Replication-enhanced oncolytic adenovirus

Abstract

Replication-promoting oncolytic adenoviruses are disclosed. These oncolytic adenoviruses have tumor-specific replication that can promote tumor lysis and promote the expression of therapeutic transgenes. Also disclosed are methods for patients suffering from cancer, including administering the replication-promoting oncolytic adenoviruses.

Description

Technical Field

[0001] Joint Research Agreement This invention was made as a result of activities performed within the scope of a joint research agreement that was in effect at the time the invention was made. The parties to the joint research agreement are Memgen, Inc. (formerly Memgen, LLC) and H. Lee Moffitt Cancer Center and Research Institute, Inc.

[0002] 1. Field of the Invention The present invention generally relates to the fields of oncolytic viruses and oncology. More particularly, the present invention relates to compositions of oncolytic viruses in which viral replication is enhanced in tumor cells for the treatment of cancer.

Background Art

[0003] 2. Description of Related Art Oncolytic viruses are a type of cancer therapeutic agent having a dual mechanism of action: 1) killing of tumor cells by direct oncolysis resulting from selective viral replication in tumor cells, and 2) induction of systemic antitumor immunity by releasing antigens from destroyed tumor cells. Both natural viruses and genetically modified viruses are under development. The U.S. FDA approved in 2015 the first oncolytic virus, talimogene laherparepvec (IMLYGIC®, Amgen Inc., Thousand Oaks, CA), a genetically modified herpesvirus encoding granulocyte macrophage colony-stimulating factor (GM-CSF) for the local treatment of melanoma, as described by Kohlhapp et al., 2016 Clinical Cancer Research. However, herpesvirus is just one of many oncolytic viruses being studied with respect to oncolytic properties.

[0004] Oncolytic adenovirus vectors are widely used in cancer gene therapy for several reasons, such as broad affinity for the infection of multiple types of tumors, vector stability, the ability to produce and purify the virus to high titers, the ability to carry transgenes, lack of integration into the host genome, and good safety.

[0005] Despite these desirable attributes of oncolytic adenovirus vectors, these vectors can sometimes be restricted in their use for cancer therapy for many reasons, such as non-specific infection and replication in normal cells, virus replication rate caused by anti-adenovirus immunogenicity, vector clearance, and deceleration or reduction of neutralization, as well as low oncolytic ability. These limitations can also contribute to inefficient gene introduction or expression when used as a delivery vehicle.

[0006] To address these limitations, efforts have been made to improve replication-competent adenoviruses. These efforts include modification of adenovirus genome components to confer selective replication in tumor cells. Well-characterized modifications include deletion of 24 base pairs of the adenovirus E1A gene (delta-24 E1A), deletion of the E1B 55K viral gene, and substitution of viral promoters such as E1A with tumor-associated antigen promoters such as the alpha-fetoprotein promoter or prostate antigen promoter.

[0007] Despite these examples of selective tumor targeting, there is a lack of recombinant adenovirus vectors in the prior art that replicate at a substantially improved rate or level in tumor cells.

[0008] Therefore, there is still a need for an oncolytic virus vector that improves one of its main mechanisms of action, namely selectively promoting virus replication in tumor cells to result in improved oncolysis. The present invention addresses this need for an oncolytic adenovirus vector that promotes virus replication in tumor cells, improves oncolysis, and improves the expression of therapeutic transgenes.

[0009] All of the topics discussed in the background are not necessarily prior art, and it should not be assumed that they are prior art only based on the results of the discussions in the background section. In line with these concepts, any recognition of prior art problems discussed in the background or related to such topics should not be treated as prior art unless it is explicitly stated as such. Instead, any discussion of a topic in the background should be treated as part of the inventors' efforts towards a specific problem and may itself be inventive.

Prior Art Documents

Patent Documents

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Patent Document 1

Non-Patent Documents

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Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

[0012] The following presents a simplified summary of the present disclosure to provide a basic understanding of some aspects of the present disclosure. This summary is not a complete overview of the disclosure. It is not intended to identify key or critical elements of the disclosure or to delineate the scope of the disclosure. Its sole purpose is to present some concepts in a simplified form as a prelude to a more detailed description that will be discussed later.

