Modified adenovirus

Abstract

The present invention relates to a modified oncolytic adenovirus of serotype Ad5; a pharmaceutical composition comprising the same; and a method for treating cancer using the same, wherein the modified adenovirus comprises at least one point mutation in hexon hypervariable region 7 (HVR7 mutation) that prevents viral binding to coagulation factor 10 (FX); at least one point mutation in the fiber knob region AB loop (KO1 mutation) that prevents viral binding to the coxsackievirus-adenovirus receptor (CAR); and at least one point mutation in the penton integrin binding motif Arg-Gly-Asp (RGD) that prevents viral binding to ανβ3 / ανβ5 integrin. [Selection diagram] Figure 7

Description

[Technical Field]

[0001] The present invention relates to a modified oncolytic adenovirus of serotype Ad5; a pharmaceutical composition comprising the adenovirus; and a method for treating cancer using the adenovirus, wherein the modified adenovirus comprises at least one point mutation in the hexon hypervariable region 7 (HVR7 mutation) that prevents viral binding to coagulation factor X (FX); at least one point mutation in the fiber knob region AB loop (KO1 mutation) that prevents viral binding to the coxsackievirus adenovirus receptor (CAR); and at least one point mutation in the penton integrin binding motif Arg-Gly-Asp (RGD) that prevents viral binding to αVβ3 / αVβ5 integrin. [Background technology]

[0002] Cancer viral therapy is an emerging field, with the herpes simplex virus type 1-based tarimodine-laharpalebeck (T-VEC) being the first oncolytic immunotherapy for advanced melanoma. Oncolytic adenoviruses are highly immunogenic viruses often used in various vaccine approaches to infectious diseases. Importantly, they possess an extraordinary ability to stimulate and enhance the immune response. Furthermore, the presence of oncolytic adenoviruses within tumors and the immunogenic cell death they induce can shape the tumor microenvironment toward a more susceptible state toward clinically relevant antitumor immunity by triggering the expression of TH1-type immune modulators such as IFNg. However, the immunogenicity of oncolytic adenoviruses is a double-edged sword; the antiviral immunity is often too overwhelming, thus neutralizing the much weaker immune response induced against autoantigens expressed by the tumor.

[0003] Adenovirus 5 (Ad5) is a common vector deployed in numerous clinical trials of cancer and gene therapy (clinicaltrials.gov, 2016) due to its genetically engineerable nature and tolerance for large transgenes. However, this serotype has several suboptimal features that hinder its widespread clinical use. Ad5 is a common respiratory virus with a seroprevalence of nearly 100% in certain populations, and neutralizing antibodies (nAbs) rapidly inactivate systemically delivered therapeutic vectors. Other suboptimal features include extensive off-target sequestration to the spleen and liver via cross-linking of the viral hexone protein with heparan sulfate proteoglycan (HSPG), and tropic directing interactions mediated by human coagulation factor X (FX). In vitro, Ad5 enters host cells via interactions between the viral fiber protein and the coxsackievirus adenovirus receptor (CAR), which is ubiquitously expressed within tight junctions on polarized epithelial cells but downregulated in progressive cancer. Subsequently, Ad5 is translocated into host cells via αvβ3 / 5 integrins through clathrin-mediated endocytosis mediated by viral penton-based proteins.

[0004] The perception of the role of oncolytic viruses in cancer treatment has changed dramatically over the past decade, as immunotherapy and stimulating the patient's own immune system to target and attack cancer have gained popularity. At the beginning of the century, oncolytic viruses were recognized as active agents in cancer treatment, acting solely through their intrinsic ability to lyse tumor cells via oncolysis. In recent years, their use as cancer vaccines has attracted attention, and their ability to release tumor antigens from cancer cells upon oncolysis to activate the immune system is recognized as a key feature in designing the ultimate immunotherapy against cancer. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, while viral vectors are becoming increasingly attractive in clinical settings, the clinical efficacy of common serotypes—Ad5—is hindered by poor tumor specificity, widespread off-target delivery, and inactivation by innate immunity, necessitating innovative manipulation strategies. [Means for solving the problem]

[0006] This specification discloses a modified adenovirus vector designed to overcome the aforementioned drawbacks.

[0007] According to a first aspect of the present invention, a) At least one of the following point mutations in the hexon hypervariable region 7 (HVR7 mutations) that prevents viral binding to coagulation factor X (FX); b) at least one of the S408E or P409A point mutations (KO1 mutations) in the fiber knob region AB loop that interfere with viral binding to the coxsackievirus adenovirus receptor (CAR); and c)α V β3 / α V At least one of the D342E or D342A point mutations in the penton integrin-binding motif Arg-Gly-Asp(RGD) that prevents viral binding to β5 integrin. A modified Ad5 serotype adenovirus containing the above is provided.

[0008] The modified Ad5 serotype adenovirus according to the present invention is advantageous because its ability to infect off-target tissues is severely impaired, and in fact, its ability to infect the liver and spleen is prevented / inhibited, and its ability to infect a wide range of body cells is also compromised. Thus, the modified adenovirus is advantageous in terms of the tissues it can infect.

[0009] Adenoviruses are medium-sized (90-100 nm) non-enveloped viruses (lacking an outer lipid bilayer) that possess an icosahedral nucleocapsid containing a double-stranded DNA genome. In humans, there are 57 commonly recognized human adenovirus serotypes (Ad-1 to 57) classified into seven species (human adenovirus A to G): A-12, 18, 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-40, 41; and G-52. Therefore, references to Ad5 in this specification refer to adenovirus serotype 5, which belongs to the C subclass of adenovirus.

[0010] As is known to those skilled in the art, an adenovirus virion consists of a non-enveloped icosahedral capsid surrounding a viral DNA-protein core complex. Hexon is the most abundant structural protein, with 240 copies of trimer molecules per capsid. Twelve copies of hexon trimers form each of the 20 triangular facets of the capsid. A complex of penton base and fiber proteins seals the vertices of the capsid and facilitates viral attachment (fiber) and internal migration (penton base).

[0011] Hexon proteins are highly conserved across various adenovirus serotypes, with the exception of nine hypervariable regions (HVRs). These HVRs reside in two distinct loops that form the exposed surface of the hexon protein: HVR1-6 are located in the DE1 loop, and HVR7-9 are located in the FG1 loop.

[0012] Therefore, references to at least one mutation in HVR7 in this specification refer to mutations in hypervariable region 7. Specifically, the at least one HVR7 mutation interferes with the interaction with coagulation factor X, thereby limiting off-target sequester of the modified adenovirus to the liver and improving targeting to target cancer cells.

[0013] In a preferred embodiment, the at least one HVR7 mutation includes at least one amino substitution mutation that interferes with FX interaction, selected from one or more of the group including I421G, T423N, E424S, E450Q, or L426Y. Most preferably, the at least one HVR7 mutation includes, additionally or alternatively, at least one of the I421G, T423N, E424S, and L426Y point mutations.

[0014] As is known to those skilled in the art, adenovirus vectors possess innate tropisms that result in the broad distribution of the vector. The entry of group C adenoviruses, such as Ad5, into cells is thought to involve high-affinity binding of the virus to the cell through the interaction of viral fiber proteins with coxsackievirus adenovirus receptors (CARs). Therefore, targeting adenovirus vectors to specific tissues or cell types for anti-cancer therapy purposes requires modification of the vector's normal tropisms to improve specificity.

