Adenoviruses for Cancer Treatment

Abstract

The present invention relates to an adenovirus comprising a nucleic acid sequence encoding Helicobacter pylori neutrophil-activating protein (NAP) and / or an immunologically equivalent fragment of NAP and a nucleic acid sequence encoding an immunomodulator capable of inducing an immune response in a subject, which enhances the clinical efficacy in terms of delaying tumor growth and prolonging survival.

Description

[Technical field]

[0001] The present invention relates generally to adenoviruses, and in particular to recombinant adenoviruses useful in cancer therapy. [Background technology]

[0002] Current cancer treatments are mainly based on chemotherapy, radiotherapy and / or surgery. Although the cure rate is increasing in early stage cancers, many advanced cancers are incurable; either because radical surgical cure is not possible or because radiotherapy or chemotherapy are dose- or dosage-limited in their implementation due to toxicity to normal cells.

[0003] As an alternative or complement to conventional cancer treatments, virotherapy using oncolytic viruses has been proposed. Virotherapy uses oncolytic viruses that can selectively replicate and grow in tumor cells. These oncolytic viruses then selectively infect and lyse tumor cells; the released progeny virus particles reinfect neighboring tumor cells, and some even enter the bloodstream and infect metastatic tumor cells.

[0004] Virotherapy using oncolytic viruses must meet two major requirements: selectivity and efficacy. There are different proposed strategies to achieve selectivity for tumor cells, including removing viral functions required for replication in normal cells but not required for replication in tumor cells, controlling viral genes that initiate replication by using tumor-selective promoters, and modifying viral capsid proteins involved in the infection of host cells.

[0005] However, the antitumor immune response initiated by oncolytic viruses is generally insufficient to bring about a good therapeutic effect on clinical symptoms, i.e., its efficacy is too low. For this reason, it has been suggested to insert a therapeutic gene into the genome of oncolytic viruses in order to enhance their efficacy. In the art, various therapeutic genes have been proposed, including those that activate prodrugs with bystander effects, activate the immune system against tumors, induce apoptosis, and inhibit angiogenesis.

[0006] Ramachandran et al., "An infection-enhanced oncolytic adenovirus secreting H. pylori neutrophil-activating protein with therapeutic effects on neuroendocrine tumors," Molecular Therapy (2013) 21(11): 2008-2018, and Ramachandran et al., "Vector-encoded Helicobacter pylori neutrophil-activating protein promotes maturation of dendritic cells with Th1 polarization and improved migration," The Journal of Immunology (2014) 193(5):2287-2296; these two publications disclose a replication-selective infection-enhancing adenovirus carrying secreted neutrophil-activating protein (NAP). NAP adenovirus promoted dendritic cell (DC) maturation and migration and improved median survival in mice. Zhang et al., "Recombinant adenovirus expressing a soluble fusion protein PD-1 / CD137L subverts the suppression of CD8+ T cells in HCC," Molecular Therapy (2019) 27(11):1906-1918, discloses that recombinant adenovirus induced tumor-specific systemic protection against tumor reimplantation.

[0007] There remains a need to improve the efficacy of oncolytic viruses. Summary of the Invention

[0008] A general goal of the present invention is to provide adenoviruses that have therapeutic efficacy in the treatment of cancer.

[0009] This and other objectives are accomplished by the embodiments disclosed herein.

[0010] The invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0011] One aspect of the present invention relates to an adenovirus comprising a nucleic acid sequence encoding Helicobacter pylori neutrophil-activating protein (NAP) and / or an immunologically equivalent fragment of NAP. An immunologically equivalent fragment of NAP is a fragment comprising at least one polypeptide domain of at least 20 amino acid residues of NAP. The adenovirus also comprises a nucleic acid sequence encoding an immunomodulator capable of inducing an immune response in a subject.

[0012] A further aspect of the present invention relates to the above-described adenoviruses for use as therapeutic agents in the treatment of cancer.

[0013] The recombinant adenovirus of the embodiment has enhanced therapeutic effect in terms of significantly inhibiting tumor growth and significantly prolonging the survival of subjects after tumor implantation.This enhanced therapeutic effect is achieved by engineering the adenovirus to co-express NAP and / or its immunologically equivalent fragment and immunomodulatory factor.The combination of NAP and immunomodulatory factor allows the adenovirus to induce immunogenic cell death of cancer cells, and further allows the inducible maturation of dendritic cells and the activation of T cells and NK cells when administered to subjects suffering from cancer.

[0014] The embodiments, together with further objects and advantages thereof, will be best understood by reference to the following description taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0015] [Figure 1] 1 is a schematic representation of the adenovirus construct used in the examples, showing the gene arrangement and gene sites. [Diagram 2] Relative cell viability of Panc01 and MiaPaCa-2 cells after infection with various recombinant adenoviruses at different multiplicities of infection (MOI) is shown, measured 4 days post-infection (dpi) and expressed as a percentage of uninfected control cells. [Diagram 3] Figure 1 shows the replication of recombinant viruses in Panc01 and MiaPaCa-2 cells. Cells were transduced with different viruses at MOI=50. Viral genomic DNA was isolated and quantified using real-time PCR at days 0, 1, 2, and 3 post-transduction. Numbers indicate viral genome copy numbers calculated based on a standard curve. Data are presented as mean ± standard deviation (SD) of triplicate samples. [Figure 4] Figure 2 shows the expression levels of the transgenes TNFSF9 or TNFSF18 2 days after transduction in Panc01 and MiaPaCa-2 cells. Expression levels were reported as mean fluorescence intensity (MFI). [Diagram 5] Calreticulin (CRT) exposure on the cell surface and ATP release 2 days after viral transduction are shown. CRT was measured by flow cytometry and expressed as MFI; ATP was measured using an ATP measurement kit (Invitrogen) and expressed in artificial units (au). [Figure 6] Expression levels of human dendritic cell (DC) maturation markers (CD80, CD86, CD40 and CCR7) analyzed by flow cytometry and expressed as MFI are shown after 18 h of co-culture of immature DCs with virus-transduced cells. [Figure 7A] Expression levels of maturation markers (CD69 and CD107a) of tumor-infiltrating CD8+ T cells, CD4+ T cells, and CD56+ NK cells analyzed by flow cytometry and expressed as MFI are shown 3 days after intratumoral treatment with the different viruses. [Figure 7B] IFN-γ release from splenocytes harvested from different virus-treated mice and splenocytes restimulated with Panc02 cells is shown. [Figure 8] 1 shows subcutaneous Panc02 tumor growth and mouse survival after treatment with different adenoviruses. [Figure 9] 1 shows subcutaneous Panc01 tumor (human xenograft) growth in immunodeficient mice and mouse survival after treatment. [Figure 10] 1 shows subcutaneous NSX2 tumor growth in mice and mouse survival after treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention relates generally to adenoviruses, and in particular to recombinant adenoviruses useful in cancer therapy.

