described (Uusi-Kerttula et al. , 2016) and assessed in a competitive inhibition assay. Neutralization assays were performed using two-fold consecutive inhibition of cell-free OAS. Preincubation with dilutions (1:40 to 1:2.5, corresponding to final concentrations of 2.5 to 40%) The procedure included a step

[0052] In vivo testing All animal studies were performed at the Mayo Clinic, Rochester, USA. For consistency, all animals were 7 weeks old and sex-matched; females were used for ease of care. All animal handling and injections were performed by experienced staff in accordance with local regulations. The study was conducted by Mrs. Jill M. Thompson, a Veterinary Technician.

[0053] The feasibility of white fur for luciferase tracking allowed the development of wild-type B6 albino mice. S(B6N-Tyr c-Brd / BrdCrCrl) (n=5 / group) and replication-deficient vector A biodistribution study was performed using 1×10 11 Injected into the lateral tail vein with vp All mice were incubated with CO 2 After IVIS imaging 72 hours post-infection, mice were sacrificed. Organs were harvested for analysis. Efficacy studies were performed in immunodeficient NOD / SCID mice (n=5 / Treatment schedules were first optimized in a pilot study (n=8). 1×10 7 SKOV3-β6 cells were implanted i.p. on day 0 and then Two mice were sacrificed on days 1 and 48 / 49 (final endpoint). CAR and αvβ6 expression in tumors was assessed by flow cytometry. For tumor lytic efficacy studies, NOD / SCID mice were administered 1 × 10 7 SKOs V3 cells or SKOV3-β6 cells were xenografted ip. Mice (n=5 / group) on days 14, 16 and 18, 10 vp OAd (PBS, OAd The primary endpoint was overall survival (OSF) of 14.0–18.5% (OAd5.3D.A20) and 18.0–19.5% (OAd5.3D.A20). The survival rate was calculated as %. Quantifying the luminescence signal emitted by the luciferase transgene using the 1M mer Vector uptake was monitored by measuring the major off-target organs and end points. Viral genome copy numbers in end tumors were quantified by qPCR. The levels of αvβ6 gene expression in intratumor tissue were quantified by qPCR.

[0054] A brief protocol for cell viability assays For cell viability assays, cells were cultured using CellTiter 9 according to the manufacturer's recommended protocol. 6 AQueous One Solution cell proliferation assay (Promega) 20,000 or 30,000 cells were used in each well of a 96-well plate. Cells were seeded at 5,000 / cell in serum-free medium and incubated overnight. The cells were infected with 10 ... and 144 hours, 20 μl of CellTiter 96 AQueou Viable cells were determined by adding One Solution reagent. After 2 hours of incubation in a 5% CO2 atmosphere, the absorbance was measured at 490 nm. The % of live cells was calculated relative to treated cells. Results are means, n=3, error bars are standard deviations. Represents the deviation.

[0055] statistical analysis All numerical and statistical analyses were performed in GraphPad Prism version 6.03. In vitro and ex vivo assays were analyzed using two-tailed unpaired t-tests or one-way Analyses were performed using ANOVA and Dunnett's multiple comparison post-hoc test. Normalization was performed and analyzed by one-way ANOVA with Sidak's multiple comparison post-hoc test. Overall survival (%) after anaplastic surgery is shown as Kaplan-Meier survival curves; survival was gated. Analyses were performed using the Hann-Breslow-Wilcoxon test. All tests: ns, p>0. 05;*, p<0.05;**, p<0.01;***p<0.001;****p<0 .0001.

[0056] result Three detargeting mutations and vector retargeting to αvβ6 integrin-expressing cells High viral titers of the novel Ad5.3D.A20 vector carrying the A20 peptide insert Replication-deficient and oncolytic variants were engineered and produced (Figure 1). The treatment did not significantly affect the titer. Predictive modeling in the NIH showed the prominence of the A20 peptide within the immunodominant HI loop (Figure 1). C).