[0015] Adenovirus infection begins with the recognition of host cell receptors by a special protein on the viral surface, namely the adenovirus fiber protein, and in particular, the globular carboxy-terminal domain of the adenovirus fiber protein, known as the carboxy-terminal knob domain. Therefore, references to the knob of the adenovirus fiber protein herein refer to the globular carboxy-terminal domain of the adenovirus fiber protein.

[0016] Therefore, reference to at least one KO1 mutation refers to at least one mutation in the AB loop of the fiber knob region. Specifically, said at least one KO1 mutation prevents viral binding to the CAR. In a preferred embodiment, said at least one KO1 mutation comprises at least one point mutation selected from one or more of the group comprising S408E or P409A that prevents CAR binding. Most preferably, said point mutation comprises S408E and P409A point mutations.

[0017] Adenovirus penton base contains five Arg-Gly-Asp sequences and binds to integrin alpha v beta 3 and alpha v beta 5 (α V β3 / α V β5) to promote virus infection by enabling virus internalization. By interfering with this interaction, off-site targeting to the spleen is reduced, thereby promoting tumor-specific targeting, and further, when used in the context of anti-cancer therapy, it has been found that the release of inflammatory cytokines that would otherwise result in a harmful immune host response is attenuated.

[0018] Therefore, reference to at least one RGD mutation refers to at least at least one mutation (RGD mutation) of the penton integrin binding motif Arg-Gly-Asp that prevents viral binding to α V β3 / α V β5 integrin. In a preferred embodiment, said at least one RGD mutation comprises at least one point mutation selected from one or more of the group comprising D342E or D342A that result in RGE or RGA, respectively. Most preferably, said point mutation is D342E that results in RGE.

[0019] In a more preferred embodiment, the adenovirus is further modified to include at least one cancer-targeting modification that selectively targets tumor cells, particularly certain types of tumor cells. As will be understood by those skilled in the art, through the incorporation of the mutations described herein to alter the innate tropism of the adenovirus, and also through the introduction of at least one targeting modification / sequence, the modified virus has improved tumor specificity and reduced off-site targeting, thereby reducing unwanted side effects. Examples of cancer-targeted modifications / sequences are known to those skilled in the art and include, but are not limited to, NGR (containing) peptides that bind to aminopeptidase N, particularly adenoviruses (Ad) having NGR in the HI loop of adenovirus fiber proteins; YSA (containing) peptides that bind to the pan-cancer marker EphA2, particularly adenoviruses having YSA in chimeric fibers that result in potent transduction of EphA2-positive cancer cells but not transduction of EphA2-negative cancer cells; growth factor antibodies, particularly those chemically bound to these targeted moieties, e.g., bFGF, EGFR, antibodies against adenoviruses using avidin / biotin conjugations, e.g., cetuximab, Herceptin, Avastin); and matrix-degrading enzymes that, once attached to the outside of the virus (typically chemically bound, e.g., via hyaluronidase), degrade the extracellular membrane, allowing the virus to penetrate the tumor microenvironment more efficiently.

[0020] In a preferred embodiment, the cancer targeting modification comprises the insertion or expression, either viral or by the virus, of an αvβ6 integrin binding peptide, such as the A20 peptide sequence NAVPNLRGDLQVLAQKVART (SEQ ID NO: 1), identified using conventional techniques such as sequence binding and homology techniques, preferably into 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 a high affinity for αvβ6 integrin. αvβ6 integrin is expressed in one-third of ovarian cancers and various other epithelial cancers and is not detectable in healthy adult tissues. Thus, as will be understood 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, esophageal cancer, lung cancer, cervical cancer, head and neck cancer, oral cancer, laryngeal cancer, skin cancer, breast cancer, kidney cancer, and colorectal cancer.

[0021] Those skilled in the art will understand that homologs, orthologues, or functional derivatives of the listed mutations also find applications in the context of the present invention. Therefore, mutations involving, for example, one or more additions, deletions, substitutions, etc., are encompassed within the present invention. In addition, it may be possible to replace one amino acid with another amino acid of a similar "type." For example, replacing one hydrophobic amino acid with another can be achieved by comparing amino acid sequences using a program such as the CLUSTAL program. This program finds the optimal alignment by comparing amino acid sequences and inserting spaces in either sequence as needed. For optimal alignment, it is possible to calculate the identity or similarity of amino acids (identity meaning conservation of amino acid type). Programs such as BLASTx align the longest similar sequences and assign values ​​to the fit. Thus, it is possible to obtain a comparison in which several regions of similarity are found, each with a different score. Both types of analysis are intended in the present invention.

[0022] In a more preferred embodiment, the modified adenovirus vector (Ad5.3D.A20) is: a) At least one of the following point mutations in the hexon hypervariable region 7 (HVR7 mutations) that prevents viral binding to coagulation factor X (FX); b) At least one of the S408E or P409A point mutations (KO1 mutations) in the fiber knob region AB loop that prevents viral binding to the coxsackievirus adenovirus receptor (CAR); c)α V β3 / α V At least one of the D342E or D342A point mutations in the penton integrin-binding motif Arg-Gly-Asp(RGD) that prevents viral binding to β5 integrin; and d) Insertion or expression of the A20 peptide sequence NAVPNLRGDLQVLAQKVART (SEQ ID NO: 1) in a viral fiber knob HI loop. Includes.

[0023] More preferably, the modified adenovirus vector (Ad5.3D.A20) a) I421G, T423N, E424S, and L426Y point mutations in the hexon hypervariable region 7 (HVR7 mutations) that prevent viral binding to coagulation factor X (FX); b) S408E and P409A point mutations (KO1 mutations) in the fiber knob region AB loop that prevent viral binding to the coxsackievirus adenovirus receptor (CAR); c) A D342E point mutation in the penton integrin binding motif Arg-Gly-Asp (resulting in an RGE mutation) that prevents viral binding to αVβ3 / αVβ5 integrins; and d) Insertion or expression of the A20 peptide sequence NAVPNLRGDLQVLAQKVART (SEQ ID NO: 1) in a viral fiber knob HI loop. Includes.

[0024] In the viral vector of the present invention, the inventors successfully excised the innate tropism of Ad5 by mutating the major capsid proteins: hexon, fiber, and penton. A triple-detargeted Ad5 vector skeleton was constructed (Ad5.3D) by modifying the hexon hypervariable region 7 (HVR7 mutation), the fiber knob AB loop (KO1 mutation), and the penton integrin-binding motif Arg-Gly-Asp (RGD mutation), including substitution mutations of amino acid residues responsible for binding to coagulation factor X (FX), the coxsackievirus adenovirus receptor (CAR), and αvβ3 / 5 integrin, respectively.

[0025] The Ad5.3D vector was constructed using homologous recombination technology and delivered with high viral titers using complementary cell lines designed to rescue damaged adenoviruses. A combination of three tropic excision mutations completely blocked cell entry via αvβ3 / 5 integrins, CAR, and FX in vitro. Furthermore, as proof of principle, we further modified the adenovirus (Ad5.3D.A20) by incorporating a viral fiber knob of a heterologous αvβ6 integrin-binding peptide (A20, NAVPNLRGDLQVLAQKVART; SEQ ID NO: 1) into the HI loop, thereby preferentially targeting specific cancer tumor cells, namely αvβ6, and incorporating an additional targeting sequence for infection. The Ad5.3D.A20 adenovirus was grown using 296-β6 cells designed to overexpress β6.