[0017] The recombinant adenovirus of the embodiment has an enhanced therapeutic effect in terms of significantly inhibiting tumor growth and significantly prolonging the survival of the subject after tumor implantation. This enhanced therapeutic effect is achieved by engineering the adenovirus to contain a nucleic acid sequence encoding Helicobacter pylori neutrophil-activating protein (NAP) and / or a nucleic acid sequence encoding an immunologically equivalent fragment of NAP, in combination with a nucleic acid sequence encoding an immunomodulatory factor capable of inducing an immune response in a subject when the adenovirus is administered to the subject. Thus, the adenovirus is a recombinant or engineered virus that contains a nucleic acid sequence encoding NAP and / or an immunologically equivalent fragment thereof, and an immunomodulatory factor.

[0018] Adenoviruses containing a nucleic acid sequence encoding NAP are known, as mentioned in the "Background Art" section. However, experimental data as presented herein (Figure 8) show that adenoviruses containing a nucleic acid sequence encoding NAP, but not a nucleic acid sequence encoding an immune modulator, can slightly inhibit tumor growth, but do not result in significant survival extension in subjects after tumor implantation.

[0019] However, combining NAP or an immunologically equivalent fragment thereof with another immunomodulatory factor significantly improved the therapeutic efficacy of the adenovirus (Figure 8). This was quite surprising since it has been shown that co-expression of NAP and other heterologous genes in a virus enhances an antibody response against the gene product of the heterologous gene when administered to a subject, whereby such antibody response interferes with or inhibits the function of the gene product (Iankov et al., "Measles virus expressed Helicobacter pylori neutrophil-activating protein significantly enhances the immunogenicity of poor immunogens," Vaccine (2013) 31(42): 4795-4801). Thus, co-expression of NAP and / or an immunologically active fragment thereof in an adenovirus together with an immunomodulatory factor would be expected to elicit an antibody response against the immunomodulatory factor in the subject when administered to the subject. The antibody response generated against the immunomodulator is then expected to inhibit and block the action of the immunomodulator in the subject; i.e., to at least significantly prevent or inhibit the induction of an immune response such as that induced by the immunomodulator in the subject.

[0020] However, experimental data as presented herein show that co-expression of NAP and immunomodulatory factor in adenovirus did not negatively affect the immune response-inducing function of said immunomodulatory factor. In addition to enhancing the therapeutic effect, very clearly, co-expression of NAP and / or its immunologically active fragment and immunomodulatory factor induced immunogenic cell death (ICD) of cancer cells.

[0021] ICD, also called immunogenic apoptosis, is a form of cell death caused by the controlled activation of immune response. This cell death is characterized by an apoptotic morphology and maintains membrane integrity. The immunogenic cell death of cancer cells induces effective antitumor immune responses by activating dendritic cells (DCs) and the resulting activation of specific T cell responses. Therefore, the induction of ICD by the adenovirus of the present invention means that the adenovirus is highly effective in antitumor therapy.

[0022] Furthermore, the adenoviruses of the present invention co-expressing NAP, and / or its immunologically active fragments, and immunomodulatory factors induced DC maturation and activation of T cells, including CD4+ T cells and CD8+ T cells, and activation of natural killer (NK) cells, including CD56+ NK cells. Furthermore, treatment with the adenoviruses of the present invention led to the generation of immune memory, as evidenced by the endogenous splenocytes of treated subjects also responding to tumor cells and releasing significant amounts of interferon gamma (IFN-γ).

[0023] Co-expression of NAP and / or its immunologically active fragment and immunomodulator in the adenovirus of the present invention did not negatively affect the expression of said immunomodulator in transduced cancer cells.Furthermore, said adenovirus could efficiently replicate in and specifically kill cancer cells.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide general definitions for many of the terms used in this invention: Singleton et al., Dictionary of microbiology and molecular biology, 3rd ed., revised edition, 2007, ISBN: 9780470035450; Walker, The Cambridge dictionary of science and technology, 1990, ISBN: 9780521394413; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., 1991, ISBN: 9783540520542; Hale, HarperCollins dictionary of biology, 1991, ISBN: 9780064610155; Lewin, Gene XII, 1993, ISBN: 9780064610155; XII), 2017, ISBN: 978-1-2841-0449-3; Knipe et al., Fields Virology, 6th Edition, 2013, ISBN: 978-1-4511-0563-6. For clarity, the following definitions are used herein:

[0025] The term "nucleic acid sequence" or "nucleotide sequence" refers to a polymer composed of nucleotides, such as ribonucleotides, deoxyribonucleotides, variants of related natural structures and / or synthetic non-natural analogues thereof linked via phosphodiester bonds, variants of related natural structures and / or synthetic non-natural analogues thereof. Examples of such nucleic acid or nucleotide sequences include deoxyribonucleic acid (DNA) sequences and ribonucleic acid (RNA) sequences. In a particular embodiment, the nucleic acid or nucleotide sequence is a DNA sequence.

[0026] One aspect of the present invention relates to an adenovirus comprising a nucleic acid sequence encoding Helicobacter pylori neutrophil-activating protein (NAP) and / or an immunologically equivalent fragment of NAP, said adenovirus also comprising a nucleic acid sequence encoding an immunomodulator capable of inducing an immune response in a subject.

[0027] The adenovirus is thereby capable of co-expressing a NAP and / or an immunologically equivalent fragment thereof in addition to an immunomodulatory agent, which is therefore distinct from a NAP or an immunologically active fragment of a NAP.

[0028] When the adenovirus is administered to a subject, the immunomodulator can induce immune response in the subject.In one embodiment, the immunomodulator can induce DC maturation.The cancer cells transduced with the adenovirus of the present invention co-cultured can mature and activate DC, as indicated by the surface upregulation of maturation and activation markers, including cluster of differentiation 80 (CD80) (also called B7-1), CD40, CD86 (also called B7-2) and CC chemokine receptor type 7 (CCR7).

[0029] In another embodiment, the immunomodulator can induce T cell activation, in particular CD4+ T cell activation, CD8+ T cell activation, or both CD4+ T cell activation and CD8+ T cell activation. In subjects transplanted with cancer cells and treated with the adenovirus of the present invention, the activation of tumor-infiltrating CD4+ and CD8+ T cells is enhanced. The adenovirus treatment results in the activation of CD4+ and CD8+ T cells, as indicated by the upregulation of surface markers CD69 and CD107a (also called lysosomal-associated membrane protein 1 (LAMP-1) or lysosome-associated membrane protein 1).