[0013] The inventors have surprisingly found that inserting two exogenous promoter / enhancer elements (CMV promoter / enhancer and SV40 early promoter / enhancer) into the adenovirus genome, along with the aforementioned modifications of the adenovirus E1A, E1B, and E3 gene components, dramatically promotes virus replication and oncolysis in tumor cells. Further, the inventors have surprisingly found that this newly described recombinant adenovirus functions as a vehicle for delivering one or more therapeutic transgenes and dramatically enhances the expression of these transgenes in tumor cells. In the generation of recombinant oncolytic adenoviruses, either the CMV promoter / enhancer or the SV40 promoter / enhancer has been individually used alone, but the inventors are not aware of any prior art describing the dual use of promoters in oncolytic viruses, nor are they aware of any prior art describing the unexpected finding that this dual-promoter oncolytic adenovirus results in enhanced tumor-specific virus replication.

[0014] In some embodiments, the disclosure relates to an oncolytic adenovirus having a viral genome modification that confers enhanced virus replication.

[0015] In some embodiments, the recombinant oncolytic adenovirus contains both a CMV promoter / enhancer and an SV40 promoter / enhancer inserted into the viral genome.

[0016] In some embodiments, the recombinant oncolytic adenovirus contains both a CMV promoter / enhancer and an SV40 promoter / enhancer inserted into the viral genome, has a partial or complete deletion of the E3 coding region, has an E1A gene containing a delta-24 deletion, and contains an E1B 55K deletion.

[0017] In a preferred embodiment, the recombinant oncolytic adenovirus contains a CMV promoter / enhancer inserted into the non-coding region upstream of the adenovirus E1 coding region, contains an SV40 promoter / enhancer inserted into the adenovirus E3 region, a part or all of the E3 coding region is deleted, has an E1A gene containing a delta-24 deletion, and contains an E1B 55K deletion.

[0018] A further embodiment of the present invention includes a heterologous nucleic acid sequence encoding one or more therapeutic proteins.

[0019] In some embodiments, the present disclosure provides an oncolytic adenovirus having enhanced virus replication specific to tumor cells.

[0020] In a further embodiment, the present invention provides a method for treating a malignant tumor, preferably cancer, by administering a replication-promoting recombinant oncolytic adenovirus.

[0021] Details of one or more embodiments are set forth in the following description. Features exemplified or described in connection with one exemplary embodiment can be combined with features of other embodiments. Accordingly, any combination of the various embodiments described herein can be provided to form further embodiments. Aspects of the embodiments can be modified, as necessary, using concepts from various patents, applications, and publications as specified herein to provide further embodiments. Other features, objects, and advantages will be apparent from the description, drawings, and claims. The present disclosure can be understood by reference to the following description in conjunction with the accompanying drawings.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0023] Brief Description of the Sequences SEQ ID NO: 1 shows the nucleotide sequence of the CMV promoter / enhancer inserted between the left - terminal inverted repeat (ITR) and the 5' end of the functional E1A region.

[0024] Array number 2 shows the nucleotide sequence of the SV40 early promoter / enhancer inserted between E3 12.5K and the E3 RID-alpha region.

[0025] Various exemplary embodiments of the present disclosure will be described below. For the sake of clarity, not all features of actual implementations are described herein. Of course, in the development of any such actual implementation, it will be understood that numerous implementation-specific decisions need to be made in order to achieve the developer's specific goals, such as compliance with system-related and business-related constraints that will vary depending on the implementation. Further, although such development efforts may be complex and time-consuming, it will be understood that they would be routine work for those skilled in the art having the benefit of the present disclosure.

[0026] The problems disclosed herein are easy to make various modifications and take alternative forms, but the specific embodiments are shown in the drawings by way of example and described in detail herein. However, the description herein of the specific embodiments is not intended to limit the present disclosure to the specific forms disclosed, but rather, on the contrary, is intended to cover all modifications, equivalents, and alternatives that fall within the spirit and scope of the present disclosure as defined by the appended claims.

[0027] Adenovirus An "adenovirus" (Ad) is a large (about 36 kb) DNA virus that infects humans and also exhibits a broad host range. Physically, adenoviruses are icosahedral viruses and contain a double-stranded linear DNA genome. There are approximately 57 serotypes of human adenoviruses, which are classified into six families based on molecular, immunological, and functional criteria. By adulthood, virtually all humans have been infected with the more common adenovirus serotypes, and the main effects are symptoms such as colds.

[0028] As a result of adenovirus infection of host cells, adenovirus DNA is maintained as an episome, with low genotoxicity that can occur in relation to vector integration. Furthermore, adenoviruses are structurally stable, and no genomic rearrangements have been detected even after large-scale amplification. Adenoviruses can infect most epithelial cells regardless of the stage of the cell cycle. So far, adenovirus infection seems to be associated only with mild diseases such as human acute respiratory diseases.