[0026] αvβ6 integrin is expressed in one-third of ovarian cancers and various other epithelial cancers, and is undetectable in healthy adult tissue. Ad5.3D.A20 efficiently transduced αvβ6+ ovarian cancer cell lines and primary clinical ascites-derived EOC exovivo cultures, even in the presence of highly neutralized ascites. The in vivo in vivo distribution profile of Ad5.3D.A20 after systemic delivery in non-tumor-bearing mice was significantly altered compared to Ad5, with decreased luciferase expression in all off-target organs and a 7-log reduction in viral genomic load in the liver. Furthermore, the antitumor efficacy of oncolytic Ad5.3D.A20 (OAd5.3D.A20) after intraperitoneal delivery was evaluated in immunodeficient mice carrying intraperitoneal SKOV3(-β6)EOC xenografts. Oncolytic treatment with OAd5.3D.A20 improved overall survival compared to Ad5 treatment.

[0027] Therefore, the Ad5.3D modified adenovirus is a viral vector that provides an exciting targeting platform for cancer treatment and adenovirus therapy through the incorporation of further cancer-specific targeting mutations.

[0028] In a more preferred embodiment, the adenovirus is further modified to include at least one transgene encoding a molecule or drug, such as a therapeutic agent, though not limited to these. Thus, this embodiment relates to intracellular drug delivery for exerting a therapeutic effect on targeted cancer cells. Examples of drugs include drugs that directly stimulate the immune response, e.g., GM-CSF, IL-12; drugs that indirectly stimulate the immune system, e.g., antibodies (or fragments of antibodies) encoding immune checkpoint inhibitors that inhibit corepressors such as CTLA-4, PD-L1, PD1, or Lag3; bispecific T cell-binding (BiTE) antibody constructs; bispecific natural killer cell-binding (BiKE) antibody constructs; drugs that sensitize tumors to cell-based immunotherapy, e.g., encoding CD19; drugs that deplete regulatory T cells in the tumor microenvironment, e.g., encoding anti-CD25 antibodies; and drugs that sensitize tumors to radiotherapy or imaging by encoding, e.g., sodium / iodine cotransporter (NIS) or somatostatin receptor 2 (SSTR2). Alternatively, the transgene may encode a therapeutic agent that is directly toxic to tumor cells, for example, by encoding the transgene Reduced Expression in Immortalized Cells (REIC / DKK3), or by encoding an enzyme that sensitizes cancer cells through the conversion of a non-toxic prodrug into a toxic drug, such as cytosine deaminase, nitroreductase, or thymidine kinase. Other transgenes known in the art and useful for treating cancer can be used in carrying out the present invention.

[0029] In a more preferred embodiment, the adenovirus is further modified to include a 24-base pair deletion dl922~947 (Δ24 mutation) in the E1A gene in order to restrict viral replication to pRB-deficient cells.

[0030] More preferably, the adenovirus is further modified to include a single adenine base addition (T1 mutation) at position 445 within the endoplasmic reticulum (ER) retaining domain of E3 / 19K in order to enhance its oncolytic ability.

[0031] According to a second aspect of the present invention, a modified adenovirus as defined herein is provided for use as a pharmaceutical.

[0032] According to a third aspect of the present invention, a modified adenovirus as defined herein is provided for use in the treatment of cancer.

[0033] A fourth aspect of the present invention provides a modified adenovirus as defined herein for use in the manufacture of a pharmaceutical product for treating cancer.

[0034] Most preferably, the cancers referred to herein include one or more of the following cancers: nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, connective tissue cancer, 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, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary origin, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer. Rectal cancer, esophageal cancer, 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 lymphoma, oral cancer, 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, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, heart cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

[0035] Compounds for use in pharmaceuticals are generally provided in pharmaceutical or veterinary compositions, and therefore, according to a further fifth aspect of the present invention, a pharmaceutical composition comprising an adenovirus as defined herein and a pharmaceutically acceptable carrier, adjuvant, diluent or excipient is provided.

[0036] Suitable pharmaceutical excipients are well known to those skilled in the art. Pharmaceutical compositions can be formulated for administration via any suitable route, e.g., orally, buccally, nasally or bronchially (inhalation), transdermally or parenterally, and can be prepared by any method well known in the field of pharmacy.

[0037] The composition can be prepared by associating the adenovirus defined above with a carrier. Generally, formulations are prepared by homogeneously and closely associating the adenovirus with a liquid carrier, a fine powder solid carrier, or both, and then, if necessary, shaping the product. The present invention relates to a method for preparing a pharmaceutical composition, comprising the step of combining or associating the adenovirus defined above with a pharmaceutically or veterinarily acceptable carrier or vehicle.

[0038] A further aspect of the present invention provides a method for treating cancer, comprising the step of administering an effective amount of a modified adenovirus or pharmaceutical composition as defined herein to a patient in need.

[0039] In this specification, the “effective dose” of adenovirus or a composition containing adenovirus is an amount sufficient to achieve the desired biological effect, such as cancer cell death. It is understood that the effective dose depends on the recipient’s age, sex, health condition and weight, the type of concomitant therapy if any, the frequency of treatment, and the nature of the desired effect. Typically, the effective dose is determined by the person administering the treatment.

[0040] In the following claims and the above description of the invention, the word “comprises,” or variations such as “comprises” or “comprising,” is used in a comprehensive sense, that is, to specify the presence of the described features, but without precluding the presence or addition of further features in the various embodiments of the invention, unless the context otherwise requires explicit language or necessary implications.

[0041] All references, including any patents or patent applications, cited herein are incorporated herein by reference. No reference is permitted to constitute prior art. Furthermore, no prior art is permitted to constitute part of the general knowledge in the art.

[0042] Preferred features of each aspect of the present invention may be described in relation to any of the other aspects.

[0043] Other features of the present invention will become apparent from the following examples. Generally speaking, the present invention extends to any novel features or any novel combination of features disclosed herein (including the appended claims and drawings). Accordingly, any features, integers, properties, compounds or chemical parts described in conjunction with a particular aspect, embodiment or example of the present invention should be understood to be applicable to any other aspect, embodiment or example described herein, insofar as they do not conflict.

[0044] Furthermore, unless otherwise specified, features disclosed herein may be replaced by alternative features that serve the same or similar purposes.

[0045] Throughout this specification and in the claims, unless otherwise specified in the context, the singular form encompasses the plural form. In particular, where the indefinite article is used, the specification should be understood as intending both plural and singular forms, unless otherwise specified in the context.

[0046] Herein, embodiments of the present invention will be described merely as examples with reference to the following. [Brief explanation of the drawing]

