Immunotherapy using GGTA and nap

Abstract

The present invention relates to a vector, such as a viral vector, comprising a first nucleic acid sequence encoding a glycosyltransferase (such as a galactosyltransferase), an enzymatically active variant or truncated form thereof; and a second nucleic acid sequence encoding a Helicobacter pylori neutrophil-activating protein (NAP), an immunologically equivalent variant or fragment thereof. The invention also relates to a vector system, an isolated cell population, a polynucleotide and relating gene therapy systems comprising said first and said second nucleic acid sequence. Related methods, kits, pharmaceutical compositions and medical uses, in particular, in the treatment of cancer, such as pancreatic cancer, are also disclosed.

Description

[0001] IMMUNOTHERAPY USING GGTA AND NAP

[0002] TECHNICAL FIELD

[0003] The present invention relates to a vector, such as a viral vector, comprising a first nucleic acid sequence encoding a glycosyltransferase (such as a galactosyltransferase), an enzymatically active variant or truncated form thereof; and a second nucleic acid sequence encoding a Helicobacter pylori neutrophilactivating protein (NAP), an immunologically equivalent variant or fragment thereof. The invention also relates to a vector system, an isolated cell population, a polynucleotide and relating gene therapy systems comprising said first and said second nucleic acid sequence. Related methods, kits, pharmaceutical compositions and medical uses, in particular, in the treatment of cancer, such as pancreatic cancer, are also disclosed.

[0004] BACKGROUND

[0005] Cancer is the second leading cause of death in the world, and pancreatic cancer is the fourth leading cause of cancer-related deaths in Europe and North- America. Survival rates vary by cancer type and by the stage at which it is diagnosed, ranging from majority survival to complete mortality five years after diagnosis. Once a cancer has metastasized, prognosis normally becomes much worse. A majority of cancer deaths are due to metastases of the primary tumor.

[0006] Low overall survival of cancer patients may be attributed to that existing treatment options for cancer are often not curative nor lead to the remission or regression of the disease. The primary treatment alternatives include surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy and palliative care. Cancer immunotherapies exploiting the fact that cancer cells often have tumor antigens are also emerging treatment options, but the clinical success of existing cancer immunotherapies is highly variable between different forms of cancer. Moreover, available treatment options are often not well-tolerated by cancer patients and are accompanied by prominent adverse effects. The choice of treatment alternatives, in general, depends on the type, location and grade of the cancer as well as the patient's health and preferences. For example, in case of pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC), surgical resection remains the only potentially curative treatment. However, only 20% of diagnosed patients have resectable disease and are eligible for surgery. Even so, most of these patients still relapse with a median survival time of only 25-28 months (Neoptolemos JP, et al. The Lancet 2017, 389(10073), 1011-1024). For the remaining 80% of patients, chemotherapy only provides marginal improvement of overall survival to about five months (Nymo LS, et al. BJS Open 2022, 6(2)). For patients with resectable PDAC, surgery followed by combinations of chemotherapy (Neoptolemos JP, et al. Lancet 2017, 389(10073):1011-1024), is the most universally accepted approach, and it is at present the standard of care in the Nordic countries. However, it is well known that initiation and completion of adjuvant chemotherapy can be precluded by treatment complications (Tzeng CW, et al. J gastrointestinal surgery 2014, 18(1) : 16-24). Complications after pancreatectomy affect 40-50 % of the patients and perioperative mortality rate is high (2-4 %) (Ziegler KM, et al. Surgery 2010, 148(4):702-710). Complications are both surgical (bleeding, anastomotic leak, obstruction, etc. necessitating reinterventions) and medical (infection, pneumonia, myocardial infarction, and venous thromboembolic events etc.). The technical complexity of the operation and the commonly frail and co- morbid patient population contribute to the high rate of complications, leaving less than 70% of the resected patient population to complete the intended adjuvant chemotherapy, and many patients have early disease progression within a few months after resection (Tzeng CW, et al. J gastrointestinal surgery 2014, 18( 1) : 16- 24). Early distant recurrence in patients with early-stage settings support the concept that pancreatic cancer is a systemic disease (Sohal DP, et al. Journal of the National Cancer Institute 2014, 106(3):dju011) and for most patients affected with PDAC (80%) only palliative care can be offered.

[0007] Accordingly, despite of the existence of current treatment options, cancer, especially cancer types that are difficult-to-treat, and / or treatment methods that are associated with severe adverse side effects remain a challenge in the field of oncology. Hence, there is a huge urgent need for novel efficacious treatment modalities for cancer, such as pancreatic cancer (e.g. PDAC), and in particular, treatment alternatives which are well-tolerated by cancer patients.

[0008] SUMMARY

[0009] An object of the present disclosure is to provide novel means and medical uses thereof that reduce, or at least partially overcome, challenges in the prior art. It is an object of the present disclosure to provide means that are beneficial in the treatment of cancer. In particular, it is an object of the present disclosure to provide means that are beneficial in the treatment of certain cancer types that are difficult to treat using treatment alternatives available in the prior art, such as pancreatic cancer (e.g. PDAC). Moreover, it is also an object to provide means that are safe for medical use, in particular, that are well-tolerated by cancer patients. It is also an object of the present disclosure to provide components and methods that are useful for the preparation of such means. These and other objects are achieved in full, or at least in part, by aspects of the inventive concepts as disclosed herein.

[0010] In a first aspect of the present disclosure is provided a vector comprising

[0011] (i) a first nucleic acid sequence encoding a glycosyltransferase (such as a galactosyltransferase), an enzymatically active variant thereof, or an enzymatically active truncated form thereof; and

[0012] (ii) a second nucleic acid sequence encoding a Helicobacter pylori neutrophilactivating protein (NAP), an immunologically equivalent variant thereof, or an immunologically equivalent fragment thereof.

[0013] In some embodiments, said glycosyltransferase is galactosyltransferase, such as an N-acetyllactosaminide alpha-1, 3-galactosyltransferase. In some embodiments, said vector is a viral vector, such as an adenovirus. In some embodiments, said vector is formulated for in vitro transfection or transduction of cells or for in vivo administration to a subject. In one embodiment, said vector is formulated for in vitro transfection or transduction of cells. In one particular embodiment, said vector is formulated for in vivo administration to a subject.

[0014] In a second aspect is provided a vector system for simultaneous or subsequent delivery of a first and a second vector to a cell and / or for generating an isolated cell population as defined in a following aspect of the present disclosure, said vector system comprising

[0015] (a) the first vector comprising said first nucleic acid sequence as defined above, and

[0016] (b) the second vector comprising said second nucleic acid sequence as defined above; wherein said first and said second vectors are separate vectors.

[0017] In some embodiments, said first and / or said second vector is a viral vector, such as an adenovirus. In some embodiments, the vector system is formulated for in vitro transfection or transduction of cells or for in vivo administration to a subject.

[0018] In a third aspect, there is provided a method of in vitro transfection or transduction of cells with the vector or the vector system as defined above.

[0019] In a related fourth aspect of the present disclosure, is provided a use of the vector or the vector system, as defined above, in a method of in vitro transfection or transduction of cells.

[0020] In a fifth aspect, there is provided an isolated cell population comprising said first nucleic acid sequence as defined above and / or a glycosyltransferase (such as a galactosyltransferase), an enzymatically active variant thereof, or an enzymatically active truncated form thereof encoded by said first nucleic acid sequence; and said second nucleic acid sequence as defined above and / or a NAP, an immunologically equivalent variant thereof or an immunologically equivalent fragment thereof encoded by said second nucleic acid sequence.

[0021] In some embodiments each cell of the isolated cell population comprises said first nucleic acid sequence as defined above and / or the glycosyltransferase (such as the galactosyltransferase), the enzymatically active variant thereof, or the enzymatically active truncated form thereof, encoded by said first nucleic acid sequence; and said second nucleic acid sequence as defined above and / or the NAP, the immunologically equivalent variant thereof or the immunologically equivalent fragment thereof, encoded by said second nucleic acid sequence. In some embodiments, the isolated cell population comprises two cell type populations, wherein

[0022] (c) a first cell type population comprises said first nucleic acid sequence as defined above and / or said glycosyltransferase (such as said galactosyltransferase), the enzymatically active variant thereof, or the enzymatically active truncated form thereof, encoded by said first nucleic acid sequence; and

[0023] (d) a second cell type population comprises said second nucleic acid sequence as defined above and / or said NAP, the immunologically equivalent variant thereof, or the immunologically equivalent fragment thereof, encoded by said second nucleic acid sequence.

[0024] In some embodiments, the isolated cell population is obtained by the above method of in vitro transfection or transduction of cells. In some embodiments, said isolated cell population is formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, intralymphatic or intratumoral administration to a subject.

[0025] In a sixth aspect is provided a polynucleotide comprising said first and said second nucleic acid sequences as defined above.

[0026] In a seventh aspect, there is provided a method for producing the vector as defined above, wherein said vector is a viral vector, said method comprising generating a modified viral vector construct by operably linking a viral backbone to the first and the second nucleic acid sequence as defined above, transfecting mammalian cells with the modified viral vector construct, culturing the mammalian cells in conditions suitable for viral replication, and harvesting the viral particles. In an eighth aspect, there is provided an RNA system, such as an mRNA system, for simultaneous or subsequent delivery of a first and a second RNA to a cell, said RNA system comprising the first RNA, such as mRNA, encoding a glycosyltransferase (such as a galactosyltransferase), an enzymatically active variant thereof, or an enzymatically active truncated form thereof, as defined herein, and the second RNA, such as mRNA, encoding a Helicobacter pylori neutrophil-activating protein (NAP), an immunologically equivalent variant thereof, or an immunologically equivalent fragment thereof, as defined herein; wherein optionally said first and said second RNA are separate RNAs.

[0027] In a ninth aspect, there is provided a kit comprising the vector, the vector system, the polynucleotide, or the RNA system (such as the mRNA system), as defined above; and instructions for use. In some embodiments, the kit comprises the vector, the vector system, or the polynucleotide, as defined above; and instructions for use.

[0028] In a tenth aspect, a pharmaceutical composition is provided, said pharmaceutical composition comprising the vector, the vector system, the isolated cell population, or the RNA system (such as the mRNA system), as defined above; and at least one pharmaceutically acceptable salt, carrier and / or excipient. In some embodiments, the pharmaceutical composition comprises the vector, the vector system, or the isolated cell population, as defined above; and at least one pharmaceutically acceptable salt, carrier and / or excipient. In some embodiments, said pharmaceutical composition is formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, intralymphatic or intratumoral administration to a subject.

[0029] In an eleventh aspect is provided the vector, the vector system, the isolated cell population, and / or the RNA system (such as the mRNA system), as defined above, for use as a medicament. In some embodiments is provided the vector, the vector system, and / or the isolated cell population, as defined above, for use as a medicament.

[0030] In a twelfth aspect is provided the vector, the vector system, the isolated cell population, and / or the RNA system (such as the mRNA system), as defined above, for use in the treatment of cancer. In one embodiment is provided the vector, the vector system, and / or the isolated cell population, as defined above, for use in the treatment of cancer. In some embodiments, said cancer is carcinoma or melanoma. In some embodiments, said cancer is carcinoma, such as pancreatic cancer, such as PDAC, such as stage IV PDAC. I some embodiments, said cancer is melanoma.

[0031] In a thirteenth aspect, there is provided a gene therapy system for simultaneous or subsequent administration thereof to a subject, comprising

[0032] (h) the first and the second cell type population as defined above;

[0033] (i) the first and the second vector as defined above;

[0034] (j) the first cell type population and the second vector as defined above; or

[0035] (k) the second cell type population and the first vector as defined above.

[0036] In some embodiments, said first cell type population, said second vector, said second cell type population and / or said first vector of the gene therapy system is formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, intra lymphatic or intratumoral administration to the subject.

[0037] In a fourteenth aspect is provided the gene therapy system as defined above, for use as a medicament.

[0038] In a fifteenth aspect is provided the gene therapy system as defined above, for use in the treatment of cancer. In some embodiments, said cancer is carcinoma or melanoma. In some embodiments, said cancer is carcinoma, such as pancreatic cancer, such as PDAC, such as stage IV PDAC. I some embodiments, said cancer is melanoma.

[0039] In a sixteenth aspect, there is provided a method of treatment of a subject in need thereof, comprising administration of the vector, the vector system, the isolated cell population, and / or the RNA system (such as the mRNA system), as defined above, to the subject in need thereof. In one embodiment, the method comprises administration of the vector, the vector system, and / or the isolated cell population, as defined above, to the subject in need thereof.

[0040] In a seventeenth aspect, there is provided a method of treatment of cancer, comprising administration of the vector, the vector system, the isolated cell population, and / or the RNA system (such as the mRNA system), as defined above, to a subject in need thereof. In one embodiment, the method comprises administration of the vector, the vector system, and / or the isolated cell population, as defined above, to the subject in need thereof.

[0041] In an eighteenth aspect, there is provided a method of treatment of a subject in need thereof, comprising administration of the gene therapy system as defined above, to the subject in need thereof.

[0042] In a nineteenth aspect, there is provided a method of treatment of cancer, comprising administration of the gene therapy system as defined above, to a subject in need thereof.

[0043] In a twentieth aspect, is provided a use of the vector, the vector system, the isolated cell population, and / or the RNA system (such as the mRNA system), as defined above, in the manufacture of a medicament. In one embodiment is provided the use of the vector, the vector system, and / or the isolated cell population, as defined above, in the manufacture of a medicament.

[0044] In a twenty-first aspect, is provided a use of the vector, the vector system, the isolated cell population, and / or the RNA system (such as the mRNA system), as defined above, in the manufacture of a medicament for the treatment of cancer. In one embodiment, is provided the use of the vector, the vector system, and / or the isolated cell population, as defined above, in the manufacture of a medicament for the treatment of cancer.

[0045] In a twenty-second aspect, is provided a use of the gene therapy system as defined above, in the manufacture of a medicament.

[0046] In a twenty-third aspect, is provided a use of the gene therapy system as defined above, in the manufacture of a medicament for the treatment of cancer. Brief description of the drawings

[0047] Fig. 1 shows schematic representation of adenoviral constructs used in the appended Examples as defined in Example 1.

[0048] Fig. 2 presents oncolytic ability of adenoviruses as defined in Example 1 using different cancer cell lines, as described in Example 2.

[0049] Fig. 3 shows assessment of the presence of a-Gal epitopes (Fig. 3A) and NAP expression (Fig. 3B) in different cancer cell lines infected by adenoviruses as defined in Example 1. (MFI: Mean Fluorescence Intensity, *upper band corresponding to NAP; statistical analysis: one-way ANOVA with Tukey's multiple comparison test, p*<0.05, p**<0.01, ****p<0.0001)

[0050] Fig. 4 presents evaluation of calreticulin (CRT) exposure on different cancer cell lines infected by adenoviruses as defined in Example 1. (MFI: Mean Fluorescence Intensity; statistical analysis: one-way ANOVA with Tukey's multiple comparison test, p*<0.05, p**<0.01)

[0051] Fig. 5A and 5B shows assessment of molecular markers indicative of dendric cell (DC) phenotypic maturation, when immature CDllc+DCs are co-cultured with different cancer cell lines infected by adenoviruses as defined in Example 1. (MFI: Mean Fluorescence Intensity; statistical analysis: one-way ANOVA with Tukey's multiple comparison test, p**<0.01)

[0052] Fig. 6 presents evaluation of a-Gal-IgG binding on different cancer cell lines infected by adenoviruses as defined in Example 1, wherein the percentage of IgG- bound cells out of live cells was measured (statistical analysis: one-way ANOVA with Tukey's multiple comparison test, p*<0.05, p**<0.01, p***<0.001, ****p<0.0001).

[0053] Fig. 7 shows experimental design of Example 7, wherein Panc-01 cells were infected with adenoviruses as defined in Example 1 and were tested in co-culture with serum (Fig. 7A) as well as evaluation of C3b deposition (Fig. 7B) and complement-dependent cytotoxicity (CDC, Fig. 7C) following exposure of the virus- infected cells to rabbit complement, wherein the percentage of C3b+cells out of live cells (Fig. 7B) and the percentage of dead cells (Fig. 7C) were measured (statistical analysis: one-way ANOVA with Tukey's multiple comparison test or t-test, p*** <0.001, ****p<0.0001).

[0054] Fig. 8 shows experimental design of Example 8, wherein Panc-01 cells were infected with adenoviruses as defined in Example 1 and were tested in co-culture with serum and isolated natural killer (NK) cells (Fig. 8A) as well as evaluation of CD107a expression by NK cells (Fig. 8B) indicative of antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells against antibody labeled target cells. (MFI: Mean Fluorescence Intensity; statistical analysis: t-test, p*<0.05)

[0055] Fig. 9 presents experimental design of Example 9 (Fig. 9A) and results (Fig. 9B- 9J) for the evaluation of immune response induced in vitro by Panc-01 cells which have been transduced by either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1, and were co-cultured with human serum and isolated human peripheral blood mononuclear cells (PBMCs). Fig. 9B and 9C show assessments of anti-a-Gal IgG antibody binding and C3b deposition, respectively, on Panc-01 cells, Fig. 9D-9F presents the assessment of phagocytic monocytes (Fig. 9D) and molecular markers of monocyte activation (Fig. 9E and 9F), Fig. 9G and 9H-9I show evaluation of molecular markers indicative of B cell (Fig. 9G) and NK cell maturation (Fig. 9H and 91) and Fig. 9J presents the assessment of relative viability of the cancer cells. (MFI: Mean Fluorescence Intensity; statistical analysis: one-way ANOVA with Tukey's multiple comparison test, p*<0.05, p**<0.01, p***<0.001, ****p<0.0001)

[0056] Fig. 10 presents the outline of the in vivo experiment described in Example 10 (Fig. 10A), wherein tumor growth over time and mice survival were evaluated (Fig. 10B and Fig. 10C) in a-Gal knock-out (KO) mice treated either by Ad(onco), Ad(onco- A), Ad(onco-B) or Ad(onco-AB) (statistical analysis: tumor size was analyzed by two- way ANOVA with Tukey's multiple comparisons and mice survival was analyzed by long-rank (Mantel-Cox) text, p*<0.05, p**<0.01, p***<0.001, ****p<0.0001).

[0057] Fig. 11 shows serum levels of anti-adenovirus IgG at necropsy of Syrian hamsters treated with different batches and dosages of Ad(onco-AB). Untreated, PBS-treated and Ad(Mock)-treated animals served as internal reference. The treatment groups correspond to the treatment groups shown in Table 6. IgG levels were measured by ELISA, at OD450 (optical density at 450 nm) as explained in Example 12 (statistical analysis: Mann Whitney U test, p*<0.05).

[0058] Fig. 12 presents evaluation of biochemical (Fig. 12A) and hematological (Fig.

[0059] 12B) analysis of blood samples collected at necropsy from hamsters of different treatment groups, as explained in Example 16 and Table 6. Individuals with hemolysis index above three are marked with a black circle where relevant. (B-: blood-, Hb: hemoglobin, EVF: erythrocyte volume fraction, LPC: leukocyte particle count, TPC: thrombocyte particle count, ALT: alanine aminotransferase, AST: aspartate aminotransferase and ALP: alkaline phosphatase; statistical analysis: Kruskal-Wallis and Mann-Whitney U-test)

[0060] Fig. 13 shows results of a comparative analysis of a viral construct according to the present disclosure (Ad(onco-AB) in comparison to controls and viral constructs anticipated by the prior art (such as AD(onco-AG)), as described in Example 17, wherein cell lytic effect (Fig. 13A), transgene expression (Fig. 13B) and the effect on Toll-like receptor 2 (TLR2) activation are measured (Fig. 13C). (MOI: multiplicity of infection; SEAP: secreted embryonic alkaline phosphatase)

[0061] Fig. 14 shows nucleic acid sequences corresponding to SEQ ID NO:l-4 as described in Example 1.

[0062] DETAILED DESCRIPTION

[0063] The present disclosure relates to new vectors, vector systems, isolated cell populations and gene therapy systems that are advantageous for a therapeutic use, especially in the treatment of cancer, such as the treatment of pancreatic cancer (e.g. PDAC). As disclosed herein, the new vectors, vector systems, isolated cell populations and gene therapy systems are suitable for various gene therapy modalities for the treatment of cancer.

[0064] The terminology used herein is for the purpose of describing particular aspects of the disclosure only, and is not intended to limit the disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0065] As used herein, when the term "about" or "approximately" is used in relation to a numerical value, it is to be interpreted as a range of ± 10 %, such as ± 9 %, such as ± 8 %, such as ± 7 %, such as ± 6 %, such as ± 5 %, such as ± 4 50%, such as ± 3 %, such as ± 2 %, such as ± 1 %. For example, when the value is stated to be about 10, this means that the value is in fact in the range of from 9 to 11, such as in the range of from 9.9 to 10.9, such as in the range of from 9.8 to 10.8, such as in the range of from 9.7 to 10.7, such as in the range of from 9.6 to 10.6, such as in the range of from 9.5 to 10.5, such as in the range of from 9.4 to 10.4, such as in the range of from 9.3 to 10.3, such as in the range of from 9.2 to 10.2, such as in the range of from 9.1 to 10.1.

[0066] The term "vector" as used herein refers to a gene delivery vehicle for transferring recombinant genetic material, such as one or more nucleic acid sequences encoding one or more transgenes, into a target cell and / or tissue. The vector may be a vehicle comprising a nucleic acid molecule, such as a viral vector, or a nucleic acid molecule used as vehicle, such as a non-viral vector. The nucleic acid molecule may be a DNA or an RNA molecule. Vectors may be used to elicit manipulation of gene expression. Vectors may for example be plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. For example, a vector may be specifically adapted for the expression of a heterologous sequence (i.e. a transgene) in the target cell, and generally has a promoter sequence that drives expression of the heterologous sequences. Some vectors may only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed, unlike expression vectors. Transcription vectors are used to amplify the inserted heterologous sequences. The purpose of a vector according to present disclosure is typically to express transgenes in the target cell. The choice of vector employed in embodiments of the present invention depends on the specific application of the vector comprising the nucleic acid sequences of the invention. The term "non-viral vector" as used herein refers to a nucleic acid molecule used as vehicle suitable to transfer recombinant genetic material, such as one or more nucleic acid sequences encoding one or more transgenes, into a target cell and / or tissue. Non-limiting examples of non-viral vectors include naked DNA that is alone able to transfer an insert into a cell and / or a tissue, a plasmid DNA, an in vitro transcribed RNA and / or a modified RNA. Non-viral vectors as disclosed herein comprise one or more transgenes (i.e. one or more heterologous nucleic acid sequences) and a larger sequence region that serves as the "backbone" of the non- viral vector. Non-viral vectors may be delivered using and physical and / or chemical systems except viral systems. Non-limiting examples of chemical methods include cationic liposomes, lipid nanoparticles and cationic polymers. Non-limiting examples of physical methods include gene gun, electroporation, hydrodynamic, particle bombardment, ultrasound utilization and magnetofection. Introducing nucleic acids into a target cell by a non-viral vector is primarily referred to herein as a "transfection" of the target cell. Said transfection may be an in vitro or an in vivo transfection.

[0067] The term "viral vector" as used herein refers to a vehicle comprising a nucleic acid molecule suitable to transfer recombinant genetic material, such as one or more nucleic acid sequences encoding one or more transgenes (i.e. one or more heterologous nucleic acid sequences), into a target cell and / or tissue. Typically, a viral vector comprises three components: a protein capsid and / or envelope that encapsulates the genetic payload, one or more transgenes of interest and a "regulatory cassette", i.e. the combined enhancer / promoter / auxiliary elements that controls stable or transient somatic expression of the transgene(s) as an episome or as a chromosomal integrant. Non-limiting examples of viral vectors include retroviruses, lentiviruses, vaccinia viruses, Semliki Forest viruses, reoviruses, Newcastle disease viruses, herpes viruses, adenoviruses and adeno-associated viruses. Introducing nucleic acids into a target cell by a viral vector is primarily referred to herein as a "transduction" of the target cell. Said transduction may be an in vitro or an in vivo transduction. The terms "viral vector" and "virus" are used herein interchangeably.

[0068] The term "oncolytic virus" as used herein refers to a viral vector that preferentially infects and kills malignant cells, such as cancer cells. As the infected malignant cells, such as cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor.

[0069] The term "viral vector construct" is used herein with reference to a nucleic acid molecule, such as a DNA or an RNA molecule, that comprises one or more transgenes (i.e. one or more heterologous nucleic acid sequences) and a larger sequence region that serves as the "backbone" of the viral vector construct. A viral vector construct as referred to herein encodes a viral vector.

[0070] The term "foreign X" as used herein refers to a moiety ("X") that is not identical to any moiety of a subject's own cells and is recognized as being foreign by the subject's immune system. Such moiety may be a molecule expressed on the surface of a cell of the subject. The moiety may be a carbohydrate moiety. Accordingly, such foreign moieties are considered epitopes, which may be recognized by an antibody, such as a natural antibody, present in the subject. The terms "foreign" and "non-self" are used herein interchangeably.

[0071] The term "natural antibody" as used herein refers to immunoglobulin found in a subject without the requirement of prior antigenic experience. These antibodies are produced naturally in a subject as a response to the natural environment, e.g. the colonization by microbes of the airways and the gastrointestinal tract occurring after birth. The expression of natural antibodies is conserved in phylogeny.

[0072] The term "natural xenoreactive antibody" as used herein refers to a natural antibody in a subject that recognizes an antigen present on a xenogeneic tissue. Xenoreactive natural antibodies are for example thought to be responsible for initiating a hyperacute rejection of porcine organs transplanted into primates, thus appear to present an immune barrier to viral transmission and organ transplantation across species. The term "enzymatically active variant or truncated form of X" as used herein refers to a variant or a truncated form of X which is capable of at least the same or a similar degree of an enzymatic function corresponding to the enzymatic function of X. The enzymatic function may be measured by in vitro or in vivo experimental methods, for example as disclosed in the appended Examples, and as further explained below in relation to the first aspect of the present disclosure.

[0073] The term "immunologically equivalent to X" as used herein refers to a variant or a fragment of X that induces an immune response equivalent to, i.e. at least the same or stronger than, an immune response induced by said X. The immune response may be measured by in vitro or in vivo experimental methods, for example as disclosed in the appended Examples, and as further explained below in relation to the first aspect of the present disclosure.

[0074] The term "codon-optimized nucleic acid sequence" as used herein refers to a nucleic acid sequence designed for improved codon composition of a recombinant gene, such as a transgene, based on various criteria without altering the amino acid sequence of the recombinant gene. This is possible because most amino acids are encoded by more than one codon. In the context of the present disclosure, a codon- optimized nucleic acid sequence is optimized for improved translation in a cell, such as a human cell, such as a human malignant cell, such as a human cancer cell, into which the codon-optimized nucleic acid sequence has been delivered.

[0075] The term "upstream" as referred to herein is to be understood in a context wherein a nucleic acid sequence is positioned upstream from another nucleic acid sequence in a nucleic acid molecule, e.g. a nucleic acid sequence is located 5' of the 5'-end of another nucleic acid sequence in the nucleic acid molecule.

[0076] The term "autologous cell population" as used herein refers to a subject's, such as a patient's, cells (such as malignant cells, for example, cancer cells), which are cultured and expanded outside the body (i.e. ex vivo) and are reintroduced into the same subject.

[0077] The term "allogeneic cell population" as used herein refers to a cell population that is introduced into a subject, wherein the cell population is genetically dissimilar from said subject. The allogeneic cell population may be introduced to any subject. The allogeneic cell population is adept at satisfying an "off-the-shelf" supply. A cell population is considered allogeneic if the cell population belongs to the same species as the subject that receives the cell population but is genetically dissimilar from the subject. Non-limiting examples of allogeneic cell populations include cell lines and donor-derived cell populations.

[0078] For the sake of clarity, the terms "cancer" and "tumor" are used herein interchangeably. As used herein, the term "malignant cell" includes malignant cells which originate from epithelial tissues (in other words cells of carcinomas), as well as malignant cells which originate from non-epithelial tissues. To illustrate by example, the term "malignant cell" includes cancer cells and other malignant cells, such as cells from sarcoma, which is a rare type of malignancy that arises from cells of mesenchymal origin.

[0079] Although the application of fundamental research of cancer immunology is a growing subspecialty of oncology, available clinical experience utilizing a xenogeneic-like response in immune oncology remains limited. The present inventors have unexpectedly found a clinical translation of a conceptually new form of immunotherapy for cancer patients, such as patients with disseminated PDAC, harnessing the pre-existing natural immunity against the different glycosylation pattern across species. The present inventors herein demonstrate that immunity against foreign carbohydrate moieties, such as anti-a-Gal (anti-galactose-a-1,3- galactose) immunity, by in vivo or in vitro decoration of malignant cells, such as cancer cells, with such carbohydrate moieties, such as a-Gal epitopes, is surprisingly beneficial in cancer therapy in combination with transforming the immunosuppressive tumor microenvironment to a highly inflammatory environment which markedly boosts antigen-presenting cell (APC) activity to induce a broad multiclonal acquired anti-tumor T cell response. Based on the in vitro and in vivo experimental data demonstrated herein in the appended Examples, the inventors envision that a therapy utilizing this novel approach is specific, efficient and can be applied without major side effects. This novel approach is expected to be useful for the treatment of any form of cancer, and in particular, of cancer types that are difficult to treat. Such cancer type for example is PDAC, for which there are no curative treatments available to date.