[0029] Oncolytic adenovirus There is a wide variety of oncolytic viruses being developed as anticancer agents that include adenoviruses (see Russell et al., 2014 Nature Biotechnology and Lawler et al., 2017 JAMA Oncology).

[0030] In the selection or design of therapeutic oncolytic adenoviruses for desired therapeutic activity, selective targeting of cancer cells to be infected, through the natural tropism of cell surface proteins or manipulation of adenoviruses to directly target cancer cells, selective replication in cancer cells, attenuation of viral pathogenicity, promotion of lytic activity, modification of antiviral immune responses that can cause rapid clearance of adenoviruses, and modification of systemic antitumor immunity by genetic modification of adenoviruses to incorporate cytokines, immune agonists, or immune checkpoint inhibitors, among other biological properties, can be considered.

[0031] Replication-competent oncolytic adenovirus vectors have several properties that are ideal for therapeutic applications, such as infectivity for a wide range of cells and tumor types, infection of non-dividing cells, lack of genomic integration, high titers, the ability to carry transgenes, stability in vitro and in vivo, and expression of transgenes. Adenovirus expression vectors include constructs that (a) assist in packaging the construct and (b) contain sufficient adenovirus sequences to ultimately express the recombinant gene construct cloned therein, if necessary.

[0032] Regulation of the biological properties of oncolytic adenoviruses can affect a series of immune interactions that may be beneficial or harmful to the effect on cancer treatment. This interaction depends on a specific tumor, the site and degree of the disease, the immunosuppressive tumor microenvironment, the oncolytic virus platform, dosage, time, and delivery conditions, as well as the response of individual patients (generally, see Aurelian L., "Oncolytic viruses as immunotherapy: progress and remaining challenges," Onco. Targets Ther. 2016, 9:2627-2637). For example, the presence of the adenovirus E3 gene has been reported to increase the oncolytic ability of adenoviruses to replicate in vitro and in vivo depending on the conditions (see Suzuki K, Alemany R, Yamamoto M and Curiel DT, "The presence of the adenovirus E3 region improves the oncolytic potency of conditionally replicative adenoviruses" Clin. Cancer Res. 2002 Nov., 8(11):3348-59). In particular, the adenovirus death protein (ADP) of E3-11.6 kDa is considered necessary for efficient cell death (see Tollefson A, Ryerse J, and Scaria A et al., "The E3-11.6-kDa Adenovirus Death Protein (ADP) is Required for Efficient Cell Death: Characterization of Cells Infected with adp Mutants" Virology 1996, 220:152-162).

[0033] Notwithstanding these aforementioned advancements, in the prior art, there is not enough of an adenoviral vector that replicates at a substantially improved rate or level in tumor cells to effectuate viral replication, which causes tumor lysis, which is one of the main mechanisms of action of oncolytic adenoviruses. Thus, there remains a need for an oncolytic viral vector that selectively promotes viral replication in tumor cells, thereby resulting in an improvement in oncolysis, which improves this main mechanism of action.

[0034] The present disclosure provides such oncolytic adenoviruses. The inventors have surprisingly found that inserting two exogenous promoter / enhancer elements (CMV promoter / enhancer and SV40 promoter / enhancer) at defined positions of the adenoviral genome, along with the modification of the aforementioned adenoviral E1A, E1B, and E3 gene components, dramatically promotes viral replication in tumor cells and promotes oncolysis (Figure 2).

[0035] Furthermore, the inventors have surprisingly found that this newly described recombinant adenovirus functions as an ideal vehicle for delivering one or more therapeutic transgenes and can promote the expression of these transgenes in tumor cells. Any member of 57 human adenovirus serotypes (HAdV-1 to 57) can incorporate a heterologous nucleic acid encoding a therapeutic protein. Human Ad5 is well genetically and biochemically characterized (GenBank M73260; AC000008). Thus, in certain embodiments, the oncolytic adenovirus is a replication-competent Ad5 serotype or a hybrid serotype containing Ad5 components. Within some embodiments of the present disclosure, one or more heterologous sequences can be incorporated into non-essential regions of the adenovirus. Within some embodiments of the present disclosure, one or more heterologous sequences can be incorporated downstream of the CMV promoter / enhancer and / or downstream of the SV40 promoter / enhancer. Representative examples of therapeutic proteins encoded by these heterologous genes include cytokines, chemokines, antibodies, and checkpoint inhibitors.

[0036] Promoter / Enhancer Many promoters function with heterologous enhancer elements separated by DNA. The CMV and SV40 "promoters" described herein constitute both enhancer and promoter elements. The enhancers of the CMV and SV40 "promoters" can function to increase the expression of adenoviral gene promoters in a manner typical of enhancer action, which can be a mechanism for increased replication.