[0047] [Figure 1] The constructed vectors. (A) A figure showing the viral titer and expected tropism of the Ad5 and triple detargeted avβ6 integrin retargeting vector, Ad5.3D.A20. (B) A figure showing the vector map of oncolytic Ad5.3D.A20. (C) A figure showing comparative predictive 3D modeling of a modified Ad5.3D.A20 fiber knob with an A20 peptide (NAVPNLRGDLQVLAQKVART; SEQ ID NO: 1) insertion in the adenovirus serotype 5 (Ad5) fiber knob and HI loop (green). CAR, coxsackievirus adenovirus receptor; FX, coagulation factor X; HVR7, FX binding mutation in hexon hypervariable region 7; KO1, CAR binding mutation in fiber knob AB loop; Luc, luciferase transgene; vp, viral particle. [Figure 2] Innate receptor-tropic excision. (A) Figure showing the 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) Figure showing the binding of replication-deficient Ad5 and HVR7 mutant Ad5 variants to coagulation factor X (FX0 was evaluated by luciferase assay by infecting cells with (+) or (-) human FX containing the anticoagulant X-bp at 37°C for 3 hours). HVR7, FX-binding mutant. Statistical significance: ns, p ≤ 0.05; **, p < 0.01. [Figure 3]In vitro evaluation of αvβ6 integrin targeting. (A) Figure showing the transduction efficiency of replication-deficient wild-type (Ad5) and triple detargeting, integrin retargeting (Ad5.3D.A20) vectors in αvβ6+ BT-20 breast cancer cells. (B) Figure showing the transduction efficiency of replication-deficient wild-type (Ad5) vector and triple detargeting, integrin retargeting (Ad5.3D.A20) vectors in αvβ6+ primary epithelial ovarian cancer (EOC) cells from patient 004. (C) Figure showing luciferase expression by oncolytic vector (T1 / Δ24) in infected avβ6-low / CAR+SKOV3 and avβ6-high / CAR+SKOV3-β6 cells (proprietary SKOV3 cells with retroviral expression of avβ6). (D) Figure showing competitive inhibition of αvβ6 integrin-mediated cell entry. The best 10% αvβ6-expressing SKOV3-β6 cells were selected by FACS, subcultured, and infected. IgG, normal mouse IgG control; 10D5, anti-αvβ6 functional blocking antibody. The ratio of viral transgene expression is shown above the bar. Statistical significance: ns, p>0.05;*, p<0.05;**, p<0.01;***, p<0.001;****, p<0.0001. [Figure 4] Effect of vector transduction in malignant ovarian ascites via ex vivo. (A) Figure showing quantification of anti-Ad5 antibodies in 20 clinical ovarian ascites (OAS) samples and control serum (black solid line) from healthy male volunteers by ELISA. Horizontal lines indicate 50% and 100% binding of anti-Ad5 ab in control serum. (B) Figure showing antigen specificity of anti-Ad5 antibodies in ascites and serum by Western blotting. (C) Figure showing vector transduction efficiency of replication-deficient (Ad5) vector and Ad5.3D.A20 vector in the absence and presence of various dilutions of ascites from ovarian cancer patient 004 in BT-20 cells. (D) Figure showing vector transduction efficiency of replication-deficient (Ad5) vector and Ad5.3D.A20 vector 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 pre-incubated and infected with increasing concentrations of ascites. [Figure 5] In vivo distribution of replication-deficient vectors 72 hours after systemic delivery. (A) Figure showing the in vivo distribution study schedule. (B) Figure showing in vivo imaging of replication-deficient (Ad5) virus and triple-detargeted Ad5.3D.A20 virus 3 days after intravenous injection. (C) Figure showing quantification of total luminescence signals from panel B throughout the body. (D) Figure showing quantification of total luminescence signals from panel B in the liver, 335. (E) Figure showing quantification of total luminescence signals from panel B in the spleen. (F) Figure showing quantification of total luminescence signals from panel B in the lungs. (G) Figure showing quantification of total luminescence signals from panel B in the ovaries. (H) Figure showing quantification of total luminescence signals from panel B in the heart. ip, intraperitoneal; IVIS, in vivo imaging system; pi, post-infection; vp, viral particle. Error bars represent the 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) Figure showing the adenovirus genome copy number from the liver, excised in Figure 5, as determined by qPCR for the hexon gene after systemic vector delivery. (B) Figure showing the adenovirus genome copy number from the spleen, excised in Figure 5, as determined by qPCR for the hexon gene after systemic vector delivery. (C) Figure showing the adenovirus genome copy number from the lung, excised in Figure 5, as determined by qPCR for the hexon gene after systemic vector delivery. (D) Figure showing the adenovirus genome copy number from the ovary, excised in Figure 5, as determined by qPCR for the hexon gene after systemic vector delivery. (E) Figure showing the adenovirus genome copy number from the heart, excised in Figure 5, as determined 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 the 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. The numbers below the graph show the reduction ratio of the Ad5.3D.A20 group compared to the Ad5 group. [Figure 7]Oncolytic efficacy study: Intraperitoneal delivery of oncolytic vectors in an ovarian cancer xenograft model. (A) Figure shows the study schedule. Intraperitoneal xenografts of human ovarian cancer cells (SKOV3 and SKOV3-6) were transplanted into immunodeficient mice (n=5 / group) and then treated with three doses of intravenous oncolytic Ad5 or triple detargeting, integrin retargeting Ad5.3D.A20 on days 14, 16, and 18. (B) Figure shows emission heatmap image measured with 349 48 hours after the first treatment (day 16). (C) Figure shows quantification of whole-body emission measured with 349 48 hours after the first treatment (day 16). (D) Figure shows emission heatmap image measured with 349 7 days after the first treatment (day 21). (E) Figure shows quantification of whole-body emission measured with 349 7 days after the first treatment (day 21). (F) This figure shows the overall survival rate of animals inoculated with SKOV3(αvβ6-low / CAR+) cells and then treated with the virus, as shown above, as a Kaplan-Meier survival curve, up to the final trial endpoint at 101 days. (G) This figure shows the overall survival rate of animals inoculated with SKOV3-β6(αvβ6-high / CAR+) cells and then treated with the virus, as shown above, as a Kaplan-Meier survival curve, up to the final trial endpoint at 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]In vivo distribution study: Heatmap images of luminescence intensity in ex vivo. (A) Figure showing liver, spleen, lung, ovary, and heart collected immediately postmortem from animals intravenously inoculated with PBS (control). (B) Figure showing liver, spleen, lung, ovary, and heart collected immediately postmortem from animals intravenously inoculated with the Ad5.Luc vector. (C) Figure showing liver, spleen, lung, ovary, and heart collected immediately postmortem from animals intravenously inoculated with the Ad5.3D.A20 vector. Organs were immersed in D-luciferin and imaged with an IVIS imager. Tissue color indicates the relative luminescence intensity emitted by the luciferase transgene; scale normalized to exclude background luminescence. (D) Figure showing the reduction ratio of total luminescence (photons / second) compared to Ad5.Luc. The mean luminescence intensity of the Ad5.Luc group was divided by the mean luminescence of each organ in the Ad5.3D.A20 group. The mean of the PBS control group was subtracted from all values. [Figure 9] Biodistribution study: Immunohistochemistry of formalin-fixed, paraffin-embedded liver sections. (A) Figure showing hematoxylin-eosin staining for visualization of cellular structure, as well as 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) Figure showing staining of Ad5-infected hepatocytes from animals infected with Ad5 vector or Ad5.3D.A20 vector using primary rabbit anti-Ad5 antibody (1 μg / mL). Sections were counterstained with hematoxylin using DAB as a substrate, mounted on coverslips, and observed under a light microscope. [Figure 10] Pilot study: This figure shows tumor localization and uptake rates in NOD / SCID mice. 1 × 10⁷ SKOV3-β6 cells / animal were transplanted as an iP on day 0, and two mice were sacrificed at each time point: days 7, 14, 21, and 48 / 49 (final endpoint). Approximate tumor size was measured, and ascites volume was quantified. [Figure 11]Oncolytic efficacy study: Characterization of endpoint tumors. (A) Figure showing viral genome copy number (per 40 ng of DNA) in postmortem tumors of OAd5 and OAd5.3D.A20 from SKOV3 and SKOV3-β6 cohorts by qPCR. (B) Figure showing 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 compared to mouse number 1 from the negative control PBS group in the SKOV3 cohort, using human ACTB (β-actin) as an endogenous control. [Figure 12] Transduction activity of Ad5.3D.A20 and Ad5-expressing luciferases in pancreatic cancer cell lines. (A) Figure showing the expression levels of αvβ6 and hCAR determined in the ASPC-1 pancreatic cancer cell line. (B) Figure showing the expression levels of αvβ6 and hCAR determined in the BxPc pancreatic cancer cell line. (C) Figure showing the expression levels of αvβ6 and hCAR determined in the CFPAC pancreatic cancer cell line. (D) Figure showing the expression levels of αvβ6 and hCAR determined in the PANC10-05 pancreatic cancer cell line. (E) Figure showing the expression levels of αvβ6 and hCAR determined in the SW1990 pancreatic cancer cell line. (F) Figure showing the expression levels of αvβ6 and hCAR determined in the PANC0403 pancreatic cancer cell line. (G) Figure showing the expression levels of αvβ6 and hCAR determined in the SUIT-2 pancreatic cancer cell line. (H) Figure showing the expression levels of αvβ6 and hCAR determined in the MiPaCa2 pancreatic cancer cell line. (I) This figure shows the expression levels of αvβ6 and hCAR determined in the PT45 pancreatic cancer cell line. Cells were infected with 5,000 vp / cell viral luciferase, and transgene expression was quantified 48 hours after infection, with corrections made for total cell protein. [Figure 13]This figure shows the transduction activity of Ad5.3D.A20 and Ad5-expressing luciferases in esophageal cancer cell lines. αvβ6 and hCAR expression levels were determined in Kyse-30 esophageal cancer cells. Cells were infected with 5,000 vp / cell viral luciferase, and transgene expression was quantified 48 hours post-infection, corrected for total cellular protein. [Figure 14] Transduction activity of Ad5.3D.A20 and Ad5-expressing luciferases in breast cancer cell lines. (A) Figure showing the expression levels of αvβ6 and hCAR determined in BT-20 breast cancer cells. (B) Figure showing the expression levels of αvβ6 and hCAR determined in BT-474 breast cancer cells. (C) Figure showing the expression levels of αvβ6 and hCAR determined in MDA-MB-361 breast cancer cells. (D) Figure showing the expression levels of αvβ6 and hCAR determined in MDA-MB-231 breast cancer cells. Cells were infected with 5,000 vp / cell viral luciferase, and transgene expression was quantified 48 hours post-infection, corrected for total cellular protein. [Figure 15] Transduction activity of Ad5.3D.A20 and Ad5-expressing luciferases in lung cancer cell lines. (A) Figure showing the expression levels of αvβ6 and hCAR determined in A427 lung cancer cells. (B) Figure showing the expression levels of αvβ6 and hCAR determined in A549 lung cancer cells. (C) Figure showing the expression levels of αvβ6 and hCAR determined in NCI-H460 lung cancer cells. Cells were infected with 5,000 vp / cell viral luciferase, and transgene expression was quantified 48 hours post-infection, corrected for total cellular protein. [Figure 16]This figure shows the replication defects and oncolytic activity of (O)Ad5.3D.A20 and Ad5 in pancreatic cancer cell lines and breast cancer cell lines. Pancreatic cancer cell lines Suit 2 (αvβ6high / hCARhigh), MiCaPa2 (αvβ6low / hCARhigh), PANC0403 (αvβ6vvhigh / hCARhigh), and PT45 (αvβ6neg / hCARhigh), as well as breast cancer cell lines BT-20 (αvβ6high / hCARneg) and MDA-MB-231 (αvβ6neg / hCARhigh) were seeded 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 did not show any adverse effects on cell viability, but the cytotoxic (tumor-lysic) activity of OAd5.3D.A20 and OAd5 was directly related to the presence or absence of cellular αvβ6 and hCARs. [Modes for carrying out the invention]