[0080] In particular, the present inventors have surprisingly found that glycomic differences in phylogeny and between species that allow for molecular discrimination between self and non-self (i.e. foreign) are particularly advantageous in an immunotherapy according to the present disclosure. The inventors demonstrate that differences in glycan structures are powerful activators of the innate immune system and constitute a foundation in the communication between innate and acquired immunity. The immune system of a subject, such as a human subject, recognizes non-self carbohydrate moieties, such as non-self glycan structures, as foreign and produces antibodies against such foreign moieties. In vivo or in vitro decoration of malignant cells, such as cancer cells, with such carbohydrate moieties will thus be recognized as foreign by a patient's own immune system.

[0081] Furthermore, the present inventors have found that reducing local tumor immune suppression is particularly beneficial in an immunotherapy as disclosed herein, using the virulence factor neutrophil-activating protein of Helicobacter pylori (NAP or HP-NAP), an immunologically equivalent variant thereof, or an immunologically equivalent fragment thereof. The inventors hereby demonstrate that NAP according to the present disclosure is chemotactic for neutrophils and monocytes and activates them to produce T helper type 1 (Thl)-associated cytokines, thereby expanding cytolytic T cells. The microenvironment of most solid tumors, such as pancreatic cancer, is highly immune suppressive and antiinflammatory, such as T helper type 2 (Th2) cell polarized. The inventors have found that an inflammatory Thl polarized immune response induced by NAP and the activation of cytolytic T cells lead to a surprisingly efficient anti-tumor immune activity in combination with the decoration of malignant cells, such as cancer cells, with the above described foreign carbohydrate moieties. The unexpectedly beneficial character of the herein disclosed novel approach utilizing the decoration of malignant cells, such as cancer cells, with foreign carbohydrate moieties and the secretion of NAP, or an immunologically equivalent variant or fragment thereof, is demonstrated in the appended Examples and is described below in more detail. The inventors show that a foreign carbohydrate moiety (i.e. epitope), such as a-Gal, decorated malignant cells (e.g. cancer cells) and NAP secretion by the malignant cells (e.g. cancer cells) according to the present disclosure induce naturally occurring antibody binding to the foreign epitopes and cause cell-lysis via complement activation and NK-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC). By so, the immune protective microenvironment in the tumor changes to a proinflammatory environment with optimal conditions for immunosurveillance. Moreover, proteins and lipids decorated with the epitope are subjected to markedly enhanced antigen presentation and trigger acquired immune responses to all foreign epitopes. Indirect-antigen presentation of these by endogenous APCs activate CD4+ T cells, and these highly activated CD4+ T cells subsequently accumulate in the target tissue and induce a vigorous delayed-type hypersensitivity (DTH)-like response by activating monocytes and macrophages. NAP activity increases pro-inflammatory cytokines in the systemic circulation and neutrophil infiltration in the tumor bed leading to severe necrosis in tumors. NAP activity induces maturing of dendritic cells (DCs) and induces them to secrete Thl- type immune polarizing cytokines. In brief, the herein disclosed conceptually new form of immunotherapy as demonstrated in the appended Examples, enhances immune surveillance of the tumor microenvironment by deposition of natural antibodies, such as natural xenoreactive antibodies, and induces complement activation and create optimal conditions for antigen-presentation leading to forceful activation of all types of immune cells against the malignant cells, such as cancer cells.

[0082] The in vivo or in vitro decoration of malignant cells, such as cancer cells, may be achieved by transduction or transfection of the cells with a nucleic acid sequence encoding a glycosyltransferase, such as a galactosyltransferase. Said glycosyltransferase or said galactosyltransferase may be capable of transferring a foreign carbohydrate moiety to lipids and / or proteins of a cell expressing the glycosyltransferase or the galactosyltransferase, respectively. As apparent from the above description, said foreign carbohydrate moiety is foreign with respect to a subject, such as a primate subject, such as an old-world primate subject or a human subject, such as a human subject, comprising said cell. Accordingly, the immune system of said subject produces antibodies, such as natural antibodies, such as xenoreactive natural antibodies, against such foreign carbohydrate moieties. Without being bound by theory, such xenoreactive natural antibodies are understood to be responsible for an immune response in the subject similar to an immune response initiating a hyperacute rejection of an organ transplanted into the subject, wherein the organ expresses epitopes recognized by these antibodies. The present inventors envision that an enzymatically active variant or an enzymatically active truncated form of the glycosyltransferase (such as of the galactosyltransferase) is also suitable for the in vivo or in vitro decoration of malignant cells, such as cancer cells, as disclosed herein.

[0083] Secretion of NAP by malignant cells, such as cancer cells, may be achieved by the transduction or transfection of the cells with a nucleic acid sequence encoding a Helicobacter pylori neutrophil-activating protein (NAP). Furthermore, the inventors envision that an immunologically equivalent variant or an immunologically equivalent fragment of NAP are also suitable as virulence factors according to the present disclosure.

[0084] As defined above, transfection or transduction of cells, such as malignant cells, such as cancer cells, may be achieved by a vector that is capable to transfer recombinant genetic material, such as one or more nucleic acid sequences, into a host cell.

[0085] Thus, in a first aspect of the present disclosure is provided a vector comprising (i) a first nucleic acid sequence encoding a glycosyltransferase (such as a galactosyltransferase), an enzymatically active variant thereof, or an enzymatically active truncated form thereof; and (ii) a second nucleic acid sequence encoding a Helicobacter pylori neutrophilactivating protein (NAP), an immunologically equivalent variant thereof, or an immunologically equivalent fragment thereof.

[0086] In one particular embodiment of the first aspect of the present disclosure is provided the vector comprising (i) the first nucleic acid sequence encoding the glycosyltransferase (such as the galactosyltransferase); and (ii) the second nucleic acid sequence encoding the Helicobacter pylori neutrophil-activating protein (NAP). In another particular embodiment, is provided the vector comprising (i) the first nucleic acid sequence encoding the galactosyltransferase; and (ii) the second nucleic acid sequence encoding the Helicobacter pylori neutrophil-activating protein (NAP).

[0087] It is appreciated that said glycosyltransferase (such as the galactosyltransferase) is enzymatically active. In one embodiment, said glycosyltransferase or said galactosyltransferase is able to transfer a foreign carbohydrate moiety to lipids and / or proteins of a cell expressing the glycosyltransferase or the galactosyltransferase, respectively. Said cell may be a malignant cell, such as a cancer cell. Said cell may be a human malignant cell, such as a human cancer cell. As discussed above, said foreign carbohydrate moiety is foreign (i.e. non-self) with respect to a subject, such as a primate subject, such as an old- world primate subject or a human subject, such as a human subject. In other words, said cell prior to expressing the glycosyltransferase or the galactosyltransferase does not comprise the foreign carbohydrate moiety. Moreover, none of the cells of said subject comprises the glycosyltransferase or the galactosyltransferase, nor the foreign carbohydrate moiety. Consequently, the cell and the subject do not encode the glycosyltransferase or the galactosyltransferase prior to the cell being engineered for the expression thereof. As explained above, said carbohydrate moiety may be recognized by antibodies present in a subject comprising said cell. Said subject may be a primate subject, such as an old-world primate subject or a human subject, such as a human subject. Said antibodies may be natural antibodies, such as natural xenoreactive antibodies. According to the above principle, the present inventors envision that several glycosyltransferases (such as a galactosyltransferase) are suitable for the herein disclosed purpose, wherein the natural capacity of the immune system is reinforced to identify non-self molecules (such as non-self moieties) engineered according to the present disclosure. The present inventors expect that selectivity is obtained due to established tolerance towards self molecules (such as self moieties) of the subject (i.e. molecules, such as moieties, that do not induce an immune response in the subject and / or are not recognized by antibodies present in the subject) but not towards non-self molecules, such as non-self moieties, of the subject. In some embodiments, said glycosyltransferase is selected from a group consisting of hexosyltransferases and pentosyltransferases. For example, the foreign carbohydrate moiety may be a-Gal or a carbohydrate related to the blood group A and blood group B antigens. These foreign carbohydrate moieties are expected by the present inventors to be recognized by antibodies, such as by xenoreactive natural antibodies, of the subject comprising the cell that expresses the glycosyltransferase. The human ABO(H) blood group antigens are produced by specific glucosyltransferase enzymes (GTs) and the ABO blood group system is determined by what type of glucosyltransferases are expressed in the body: N-acetylgalactosaminyltransferase (GTA) or Galactosyltransferase (GTB). The final step in the enzymatic synthesis of the ABO(H) blood group A and B antigens is catalyzed by two closely related glycosyltransferases, an alpha-(l->3)-N-acetylgalactosaminyltransferase (GTA) and an alpha-(l->3)-galactosyltransferase (GTB). The A antigen on red blood cells is synthesized by the A glycosyltransferase (al,3-N-acetylgalactosaminyltransferase) adding GalNAc in an a(l,3)-linkage to Gal within the H antigen. The B antigen arises when the B glycosyltransferase (al,3-galactosyltransferase) adds Gal in an a(l,3)- linkage to the H antigen terminal Gal. Thus, in some embodiments, said glycosyltransferase is a glucosyltransferase, such as a glucosyltransferase selected from a group consisting of al,3-N-acetylgalactosaminyltransferase and al, 3- galactosyltransferase. As it is appreciated by those skilled in the art, in case of foreign carbohydrate moieties relating to the blood group A and blood group B antigens, the foreign carbohydrate moiety may be selected for non-compatibility with the subject according to the blood type of said subject. In view of the above, in one embodiment, said glycosyltransferase is selected from a group consisting of al,3-N-acetylgalactosaminyltransferase, al,3-galactosyltransferase and N- acetyllactosaminide alpha-1, 3-galactosyltransferase (a-l,3-GT). The inventors herein demonstrate that a-l,3-GT and a-Gal produced by a-l,3-GT may be particularly beneficial in a vector as disclosed herein. Thus, in one particular embodiment, said foreign carbohydrate moiety is a-Gal. Galactose-alpha-l,3-galactose, commonly known as a-Gal, is a sugar structure found in glycoproteins and glycolipids from all mammals except the old-world primates, e.g. chimpanzees and gorillas, and humans. Most mammals, except humans and old-world non-human primates, express a Ggtal gene encoding the N-acetyllactosaminide a-1,3- galactosyltransferase enzyme, which synthesizes Gal-al-3Gal-|31-4GlcNAc (a-Gal). Thus the carbohydrate moiety may be produced by an enzyme transcribed from a Ggtal gene encoding a-l,3-GT. In humans, the immune system recognizes a-Gal as foreign and produces xenoreactive antibodies against a-Gal. This immune response is considered for example a leading cause of organ hyperacute rejection after xenotransplantation. Anti a-Gal antibodies are some of the most common in humans. Regular stimulation from gut flora, typically initiated within the first six months of life, leads to an exceptionally high titer of anti-a-Gal antibodies representing around 1% of all circulating immunoglobulin G (IgG) and an even larger proportion of immunoglobulin M (IgM ) . Thus, in some embodiments, antibodies that recognize the foreign carbohydrate moiety, such as a-Gal, are present in the subject prior to the cell expressing the glycosyltransferase, such as a-l,3-GT. In one embodiment, said glycosyltransferase is thus a galactosyltransferase. In a particular embodiment, said galactosyltransferase is N-acetyllactosaminide alpha-1, 3- galactosyltransferase. It is appreciated that said N-acetyllactosaminide alpha-1, 3- galactosyltransferase is enzymatically active. Accordingly, in one embodiment, said first nucleic acid sequence is a Ggtal gene. Said N-acetyllactosaminide alpha-1, 3- galactosyltransferase may derive from a non-primate species, such as non-old-word- primate species. It may derive from mammals that are not humans or non-old-word- primate species. For example, it may derive from a species of the family Suidae, such as from a species of the genus Sus. Said species may be Sus scrofa. As demonstrated in the appended Examples, such as Examples 3 and 6, a vector comprising a nucleic acid sequence derived from the Ggtal gene of the species Sus scrofa encoding an a-

[0088] 1.3-GT according to the present disclosure induces a high expression of a-Gal on most proteins and lipids in infected cells. Moreover, the modified cells are recognized as foreign by the immune system of the subject that comprises said cells. A-Gal-decorated proteins on the surface of malignant cells, such as cancer cells, bind naturally occurring a-Gal antibodies. The present inventors envision that a-l,3-GT encoding nucleic acid sequences engineered based on genes of species other than Sus scrofa corresponding to the Ggtal gene of the species Sus scrofa are also applicable according to the present disclosure provided that they encode an enzymatically active form of a-l,3-GT that enables the decoration of cells with a foreign carbohydrate moiety according to the present disclosure. The skilled person is aware of this concept and is in the possession of the knowledge that such a-l,3-GT enzymes may comprise substantially different amino acid sequences and may be encoded by substantially different nucleic acid sequences in different species but are equally suitable for the herein demonstrated function. The skilled person appreciates that using bioinformatic analysis (e.g. BLAST analysis) of the herein disclosed amino acid sequences of a-l,3-GT or a variant thereof, such as SEQ ID NO:2, polypeptides from different species may be identified with a similar or the same enzymatic activity as that of a-l,3-GT from the species Sus scrofa. For example, the inventors have identified N-acetyllactosaminide alpha-1, 3- galactosyltransferase-like protein from Phascolarctos cinereus with a sequence identity of about 67% to SEQ ID NO:2. Moreover, N-acetyllactosaminide alpha-1, 3- galactosyltransferase-like 1 protein from Sciurus carolinensis with a sequence identity of about 50% to SEQ ID NO:2 was identified. N-acetyllactosaminide alpha-

[0089] 1.3-galactosyltransferase-like protein from Spec bombifrons was also found with a sequence identity of about 43% to SEQ ID NO:2. These are non-limiting examples of potentially suitable enzyme variants contemplated by the present inventors. Independently of the level of the amino acid sequence identity, these polypeptides and variants thereof may have a similar or the same enzymatic function as that of a- 1,3-GT from the species Sus scrofa. The present inventors envision that these enzymes and variants thereof are equally suitable in the context of the present disclosure provided that they are enzymatically active and perform a similar or the same enzymatic function as it is demonstrated herein. In some embodiments, said N-acetyllactosaminide alpha-1, 3-galactosyltransferase comprises an amino acid sequence selected from a group consisting of an amino acid sequence according to SEQ ID NO:2 and amino acid sequences having at least 38%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70% sequence identity to SEQ ID NO:2. It is to be understood that said N-acetyllactosaminide alpha-1, 3-galactosyltransferase comprising an amino acid sequence having at least 38%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70% sequence identity to SEQ ID NO:2 is enzymatically active. As discussed above, the inventors envision that a variant or a truncated form of the glycosyltransferase, such as of said galactosyltransferase or of said N-acetyllactosaminide alpha-1, 3-galactosyltransferase, is also suitable for the in vivo or in vitro decoration of malignant cells, such as cancer cells, according to the present disclosure, provided that said variant or said truncated form is enzymatically active. Such enzymatic activity is to be understood in terms of the enzymatic function of the glycosyltransferase, such as of said galactosyltransferase or of said N- acetyllactosaminide alpha-1, 3-galactosyltransferase. Thus, in the context of the present disclosure, the enzymatic activity may be assessed by the presence of a foreign carbohydrate moiety on the surface of a cell that has been transfected or transduced to express the glycosyltransferase (such as the galactosyltransferase), wherein the assessed foreign carbohydrate moiety is produced by the enzymatic activity of the glycosyltransferase (or the galactosyltransferase, respectively). As demonstrated in Example 3 below, the presence of a-Gal may for example be assessed by an a-Gal-specific fluorescent staining of the transduced cells followed by a FACS analysis. The presence of a-Gal on the cells is indicative of the enzymatic activity of a-l,3-GT expressed by the transduced cells. The skilled person will appreciate that such method may readily be adapted for any given foreign carbohydrate moiety that is suitable for the decoration of malignant cells, such as cancer cells, according to the present disclosure. As defined above, an enzymatically active variant or truncated form of the glycosyltransferase (such as the galactosyltransferase) is capable of at least the same or a similar degree of an enzymatic function as the enzymatic function of said glycosyltransferase (or said galactosyltransferase, respectively). Such enzymatic function is considered the same or at a similar degree in the context of the present disclosure, if at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, of the parameter value measured for the glycosyltransferase (or the galactosyltransferase, respectively) in intact form is reached using one or more, such as at least two or more, analysis methods. In certain cases, such enzymatic function is considered the same or at a similar degree in the context of the present disclosure, if at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, of the parameter value measured for the galactosyltransferase, such as a-l,3-GT, such as a-l,3-GT comprising the amino acid sequence according to SEQ ID NO:2, is reached using one or more, such as at least two or more, analysis methods. In particular, said enzymatic function is considered the same or at a similar degree in the context of the present disclosure if the percentage as recited above of the parameter value measured for a-l,3-GT comprising the amino acid sequence according to SEQ ID NO:2, is reached using one or more, such as at least two or more, analysis methods. Said analysis methods may be in vitro methods, for example as described in the appended Examples. It is to be understood, that said enzymatic function may be measured in isolation for the glycosyltransferase, such as the galactosyltransferase, such as a-l,3-GT, such as a- 1,3-GT comprising the amino acid sequence according to SEQ ID NO:2, an enzymatically active variant thereof or an enzymatically active truncated form thereof, such as demonstrated for the viral vector, Ad(onco-A) described in the Examples. It will be appreciated that for the purpose of the herein described comparison, the vector backbone, such as the viral vector backbone, may be selected to be the same between the compared variants comprising either the glycosyltransferase, such as the galactosyltransferase, such as a-l,3-GT, such as a- 1,3-GT comprising the amino acid sequence according to SEQ ID NO:2, an enzymatically active variant thereof or an enzymatically active truncated form thereof. In some embodiments, said N-acetyllactosaminide alpha-1, 3- galactosyltransferase comprises an amino acid sequence selected from a group consisting of an amino acid sequence according to SEQ ID NO:2 and amino acid sequences having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO:2. In some embodiments, said N-acetyllactosaminide alpha-1, 3- galactosyltransferase comprises an amino acid sequence selected from a group consisting of an amino acid sequence according to SEQ ID NO:2 and amino acid sequences having at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO:2.ln one embodiment, said N-acetyllactosaminide alpha-1, 3-galactosyltransferase comprises the amino acid sequence according to SEQ ID NO:2. In some embodiments, said first nucleic acid sequence comprises a nucleic acid sequence selected from a group consisting of a nucleic acid sequence according to SEQ ID NQ:40, any nucleic acid sequence having at least 70% sequence identity to SEQ ID NQ:40 and any codon- optimized version thereof. In some embodiments, said first nucleic acid sequence comprises a nucleic acid sequence selected from a group consisting of a nucleic acid sequence according to SEQ ID NQ:40, any nucleic acid sequence having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NQ:40 and any codon-optimized version thereof. As explained above, codon-optimization may be advantageous to facilitate protein translation. In some embodiments, said first nucleic acid sequence comprises a nucleic acid sequence selected from a group consisting of a nucleic acid sequence according to SEQ ID NO:1 and nucleic acid sequences having at least 70% sequence identity to SEQ ID NO:1. In some embodiments, said first nucleic acid sequence comprises a nucleic acid sequence selected from a group consisting of a nucleic acid sequence according to SEQ ID NO:1 and nucleic acid sequences having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO:1. In one embodiment, said first nucleic acid sequence comprises a nucleic acid sequence according to SEQ ID NO:1.

[0090] In some embodiments, said NAP comprises an amino acid sequence selected from a group consisting of an amino acid sequence according to SEQ ID NO:4 and amino acid sequences having at least 70% sequence identity to SEQ ID NO:4. In some embodiments, said NAP comprises an amino acid sequence selected from a group consisting of an amino acid sequence according to SEQ ID NO:4 and amino acid sequences having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO:4. In some embodiments, said NAP comprises an amino acid sequence selected from a group consisting of an amino acid sequence according to SEQ ID NO:4 and amino acid sequences having at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO:4. As demonstrated in the appended Examples, NAP according to the present disclosure induces an immune response in a subject. Moreover, the Examples show several methods equally relevant for assessing the immune response elicited by NAP. An immunologically equivalent fragment or variant of NAP is to be understood to induce an immune response equivalent to, i.e. at least the same or stronger than, an immune response induced by said NAP, such as NAP comprising the amino acid sequence according to SEQ ID NO:4. Such immune response is considered the same in the context of the present disclosure, if at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, of the parameter value measured for NAP, such as NAP comprising the amino acid sequence according to SEQ ID NO:4, is reached using one or more, such as at least two or more, analysis methods. Said analysis methods may be in vitro methods, for example as described in the appended Examples. In particular, said immune response is considered the same in the context of the present disclosure if the percentage as recited above of the parameter value measured for NAP comprising the amino acid sequence according to SEQ ID NO:4. is reached using one or more, such as at least two or more, analysis methods. It is to be understood, that said immune response may be measured in isolation as induced by NAP, such as NAP comprising the amino acid sequence according to SEQ ID NO:4, an immunologically equivalent fragment thereof or an immunologically equivalent variant thereof, such as demonstrated for the viral vector, Ad(onco-B) described in the Examples. It will be appreciated that for the purpose of the herein described comparison, the vector backbone, such as the viral vector backbone, may be selected to be the same between the compared variants comprising either NAP, such as NAP comprising the amino acid sequence according to SEQ ID NO:4, an immunologically equivalent fragment thereof or an immunologically equivalent variant thereof. In one embodiment, said NAP comprising any one of said amino acid sequences having at least 70% sequence identity to SEQ ID NO:4 is capable of inducing an immune response equivalent to an immune response induced by said NAP comprising the amino acid sequence according to SEQ ID NO:4. In some embodiments, said NAP comprises an amino acid sequence according to SEQ ID NO:5, wherein

[0091] XI is selected from E and G,

[0092] X2 is selected from I and L, and

[0093] X3 is selected from Y and H. In some embodiments, said NAP comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:4 and SEQ ID NO:6-12. In a particular embodiment, said NAP comprises the amino acid sequence according to SEQ ID NO:4. In some embodiments, the immunologically equivalent fragment of NAP has a length of at least 20, such as at least 25, such as at least 30, such as at least 34, amino acid residues. In some embodiments, the immunologically equivalent fragment of NAP comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NQ:13-20 and amino acid sequences having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NQ:13-20. As discussed above, the second nucleic acid sequence may encode an immunologically equivalent fragment of NAP. Without being bound by theory, the inventors envision that NAP fragments which correspond to dominant T-cell epitopes of NAP recognized by HP-NAP specific T-cells, may be suitable to be considered as immunologically equivalent fragments of NAP according to the present disclosure. Such dominant T cell epitopes of NAP are known in the art, and will be appreciated by the skilled person to be suitable in the present context. In one embodiment, the immunologically equivalent fragment of NAP comprises an amino acid sequence selected from a group consisting of the amino acid sequences according to SEQ ID NQ:13-20, such as a group consisting of the amino acid sequences according to SEQ ID NO:13-15 and SEQ ID NQ:20. In some embodiments, said second nucleic acid sequence comprises a nucleic acid sequence selected from a group consisting of a nucleic acid sequence according to SEQ ID NO:3, any nucleic acid sequence having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO:3 and any codon-optimized version thereof; such as a group consisting of a nucleic acid sequence according to SEQ ID NO:3 and any nucleic acid sequence having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO:3. In one embodiment, said second nucleic acid sequence comprises a nucleic acid sequence according to SEQ ID NO:3.

[0094] In some embodiments, the vector according to the first aspect of the present disclosure is a non-viral vector or a viral vector. In some embodiments, said vector is a non-viral vector. Said non-viral vector may be a DNA vector or an RNA vector.

[0095] In some embodiments, said vector is a viral vector. Said viral vector may be an RNA vector or a DNA vector. In one embodiment, said viral vector is a DNA vector. In some embodiments, said viral vector is a retrovirus, a lentivirus, a vaccinia virus, a Semliki Forest virus, a reovirus, a Newcastle disease virus, a herpes virus, an adenovirus or an adeno-associated virus. In some embodiments, the viral vector is an engineered viral vector for reduced immunogenicity, wherein the engineered viral vector exhibits reduced immunogenicity in comparison to the immunogenicity of the native viral vector from which the engineered viral vector derives from. Such reduced immunogenicity may for example be demonstrated by lower neutralizing antibody titers against the engineered viral vector in comparison to the native viral vector when administered to a subject. This reduced immunogenicity is considered advantageous for repeated administration of the viral vector to the subject. In some embodiments, said viral vector is a retrovirus, a lentivirus, a vaccinia virus, an adenovirus or an adeno-associated virus. In one embodiment, said viral vector is a retrovirus, a vaccinia virus, an adenovirus or an adeno-associated virus. In one embodiment, said viral vector is a retrovirus, an adenovirus or an adeno-associated virus. In one embodiment, said viral vector is a retrovirus. In one embodiment, said viral vector is a lentivirus, a vaccinia virus, an adenovirus or an adeno-associated virus. In one embodiment, said viral vector is a lentivirus, an adenovirus or an adeno-associated virus. In one embodiment, said viral vector is a lentivirus. In one embodiment, said viral vector is a vaccinia virus, an adenovirus or an adeno- associated virus. In one embodiment, said viral vector is a vaccinia virus. In some embodiments, said viral vector is an adenovirus or an adeno-associated virus. In one embodiment, said viral vector is an adeno-associated virus. In a particular embodiment, said viral vector is an adenovirus. In one embodiment, said viral vector is a human adenovirus type 5, such as a genetically modified variant of human adenovirus type 5. The present inventors found that certain genetic modifications of the viral vector as disclosed herein are beneficial, for example for the possession of an enhanced capacity to infect exocrine pancreas cells. Genetic modifications may also or alternatively lead to limiting the replicative ability of the viral vector. The replicative ability of the viral vector may be limited to malignant cells, such as cancer cells. Such genetic modification may be a modification which allows viral entry through interaction with CD46, a cell surface protein abundantly expressed on many tumor types. This may be for example achieved by a viral vector that is modified to expresses a CD46-binding fiber knob of human adenovirus type 35. Thus, in some embodiments, said viral vector is modified to expresses a CD46-binding fiber knob of human adenovirus type 35. As described in Example 1, in one embodiment, said human adenovirus type 5 vector is modified to expresses a CD46-binding fiber knob of human adenovirus type 35. As discussed above, a viral vector according to the present disclosure may be a viral vector that is genetically modified, for example to limit its replicative ability, such as to limit its replicative ability to malignant cells, such as cancer cells. As described in Example 1, a viral vector as disclosed herein may be restricted to replication in malignant cells, such as cancer cells. The viral vector in some embodiments is restricted to replication in malignant cells with aberrant p53 and Rb-E2F mediated cell cycle control, such as in cancer cells with aberrant p53 and Rb-E2F mediated cell cycle control. This may be achieved by a 24 bp deletion in the E1A gene (E1AA24) and full deletion of the E1B gene in the viral vector. Said partial deletion of the E1A gene and full deletion of the E1B gene is expected to prevent viral replication in normally functioning cells of a viral vector of the present disclosure. In some embodiments, the viral vector, thus, comprises a mutated adenovirus early region 1A (E1A) gene encoding a mutated E1A protein having a substantially reduced Rb protein binding capability as compared to a wildtype E1A protein (Fueyo J, et al., Oncogene 2000, 19(2-23). As mentioned above, introducing a 24 bp deletion in the wild-type E1A gene, wherein said 24 bp encodes an amino acid sequence according to SEQ ID NO:35, said mutated E1A gene may be obtained. The wild-type E1A gene encodes several overlapping mRNAs, thus several wild-type E1A proteins are derivable from this gene. Said wild-type E1A gene for example encodes two splice variants, a 32-kDa adenovirus E1A protein and a 26-kDa adenovirus E1A protein. Said wild-type E1A protein may thus comprise an amino acid sequence according to SEQ ID NO:21 or SEQ ID NO:22. Accordingly, said mutated E1A protein may comprise an amino acid sequence according to SEQ ID NO:23 or SEQ ID NO:24. Moreover, it is known that additional splice variants may derive from the wild-type E1A gene. These splice variants differ from each other with regard to the length of the intron which is removed by RNA splicing. Said wildtype E1A protein may thus comprise an amino acid sequence according to SEQ ID NO:36 or SEQ ID NO:37 and accordingly, said mutated E1A protein may comprise an amino acid sequence according to SEQ ID NO:38 or SEQ ID NO:39. Moreover, in some embodiments, an adenovirus early region IB (E1B) gene is deleted in the viral vector. Said E1B gene encodes two splice variants, a 19-kDa E1B protein and a 55- kDa E1B protein. In a particular embodiment, said viral vector is an oncolytic virus, such as an oncolytic adenovirus. An oncolytic virus, as disclosed herein, is demonstrated in the appended Examples to stimulate host anti-tumor immune system responses. As defined above, an oncolytic virus according to the present disclosure exhibits preferential viral replication in malignant cells, such as cancer cells, and oncolysis of the infected cells (e.g. as shown in Fig. 2). Besides direct killing, oncolytic viruses, as defined herein, induce an immunogenic cell death, which constitute one of the main triggers of an anti-cancer T-cell response in vivo. For example, membrane expression of calreticulin and the release of high mobility group box 1 (HMGB1) by the dying tumor cells are signs of immunogenic cell death. As shown in Fig. 4, an oncolytic virus as disclosed herein induces a marked expression of calreticulin on infected cells.