[0037] Heterologous nucleic acid expression may be under the control of a promoter that functions in mammalian cells, preferably human tumor cells. In one embodiment, the promoter that directs the expression of the heterologous nucleic acid encoding the therapeutic protein is the cytomegalovirus (CMV) promoter / enhancer. In a further embodiment, the promoter that directs the expression of the heterologous nucleic acid encoding the therapeutic protein is the SV40 promoter / enhancer.

[0038] The invention provided in this specification includes two promoters / enhancers having the ability to drive the expression of heterologous nucleic acids encoding proteins. A promoter generally includes a sequence that functions to identify the position of the start site of RNA synthesis. The best example of this is the TATA box, but in some promoters lacking the TATA box, such as the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 early gene (preferred embodiments of the present invention), a separate element covering the start site itself serves to fix the starting location. Additional promoter elements regulate the frequency of transcription initiation. These are usually in the region 30 to 110 bp upstream of the start site, but promoters have been shown to contain functional elements downstream of the start site as well. One identifies the position at the 5' end of the transcription start site of the transcription reading frame "downstream" (i.e., 3' of it) of a selected promoter in order to bring about a coding sequence "under the control" of the promoter. The "upstream" promoter stimulates the transcription of DNA and promotes the expression of the encoded RNA.

[0039] Since the spacing between promoter elements is often flexible, the promoter function is maintained even if the elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased to 50 bp intervals before the activity begins to decline. Depending on the promoter, the individual elements appear to be able to function either cooperatively or independently to activate transcription. A promoter may or may not be used in combination with an "enhancer", which means a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. In a preferred embodiment of the present invention, the CMV promoter incorporated into a replication-promoting adenovirus is used in combination with the CMV enhancer region. In an even more preferred embodiment of the present invention, the SV40 promoter incorporated into a replication-promoting adenovirus is used in combination with the SV40 enhancer region.

[0040] A promoter may originally be associated with a nucleic acid sequence and can be obtained by isolating a 5' non-coding sequence located upstream of a coding segment and / or exon. Such a promoter can be referred to as "endogenous". Similarly, an enhancer may also be associated with a nucleic acid sequence and can be located either downstream or upstream of that sequence. Alternatively, certain advantages will be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which usually means a promoter that is not associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also usually means an enhancer that is not associated with a nucleic acid sequence in its natural environment. Such a promoter or enhancer can include the promoter or enhancer of other genes, and the promoter or enhancer isolated from any other adenovirus or prokaryotic or eukaryotic cell, and a promoter or enhancer that is not "naturally occurring", that is, contains different elements of different transcriptional regulatory regions and / or mutations that change expression.

[0041] It may be important to use a promoter and / or enhancer that effectively directs the expression of a DNA segment in an organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology generally know the use of promoters, enhancers, and combinations of cell types for protein expression. The promoter used can be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions for directing high-level expression of the introduced DNA segment, for example, advantageous in the large-scale production of recombinant proteins and / or peptides. This promoter may be heterologous or endogenous. The inventors have surprisingly found that simultaneous inclusion of both the CMV promoter / enhancer upstream of the E1A delta-24 region and the SV40 promoter / enhancer in a partially deleted region of the E3 region results in at least a 10- to 100-fold dramatic increase in tumor-specific viral replication and tumor-specific oncolysis compared to a control virus lacking the characteristics of the dual promoter.

[0042] Method for screening adenoviruses for therapeutic utility The oncolytic adenoviruses of the present disclosure, or variants or derivatives thereof, can be evaluated for their therapeutic utility by examining their lytic ability in tumor cells. The tumor cells can include primary tumor cells obtained from a patient's biopsy or surgical resection. Alternatively, the tumor cells can include a tumor cell line. The cytolytic activity of the adenoviruses of the present disclosure can be measured in vitro in a tumor cell line by infecting the cells with serial dilutions of the adenovirus and measuring the cytolytic ability (i.e., IC 50 ). Specific methods for measuring cytolytic activity include, but are not limited to, MTS, MTT, and ATP colorimetric assays. Real-time cytotoxicity assays can also be used to measure cytolytic activity.

[0043] The therapeutic index of the oncolytic adenoviruses of the present disclosure, i.e., the comparison of the amount of therapeutic agent that causes a therapeutic effect and the amount that causes toxicity, can be calculated by comparing the efficacy of the adenovirus's cytolytic ability in tumor cell lines with that of corresponding normal cells.