[0030] In a further embodiment, the immunomodulator can induce NK cell activation, particularly CD56+ NK cell activation.In subjects transplanted with cancer cells and treated with the adenovirus of the present invention, the activation of tumor-infiltrating NK cells is enhanced.The adenovirus treatment leads to the activation of these NK cells, as shown by the upregulation of surface markers CD69 and CD107a.

[0031] In a preferred embodiment, the immunomodulatory agent is capable of inducing DC maturation and T cell activation; capable of inducing DC maturation and NK cell activation, or capable of inducing T cell activation and NK cell activation in a subject. In a currently preferred embodiment, the immunomodulatory agent is capable of inducing DC maturation, T cell activation, and NK cell activation in a subject.

[0032] In one embodiment, the immunomodulatory factor is a member of the tumor necrosis factor (TNF) superfamily (TNFSF).

[0033] TNFSF is a protein superfamily of type II transmembrane proteins that contain TNF homology domains and form trimers. Members of TNFSF can be released from the cell membrane by exoproteolytic cleavage and function as cytokines. These proteins are expressed exclusively by immune cells and regulate a variety of cellular functions, including not only immune responses and inflammation, but also proliferation, differentiation, apoptosis, and embryonic development.

[0034] In one embodiment, the TNFSF member is selected from the group consisting of TNFSF1, TNFSF2, TNFSF4, TNFSF5, TNFSF7, TNFSF9, TNFSF14, TNFSF18, and combinations thereof.

[0035] TNFSF1, also called lymphotoxin-alpha (LT-α) or TNF-beta (TNF-β), exhibits antiproliferative activity against tumor cells and induces cell destruction in tumor cells. TNFSF1 is involved in the induction of inflammation and antiviral responses, the development of secondary lymphoid organs, and the control of cell survival, proliferation, differentiation, and apoptosis.

[0036] TNFSF2, also called tumor necrosis factor (TNF), TNF-α, cachexin, or cachectin, plays a role in immune cell regulation, induction of fever, cachexia, inflammation, and apoptosis. TNFSF2 also inhibits tumor formation.

[0037] TNFSF4, also known as OX40 ligand, CD252, Gp34 or CD134L, induces activation of T cell immune responses by T cell costimulation.

[0038] TNFSF5, also known as CD40 ligand (CD40L), regulates adaptive immune responses by activating antigen-presenting cells (APCs).

[0039] TNFSF7, also called CD27 ligand (CD27L) or CD70, regulates B cell activation and T cell homeostasis.

[0040] TNFSF9, also called CD137 ligand or 4-1BB ligand (4-1BBL), is present on APCs and binds to CD137 (also called 4-1BB) expressed on activated T cells.

[0041] TNFSF14, also known as LIGHT, CD258 or HVEML, regulates B cell activation and T cell homeostasis.

[0042] TNFS18, also called glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) ligand (GITRL), activation-induced TNFR member ligand (AITRL) or TL-6, is a cytokine that is a ligand for the receptor TNF receptor superfamily 18 (TNFRSF18) (also called GITR or AITR). TNFS18 is thought to regulate T cell survival and to be important for the interaction between T cells and endothelial cells.

[0043] In one embodiment, the immunomodulator is a membrane-bound immunomodulator.In such a case, the immunomodulator expressed in cancer cells infected with the adenovirus of the present invention is preferably bound to the cell membrane of the infected cancer cells.Membrane-bound immunomodulator can induce local immune response at the site of viral infection of cancer cells, i.e., the site of cancer cells or tumors, in a subject.Such membrane-bound immunomodulator is generally preferred over soluble immunomodulator; this is because soluble immunomodulator migrates away from the site of cancer cells, thereby reducing the effectiveness of inducing immune response at the desired site of a subject.

[0044] In one embodiment, the adenovirus comprises a nucleotide sequence encoding one TNFSF member, preferably a TNFSF member selected from the group above. In such a case, the adenovirus expresses a single TNFSF protein. In another embodiment, the adenovirus comprises a single nucleotide sequence encoding multiple, i.e., at least two different TNFSF members, or multiple nucleotide sequences encoding different TNFSF members.

[0045] In a preferred embodiment, the TNFSF member is selected from the group consisting of TNFSF5, TNFSF9, TNFSF14, TNFSF18, and combinations thereof.

[0046] In a more preferred embodiment, the TNFSF member is selected from the group consisting of TNFSF9, TNFSF18, and combinations thereof.

[0047] In one embodiment, the adenovirus comprises a nucleic acid sequence encoding TNFSF9. In another embodiment, the adenovirus comprises a nucleic acid sequence encoding TNFSF18. In a further embodiment, the adenovirus comprises a nucleic acid sequence encoding TNFSF9 and a nucleic acid sequence encoding TNFSF18, or a nucleic acid sequence encoding TNFSF9 and TNFSF18.

[0048] Helicobacter pylori neutrophil-activating protein, or NAP or HP-NAP for short, is a dodecameric protein that acts as a virulence factor in Helicobacter pylori infection. It is composed of 12 monomeric subunits, each of which consists of four α-helices. The surface of NAP is highly positively charged and it can interact with and activate human white blood cells (WBCs; also called leukocytes).

[0049] NAP plays an important role in neutrophil migration into inflamed tissues in Helicobacter pylori infection. NAP promotes strong binding of neutrophils and monocytes to endothelium and extravasation of neutrophils and monocytes by upregulating the surface expression of β2 integrins. NAP can also activate neutrophils in terms of producing reactive oxygen species (ROS) and myeloperoxidase. NAP also activates the secretion of other inflammatory cytokines such as TNF-α and interleukin 8 (IL-8) (also called chemokine (CXC motif) ligand 8 (CXCL8)), thereby inducing the expression of adhesion molecules such as vascular cell adhesion molecule (V-CAM), intercellular adhesion molecule (I-CAM), and secretion of IL-8 by endothelial cells. In addition, NAPs can also induce the expression and secretion of several cytokines and chemokines by neutrophils, such as IL-8, macrophage inflammatory protein 1 alpha (MIP-1α) and MIP-1β (also called chemokine (CC motif) ligand 4 (CCL4)). These cytokines and chemokines further attract neutrophils to the inflammatory site by chemotaxis.

[0050] NAP is a toll-like receptor 2 (TLR-2) agonist, is chemotactic for neutrophils and monocytes, and can mature DCs both in vitro and in vivo. NAP can also stimulate the secretion of Th1-polarizing cytokines IL-12 and IL-23. NAP stimulates monocytes to differentiate and mature into DCs by upregulating the expression of HLA-antigen D-related (HLA-DR), CD80, and CD86. NAP also has immune regulatory functions that are crucial for helping cytotoxic T cells and NK cell activation. NAP can induce high levels of IFN-γ and low levels of IL-4 secretion by T cells, also suggesting a Th1-polarizing response. This is consistent with reports that humans infected with Helicobacter pylori exhibit a strong Th1-polarizing response.