[0096] In some embodiments, as demonstrated in Example 1 and Fig. 1, said first nucleic acid sequence is arranged upstream of said second nucleic acid sequence in the non-viral vector or a viral vector construct encoding said viral vector according to the first aspect of the present disclosure. However, the present inventors envision that the order of the first and the second nucleic acid sequence may be swapped. The skilled person will appreciate that the order of said nucleic acid sequences is not expected to have any significant effect on the function of the encoded polypeptides according to the present disclosure.

[0097] In some embodiments, the vector as disclosed herein further comprises a third nucleic acid sequence. In one embodiment, said third nucleic acid sequence is arranged between the first and the second nucleic acid sequence in the non-viral vector or a vector construct encoding said viral vector. In some embodiments, said third nucleic acid sequence allows for obtaining individual polypeptides encoded by said first and said second nucleic acid sequences, respectively. Moreover, said third nucleic acid sequence may be advantageous for facilitating correct folding of said polypeptides. The present inventors envision several alternatives for obtaining isolated translation of the polypeptides encoded by said first and said second nucleic acid sequences. These alternatives are considered known by those skilled in the art. For example, in one embodiment, said third nucleic acid sequence is an Internal Ribosome Entry Site (IRES) sequence. As it will be appreciated, an IRES sequence enables transcription of two separate mRNA molecules based on the first and the second nucleic acid sequence and subsequent separate translation thereof into the encoded polypeptides. In another embodiment, said third nucleic acid sequence encodes a self-cleaving peptide. Such self-cleaving peptide allows for transcription of one mRNA molecule based on the first and the second nucleic acid sequences and subsequently, for a separation of the encoded polypeptides post-transcriptionally by for example inducing a ribosomal skipping during protein translation. Ribosomal skipping is triggered by the peptide bond between a proline (P) and a glycine (G) amino acid residue in the C-terminal of the self-cleaving peptide. This results in that the peptide or protein which is located upstream of the self-cleaving peptide has extra amino acids on its C-terminal end (corresponding to the amino acid sequence of the self-cleaving peptide from its N-terminal to said C-terminally located glycine), while the peptide or protein which is located downstream of the self-cleaving peptide will have an extra proline on its N-terminal end. Such self-cleaving peptide may be a 2A self-cleaving peptide, also known as a 2A peptide. The self-cleaving 2A peptide may comprise a core sequence motif of DxExNPGP (SEQ ID NO:34). In an embodiment, the self-cleaving 2A peptide is selected from a 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). The selfcleaving peptide may thus comprise an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:25-28 and amino acid sequences having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of SEQ ID NO:25-28. In some embodiments, the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:25-28. In some embodiments, the self-cleaving peptide further comprises a linker sequence region at the N-terminal thereof. Thus, in some embodiments, the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:29-32 and amino acid sequences having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of SEQ ID NO:29-32. In one embodiment, the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:29-32. In a particular embodiment, the selfcleaving peptide comprises an amino acid sequence according to SEQ ID NO:25, such as an amino acid sequence according to SEQ ID NO:29.

[0098] In some embodiments, the vector as defined in the first aspect of the present disclosure is formulated for in vitro transfection or transduction of cells or for in vivo administration to a subject. In some particular embodiments, the vector is formulated for in vitro transfection or transduction of cells, such as for ex vivo gene therapy. In other particular embodiments, the vector is formulated for in vivo administration to a subject. In some embodiments, the vector is formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, intralymphatic or intratumoral administration to a subject. In some embodiments, the vector is formulated for intratumoral or intralymphatic administration to a subject. In one embodiment, said vector is formulated for intratumoral, subcutaneous or intra lymphatic administration to a subject. In one embodiment, said vector is formulated for intratumoral or intra lymphatic administration to a subject. In one embodiment, said vector is formulated for intratumoral or subcutaneous administration to a subject. In one embodiment, said vector is formulated for subcutaneous or intra lymphatic administration to a subject. In one embodiment, said vector is formulated for intra lymphatic administration to a subject. In one embodiment, said vector is formulated for subcutaneous administration to a subject. In one embodiment, said vector is formulated for intratumoral administration to a subject. The term "Intralymphatic administration", as used herein, refers to direct administration (e.g. injection) to a lymph node of a subject. The lymph node may for example be a draining lymph node or a lymph node located in closed proximity to a tumor in the subject.

[0099] As it will be appreciated based on the above description, the herein disclosed conceptually new form of immunotherapy by in vivo or in vitro decoration of malignant cells, such as cancer cells, with foreign carbohydrate moieties, such as a- Gal epitopes, in combination with the secretion of NAP or an immunologically equivalent variant or fragment thereof, may be achieved by solutions alternative to the vector according to the first aspect of the present disclosure.

[0100] Thus, in a second aspect of the present disclosure is provided a vector system for simultaneous or subsequent delivery of a first and a second vector to a cell and / or for generating an isolated cell population as defined in any embodiment of a fourth aspect of the present disclosure, said vector system comprising

[0101] (a) the first vector comprising said first nucleic acid sequence, as defined above, and (b) the second vector comprising said second nucleic acid sequence, as defined above; wherein said first and said second vectors are separate vectors.

[0102] The first nucleic acid sequence may be as defined in any one of the embodiments discussed in relation to the first aspect of disclosure. The second nucleic acid sequence may be as defined in any one of the embodiments discussed in relation to the first aspect of disclosure. It is to be understood that that embodiments discussed above in relation to the first aspect of the disclosure relating to the first nucleic acid sequence and the second nucleic acid sequence as disclosed herein are equally relevant for the second aspect of the disclosure. For the sake of brevity, these will not be repeated herein or just briefly mentioned below.

[0103] In some embodiments, said first and / or said second vector is a non-viral vector or a viral vector. In said embodiments, said first and / or said second vector is a non-viral vector as defined in relation to the first aspect of disclosure. In some embodiments, said first and / or said second vector is a viral vector as defined in relation to the first aspect of disclosure. In some embodiments, the vector system is formulated for in vitro transfection or transduction of cells or for in vivo administration to a subject. In some particular embodiments, the vector system is formulated for in vitro transfection or transduction of cells, such as for ex vivo gene therapy. In other particular embodiments, the vector system is formulated for in vivo administration to a subject. Embodiments discussed above in relation to the first aspect of the disclosure relating to formulation for in vitro transfection or transduction of cells or for in vivo administration to a subject are to be understood equally relevant for the second aspect of the disclosure. Thus, in some embodiments, the first and / or the second vector is formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, intralymphatic or intratumoral administration to a subject. In some embodiments, the first and / or the second vector is formulated for intratumoral, subcutaneous or intralymphatic administration to a subject. In one embodiment, the first and / or the second vector is formulated for intratumoral or intra lymphatic administration to a subject. In one embodiment, the first and / or the second vector is formulated for intratumoral or subcutaneous administration to a subject. In one embodiment, the first and / or the second vector is formulated for subcutaneous or intra lymphatic administration to a subject. In one embodiment, the first and / or the second vector is formulated for intralymphatic administration to a subject. In one embodiment, the first and / or the second vector is formulated for subcutaneous administration to a subject. In one embodiment, the first and / or the second vector is formulated for intratumoral administration to a subject.

[0104] The present inventors envision that the herein disclosed conceptually new form of immunotherapy may be achieved by various gene therapy modalities. For example, the vector and / or the vector system as discussed above in relation to the first and the second aspect of the present disclosure, is expected to be suitable for in vivo delivery to a subject, and alternatively, may be delivered ex vivo. In an ex vivo gene therapy modality, cells of a subject, such as a patient, are extracted and cultured outside of the body. Cells are then genetically modified by introduction of one or more therapeutic transgenes (i.e. one or more nucleic acid sequences encoding one or more desired polypeptides) and are then re-introduced back into the same subject (e.g. patient). These genetically modified cells may also be suitable for introduction into another subject, e.g. patient. Accordingly, these cells may be autologous or allogeneic to a subject. Ex vivo genetic modification of the cells may be achieved by in vitro transfection or transduction of the cells.

[0105] Thus, in a third aspect of the present disclosure is provided a method of in vitro transfection or transduction of cells with the vector as defined in any one of the embodiments discussed in relation to the first aspect of disclosure or the vector system as defined in any one of the embodiments discussed in relation to the second aspect of disclosure. In one embodiment of the third aspect of the disclosure is provided the method of in vitro transfection or transduction of cells with the vector as defined in any one of the embodiments discussed in relation to the first aspect of disclosure. As it will be appreciated based on the above described definitions, non-viral vectors according to the present disclosure are suitable for in vitro transfection of the cells, while viral vectors as disclosed herein may be used for in vitro transduction of the cells.

[0106] In a related fourth aspect of the present disclosure, is thereby also provided a use of the vector as defined in any one of the embodiments discussed in relation to the first aspect of disclosure or the vector system as defined in any one of the embodiments discussed in relation to the second aspect of disclosure in a method of in vitro transfection or transduction of cells, such as the method according to the third aspect of the present disclosure. In one embodiment of the fourth aspect of the disclosure is provided the use of the vector as defined in any one of the embodiments discussed in relation to the first aspect of disclosure in a method of in vitro transfection or transduction of cells, such as the method according to the third aspect of the present disclosure.

[0107] Furthermore, in a fifth aspect of the disclosure, is provided an isolated cell population comprising said first nucleic acid sequence as defined above and / or a glycosyltransferase (such as a galactosyltransferase), an enzymatically active variant thereof, or an enzymatically active truncated form thereof, encoded by said first nucleic acid sequence; and said second nucleic acid sequence as defined above and / or a NAP, an immunologically equivalent variant thereof, or an immunologically equivalent fragment thereof, encoded by said second nucleic acid sequence.

[0108] As further explained below, the term "isolated cell population" in the present context refers to either a plurality of cells in vitro, wherein each cell comprises the first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase), the enzymatically active variant thereof, or the enzymatically active truncated form thereof; and the second nucleic acid sequence and / or said NAP, the immunologically equivalent variant thereof, or the immunologically equivalent fragment thereof; or alternatively, a plurality of cells in vitro comprising at least one cell of a first cell type comprising said first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase), the enzymatically active variant thereof, or the enzymatically active truncated form thereof; and at least one cell of a second cell type comprising said second nucleic acid sequence and / or said NAP, the immunologically equivalent variant thereof, or the immunologically equivalent fragment thereof.

[0109] It is to be understood that, in the context of a plurality of cells in vitro comprising at least one cell of a first cell type and at least one cell of a second cell type mentioned above, said first cell type and second cell type differ from each other at least in terms of presence of the first nucleic acid sequence as defined herein and second nucleic acid sequence as defined herein. Thus, said first and second cell type may originate from the same ancestor cell type, such as, but not limited to, malignant cells from a patient, or a cell line. Alternatively, said first and second cell type may originate from the different ancestor cell types, such as but not limited to different cell lines, cells derived from different subjects or cells derived from different tissues of the same subject.

[0110] Thus, the term "cell population", as used herein, refers to a "plurality of cells" and these terms may be used interchangeably in all aspects of the present disclosure. The terms "isolated" and "in vitro” refer to that the plurality of cells or the cell population are present outside an organism.

[0111] In other words, in a related embodiment, is thus provided a plurality of cells in vitro comprising said first nucleic acid sequence as defined above and / or a glycosyltransferase (such as a galactosyltransferase), an enzymatically active variant thereof, or an enzymatically active truncated form thereof, encoded by said first nucleic acid sequence; and said second nucleic acid sequence as defined above and / or a NAP, an immunologically equivalent variant thereof, or an immunologically equivalent fragment thereof, encoded by said second nucleic acid sequence.

[0112] The first nucleic acid sequence may be as defined in any one of the embodiments discussed in relation to the first aspect of the disclosure. The second nucleic acid sequence may be as defined in any one of the embodiments discussed in relation to the first aspect of the disclosure. Moreover, said glycosyltransferase (such as the galactosyltransferase), the enzymatically active variant thereof, and the enzymatically active truncated form thereof may be as defined in embodiments discussed in relation to the first aspect of the disclosure. Said NAP, the immunologically equivalent variant thereof, or the immunologically equivalent fragment thereof may be as defined in embodiments discussed in relation to the first aspect of the disclosure. In one particular embodiment, the isolated cell population comprises said first nucleic acid sequence and / or the glycosyltransferase (such as said galactosyltransferase); and said second nucleic acid sequence and / or said NAP.

[0113] The isolated cell population may be obtained using the method of in vitro transfection or transduction of cells according to the third aspect of the present disclosure. The present inventors envision that the isolated cell population may be formulated at least by two alternative ways. For example, it may be that each cell of the isolated cell population comprises the nucleic acid sequences and / or encoded polypeptides as described above. Alternatively, the isolated cell population may be a pooled cell population comprising cell populations of two different cell types, wherein one cell type population comprises the first nucleic acid sequence and / or the polypeptide encoded by the first nucleic acid sequence and one cell type population comprises the second nucleic acid sequence and / or the polypeptide encoded by the second nucleic acid sequence. Thus, in one embodiment, each cell of the isolated cell population comprises said first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase), the enzymatically active variant thereof, or the enzymatically active truncated form thereof; and said second nucleic acid sequence and / or said NAP, the immunologically equivalent variant thereof, or the immunologically equivalent fragment thereof.

[0114] In other words, in a related embodiment, is thus provided the plurality of cells in vitro, wherein each cell comprises said first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase), the enzymatically active variant thereof, or the enzymatically active truncated form thereof; and said second nucleic acid sequence and / or said NAP, the immunologically equivalent variant thereof, or the immunologically equivalent fragment thereof.

[0115] It will be appreciated that in this case, the plurality of cells in vitro comprises two or more cells according to the following related embodiment:

[0116] In a related embodiment, is thus provided an isolated cell that comprises said first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase), the enzymatically active variant thereof, or the enzymatically active truncated form thereof; and said second nucleic acid sequence and / or said NAP, the immunologically equivalent variant thereof, or the immunologically equivalent fragment thereof.

[0117] It is to be understood that said isolated cell may be comprised in the plurality of cells in vitro, i.e. in the isolated cell population, as defined above.

[0118] In one embodiment, each cell of the isolated cell population comprises said first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase); and said second nucleic acid sequence and / or said NAP. In a related embodiment, is thus provided the plurality of cells in vitro, wherein each cell comprises said first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase); and said second nucleic acid sequence and / or said NAP.

[0119] In another related embodiment, the isolated cell thus comprises said first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase); and said second nucleic acid sequence and / or said NAP.

[0120] In another embodiment, the isolated cell population comprises two cell type populations, wherein

[0121] (c) a first cell type population comprises said first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase), the enzymatically active variant thereof, or the enzymatically active truncated form thereof; and

[0122] (d) a second cell type population comprises said second nucleic acid sequence and / or said NAP, the immunologically equivalent variant thereof, or the immunologically equivalent fragment thereof.

[0123] In a related embodiment, is thus provided the plurality of cells in vitro comprising

[0124] (c) at least one cell of a first cell type comprising said first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase), the enzymatically active variant thereof, or the enzymatically active truncated form thereof; and

[0125] (d) at least one cell of a second cell type comprising said second nucleic acid sequence and / or said NAP, the immunologically equivalent variant thereof, or the immunologically equivalent fragment thereof.

[0126] In one embodiment, the first cell type population (i.e. one or more cells of the first cell type) comprises said first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase); and the second cell type population (i.e. one or more cells of the second cell type) comprises said second nucleic acid sequence and / or said NAP. In a related embodiment, is thus provided the plurality of cells in vitro comprising at least one cell of the first cell type comprising said first nucleic acid sequence and / or said glycosyltransferase (such as said galactosyltransferase); and at least one cell of the second cell type comprising said second nucleic acid sequence and / or said NAP.

[0127] The isolated cell population may be a malignant cell population, such as a cancer cell population. Accordingly, plurality of cells in vitro may be a plurality of malignant cells in vitro, such as a plurality of cancer cells in vitro. Moreover, the isolated cell may be a malignant cell, such as a cancer cell. The isolated cell population may be a human cell population, such as a human malignant cell population, such as a human cancer cell population. Accordingly, plurality of cells in vitro may be a plurality of human cells in vitro, such as a plurality of human malignant cells in vitro, such as a plurality of human cancer cells in vitro. Moreover, the isolated cell may be a human cell, such as a human malignant cell, such as a human cancer cell. Moreover, the isolated cell population (i.e. the plurality of cells in vitro) may be a primary cell population or a stable cell line. In some embodiments, the isolated cell population (i.e. the plurality of cells in vitro) is formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, intralymphatic or intratumoral administration to a subject. In some embodiments, the isolated cell population (i.e. the plurality of cells in vitro) is formulated for subcutaneous, intratumoral or intra lymphatic administration to a subject. In one embodiment, said isolated cell population is formulated for subcutaneous or intratumoral administration to a subject. In another embodiment, the isolated cell population (i.e. the plurality of cells in vitro) is formulated for intralymphatic or intratumoral administration to a subject. In one embodiment, said isolated cell population (i.e. the plurality of cells in vitro) is formulated for subcutaneous or intralymphatic administration to a subject. In one embodiment, the isolated cell population (i.e. the plurality of cells in vitro) is formulated for subcutaneous administration to a subject. In one embodiment, the isolated cell population (i.e. the plurality of cells in vitro) is formulated for intratumoral administration to a subject. In one embodiment, the isolated cell population (i.e. the plurality of cells in vitro) is formulated for intra lymphatic administration to a subject.

[0128] In a sixth aspect of the present disclosure is provided a polynucleotide comprising said first and said second nucleic acid sequences as defined in any one of the embodiments discussed in relation to the first aspect of the present disclosure. In some embodiments, the polynucleotide is comprised in a non-viral vector or a viral vector construct. It is to be understood that embodiments discussed above in relation to the first aspect of the disclosure relating to the arrangement of the first nucleic acid sequence and the second nucleic acid sequence as well as to the third nucleic acid sequence, as disclosed herein, are equally relevant for the sixth aspect of the disclosure. For the sake of brevity, these will not be repeated herein or just briefly mentioned below. In some embodiments, said first nucleic acid sequence is arranged upstream of said second nucleic acid sequence in said polynucleotide. In one embodiment, said polynucleotide further comprises a third nucleic acid sequence. In some embodiments, said third nucleic acid sequence is arranged between the first and the second nucleic acid sequence in said polynucleotide. In some embodiments, said third nucleic acid sequence allows for obtaining individual polypeptides encoded by said first and said second nucleic acid sequences, respectively. In some embodiments, said third nucleic acid sequence is an IRES sequence. In some embodiments, said third nucleic acid sequence encodes a selfcleaving peptide. The self-cleaving peptide may be a 2A peptide, such as a 2A peptide comprising a core sequence motif of DxExNPGP (SEQ ID NO:34). In an embodiment, the self-cleaving 2A peptide is selected from a 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 some embodiments, the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:25-28 and amino acid sequences having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of SEQ ID NO:25-28. In some embodiments, the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:25-28. In some embodiments, the self-cleaving peptide further comprises a linker region at the N-terminal thereof. In some embodiments, the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:29-32 and amino acid sequences having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of SEQ ID NO:29-32. In some embodiments, the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:29-32. In one particular embodiment, the selfcleaving peptide comprises an amino acid sequence according to SEQ ID NO:25, such as an amino acid sequence according to SEQ ID NO:29. In another particular embodiment, said polynucleotide encodes a viral vector as defined in any one of the embodiments discussed in relation to the first aspect of the present disclosure.

[0129] In a seventh aspect of the disclosure, there is provided a method for producing the vector as defined in any one of the embodiments discussed in relation to the first aspect herein, wherein said vector is a viral vector, said method comprising generating a modified viral vector construct by operably linking a viral backbone to the first and the second nucleic acid sequence as defined above, transfecting mammalian cells with the modified viral vector construct, culturing the mammalian cells in conditions suitable for viral replication, and harvesting the viral particles.

[0130] The first nucleic acid sequence may be as defined in any one of the embodiments discussed in relation to the first aspect of the disclosure. The second nucleic acid sequence may be as defined in any one of the embodiments discussed in relation to the first aspect of the disclosure. Said modified viral vector construct may be a viral vector construct as defined in any one of the embodiments discussed in relation to the sixth aspect of the present disclosure. In some embodiments, the modified viral vector construct encodes the viral vector as defined according to the first aspect of the disclosure. Moreover, it is to be understood that embodiments discussed above in relation to the first aspect of the disclosure relating to the arrangement of the first nucleic acid sequence and the second nucleic acid sequence as well as to the third nucleic acid sequence, as disclosed herein, are equally relevant for the seventh aspect of the disclosure. For the sake of brevity, these will not be repeated herein.

[0131] As it will be appreciated based on the above description, the herein disclosed conceptually new form of immunotherapy by in vivo or in vitro decoration of malignant cells, such as cancer cells, with foreign carbohydrate moieties, such as a- Gal epitopes, in combination with the secretion of NAP or an immunologically equivalent variant or fragment thereof, may be achieved by solutions alternative to the vector according to the first aspect, the vector system according to the second aspect or the isolated cell populations according to the fifth aspect of the present disclosure.

[0132] Thus, in an eighth aspect of the present disclosure there is provided an RNA system, such as an mRNA system, for simultaneous or subsequent delivery of a first and a second RNA to a cell, said RNA system comprising the first RNA, such as mRNA, encoding a glycosyltransferase (such as a galactosyltransferase), an enzymatically active variant thereof, or an enzymatically active truncated form thereof, as defined herein, and the second RNA, such as mRNA, encoding a Helicobacter pylori neutrophil-activating protein (NAP), an immunologically equivalent variant thereof, or an immunologically equivalent fragment thereof, as defined; wherein optionally said first and said second RNA are separate RNAs. In one embodiment, said fist and said second RNA, such as first and said second mRNA, as separate RNAs, such as separate mRNAs. The first nucleic acid sequence may be as defined in any one of the embodiments discussed in relation to the first aspect of disclosure. The second nucleic acid sequence may be as defined in any one of the embodiments discussed in relation to the first aspect of disclosure. It is to be understood that that embodiments discussed above in relation to the first aspect of the disclosure relating to the first nucleic acid sequence and the second nucleic acid sequence as disclosed herein are equally relevant for the eighth aspect of the disclosure. For the sake of brevity, these will not be repeated herein or just briefly mentioned below.

[0133] In some embodiments, said first and said second RNA, such as mRNA, are modified, for example for increased stability, reduced immunogenicity and / or targeted delivery.

[0134] In some embodiments, the RNA systemis formulated for in vitro delivery to cells or for in vivo administration to a subject. In some particular embodiments, the RNA system is formulated for in vitro delivery to cells. In other particular embodiments, the RNA system is formulated for in vivo administration to a subject. In some embodiments, said RNA system is a mRNA system. Thus, in some embodiments, the first and / or the second RNA is formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, intralymphatic or intratumoral administration to a subject. In some embodiments, the first and / or the second RNA is formulated for intratumoral, subcutaneous or intra lymphatic administration to a subject. In one embodiment, the first and / or the second RNA is formulated for intratumoral or intralymphatic administration to a subject. In one embodiment, the first and / or the second RNA is formulated for intratumoral or subcutaneous administration to a subject. In one embodiment, the first and / or the second RNA is formulated for subcutaneous or intralymphatic administration to a subject. In one embodiment, the first and / or the second RNA is formulated for intralymphatic administration to a subject. In one embodiment, the first and / or the second RNA is formulated for subcutaneous administration to a subject. In one embodiment, the first and / or the second RNA is formulated for intratumoral administration to a subject. In some embodiments, said first RNA and said second RNA are a first mRNA and a second mRNA.

[0135] The inventors envision that there are several manners suitable for the delivery of the herein disclosed RNA system, such as a mRNA system, and that the RNA system may be formulated according to the chosen alternative. The skilled person is aware of potential manners suitable for the delivery of RNA, such as mRNA, for example, based on Hou et al, Nature Review Materials, 6: 1078-1094 (2021), Kowalski et al, Molecular Therapy, 27(4): 710-728 (2019), Kimura et al, Molecular Therapy, 31(8): 2360-2375 (2023) and Pardi et al, Nature Reviews, Drug Discovery, 17(4): 261-279 (2018). Accordingly, in one embodiment of the eighth aspect of the disclosure, the RNA system, such as mRNA, is encapsulated, attached to a carrier surface or is formulated for delivery to by electroporation. In one embodiment, the first and / or the second RNA, such as the first and / or the second mRNA, are encapsulated separately, both attached to a carrier surface or is formulated for delivery to by electroporation. In one embodiment, said RNA system, such as mRNA system, is encapsulated, such as encapsulated in a particle. In one embodiment, said particle is made of a lipid, a polymer, cholesterol and / or a cell penetrating peptide. In one embodiment, said particle is a lipid particle and / or a liposome. In one embodiment, said particle is a nanoparticle, such as a lipid nanoparticle. In one embodiment, the first and / or the second RNA, such as mRNA, are encapsulated in separate particles. In some embodiments, said particles are made of a lipid, a polymer, cholesterol and / or a cell penetrating peptide. In one embodiment, said particles are lipid particles and / or liposomes. In one embodiment, said particles are nanoparticles, such as lipid nanoparticles.

[0136] In a ninth aspect of the disclosure, there is provided a kit comprising the vector, the vector system, the polynucleotide, or the RNA system, as defined above; and instructions for use. In one embodiment is provided the kit comprising the vector, the vector system, or the polynucleotide, as defined above; and instructions for use. The vector, the vector system, the polynucleotide and the RNA system may be as defined in any one of the embodiments discussed in relation to the first, the second, the sixth and the eighth aspect of the present disclosure, respectively. In one embodiment, the kit comprises the vector or the vector system; and instructions for use. In one embodiment, the kit comprises the vector; and instructions for use. The kit, such as the kit comprising the vector or the vector system, may further comprise one or more reagents that are useful in the method of in vitro transfection or transduction of cells according to the third aspect of the disclosure, such as reagents suitable for aiding or enhancing transduction and / or reagents aiding or enhancing transfection. For example, as discussed above with respect to the definition of the term "non-viral vector", said reagents may be applicable in the chemical and / or physical methods useful in the delivery of non-viral vectors to a target cell and / or tissue. In one embodiment, the kit comprises the polynucleotide; and instructions for use. The kit comprising the polynucleotide may further comprise one or more reagents that are useful in the method according to the seventh aspect of the disclosure, for producing the vector as defined in any one of the embodiments discussed in relation to the first aspect herein. In one embodiment, the kit comprises the RNA system, such as the mRNA system; and instructions for use. The kit comprising the RNA system may further comprise one or more reagents that are useful for delivery of the first and the second mRNA to a cell, as discussed in relation to the eighth aspect above.

[0137] In a tenth aspect of the present disclosure, a pharmaceutical composition is provided, said pharmaceutical composition comprising the vector, the vector system, the isolated cell population, or the RNA system, as defined above; and at least one pharmaceutically acceptable salt, carrier and / or excipient. In one embodiment, the pharmaceutical composition comprises the vector, the vector system, or the isolated cell population, as defined above; and at least one pharmaceutically acceptable salt, carrier and / or excipient. The vector, the vector system, the isolated cell population and the RNA system may be as defined in any one of the embodiments discussed in relation to the first, the second, the fifth and the eighth aspect of the present disclosure, respectively. In one embodiment, the pharmaceutical composition comprises the vector or the isolated cell population; and at least one pharmaceutically acceptable salt, carrier and / or excipient. In one embodiment, the pharmaceutical composition comprises the vector; and at least one pharmaceutically acceptable salt, carrier and / or excipient. In one embodiment, the pharmaceutical composition comprises the isolated cell population; and at least one pharmaceutically acceptable salt, carrier and / or excipient. In one embodiment, the pharmaceutical composition comprises the RNA system; and at least one pharmaceutically acceptable salt, carrier and / or excipient. In one embodiment of such pharmaceutical compositions, the first and the second RNA are encapsulated, such as encapsulated in the same or separate particles, such as lipid nanoparticles. As discussed in relation to the a twelfth aspect of the present disclosure, the first and the second RNA may be administered simultaneously or subsequently, such as simultaneously, to a subject. In related embodiments, said first and said second RNA may thus be formulated in separate pharmaceutical compositions for simultaneous or subsequent administration to the subject. Accordingly, in related aspects, it is provided a pharmaceutical composition combination comprising a first pharmaceutical composition comprising the first RNA, and at least one pharmaceutically acceptable salt, carrier and / or excipient; and a second pharmaceutical composition comprising the second RNA and at least one pharmaceutically acceptable salt, carrier and / or excipient; for simultaneous or subsequent administration to the subject. It is to be understood that embodiments relating to the formulation of the pharmaceutical composition comprising the RNA system are equally relevant for the first and the second pharmaceutical compositions of the combination. In some embodiments, said pharmaceutical composition is formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, intralymphatic or intratumoral administration to a subject. In some embodiments, the pharmaceutical composition is formulated for subcutaneous, intratumoral or intralymphatic administration to a subject. In one embodiment, said pharmaceutical composition is formulated for subcutaneous or intratumoral administration to a subject. In another embodiment, the pharmaceutical composition is formulated for intralymphatic or intratumoral administration to a subject. In one embodiment, said pharmaceutical composition is formulated for subcutaneous or intralymphatic administration to a subject. In one embodiment, the pharmaceutical composition is formulated for subcutaneous administration to a subject. In one embodiment, the pharmaceutical composition is formulated for intratumoral administration to a subject. In one embodiment, the pharmaceutical composition is formulated for intralymphatic administration to a subject. The present inventors envision that said at least one pharmaceutically acceptable salt, carrier and / or excipient as well as the formulation of the pharmaceutical composition may be selected depending on whether the pharmaceutical composition comprises a vector, a vector system or an isolated cell population as disclosed herein. Accordingly, the pharmaceutical composition may be formulated to suit alternative gene therapy modalities according to the present disclosure, for example, to suit in vivo or ex vivo therapeutic use. For example, the pharmaceutical composition comprising the vector or the vector system may be formulated for in vivo administration to the subject in an in vivo gene therapy. In certain embodiments, the pharmaceutical composition comprising the vector or the vector system is formulated for intra lymphatic or intratumoral administration to a subject. In one particular embodiment, the pharmaceutical composition comprising the vector or the vector system is formulated for intratumoral administration to a subject. Moreover, the pharmaceutical composition comprising the isolated cell population may be formulated for in vivo administration to the subject, for example in context of an ex vivo gene therapy. In certain embodiments, the pharmaceutical composition comprising the isolated cell population is formulated for subcutaneous, intratumoral or intra lymphatic administration to a subject In certain embodiments, the pharmaceutical composition comprising the isolated cell population is formulated for intra lymphatic or subcutaneous administration to a subject. In certain embodiments, the pharmaceutical composition comprising the isolated cell population is formulated for intratumoral or subcutaneous administration to a subject. In one embodiment, said pharmaceutical composition comprising the isolated cell population is formulated for intratumoral or intralymphatic administration to a subject. In one particular embodiment, the pharmaceutical composition comprising the isolated cell population is formulated for subcutaneous administration to a subject. In one particular embodiment, the pharmaceutical composition comprising the isolated cell population is formulated for intratumoral administration to a subject. In one particular embodiment, the pharmaceutical composition comprising the isolated cell population is formulated for intralymphatic administration to a subject. In certain embodiments, the pharmaceutical composition comprising the RNA system is formulated for intra lymphatic or intratumoral administration to a subject. In one particular embodiment, the pharmaceutical composition comprising the RNA system is formulated for intratumoral administration to a subject. As used herein, "pharmaceutically acceptable salt, carrier or excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible.