[0044] The oncolytic adenoviruses of the present disclosure can be further evaluated for therapeutic utility by infecting tumor cells and / or normal cells and assessing their ability to express the functional proteins encoded by the oncolytic adenoviruses. An example of the present invention is a replication - promoting oncolytic virus (MEM - 288) that encodes both a chimeric CD40 ligand (MEM40) under the control of the CMV promoter / enhancer and IFNβ under the control of the SV40 early promoter / enhancer (Figure 3). These proteins can be detected by antibodies that specifically recognize each protein expressed by the cells after infection with MEM - 288. The nucleic acid sequence of the chimeric CD40 ligand (MEM40) was described as ISF35 in U.S. Patent No. 7,495,090, which is incorporated herein by reference.

[0045] The oncolytic adenoviruses of the present disclosure can be further evaluated for their ability to target tumor cell growth and their ability to reduce tumor formation or tumor cell burden in mice with autologous or heterologous tumor models, in mice having naturally - derived tumors or transplanted tumors. Tumor burden, measured by tumor size, immune protection from tumor relapse, and animal survival, can all serve as criteria for therapeutic utility and animal tumor models.

[0046] Methods of treatment and administration Within various embodiments of the present disclosure, methods for treating cancer are also provided that include administering to a subject having cancer an adenovirus described herein. The oncolytic adenovirus of the invention can be administered by intratumoral injection. However, other delivery routes can also be considered, including intravenous, intraperitoneal, intratracheal, intramuscular, intracranial, endoscopic, intralesional, percutaneous, subcutaneous, topical, or direct injection or perfusion.

[0047] In certain embodiments, cancer is treated using a composition described herein (e.g., a pharmaceutical composition). As noted above, the term "cancer" as used herein refers to a large family of diseases characterized by uncontrolled cell growth in the body. Representative forms of cancer include carcinomas, sarcomas, myelomas, leukemias, lymphomas, and the above-mentioned mixed types. Further examples include bile duct cancer, bladder cancer, brain cancer such as glioblastoma, breast cancer, cervical cancer, CNS tumors (glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma, etc.), colorectal cancer, endometrial cancer, hematopoietic cell cancers including leukemia and lymphoma, hepatocellular cancer, kidney cancer, laryngeal cancer, lung cancer, melanoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, squamous cell carcinoma, and thyroid cancer, but are not limited thereto. Cancer can be disseminated (e.g., leukemia), a solid tumor (e.g., sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, and osteosarcoma), or some combination thereof (e.g., metastatic cancer having both solid tumors and disseminated or disseminated cancer cells). For example, any cancer patient who can undergo autologous or allogeneic stem cell transplantation is considered a candidate for this therapy.

[0048] In some embodiments, administration can be achieved by direct administration to the tumor or the site where the tumor was (e.g., after surgical resection or ablation therapy). Administration can be by direct injection or infusion over a selected period.

[0049] Direct injection into the tumor (intratumoral injection) can be achieved by a thin catheter or cannula. In certain embodiments, the pharmaceutical compositions provided herein can be delivered by a microelectromechanical (MEMS) system under MR-guided procedures.

[0050] When administered to a subject, an effective amount of the compositions described herein is given to treat cancer (e.g., to alleviate, ameliorate, reduce, restore, stabilize, prevent expansion, slow or delay progression, or cure). For example, an amount sufficient to achieve the effect of reducing or destroying cancerous or tumorous cells, or inhibiting the growth and / or proliferation of such cells may be used. To be clinically effective, the compositions provided herein may be administered one or more times depending on the treatment regimen.

[0051] Treatment regimens using the replication-promoting oncolytic adenoviruses of the invention may include single or multiple administrations. Multiple administrations can be carried out on an alternating schedule and / or in response to one or more indicators of the effectiveness of one or more pre-administrations, or the side effects of one or more pre-administrations that are apparent to those of ordinary skill in the art having the benefit of the present disclosure in particular.

[0052] The dosage of the pharmaceutical composition used can depend on individual patient parameters including the particular condition being treated, the severity of the condition, age, health status, build, and weight, the duration of treatment, the nature of any concomitant therapy (if any), the particular route of administration, and other similar factors within the knowledge and expertise of the medical practitioner. Further, the dosage can depend on the availability of the product.