[0051] In one embodiment, the NAP preferably comprises or consists of an amino acid sequence selected from any of the following sequences: MKTFEILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKATEEIYEEFADMFDDLAERIVQLGHHPLVTLSEAIKLTRVKEETKTSFHSKDIFKEILEDYKYLEKEFKELSNTAEKEGDKVTVTYADDQLAKLQKSIWMLQAHLA (SEQ ID NO: 3) MKTFEILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKATEEIYEEFADMFDDLAERIVQLGHHPLVTLSEAIKLTRVKEETKTSFHSKDIFKEILEDYKHLEKEFKELSNTAEKEGDKVTVTYADDQLAKLQKSIWMLQAHLA (SEQ ID NO: 4) MKTFEILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKATEEIYEEFADMFDDLAERIVQLGHHPLVTLSEALKLTRVKEETKTSFHSKDIFKEILEDYKYLEKEFKELSNTAEKEGDKVTVTYADDQLAKLQKSIWMLQAHLA (SEQ ID NO:5) MKTFEILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKATEEIYEEFADMFDDLAERIVQLGHHPLVTLSEALKLTRVKEETKTSFHSKDIFKEILEDYKHLEKEFKELSNTAEKEGDKVTVTYADDQLAKLQKSIWMLQAHLA (SEQ ID NO: 6) MKTFEILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKATEEIYEGFADMFDDLAERIVQLGHHPLVTLSEAIKLTRVKEETKTSFHSKDIFKEILEDYKYLEKEFKELSNTAEKEGDKVTVTYADDQLAKLQKSIWMLQAHLA (SEQ ID NO: 7) MKTFEILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKATEEIYEGFADMFDDLAERIVQLGHHPLVTLSEALKLTRVKEETKTSFHSKDIFKEILEDYKYLEKEFKELSNTAEKEGDKVTVTYADDQLAKLQKSIWMLQAHLA (SEQ ID NO: 8) MKTFEILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKATEEIYEGFADMFDDLAERIVQLGHHPLVTLSEAIKLTRVKEETKTSFHSKDIFKEILEDYKHLEKEFKELSNTAEKEGDKVTVTYADDQLAKLQKSIWMLQAHLA (SEQ ID NO: 9) MKTFEILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKATEEIYEGFADMFDDLAERIVQLGHHPLVTLSEALKLTRVKEETKTSFHSKDIFKEILEDYKHLEKEFKELSNTAEKEGDKVTVTYADDQLAKLQKSIWMLQAHLA (SEQ ID NO: 10)

[0052] An immunologically equivalent fragment of NAP is a fragment that comprises at least one polypeptide domain of at least 20 amino acid residues (such as at least 20 contiguous amino acid residues), preferably at least 30 amino acid residues (such as at least 30 contiguous amino acid residues), and more preferably at least 40 amino acid residues (such as at least 40 contiguous amino acid residues) of NAP.

[0053] Specific, non-limiting examples of immunologically equivalent fragments of NAP include: Corresponding to amino acid numbers 5-40 of SEQ ID NO: 3-10, EILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKAT (SEQ ID NO:11) Corresponding to amino acid numbers 110-144 of SEQ ID NO: 3-10, NTAEKEGDKVTVTYADDQLAKLQKSIWMLQAHLA (SEQ ID NO:12) Corresponding to amino acid numbers 39-74 of SEQ ID NO: 5-6, ATEEIYEEFADMFDDLAERIVQLGHHPLVTLSEALK (SEQ ID NO: 13) SEQ ID NO: 5, corresponding to amino acid numbers 75-110 of 10, LTRVKEETKTSFHSKDIFKEILEDYKHLEKEFKELS (SEQ ID NO: 14)

[0054] Further information regarding immunologically equivalent fragments of NAP is provided in EP1767214B1, the description of immunologically equivalent fragments of HP-NAP disclosed in paragraph 32 thereof, "Definition of dominant T cell epitopes of HP-NAP recognised by HP-NAP-specific T cells derived from Helicobacter pylori-induced gastric infiltrates", is incorporated herein by reference.

[0055] In one embodiment, the adenovirus comprises a nucleic acid sequence encoding a single NAP, a nucleic acid sequence encoding multiple different NAPs, a nucleic acid sequence encoding a single immunologically active fragment of a NAP, a nucleic acid sequence encoding multiple different immunologically active fragments of a NAP, or at least one nucleic acid sequence encoding at least one NAP and at least one nucleic acid sequence encoding at least one immunologically active fragment of a NAP.

[0056] In one embodiment, the adenovirus further comprises a nucleic acid sequence encoding a self-cleaving peptide located between the nucleic acid sequence encoding a NAP and / or an immunologically active fragment of a NAP and the nucleic acid sequence encoding the immunomodulator.

[0057] As used herein, a "self-cleaving peptide" is a peptide that can induce ribosome skipping during protein translation in a cell. Apparent cleavage occurs by ribosome skipping at the peptide bond between proline (P) and glycine (G) located at the C-terminus of the self-cleaving peptide, resulting in a peptide or protein located upstream of the self-cleaving peptide containing an extra amino acid at its C-terminus, and a peptide or protein located downstream of the self-cleaving peptide containing an extra proline at its N-terminus.

[0058] In one embodiment, the self-cleaving peptide is a self-cleaving 2A peptide, also referred to as a 2A peptide. Such a self-cleaving 2A peptide comprises the core sequence motif of DxExNPGP (SEQ ID NO: 15). In one embodiment, the self-cleaving 2A peptide is selected from the group consisting of Thosea asigna virus 2A peptide (T2A), Porcine Teschovirus-1 2A peptide (P2A), Equine Rhinitis A virus 2A peptide (E2A) and Foot and Mouth Disease virus 2A (F2A). In one embodiment, T2A has the amino acid sequence: EGRGSLLTCGDVEENPGP (SEQ ID NO: 16) or GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 17). In one embodiment, P2A consists of the amino acid sequence: ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18) or GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 19). In one embodiment, E2A consists of the amino acid sequence: QCTNYALLKLAGDVESNPGP (SEQ ID NO:20) or GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:21). In one embodiment, F2A consists of the amino acid sequence: VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 22) or GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23).

[0059] By including a nucleic acid sequence encoding a self-cleaving peptide between the nucleic acid sequence encoding NAP and / or its immunologically equivalent fragment and the nucleic acid sequence encoding the immunomodulator, correct translation and folding of NAP and / or its immunologically equivalent fragment and the immunomodulator is facilitated independent of each other and independent of other proteins of the adenoviral genome.