[0048] material and method Adenovirus vectors, cell lines, and clinical ascites All constructed vectors expressed luciferase (Luc) and were based on a wild-type Ad5 genome captured on a bacterial artificial chromosome (BAC). As previously described (Uusi-Kerttula et al., 2016), all genetic modifications were introduced into the BAC by AdZ homologous recombination (Stanton et al., 2008). The virus was produced in T-Rex 293 or HEK293-β6 cells (A20 modified virus). The replication-deficient vector has an E1 / E3 gene deletion, while the oncolytic vector has a 24-base pair deletion dl922-947(Δ24) in the E1A gene (Fueyo et al., 2000) to restrict viral replication to pRB-deficient cells (Sherr, 1996), and a single adenine base addition, which is a T1 mutation at position 445 in the endoplasmic reticulum (ER)-retaining domain of E3 / 19K (Gros et al., 2008). A heterologous A20 peptide sequence from FMDV (NAVPNLRGDLQVLAQKVART; SEQ ID NO: 1) was genetically inserted into the fiber knob HI loop. High-titer viruses were produced in T-REx-293 cells or HEK293-β6 cells, essentially as previously described (Uusi-Kerttula et al., 2015, Uusi-Kerttula et al., 2016).

[0049] The SKOV3-β6 cell line was self-generated. A puromycin-selective pBABE-β6 plasmid with a β6 gene insertion (number 13596; Addgene) was transfected into a 293 Phoenix packaging cell line using Effectene. After 48 hours, the retrovirus was collected, 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 fluid was granted through a Wales Cancer Bank application for biomaterials (see WCB 14 / 004). All patients gave written informed consent before collection. Ascites clinical samples were collected and anonymized from patients being treated for advanced ovarian cancer at the Velindre Cancer Centre in Cardiff. Cells were processed and subcultured as previously described (Uusi-Kerttula et al., 2015; Uusi-Kerttula et al., 2016).

[0050] In vitro assay Cell surface receptor expression was assessed by flow cytometry using anti-αvβ6 clone 10D5 and anti-CAR antibody clone RmcB, followed by secondary F(ab')2 goat α-mouse IgG(H+L)IgG AlexaFluor647, as previously described (Uusi-Kerttula et al., 2016). The presence of anti-Ad5 antibodies in ovarian ascites and serum was determined according to essentially previously reported ELISA methods (Stallwood et al., 2000). Antigen specificity of the antibodies was assessed by Western blotting.

[0051] The in vitro transduction efficiency was evaluated by a luciferase reporter gene assay using a multimode plate reader essentially as previously described (Uusi-Kerttula et al., 2015, Uusi-Kerttula et al., 2016), and the relative light units (RLU) were normalized to the total protein concentration (RLU / mg) of each well. To evaluate the effect of FX on transduction efficiency, the transduction medium was supplemented with 10 μg / mL of human FX. The vector tropism of cell receptors was evaluated by a competitive inhibition assay as previously described (Uusi-Kerttula et al., 2016) using an anti-αvβ6 antibody (10 μg / mL; clone 10D5, Millipore) or normal anti-mouse control IgG (10 μg / mL; Santa Cruz). The neutralization assay included a pre-incubation step with serial two-fold dilutions of cell-free OAS (1:40 to 1:2.5, corresponding to final concentrations of 2.5 to 40%).

[0052] In vivo study All animal experiments were performed at the Mayo Clinic in Rochester, USA. To maintain consistency in the results, all animals were 7 weeks old and sex-matched; female mice were selected for ease of breeding. The handling and injection of all animals were performed by Mrs. Jill M. Thompson, an experienced veterinary technician, in accordance with local regulations.