[0138] As discussed above, the herein disclosed means according to any one of the embodiments discussed in relation to the first, the second, the fifth aspect or the eighth aspect of the present disclosure, such as embodiments discussed in relation to the first, the second or the fifth aspect, are exceptionally beneficial for a conceptually new form of immunotherapy, such as an immunotherapy for cancer treatment, according to the present disclosure. The vector, the vector system, the isolated cell population, or the RNA system, as defined above in any one of the embodiments discussed in relation to the first, the second, the fifth or the eighth aspect of the present disclosure, such as embodiments discussed in relation to the first, the second or the fifth aspect, provide means suitable for alternative immunotherapy modalities, including in vivo and ex vivo gene therapies. As discussed above, in vivo gene therapy entails the direct administration of the vector or the vector system as disclosed herein into a subject, such as a patient, such as a cancer patient. Ex vivo gene therapy involves the extraction of a subject's, such as a patient's, cells, such as malignant cells (e.g. cancer cells), or obtaining cells from an allogeneic source. Thus, for example the cells may be obtained from a different subject or an established cell line. The cells are then genetically modified using the vector or the vector system as disclosed herein. This process typically involves selection and expansion of the genetically modified cells in culture. The genetically modified cells are then introduced or re-introduced into the subject, e.g. patient. In view of the above, it will be appreciated that the administration route may be selected in view of the chosen gene therapy modality. Furthermore, the immunotherapy may involve the direct administration of the RNA system as disclosed herein into a subject, such as a patient, such as a cancer patient. Direct administration of the RNA system may occur via an administration route suitable for RNA delivery.

[0139] Thus, in an eleventh aspect of the present disclosure is provided the vector, the vector system, the isolated cell population, and / or the RNA system, as defined above in any one of the embodiments discussed in relation to the first, the second, the fifth, or the eighth aspect of the present disclosure, for use as a medicament. In some embodiments there is provided the vector, the vector system, and / or the isolated cell population, as defined above, for use as a medicament. In one embodiment is provided the vector, the vector system, the isolated cell population, or the RNA system, for use as a medicament. In some embodiments there is provided the vector, the vector system, or the isolated cell population, as defined above, for use as a medicament. In one embodiment of the eleventh aspect of the disclosure is provided the vector or the isolated cell population for use as a medicament. In another embodiment is provided the vector for use as a medicament. In yet another embodiment is provided the isolated cell population for use as a medicament. In one embodiment is provided the vector, the vector system, or the RNA system, for use as a medicament. In one embodiment is provided the vector, or the RNA system, for use as a medicament. In yet another embodiment is provided the RNA system for use as a medicament.

[0140] Moreover, in a twelfth aspect is provided the vector, the vector system, the isolated cell population, and / or the RNA system, as defined above, for use in the treatment of cancer. As used herein, the term "cancer" encompasses cancers (which arise from epithelial tissues) as well as malignancies arising from non-epithelial tissues. Thus, for brevity, when the term cancer is used in the context of the present disclosure it is to be understood to refer to both malignancies (such as tumors) that originate from epithelial tissues (also known as carcinomas), as well as malignancies from non-epithelial tissues. Non-limiting examples of malignancies arising from non- epithelial tissues are melanoma, sarcoma, lymphoma, leukemia, seminoma, germinoma, dysgerminoma, blastoma, brain and central nervous system cancers as well as mixed-type cancers. The skilled person will appreciate that the term "cancer" as defined in the context of this twelfth aspect of the present disclosure is equally relevant for the complete present disclosure, including the fifteenth, seventeenth, nineteenth, twenty-first and twenty-third aspects disclosed herein which relate to medical uses.

[0141] In one embodiment is provided the vector, the vector system, the isolated cell population, and / or the RNA system, as defined above, for use in the treatment of cancer. In one embodiment is provided the vector, the vector system, and / or the isolated cell population, as defined above, for use in the treatment of cancer. In one embodiment, said cancer is a malignancy arising from epithelial tissue or a malignancy arising from non-epithelial tissue. The vector, the vector system, the isolated cell population and the RNA system may be as defined in any one of the embodiments discussed in relation to the first, the second, the fifth and the eighth aspect of the present disclosure, respectively. The skilled person will appreciate that various means may be suitable for the herein disclosed novel immunotherapy, provided that said means comprise both of said first and said second nucleic acid sequence or said first and said second RNA encoded by said first and said second nucleic acid sequences, respectively. Such suitable means include the vector, the vector system, the isolated cell population and / or the RNA system, such as the vector, the vector system, and / or the isolated cell population, of the present disclosure. It is to be understood that said use may comprise the administration of each of the vector, the vector system, the isolated cell population and the RNA system, or a selection of these, for example, said use may comprise the administration of the vector and the isolated cell population. In some embodiments, said use comprises the administration of each of the vector, the vector system, and the isolated cell population, or a selection of these, for example, the administration of the vector and the isolated cell population. In some embodiments, is provided the vector, the vector system, the isolated cell population, or the RNA system, as defined above, for use in the treatment of cancer. In one embodiment is provided the vector, the vector system, or the isolated cell population, as defined above, for use in the treatment of cancer. In one embodiment is provided the vector or the isolated cell population for use in the treatment of cancer. In another embodiment is provided the vector for use in the treatment of cancer. In yet another embodiment is provided the isolated cell population for use in the treatment of cancer. In one embodiment is provided the vector, the vector system, or the RNA system, for use in the treatment of cancer. In one embodiment is provided the vector, or the RNA system, for use in the treatment of cancer. In yet another embodiment is provided the RNA system for use in the treatment of cancer. In some embodiments, said cancer is selected from a group consisting of carcinoma, melanoma, sarcoma, lymphoma, leukemia, seminoma, germinoma, dysgerminoma, blastoma, brain and central nervous system cancers, mixed-type cancers and pediatric cancers. In some embodiments, said cancer is selected from a group consisting of carcinoma, melanoma, sarcoma, lymphoma, leukemia, seminoma, germinoma, dysgerminoma, blastoma, brain and central nervous system cancers and mixed-type cancers. In some embodiments, said cancer is selected from a group consisting of carcinoma, melanoma, sarcoma, lymphoma, leukemia, seminoma, germinoma, dysgerminoma and blastoma. In some embodiments, said cancer is carcinoma or melanoma. In one embodiment, said cancer is carcinoma. The skilled person knows that a carcinoma is a type of cancer that arises in epithelial tissue. In some embodiments, said carcinoma is selected from a group consisting of pancreatic cancer, breast cancer, lung cancer, liver cancer, bile duct cancer, gallbladder cancer, intestinal cancer, urogenital cancer, prostate cancer, head-neck cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, skin cancer, thyroid cancer, neuroendocrine tumors (NET) and uterine cancer. In some embodiments, said carcinoma is selected from a group consisting of pancreatic cancer, breast cancer, lung cancer, liver cancer and kidney cancer. In some embodiments, said cancer is pancreatic cancer. In one particular embodiment, said cancer is pancreatic ductal adenocarcinoma (PDAC), such as stage IV PDAC. In one embodiment, said carcinoma is breast cancer, such as ductal carcinoma in situ (DCIS). In one embodiment, said carcinoma is lung cancer, such as non-small cell lung cancer or small cell lung cancer. In one embodiment, said carcinoma is liver cancer, such as hepatocellular carcinoma. In one embodiment, said carcinoma is bile duct cancer (also known as cholangiocarcinoma). In one embodiment, said carcinoma is gallbladder cancer. In one embodiment, said carcinoma is intestinal cancer, such as intestinal cancer selected from the group consisting of small bowel cancer, colon cancer and rectal cancer. In one embodiment, said carcinoma is urogenital cancer, such as urogenital cancer selected from the group consisting of bladder cancer, ureter and renal pelvis transitional cell cancer, kidney cancer, penile cancer and testicular cancer. In one embodiment said carcinoma is skin cancer, such as squamous cell carcinoma of the skin.

[0142] In some embodiments, said cancer is melanoma. In some embodiments, said sarcoma is selected from a group consisting of osteosarcoma and liposarcoma. In some embodiments, said lymphoma is selected from a group consisting of nonHodgkin lymphoma and Hodgkin lymphoma. In some embodiments, said leukemia is selected from a group consisting of acute leukemia and chronic leukemia. In some embodiments said germinoma is selected from the group consisting of germinoma, germ cell tumor, such as a intracranial germ cell tumor (such as childhood intracranial germ cell tumor), an extracranial germ cell tumor or an extragonadal germ cell tumor. In some embodiments, said blastoma is selected from a group consisting of glioblastoma and neuroblastoma. In some embodiments said brain and central nervous system cancer is selected from the group consisting of astrocytomas (such as childhood astrocytomas), glioblastomas and neuroblastomas. In some embodiments, said pediatric cancer is selected from the group consisting of leukemias, brain and central nervous system tumors, lymphomas, retinoblastomas, sarcomas, Wilms tumor, neuroblastoma and glioblastoma.

[0143] In certain embodiments, said cancer is in a form of a solid tumor. In some embodiments, said cancer is a primary cancer or a metastatic cancer. In some embodiments, said treatment is a prophylactic and / or a therapeutic treatment. In certain embodiments, said treatment is a therapeutic treatment.

[0144] In some embodiments, said use comprises administration of the vector, as defined in any one of the embodiments discussed in relation to the first aspect of the disclosure, to a subject in need thereof. In some embodiments, wherein said vector is a viral vector as defined in embodiments discussed in relation to the first aspect of the disclosure, said use comprises administration of the vector to the subject in a dose comprising at least about 1 x 1011viral particles, such as at least about 3 x 1011viral particles, such as at least about 5 x 1011viral particles. For the sake of clarity, 1011and Ell are used herein interchangeably. In some embodiments, wherein said vector is a viral vector as defined in embodiments discussed in relation to the first aspect of the disclosure, said use comprises administration of the vector to the subject in a dose comprising from about 1 x 1011viral particles to about 10 x 1011viral particles, such as a dose comprising from about 1 x 1011viral particles to about 5 x 1011viral particles. As demonstrated in the appended Examples in vivo, e.g. in Examples 11-16, the vector as disclosed herein is safe, at least the dose range of from about 1 x 1011viral particles to about 5 x 1011viral particles. This is illustrated in the recited examples by at least the absence of toxicity, sparse shedding and effective viral clearance. In some embodiments, said use comprises a single administration of the vector to the subject. In other embodiments, said use comprises repeated administration of the vector to the subject. In one embodiment, wherein said vector is a viral vector, said use comprises administration of the vector to the subject in a dose comprising about 1 x 1011viral particles. In another embodiment, wherein said vector is a viral vector, said use comprises administration of the vector to the subject in a dose comprising about 5 x 1011viral particles. In some embodiments, wherein said vector is a viral vector as defined in embodiments discussed in relation to the first aspect of the disclosure, each administration of said repeated administration comprises the administration of the vector in a dose as defined in the embodiments immediately above. In some embodiments, a time interval between consecutive administrations of said repeated administration is at least about 1 week, such as at least about 1 month, such as at least about 6 months, such as at least about 1 year. In some embodiments, a time interval between consecutive administrations of said repeated administration is from about 1 week to about 6 week. In some embodiments, said administration, such as said single or said repeated administration, of the vector is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration. In some particular embodiments, said administration, such as said single or said repeated administration, of the vector is an intra lymphatic administration or an intratumoral administration, such as an intratumoral administration. Previous studies known in the art have administered conditionally replicative adenoviruses (CRAds) intravenously, intraperitoneally or in some cases intracranially. These administration routes may lead to fast systemic spread of the virus according to biodistribution analyses, where viral particles may be abundantly detected in blood and tissues. In these cases, hepatic uptake may be extensive and serum transaminases may be elevated. The fast systemic viral spread may lead to toxicity and acute systemic immune responses that would not be provoked in a setting with intratumoral injections. An intratumoral administration route as disclosed herein likely causes a slower systemic spread of viral particles from the injection site since extensive tumor stroma and high intratumoral tissue pressure are well known to limit viral spread within the solid tumor tissue. To mimic this situation, the present inventors employed in the appended Examples subcutaneous injection that causes a similar slow systemic release of viral particles. Moreover, the present inventors envision that an intralymaphtic administration route may be advantageous in the context of the present disclosure for efficiency and safety as well as for reducing any potential systematic adverse effect. Thus, in some embodiments, said administration, such as said single or said repeated administration, of the vector is a subcutaneous administration, an intratumoral administration or an intralymphatic administration. In some embodiments, said administration, such as said single or said repeated administration, of the vector is a subcutaneous administration or an intralymphatic administration. In some embodiments, said administration, such as said single or said repeated administration, of the vector is a subcutaneous administration or an intratumoral administration. In some embodiments, said administration, such as said single or said repeated administration, of the vector is an intratumoral administration or an intralymphatic administration. In one embodiment, said administration, such as said single or said repeated administration, of the vector is a subcutaneous administration. In one embodiment, said administration, such as said single or said repeated administration, of the vector is a intratumoral administration. In one embodiment, said administration, such as said single or said repeated administration, of the vector is a intralymphatic administration. It is to be understood that the above discussed benefits of certain administration routes are equally relevant for embodiments discussed in relation to administration routes in each aspect of the present disclosure.

[0145] In some embodiments, said use comprises administration of the vector system comprising the first and the second vector, as defined in any one of the embodiments discussed in relation to the second aspect of the disclosure, to a subject in need thereof. In some embodiments, said first and said second vector are administered simultaneously or subsequently, such as simultaneously, to the subject. In some embodiments, the first and the second vector of said vector system are viral vectors as defined in embodiments discussed in relation to the second aspect of the disclosure, said use comprises administration of the first vector to the subject in a dose comprising at least about 1 x 1011viral particles, such as at least about 3 x 1011viral particles, such as at least about 5 x 1011viral particles; and / or administration of the second vector to the subject in a dose comprising at least about 1 x 1011viral particles, such as at least about 3 x 1011viral particles, such as at least about 5 x 1011viral particles. In some embodiments, wherein the first and the second vector of said vector system are viral vectors as defined in embodiments discussed in relation to the second aspect of the disclosure, said use comprises administration of the first vector to the subject in a dose comprising from about 1 x 1011viral particles to about 10 x 1011viral particles, such as a dose comprising from about 1 x 1011viral particles to about 5 x 1011viral particles; and / or administration of the second vector to the subject in a dose comprising from about 1 x 1011viral particles to about 10 x 1011viral particles, such as a dose comprising from about 1 x 1011viral particles to about 5 x 1011viral particles. In one embodiment, wherein the first and the second vector of said vector system are viral vectors, said use comprises administration of the first vector to the subject in a dose comprising about 1 x 1011viral particles and / or administration of the second vector to the subject in a dose comprising about 1 x 1011viral particles. In another embodiment, wherein the first and the second vector of said vector system are viral vectors, said use comprises administration of the first vector to the subject in a dose comprising about 5 x 1011viral particles and / or administration of the second vector to the subject in a dose comprising about 5 x 1011viral particles. In some embodiments, said use comprises a single administration of the first vector and / or a single administration of the second vector to the subject. In some embodiments, said use comprises repeated administration of the first vector and / or repeated administration of the second vector to the subject. In some embodiments, wherein the first and the second vector of said vector system are viral vectors as defined in embodiments discussed in relation to the second aspect of the disclosure, each administration of said repeated administration of the first vector comprises the administration of the first vector in a dose as defined in the embodiments discussed immediately above; and / or each administration of said repeated administration of the second vector comprises the administration of the second vector in a dose as defined in the embodiments discussed immediately above. In some embodiments, a time interval between consecutive administrations of said repeated administration of the first and / or the second vector is at least about 1 week, such as at least about 1 month, such as at least about 6 months, such as at least about 1 year. In some embodiments, a time interval between consecutive administrations of said repeated administration of the first and / or the second vector is from about 1 week to about 6 week. In some embodiments, said administration, such as said single or said repeated administration, of the first and / or the second vector is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration. In some embodiments, said administration, such as said single or said repeated administration, of the first and / or the second vector is an intra lymphatic administration or an intratumoral administration, such as an intratumoral administration.

[0146] In some embodiments, said use comprises administration of the isolated cell population to a subject in need thereof. In some embodiments, the isolated cell population administered to the subject is an allogeneic or an autologous cell population. As discussed above in relation to ex vivo gene therapy modalities, in some embodiments, said use comprises

[0147] (e) obtaining a precursor cell population, such as a malignant cell population (such as a cancer cell population), from a subject suffering from cancer, such as cancer as defined above,

[0148] (f) ex vivo transfecting or transducing the precursor cell population obtained in step (e) to obtain the isolated cell population, as defined above,

[0149] (g) administering the isolated cell population obtained in step (f) to the subject from which the precursor cell population was obtained in step (e).

[0150] In relation to ex vivo gene therapy modalities, in some embodiments, said use comprises the steps of ex vivo transfecting or transducing a precursor cell population, such as a malignant cell population (such as a cancer cell population), to obtain an isolated cell population, as defined above; and administering said isolated cell population to subject in need thereof. It will be appreciated that the cancer cell population may from a cancer as defined herein, in other word may be from a malignancy arising from epithelial tissue or a malignancy arising from non-epithelial tissue.

[0151] The isolated cell population may be as defined in any one of the embodiments discussed in relation to the fifth aspect of the disclosure. Said ex vivo transfection or transduction of the precursor cell population is to be understood as performed outside the body of the subject, i.e. it refers to an in vitro transfection or transduction of the precursor cell population. Said in vitro transfection or transduction of the precursor cell population may be performed using the method according to the third aspect of the present disclosure. Accordingly, said isolated cell population may be obtained using the vector or the vector system as disclosed in any one of the embodiments discussed in relation to the first or the second aspects of the present disclosure. In one embodiment, the precursor cell population is transfected or transduced ex vivo in step (f) using the vector, such as the viral vector, according to the first aspect of the disclosure. In one embodiment, the precursor cell population is transduced ex vivo in step (f) using the viral vector. In view of the herein described ex vivo gene therapy modalities, in some embodiments, the isolated cell population administered to the subject is thus an autologous cell population. In other embodiments, the isolated cell population administered to the subject is an allogeneic cell population. The present inventors envision that the isolated cell population administered to a subject according to the present disclosure, may be allogeneic to a subject. This may be especially the case in the therapy of certain cancer types, such as melanoma. Thus, in one embodiment, the use comprises administration of the isolated cell population to a subject suffering from melanoma, wherein the isolated cell population is an allogeneic cell population. The present inventors expect that any potential immune response that may be induced against the allogeneic cell population will be dominated by the herein disclosed advantageous immune response. In some embodiments, the isolated cell population administered to the subject is stable cell line, such as a stable malignant cell line, such as a stable cancer cell line, such as a stable melanoma cell line. In one embodiment, said stable malignant cell line is a stable carcinoma cell line. In one embodiment, said stable malignant cell line is a stable melanoma cell line. In some embodiments, said use comprises a single administration of the isolated cell population to the subject. In other embodiments, said use comprises repeated administration of the isolated cell population to the subject. A time interval between consecutive administrations of said repeated administration of the isolated cell population may be at least about 1 week, such as at least about 1 month, such as at least about 6 months, such as at least about 1 year. In some embodiments, a time interval between consecutive administrations of said repeated administration of the isolated cell population is from about 1 week to about 6 week. In some embodiments, said administration, such as said single or said repeated administration, of the isolated cell population is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration. In some embodiments, said administration, such as said single or said repeated administration, of the isolated cell population is a subcutaneous administration, an intratumoral administration or an intralymphatic administration. In some embodiments, said administration, such as said single or said repeated administration, of the isolated cell population is a subcutaneous administration or an intralymphatic administration. In some embodiments, said administration, such as said single or said repeated administration, of the isolated cell population is a subcutaneous administration or an intratumoral administration. In some embodiments, said administration, such as said single or said repeated administration, of the isolated cell population is an intratumoral administration or an intralymphatic administration. In one embodiment, said administration, such as said single or said repeated administration, of the isolated cell population is a subcutaneous administration. In one embodiment, said administration, such as said single or said repeated administration, of the isolated cell population is an intratumoral administration. In one embodiment, said administration, such as said single or said repeated administration, of the isolated cell population is an intra lymphatic administration.

[0152] In some embodiments, said use comprises the administration of the RNA system comprising the first and the second RNA, such as administration of the RNA system comprising the first and the second mRNA , as defined in any one of the embodiments discussed in relation to the eighth aspect of the disclosure, to a subject in need thereof. In some embodiments, said first and said second RNA are administered simultaneously or subsequently, such as simultaneously, to the subject. In some embodiments, said use comprises a single administration of the first RNA and / or a single administration of the second RNA to the subject. In some embodiments, said use comprises repeated administration of the first RNA and / or repeated administration of the second RNA to the subject. In some embodiments, a time interval between consecutive administrations of said repeated administration of the first and / or the second RNA is at least about 1 week, such as at least about 1 month, such as at least about 6 months, such as at least about 1 year. In some embodiments, a time interval between consecutive administrations of said repeated administration of the first and / or the second RNA is from about 1 week to about 6 week. In some embodiments, said administration, such as said single or said repeated administration, of the first and / or the second RNA is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration. In some embodiments, said administration, such as said single or said repeated administration, of the first and / or the second RNA is an intralymphatic administration or an intratumoral administration, such as an intratumoral administration.

[0153] In light of the above discussed gene therapy modalities and in view of that the herein disclosed conceptually new form of immunotherapy may be achieved by alternative means and manners, the present inventors envision additional gene therapy systems applicable in the context of the present disclosure. Thus, in a thirteenth aspect of the present disclosure is provided a gene therapy system for simultaneous or subsequent administration thereof to a subject, comprising

[0154] (h) the first and the second cell type population as defined according to the fifth aspect of the present disclosure;

[0155] (i) the first and the second vector as defined according to the second aspect of the present disclosure;

[0156] (j) the first cell type population as defined according to the fifth aspect of the present disclosure and the second vector as defined according to the second aspect of the present disclosure; or

[0157] (k) the second cell type population as defined according to the fifth aspect of the present disclosure and the first vector as defined according to the second aspect of the present disclosure.

[0158] In some embodiments, said first cell type population, said second vector, said second cell type population and / or said first vector is formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, intralymphatic or intratumoral administration to the subject. It will be appreciated that the administration route as well as the formulation of the above referred components of the gene therapy systems may be selected for suitability for the given components. Moreover, it is to be understood that embodiments discussed above in relation to the second and fifth aspect of the present disclosure relating to the formulation of the first vector and the second vector as well as relating to the formulation of the isolated cell population, as disclosed herein, are equally relevant for the thirteenth aspect of the disclosure. For the sake of brevity, these will not be repeated herein.

[0159] In a fourteenth aspect of the present disclosure is provided the gene therapy system as defined in any one of the embodiments discussed above in relation to the thirteenth aspect of the present disclosure, for use as a medicament.

[0160] In a fifteenth aspect of the present disclosure is provided the gene therapy system as defined in any one of the embodiments discussed above in relation to the thirteenth aspect of the present disclosure, for use in the treatment of cancer. In one embodiment, said cancer is a malignancy arising from epithelial tissue or a malignancy arising from non-epithelial tissue. It is to be understood that embodiments discussed above in relation to the twelfth aspect of the present disclosure relating to the cancer types as well as treatment types, as disclosed herein, are equally relevant for the fifteenth aspect of the disclosure. For the sake of brevity, these will not be repeated herein or will be just briefly mentioned below. The skilled person will appreciate that the term cancer in the present context encompasses malignancies that originate from epithelial tissues as well as malignancies from non-epithelial tissues, as discussed in relation to said twelfth aspect. Accordingly, said cancer may be as defined in embodiments discussed above in relation to the twelfth aspect of the present disclosure. Said treatment may be a prophylactic and / or a therapeutic treatment. Said treatment may be a therapeutic treatment. Moreover, in some embodiments, said use comprises administration of the gene therapy system as defined in any one of the embodiments discussed in relation to the thirteenth aspect of the present disclosure. For the sake of clarity, several gene therapy systems according to the thirteenth aspect of the present disclosure are disclosed, each comprising at least the recited two components, as described above. It is to be understood that both components of the gene therapy systems are administered to the subject. In some embodiments, said first cell population is administered to the subject simultaneously, a priori or subsequently to the administration of the second cell population; said first vector is administered to the subject simultaneously, a priori or subsequently to the administration of the second vector; said second vector is administered to the subject simultaneously, a priori or subsequently to the administration of the first cell population; or said first vector is administered to the subject simultaneously, a priori or subsequently to the administration of the second cell population.

[0161] In some embodiments, said administration of said first cell type population, said second vector, said second cell type population and / or said first vector is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration. It is to be understood that embodiments discussed above in relation to the twelfth aspect of the present disclosure relating to the first vector and the second vector as well as relating to the isolated cell population, as disclosed herein, are equally relevant for the fifteenth aspect of the disclosure. These include for example embodiments relating to dosage, the number of administrations and administration routes. For the sake of brevity, these will not be repeated herein.

[0162] In a sixteenth aspect of the present disclosure is provided a method of treatment of a subject in need thereof, comprising administration of the vector, the vector system, the isolated cell population, and / or the RNA system, to the subject in need thereof. In one embodiment is provided the method of treatment of a subject in need thereof, comprising administration of the vector, the vector system, and / or the isolated cell population, as defined above, to the subject in need thereof. The vector, the vector system, the isolated cell population and the RNA system may be as defined in any one of the embodiments discussed in relation to the first, the second, the fifth and the eighth aspect of the present disclosure, respectively. In one embodiment is provided the method of treatment of a subject in need thereof, comprising administration of the vector, the vector system, the isolated cell population, or the RNA system, to the subject in need thereof. In one embodiment is provided the method of treatment of a subject in need thereof, comprising administration of the vector, the vector system, or the isolated cell population, to the subject in need thereof. In one embodiment is provided the method of treatment of a subject in need thereof, comprising administration of the vector or the isolated cell population to the subject in need thereof. In another embodiment is provided the method of treatment of a subject in need thereof, comprising administration of the vector to the subject in need thereof. In yet another embodiment is provided the method of treatment of a subject in need thereof, comprising administration of the isolated cell population to the subject in need thereof. In one embodiment is provided the method of treatment of a subject in need thereof, comprising administration of the vector, the vector system, or the RNA system, to the subject in need thereof. In one embodiment is provided the method of treatment of a subject in need thereof, comprising administration of the vector, or the RNA system, to the subject in need thereof. In yet another embodiment is provided the method of treatment of a subject in need thereof, comprising administration of the RNA system to the subject in need thereof.