Examples

[0053] (Example 1) Dual promoter replication-promoting oncolytic adenovirus A typical replication-enhanced oncolytic adenovirus type 5 (Figure 1) has been shown to have the following features: it has a CMV promoter / enhancer represented by SEQ ID NO: 1 and an SV40 promoter / enhancer represented by SEQ ID NO: 2. A preferred embodiment of this generalized vector construct (Figure 2) has the following features: a CMV promoter / enhancer represented by SEQ ID NO: 1 downstream of the left terminal inverted repeat (ITR) and upstream of the functional E1A region, an SV40 promoter / enhancer represented by SEQ ID NO: 2 within the E3 region, an E1A gene region containing a 24-nucleotide deletion, a partial or complete deletion of the E3 region, and a deletion of the E1b 55k region. Further modifications are possible for this template replication-enhanced oncolytic virus, including adding a heterologous nucleic acid downstream of either the CMV promoter / enhancer or the SV40 promoter / enhancer for the expression of a therapeutic protein.

[0054] (Example 2) Construction of a Dual-Promoter Replication-Enhanced Oncolytic Adenovirus Encoding Two Transgenes The following features: the CMV promoter / enhancer represented by SEQ ID NO: 1 downstream of the left terminal inverted repeat (ITR) and upstream of the functional E1A region, the SV40 promoter / enhancer represented by SEQ ID NO: 2 within the E3 region, an E1A gene region containing a 24-nucleotide deletion, a partial or complete deletion of the E3 region, and a deletion of the E1b 55k region, a replication-promoting oncolytic adenovirus type 5 (Figure 3) was constructed. Furthermore, a chimeric CD40 ligand (MEM40) cloned downstream of the CMV promoter / enhancer and a heterologous nucleic acid encoding IFNβ cloned downstream of the SV40 promoter / enhancer were incorporated. This replication-promoting oncolytic adenovirus is called MEM-288 and can express two proteins that can be easily expressed and detected from tumor cells after infection (Figure 4). In addition, the present inventors directly compared the expression of MEM40 in tumor cells after infection with either MEM-288 or a similar oncolytic adenovirus vector (MEM-188) containing a heterologous nucleic acid encoding a chimeric CD40 ligand (MEM40) cloned downstream of the CMV promoter / enhancer but not containing the SV40 promoter / enhancer and the IFNβ transgene insert at an equivalent infectious titer. MEM-288 expressed a higher level of the MEM40 transgene than MEM-188 across the panel of various lung tumor types (Figure 5).

[0055] (Example 3) Promotion of viral replication in tumor cells A549 tumor cells were increased in titer and infected with MEM-288, a replication-promoting oncolytic adenovirus containing double CMV and SV40 promoter / enhancer elements (Figure 6). It was directly compared with control viruses (Ad-GFP and MEM-188) with a similar set of features except for the lack of the SV40 promoter / enhancer element. MEM-288 showed a significant increase in the expression of MEM40, which was particularly prominent at MOI 10 and MOI 1. Even at MOI 1 where MEM-188 did not show transgene expression, MEM-288 gradually showed an increase in MEM40 expression, which became particularly prominent on day 6 after infection. These findings indicate that MEM-288 has excellent replication ability. To more directly measure whether the replication ability of MEM-288 is higher, the inventors performed a standard replication assay based on the release of intracellular virus by freeze-thaw lysis. After A549 cells were infected with Ad-GFP, MEM-188, and MEM-288 at different MOIs, the intracellular levels of the virus were measured using 293 cells 2 days later using a hexon staining kit from Cell Biolabs, Inc. to calculate the infectious virus titer. Ad-GFP infection of A549 resulted in detectable levels of virus replication from MOI 10 (Figure 7). The MEM-188 virus resulted in detectable virus replication from MOI 1. In contrast, MEM-288 caused high levels of virus replication even at MOI 0.01 and MOI 0.1. Furthermore, infection with MEM-288 at one or more MOIs caused a hexon staining signal that was too high to quantify. These results indicate that the replication ability of MEM-288 is probably 100 to 1000 times or more higher than that of the other tested viruses lacking the double promoter / enhancer element of the present invention.

[0056] MEM-288 differs from MEM-188 in that it has the SV40 promoter / enhancer and the IFNβ transgene. To determine whether either the SV40 or IFNβ element contributes to the higher replication of MEM-288, we added recombinant human IFNβ to MEM-188-infected A549 and then measured virus replication as described above. Importantly, instead of increasing replication, IFNβ addition decreased it to about one-fourteenth (Figure 8). This result suggests that the SV40 promoter / enhancer within the E3 region mediates the oncolytic effect and increased replication of MEM-288.