[0053] Due to the feasibility of luciferase tracking in white fur, the in vivo distribution study of the replication-deficient vector was performed in wild-type B6 albino mice (B6N-Tyr c-Brd / BrdCrCrl) (n = 5 / group). The virus was injected into the lateral tail vein at 1 × 10 11 vp. All mice were sacrificed by CO2 inhalation after IVIS imaging 72 hours post-infection, and the organs were excised for analysis. The efficacy study was performed in immunodeficient NOD / SCID mice (n = 5 / group). The treatment schedule was first optimized in a pilot study (n = 8). 1 × 10 7SKOV3-β6 cells were transplanted as an i-infusion on day 0, and two mice were sacrificed on days 7, 14, 21, and 48 / 49 (final endpoint). CAR and αvβ6 expression in the tumors at each time point was evaluated by flow cytometry. In the oncolytic efficacy study, NOD / SCID mice were injected with 1 × 10¹⁶ cells on day 0. 7 Individual SKOV3 cells or SKOV3-β6 cells were xenotransplanted into mice (iP). Then, mice (n=5 / group) were 1×10⁶ on days 14, 16, and 18. 10 Patients were treated with intravenous injections of vp OAd (PBS, OAd5, and OAd5.3D.A20). The primary endpoint was overall survival (%). Vector uptake was monitored by quantifying the luminescence signal released by the luciferase transgene using a Xenogen IVIS 200 imager (PerkinElmer). Viral genome copy number in major off-target organs and endpoint tumors was quantified by qPCR. The level of αvβ6 gene expression in endpoint tumors was quantified by qPCR.

[0054] A concise protocol for cell viability assays For the cell viability assay, the CellTiter 96 AQueous One Solution cell proliferation assay (Promega) was used, following the manufacturer's recommended protocol. 20,000 or 30,000 cells were seeded in each well of a 96-well plate and incubated overnight. Cells were infected with 5,000 virus particles per cell (vp / cell) in serum-free medium for 3 hours. Viability was determined by adding 20 μl of CellTiter 96 AQueous One Solution reagent per well at 24, 48, 72, 96, and 144 hours post-infection. After incubation for 2 hours in a humidified 5% CO2 atmosphere, absorbance was measured at 490 nm. The percentage of viable cells relative to untreated cells was calculated. Results are mean, n=3, and error bars represent the standard deviation.

[0055] statistical analysis All numerical and statistical analyses were performed using GraphPad Prism version 6.03. In vitro and exovivo assays were analyzed by two-sided unpaired t-tests or one-way ANOVA and Dunnett's multiple comparison post-hoc test. In vivo data were normalized and analyzed by one-way ANOVA and Sidak's multiple comparison post-hoc test. Overall survival (%) after tumor lysis is shown as Kaplan-Meier survival curves; survival rates were analyzed by Gehan-Breslow-Wilcoxon test. All trials: ns, p>0.05;*, p<0.05;**, p<0.01;***p<0.001;****p<0.0001.

[0056] result We constructed and produced high-viral-titer replication-deficient and oncolytic variants of a novel Ad5.3D.A20 vector containing three detargeting mutations and an A20 peptide insertion that retargets the vector to αvβ6 integrin-expressing cells (Figure 1). Multiple genetic manipulations did not significantly affect titer. Predictive modeling on the SWISS147 MODEL platform showed A20 peptide prominence within the immune-dominant HI loop (Figure 1C).

[0057] The transduction efficiency of replication-deficient vectors was evaluated in cell lines expressing variable amounts of CAR and αvβ6 integrin. Detargeting mutations in Ad5.3D.A20 completely abolished CAR-mediated entry in CHO-CAR cells (CAR+), but Ad5 efficiently transduced these cells (Figure 2A). HVR7 mutations abolished FX-mediated vector transduction (Figure 2B). As expected, FX significantly increased Ad5 transduction into these cells compared to culture conditions without FX (Figure 2B; right panel). Conversely, the addition of human FX to the culture medium had no effect on the transduction efficiency of the FX-bound excised Ad5.HVR7 control vector in CHO-K1 cells (Figure 2B; left panel). Furthermore, the transduction enhancement observed for Ad5 was reversed by the addition of the anticoagulant X-bp, a Gla domain interacting protein that binds to and inactivates FX in a 3:1 molar excess in the culture medium (Figure 2B, right panel). In contrast, FX depletion did not affect the transduction of the Ad5.HVR7 vector (Figure 2B, left panel).

[0058] αvβ6 integrin was identified as the primary entry receptor for triple detargeting and integrin retargeting Ad5.3D.A20 (Figure 3). Compared to Ad5, Ad5.3D.A20 transduced αvβ6+ / CAR-BT-20 breast cancer cells with 305-fold higher efficiency (Figure 3A; p=0.0270) and transduced primary EOC004 cells (αvβ6+ / CAR-) with 69-fold increased efficiency (Figure 3B; p=0.0090). In addition, the oncolytic variant of the Ad5.3D.A20 vector transduced SKOV3-β6 cells (αvβ6-high / CAR+) with approximately 5-fold increased efficiency compared to SKOV3 cells expressing low levels of αvβ6 (αvβ6-low / CAR+, Figure 3C; p<0.0001), confirming that the oncolytic modification did not impair the A20 peptide:αvβ6 interaction. A competitive assay using an anti-αvβ6 antibody (10D5) significantly inhibited transduction by the Ad5.3D.A20 vector (169 Figure 3D; p=0.0010), confirming the selectivity for αvβ6.

[0059] Clinical ovarian ascites (OAS) samples from 20 patients were screened for the presence of anti-Ad5 antibodies by ELISA. The titer of anti-Ad5 ab in malignant ovarian ascites was examined against the serum anti-Ad5 antibody titer of healthy adult male volunteers (Figure 4A). A similar proportion of patients were found to have lower and higher antibody titers than the control serum (Figure 4A, black dotted line). Ascites from patient 001 (OAS001) was selected for the subsequent neutralization assay because its antibody titer was similar to that of the control serum. Antibodies in OAS001 and the control serum appeared to be specific to fiber proteins, but the most abundant capsid protein hexone was recognized only at very low levels by Western blotting using denatured viral particles (Figure 4B). The neutralizing effect of OAS001 on the transduction efficiency of Ad5.3D.A20 was evaluated in αvβ6+ / CAR-EOC004 primary cells. Ad5.3D.A20 showed superior transduction efficiency (up to 902 times higher) compared to Ad5 at OAS concentrations of 2.5, 5, and 10%, while Ad5 did not transduce these cells to a detectable level (Figure 4C).

[0060] The vectors were intravenously administered to non-tumor-carrying mice, and the effects of three detargeting mutations on the vector's tropism, particularly its in vivo distribution, were evaluated (Figure 5A). Ad5 showed strong localization to the liver and spleen, but luminescence from the Ad5.3D.A20 vector was undetectable at 72 hours (Figure 5B). Animals inoculated with Ad5 showed significantly higher whole-body luminescence than control animals treated with PBS (p<0.0001) or Ad5.3D.A20 (p<0.0001) (Figure 5C). The liver, spleen, lungs, ovaries, and heart were excised, and ex vivo luminescence was quantified (see Figures 8A-8C for luminescence heatmaps). The livers of Ad5-loaded animals showed significantly stronger luminescence than the PBS control or Ad5.3D.A20 groups (both p<0.0001) (Figure 5D). Similarly, Ad5.3D.A20 significantly reduced transgene expression in the spleen, lungs, ovaries, and heart compared to Ad5 (Figures 5E-5H; p<0.0001 in all cases). See Figure 8D for the magnification changes in luminescence intensity in each organ.