[0060] In one embodiment, the adenovirus is an oncolytic adenovirus.Oncolytic adenovirus is an adenovirus that can selectively replicate in cancer cells.Such oncolytic adenovirus can selectively replicate in cancer cells, i.e., has tumor tropism, and preferably can lyse cancer cells; i.e., can lyse oncolytic.

[0061] In one embodiment, the adenovirus is engineered to be oncolytic, i.e., engineered to selectively replicate in cancer cells. In a preferred embodiment, the oncolytic adenovirus comprises a mutant adenoviral early region 1A (E1A) gene encoding a mutant E1A protein that has a lower Rb binding ability compared to the wild-type E1A protein.

[0062] The adenovirus E1A protein binds to the intracellular Rb protein of the infected host cell. Binding of the E1A protein to the intracellular Rb protein releases E2F, which activates the transcription of other viral genes, such as E2 (encoding proteins involved in viral replication), E3 (encoding proteins that inhibit the host's antiviral immune response), and E4 (encoding proteins involved in viral ribonucleic acid (RNA) transport), as well as cellular genes that activate the cell cycle.

[0063] By weakening, such as by inhibiting or completely preventing, the E1A protein from binding to the intracellular Rb protein, the adenovirus becomes a cancer cell-selective or tumor cell-selective virus. Thus, an adenovirus containing a mutant E1A gene encoding a mutant E1A protein can replicate in and lyse cancer cells, but not in healthy or normal cells, i.e., non-cancerous cells.

[0064] In one embodiment, the mutant E1A gene comprises a 24 base pair (bp) deletion corresponding to nucleotides 919 to 943 of the wild-type E1A gene. This 24 bp deletion corresponds to the nucleotides cttacctgccaggaggctggcttt (SEQ ID NO: 24). These 24 nucleotides encode amino acids 121 to 128 of the E1A protein. Thus, the mutant E1A protein lacks amino acids 121 to 128, which correspond to LTCHEACF (SEQ ID NO: 25) of the wild-type E1A protein.

[0065] In one embodiment, the adenovirus lacks a nucleic acid sequence encoding the 19 kDa adenoviral E1B protein and a nucleic acid sequence encoding the 55 kDa adenoviral E1B protein.

[0066] In one embodiment, one of the nucleic acid sequences encoding the 19 kDa adenoviral E1B protein and the 55 kDa adenoviral E1B protein is replaced with a nucleic acid sequence encoding a NAP and / or a nucleic acid sequence encoding an immunologically equivalent fragment of a NAP, and the other of the nucleic acid sequences encoding the 19 kDa adenoviral E1B protein and the 55 kDa adenoviral E1B protein is replaced with a nucleic acid sequence encoding the immune modulator.

[0067] Thus, in a preferred embodiment, the adenoviral nucleic acid sequences encoding the 19-kDa E1B protein and the 55-kDa adenoviral E1B protein are replaced with NAP and / or its immunologically equivalent fragments, and with nucleic acid sequences encoding the immune modulator.

[0068] In one embodiment, the adenovirus comprises a nucleic acid sequence encoding the immunomodulatory agent, followed by a nucleic acid sequence encoding a self-cleaving peptide, followed by a nucleic acid sequence encoding a NAP and / or an immunologically active fragment thereof, as shown in Figure 1. In another embodiment, the adenovirus comprises a nucleic acid sequence encoding a NAP and / or an immunologically active fragment thereof, followed by a nucleic acid sequence encoding a self-cleaving peptide, followed by a nucleic acid sequence encoding the immunomodulatory agent.

[0069] In one embodiment, the adenovirus is human adenovirus type 5, preferably oncolytic human adenovirus type 5.

[0070] Human adenovirus type 5 (Ad5) belongs to group C and is a virus formed by a proteinaceous icosahedral capsid that packages a 36 kilobase linear deoxyribonucleic acid (DNA). In adults, Ad5 infection is usually asymptomatic, whereas in children, it causes the common cold and conjunctivitis. Generally, Ad5 infects epithelial cells, which in the natural course of infection are cells of the bronchial epithelium. Ad5 enters cells by interacting with the Coxsackie adenovirus receptor (CAR), a cellular protein involved in cell-cell adhesion, through the interaction of the viral fiber, a viral protein that extends as an antenna at the apex of the capsid. When the viral DNA reaches the cell nucleus, the viral early genes (E1 to E4) begin to transcribe in an orderly and sequential manner. The first viral genes to be expressed are those of the early region 1A (E1A). E1A binds to the intracellular protein Rb and releases E2F, which activates the transcription of other viral genes such as E2 (encoding proteins involved in viral replication), E3 (encoding proteins that inhibit the host's antiviral immune response), and E4 (encoding proteins involved in viral ribonucleic acid (RNA) transport) as well as cellular genes that activate the cell cycle. In addition, E1B binds to p53, activating the cell cycle and preventing apoptosis of infected cells. Expression of early genes leads to viral DNA replication, and once DNA replication has occurred, its major late promoter is activated to direct the transcription of messenger RNAs (mRNAs) that, by differential splicing, generate all the RNAs that code for the encapsidation structural proteins.

[0071] Another aspect of the invention pertains to adenoviruses according to an embodiment for use as therapeutic agents.

[0072] A further aspect of the invention relates to an adenovirus according to an embodiment for use in cancer treatment.

[0073] A related aspect of the invention defines the use of an adenovirus according to this embodiment for the manufacture of a therapeutic medicament for the treatment of cancer.

[0074] The present invention further relates to a method of treating cancer, comprising administering to a subject suffering from cancer an effective amount of an adenovirus according to the present embodiments.

[0075] "Treating" or "treatment" herein is well understood in the art and refers to an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, for example, alleviation or amelioration of one or more symptoms or conditions, reduction in the extent of cancer disease, stabilization of cancer disease, i.e., prevention of exacerbation, prevention of the spread of cancer disease, delay or slowing of disease progression, improvement or alleviation of cancer symptoms, reduction of recurrence of cancer disease, and remission. "Treating" or "treatment" may also prolong survival compared to expected survival in the absence of any treatment. "Cancer treatment" herein includes inhibiting cancer in a subject as well as prophylactic treatment.

[0076] "Prevent" or "prevention" as used herein is well understood in the art and refers to an approach that reduces or eliminates the risk of developing a cancer disease or condition, including postponing or delaying the onset of a cancer disease. For example, a patient with a predisposition to developing a cancer disease (such as due to a genetic or hereditary predisposition) may benefit from administration of the adenovirus of this embodiment in preventing cancer, reducing the risk of a cancer disease, delaying and / or slowing the onset of a cancer disease.