[0163] In a seventeenth aspect of the present disclosure is provided a method of treatment of cancer, comprising administration of the vector, the vector system, the isolated cell population, and / or the RNA system, to a subject in need thereof. In one embodiment, the method comprises administration of the vector, the vector system, and / or the isolated cell population, as defined above, to the subject in need thereof. In one embodiment, said cancer is a malignancy arising from epithelial tissue or a malignancy arising from non-epithelial tissue. The vector, the vector system, the isolated cell population and the RNA system may be as defined in any one of the embodiments discussed in relation to the first, the second, the fifth and the eighth aspect of the present disclosure, respectively. In one embodiment, the method comprises administration of the vector, the vector system, or the isolated cell population, as defined above, to the subject in need thereof. In one embodiment, is provided the method of treatment of cancer, comprising administration of the vector, the vector system, or the isolated cell population, to the subject in need thereof. In one embodiment is provided the method of treatment of cancer comprising administration of the vector or the isolated cell population to the subject in need thereof. In another embodiment is provided the method of treatment of cancer comprising administration of the vector to the subject in need thereof. In yet another embodiment is provided the method of treatment of cancer comprising administration of the isolated cell population to the subject in need thereof. In one embodiment, is provided the method of treatment of cancer, comprising administration of the vector, the vector system, or the RNA system, to the subject in need thereof. In one embodiment, is provided the method of treatment of cancer, comprising administration of the vector, or the RNA system, to the subject in need thereof. In yet another embodiment is provided the method of treatment of cancer comprising administration of the RNA system to the subject in need thereof. In certain embodiments, the method of treatment of cancer, comprises administration of the vector, the vector system, the isolated cell population and the RNA system, such as administration of the vector, the vector system and the isolated cell population. In one embodiment, said RNA system is an mRNA system. In certain embodiments, the method of treatment of cancer, comprises administration of the vector and the isolated cell population. It is to be understood that embodiments discussed above in relation to the twelfth aspect of the present disclosure relating to the cancer types as well as treatment types, as disclosed herein, are equally relevant for the seventeenth aspect of the disclosure. For the sake of brevity, these will not be repeated herein. Moreover, it is to be understood that embodiments discussed above in relation to the twelfth aspect of the present disclosure relating to the vector, the vector system, the isolated cell population, as well as the RNA system, as disclosed herein, are equally relevant for the seventeenth aspect of the disclosure. These include for example embodiments relating to dosage, the number of administrations and administration routes. For the sake of brevity, these will not be repeated herein.

[0164] In an eighteenth aspect of the present disclosure is provided a method of treatment of a subject in need thereof, comprising administration of the gene therapy system as defined in any one of the embodiments discussed in relation to the thirteenth aspect of the present disclosure, to the subject in need thereof.

[0165] In a nineteenth aspect of the present disclosure is provided a method of treatment of cancer, comprising administration of the gene therapy system as defined in any one of the embodiments discussed in relation to the thirteenth aspect of the present disclosure, to a subject in need thereof. It is to be understood that embodiments discussed above in relation to the twelfth aspect of the present disclosure relating to the cancer types as well as treatment types, as disclosed herein, are equally relevant for the nineteenth aspect of the disclosure. For the sake of brevity, these will not be repeated herein. In addition, the skilled person will appreciate that the term cancer in the present context encompasses malignancies that originate from epithelial tissues as well as malignancies from non-epithelial tissues, as discussed in relation to said twelfth aspect. Moreover, it is to be understood that embodiments discussed above in relation to the twelfth and fifteenth aspects of the present disclosure relating to the gene therapy systems and / or the components thereof, as disclosed herein, are equally relevant for the nineteenth aspect of the disclosure. These include for example embodiments relating to dosage, the number of administrations and administration routes. For the sake of brevity, these will not be repeated herein.

[0166] In a twentieth aspect of the present disclosure is provided a use of the vector, the vector system, the isolated cell population, and / or the RNA system, in the manufacture of a medicament. In one embodiment is provided the use of the vector, the vector system, and / or the isolated cell population, in the manufacture of a medicament. The vector, the vector system, the isolated cell population and the RNA system may be as defined in any one of the embodiments discussed in relation to the first, the second, the fifth and the eighth aspect of the present disclosure, respectively. In one embodiment, is provided the use of the vector, the vector system, the isolated cell population, or the RNA system, in the manufacture of a medicament. In one embodiment, said RNA system is an mRNA system. In one embodiment, is provided the use of the vector, the vector system, or the isolated cell population, in the manufacture of a medicament. In one embodiment is provided the use of the vector or the isolated cell population in the manufacture of a medicament. In one embodiment is provided the use of the vector in the manufacture of a medicament. In yet another embodiment is provided the use of the isolated cell population in the manufacture of a medicament. In one embodiment, is provided the use of the vector, the vector system, or the RNA system, in the manufacture of a medicament. In one embodiment, is provided the use of the vector, or the RNA system, in the manufacture of a medicament. In yet another embodiment is provided the use of the mRNA system in the manufacture of a medicament.

[0167] In a twenty-first aspect of the present disclosure is provided a use of the vector, the vector system, the isolated cell population, and / or the RNA system in the manufacture of a medicament for the treatment of cancer. In one embodiment, is provided the use of the vector, the vector system, and / or the isolated cell population, in the manufacture of a medicament for the treatment of cancer. In one embodiment of the twenty-first aspect of the disclosure is provided the use of the vector, the vector system, the isolated cell population, or the RNA system, in the manufacture of a medicament for the treatment of cancer. In one embodiment of the twenty-first aspect of the disclosure is provided the use of the vector, the vector system, or the isolated cell population, in the manufacture of a medicament for the treatment of cancer. In one embodiment is provided the use of the vector or the isolated cell population in the manufacture of a medicament for the treatment of cancer. In one embodiment is provided the use of the vector in the manufacture of a medicament for the treatment of cancer. In yet another embodiment is provided the use of the isolated cell population in the manufacture of a medicament for the treatment of cancer. In one embodiment, is provided the use of the vector, the vector system, or the RNA system, in the manufacture of a medicament for the treatment of cancer. In one embodiment, is provided the use of the vector, or the RNA system, in the manufacture of a medicament for the treatment of cancer. In yet another embodiment is provided the use of the RNA system in the manufacture of a medicament for the treatment of cancer. The vector, the vector system, the isolated cell population and the mRNA may be as defined in any one of the embodiments discussed in relation to the first, the second, the fifth and the eighth aspect of the present disclosure, respectively. It is to be understood that embodiments discussed above in relation to the twelfth aspect of the present disclosure relating to the cancer types as well as treatment types, as disclosed herein, are equally relevant for the twenty-first aspect of the disclosure. For the sake of brevity, these will not be repeated herein. The skilled person will appreciate that the term cancer also in this context encompasses malignancies that originate from epithelial tissues as well as malignancies from non-epithelial tissues, as discussed in relation to said twelfth aspect. Moreover, it is to be understood that embodiments discussed above in relation to the twelfth aspect of the present disclosure relating to the vector, the vector system, the isolated cell population as well as the RNA system, as disclosed herein, are equally relevant for the twenty-first aspect of the disclosure. These include for example embodiments relating to dosage, the number of administrations and administration routes. For the sake of brevity, these will not be repeated herein.

[0168] In a twenty-second aspect of the present disclosure is provided a use of the gene therapy system as defined in any one of the embodiments discussed in relation to the thirteenth aspect of the present disclosure, in the manufacture of a medicament. It is to be understood that said medicament may comprise two separate components corresponding to the components of the gene therapy systems, as disclosed in the thirteenth aspect herein. These components of the medicament are formulated for simultaneous or subsequent administration thereof to a subject, and are considered constituting the medicament in combination.

[0169] In a twenty-third aspect of the present disclosure is provided a use of the gene therapy system as defined in any one of the embodiments discussed in relation to the thirteenth aspect of the present disclosure, in the manufacture of a medicament for the treatment of cancer. It is to be understood that said medicament may comprise two separate components corresponding to the components of the gene therapy systems, as disclosed in the thirteenth aspect herein. These components of the medicament are formulated for simultaneous or subsequent administration thereof to a subject, and are considered constituting the medicament in combination. Moreover, it is to be understood that embodiments discussed above in relation to the twelfth aspect of the present disclosure relating to the cancer types as well as treatment types, as disclosed herein, are equally relevant for the twenty- third aspect of the disclosure. For the sake of brevity, these will not be repeated herein. The skilled person will appreciate that the term cancer also in this context encompasses malignancies that originate from epithelial tissues as well as malignancies from non-epithelial tissues, as discussed in relation to said twelfth aspect. In addition, it is to be understood that embodiments discussed above in relation to the twelfth and fifteenth aspects of the present disclosure relating to the gene therapy systems and / or the components thereof, as disclosed herein, are equally relevant for the twenty-third aspect of the disclosure. These include for example embodiments relating to dosage, the number of administrations and administration routes. For the sake of brevity, these will not be repeated herein.

[0170] While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention is not to be limited to any particular embodiment, but that the invention will include all embodiments falling within the scope of the appended claims.

[0171] The invention will be further illustrated by the following non-limiting Examples. They are offered for illustrative purposes only and are not intended to limit the invention in any manner. Those of skilled in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperatures, etc.), but some experimental error and deviations may be present. Unless otherwise indicated, the practice of the invention employs conventional methods of cell biology, biochemistry, recombinant gene tecnology and molecular biology, in vitro and in vivo experimental methods and bioinformatics, within the skill of the art. Such techniques are explained fully in the existing literature. Additionally, it will be apparent to one of skilled in the art that the methods applied herein for recombinant viral vector engineering and functional testing can also be applied to other vectors described herein and contemplated by the present inventors to fall within the scope of the disclosure.

[0172] Incorporation by reference

[0173] Various publications are cited in the present application, each of which is incorporated by reference herein in its entirety.

[0174] EXAMPLES

[0175] Example 1 Adenoviral constructs and cancer cell lines

[0176] Example 1 describes adenoviral constructs and cancer cell lines used in the following Examples.

[0177] Adenoviral constructs

[0178] The adenoviral constructs have a backbone from Adenovirus serotype 5. To facilitate entry into tumor cells, the coxsackie-adenovirus receptor (CAR)-binding fiber knob of serotype 5 is replaced by the CD46-binding fiber knob of serotype 35, which allows viral entry through interaction with CD46, a cell surface protein abundantly expressed on many tumor types.

[0179] As demonstrated in Fig. 1, the E1B gene is fully deleted in each construct. Ad(Mock) corresponds to a non-replicative adenovirus, wherein the both E1A and E1B genes are fully deleted. Ad(onco) viruses (Ad(onco), Ad(onco-A), Ad(onco-B) and Ad(onco-AB)) are restricted to replication in cancer cells, such as cancer cells with aberrant p53 and Rb-E2F mediated cell cycle control, by a 24 bp deletion in the E1A gene (E1AA24) and full deletion of the E1B gene. Said partial deletion of the E1A gene and full deletion of the E1B gene prevent viral replication in normally functioning cells.

[0180] Ad(onco-A), Ad(onco-B) and Ad(onco-AB) are based on said Ad(onco) virus that additionally comprises either - A: a codon-optimized Ggtal gene of Sus scrofa optimized for protein translation in human according to SEQ ID NO:1 encoding a glycoprotein galactosyltransferase alpha 1,3 (a-l,3-GT) according to SEQ ID NO:2 which enables synthesis of galactose-a-l,3-galactose (a-Gal) on glycoproteins and glycolipids,

[0181] - B: a Nap A gene (Helicobacter pylori) according to SEQ ID NO:3 encoding a Helicobacter pylori (H. pylori) neutrophil-activating protein (NAP) according to SEQ ID NO:4, or

[0182] - AB: both of the above two genes (see Fig. 14).

[0183] The adenoviral construct of Ad(onco-AB) as used in the herein presented Examples comprises a nucleic acid sequence according to SEQ ID NO:33.

[0184] Virus production and amplification

[0185] Expression cassettes corresponding to the above constructs were synthesized at GenScript (Piscataway, New Jersey, USA) and cloned into empty shuttle plasmids. To generate Ad(Mock), Ad(onco-A), Ad(onco-B) and Ad(onco-AB), the corresponding shuttle plasmids, either empty for Mock or containing either E1AA24 (onco), E1AA24 and Ggtal (onco-A), E1AA24 and NAP (onco-B) or E1AA24, Ggtal and NAP (onco-AB) were used for genetic recombination with pAd5f35(E3) backbone plasmid (as described in Yu D, et al. Pios One 2013, 8(1): e54952). The resulting plasmids were purified, linearized and transfected into 911 cells to produce the corresponding viruses.

[0186] The viruses were further amplified in several rounds with an increasing number of cells at each round. The final harvests of viruses were purified by CsCI gradient centrifugation, dialyzed against a buffer containing 10 mM Tris-HCI (pH 8.0), 2 mM MgCl2 and 4% sucrose and stored in aliquots at -80°C. Virus titers were also determined by an FFU (fluorescent focus units) assay. Each virus was produced once and titrated in parallel.

[0187] Cancer cell lines Panc-01, Miacapa-2, SKNFI and U251 cancer cell lines were used in the following Examples.

[0188] Panc-01 and Miacapa-2 are human pancreatic cancer cell lines, SKNFI is a human neuroblastoma cell line and U251 is a human glioblastoma cell line, which were cultured according to standard laboratory protocols. Briefly, all cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 1 mM sodium pyruvate, 100 units / mL penicillin, 100 pg / mL streptomycin (1% PeSt), and 10% (vol / vol) heat-inactivated fetal bovine serum (FBS). The cells were cultured in tissue culture flasks in a humidified incubator at 37°C, in an atmosphere of 5% CO2.

[0189] Example 2

[0190] Evaluation of cell viability of virus-transduced cancer cell lines

[0191] Example 2 presents evaluation of cell viability of Panc-01, Miacapa-2, SKNFI and U251 cancer cell lines which have been transduced by either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1.

[0192] Materials and Methods

[0193] Panc-01, Miacapa-2, SKNFI and U251 cancer cell lines were cultured as described in Example 1 and infected at increasing MOI (i.e. multiplicity of infection) of Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) and Ad(onco-AB) viruses. Cytotoxicity (i.e. cell viability) was measured by MTS assay (Promega, Madison, Wl) after 4 days of infection according to the manufacturer's instructions. Relative cell viability was analyzed by normalization with non-infected cells.

[0194] Results and Conclusions

[0195] As demonstrated in Fig. 2, Ad(Mock) showed no, and in case of SKNF cells, limited oncolytic ability. Ad(onco), Ad(onco-A), Ad(onco-B) and Ad(onco-AB) induced oncolysis of the infected cells, wherein the degree of cell viability correlated negatively with increasing MOI of the viruses. Ad(onco-AB) showed the same oncolytic ability as other oncolytic adenovirus controls (i.e. Ad(onco), Ad(onco-A) and Ad(onco-B)). This indicates that the two transgenes do not affect the oncolytic ability of Ad(onco-AB). Consequently, the present Example demonstrates that an adenovirus according to present invention induces oncolysis of various cancer cells.

[0196] Example 3

[0197] Evaluation of the presence of a-Gal epitopes and NAP expression by virus- transduced cancer cell lines

[0198] Example 3 shows evaluation of the presence a-Gal epitopes in Panc-01, Miacapa-2, SKNFI and U251 cancer cell lines and NAP expression levels in Panc-01 cells, wherein the cells have been transduced by either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1.

[0199] Materials and Methods

[0200] Panc-01, Miacapa-2, SKNFI and U251 cancer cell lines were cultured as described in Example 1.

[0201] For analyzing the presence of a-Gal (A), the cells were infected with Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB) at MOI 50 for 48 hours. Afterwards, the cells were collected and a-Gal (A) epitopes were stained using FITC- conjugated Isolectin B4 (Bandeiraea simplicifolia, Enzo Life Science, NY, USA). The cells and data were analyzed by FACS.

[0202] NAP (B) expression was analyzed by Western blot (WB) using standard protocols, wherein Panc-01 cells were infected with either Ad(Mock) or Ad(onco-AB) virus. Infected cells were collected after 24h, 48h and 72h. NAP expression was detected using a primary a-NAP antibody (a gift from Dr. Marina de Bernard, University of Padova, Italy, 1:10) and a secondary monkey anti-rabbit IgG antibody (Thermo Fisher Scientific, Waltham, Massachusetts, USA, 1:500).

[0203] Results and conclusions As shown in Fig. 3A, a-Gal (A) was functionally synthetized and present on the cell surface in all tested cancer cells lines infected by either Ad(onco-A) or Ad(onco- AB), which comprise the Ggtal gene. a-Gal (A) epitopes were not detectable in any of the tested cancer cell lines which were infected by either Ad(Mock), Ad(onco) or Ad(onco-B), which do not comprise the Ggtal gene. Ad(onco-AB) induced a-Gal (A) epitope levels similar to that induced by Ad(onco-A).

[0204] As demonstrated in Fig. 3B, Ad (onco-AB), but not Ad(Mock), induced NAP (B) expression in Panc-01 cells. NAP (B) expression was already detectable at 48h after infection using Ad (onco-AB), as shown by the appearance of the upper band in WB analysis of the corresponding Ad (onco-AB) samples.

[0205] In conclusion, the present data demonstrate that both a-Gal (A) decoration of cell surface proteins and lipids as well as NAP (B) expression was induced in cancer cells infected by Ad (onco-AB). Thus, Example 2 illustrates that an adenovirus comprising a viral vector according to the present invention induces simultaneous expression of a-Gal epitopes and NAP in infected cancer cells.

[0206] Example 4

[0207] Evaluation of calreticulin exposure on virus-transduced cancer cell lines

[0208] Oncolytic adenoviruses may induce immunogenic cell death (ICD), a type of cell death eliciting an immune response. ICD involves changes in the composition of the cell surface of dying and / or dead cells and the release of soluble danger- associated molecular patterns (DAMPs) in a defined time sequence. Calreticulin (CRT) surface exposure is one of the ICD markers.

[0209] Example 4 demonstrates CRT surface exposure on Panc-01, Miacapa-2, SKNFI and U251 cancer cells which have been transduced by either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1.

[0210] Materials and Methods

[0211] Panc-01, Miacapa-2, SKNFI and U251 cancer cell lines were infected with either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB)at MOI 10 for 48 hours. Infected cells were stained with an anti-calreticulin antibody (PA3-900, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and secondary monkey Alexa-Fluro633 conjugated anti-rabbit IgG (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The cells and data were analyzed by FACS.

[0212] Results and conclusions

[0213] Evaluation of calreticulin exposure on virus-transduced cancer cell lines is shown in Fig. 4.

[0214] As shown in Fig. 4, Ad(onco-AB) caused an increase in surface exposure of CRT on the infected cells compared to that induced by the non-replicating Ad(Mock) virus. This increased exposure was significant in case of Panc-01, Miacapa-2 and SKNFI cells.

[0215] A tendency of higher CRT exposure levels induced by Ad(onco-AB) was observed in comparison to that induced by the oncolytic control virus Ad(onco), which indicates that the presence of a-l,3-GT (A) and NAP (B) transgenes in the Ad(onco-AB) viral vector does not affect negatively oncolytic virus-induced ICD, in fact, has the potential to facilitate oncolytic virus-induced ICD.

[0216] A tendency of higher CRT exposure levels induced by Ad(onco-AB) was observed in most of the cases in comparison to that induced by Ad(onco-A) or Ad(onco-B). For example, Ad(onco-AB) induced higher CRT exposure levels in Panc- 01 cells in comparison that induced by Ad(onco-A) or Ad(onco-B). This indicates that the presence of a-l,3-GT (A) and NAP (B) transgenes in the Ad(onco-AB) viral vector has the potential to facilitate oncolytic virus-induced ICD in comparison to a vector which comprises the a-l,3-GT (A) transgene or the NAP (B) transgene alone.

[0217] In conclusion, an adenovirus comprising a viral vector according to the present invention has the ability to induce elevated CRT exposure levels in cancer cells, and thus induce ICD, which is known to provoke potent and long-lasting anti-cancer immunity. Additionally, the present data surprisingly show that CRT exposure levels induced by an adenovirus comprising both a-l,3-GT (A) and NAP (B) transgenes has the potential to be more prominent on cancer cells, such as on pancreatic cancer cells, in comparison to that induced by an adenovirus comprising only one of a-1,3- GT and NAP transgenes.

[0218] Example 5

[0219] Evaluation of phenotypic maturation of DCs when coculturing with virus- transduced cancer cell lines

[0220] Example 5 presents evaluation of phenotypic maturation of CDllc+immature dendritic cells (DCs) co-cultured with Panc-01, Miacapa-2, SKNFI or U251 cancer cells which have been transduced by either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1. The assessment was performed using phenotypic markers of DC maturation, such as CD80, CD86, CD83, CD40 and CD70.

[0221] Materials and Methods

[0222] Panc-01, Miacapa-2, SKNFI or U251 cancer cell lines were infected with either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB) at MOI 50 for 48 hours. The infected cells were collected, washed in culture medium and co-cultured with 2 x 105immature DCs in a ratio 1:1 for 48 h. Maturation of DCs was analyzed by flow cytometry. Antibodies used to detect maturation markers were, APC-anti-CD80, PE- Cy7-anti-CD83, BV421-anti-CD86 and PE-anti-CD40, BV510-anti-CD70. All antibodies were purchased from BD BioLegend (San Diego, CA). The cells and data were analyzed by FACS.

[0223] Results and conclusions

[0224] As demonstrated in Fig. 5A and 5B, all maturation markers were upregulated on DCs when co-cultured with tumor cells infected by an oncolytic virus, such as Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), in comparison to the nonreplicate virus control group. The presented results in Table 1 and Fig. 5A also reveal significant upregulation of certain maturation markers, such as CD80, on DCs specifically when co-cultured with in Panc-01 cells infected by Ad(onco-AB). A trend for prominent upregulation of other tested markers in this experimental group was also observed. Comparative analysis between replicating viral constructs (i.e. Ad(onco), Ad(onco-A), Ad(onco-B) and Ad(onco-AB)) in cell lines other than Panc-01, as shown in Fig. 5, correspond to the data presented in Table 1, i.e. NS difference, (numerical data not shown). These data all together suggest a beneficial effect on DC maturation when immature DCs are co-cultured with a pancreatic cancer line infected with Ad(onco-AB).

[0225] Table 1. Mean fluorescence intensity (MFI) difference between the different experimental groups tested in Example 5 and presented in Fig. 5A. (Statistical analysis: one-way ANOVA with Tukey's multiple comparison test; NS: no significance, p*<0.05, p**<0.01)

[0226] In conclusion, this Example further supports that a-l,3-GT (A) and NAP (B) transgenes do not affect oncolytic virus-induced ICD. Moreover, co-expression of the two transgenes appears to have an advantageous effect on DC maturation.

[0227] Example 6

[0228] Evaluation of a-Gal-IgG binding on virus-transduced cancer cells

[0229] Humans lack expression of functional a-l,3-GT and thus, a-Gal. Instead, anti-a- Gal antibodies naturally occur in substantial titers in human serum. Thus, a-Gal expression on cells in vitro may be evaluated by co-culturing these with human serum comprising anti-a-Gal antibodies with subsequent detection of the antigenantibody complexes by an anti-IgG antibody. Example 6 presents evaluation of a- Gal-IgG binding on Panc-01, Miacapa-2, SKNFI or U251 cancer cells which have been transduced by either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1, and were incubated with human serum.

[0230] Materials and Methods

[0231] Panc-01, Miacapa-2, SKNFI and U251 cancer cells were infected with either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB) at MOI 10 for 48 hours. Virus-transduced cells were incubated with human serum mixed from 3 different donors for 30 minutes. Then the collected cells were stained with APC-anti-IgG antibody (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and evaluated by FACS.

[0232] Results and conclusions

[0233] As shown in Fig. 6, Ad(onco-A) or Ad(onco-AB) transduced cells have functional a-l,3-GT expression and thus, a-Gal epitope modifications that can be bound with human pre-existing anti-a-Gal antibodies and detected by anti-IgG antibodies. Ad(Mock), Ad(onco) or Ad(onco-B) transduced cells do not have functional a-l,3-GT expression and thus, a-Gal epitope modifications. Consequently, Ad(Mock), Ad(onco) or Ad(onco-B) transduced cells lack a similar degree of a-Gal-IgG binding.

[0234] In conclusion, Ad(onco-AB) is capable of inducing a-Gal decoration of proteins and lipids on cancer cells as well as inducing the binding of naturally occurring anti- a-Gal antibodies to the a-Gal epitope in humans, which in turn enables subsequent cell-lysis of a-l,3-GT-expressing cancer cells via complement activation and NK-cell mediated antibody-dependent cellular cytotoxicity (ADCC). Moreover, binding of anti-a-Gal antibodies to proteins and / or lipids decorated with a-Gal enable markedly enhanced antigen presentation and triggering immune responses.

[0235] Example 7

[0236] Evaluation of C3b deposition on virus-transduced Panc-01 cells and virus-induced complement-dependent cytotoxicity Complement-dependent cytotoxicity (CDC) is an immune response in which target cells are lysed through activation and recruitment of the complement cascade to the targeted cell surface. C3 is the major complement component which plays a central role in the activation of the complement system. Its processing by C3 convertase is the central reaction in both classical and alternative complement pathways. It is cleaved into two fragments, C3a and C3b. After activation, C3b can bind covalently to cell surface carbohydrates or immune aggregates.

[0237] Example 7 presents evaluation of C3b deposition on Panc-01 cells which have been transduced by either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco- AB), as defined in Example 1, and were incubated with human serum. Example 7 also demonstrates virus-induced CDC by adding rabbit complement into the co-cultures of virus-transduced Panc-01 cells and human serum.

[0238] Materials and Methods

[0239] Panc-01 cells have been transduced by either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1, and were co-cultured with serum mixed from 3 different donors for 20 mins (Fig. 7A). C3b deposition was stained by PE-anti-C3b antibody (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and evaluated by FACS. CDC was evaluated by adding rabbit complement (Sigma-Aldrich, St. Louis, Missouri, USA) into co-culture and dead cells were assessed by EthD-1 staining according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The cell and data were evaluated by FACS.

[0240] Results and conclusions

[0241] The data presented in Fig. 7B show C3b-positive cells out of total live cells, wherein the cells were infected with either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB) and wherein two conditions, with and without coculturing with human serum, were tested. As demonstrated in Example 6, coculturing with human serum enables Ad(onco-A) or Ad(onco-AB) transduced cells to have functional a-l,3-GT expression and thus, a-Gal epitope modifications that can be bound with human pre-existing anti-a-Gal antibodies present in human serum. As presented in Table 2 and Fig. 7B, the present inventors have surprisingly found that Ad(onco-AB) induces a prominently and significantly enhanced deposition of C3b on Panc-01 cells when co-cultured with human serum in comparison to that induced by either Ad(onco-A) or Ad(onco-B), demonstrating an unexpected synergistic effect of the two transgenes (a-l,3-GT (A) and NAP (B)) in a viral construct according to the present invention.

[0242] Table 2. Percentage difference ( %) between the different experimental groups tested in Example 7 and presented in Fig. 7. (Statistical analysis: one-way ANOVA with Tukey's multiple comparison test; NS: no significance, p*<0.05, p**<0.01, ****p<0.0001)

[0243] The data presented in Fig. 7C show the percentage of EthD-1 positive cells as dead cells out of total cells wherein rabbit complement was added to Panc-01 cells infected with either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB) and wherein two conditions, with and without co-culturing with human serum, were tested. In line with the results presented in Table 2 and Fig. 7B, the results clearly indicate a superior effect on CDC achieved by Ad(onco-AB) in comparison to Ad(onco-A) or Ad(onco-B) alone. In other words, Ad(onco-AB) induced higher C3b deposition resulted in enhanced CDC in comparison to any of the control viruses (Ad(Mock) and Ad(onco)) as well as in comparison to viruses which only comprise a- 1,3-GT (A) or NAP (B) transgenes (Ad(onco-A) and Ad(onco-B)). In conclusion, the data shown in Example 7 unambiguously reveal an unexpected synergetic effect in the activation of CDC using a viral vector according to present invention, which comprises both a-l,3-GT (A) and NAP (B) transgenes, i.e. wherein infection of cells using a virus comprising said vector induces decoration of cancer cells with a-Gal epitopes and, at the same time, induces NAP expression by said cells. Importantly, said remarkable CDC response cannot be achieved using a viral vector which enables only one of these, i.e. either the decoration of cancer cells with a-Gal epitopes or NAP expression by cancer cells.

[0244] Example 8 Evaluation of ADCC from virus-transduced Panc-01 cells co-cultured with isolated NK cells

[0245] Natural killer (NK) cells, a small set of lymphocytes, are considered important players in inducing antibody-dependent cellular cytotoxicity (ADCC) by provoking innate immune response and harmonizing spontaneous cytotoxicity activated by antibodies linked to target cells. ADCC activity is defined as the percentage of NK cells expressing CD107a and increased number of CD107a+NK cells and / or elevated CD107a expression are indicative of enhanced ADCC against antibody linked target cells.

[0246] Example 8 presents evaluation of CD107a expression on NK cells which have been co-cultured with Panc-01 cells transduced by either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1, and were co- cultured with human serum.

[0247] Materials and Methods

[0248] Panc-01 cells were transfected with either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1, and were co-cultured with serum mixed from 3 different donors for 45 mins, followed by washing once and adding isolated NKs (in 1:1 ratio) for 24h (Fig. 8A). ADCC was evaluated by measuring CD107a expression using BV421-conjugated anti-CD107a (BioLegend, San Diego, CA) on NK cells.

[0249] Results and conclusions The data presented in Table 3 and Fig. 8B show the level of CD107a expression on NK cells, wherein Panc-01 cells infected with either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB) were co-cultured with NK cells and wherein two conditions, with and without co-culturing with human serum, were tested.