[0061] Modification of Ad5.3D.A20 confirmed a 196-fold reduction in viral isolation in multiple normal tissues, confirmed by quantification of off-target organ viral load by qPCR. The Ad5.3D.A20 genome copy number was 10 million times lower in the liver compared to Ad5 (Figure 6A; p<0.0001). Similarly, the Ad5.3D.A20 genome copy number was more than 700 times lower in the spleen compared to Ad5 (Figure 6B; p<0.0001). In addition, the Ad5.3D.A20 vector showed improved off-target profiles in all organs compared to Ad5, with viral loads reduced by 10 times in the lungs, heart, and ovaries, respectively. 5 , 10 4 , 10 3 The levels were low (Figures 6C-6E). The success of liver detargeting was attributed to Ad5 gene modification, as evidenced by immunohistochemical staining of liver sections showing high CAR expression levels, while αvβ6 was undetectable (Figure 9A). The detargeting effect of Ad5.3D.A20 gene modification is confirmed by the observation that liver sections of mice in the Ad5 group showed positive staining for Ad capsid protein, but livers of mice loaded with the Ad5.3D.A20 vector did not (Figure 9B).

[0062] To evaluate the efficacy of αvβ6 retargeting in an in vivo cancer model, αvβ6-high / CAR-SKOV3-β6 human ovarian cancer xenografts were established in immunodeficient NOD / SCID mice. Within 14 days of intraperitoneal transplantation of SKOV3-β6 cells, the animals developed large solid tumors at the cell injection site and various locations within the peritoneal cavity, and by day 49, the tumors had spread throughout the peritoneal cavity, accumulating large amounts of ascites. The tumors retained high αvβ6 expression (see Figure 10 for flow cytometry). Based on these observations, a viral therapy efficacy study was conducted by delivering oncolytic variants of Ad5 and Ad5.3D.A20 intravenously three times on days 14, 16, and 18 post-transplantation of αvβ6-low / CAR-SKOV3 and αvβ6-high / CAR-SKOV3-β6.

[0063] IVIS imaging 48 hours after the initial viral therapy administration (day 16) showed widespread luminescence throughout the abdominal region of animals treated with the oncolytic Ad5 vector, with the highest intensity in the liver / spleen region in both SKOV3 and SKOV3-β6 xenograft models (Figure 7B). This distribution was maintained, but the intensity decreased after 5 days, on day 21 (Figure 7D). In contrast, the Ad5.3D.A20 223 vector showed highly selective localization, consistent with successful detargeting of non-tumor tissues, and resulted in significantly reduced overall luminescence compared to Ad5. In both SKOV3 and SKOV3-β6 models, quantitative whole-body luminescence indicated that Ad5.3D.A20 vector uptake was significantly lower than Ad5 at both day 16 (Figure 7C; p<0.05 and <0.01, respectively) and day 21 (Figure 7E; p<0.0001).

[0064] Antitumor activity was observed for both oncolytic Ad5 and oncolytic Ad5.3D.A20 in the SKOV3 xenograft model (Figure 7F). Consistent with the enhanced tumor-selective effect of Ad5.3D.A20, all five mice treated with Ad5.3D.A20 were still alive at the final 101-day mark, while animals treated with Ad5 survived for a maximum of only 70 days.

[0065] Additional transduction assays were performed on a series of cancer cell lines originating from the pancreas (Figure 12), esophagus (Figure 13), breast (Figure 14), and lung (Figure 15). All cell types were first analyzed for αvβ6 and hCAR expression (histograms in each figure). 7 / 9 pancreatic cell lines (ASPC-1, BxPc, CFPAC, PANC 10.05, SW1990, PANC 0403, and Suit2) expressed αvβ6 at varying levels and were efficiently transducible with Ad5.3D.A20 (Figures 12A-12G). Conversely, as expected, MiPaCa2 (Figure 12H) and PT45 (Figure 12I) cells expressed αvβ6 at very low or no levels and were less tolerant of Ad5.3D.A20-mediated transduction. The esophageal cell line Kyse-30 expressed high levels of αvβ6 and was highly tolerant of Ad5.3D.A20-mediated transduction (Figure 13). Of the breast cancer cell lines tested, three of the four cell lines (BT-20, BT-474, and MDA-MB361) expressed αvβ6 to varying degrees and were tolerant of Ad5.3D.A20-mediated transduction (Figures 14A-14C). Conversely, in MDA-MB-231 cells, the lack of αvβ6 expression made the cells non-infectious to Ad5.3D.A20 (Figure 14D). In all three lung cancer cell lines tested (A427, A549, and NCI-H460, Figures 15A-15C), αvβ6 was absent, and the cells were refractive against Ad5.3D.A20-mediated transduction.

[0066] To evaluate the cytotoxic activity of the oncolytic version of Ad5.3D.A20, αvβ6 high (Suit2, Panc0403) and αvβ6 low (MiPaCa2) or αvβ6 negA cell viability assay was performed using the (PT45) pancreatic cancer cell line (Figure 16). Cells were infected with either replication-deficient Ad5 or Ad5.3D.A20, or oncolytic (O)Ad5 or Ad5.3D.A20, at a rate of 5,000 vp / cell. As expected, replication-deficient vectors did not mediate a significant effect on cell viability, while the cytotoxic activity of oncolytic vectors was shown to correlate well with αvβ6 expression. Similarly, αvβ6 high / hCAR neg In the triple-negative breast cancer cell line BT-20, the combination of high levels of αvβ6 and the absence of hCAR allowed only OAd5.3D.A20 to efficiently mediate cell killing. Conversely, αvβ6 neg / hCAR high In the breast cancer cell line MDA-MB-231, the presence of hCAR and the absence of αvβ6 allowed only OAd5 to efficiently kill cells.

[0067] Consideration This paper describes Ad5.3D.A20, a novel tumor-selective oncolytic adenovirus vector in which all known tropisms have been resected and retargeted to the overexpressed prognostic cancer marker αvβ6 integrin. Integrin αvβ6 is a promising target for therapeutic cancer applications because it is expressed in aggressively transformed cancers.

[0068] In this study, a replication-deficient variant of the Ad5.3D.A20 vector successfully detargeted cellular viral uptake via the innate viral uptake pathway (Figure 2) and instead selectively retargeted αvβ6+ cells in vitro and ex vivo (Figure 3). While interactions that limit the efficacy of systemic delivery of adenovirus vectors can theoretically be bypassed by intracavitary administration of the vector via ip administration, this approach presents challenges in practice because wild-type Ad5 is sequestered by anti-Ad5 nAbs in ascites fluid. Therefore, we evaluated the transduction efficiency of Ad5.3D.A20 in the presence of OAS containing high levels of existing anti-Ad5 nAbs (Figure 4A). Unlike Ad5, Ad5.3D.A20 retained its ability to transduce αvβ6+ cells even at relatively high OAS concentrations (Figure 4C).

[0069] The clinical efficacy of Ad5 vectors with unmodified capsids is also significantly limited by off-target tissue isolation, particularly in the liver. We demonstrate that Ad5.3D.A20 successfully alters the in vivo distribution of the Ad5 vector in vivo. In tumor-free mice, replication-deficient Ad5.3D.A20 showed improved in vivo distribution compared to parental Ad5, with significantly reduced viral transgene expression in the liver, spleen, and lungs (Figure 5), and reduced viral genome copy number in all 274 off-target organs compared to Ad5 (Figure 6).