[0077] The cancer or cancer disease is preferably selected from the group consisting of carcinoma (such as pancreatic cancer, breast cancer, lung cancer, liver cancer, or kidney cancer); sarcoma (such as osteosarcoma or liposarcoma); lymphoma (such as non-Hodgkin's lymphoma or Hodgkin's lymphoma); leukemia (such as acute leukemia or chronic leukemia); seminoma; germinoma; dysgerminoma; and blastoma (such as glioblastoma or neuroblastoma). In a particular embodiment, the cancer is a carcinoma, such as pancreatic cancer.

[0078] The patient is preferably a human patient, however, the embodiments also have veterinary applications, i.e., non-human patients such as non-human mammals including, for example, primates, monkeys, apes, cows, sheep, pigs, goats, horses, cats, dogs, mice, rats and guinea pigs.

[0079] The adenovirus may be administered to the patient by a variety of routes including, for example, intravenous, subcutaneous, intraperitoneal, intramuscular, or intratumoral administration.

[0080] The adenovirus is typically administered in the form of a pharmaceutical composition comprising the adenovirus.The pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, vehicles and / or additives.The non-limiting examples of such pharmaceutically acceptable carriers, vehicles and additives include injection solutions (such as saline or buffer injection solutions).

[0081] The preferred pharmaceutical composition comprises an effective amount of adenovirus.In this specification, "effective amount" refers to the amount that is effective in the dosage and period required to achieve the desired result.For example, in terms of inhibiting tumor growth, the effective amount is the amount that induces remission, reduces tumor load, and / or prevents tumor spread or growth, compared to the response obtained without administering cells.The effective amount may vary depending on factors such as the patient's condition, age, sex, weight, etc.

[0082] Working Example Example 1: Construction of adenovirus Materials and Methods Adenoviral Plasmids and Viruses The pAdEasy system was used to engineer recombinant adenovirus genomes. DNA constructs were synthesized that contained sequences corresponding to the wild-type adenovirus genome, but in which the E1A coding sequence was mutated and had a 24-bp deletion (E1a-Δ24) (Fueyo, J. et al., "A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo," Oncogene (2000) 19(1): 2-12); and the native p19K and p55K coding sequences were replaced with those of TNFSF9 (SF9) or TNFSF18 (SF18), the self-cleaving 2A peptide of Thosea asigna virus 2A (T2A), and the neutrophil-activating protein (NAP) of Helicobacter pylori. Both human and mouse TNFSF9 or TNFSF18 were constructed and used for the experimental conditions (i.e., human genes were used for human cell lines, whereas mouse genes were used for mouse models). These constructs were cloned into empty pShuttle to generate pSh(O9B) and pSh(O18B). pShuttle plasmids were further recombined with pAdEasyf35 to generate pAd(O9B) and pAd(O18B); these were used to generate oncolytic viruses Ad(O9B) and Ad(O18B). In a similar manner, a non-replicating adenovirus Ad(Luc) was generated and used as a negative control. In addition, two control viruses were generated in a similar manner; Ad(O) contained mutant E1A with a 24-bp deletion but lacked transgene expression, whereas Ad(OB) contained mutant E1A with a 24-bp deletion and only NAP as transgene expression.

[0083] Cell lines and culture conditions Human embryonic retinoblastoma 911 cells (Crucell, Leiden, The Netherlands) were cultured in a humidified incubator (5% CO2, 37°C). The cell line was maintained in Dulbecco's Modified Eagle's Medium (DMEM) Glutamax containing 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate and 100 U / ml penicillin / streptomycin (PEST). All materials used were purchased from Thermo Fisher Scientific.

[0084] Virus production and amplification Recombinant adenovirus was produced after transduction of 90% confluent 911 cells with 12 μg of PacI-digested pAd5(E1AD24-AB) DNA with the addition of polyethyleneimine (PEI, Polysciences). Cytopathic effect (CPE) was evident within 5 days, as approximately half of the cells exhibited round nuclei and detachment. The transduced cells were harvested on day 6 and lysed by repeated freeze / thaw cycles to release cytoplasmic virus particles. Adenovirus titers increased with successive transduction cycles of 911 cells. Virus was purified by CsCl gradient ultracentrifugation at 25,000 revolutions per minute (rpm) at 4°C for 2 h and dialyzed against storage solution (10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, and 4% w / v sucrose). Purified virus was stored in aliquots at -80°C.

[0085] result All viral DNA constructs can be used to produce high titers of virus. The viral constructs, their names and genome arrangements are shown in Figure 1. The transgenes (Luc, SF9, SF18, NAP) and mutations (E1a-Δ24) are indicated, and each modified position in the genome is shown relative to the wild-type genome arrangement.

[0086] Example 2: Oncolytic viruses co-expressing TNFSF9 or TNFSF18 with NAP specifically killed cancer cells and replicated efficiently Cell lines and culture conditions Human pancreatic cancer cell lines Panc01 (purchased from ATCC, USA) and MiaPaCa-2 (purchased from ATCC, USA) were cultured in a humidified incubator (5% CO2, 37°C). These cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM) Glutamax containing 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, and 100 U / ml penicillin / streptomycin (PEST). All materials used were purchased from Thermo Fisher Scientific.

[0087] MTS killing assay Panc01 and MiaPaCa-2 cells (1x10 4 Cells) were transduced with different viruses at a multiplicity of infection (MOI) of 0.1 to 1000 (corresponding number / volume of 200 μL of medium mixed with cells and viruses) and seeded in 96-well plates; cell viability was measured after 5 days with Alamar Blue compound. Cell viability was calculated as the percentage of live cells compared to untreated control cells. Results are expressed as the mean of three independent experiments.

[0088] The control virus Ad(Luc) did not show any cell killing, while both engineered Ad(O9B) and Ad(O18B) showed similar cell killing potential compared to Ad(O) and Ad(OB); indicating that the insertion of additional transgenes, i.e., TNFSF9 or TNFSF18, did not negatively affect the virus killing potential (Figure 2).

[0089] Replication assay Panc01 and MiaPaCa-2 cells (1x10 4Cells) were transduced with IgG and the cells were seeded in 96-well plates. Viral DNA was extracted from the cells at different time points using the High Pure Viral Nucleic Acid kit (Roche), and quantitative polymerase chain reaction (qPCR) was performed using adenovirus-specific primers (forward primer: CATCAGGTTGATTCACATCGG (SEQ ID NO: 1), reverse primer: GAAGCGCTGTATGTTGTTCTG (SEQ ID NO: 2)).

[0090] The control virus Ad(Luc) did not replicate in either cell line, whereas the recombinant viruses Ad(O9B) and Ad(O18B) replicated in both cell lines (Figure 3). In the Panc01 cell line, the insertion of either transgene (TNFSF9 or TNFSF18) actually increased viral replication compared to the viruses Ad(O) and Ad(OB). In the MiaPaCa-2 cell line, replication was comparable for all oncolytic viruses.