[0250] Table 3. Percentage difference ( %) between the different experimental groups tested in Example 8 and presented in Fig. 8B. (Statistical analysis: one-way ANOVA with Tukey's multiple comparison test or t-test; NS: no significance, p*<0.05)

[0251] As demonstrated in Example 6, co-culturing with human serum leads to Ad(onco-A) or Ad(onco-AB) transduced cells to have functional a-l,3-GT expression and thus, a-Gal epitope modifications that can be bound with human pre-existing anti-a-Gal antibodies present in human serum. These a-Gal-anti-a-Gal complexes predispose the labeled cancer cells for ADCC mediated by NK cells. As presented in Fig. 8B, the present inventors have surprisingly found significantly enhanced CD107a expression on NK cells when co-cultured with Panc-01 cells infected with Ad(onco- AB) virus in the presence of human serum. In comparison, the degree of CD107a expression on NK cells when co-cultured with Panc-01 cells infected with either Ad(onco-A) or Ad(onco-B) in presence of human serum was less prominent, demonstrating an unexpected beneficial effect of the two transgenes (a-l,3-GT (A) and NAP (B)) in a viral construct according to the present invention.

[0252] In conclusion, the present data demonstrate a surprising enhancing effect for NK-cell mediated ADCC, targeting cancer cells labeled with a-Gal-anti-a-Gal antibody immunocomplexes. This unexpected effect is achieved by using a viral vector according to present invention, which comprises both a-l,3-GT (A) and NAP (B) transgenes, i.e. wherein infection of cells using a virus comprising said vector induces decoration of cancer cells with a-Gal epitopes and, at the same time, induces NAP expression by said cells. Importantly, said remarkable ADCC response cannot be achieved using a viral vector which enables only one of these, i.e. either the decoration of cancer cells with a-Gal epitopes or NAP expression by cancer cells.

[0253] Example 9

[0254] Evaluation of virus-induced immune response in vitro

[0255] Human peripheral blood mononuclear cells (PBMCs) are isolated from peripheral blood and identified as any blood cell with a round nucleus including lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. PBMCs are a well-established and useful test system for the investigation of immune modulatory effects and immune response in vitro.

[0256] Example 9 presents evaluation of immune response in vitro induced by Panc- 01 cells which have been transduced by either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1, and were co-cultured with human serum and isolated PBMCs.

[0257] Materials and Methods

[0258] Panc-01 cells were transfected with either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1. Infected cells were stained with Ph rodo (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and were co- cultured with human serum mixed from 3 different donors for 20 mins, followed by adding isolated PBMCs for 15 min (for evaluating a-Gal-IgG binding, C3b deposition and CDC) or for 48h (for evaluating phagocytosis by monocytes and activation of immune cells) (Fig. 9A). a-Gal-IgG binding was evaluated by APC-anti-IgG antibody and C3b deposition was evaluated by PE-anti-C3b antibody (Thermo Fisher Scientific, Waltham, Massachusetts, USA). CDC cytotoxicity was assessed by evaluating pH rodo by FACS and normalized to non-infected control tumor cells. Phagocytosis by monocytes was elevated by FACS and calculated as the % of Ph rodo positive monocytes out of total monocytes (gated as CD14+cells). Immune cell activation was evaluated by staining with antibodies (BV421-conjugated anti-human CD45, BUV395-conjugated antihuman CD14, APC-conjugated anti-human CD83, BV605-conjugated anti-human MHC II, PE-conjugated anti-human CD16, APC-conjugated anti-human CD69 and BB515-conjugated anti-human CD19; all antibodies were purchased from BioLegend, San Diego, CA). The cells and data were analyzed by FACS.

[0259] Results and conclusions

[0260] The results presented in Table 4 below are illustrated in Fig. 9.

[0261] Table 4. Percentage difference ( %) between the different experimental groups tested in Example 9 and presented in Fig. 9. (Statistical analysis: one-way ANOVA with Tukey's multiple comparison test; NS: no significance, p*<0.05, p**<0.01, p* **<0.001, ****p<0.0001) As shown in Table 4 and Fig. 9B, Ad(onco-AB) transduced Panc-01 cells exhibited significantly higher anti-a-Gal IgG antibody binding than Panc-01 cells infected with either Ad(onco-A) or Ad(onco-B) in the co-culture. Accordingly, Ad(onco-AB) transduced Panc-01 cells are outstandingly decorated with a-Gal-anti- a-Gal antibody complexes in comparison to cancer cells infected by any of the control viruses (Ad(Mock) and Ad(onco)) and / or viruses which only harbor one of the two transgenes in question (Ad(onco-A) or Ad(onco-B)). As presented in Table 4 and Fig. 9C Ad(onco-AB) transduced Panc-01 cells exhibited elevated C3b deposition in comparison to Panc-01 cells infected with either Ad(onco-A) or Ad(onco-B) in the co-culture. Accordingly, Ad(onco-AB) transduced Panc-01 cells are predisposed to increased CDC in comparison to cancer cells infected by any of the control viruses (Ad(Mock) and Ad(onco)) and / or viruses which only harbor one of the two transgenes in question (Ad(onco-A) or Ad(onco- B)).

[0262] As apparent from Table 4, Fig. 9B and 9C, the prominent increase in the number of a-Gal-lgG+and C3b+cells out of all tumor cells when infected with Ad(onco-AB) demonstrates a synergistic effect achieved by using a virus comprising a viral vector according to present invention to infect cancer cells, which comprises both a-l,3-GT (A) and NAP (B) transgenes, i.e. wherein decoration of cancer cells with a-Gal epitopes and, at the same time, NAP expression by said cells are induced upon infection.

[0263] Table 4 together with Fig. 9D, 9E and 9F presents the assessment of phagocytic monocytes in the co-culture. Ad(onco-A) and Ad(onco-AB) transduced cells showed increased phagocytosis by monocytes (Fig. 9D) in comparison to cancer cells infected either by Ad(Mock), Ad(onco)) or Ad(onco-B). Phagocytosis of Ad(onco-A) and Ad(onco-AB) transduced cancer cells was comparable, however monocytes which have been co-cultured with Panc-01 cells infected by Ad(onco-AB) showed a unique upregulation of activation marker, such as CD86 (Fig. 9E) and MHC II (Fig. 9F), expression. Taken together, these results suggest a surprisingly advantageous effect on monocyte activation of a viral vector according to present invention, which comprises a-l,3-GT (A) and NAP (B) transgenes, i.e. wherein infection of cells using a virus comprising said vector induces decoration of cancer cells with a-Gal epitopes and, at the same time, induces NAP expression by said cells.

[0264] CD69+B cells are known as activated B cells which may be induced by the activation of the B cell receptor (BCR). Upregulated expression of CD69 indicates NK cell activation while decreased expression of CD16 indicates NK-cell mediated target cell cytotoxicity through ADCC and cytokine production. As shown in Table 4, Fig. 9G, 9H and 91, B cells had higher level of CD69 expression and NK cells exhibited upregulated CD69 and down-regulated CD16 expression in Ad(onco-AB) transduced cells in the co-culture when compared to cancer cells infected by any of the control viruses (Ad(Mock) and Ad(onco)) and / or viruses which only harbor one of the two transgenes in question (Ad(onco-A) or Ad(onco-B)). On the contrary and surprisingly, cancer cells infected by viruses which comprise only one of the two transgenes, a- 1,3-GT (A) or NAP (B), do not exhibit any difference on the expression of the tested B cell and NK cell markers in comparison to the control viruses (Ad(Mock) and Ad(onco)). Taken together, these results suggest a synergistic effect on B cell and NK cell maturation and activation achieved by a viral vector according to the present invention, which comprises a-l,3-GT (A) and NAP (B) transgenes, i.e. wherein infection of cells using a virus comprising said vector induces decoration of cancer cells with a-Gal epitopes and, at the same time, induces NAP expression by said cells.

[0265] Table 4 and Fig. 9J shows relative viability of cancer cells infected either Ad(Mock), Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB) co-cultured with PBMCs in presence of human serum. As apparent, each oncolytic adenovirus reduced cancer cell viability in contrast to the Ad(Mock) control. Infection with Ad(onco-AB) virus induced the most prominent decrease in cell viability of cancer cells in comparison to any of Ad(onco), Ad(onco-A) and Ad(onco-B), indicating a superior performance of Ad(onco-AB) in inducing immune response mediated cell death of the infected cells. Taken together, these data indicate that using a virus comprising a viral vector according to the present invention, which comprises a-1,3- GT (A) and NAP (B) transgenes, exerts enhanced cytotoxicity targeting cancer cells by inducing decoration of these cells with a-Gal epitopes and, at the same time, inducing NAP expression by said cells. Importantly, such prominent effect on cytotoxicity cannot be achieved for cancer cells infected by viruses which comprise only one of the two transgenes, a-l,3-GT (A) or NAP (B),

[0266] In summary, the results presented in Example 9 demonstrate a superior immune response induced in vitro by the infection of cancer cells using a virus comprising a viral vector according to the present invention, which comprises a-1,3- GT (A) and NAP (B) transgenes. Said superior immune response manifests in profound cytotoxicity targeting infected cancer cells and is achieved by simultaneous decoration of the cancer cells with a-Gal epitopes and induction of NAP expression by said cells. Consequently, the present inventors have found that a viral vector according to the present invention as well as viral infection of cancer cells which induces decoration of the cells with a-Gal epitopes and induces NAP expression by said cells is surprisingly beneficial for cancer therapy.

[0267] Example 10 Evaluation of tumor growth in vivo

[0268] Example 10 presents evaluation of tumor growth in an in vivo model using a-Gal knock-out (KO) mice, also referred to herein as GGTA1 KO mice. Tumors have been generated in said mice using a pancreatic ductal adenocarcinoma (PDAC) cell line and either Ad(onco), Ad(onco-A), Ad(onco-B) or Ad(onco-AB), as defined in Example 1, was injected intratumorally to evaluate their therapeutic effect on tumor growth.

[0269] Materials and Methods a-Gal KO mice were bread at the animal facility of Rudbeck Laboratory, Uppsala University, Sweden. They were housed in groups of 3-5 individuals in single ventilated cages under controlled climate conditions with a 12 h light, 12 h dark cycle and monitored at least once daily. The cages were enriched with bedding material, polycarbonate houses and paper wool, with free access to food and water.

[0270] The experiment timeline including the vaccination regimen is outlined in Fig. 10A. In brief, GGTA1 knock mouse were primed and boosted with Gal-BSA (10 pg / mouse), CpG (5 nmol / mouse) and Incomplete Freund's adjuvant (IFA, 50 pl / mouse) in a 1:1 ratio diluted in PBS on day -14 and day -7. Ggtal-knock out pancreatic ductal adenocarcinoma cell line (Panc-02-KO) were injected subcutaneously in the right flank. On day 10, Ad(onco), Ad(onco-A), Ad(onco-B) and Ad(onco-AB) viruses, respectively, (l*108FFU / mouse) were injected intratumorally in total 6 times, every 2 days, as shown in Fig. 10A. The tumor size was measured over time using caliper measurement and the tumor size was calculated as volume = length x width2x n / 6. When tumor size reached 1000 mm3in a mouse, a so-called humane end point of the experiment as explained below, the mouse was euthanized.

[0271] Results and conclusions Tumor growth was monitored from day 10 following tumor implantation. As shown in Table 5 and Fig. 10B, tumor growth in Ad(onco-AB)-treated mice was substantially and significantly delayed in comparison to the controls (Ad(onco)) and mice treated with either Ad(onco-A) or Ad(onco-B). Furthermore, mice treated with Ad(onco-AB) showed prolonged and significantly longer survival than the other control and treatment groups, as shown in Fig. 10C). These data together demonstrate that the advantageous effect of Ad(onco-AB) on tumor growth and survival was exceptionally superior in comparison to the effect observed in cases wherein mice were treated with either Ad(onco-AB) or Ad(onco-AB).

[0272] Table 5. Comparison of tumor growth over time and survival of mice between the different experimental groups tested in Example 10 and presented in Fig. 10. (Statistical analysis: tumor size was analyzed by two-way ANOVA with Tukey's multiple comparisons and mice survival was analyzed by long-rank (Man tel -Cox) text; p*<0.05, p**<0.01, p***<0.001, ****p<0.0001.)

[0273] Accordingly, the present inventors have demonstrated that a superior therapeutic effect may be achieved in vivo by the infection of cancer cells using a viral vector according to the present invention, which comprises a-l,3-GT (A) and NAP (B) transgenes. Consequently, the present inventors have surprisingly found that a viral vector according to the present invention as well as viral infection of cancer cells which induces decoration of the cells with a-Gal epitopes and induces NAP expression by said cells is surprisingly beneficial for cancer therapy.

[0274] Example 11 Viruses, animal models, treatment and sampling protocols for in vivo studies described in Examples 12-16

[0275] Example 11 describes viruses, animal models and sampling protocols used in the herein presented in vivo experiments according to Examples 12-16. Viruses

[0276] Two batches of Ad(onco-AB) were used for the hamster study: Ad(onco-AB)- GMP and non-GMP Ad(onco-AB). Ad(onco-AB)-GMP was produced under GMP (good manufacturing practice) conditions and is intended for clinical use. Ad(onco- AB), produced under non-GMP conditions, was also used for some groups in the study. For the sake of clarity, Ad(onco-AB) or non-GMP Ad(onco-AB), as used herein, both refer to Ad(onco-AB) produced under non-GMP conditions. As a nonreplicating control, another virus, Ad(Mock), was used. Its surface structure is identical to Ad(onco-AB) with backbone of serotype 5 and CD46-binding fiber knob of serotype 35. It has a complete deletion of the E1A and E1B genes, inhibiting any viral replication, and does not carry any transgenes.

[0277] Similarly as explained in Example 1, for the mouse experiment, Ad(onco-AB) and three other control viruses, Ad(onco), Ad(onco-A) and Ad(onco-B), were used. A non-replicating adenoviral vector, Ad(luc), expressing the transgenes green fluorescent protein and luciferase was used as a negative control.

[0278] Hamsters

[0279] Seven weeks old male and female Syrian / Golden hamsters were purchased from Janvier-Labs and housed at the National Veterinary Institute, Uppsala, Sweden. The hamsters were single housed in Sealsafe GR900 (Tecniplast) on aspen bedding (Tapvei) under controlled climate conditions with a 12 h light, 12 h dark cycle and monitored at least once daily. The cages were enriched with bedding material, polycarbonate houses, chewing sticks and sand baths. They had unlimited access to drinking water and were fed ad lib with Nature Hamster (Versele-Laga), Complete Hamster & Gerbil (Versele-Laga) and Harry Hamster tasty mix (Tiny Friends Farm). During the seven-day acclimatization the hamsters were trained to be accustomed to the handler and prepared for the experimental procedures.

[0280] The hamsters were randomized into treatment groups with three males and three females per group receiving two subcutaneous injections on days 0 and 21 with PBS, Ad-Mock (5E11 viral particles (vp) / kg), Ad(onco-AB)-GMP (1E11 vp / kg) or Ad(onco-AB)-GMP (5 Ell vp / kg). At day 24 and 25, in total three hamsters per group were euthanized and the remaining hamsters were euthanized at day 42. In addition, one male and one female received 5E11 vp / kg of non-GMP Ad(onco-AB) and were euthanized at day 25. Another group of two males and two females were treated with 5E11 vp / kg non-GMP Ad(onco-AB) in an intensified treatment schedule with three subcutaneous injections at day 1, 17 and 31 followed by termination at day 34. Two males and two females were left untreated as negative controls and euthanized at day 17.

[0281] Treatment groups and schedule for hamsters is presented below in Table 6:

[0282] Table 6. Overview of treatment groups and treatment schedules of Syrian hamsters

[0283] (5E11 and 1E11 correspond to 5,00E+ll vp / kg and 1,OOE+11 vp / kg, respectively). a-Gal knock-out (KO) mice a-Gal KO mice, also referred to herein as GGTA1 KO mice, were bread at the animal facility of Rudbeck Laboratory, Uppsala University, Sweden. They were housed in groups of 3-5 individuals in single ventilated cages under controlled climate conditions with a 12 h light, 12 h dark cycle and monitored at least once daily. The cages were enriched with bedding material, polycarbonate houses and paper wool, with free access to food and water. Mice were immunized against a-Gal by two injections of mucin and polyinosinic-polycytidylic (Poly I :C), wherein mucin is a source of aGal, as antigen. Tumor cells from a mouse GGTAl-knock out pancreatic ductal adenocarcinoma cell line (Panc-02-KO), were injected subcutaneously in the right flank. When palpable tumors were formed virus (Ad(onco-AB), Ad(onco), Ad(onco-A) or Ad(onco-B)) was injected intratumorally on day 10 and 15 as shown in Table 7. When tumor size reached 1000 mm3, a so-called humane end point of the experiment, mice were euthanized and organs were collected. Accordingly, mice were not terminated due to e.g. the nature of tumor growth or organ failure. Termination dates ranged from 11 to 29 days after the second viral injection.

[0284] Table 7. Overview of treatment groups and treatment schedules ofa-Gal KO mice.

[0285] Sampling protocols

[0286] From the hamsters, bio fluids including feces, urine-soaked bedding and buccal swabs were collected on day 1, 3, 6, 12 and 21 after the first treatment and at termination. The cages, including the interior materials and food, were changed on the day before the sampling to capture the actual shedding at the sampling timepoint.

[0287] At termination, the hamsters were anesthetized with isoflurane followed by collection of blood through heart puncture after which cervical dislocation was performed. Tissue samples of approximately 2x2x2 mm for biodistribution analysis were collected from brain, spinal cord, heart, lungs, mammary gland / skin, ventricle, small intestine, large intestine, liver, pancreas, femoral bone marrow, adrenal gland, mesenteric lymph nodes, testicles, epididymides, ovaries, urinary bladder and prostate. Mammary gland tissue was difficult to isolate during necropsy and these biopsies represent a combination of mammary gland tissue, skin and subcutaneous tissue. Bio fluids including feces, saliva and urine was also collected. In case the urinary bladder contained urine a sample was taken by cystocentesis, otherwise urine was collected from the bedding. All samples intended for biodistribution were stored at -80°C until analysis. The remaining tissue from the respective organs and in addition the sternum and optic nerve were fixed in 4% buffered formaldehyde or in Davidson's solution and later used for histopathological analysis. From mice, heart, lung, spleen, liver, kidney, ventricle, brain (except three animals) and tumor were collected and fixed in 4% buffered formaldehyde for histopathological analysis.

[0288] Example 12

[0289] Evaluation of adverse side effects and humoral immune response in vivo

[0290] Example 12 presents evaluation of adverse side effects as well as serum IgG levels upon administration of Ad(onco-AB)-GMP or non-GMP Ad(onco-AB) to hamsters in comparison to untreated controls and controls which were administered PBS or Ad(Mock) as described in Example 11.

[0291] Materials and Methods

[0292] In order to monitor the humoral immune response against Ad(onco-AB)-GMP or non-GMP Ad(onco-AB), anti-adenovirus IgG titration was performed by ELISA on serum collected at necropsy, according to standard protocols.

[0293] Briefly, polystyrene high-bind ELISA plates (Costar 3361, Sigma-Aldrich, St.

[0294] Louis, Missouri, USA) were coated with viral particles through the incubation of 4E11 vp / mL Ad(onco-AB) in PBS at 4°C overnight. 2% BSA diluted in PBS was used for blocking for 2 h. Hamster serum collected at necropsy and diluted at 1:1000 in PBS was applied and incubated for 1 h at room temperature. Secondary antibody, goat anti-Syrian hamster IgG, conjugated with horse reddish peroxidase (HRP, ab6892, Abeam, Cambridge, United Kingdom) diluted at 1:8000 was applied and incubated for 1 h at room temperature. Substrate (#34028, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was applied for HRP detection and the signal was analyzed at 450 nm (Imark Microplate reader, Bio-Rad, Hercules, California, USA). Mann Whitney U test was used to compare pooled virus treated groups to pooled untreated and PBS treated groups.

[0295] Results and conclusions Syrian hamsters were treated with repeated injections of virus according to

[0296] Example 11. Throughout the experiment no adverse effects were observed by visual inspection.

[0297] Treatment with Ad(Mock), Ad(onco-AB)-GMP or non-GMP Ad(onco-AB) stimulated production of anti-adenovirus IgG antibodies, as indicated by a significantly higher titer in virus treated animals in comparison to untreated controls and PBS treated animals (Fig. 11, p*=1.5E-6). A trend of increasing titers was observed from day 3 to day 21 after the second treatment injection (Fig. 11). The titers were similar between the treated groups regardless of dose (1E11 vs 5E11 vp / kg) and the number of injections. The results indicate that treatment with Ad(onco-AB)-GMP or non-GMP Ad(onco-AB) is well-tolerated and stimulates a humoral immune response.

[0298] Example 13 Evaluation of viral particle tissue distribution in vivo

[0299] Example 13 presents evaluation of viral particle tissue distribution upon administration of Ad(onco-AB)-GMP or non-GMP Ad(onco-AB) to hamsters in comparison to untreated controls and controls which were administered PBS or Ad(Mock) as described in Example 11.

[0300] Materials and Methods

[0301] An ISO17025 accredited method for extracting viral DNA (Adenovirus sp.) from Syrian hamster (Mesocricetus auratus) tissues and an ISO17025 accredited qPCR Probe assay for determining the concentration of viral particles was developed by TATAA Biocenter (Gothenburg, Sweden). In addition, the ValidPrime® Vertebrate assay (TATAA Biocenter, #A105P10) was verified for use on golden hamster DNA. Once finished, a biodistribution analysis was performed using the validated methods. A total of 1205 samples (including a few duplicate samples that had to be diluted to prevent inhibition) was assayed for presence of virus particles, as well as determining the number of hamster genomes in each sample. For DNA isolation, samples were processed mechanically with beads and chemically with proteinase K, RNase A (Qiagen, Hilden, Germany; cat. nr. 19101) and Buffer ATL (Qiagen, Hilden, Germany; cat. nr. 939016). Saliva, serum and urine were processed by QIAsymphony DSP Virus / Pathogen Kit (Qiagen, Hilden, Germany; cat. nr. 937036). Feces was processed by QIAsymphony PowerFecal Pro DNA kit (Qiagen, Hilden, Germany; cat. nr. 938036). Blood clot and tissues were processed by QIAsymphony DSP DNA Mini Kit (Qiagen, Hilden, Germany; cat. nr. 937236).

[0302] Extracted DNA samples were subjected to qPCR using both TATAA's proprietary ValidPrime Universal Vertebrate assay (WA105P10, TATAA Biocenter) and the validated E4orfl assay. qPCR reactions were setup as described in Table 8 and cycled according to Table 9. Positive controls were included using hamster gDNA, and E4orfl vector. Negative control reactions, NTCs, were also included on each plate for each reaction type. In addition, four inter-plate calibrators (IPC), (IPC250S, TATAA Biocenter) were included on each plate. Plates were cycled on QuantStudio 7 Pro (Thermo Fisher Scientific) and Cq values were determined by the Design & Analysis Software (v. 2.6.0, Thermo Fisher Scientific, Waltham, Massachusetts, USA) using the built-in relative threshold algorithm that uses a fitted efficiency model for determination of Cq values. qPCR data was analyzed with GenEx (MultiD) using the integrated functions for IPC correction and reverse calibration to the standard curve.

[0303] Table 8. Mastermix protocol for one qPCR reaction

[0304] Table d. Thermal protocol for qPCR

[0305] The assay limit of detection and quantification (LOD / LOQ, respectively) was determined to 20 vp / reaction. Samples were included in further analyses if at least one replicate had a viral particle number above 20. If no signal was detected in the second replicate, the mean viral particle number of the sample was set to the number of the first sample. If viral particles were detected (below or above LOD) in both samples, the average was used for the further analysis.

[0306] Results and conclusions To study the tissue distribution of Ad(onco-AB)-GMP and non-GMP Ad(onco- AB), tissue samples obtained from Syrian hamsters according to the treatment groups presented in Table 6 were analyzed by qPCR for detection and quantification of viral particles. The results are presented in Table 10.

[0307]

[0308] Table 10. Viral genome copies in tissue samples at early (3) and late (21) termination,

[0309] 3 and 21 days after last treatment injection, respectively. Analyzed tissues included coagulated blood, serum, brain, spinal cord, optic nerve, heart, lung, mammary gland, ventricle, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenal gland, mesenteric lymph node, bone marrow, bladder, ovary, uterus, testis, epididymis and prostate. Only tissues where virus have been detected in at least one sample are shown. Each grid in the table shows the number of positive samples per group and tissue type and the total number of animals (x:n) in the group (top row) and viral genome copy number normalized to one million hamster genome copies (bottom row).

[0310] Viral particles (vp) above detection limit were found in only 17 of 343 samples from animals treated with Ad(onco-AB)-GMP or Ad(onco-AB), (Group 5-10) (see treatment groups in Table 6), originating from mammary glands / skin, bone marrow, spleen, ventricle and bladder. The viral numbers ranged from 13 to 1170 vp per 1E6 hamster genome copies.

[0311] The distribution of positive samples in different treatment groups, tissues and termination time points is shown in Table 10. No virus was found in the PBS and Ad(Mock) group. Five samples were positive in the low dose Ad(onco-AB)-GMP group (1E11) and six samples were positive in the corresponding high dose group (5 Ell). Out of these 11 samples, 8 were found at the early termination time point and three were found at the late termination time point. This indicates that viral particles are gradually cleared from the tissues without significant replication. Ad(onco-AB) was administered in a 2-dose (n=2) or a 3-dose (n=4) schedule, as shown in Table 6. Two samples were positive in the 2-dose group and an equivalent proportion, four samples, were found in the 3-dose group.

[0312] In summary, viral particles were in most samples not detected or detected only in low copy numbers, indicating that neither Ad(onco-AB)-GMP nor Ad(onco- AB) replicates in the investigated tissues.

[0313] Example 14 Evaluation of viral particle shedding in vivo

[0314] Example 14 presents evaluation of viral particle shedding upon administration of Ad(onco-AB)-GMP or non-GMP Ad(onco-AB) to hamsters in comparison to untreated controls and controls which were administered PBS or Ad(Mock) as described in Example 11.

[0315] Materials and Methods qPCR was utilized to detect and quantify viral genome copies in feces, saliva and urine using essentially the same materials and methods as described in Example 12 with the respective DNA isolation kits applicable for the given sample types as indicated in Example 12. Feces, urine and saliva samples were collected at 1, 3, 6, 12 and 21 days after the first treatment injection. Additionally shedding samples were collected at the day of termination after two or three treatment injections, as shown in Table 6.

[0316] Results and conclusions To study viral shedding into the surrounding environment, biofluids were collected and analyzed. Out of 420 samples from virus treated animals (group 3-10, as shown in Table 6) only ten contained detectable viral levels which are shown in Table 11. Table 11. Viral genome copies in feces, urine and saliva samples obtained from hamsters 1, 3, 6, 12 and 21 days after the first treatment injection or at termination. At termination, two or three injections have been given, as shown in Table 6. Each grid in the table shows the number of positive samples per group and tissue type and the number of animals (x:n) in the group (top row), and viral genome copy number normalized to one hamster genome copy (bottom row).

[0317] Nine out of these positive samples were found one or three days after treatment injection. Only one positive sample was found among the samples collected later (day 6, 12, 21 and at termination). In most cases the positive samples were retrieved from different individuals. The hamster with ID:5A is an exception with detectible virus in three different samples. In accordance with the tissue samples, most positive biofluid samples were found in groups treated with Ad(onco- AB)-GMP and Ad(onco-AB).

[0318] In summary, viral particles were in most samples not detected or detected only in low copy numbers, indicating that viral shredding of Ad(onco-AB)-GMP and Ad(onco-AB) into the surrounding environment is below biologically relevant levels, without risk of secondary infections.

[0319] Example 15 Histopathological evaluation in vivo

[0320] Example 15 presents histopathological evaluation of various tissues collected from hamsters and mice at necropsy to examine treatment related toxicity upon administration of Ad(onco-AB)-GMP, non-GMP Ad(onco-AB), Ad(onco), Ad(onco-A), or Ad(onco-B) in comparison to untreated controls and controls which were administered PBS or Ad(Mock), as described in Example 11.

[0321] Materials and Methods

[0322] Biopsies were fixed and stored in 4% formaldehyde until further processing.

[0323] The optic nerves, testes and epididymis were fixed in Davidsons's solution for 48 h and then transferred to 4% buffered formaldehyde. The preparation and examination of tissues were performed under GLP-standard by BioVet Veterinary Medical Laboratory, Sollentuna. Tissues were paraffin embedded, sectioned (4-6 pm), stained with hematoxylin-eosin and examined by light microscopy. Results and conclusions

[0324] The results of the histopathological evaluation in tissue samples from hamsters and mice according to the treatment and control groups as explained in Example 11 are presented in Table 12 and 13.

[0325] Table 12. Histopathological alterations in hamster tissue samples according to the treatment groups shown in Table 6, at early (3 d) and late (21 d) termination after last treatment injection. Analyzed tissues include brain, spinal cord, optic nerve, heart, lung, mammary gland, ventricle, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenal gland, mesenteric lymph node, bone marrow, bladder, ovary, uterus, testis, epididymis and prostate. Only tissues where alterations were found in at least one sample are shown. The values show number of affected samples (x) and number of animals (n) in the group (x:n, top row) and grade of the alterations in the respective samples (bottom row). (S.c. = subcutaneous, 1 = minimal, 2 = slight, 3 = moderate, 4 = marked, 5 = severe, P= present, MNC = mononuclear cells (*mainly lymphocytes, scattered macrophages and plasma cells))

[0326] Table 13. Histopathological alterations in mouse tissue samples according to the treatment groups shown in Table 7 at termination. Analyzed tissues include brain (missing from three animals), heart, lung, stomach, liver, spleen and kidney. Only tissues where alterations were found in at least one sample are shown. The values show number of affected samples (x) and number of animals (n) in the group (x:n, top row) and grade of the alterations in the respective samples (bottom row). (I.t. = intratumoral, 1 = minimal, 2 = slight, 3 = moderate, 4 = marked, 5 = severe; *lnflammatory cells: mainly lymphocytes, macrophages and neutrophilic granulocytes; **lnflammatory cells: mainly lymphocytes and macrophages).