[0070] To test the efficacy of the oncolytic form of the detargeted / retargeted Ad5.3D.A20 vector, an orthotopic iP xenograft model of human ovarian cancer was established in immunodeficient mice. The more localized in vivo distribution of viral encoding transgene expression of oncolytic Ad5.3D.A20 after intraperitoneal administration was consistent with reduced off-target isolation and / or tumor-selective viral uptake (Figures 7B–7E). This was supported by superior survival rates of animals treated with Ad5.3D.A20 compared to Ad5 in an SKOV3 xenograft model (Figure 7F).

[0071] Administration of Ad5.3D or Ad5.3D.A20, respectively, offers promising treatment options for advanced chemotherapy-resistant or αvβ6+ cancers, particularly ovarian, pancreatic, esophageal, and breast cancers, though not exclusively. This vector provides a crucial platform that can ultimately be modified for highly accurate viral therapy applications.

[0072] References FUEYO,J.,GOMEZ-MANZANO,C.,ALEMANY,R.,LEE,PSY,MCDONNELL,TJ,MITLIANGA,P.,SHI,YX,LEVIN,VA,YUNG,WKA&KYRITSIS,AP2000.A mutant oncolytic adenovirus targeting the Rb pathway producing anti-glioma effect in vivo.Oncogene,19,2-12. GROS, A., MARTINEZ-QUINTANILLA, J., PUIG, C., GUEDAN, S., MOLLEVI, DG, ALEMANY, R. & CASCALLO, M. 2008. Bioselection of a gain of functional mutation that enhances adenovirus 5 release and improves its antitumoral potency. Cancer Research, 68, 8928-8937. SHERR,CJ1996.Cancer cell cycles.Science,274,1672-1674. STALLWOOD, Y., FISHER, KD, GALLIMORE, PH&MAUTNER, V.2000. Neutralization of adenovirus infectivity by ascitic fluid from ovarian cancer patients.Gene Therapy,7,637-643. STANTON,R.J.,MCSHARRY,B.P.,ARMSTRONG,M.,TOMASEC,P.&WILKINSON,G.W.G.2008.Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.BioTechniques,45,659-668. UUSI-KERTTULA,H.,DAVIES,J.,COUGHLAN,L.,HULIN-CURTIS,S.,JONES,R.,HANNA,L.,CHESTER,J.D.&PARKER,A.L.2016.Pseudotyped alphavbeta6 integrin-targeted adenovirus vectors for ovarian cancer therapies.Oncotarget. UUSI-KERTTULA,H.,LEGUT,M.,DAVIES,J.,JONES,R.,HUDSON,E.,HANNA,L.,STANTON,R.J.,CHESTER,J.D.&PARKER,A.L.2015.Incorporation of Peptides Targeting EGFR and FGFR1 into the Adenoviral Fiber Knob Domain and Their Evaluation as Targeted Cancer Therapies.Hum Gene Ther,26,320-9.

Claims

1. A modified Ad5 serotype adenovirus, which includes mutations to alter the natural tropism of the adenovirus, Here, mutation occurs, a) I421G, T423N, E424S, E450Q, and L426Y point mutations in the hexon hypervariable region 7 (HVR7 mutations) that prevent viral binding to coagulation factor X (FX); b) S408E and P409A point mutations (KO1 mutations) in the fiber knob region AB loop that prevent viral binding to the coxsackievirus adenovirus receptor (CAR); and c) α Ｖ β ３ / α Ｖ β ５ A D342E point mutation in the penton integrin-binding motif Arg-Gly-Asp (RGD) that prevents viral binding to integrins. It consists of, Furthermore, the adenovirus is further modified to include at least one cancer-targeting modification or sequence that selectively targets tumor cells. Modified Ad5 serotype adenovirus.

2. The modified adenovirus according to claim 1, wherein the adenovirus comprises at least one NGR (containing) peptide motif that binds to aminopeptidase N, wherein the NGR is located in the HI loop of the adenovirus fiber protein; or in at least one YSA (containing) peptide motif that binds to the pan-cancer marker EphA2, wherein the YSA is located in the chimeric fiber, or in at least one cancer-targeting antibody, or at least one growth factor antibody, or at least one matrix-degrading enzyme.

3. The modified adenovirus according to claim 1, wherein the cancer-targeting modification comprises insertion into or by the virus of an αvβ6 integrin-binding peptide or an A20 peptide sequence NAVPNLRGDLQVLAQKVART (SEQ ID NO: 1).

4. The modified adenovirus according to claim 3, wherein the A20 peptide sequence is inserted into or expressed in the viral fiber knob HI loop.

5. Modified adenovirus (Ad5.3D.A20) a) I421G, T423N, E424S, E450Q, and L426Y point mutations in the hexon hypervariable region 7 (HVR7 mutations) that prevent viral binding to coagulation factor X (FX); b) S408E and P409A point mutations (KO1 mutations) in the fiber knob region AB loop that prevent viral binding to the coxsackievirus adenovirus receptor (CAR); and c) A D342E point mutation in the penton integrin-binding motif Arg-Gly-Asp (RGD) that results in an RGE mutation that prevents viral binding to αVβ3 / αVβ5 integrins; This includes mutations to alter the natural tropism of adenoviruses, Furthermore, the adenovirus is further modified to include at least one cancer-targeting modification or sequence that selectively targets tumor cells, comprising the insertion or expression of the A20 peptide sequence NAVPNLRGDLQVLAQKVART (SEQ ID NO: 1) in the viral fiber knob HI loop. A modified adenovirus according to any one of claims 1 to 4.

6. The modified adenovirus according to any one of claims 1 to 5, wherein the adenovirus is further modified to include at least one transgene encoding a therapeutic molecule or drug.

7. The modified adenovirus according to any one of claims 1 to 6, wherein the adenovirus is further modified to include a 24-base pair deletion dl922-947 (Δ24 mutation) in the E1A gene in order to restrict viral replication to pRB-deficient cells.

8. The modified adenovirus according to any one of claims 1 to 7, wherein the adenovirus is further modified to include a single adenine base addition (T1 mutation) at position 445 in the E3 / 19K endoplasmic reticulum (ER) retaining domain in order to enhance its oncolytic ability.

9. A modified adenovirus according to any one of claims 1 to 8, for use as a pharmaceutical.

10. A modified adenovirus according to any one of claims 1 to 9, for use in the treatment of cancer.

11. A modified adenovirus according to any one of claims 1 to 9, for use in the manufacture of a pharmaceutical product for treating cancer.

12. The aforementioned cancers include nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, kidney cancer, connective tissue cancer, 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, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary origin, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, cervical cancer, and kidney cancer. Visceral cancer, Wilms' tumor, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine and pancreatic cancer, glucagonoma, 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, somatostatinoma, gingival cancer, heart cancer, lip cancer, meningeal cancer, oral cancer (mouth) A modified adenovirus according to claim 10 or 11, selected from the group including cancer, nerve cancer, palatine cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer (pharynx cancer), pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

13. The modified adenovirus according to claim 12, wherein the cancer is selected from the group including ovarian cancer, pancreatic cancer, esophageal cancer, lung cancer, cervical cancer, head and neck cancer, oral cancer, laryngeal cancer, skin cancer, breast cancer, kidney cancer, and colorectal cancer.

14. A pharmaceutical composition comprising a modified adenovirus according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier, adjuvant, diluent, or excipient.

15. A method for preparing a pharmaceutical composition, comprising combining or associating a modified adenovirus according to any one of claims 1 to 8 with a pharmaceutically or veterinarily acceptable carrier or vehicle.

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