[0091] Example 3: Co-expression of TNFSF9 or TNFSF18 with NAP did not negatively affect transgene expression Transgene expression Various viruses were used at an MOI of 50 to infect Panc01 and MiaPaCa-2 cells (1x10 6 TNFSF9 or TNFSF18 transgene expression was assessed by flow cytometry 2 days after transduction.

[0092] As expected, none of the cells transduced with the control virus expressed the transgene TNFSF9, nor did they express TNFSF18. However, cells transduced with Ad(O9B) virus expressed high levels of TNFSF9, and cells transduced with Ad(O18B) virus expressed high levels of TNFSF18 (Fig. 4). These data indicate that coexpression of either TNFSF9 or TNFSF18 with NAP does not negatively affect the expression of TNFSF9 or TNFSF18.

[0093] Example 4: Co-expression of TNFSF9 or TNFSF18 with NAP results in induction of immunogenic cell death Panc01 and MiaPaCa-2 cells (5x10 5 Cells) were transduced with Ad(O9B) and the cells were seeded in 48-well plates. Since the transgene was confirmed in the above example, only oncolytic viruses co-expressing the transgene TNFSF9 or TNFSF18 with NAP, i.e., Ad(O9B) and Ad(O18B), were tested from this point on. 48 hours after transduction, the cell surface calreticulin (CRT) level was observed by flow cytometry, and the immunogenic cell death (ICD) was examined by measuring the adenosine triphosphate (ATP) release in the supernatant using an ATP measurement kit (Invitrogen).

[0094] The non-oncolytic control virus Ad(Luc) did not induce any signs of ICD compared to untreated cells; it did not increase CRT levels or ATP release. On the other hand, all cells transduced with oncolytic viruses showed high levels of CRT and released high levels of ATP (Figure 5), indicating that ICD occurred in the transduced cells.

[0095] Example 5: Co-expression of TNFSF9 or TNFSF18 with NAP induced dendritic cell maturation Panc01 and MiaPaCa-2 cells (5x10 5 ) were transduced into the co-cultures and the cells were seeded in 48-well plates. 48 h after transduction, immature dendritic cells (DCs) derived from different donors were added to the co-cultures, and DC maturation was examined 18 h later by measuring the surface expression levels of DC activation markers (cluster of differentiation 80 (CD80), CD40, CD86, and CC chemokine receptor type 7 (CCR7)). Results are expressed as the mean of three independent experiments.

[0096] Cell transduction with recombinant oncolytic viruses Ad(O9B) or Ad(O18B) was able to mature and activate DCs in coculture, as shown by enhanced surface expression of maturation / activation markers (CD80, CD40, CD86, CCR7) in Figure 6. Coexpression of transgenes TNFSF9 or TNFSF18 with NAP was able to induce DC maturation and activation.

[0097] Example 6: Oncolytic viruses co-expressing TNFSF9 or TNFSF18 with NAP induced activation of T cells and NK cells and exerted cytotoxic function Panc02 cells (1 x 10 in 100 μl Dulbecco's phosphate-buffered saline (DPBS) 6 Cells) were implanted subcutaneously (sc) into the right hind flank of 6- to 8-week-old female C57Bl / 6 mice (Taconic, Silkeborg, Denmark). In these mice, tumors were palpable (approximately 50 mm in size) 12 days after tumor inoculation. 3 ), PBS (50 μl) or various viruses (1 × 10 in 50 μl PBS) were used. 11 Viral particles (VP) were used to treat tumors intratumorally (it). Activation of both tumor-infiltrating CD8 and CD4 T cells, and NK cells, was examined 3 days after treatment. In addition, splenocytes were isolated after virus treatment, mixed with Panc02, and IFN-γ release into the supernatant was examined.

[0098] Control virus treatment did not activate either T or NK cells. On the other hand, both Ad(O9B) and Ad(O18B) treatments activated T and NK cells, as shown by upregulation of surface markers CD69 and CD107a (Fig. 7A). Furthermore, Ad(O9B) and Ad(O18B) treatments also generated memory, as shown by resident splenocytes releasing significant amounts of IFN-γ in response to tumor cells (Fig. 7B).

[0099] Example 7: Oncolytic viruses co-expressing TNFSF9 or TNFSF18 with NAP resulted in enhanced therapeutic efficacy Panc02 cells (1x10 in 100 μl DPBS) 6 Cells) were implanted subcutaneously (sc) into the right hind flank of 6- to 8-week-old female C57Bl / 6 mice (Taconic, Silkeborg, Denmark). In these mice, tumors were palpable (approximately 50 mm in size) 7, 10, and 12 days after tumor inoculation. 3 ), PBS (50 μl) or various viruses (1 × 10 in 50 μl PBS) were used. 11 The subjects were treated intratumorally (it) with VP.

[0100] In another set of experiments, Panc01 cells (5x10 in 100 μl of a 1:1 mixture of DPBS and Matrigel) were 6 Cells) were implanted subcutaneously (sc) into the right hind flank of 6- to 8-week-old female athymic nude mice (JANVIER Labs, France). In these mice, tumors were palpable (approximately 50 mm in size) 7, 10, and 12 days after tumor inoculation. 3 ), PBS (50 μl) or various viruses (1 × 10 in 50 μl PBS) were used. 11 The subjects were treated intratumorally (it) with VP.

[0101] Tumor volume was the endpoint volume of the study (EPV, 1000 mm 3 These animals were individually monitored for tumor growth until the tumor size exceeded 100 mg / kg / day. Tumor size was calculated using the ellipsoid volume formula: Tumor volume = (length x width 2 x π) / 6 The calculation was carried out using

[0102] Time to endpoint (TTE) for each mouse: TTE = [log(EPV) - b] / m where the constant b is the intercept of the line obtained by linear regression of time and m is the slope. The log-transformed tumor growth data set consisted of the first measured tumor volume that exceeded the EPV, and the three consecutive measurements of tumor volume just before the EPV was reached. Survival curves were constructed based on TTE values ​​using the Kaplan-Meier method and comparisons were made using the log-rank (Mantel-Cox) test.

[0103] In both the Panc02 and Panc01 xenograft models, oncolytic viruses Ad(O) and Ad(OB) were able to slightly inhibit tumor growth and slightly extend mouse survival (Figures 8 and 9). On the other hand, Ad(O9B) and Ad(O18B) significantly delayed tumor growth (only in the Panc02 model) and extended mouse survival (Figures 8 and 9). The data from the Panc02 model indicate that co-expression of TNFSF9 or TNFSF18 with NAP significantly activated the host immune response and achieved therapeutic antitumor effects. The data from the Panc01 model indicate that co-expression of NAP and TNFSF9 resulted in a strong antitumor effect, especially in this human xenograft model, where adenovirus replication is permissive.