[0327] Histopathological examination was performed to examine if treatment with Ad(onco-AB)-GMP or non-GMP Ad(onco-AB) induced tissue lesions. In hamsters, periportal and / or lobular infiltrates of mononuclear cells were found in 10 out of 34 liver biopsies, from untreated, PBS treated and virus treated groups. No dose response correlation was seen and the finding was regarded incidental without relation to the treatment. Discrete immunological or histological alterations was observed in kidney, heart, stomach bladder and prostate. None of them, however, was considered related to the treatment. No microscopic alterations were observed in the brain, optic nerve, stomach, intestines, lungs, pancreas, sternum, lymph node, adrenal gland, spleen, mammary gland, ovary, uterus, testes, or epididymis.

[0328] In mice, two animals per group were treated with PBS, Ad(luc), Ad(onco), Ad(onco-A), Ad(onco-B) and Ad(onco-AB), respectively. Liver biopsies showed slight focal centrilobular perivascular inflammatory cell foci in two out of two animals treated with Ad(onco-A). Minimal to slight centrilobular hypertrophy was found in five out of 10 animals across different groups. Minimal to slight multifocal inflammatory cell foci were observed in all animals except one animal from the PBS group. In the ventricle, slight infiltration of inflammatory cells, mainly granulocytes, were observed in the lamina propria of one animal treated with Ad(onco-AB). In kidneys, minimal multifocal tubular dilatation in cortex was observed in all animals except three animals treated with PBS, Ad(onco) and Ad(onco-B), respectively. In the spleen, slight to moderate diffuse extramedullary hematopoiesis were observed in all evaluated animals. No microscopic alterations were observed in the heart, lung, and brain.

[0329] In summary, the histopathological examination in hamster and GGTA1- knockout mouse could not find any macroscopic or microscopic lesions or alterations that could be related to the given treatment.

[0330] Example 16

[0331] Evaluation of hematological and biochemical homeostasis in vivo

[0332] Example 16 presents hematological and biochemical evaluation in hamsters at necropsy to examine treatment related toxicity upon administration of Ad(onco-AB)- GMP or non-GMP Ad(onco-AB) in comparison to untreated controls and controls which were administered PBS or Ad(Mock), as described in Example 11.

[0333] Materials and Methods

[0334] Whole blood was collected in EDTA-tubes at necropsy and immediately transferred to Clinical Chemical Laboratory at the University Animal Hospital, Uppsala. Hematological analyses including hemoglobin (Hb), erythrocyte volume fraction (EVF), leukocyte particle count (LPC), thrombocyte particle count (TPC), neutrophils, eosinophils, lymphocytes and monocytes were performed within 8 h from necropsy according to standard laboratory procedures. Serum was extracted from untreated blood, following 10 minutes centrifugation at 1500 RPM. Serum analyses including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein, albumin, and creatinine were performed according to standard laboratory procedures by the Clinical Chemical Laboratory at the University Animal Hospital, Uppsala.

[0335] Mann-Whitney U-test were used to identify any statistically significant differences between the respective treated groups and controls. Mann-Whitney U- test was used to compare pooled controls (group 1, 2 and 11) and pooled virus- treated groups (group 3-10). In addition, pooled controls (group 1, 2 and 11) and the individual virus-treated groups were compared using Kruskal-Wallis test (nonparametric one-way ANOVA) with Bonferroni correction for multiple comparison.

[0336] Results and conclusions

[0337] Blood was collected at termination and common blood analyses were performed. Results from the respective treatment groups were compared to the internal controls of untreated and PBS-injected animals (Fig. 12A and 12B), applying Kruskal-Wallis and Mann-Whitney U-tests. No significant differences between treated groups and controls were seen for any of the analyzed parameters. Some individual values from virus-treated groups (mainly ALT and AST) were outside the range of the control group but in many cases these samples were affected by hemolysis. Overall, the results indicate that the given treatments have no significant effect on hematological and biochemical homeostasis.

[0338] Conclusive remarks on Examples 11-16

[0339] The preclinical safety, toxicity and biodistribution study described in Examples 10-15 shows a favorable safety profile of Ad(onco-AB) and Ad(onco-AB)-GMP. It is supported by mild histopathological alterations, maintained blood homeostasis, low tissue replication and sparse shedding.

[0340] The anti-virus IgG titers, stimulated by the first and further established by the second injection demonstrated successful injection of the hamsters and confirmed an expected humoral immune response. At the same time the animals showed no signs of discomfort during the study and the histopathologic analysis revealed no treatment related toxicity.

[0341] Doses, scheme and route of administration were selected to mimic a setting envisioned for clinical studies. Two doses, 1E11 and 5 Ell vp / kg, were selected based on previous studies of conditionally replicative oncolytic adenoviruses (CRAds) in Syrian hamster and clinical trials, where this dose interval has been safe. Two or three injections were given since repeated doses is a common strategy to increase virus exposure and boost the anti-tumor immune response in clinical trials.

[0342] The biodistribution results from this study shows absence of viral particles in blood cloth and serum, and occasional low numbers in the spleen and other well circulated organs. These results suggest slow release of virus from the injection site and effective clearance of circulating viruses by immune cell phagocytosis and degradation before the time point of necropsy. Low absolute viral numbers and unaffected histopathology are considered confirming a lack of viral replication and inflammation in the mammary gland tissue.

[0343] Analysis of viral particle shedding revealed viral particle numbers above limit of detection only in a very few samples, from each sample type on day 1 and 3 but not on day 6 or 12 (after the first treatment injections). This may represent minimal systemic viral spread and subsequent shedding on the first days after the first injection that decreases under quantifiable levels by day 6. Although late shedding might occur, the viral numbers are low and unlikely to cause any biologically relevant effects in the environment.

[0344] Histopathological examination revealed no tissue alterations related to the treatment. Aberrant findings were in all cases regarded as common features of the model or incidental. This interpretation is further supported by normal biochemistry, hematology and low levels of viruses in aberrant samples.

[0345] As described below in Example 18, complete safety evaluation of Ad(onco-AB) will be done in human clinical trials, in which the viral infectivity and replication is uncompromised and the immune activation, caused by transgene expression, is fully representative. Syrian hamster and GGTAl-knock out mice are yet the best available models prior to such clinical trials. The Syrian hamster contributes with representative viral infectivity and replication. GGTA1 knock out mouse gives an opportunity to study toxicity related to a-Gal expression. The intratumoral injection used in the mouse experiment may well mimic the clinical intratumoral injections in terms of access to blood vessels and cells permissive for viral infection and replication. In conclusion, the absence of toxicity, sparse shedding and effective viral clearance (as described in Examples 11-16) indicate that Ad(onco-AB) and Ad(onco- AB-GMP) are safe in repeated doses at least in a dose range between 1E11 and 5E11 vp / kg, as tested in Syrian hamster and GGTA1 knock out mice.

[0346] Example 17

[0347] Comparison of immune stimulatory transgenes in vitro

[0348] Toll like receptor (TLR) stimulation is a key trigger of the innate immune response following the administration of oncolytic virus and is important for the further development of anti-tumor immunity.

[0349] In Example 17, cell lysis effect and transgene expression was evaluated human pancreatic ductal adenocarcinoma (Panc-01) cells upon infection of the cells using Ad(onco-AB) in comparison to the controls Ad(Onco) as described in Example 1 and Ad(GFP-Luc) which is a non-replicating adenoviral vector expressing green fluorescent protein (GFP) and Luciferase (Luc). In addition, an adenoviral construct based on known constructs of the prior art expressing a-Gal (A) and the granulocytemacrophage colony-stimulating factor, GM-CSF (G), was developed using the same vector backbone used in Ad(onco-AB) and was tested in the present Example in comparison to the above viruses.

[0350] Furthermore, Toll-like receptor 2 activation was evaluated in HEK 293 cells co-transduced with human TLR2 and SEAP (secreted embryonic alkaline phosphatase) using supernatants obtained from Panc-01 cell cultures infected by either Ad(onco), Ad(onco-A), Ad(onco-AB) on Ad(oncoAG) in comparison to supernatants obtained from un-transduced cells.

[0351] Materials and Methods

[0352] Functional tests of the viruses were performed on the human pancreatic ductal adenocarcinoma cell line, Panc-01. The cells were cultured as described in Example 1. TLR receptor activation was studied through the HEK-Blue™ hTLR2 system (InvivoGen, San Diego, California, USA). These cells are engineered to respond to hTLR2 receptor stimulation by increasing expression of SEAP, which in turn can be measured by colorimetric methods using HEK-Blue™ detection culture media (InvivoGen, San Diego, California, USA). Briefly, the SEAP reporter gene is placed under the control of the IFN-P minimal promoter fused to five N F-KB and AP-1- binding sites. Additionally, the CD14 co-receptor gene is transfected into these cells to enhance the TLR2 response. Stimulation with a TLR2 ligand activates NF-KB and AP-1 which induce the production of SEAP. Thus, elevated expression levels of SEAP correlate with increased TLR2 activity. Briefly, HEK 293 cells co-transduced with human TLR2 and SEAP were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 1 mM sodium pyruvate, 100 units / mL penicillin, 100 pg / mL streptomycin (1% PeSt), and 10% (vol / vol) heat-inactivated fetal bovine serum (FBS). The cells were cultured in tissue culture flasks in a humidified incubator at 37°C, in an atmosphere of 5% CO2.

[0353] To test cell lysis effect of Ad(onco), Ad(onco-AB), Ad(onco-AG) and Ad(GFP- Luc), PancOl cells were infected by either at different infective doses, ranging from 0.1 to 10000 viral particles per cell. After five days in culture, cell viability was measured by AlamaBlue MTS assay (ThermoFisher Scientific, USA) according to the manufacturer's instructions, using excitation at 540 nm and emission at 590 nm.

[0354] Transgene expression induced by infecting PancOl cells using either Ad(onco), Ad(onco-AB) or Ad(onco-AG) was analyzed at MOI 200 vp / cell. Similarly as described in Example 3, after 48 h in culture, the cells were stained for evaluating the presence of a-Gal (A) epitopes using FITC-conjugated Isolectin B4 (Bandeiraea simplicifolia, Enzo Life Science, NY, USA). The cells and data were analyzed by FACS.

[0355] For analyzing TLR activation, Panc-01 cells were infected either by Ad(onco), Ad(onco-A), Ad(onco-AB) or Ad(oncoAG) at MOI 50 vp / cell. Supernatants from the cell cultures were collected 48 h after infection. hTLR2 ligand activity indued by cellular products secreted to the supernatants were measured using the HEK-Blue™ hTLR2 system (InvivoGen, San Diego, California, USA) described above by adding the collected supernatants to TLR2 / NF-KB-SEAP reporter HEK 293 cells in culture.

[0356] Absorbance was measured at 620 nm after 18, 24 and 48 h.

[0357] Results and conclusions

[0358] The results obtained for cell lysis analysis are presented on Fig. 13A, wherein the multiplicity of infection (MOI) indicates the number of viral particles per cell. As shown in Fig. 13A, each tested replicating virus, i.e. Ad(onco), Ad(onco-AB) and Ad(onco-AG) induced cell lysis of Panc-01 cells at increasing MOI with similar lytic efficiency. As expected, the non-replicating virus Ad(GFP-Luc) was less effective in reducing cell viability.

[0359] As shown in Fig. 13B, Ad(onco-AB) and Ad(onco-AG) induced similar levels of a-Gal epitope decoration of the infected cells, although the data obtained for Ad(onco-AG) appear with a greater deviation. As expected, Ad(onco), which lacks any a-l,3-GT encoding gene, did not induce the decoration of the infected cells with a-Gal epitopes.

[0360] As presented in Fig. 13C, Ad(Onco-AB) induced a significantly higher SEAP expression than Ad(Onco-AG), Ad(Onco-A) and the other control viruses. In fact, Surprisingly, these data unambiguously show that a viral construct according to the present invention that induces the secretion of NAP by the infected cells effectively activates TLR2, while GM-CSF does not.

[0361] In conclusion, Example 17 demonstrates that while Ad(onco-AB) and Ad(onco- AG) exert similar cell lytic effect and induce similar expression of a-Gal, Ad(onco-AB) shows a clearly increased potential to elicit hTLR2 receptor activation. Accordingly, the NAP transgene in a viral construct as disclosed herein gives Ad(onco-AB) unique mechanisms to stimulate an effective innate immune response, which could not be gained by other immune stimulatory molecules, such as GM-CSF. Example 18 Clinical trials

[0362] The present example describes a planned clinical study for the evaluation of safety and therapeutic effect of Ad(onco-AB)-GMP in patients suffering from pancreatic ductal adenocarcinoma (PDAC).

[0363] Patients

[0364] Patients with disseminated PDAC, i.e. with known metastases in the liver (verified with cytology or biopsy) and accessible for ultrasound injection are eligible to participate in the clinical studies. No modification of standard of care, including chemotherapy, is proposed.

[0365] The patients receive verbal and written information about the study during a visit at the hospital. Eligible patients are scrutinized for inclusion and exclusion criteria, as described below.

[0366] Eligibility criteria

[0367] Inclusion criteria (all of the following)

[0368] • Stage IV PDAC of the pancreatic head

[0369] • Tx, Nx, Ml (liver) (UICC 7th version, 2010)

[0370] • Age 18-70 years

[0371] • Written informed consent

[0372] • Considered able to receive the study specific immunotherapy

[0373] Exclusion criteria (one or more of the following)

[0374] • Pre-treatment work-up, including appropriate CT imaging, showing threatening bile obstruction or tumor invasion of the large vessel walls with risk for bleedings.

[0375] • Chronic neuropathy > grade 2

[0376] • WHO performance score > 2

[0377] • Granulocyte count < 1500 per mL • Platelet count < 100000 per mL

[0378] • Serum creatinine > 1.5 UNL (upper limit normal range)

[0379] • Albumin < 2,5 g / dl

[0380] • Female patients in child bearing age not using adequate contraception, pregnant or lactating women

[0381] • Mental or organic disorders which could interfere with informed consent or treatments

[0382] • Other malignancy within the past 5 years, except non-melanomatous skin or non-invasive cervical cancer

[0383] • 50% CD46 negative PDAC cells in the confirmatory percutaneous biopsy of liver metastases

[0384] • Any reason why, in the opinion of the investigator, the patient should not participate

[0385] First clinical trial - Phase I

[0386] A first clinical trial focusing on safety and dose escalation of Ad(onco-AB)- GMP in patients with advanced (stage IV) PDAC is conducted at the Department of Surgery, Sahlgrenska University Hospital, Gothenburg.

[0387] The study includes 3 study groups, each with 3 patients, and with ultrasound guided injection of escalating dosages of Ad(onco-AB)-GMP (1, 3, 10 x 10All virus particles) in liver metastases according to standard procedures and performed by a dedicated radiologist.

[0388] The study is conducted in close contact with the DSMB (Data and Safety Monitoring Board) that will be provided with all adverse events observed within the first months after injection of Ad(onco-AB)-GMP of the first patient in each group as well as after completion of all patients in a group. There is always at least one- month observation period between the first and second patient in each study group. The DSMB advises on progression to the higher dose. If clinically relevant side effects are obtained, 3 additional patients will be treated with the same dose and only if all of them tolerate the dose, the dosing is escalated. Second clinical trial - Phase II

[0389] After completion of the safety study, a multi-center safety and efficacy clinical study is performed. 20 subjects, fulfilling the same eligibility criteria as in the dose escalation study above, are included and treated with local injections of Ad(onco-AB)-GMP in liver metastases with the highest acceptable dose identified in the safety study. The study is an open, non-randomized, exploratory phase II study focusing on safety.

[0390] Indirect comparison concerning efficacy is performed by registry comparisons of a group of patients (n=60) with similar staging of their PDAC and only receiving standard of care within participating hospitals. The size of the proposed study (20 subjects) is based on experience in detecting adverse events in patients with stage IV PDAC. Using a strategy with a registry-based control group, it is considered confidently that clinically meaningful therapeutically benefits can be detected. Follow-up is scheduled for the lifetime of participating subjects with biannual progress reports. Primary outcome is safety and primary efficacy endpoint is overall survival by 12 months (intention to treat). Based on available national register-data, a < 20 % overall survival rate at 12 months in this group of patients, stage IV PDAC given standard of care is assumed (Latenstein AEJ, et al. Eur J Cancer 2020, 125:83- 93).

[0391] All requirements for cGCP, cGLP and cGMP are fulfilled, including on-site data monitoring. All study activity, including registration of side effects, is reported in study specific databases.

[0392] Adverse events

[0393] Side effects are graded by the actual version of the "Common Terminology Criteria for Adverse Events" (issued by the US National Cancer Institute (NCI). Grades 3-5 are reported. Laboratory parameters are determined at each study visit for dose adjustments (reduction or dose delays) of adjuvant chemotherapy are performed according to local guidelines at each center. Follow up

[0394] Follow-up is based on physical examination, blood samples and CT scans of chest and abdomen. The quality of life (QoL) is assessed by the QLQ-30 of the European Organization for Research and Treatment of Cancer (EORTC) at study inclusion as well as at all follow-up visits. Data required by the study-protocol is entered into Case Report Forms (CRF).

[0395] Expected Outcome, significance and conclusions

[0396] Based on the herein described clinical studies the present inventors expect to demonstrate the efficacy and tolerability of treatment of cancer using Ad(onco-AB) in human patients. Moreover, said treatment will be evaluated for safety and any potential adverse events in a clinical setting. The present inventors envision based on the herein demonstrated in vitro and in vivo studies that Ad(onco-AB) for use in a treatment according to the present disclosure will be well tolerated and therapeutic effects of Ad(onco-AB), as proven in the above Examples, will be demonstrated with a surprisingly advantageous therapeutic potential in the clinic. The inventors foresee improvements in progression free survival and / or overall survival of cancer patients administered Ad(onco-AB), as disclosed herein. Furthermore, an improvement in quality of life of patients diagnosed with cancer is expected. The present inventors envision that the outcome will be indicative of the therapeutic potential of Ad(onco- AB) in various viral gene therapy modalities, such as in vivo and ex vivo gene therapies.

[0397] PDAC represents a formidable challenge in medicine as today's treatment options have only limited success rate. Efforts must be focused on finding a treatment that can become curable, yet without major adverse effects. The herein disclosed therapeutic approach leads to a xenogeneic-based attack, directed against the patient's individual tumor neoantigens. The present inventors expect a benefit for newly diagnosed PDAC patients with disseminated disease, wherein at the time of diagnosis the patients can be offered a potentially curative treatment according to the present disclosure, potentially followed by and / or combined with a surgery. Ad(onco-AB)-GMP is expected to be suitable for offering to all patients with PDAC. Further refinement of the treatment for PDAC is also expected in conjunction with ordinary health-care, e.g. repeated administration of Ad(onco-AB)-GMP. The therapeutic approach proposed herein is expected to be not limited to pancreatic cancer as the herein disclosed therapy finds a universal way to expose tumor-specific neoantigens (unique to the patient) to the patient's immune system and thereby trigger a T cell attack on malignant cells, such as cancer cells. The expected positive results in PDAC, inherently expands of the present therapy to other difficult-to-treat cancers and thereby further increase the beneficial effects to affected individuals as well as for society.

[0398] ITEMIZED LIST OF EMBODIMENTS

[0399] 1. A vector comprising

[0400] (i) a first nucleic acid sequence encoding a glycosyltransferase, an enzymatically active variant thereof, or an enzymatically active truncated form thereof; and

[0401] (ii) a second nucleic acid sequence encoding a Helicobacter pylori neutrophilactivating protein (NAP), an immunologically equivalent variant thereof, or an immunologically equivalent fragment thereof.

[0402] 2. The vector according to item 1, wherein said glycosyltransferase is able to transfer a foreign carbohydrate moiety to lipids and / or proteins of a cell expressing the glycosyltransferase.

[0403] 3. The vector according to item 2, wherein said foreign carbohydrate moiety is recognized by antibodies present in a subject, such as a human subject, comprising said cell.

[0404] 4. The vector according to any one of items 1-3, wherein said glycosyltransferase is selected from a group consisting of hexosyltransferases and pentosyltransferases.

[0405] 5. The vector according to any one of items 1-4, wherein said glycosyltransferase is a hexosyltransferase.

[0406] 6. The vector according to any one of items 1-5, wherein said glycosyltransferase is a glucosyltransferase, such as a glucosyltransferase selected from a group consisting of al,3-N-acetylgalactosaminyltransferase and al,3-galactosyltransferase.

[0407] 7. The vector according to any one of items 1-5, wherein said glycosyltransferase is a galactosyltransferase, such as an N-acetyllactosaminide alpha-1, 3- galactosyltransferase.

[0408] 8. The vector according to item 7, wherein said N-acetyllactosaminide alpha-1, 3- galactosyltransferase comprises an amino acid sequence selected from a group consisting of an amino acid sequence according to SEQ ID NO:2 and amino acid sequences having at least 38%, such as at least 70% sequence identity to SEQ ID

[0409] NO:2. 9. The vector according to item 8, wherein said N-acetyllactosaminide alpha-1, 3- galactosyltransferase comprising an amino acid sequence having at least 38%, such as at least 70% sequence identity to SEQ ID NO:2 is enzymatically active.

[0410] 10. The vector according to any one of items 7-9, wherein said N- acetyllactosaminide alpha-1, 3-galactosyltransferase comprises the amino acid sequence according to SEQ ID NO:2.

[0411] 11. The vector according to any one of items 1-10, wherein said first nucleic acid sequence comprises a nucleic acid sequence selected from a group consisting of a nucleic acid sequence according to SEQ ID NQ:40, any nucleic acid sequence having at least 70% sequence identity to SEQ ID NQ:40 and any codon-optimized version thereof.

[0412] 12. The vector according to any one of items 1-11, wherein said first nucleic acid sequence comprises a nucleic acid sequence selected from a group consisting of a nucleic acid sequence according to SEQ ID NO:1 and nucleic acid sequences having at least 70% sequence identity to SEQ ID NO:1, such as a nucleic acid sequence according to SEQ ID NO:1.

[0413] 13. The vector according to any one of items 1-12, wherein said NAP comprises an amino acid sequence selected from a group consisting of an amino acid sequence according to SEQ ID NO:4 and amino acid sequences having at least 70% sequence identity to SEQ ID NO:4.

[0414] 14. The vector according to item 13, wherein said NAP comprising any one of said amino acid sequences having at least 70% sequence identity to SEQ ID NO:4 is capable of inducing an immune response equivalent to an immune response induced by said NAP comprising the amino acid sequence according to SEQ ID NO:4.

[0415] 15. The vector according to any one of items 1-14, wherein said NAP comprises an amino acid sequence according to SEQ ID NO:5, wherein

[0416] XI is selected from E and G, X2 is selected from I and L, and X3 is selected from Y and H.

[0417] 16. The vector according to any one of items 1-15, wherein said NAP comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:4 and SEQ ID NO:6-12.

[0418] 17. The vector according to any one of items 1-16, wherein said NAP comprises the amino acid sequence according to SEQ ID NO:4.

[0419] 18. The vector according to any one of items 1-17, wherein the immunologically equivalent fragment of NAP has a length of at least 20, such as at least 25, such as at least 30, such as at least 34, amino acid residues.

[0420] 19. The vector according to any one of items 1-18, wherein the immunologically equivalent fragment of NAP comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NQ:13-20 and amino acid sequences having at least 70% sequence identity to SEQ ID NQ:13-20.

[0421] 20. The vector according to any one of items 1-19, wherein the immunologically equivalent fragment of NAP comprises an amino acid sequence selected from a group consisting of the amino acid sequences according to SEQ ID NQ:13-20, such as a group consisting of the amino acid sequences according to SEQ ID NO:13-15 and SEQ ID NQ:20.

[0422] 21. The vector according to any one of items 1-20, wherein said second nucleic acid sequence comprises a nucleic acid sequence selected from a group consisting of a nucleic acid sequence according to SEQ ID NO:3, any nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:3 and any codon-optimized version thereof; such as a group consisting of a nucleic acid sequence according to SEQ ID NO:3 and any nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:3; such as a nucleic acid sequence according to SEQ ID NO:3.

[0423] 22. The vector according to any one of items 1-21, wherein said vector is a non-viral vector or a viral vector. 23. The vector according to any one of items 1-22, wherein said vector is a non-viral vector.

[0424] 24. The vector according to item 22 or 23, wherein said non-viral vector is a DNA vector or an RNA vector.

[0425] 25. The vector according to any one of items 1-22, wherein said vector is a viral vector.

[0426] 26. The vector according to item 22 or 25, wherein said viral vector is an RNA vector or a DNA vector.

[0427] 27. The vector according to item 26, wherein said viral vector is a DNA vector.

[0428] 28. The vector according to any one of items 22 and 25-27, wherein said viral vector is a retrovirus, a lentivirus, a vaccinia virus, a Semliki Forest virus, a reovirus, a Newcastle disease virus, a herpes virus, an adenovirus or an adeno-associated virus, such as wherein said viral vector is a retrovirus, a lentivirus, a vaccinia virus, an adenovirus or an adeno-associated virus.

[0429] 29. The vector according to any one of items 22 and 25-28, wherein said viral vector is an adenovirus or an adeno-associated virus.

[0430] 30. The vector according to any one of items 22 and 25-29, wherein said viral vector is an adenovirus.

[0431] 31. The vector according to any one of items 22 and 25-30, wherein said viral vector is a human adenovirus type 5, such as a genetically modified variant of human adenovirus type 5.

[0432] 32. The vector according to any one of items 22 and 25-31, wherein said viral vector is modified to expresses a CD46-binding fiber knob of human adenovirus type 35.

[0433] 33. The vector according to any one of items 22 and 25-32, wherein said vector comprises a mutated adenovirus early region 1A (E1A) gene encoding a mutated E1A protein having a substantially reduced Rb protein binding capability as compared to a wild-type E1A protein. 34. The vector according to any one of items 22 and 25-33, wherein said wild-type E1A protein comprises an amino acid sequence selected from the group consisting of amino acid sequences according to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:36 and SEQ ID NO:37.

[0434] 35. The vector according to any one of items 22 and 25-34, wherein said mutated E1A protein comprises an amino acid sequence selected from the group consisting of amino acid sequences according to SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:38 and SEQ ID NO:39.

[0435] 36. The vector according to any one of items 22 and 25-35, wherein an adenovirus early region IB (E1B) gene is deleted.

[0436] 37. The vector according to any one of items 22 and 25-36, wherein said viral vector is an oncolytic virus, such as an oncolytic adenovirus.

[0437] 38. The vector according to any one of items 1-37, wherein said first nucleic acid sequence is arranged upstream of said second nucleic acid sequence in the non-viral vector or a viral vector construct encoding said viral vector.

[0438] 39. The vector according to any one of items 1-38, wherein said vector further comprises a third nucleic acid sequence.

[0439] 40. The vector according to item 39, wherein said third nucleic acid sequence is arranged between the first and the second nucleic acid sequence in the non-viral vector or a viral vector construct encoding said viral vector.

[0440] 41. The vector according to item 39 or 40, wherein said third nucleic acid sequence allows for obtaining individual polypeptides encoded by said first and said second nucleic acid sequences, respectively.

[0441] 42. The vector according to any one of items 39-41, wherein said third nucleic acid sequence is an Internal Ribosome Entry Site (IRES) sequence.

[0442] 43. The vector according to any one of items 39-41, wherein said third nucleic acid sequence encodes a self-cleaving peptide. 44. The vector according to item 43, wherein the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:25-28 and amino acid sequences having at least 70% sequence identity to any one of SEQ ID NO:25-28.

[0443] 45. The vector according to item 43 or 44, wherein the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:25-28.

[0444] 46. The vector according to any one of items 44-45, wherein the self-cleaving peptide further comprises a linker sequence region at the N-terminal thereof.

[0445] 47. The vector according to any one of items 43-46, wherein the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:29-32 and amino acid sequences having at least 70% sequence identity to any one of SEQ ID NO:29-32.

[0446] 48. The vector according to any one of items 43-47, wherein the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:29-32.

[0447] 49. The vector according to any one of items 43-48, wherein the self-cleaving peptide comprises an amino acid sequence according to SEQ ID NO:25, such as an amino acid sequence according to SEQ ID NO:29.

[0448] 50. The vector as defined in any one of items 1-49, wherein said vector is formulated for in vitro transfection or transduction of cells or for in vivo administration to a subject, such as wherein said vector is formulated in vitro transfection or transduction of cells.

[0449] 51. A vector system for simultaneous or subsequent delivery of a first and a second vector to a cell and / or for generating an isolated cell population as defined in item 59, said vector system comprising

[0450] (a) the first vector comprising said first nucleic acid sequence as defined in any one of items 1-12, and (b) the second vector comprising said second nucleic acid sequence as defined in any one of items 1 and 13-21; wherein said first and said second vectors are separate vectors.