[0057] (Example 4) Promotion of Oncolytic Activity We infected A549 lung tumor cells with Ad-GFP, MEM-188, or MEM-288 at various multiplicities of infection (MOI) and then measured the viable cell count. These analyses were further extended by adding a method for measuring oncolysis, including the use of an impedance-based system (xCELLigence system) that can very accurately measure the viability of cancer cells based on their attachment to the plate over time in additional cell lines. This real-time cell analysis (RTCA) was performed by plating the tumor strain at a density of 2,500 cells per well and allowing it to attach overnight. The oncolytic virus was added to the culture, and cancer cell viability was measured every 15 minutes for up to 120 hours. We again found that in studies using four lung cell lines, A549, H23, PC9, and HCC44 (Figures 9 - 12 respectively), the oncolytic activity increased 10- to 100-fold with MEM-288.

[0058] Next, the inventors used SCID mice with subcutaneous A549 tumors expressing firefly luciferase to enable quantification of viable tumor cells. After two injections of three different viruses (10e9 infectious units / injection), MEM-288 significantly reduced tumor growth compared to Ad-GFP and MEM-188 (Figure 13). These results indicate that the higher oncolytic effect of MEM-288 is also evident in vivo.

[0059] (Example 5) Promotion of Oncolytic Activity and Tumor Specificity Lymphoid tumor cells are resistant to lysis by oncolytic viruses, including oncolytic adenoviruses. The human B cell tumor cell line Raji was used to measure the potential effect of MEM-288 on cell viability. Control Ad-GFP and MEM-188 did not show a decrease in cell viability up to an MOI of 100 (Figure 14). In contrast, a clear dose-dependent decrease in the viability of Raji cells was observed after infection with MEM-288 (Figure 14).

[0060] Most importantly, the inventors also found that no significant killing was observed in non-cancerous cells such as human lung cancer-associated fibroblasts (CAFs), indicating that this oncolytic effect was specific to tumor cells (Figure 15). MEM-288 showed a significant oncolytic effect at MOI 1 and MOI 10 using the RTCA assay, but such a clear oncolytic effect was not shown in two independently obtained CAF isolates (Figure 15).

[0061] Next, the inventors measured the potential magnification difference between A549 tumor cells and CAFs using a wide range of MOIs (Figure 16). The inventors found that when comparing the infectious dose levels (IC50) that cause 50% cell death, the IC50 of A549 tumor cells was 0.73, while the IC50 of two independently obtained CAF isolates was 45.2 and 122.9. These results are interpreted as a 61.9-fold and 168.3-fold difference in the ability of MEM-288 to induce cancer lysis compared to normal cells, respectively (Figure 16).

[0062] The inventors also measured the oncolytic effect of MEM-288 on human monocyte-derived dendritic cells (DCs) that grow steadily (i.e., cell division) but do not divide in vitro. Infection at a high multiplicity of infection (MOI) of 50, which usually completely eradicates tumor cells, did not affect the survival rate of DCs after infection with MEM-288 or the control virus (Figure 17). These results indicate the tumor cell specificity of this innovative replication-promoting oncolytic adenovirus.

[0063] (Example 6) Oncolysis promotion not affected by the adenovirus E3 regulatory domain As described above, the modulation of the biological properties of oncolytic adenoviruses can affect a series of immune interactions that may be beneficial or harmful to the efficacy of cancer treatment. This interaction is influenced by the specific tumor, the site and degree of the disease, the immunosuppressive tumor microenvironment, the oncolytic virus platform, the dose, the time, the delivery conditions, and the response of individual patients (generally, see Aurelian L., "Oncolytic viruses as immunotherapy: progress and remaining challenges", Onco. Targets Ther. 2016, 9: 2627-2637). For example, the presence of the adenovirus E3 gene has been reported to increase the oncolytic ability of adenoviruses to replicate in vitro and in vivo depending on the conditions (see Suzuki K, Alemany R, Yamamoto M, and Curiel DT, "The presence of the adenovirus E3 region improves the oncolytic potency of conditionally replicative adenoviruses", Clin. Cancer Res. 2002 Nov., 8(11): 3348-59). In particular, the E3-11.6 kDa adenovirus death protein (ADP) is thought to be required for efficient cell death (see Tollefson A, Ryerse J, and Scaria A et al., "The E3-11.6-kDa Adenovirus Death Protein (ADP) is Required for Efficient Cell Death: Characterization of Cells Infected with adp Mutants", Virology 1996, 220: 152-162).

[0064] By examining two different control GFP-encoding oncolytic adenoviruses, one GFP virus containing a nearly complete E3 deletion as described in Figures 4, 7, 9-17, and another GFP virus (Ad-GFP-2) containing a partial E3 deletion set (E3 6.7k and E3 19k deletions) found in MEM-288, the inventors investigated the potential contribution of the E3 adenoviral region of oncolytic adenoviruses.