[0104] Example 8: Oncolytic viruses co-expressing immunogenic GD2 mimotopes with NAP did not enhance therapeutic efficacy NXS2 cells (1x10 in 100 μl DPBS) 6 Cells) were implanted subcutaneously (sc) into the right hind flank of 6- to 8-week-old female A / J mice (Envigo, The Netherlands). In these mice, tumors were palpable (approximately 50 mm in size) 7 days after tumor inoculation. 3), PBS (50 μl) or various engineered oncolytic Semliki Forest viruses (1 × 10 in 50 μl PBS) were used. 11 The subjects were treated intratumorally (it) with VP.

[0105] Animal monitoring, tumor size measurements, and survival curves were performed as described in Example 7.

[0106] Both oncolytic viruses significantly inhibited tumor growth and extended survival of mice (Figure 10). On the other hand, co-expression of NAP and immunogenic GD2 mimotope in A774-NAPGD2 did not result in tumor growth inhibition or extended survival of these mice compared to oncolytic viruses expressing single factors (GD2 or NAP) (Figure 10). These data suggest that the synergistic effect obtained by co-expressing immunomodulatory factors and NAP does not occur when NAP is co-expressed with immunogens.

[0107] It should be understood that the above-mentioned embodiments are a few specific examples of the present invention. Those skilled in the art will understand that various modifications, combinations and variations can be made to the embodiments without departing from the scope of the present invention. In particular, it is possible to combine different part solutions in different embodiments using other configurations that are technically possible.

Claims

1. a nucleic acid sequence encoding the Helicobacter pylori neutrophil-activating protein (NAP) and / or an immunologically equivalent fragment of NAP; and A nucleic acid sequence encoding an immunomodulator capable of inducing an immune response in a subject; An adenovirus comprising: wherein the immunologically equivalent fragment of NAP is a fragment containing at least one polypeptide domain of at least 20 amino acid residues of NAP; Adenovirus.

2. 2. The adenovirus of claim 1, wherein the immunomodulatory agent is capable of inducing dendritic cell (DC) maturation, T cell activation and / or NK cell activation in the subject.

3. The adenovirus of claim 2, wherein the immunomodulatory agent is capable of inducing DC maturation, T cell activation and NK cell activation in the subject.

4. The adenovirus of claim 2, wherein the immunomodulatory factor is capable of inducing expression of cluster of differentiation 80 (CD80), CD40, CD86 and C-C chemokine receptor type 7 (CCR7) by dendritic cells.

5. The adenovirus of claim 2, wherein the immunomodulatory factor is capable of inducing CD4+ T cell activation and / or CD8+ T cell activation.

6. The adenovirus of claim 5, wherein the immunomodulatory factor is capable of inducing expression of cluster of differentiation 69 (CD69) and CD107a by CD4+ T cells and / or CD8+ T cells.

7. The adenovirus of claim 2, wherein the immunomodulatory factor is capable of inducing activation of CD56+ NK cells.

8. 8. The adenovirus of claim 7, wherein the immunomodulatory factor is capable of inducing expression of cluster of differentiation 69 (CD69) and CD107a by CD56+ NK cells.

9. The adenovirus according to any one of claims 1 to 8, wherein the immunomodulatory factor is a member of the tumor necrosis factor superfamily (TNFSF).

10. 10. The adenovirus of claim 9, wherein the TNFSF member is selected from the group consisting of TNFSF1, TNFSF2, TNFSF4, TNFSF5, TNFSF7, TNFSF9, TNFSF14, TNFSF18 and combinations thereof.

11. 11. The adenovirus of claim 10, wherein the TNFSF member is selected from the group consisting of TNFSF5, TNFSF9, TNFSF14, TNFSF18 and combinations thereof.

12. 12. The adenovirus of claim 11, wherein the TNFSF member is selected from the group consisting of TNFSF9, TNFSF18, and combinations thereof.

13. The adenovirus according to any one of claims 1 to 8, wherein the immunologically equivalent fragment of NAP is a fragment comprising at least one polypeptide domain of at least 30 amino acid residues of NAP, more preferably at least 40 amino acid residues.

14. The adenovirus according to any one of claims 1 to 8, wherein the immunologically equivalent fragment of NAP is selected from the group consisting of SEQ ID NOs: 11 to 14.

15. The adenovirus according to any one of claims 1 to 8, further comprising a nucleic acid sequence encoding a self-cleaving peptide located between the nucleic acid sequence encoding NAP and / or an immunologically equivalent fragment of NAP and the nucleic acid sequence encoding said immune modulator.

16. The adenovirus of claim 15, wherein the self-cleaving peptide is selected from the group consisting of SEQ ID NOs: 16-23.

17. The adenovirus according to any one of claims 1 to 8, wherein the adenovirus is an oncolytic adenovirus.

18. The adenovirus of claim 17, wherein the oncolytic adenovirus comprises a mutant adenovirus early region 1A (E1A) gene encoding a mutant E1A protein having a lower Rb protein binding ability compared to a wild-type E1A protein.

19. 20. The adenovirus of claim 18, wherein the mutant E1A gene comprises a 24 bp deletion corresponding to nucleotides 919 to 943 of the wild-type E1A gene.

20. 20. The adenovirus of claim 18, wherein the mutant E1A protein lacks amino acids 121 to 128 of the wild-type E1A protein.

21. The adenovirus according to any one of claims 1 to 8, wherein one of the nucleic acid sequences encoding the 19 kDa adenoviral E1B protein and the 55 kDa adenoviral E1B protein is replaced with a nucleic acid sequence encoding NAP and / or an immunologically equivalent fragment of NAP; and An adenovirus, wherein the other of the nucleic acid sequences encoding the 19 kDa adenoviral E1B protein and the 55 kDa adenoviral E1B protein has been replaced with a nucleic acid sequence encoding said immunomodulator.

22. The adenovirus according to any one of claims 1 to 8, wherein the adenovirus is human adenovirus type 5.

23. The adenovirus according to any one of claims 1 to 8, which is used as a therapeutic agent.

24. The adenovirus according to any one of claims 1 to 8, which is used in cancer treatment.

25. 25. The adenovirus for use according to claim 24, wherein the cancer is selected from the group consisting of carcinoma (such as pancreatic cancer, breast cancer, lung cancer, liver cancer, or renal cancer); sarcoma (such as osteosarcoma or liposarcoma); lymphoma (such as non-Hodgkin's lymphoma or Hodgkin's lymphoma); leukemia (such as acute leukemia or chronic leukemia); seminoma; germinoma; dysgerminoma; and blastoma (such as glioblastoma or neuroblastoma).

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