[0057] Transduction efficiency of replication-deficient vectors was assessed using 100-fold increase in expression of CAR and αvβ6 integrin expressing CAR- ... The detargeting mutations in Ad5.3D.A20 were evaluated in cell lines expressing CHO-CAR completely abolished CAR-mediated entry in CAR+ cells, whereas Ad5 inhibited these cells. The HVR7 mutation efficiently transduced FX-mediated vectors. As expected, FX significantly increased the expression of FX-dependent markers in the IL-16 / IL-2 cells compared to the culture conditions without FX (Figure 2B). , which significantly increased Ad5 transduction into these cells (Fig. 2B; right panel). Addition of human FX to the culture medium inhibits FX-bound excised Ad5.HV in CHO-K1 cells There was no effect on transduction efficiency of the R7 control vector (Figure 2B; left panel). The enhanced transduction seen with 5 was due to its binding to a 3:1 molar excess of FX in the culture medium. by the addition of anticoagulant X-bp, a Gla domain-interacting protein that inactivates (Fig. 2B, right panel). In contrast, FX depletion was reversed in the Ad5.HVR7 vector form. It had no effect on transduction (Fig. 2B, left panel).

[0058] αvβ6 integrin is triple detargeted, integrin retargeted Ad5.3D.A Ad5.3D.A20 was identified as the major entry receptor for Ad5 and Ad5.3D.A20 (Fig. 3). In comparison, αvβ6+ / CAR-BT-20 breast cancer cells were transduced 305-fold more efficiently (Figure 3A; p = 0.0270) and primary EOC004 cells (αvβ6+ / CAR-) were cultured for 6 9-fold increased transduction efficiency (Figure 3B; p=0.0090). An oncolytic variant of the A20 vector inhibited SKOV3 expressing low levels of αvβ6. Approximately five-fold increase compared to αvβ6-low / CAR+ cells (Fig. 3C; p<0.0001). SKOV3-β6 cells (αvβ6-high / CAR+) were transduced with increased efficiency and tumor It was confirmed that the oncolytic modifications did not impair the A20 peptide:αvβ6 interaction. Competition assays using anti-αvβ6 antibody (10D5) showed that Ad5.3D.A20 vector (169 Fig. 3D; p = 0.0010), and selectively inhibited αvβ6 Sex was confirmed.

[0059] Clinical ovarian ascites (OAS) samples from 20 patients were screened for anti-Ad5 antibodies by ELISA. The anti-Ad5 ab titers in malignant ovarian ascites were screened for the presence of Serum anti-Ad5 antibody titers were probed in healthy adult male volunteers (Fig. 4A). A proportion of patients were found to have both lower and higher antibody titers than the control serum. (Figure 4A, black dotted line). The ascites from patient 001 (OAS001) showed similar antibody titers to the control serum. The antibodies of OAS001 and the control serum were selected for the subsequent neutralization assay because they were similar to each other. appeared to be specific for the fiber protein, but not the most abundant capsid protein. Protein hexon was detected at very low levels in Western blots using denatured virus particles. The effect of OAS001 on the transduction efficiency of Ad5.3D.A20 was Neutralizing effects were evaluated in αvβ6+ / CAR-EOC004 primary cells. 20 showed superior transduction efficiency compared to Ad5 at OAS concentrations of 2.5, 5, and 10%. (up to 902-fold higher), whereas Ad5 detectably transduced these cells. did not (Figure 4C).

[0060] Non-tumor-bearing mice were inoculated intravenously to determine the tropism of the vector in vivo, specifically the vector biosynthesis. The effect of three detargeting mutations on intracellular distribution was evaluated (Fig. 5A). Ad5 was expressed in the liver and The Ad5.3D.A20 vector showed strong localization in the splenic and peritoneal regions, whereas the Ad5.3D.A20 vector showed luminescence at 72 Ad5-inoculated animals showed no significant difference from PBS (p<0.001). Overall, the overall mortality rate was significantly higher than in control animals treated with Ad5.3D.A20 (p<0.0001) or Ad5.3D.A20 (p<0.0001). The liver, spleen, lungs, ovaries and heart were excised and then excised. In vivo luminescence was quantified (see Figures 8A-C for luminescence heat maps). Livers of d5-challenged animals were significantly more luminescent than either the PBS control or Ad5.3D.A20 groups. (both p<0.0001) (Fig. 5D). Similarly, Ad5.3D.A20 inhibited Ad5 and In comparison, transgene expression was significantly reduced in the spleen, lung, ovary and heart (Figure 5 E–H; p<0.0001 for all). The fold change in luminescence intensity in each organ was , see Figure 8D.