[0065] Figure 18 shows the expression of GFP or chimeric CD40 ligand (MEM40) in A549 human lung cancer cell lines after infection with either Ad-GFP-2 or MEM-288. Panel (A) shows GFP expression in uninfected cells (untreated) or cells infected with Ad-GFP-2 at MOIs of 10 and 100. Panel (B) shows MEM40 expression in uninfected cells (untreated) or cells infected with MEM-288 at MOIs of 10 and 100. This result shows that at a low MOI of 10, MEM-288 has a higher percentage of cells containing MEM40 than Ad-GFP-2, suggesting that MEM-288 may potentially replicate more than Ad-GFP-2.

[0066] Figure 19 shows the viability of A549 human lung cancer cell lines after infection with Ad-GFP-2 or MEM-288. Cells were infected with Ad-GFP-2 and MEM-288 at the indicated MOIs of 1, 10, and 100. After 2 days, the remaining live cells were counted and presented as a percentage of untreated cells (UT). This result shows that MEM-288 has higher oncolytic activity than Ad-GFP-2 at all three MOIs. Furthermore, these results reflect the results of MEM-288 against the GFP control virus containing a nearly complete E3 region deletion, as described in Figures 4, 7, 9-17.

[0067] Therefore, these results as a whole indicate that the innovative replication-promoting adenoviral vectors of the inventors have, regardless of the regional contribution of the E3 region such as partial or total deletion of E3, mostly better viral replication and oncolytic activities, which would not be obvious to those skilled in the art from the prior art described in the above references.

[0068] The specific embodiments disclosed above are merely illustrative, as those skilled in the art having the benefit of the teachings herein can modify and practice the disclosure in different but equivalent ways that will be apparent to them. For example, the above method steps can be performed in a different order. Further, except as otherwise specified in the following claims, the details of the structures or designs shown herein are not intended to be limiting. Thus, it is apparent that the specific embodiments disclosed above can be changed or modified, and all such variations are considered to be within the scope and spirit of the disclosure. Therefore, the protection sought herein is as set forth in the following claims.

Claims

1. A recombinant adenovirus vector with enhanced viral replication, comprising a CMV promoter / enhancer and an SV40 promoter / enhancer inserted into the adenovirus genome, wherein the recombinant adenovirus vector has: a. A CMV promoter / enhancer inserted into the non-coding region upstream of the adenovirus E1 coding region; b. An SV40 promoter / enhancer inserted into the adenovirus E3 region; c. A partial or complete deletion of the E3 coding region; d. An E1A gene containing a delta-24 deletion; and e. An E1B 55K deletion, wherein the CMV promoter / enhancer region is operably linked to a heterologous nucleic acid sequence encoding MEM40, and the SV40 promoter / enhancer region is operably linked to a heterologous nucleic acid sequence encoding IFNβ. A replication-enhanced adenovirus vector.

2. The replication-enhanced adenovirus vector according to claim 1, wherein the serotype of the adenovirus is type 5.

3. The replication-enhanced adenovirus vector according to claim 1, wherein the CMV promoter / enhancer is located between the left terminal inverted repeat (ITR) and the 5' end of the functional E1A region.

4. The replication-enhanced adenovirus vector according to claim 1, wherein the SV40 promoter / enhancer is located between E3 12.5K and the E3 RID-alpha region.

5. a. The CMV promoter / enhancer contains the nucleotide sequence of SEQ ID NO: 1; b. The SV40 promoter / enhancer contains the nucleotide sequence of SEQ ID NO:

2. The replication-enhanced adenovirus vector according to claim 1.

6. The replication-enhanced adenovirus vector according to claim 1, wherein the virus has enhanced replication with respect to tumor cells.

7. The replication-enhanced adenovirus vector according to claim 1, for use in a method of treating malignant tumors.

8. The replication-enhanced adenovirus vector for use according to claim 7, wherein the malignant tumor is cancer in a subject.

9. The replication-enhanced adenovirus vector for use according to claim 8, wherein the subject is human.

10. The replication-promoting adenovirus vector according to claim 1, wherein the CMV promoter / enhancer is operably linked to a heterologous nucleic acid sequence encoding MEM40, and the SV40 promoter / enhancer is operably linked to a heterologous nucleic acid sequence encoding IFNβ.

11. a. The CMV promoter / enhancer comprises the nucleotide sequence of SEQ ID NO: 1, b. The replication-promoting adenovirus vector according to claim 10, wherein the SV40 promoter / enhancer comprises the nucleotide sequence of SEQ ID NO: 2.

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