[0061] Modifications of Ad5.3D.A20 enhance viral isolation in multiple normal tissues. Confirmation of reduction was achieved via quantification of viral load in off-target organs by qPCR. The genome copy number of Ad5.3D.A20 was 10 000,000-fold lower (Figure 6A; p<0.0001). The copy number was >700-fold lower in the spleen compared to Ad5 ( Fig. 6B ; p < 0.0001). In addition, the Ad5.3D.A20 vector was more effective in preventing off-target infection in all organs than Ad5. showed improved pulmonary, cardiac and ovarian viral loads of 10.1% and 10.2%, respectively. 5 , 10 4 , 10 3 The success of liver detargeting was due to the genetic modification of Ad5. The high expression level of CAR, whereas αvβ6 was undetectable, suggested that this was due to This is supported by immunohistochemical staining of liver sections which showed ( Fig. 9A ) Ad5. 3D. Confirmation of the detargeting effect of gene modification in A20 was confirmed by liver sections of mice in the Ad5 group. showed positive staining for Ad capsid protein, whereas Ad5.3D.A20 vector This is provided by the observation that livers of mice loaded with IgG were not affected by IgG (Figure 9B).

[0062] To evaluate the efficacy of αvβ6 retargeting in in vivo cancer models, we used αvβ6-hig h / CAR-SKOV3-β6 human ovarian cancer xenografts were cultured in immunodeficient NOD / SCID mice Animals were cultured at the site of cell injection within 14 days after intraperitoneal implantation of SKOV3-β6 cells. and developed large solid tumors at various sites in the abdominal cavity, and by day 49, the tumors had grown to The tumors accumulated ascites and spread throughout the peritoneal cavity. The tumors maintained high αvβ6 expression (flow cytometry). (For cytometry, see Figure 10.) Based on these observations, 1 day after transplantation of αvβ6-high / CAR-SKOV3 and αvβ6-high / CAR-SKOV3-β6 Three doses of Ad5 and Ad5.3D.A20 were administered intravenously on days 4, 16, and 18. Virotherapy efficacy testing was performed by delivering an oncolytic variant.

[0063] IVIS imaging 48 hours after the first virotherapy treatment administration (day 16) showed tumor lysis Animals treated with the avian Ad5 vector showed widespread luminescence throughout the abdominal region, and SKOV3 and In both SKOV3-β6 xenograft models, the highest intensity was in the liver / spleen region (Figure 7 B). This distribution was maintained, but the intensity decreased 5 days later, on day 21 (Figure 7D). Conversely, the Ad5.3D.A20 223 vector showed highly selective localization and was localized to non-tumor tissues. Consistent with successful detargeting of the tissue, there was a significant decrease in overall luminescence compared to Ad5. In both the Ad5.3D.A.3 and SKOV3-β6 models, quantification of whole-body luminescence demonstrated that Ad5.3D.A. 20 vector uptake at day 16 (Figure 7C; p<0.05 and <0.01, respectively). and 21 days (Figure 7E; p<0.0001) were significantly lower than Ad5. Ta.

[0064] Antitumor activity was determined by oncolytic Ad5 and oncolytic A in the SKOV3 xenograft model. The enhanced expression of Ad5.3D.A20 was observed in both Ad5.3D.A20 and Ad5.3D.A20 (Figure 7F). Consistent with the tumor-selective effect observed, all five mice treated with Ad5.3D.A20 had 1 Animals treated with Ad5 survived only up to 70 days, whereas animals treated with Ad6 survived only up to 70 days. It did not exist.

[0065] A series of tumors of pancreatic (Figure 12), esophageal (Figure 13), breast (Figure 14) and pulmonary (Figure 15) origin were identified. Additional transduction assays were performed in cancer cell lines. All cell types were first transduced with αvβ6 and The expression of hCAR in 7 / 9 pancreatic cell lines (ASPC -1, BxPc, CFPAC, PANC 10.05, SW1990, PANC 040 3 and Suit2), which expressed αvβ6 at various levels and Ad5.3D.A20 (Figures 12A to 12G). Conversely, as expected, However, MiPaCa2 (Fig. S12H) and PT45 (Fig. S12I) cells express very low levels of αvβ6. were poorly or not expressed at all and were less permissive to Ad5.3D.A20-mediated transduction. The esophageal cell line Kyse-30 expressed high levels of αvβ6 and was not In the breast cancer cell lines tested, Three of the four cell lines tested (BT-20, BT-474 and MDA-MB361 ) express αvβ6 to various degrees and are permissive for transduction with Ad5.3D.A20 (Fig. 14A to 14C), whereas in MDA-MB-231 cells, αvβ6 Lack of expression rendered cells noninfectible to Ad5.3D.A20 (Fig. 14D). In all three lung cancer cell lines tested (A427, A549 and NCI-H460, Fig. 15A-C), in the absence of αvβ6, cells were not susceptible to Ad5.3D.A20-mediated transduction. In contrast, it was refractive.

[0066] To evaluate the cell-killing activity of the oncolytic version of Ad5.3D.A20, αv β6 high (Suit2, Panc0403) and αvβ6 low (MiPaCa2 ) or αvβ6 neg Cell viability assays were performed on the (PT45) pancreatic cancer cell line. (Figure 16). Cells were infected with 5,000 vp / cell of replication-deficient Ad5 or Ad5.3D. Ad5.3D.A20 or oncolytic (O)Ad5 or Ad5.3D.A20. As expected, the replication-defective vector did not mediate any significant effect on cell viability. However, the cell killing activity of oncolytic vectors was shown to correlate well with αvβ6 expression. Similarly, αvβ6 high / hCAR neg Triple negative breast cancer cell line B In T-20, the presence of high levels of αvβ6 combined with the absence of hCAR led to the OAd5 Only .3D.A20 was able to efficiently mediate cell killing. Conversely, αvβ6 neg / hCAR high In breast cancer cell line MDA-MB-231, the presence of hCAR and αv In the absence of β6, only OAd5 was able to kill cells efficiently.

[0067] Consideration All known tropisms were ablated and overexpressed prognostic cancer marker αvβ6 in A novel tumor-selective oncolytic adenoviral vector, Ad, retargeted to tegrin 5.3D.A20 describes the expression of integrin αvβ6 in aggressive transformed cancers. It is therefore a promising target for therapeutic cancer applications.

[0068] In this study, a replication-defective form of the Ad5.3D.A20 vector was shown to mediate natural viral integration. pathway (Figure 2) and instead demonstrate in vitro and We successfully selectively retargeted αvβ6+ cells in vivo and ex vivo (Figure 3). Efficacy-limiting interactions that occur with systemic delivery of noviral vectors could theoretically be prevented by i. Although this can be circumvented by intraluminal administration of the vector via p. This approach poses challenges because Ad5 is sequestered by anti-Ad5 nAbs in the ascites. Thus, in the presence of OAS containing high pre-existing levels of anti-Ad5 nAbs, Ad The transduction efficiency of Ad5.3D.A20 was evaluated (Figure 4A). Unlike Ad5, Ad5.3 D.A20 retains the ability to transduce αvβ6+ cells even at relatively high OAS concentrations. The results showed that the serotonin-dependent β-blockers (Sb-blockers) were significantly higher in the control group

[0069] The clinical efficacy of Ad5 vectors with unmodified capsids has also been shown to be significantly improved, particularly in the liver. Target tissue sequestration is also significantly restricted. We demonstrate that we successfully altered the biodistribution of the d5 vector in tumor-free mice. In mice, the replication-deficient Ad5.3D.A20 showed improved biodistribution compared to the parental Ad5. and viral transgene expression in the liver, spleen, and lungs was significantly reduced (Fig. 5), compared with Ad5. In comparison, viral genome copy numbers were reduced in all off-target organs (Figure 6 ).

[0070] To test the efficacy of the oncolytic form of the detargeted / retargeted Ad5.3D.A20 vector, To investigate the mechanism of disease progression, we established an orthotopic ip xenograft model of human ovarian cancer in immunodeficient mice. Localization of virally encoded transgene expression of oncolytic Ad5.3D.A20 following intraluminal administration Localized biodistribution may prevent off-target sequestration and / or tumor-selective viral uptake. This was consistent with the reduction in sarcoma in the SKOV3 xenograft model (Figures 7B-7E). Supported by superior survival of animals treated with Ad5.3D.A20 compared to Ad5 (Figure 7F).

[0071] Administration of Ad5.3D or Ad5.3D.A20, respectively, improved progression of chemotherapy resistance or αvβ6+ cancers, particularly but not exclusively ovarian, pancreatic, esophageal and This vector offers a promising treatment option for breast cancer. It may ultimately be used to develop precision viral therapy. It provides an important platform that can be modified for use.

[0072] References FUEYO, J., GOMEZ-MANZANO, C., ALEMANY, R., LE E,PSY,MCDONNELL,TJ,MITLIANGA,P.,SHI ,YX,LEVIN,VA,YUNG,WKA&KYRITSIS,A. P.2000.A mutant oncolytic adenovirus tar getting the Rb pathway produces anti-glio ma effect in vivo.Oncogene,19,2-12. GROS, A., MARTINEZ-QUINTANILLA, J., PUIG, C. ,GUEDAN,S.,MOLLEVI,D.G.,ALEMANY,R.&CASCA LLO,M.2008.Bioselection of a gain of fun ction mutation that enhances adenovirus 5 release and improves its antitumoral p otency.Cancer Research,68,8928-8937. SHERR,C.J.1996.Cancer cell cycles.Scien ce,274,1672-1674. STALLWOOD,Y.,FISHER,K.D.,GALLIMORE,P.H. &MAUTNER,V.2000.Neutralisation of adenov irus infectivity by ascitic fluid from o varian cancer patients.Gene Therapy,7,63 7-643. STANTON,R.J.,MCSHARRY,B.P.,ARMSTRONG,M. ,TOMASEC,P.&WILKINSON,G.W.G.2008.Re-engi neering adenovirus vector systems to ena ble high-throughput analyses of gene fun ction.BioTechniques,45,659-668. UUSI-KERTTULA,H.,DAVIES,J.,COUGHLAN,L., HULIN-CURTIS,S.,JONES,R.,HANNA,L.,CHESTE R,J.D.&PARKER,A.L.2016.Pseudotyped alpha vbeta6 integrin-targeted adenovirus vect ors for ovarian cancer therapies.Oncotar get. UUSI-KERTTULA,H.,LEGUT,M.,DAVIES,J.,JON ES,R.,HUDSON,E.,HANNA,L.,STANTON,R.J.,CH ESTER,J.D.&PARKER,A.L.2015.Incorporation of Peptides Targeting EGFR and FGFR1 in to the Adenoviral Fiber Knob Domain and Their Evaluation as Targeted Cancer Ther apies.Hum Gene Ther,26,320-9.

Claims

1. a) I4 in hexon hypervariable region 7, which prevents viral binding to coagulation factor 10 (FX) 21G, T423N, E424S, E450Q or L426Y point mutations (HVR7 at least one of the following: b) Prevents viral binding to the Coxsackievirus / Adenovirus Receptor (CAR) The S408E or P409A point mutation in the AB loop of the fiber knob region (KO1 at least one of the following: c) α Ｖ β ３ / α Ｖ β ５ Penton integrin, which prevents virus binding to integrins The D342E or D342A mutation in the RGD binding motif At least one of the mutations A modified Ad5 serotype adenovirus comprising:

2. The HVR7 mutations are the following point mutations: I421G, T423N, E424S, and The modified amino acid sequence according to claim 1, which comprises or consists of at least one of L426Y and L427Y. Denovirus.

3. The KO1 mutation comprises or consists of S408E and P409A point mutations. A modified adenovirus according to claim 1 or 2.

4. 4. The method of claim 1, wherein the RGD mutation is D342E, resulting in RGE. A modified adenovirus according to any one of claims 1 to 4.

5. The adenovirus comprises at least one cancer targeting modification that selectively targets tumor cells. The method according to any one of claims 1 to 4, further modified to include a mutation or sequence. Modified adenovirus.

6. The adenovirus comprises at least one NGR that binds aminopeptidase N (including The NGR comprises a peptide motif corresponding to the adenovirus fiber protein. or at least one that binds to the pan-cancer marker EphA2; and one YSA (containing) peptide motif, wherein the YSA is a chimeric fiber or at least one cancer-targeting antibody, or at least one growth factor The modified antigen of claim 5 in an antibody or at least one matrix-degrading enzyme. Denovirus.

7. The cancer targeting modification is an αvβ6 integrin binding peptide or an A20 peptide sequence. NAVPNLRGDLQVLAQKVART (SEQ ID NO: 1) to the virus 6. The modified adenovirus of claim 5, comprising insertion or expression by a gene.

8. the A20 peptide sequence is inserted into the viral fiber knob HI loop; or The modified adenovirus of claim 7 is expressed in the viral fiber knob HI loop. Russ.

9. The adenovirus (Ad5.3D.A20), a) I42 in hexon hypervariable region 7, which prevents viral binding to coagulation factor 10 (FX) 1G, T423N, E424S, E450Q or L426Y point mutations (HVR7 mutations at least one of the following mutations: b) Prevents viral binding to the Coxsackievirus / Adenovirus Receptor (CAR) The S408E or P409A point mutation in the AB loop of the fiber knob region (KO1 at least one of the following: c) α Ｖ β ３ / α Ｖ β ５ Penton integrin, which prevents virus binding to integrins The D342E or D342A mutation in the RGD binding motif at least one of the mutations; and d) A20 peptide sequence NAVPNLRGDL in the viral fiber knob HI loop Insertion or expression of QVLAQKVART (SEQ ID NO: 1) 9. The modified adenovirus according to any one of claims 1 to 8, comprising:

10. The modified adenovirus (Ad5.3D.A20) a) I4 in hexon hypervariable region 7, which prevents viral binding to coagulation factor 10 (FX) 21G, T423N, E424S and L426Y point mutations (HVR7 mutations); b) Prevents viral binding to the Coxsackievirus / Adenovirus Receptor (CAR) The S408E and P409A point mutations in the AB loop of the fiber knob region (KO1 spontaneous mutations); c) RGE mutations that prevent viral binding to αVβ3 / αVβ5 integrins D342 of the penton integrin binding motif Arg-Gly-Asp (RGD) E point mutation; and d) A20 peptide sequence NAVPNLRGDL in the viral fiber knob HI loop Insertion or expression of QVLAQKVART (SEQ ID NO: 1) The modified adenovirus of claim 8 .

11. The adenovirus carries at least one transgene encoding a therapeutic molecule or drug. The modified adenovirus of any one of claims 1 to 10, further modified to include virus.

12. The adenovirus contains a 24 base pair nucleotide sequence to restrict viral replication to pRB-deficient cells. Further modified to include the deletion dl922-947 (Δ24 mutation) in the E1A gene A modified adenovirus according to any one of claims 1 to 11,

13. The adenovirus is designed to retain E3 / 19K in the endoplasmic reticulum (ER) to enhance its oncolytic activity. It was further modified to contain a single adenine base addition (T1 mutation) at position 445 within the carrier domain. A modified adenovirus according to any one of claims 1 to 12, which is modified.

14. A modified adenovirus according to any one of claims 1 to 13 for use as a medicament. Russ.

15. A modified adenosine monophosphate esterase (Aa ) of any one of claims 1 to 14 for use in the treatment of cancer. Ilse.

16. 15. Any one of claims 1 to 14 for use in the manufacture of a medicament for treating cancer. The modified adenovirus described in.

17. The cancer is nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, cancer of the connective tissue, melanoma, Lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer r), oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma , pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Meier syndrome Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer , ureteral cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal tumor, bone cancer, osteocarcinoma Osteoma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, Fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, biliary Bladder cancer, Head cancer, Eye cancer, Neck cancer, Kidney cancer, Wilms' tumor, Liver cancer, Kaposi's tumor tumors, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer cancer), skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagon pancreatic cancer, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine Cancer, stomach cancer, thymic cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, Vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, soma Tostatinoma, gum cancer, heart cancer, lip cancer, meningeal cancer, mouth cancer , nerve cancer, palate cancer, parotid cancer, peritoneal cancer, pharynx cancer, pleural cancer 17. The method according to claim 15 or 16, further comprising administering to the patient a cancer of the present invention, which is selected from the group consisting of salivary gland cancer, tongue cancer and tonsil cancer. A modified adenovirus as described herein.

18. The cancer is ovarian cancer, pancreatic cancer, esophageal cancer, lung cancer, cervical cancer, head and neck cancer, oral cancer, selected from the group including cancer, laryngeal cancer, skin cancer, breast cancer, kidney cancer and colorectal cancer The modified adenovirus of claim 17.

19. A modified adenovirus according to any one of claims 1 to 13, and a pharma- ceutically acceptable The pharmaceutical composition comprises a carrier, adjuvant, diluent or excipient as described above.

20. A method for preparing a pharmaceutical composition comprising the steps of: The adenovirus is combined with a pharma- ceutical or veterinarily acceptable carrier or vehicle, or The method comprises bringing into association.

21. A method for treating cancer comprising administering an effective amount of a modification according to any one of claims 1 to 13.

20. The method of claim 19, further comprising administering the adenovirus or the pharmaceutical composition of claim 19 to a patient in need thereof. The method includes:

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