[0451] 52. The vector system according to item 51, wherein said first and / or said second vector is a non-viral vector or a viral vector.

[0452] 53. The vector system according to item 51 or 52, wherein said first and / or said second vector is a non-viral vector as defined in item 23 or 24.

[0453] 54. The vector system according to item 51 or 52, wherein said first and / or said second vector is a viral vector as defined in any one of items 25-37.

[0454] 55. The vector system as defined in any one of items 51-54, wherein said vector system is formulated for in vitro transfection or transduction of cells or for in vivo administration to a subject, such as wherein said vector system is formulated in vitro transfection or transduction of cells.

[0455] 56. A method of in vitro transfection or transduction of cells with the vector as defined in any one of items 1-50 or the vector system as defined in any one of items 51-55.

[0456] 57. An isolated cell population comprising said first nucleic acid sequence as defined in any one of items 1-12 and / or a glycosyltransferase (such as a galactosyltransferase), an enzymatically active variant thereof, or an enzymatically active truncated form thereof, encoded by said first nucleic acid sequence; and said second nucleic acid sequence as defined in any one of items 1 and 13-21 and / or a NAP, an immunologically equivalent variant thereof, or an immunologically equivalent fragment thereof, encoded by said second nucleic acid sequence.

[0457] 58. The isolated cell population according to item 57, wherein each cell comprises said first nucleic acid sequence as defined in any one of items 1-12 and / or said glycosyltransferase (such as said galactosyltransferase), the enzymatically active variant thereof, or the enzymatically active truncated form thereof, as defined in item 57; and said second nucleic acid sequence as defined in any one of items 1 and 13-21 and / or said NAP, the immunologically equivalent variant thereof, or the immunologically equivalent fragment thereof, as defined in item 57.

[0458] 59. The isolated cell population according to item 57, wherein the isolated cell population comprises two cell type populations, wherein

[0459] (c) a first cell type population comprises said first nucleic acid sequence as defined in any one of items 1-12 and / or said glycosyltransferase (such as said galactosyltransferase), the enzymatically active variant thereof, or the enzymatically active truncated form thereof, as defined in item 57; and

[0460] (d) a second cell type population comprises said second nucleic acid sequence as defined in any one of items 1 and 13-21 and / or said NAP, the immunologically equivalent variant thereof, or the immunologically equivalent fragment thereof, as defined in item 57.

[0461] 60. A polynucleotide comprising said first and said second nucleic acid sequences as defined in any one of items 1-21.

[0462] 61. The polynucleotide sequence according to item 60, wherein said polynucleotide is comprised in a non-viral vector or a viral vector construct.

[0463] 62. The polynucleotide sequence according to item 60 or 61, wherein said first nucleic acid sequence is arranged upstream of said second nucleic acid sequence in said polynucleotide.

[0464] 63. The polynucleotide sequence according to any one of items 60-62, wherein said polynucleotide further comprises a third nucleic acid sequence.

[0465] 64. The polynucleotide sequence according to item 63, wherein said third nucleic acid sequence is arranged between the first and the second nucleic acid sequence in said polynucleotide. 65. The polynucleotide sequence according to item 63 or 64, wherein said third nucleic acid sequence allows for obtaining individual polypeptides encoded by said first and said second nucleic acid sequences, respectively.

[0466] 66. The polynucleotide sequence according to any one of items 63-65, wherein said third nucleic acid sequence is an IRES sequence.

[0467] 67. The polynucleotide sequence according to any one of items 63-65, wherein said third nucleic acid sequence encodes a self-cleaving peptide.

[0468] 68. The polynucleotide sequence according to item 67, wherein the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:25-28 and amino acid sequences having at least 70% sequence identity to any one of SEQ ID NO:25-28.

[0469] 69. The polynucleotide sequence according to item 67 or 68, wherein the selfcleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:25-28.

[0470] 70. The polynucleotide sequence according to any one of items 67-69, wherein the self-cleaving peptide further comprises a linker region at the N-terminal thereof.

[0471] 71. The polynucleotide sequence according to any one of items 67-70, wherein the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:29-32 and amino acid sequences having at least 70% sequence identity to any one of SEQ ID NO:29-32.

[0472] 72. The polynucleotide sequence according to any one of items 67-71, wherein the self-cleaving peptide comprises an amino acid sequence selected from a group consisting of amino acid sequences according to SEQ ID NO:29-32.

[0473] 73. The polynucleotide sequence according to any one of items 67-72, wherein the self-cleaving peptide comprises an amino acid sequence according to SEQ ID NO:25, such as an amino acid sequence according to SEQ ID NO:29. 74. The polynucleotide sequence according to any one of items 60-73, wherein said polynucleotide encodes a viral vector as defined in any one of items 25-50.

[0474] 75. A kit comprising the vector as defined in any one of items 1-50, or the vector system as defined in any one of items 51-55; and instructions for use.

[0475] 76. A pharmaceutical composition comprising the vector as defined in any one of items 1-50, the vector system as defined in any one of items 51-55, or the isolated cell population as defined in any one of items 57-59; and at least one pharmaceutically acceptable salt, carrier and / or excipient.

[0476] 77. The pharmaceutical composition according to item 76, wherein said pharmaceutical composition is formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, intralymphatic or intratumoral administration to a subject.

[0477] 78. The vector as defined in any one of items 1-50, the vector system as defined in any one of items 51-55, and / or the isolated cell population as defined in any one of items 57-59, for use as a medicament.

[0478] 79. The vector as defined in any one of items 1-50, the vector system as defined in any one of items 51-55, and / or the isolated cell population as defined in any one of items 57-59, for use in the treatment of cancer, such as wherein said cancer is a malignancy arising from epithelial tissue or a malignancy arising from non-epithelial tissue.

[0479] 80. The vector, the vector system or the isolated cell population for use according to item 79, wherein said cancer is selected from a group consisting of carcinoma, melanoma, sarcoma, lymphoma, leukemia, seminoma, germinoma, dysgerminoma, blastoma, brain and central nervous system cancers, mixed-type cancers and pediatric cancers; such as the group consisting of carcinoma, melanoma, sarcoma, lymphoma, leukemia, seminoma, germinoma, dysgerminoma, blastoma, brain and central nervous system cancers and mixed-type cancers.

[0480] 81. The vector, the vector system or the isolated cell population for use according item 79 or 80, wherein said cancer is carcinoma or melanoma.

[0481] 82. The vector, the vector system or the isolated cell population for use according to any one of items 79-81, wherein said cancer is carcinoma.

[0482] 83. The vector, the vector system or the isolated cell population for use according to any one of items 80-82, wherein said carcinoma is selected from a group consisting of pancreatic cancer, breast cancer, lung cancer, liver cancer, bile duct cancer, gallbladder cancer, intestinal cancer, urogenital cancer, prostate cancer, head-neck cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, skin cancer, thyroid cancer, neuroendocrine tumors (NET) and uterine cancer, such as the group consisting of pancreatic cancer, breast cancer, lung cancer, liver cancer and kidney cancer.

[0483] 84. The vector, the vector system or the isolated cell population for use according to any one of items 79-83, wherein said cancer is pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC), such as stage IV PDAC.

[0484] 85. The vector, the vector system or the isolated cell population for use according to any one of items 79-81, wherein said cancer is melanoma.

[0485] 86. The vector, the vector system or the isolated cell population for use according to item 80, wherein said sarcoma is selected from a group consisting of osteosarcoma and liposarcoma.

[0486] 87. The vector, the vector system or the isolated cell population for use according to item 80, wherein said lymphoma is selected from a group consisting of non-Hodgkin lymphoma and Hodgkin lymphoma. 88. The vector, the vector system or the isolated cell population for use according to item 80, wherein said leukemia is selected from a group consisting of acute leukemia and chronic leukemia.

[0487] 89. The vector, the vector system or the isolated cell population for use according to item 80, wherein said blastoma is selected from a group consisting of glioblastoma and neuroblastoma.

[0488] 90. The vector, the vector system or the isolated cell population for use according to any one of items 79-89, wherein said cancer is in a form of a solid tumor.

[0489] 91. The vector, the vector system or the isolated cell population for use according to any one of items 79-90, wherein said cancer is a primary cancer or a metastatic cancer.

[0490] 92. The vector, the vector system or the isolated cell population for use according to any one of items 79-91, wherein said treatment is a prophylactic and / or a therapeutic treatment.

[0491] 93. The vector, the vector system or the isolated cell population for use according to any one of items 79-92, wherein said treatment is a therapeutic treatment.

[0492] 94. The vector for use according to any one of items 79-93, wherein said use comprises administration of the vector to a subject in need thereof.

[0493] 95. The vector for use according to item 94, wherein said vector is a viral vector as defined in any one of items 25-50 and wherein said use comprises administration of the vector to the subject in a dose comprising at least about 1 x 1011viral particles, such as at least about 3 x 1011viral particles, such as at least about 5 x 1011viral particles.

[0494] 96. The vector for use according to item 94 or 95, wherein said vector is a viral vector as defined in any one of items 25-50 and wherein said use comprises administration of the vector to the subject in a dose comprising from about 1 x 1011 viral particles to about 10 x 1011viral particles, such as a dose comprising from about 1 x 1011viral particles to about 5 x 1011viral particles.

[0495] 97. The vector for use according to any one of items 94-96, wherein said use comprises a single administration of the vector to the subject.

[0496] 98. The vector for use according to any one of items 94-96, wherein said use comprises repeated administration of the vector to the subject.

[0497] 99. The vector for use according to item 98, wherein said vector is a viral vector as defined in any one of items 25-50 and wherein each administration of said repeated administration comprises the administration of the vector in a dose as defined in item 95 or 96.

[0498] 100. The vector for use according to item 98 or 99, wherein a time interval between consecutive administrations of said repeated administration is at least about 1 week, such as at least about 1 month, such as at least about 6 months, such as at least about 1 year.

[0499] 101. The vector for use according to any one of items 98-100, wherein a time interval between consecutive administrations of said repeated administration is from about 1 week to about 6 week.

[0500] 102. The vector for use according to any one of items 94-101, wherein said administration, such as said single or said repeated administration, of the vector is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration.

[0501] 103. The vector for use according to any one of items 94-102, wherein said administration, such as said single or said repeated administration, of the vector is an intralymphatic administration or an intratumoral administration, such as an intratumoral administration. 104. The vector system for use according to any one of items 79-93, wherein said use comprises administration of the vector system comprising the first and the second vector to a subject in need thereof.

[0502] 105. The vector system for use according to item 104, wherein said first and said second vector are administered simultaneously or subsequently, such as simultaneously, to the subject.

[0503] 106. The vector system for use according to item 104 or 105, wherein the first and the second vector of said vector system are viral vectors as defined in item 54 and wherein said use comprises administration of the first vector to the subject in a dose comprising at least about 1 x 1011viral particles, such as at least about 3 x 1011viral particles, such as at least about 5 x 1011viral particles; and / or administration of the second vector to the subject in a dose comprising at least about 1 x 1011viral particles, such as at least about 3 x 1011viral particles, such as at least about 5 x 1011viral particles.

[0504] 107. The vector system for use according to any one of items 104-106, wherein the first and the second vector of said vector system are viral vectors as defined in item 54 and wherein said use comprises administration of the first vector to the subject in a dose comprising from about 1 x 1011viral particles to about 10 x 1011viral particles, such as a dose comprising from about 1 x 1011viral particles to about 5 x 1011viral particles; and / or administration of the second vector to the subject in a dose comprising from about 1 x 1011viral particles to about 10 x 1011viral particles, such as a dose comprising from about 1 x 1011viral particles to about 5 x 1011viral particles.

[0505] 108. The vector system for use according to any one of items 104-107, wherein said use comprises a single administration of the first vector and / or a single administration of the second vector to the subject. 109. The vector system for use according to any one of items 104-107, wherein said use comprises repeated administration of the first vector and / or repeated administration of the second vector to the subject.

[0506] 110. The vector system for use according to item 109, wherein the first and the second vector of said vector system are viral vectors as defined in item 54 and wherein each administration of said repeated administration of the first vector comprises the administration of the first vector in a dose as defined in item 106 or 107; and / or each administration of said repeated administration of the second vector comprises the administration of the second vector in a dose as defined in item 106 or 107.

[0507] 111. The vector system for use according to item 109 or 110, wherein a time interval between consecutive administrations of said repeated administration of the first and / or the second vector is at least about 1 week, such as at least about 1 month, such as at least about 6 months, such as at least about 1 year.

[0508] 112. The vector system for use according to any one of items 109-111, wherein a time interval between consecutive administrations of said repeated administration of the first and / or the second vector is from about 1 week to about 6 week.

[0509] 113. The vector system for use according to any one of items 104-112, wherein said administration, such as said single or said repeated administration, of the first and / or the second vector is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration.

[0510] 114. The vector system for use according to any one of items 104-113, wherein said administration, such as said single or said repeated administration, of the first and / or the second vector is an intra lymphatic administration or an intratumoral administration, such as an intratumoral administration. 115. The isolated cell population for use according to any one of items 79-93, wherein said use comprises administration of the isolated cell population to a subject in need thereof.

[0511] 116. The isolated cell population for use according to item 115, wherein the isolated cell population administered to the subject is an allogeneic or an autologous cell population.

[0512] 117. The isolated cell population for use according to any one of items 79-93, 115 and 116, wherein said use comprises

[0513] (e) obtaining a precursor cell population, such as a malignant cell population (such as a cancer cell population), from a subject suffering from cancer, such as cancer as defined in any one of items 80-91,

[0514] (f) ex vivo transfecting or transducing the precursor cell population obtained in step (e) to obtain the isolated cell population as defined in any one of items 57-59,

[0515] (g) administering the isolated cell population obtained in step (f) to the subject from which the precursor cell population was obtained in step (e).

[0516] 118. The isolated cell population for use according to any one of items 115-117, wherein the isolated cell population administered to the subject is an autologous cell population.

[0517] 119. The isolated cell population for use according to item 115 or 116, wherein the isolated cell population administered to the subject is an allogeneic cell population.

[0518] 120. The isolated cell population for use according to any one of items 115, 116 and 119, wherein the isolated cell population administered to the subject is stable cell line, such as a stable malignant cell line, such as a stable cancer cell line, such as a stable melanoma cell line.

[0519] 121. The isolated cell population for use according to any one of items 115-120, wherein said use comprises a single administration of the isolated cell population to the subject. 122. The isolated cell population for use according to any one of items 115-120, wherein said use comprises repeated administration of the isolated cell population to the subject.

[0520] 123. The isolated cell population for use according to item 122, wherein a time interval between consecutive administrations of said repeated administration of the isolated cell population is at least about 1 week, such as at least about 1 month, such as at least about 6 months, such as at least about 1 year.

[0521] 124. The isolated cell population for use according to item 122 or 123, wherein a time interval between consecutive administrations of said repeated administration of the isolated cell population is from about 1 week to about 6 week.

[0522] 125. The isolated cell population for use according to any one of items 115-124, wherein said administration, such as said single or said repeated administration, of the isolated cell population is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration.

[0523] 126. The isolated cell population for use according to any one of items 115-125, wherein said administration, such as said single or said repeated administration, of the isolated cell population is a subcutaneous administration or an intralymphatic administration, such as an intra lymphatic administration.

[0524] 127. A gene therapy system for simultaneous or subsequent administration thereof to a subject, comprising

[0525] (h) the first and the second cell type population as defined in item 59;

[0526] (i) the first and the second vector as defined in any one of items 51-54;

[0527] (j) the first cell type population as defined in item 59 and the second vector as defined in any one of items 51-54; or

[0528] (k) the second cell type population as defined in item 59 and the first vector as defined in any one of items 51-54. 128. The gene therapy system according to item 127, wherein said first cell type population, said second vector, said second cell type population and / or said first vector is formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, intra lymphatic or intratumoral administration to the subject.

[0529] 129. The gene therapy system according to item 127 or 128, for use as a medicament.

[0530] 130. The gene therapy system according to item 127 or 128, for use in the treatment of cancer, such as wherein said cancer is a malignancy arising from epithelial tissue or a malignancy arising from non-epithelial tissue.

[0531] 131. The gene therapy system for use according to item 130, wherein said cancer is as defined in any one of items 80-91.

[0532] 132. The gene therapy system for use according to item 130 or 131, wherein said treatment is a prophylactic and / or a therapeutic treatment.

[0533] 133. The gene therapy system for use according to any one of items 130-132, wherein said treatment is a therapeutic treatment.

[0534] 136. The gene therapy system for use according to any one of items 130-135, wherein said use comprises administration of the gene therapy system as defined in item 127 or 128.

[0535] 137. The gene therapy system for use according to item 136, wherein said first cell population is administered to the subject simultaneously, a priori or subsequently to the administration of the second cell population; said first vector is administered to the subject simultaneously, a priori or subsequently to the administration of the second vector; said second vector is administered to the subject simultaneously, a priori or subsequently to the administration of the first cell population; or said first vector is administered to the subject simultaneously, a priori or subsequently to the administration of the second cell population. 138. The gene therapy system for use according to item 136 or 137, wherein said administration of said first cell type population, said second vector, said second cell type population and / or said first vector is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration.

[0536] 139. A method for producing the viral vector as defined in any one of items 25-50, said method comprising generating a modified viral vector construct by operably linking a viral backbone to the first and the second nucleic acid sequence as defined in (i) and (ii) in any one of items 1-21, transfecting mammalian cells with the modified viral vector construct, culturing the mammalian cells in conditions suitable for viral replication, and harvesting the viral particles.

[0537] 140. The method according to item 139, wherein said modified viral vector construct is a viral vector construct as defined in any one of items 61-74, such as a viral vector construct encoding a viral vector as defined in any one of items 25-50.

[0538] 141. A method of treatment of a subject in need thereof, comprising administration of the vector as defined in any one of items 1-50, the vector system as defined in any one of items 51-55, and / or the isolated cell population as defined in any one of items 57-59, to the subject in need thereof.

[0539] 142. A method of treatment of cancer, comprising administration of the vector as defined in any one of items 1-50, the vector system as defined in any one of items 51-55, and / or the isolated cell population as defined in any one of items 57-59, to a subject in need thereof, such as wherein said cancer is a malignancy arising from epithelial tissue or a malignancy arising from non-epithelial tissue.

[0540] 143. The method of treatment according to item 142, wherein said cancer is selected from a group consisting of carcinoma, melanoma, sarcoma, lymphoma, leukemia, seminoma, germinoma, dysgerminoma, blastoma, brain and central nervous system cancers, mixed-type cancers and pediatric cancers; such as the group consisting of carcinoma, melanoma, sarcoma, lymphoma, leukemia, seminoma, germinoma, dysgerminoma, blastoma, brain and central nervous system cancers and mixed-type cancers.

[0541] 144. The method of treatment according to item 142 or 143, wherein said cancer is carcinoma or melanoma.

[0542] 145. The method of treatment according to any one of items 142-144, wherein said cancer is carcinoma.

[0543] 146. The method of treatment according to any one of items 143-145, wherein said carcinoma is selected from a group consisting of pancreatic cancer, breast cancer, lung cancer, liver cancer and kidney cancer.

[0544] 147. The method of treatment according to any one of items 142-146, wherein said cancer is pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC), such as stage IV PDAC.

[0545] 148. The method of treatment according to any one of items 142-144, wherein said cancer is melanoma.

[0546] 149. The method of treatment according to item 143, wherein said sarcoma is selected from a group consisting of osteosarcoma and liposarcoma.

[0547] 150. The method of treatment according to item 143, wherein said lymphoma is selected from a group consisting of non-Hodgkin lymphoma and Hodgkin lymphoma.

[0548] 151. The method of treatment according to item 143, wherein said leukemia is selected from a group consisting of acute leukemia and chronic leukemia.

[0549] 152. The method of treatment according to item 143, wherein said blastoma is selected from a group consisting of glioblastoma and neuroblastoma.

[0550] 153. The method of treatment according to any one of items 142-152, wherein said cancer is in a form of a solid tumor. 154. The method of treatment according to any one of items 142-153, wherein said cancer is a primary cancer or a metastatic cancer.

[0551] 155. The method of treatment according to any one of items 141-154, wherein said treatment is a prophylactic and / or a therapeutic treatment.

[0552] 156. The method of treatment according to any one of items 141-155, wherein said treatment is a therapeutic treatment.

[0553] 157. The method of treatment according to any one of items 141-156, wherein said method comprises administration of the vector to the subject in need thereof.

[0554] 158. The method of treatment according to any one of items 141-157, wherein said vector is a viral vector as defined in any one of items 25-50 and wherein said method comprises administration of the vector to the subject in a dose comprising at least about 1 x 1011viral particles, such as at least about 3 x 1011viral particles, such as at least about 5 x 1011viral particles.

[0555] 159. The method of treatment according to any one of items 141-158, wherein said vector is a viral vector as defined in any one of items 25-50 and wherein said method comprises administration of the vector to the subject in a dose comprising from about 1 x 1011viral particles to about 10 x 1011viral particles, such as a dose comprising from about 1 x 1011viral particles to about 5 x 1011viral particles.

[0556] 160. The method of treatment according to any one of items 141-159, wherein said method comprises a single administration of the vector to the subject.

[0557] 161. The method of treatment according to any one of items 141-159, wherein said method comprises repeated administration of the vector to the subject.

[0558] 162. The method of treatment according to item 161, wherein said vector is a viral vector as defined in any one of items 25-50 and wherein each administration of said repeated administration comprises the administration of the vector in a dose as defined in item 158 or 159. 163. The method of treatment according to item 161 or 162, wherein a time interval between consecutive administrations of said repeated administration is at least about 1 week, such as at least about 1 month, such as at least about 6 months, such as at least about 1 year.

[0559] 164. The method of treatment according to any one of items 161-163, wherein a time interval between consecutive administrations of said repeated administration is from about 1 week to about 6 week.

[0560] 165. The method of treatment according to any one of items 141-164, wherein said administration, such as said single or repeated administration, of the vector is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration.

[0561] 166. The method of treatment according to any one of items 141-165, wherein said administration, such as said single or repeated administration, of the vector is an intra lymphatic administration or an intratumoral administration, such as an intratumoral administration.

[0562] 167. The method of treatment according to any one of items 141-156, wherein said method comprises administration of the vector system comprising the first and the second vector to the subject in need thereof.

[0563] 168. The method of treatment according to any one of items 141-156 and 167, wherein said first and said second vector are administered simultaneously or subsequently, such as simultaneously, to the subject.

[0564] 169. The method of treatment according to any one of items 141-156, 167 and 168, wherein the first and the second vector of said vector system are viral vectors as defined in item 54 and wherein said method comprises administration of the first vector to the subject in a dose comprising at least about 1 x 1011viral particles, such as at least about 3 x 1011viral particles, such as at least about 5 x 1011viral particles; and / or administration of the second vector to the subject in a dose comprising at least about 1 x 1011viral particles, such as at least about 3 x 1011viral particles, such as at least about 5 x 1011viral particles.

[0565] 170. The method of treatment according to any one of items 141-156 and 167-169, wherein the first and the second vector of said vector system are viral vectors as defined in item 54 and wherein said method comprises administration of the first vector to the subject in a dose comprising from about 1 x 1011viral particles to about 10 x 1011viral particles, such as a dose comprising from about 1 x 1011viral particles to about 5 x 1011viral particles; and / or administration of the second vector to the subject in a dose comprising from about 1 x 1011viral particles to about 10 x 1011viral particles, such as a dose comprising from about 1 x 1011viral particles to about 5 x 1011viral particles.

[0566] 171. The method of treatment according to any one of items 141-156 and 167-170, wherein said method comprises a single administration of the first vector and / or a single administration of the second vector to the subject.

[0567] 172. The method of treatment according to any one of items 141-156 and 167-170, wherein said method comprises repeated administration of the first vector and / or repeated administration of the second vector to the subject.

[0568] 173. The method of treatment according to item 172, wherein the first and the second vector of said vector system are viral vectors as defined in item 54 and wherein each administration of said repeated administration of the first vector comprises the administration of the first vector in a dose as defined in item 169 or 170; and / or each administration of said repeated administration of the second vector comprises the administration of the second vector in a dose as defined in item 169 or 170.

[0569] 174. The method of treatment according to item 172 or 173, wherein a time interval between consecutive administrations of said repeated administration of the first and / or the second vector is at least about 1 week, such as at least about 1 month, such as at least about 6 months, such as at least about 1 year. 175. The method of treatment according to any one of items 172-174, wherein a time interval between consecutive administrations of said repeated administration of the first and / or the second vector is from about 1 week to about 6 week.

[0570] 176. The method of treatment according to any one of items 141-156 and 167-175, wherein said administration, such as said single or said repeated administration, of the first and / or the second vector is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration.

[0571] 177. The method of treatment according to any one of items 141-156 and 167-176, wherein said administration, such as said single or said repeated administration, of the first and / or the second vector is an intra lymphatic administration or an intratumoral administration, such as an intratumoral administration.

[0572] 178. The method of treatment according to any one of items 141-156, wherein said method comprises administration of the isolated cell population to the subject in need thereof.

[0573] 179. The method of treatment according to any one of items 141-156 and 178, wherein the isolated cell population administered to the subject is an allogeneic or an autologous cell population.

[0574] 180. The method of treatment according to any one of items 141-156, 178 and 179, wherein said method comprises

[0575] (e) obtaining a precursor cell population, such as a malignant cell population (such as a cancer cell population), from a subject suffering from cancer, such as cancer as defined in any one of items 143-154,

[0576] (f) ex vivo transfecting or transducing the precursor cell population obtained in step (e) to obtain the isolated cell population as defined in any one of items 57-59,

[0577] (g) administering the isolated cell population obtained in step (f) to the subject from which the precursor cell population was obtained in step (e). 181. The method of treatment according to any one of items 141-156 and 178-180, wherein the isolated cell population administered to the subject is an autologous cell population.

[0578] 182. The method of treatment according to any one of items 141-156, 178 and 179, wherein the isolated cell population administered to the subject is an allogeneic cell population.

[0579] 183. The method of treatment according to any one of items 141-156, 178, 179 and 182 and 182, wherein the isolated cell population administered to the subject is stable cell line, such as a stable malignant cell line, such as a stable cancer cell line, such as a stable melanoma cell line.

[0580] 184. The method of treatment according to any one of items 141-156 and 178-183, wherein said method comprises a single administration of the isolated cell population to the subject.

[0581] 185. The method of treatment according to any one of items 141-156 and 178-183, wherein said method comprises repeated administration of the isolated cell population to the subject.

[0582] 186. The method of treatment according to item 185, wherein a time interval between consecutive administrations of said repeated administration of the isolated cell population is at least about 1 week, such as at least about 1 month, such as at least about 6 months, such as at least about 1 year.

[0583] 187. The method of treatment according to item 185 or 186, wherein a time interval between consecutive administrations of said repeated administration of the isolated cell population is from about 1 week to about 6 week.

[0584] 188. The method of treatment according to any one of items 141-156 and 178-187, wherein said administration, such as said single or said repeated administration, of the isolated cell population is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration. 189. The method of treatment according to any one of items 141-156 and 178-187, wherein said administration, such as said single or said repeated administration, of the isolated cell population is a subcutaneous administration or an intralymphatic administration, such as an intra lymphatic administration.

[0585] 190. A method of treatment of a subject in need thereof, comprising administration of the gene therapy system as defined in item 127 or 128, to the subject in need thereof.

[0586] 191. A method of treatment of cancer, comprising administration of the gene therapy system as defined in item 127 or 128, to a subject in need thereof, such as wherein said cancer is a malignancy arising from epithelial tissue or a malignancy arising from non-epithelial tissue

[0587] 192. The method of treatment according to item 191, wherein said cancer is as defined in any one of items 143-154.

[0588] 193. The method of treatment according to any one of items 190-192, wherein said treatment is a prophylactic and / or a therapeutic treatment.

[0589] 194. The method of treatment according to any one of items 190-193, wherein said treatment is a therapeutic treatment.

[0590] 195. The method of treatment according to any one of items 190-194, wherein said method comprises administration of the gene therapy system as defined in item 127 or 128.

[0591] 196. The method of treatment according to item 195, wherein said first cell population is administered to the subject simultaneously, a priori or subsequently to the administration of the second cell population; said first vector is administered to the subject simultaneously, a priori or subsequently to the administration of the second vector; said second vector is administered to the subject simultaneously, a priori or subsequently to the administration of the first cell population; or said first vector is administered to the subject simultaneously, a priori or subsequently to the administration of the second cell population.

[0592] 197. The method of treatment according to item 195 or 196, wherein said administration of said first cell type population, said second vector, said second cell type population and / or said first vector is an intravenous administration, a subcutaneous administration, an intraperitoneal administration, an intramuscular administration, an intralymphatic administration or an intratumoral administration.

[0593] 198. Use of the vector as defined in any one of items 1-50, the vector system as defined in any one of items 51-55, and / or the isolated cell population as defined in any one of items 57-59, in the manufacture of a medicament.

[0594] 199. Use of the vector as defined in any one of items 1-50, the vector system as defined in any one of items 51-55, and / or the isolated cell population as defined in any one of items 57-59, in the manufacture of a medicament for the treatment of cancer, such as wherein said cancer is a malignancy arising from epithelial tissue or a malignancy arising from non-epithelial tissue.

[0595] 200. The use according to item 199, wherein said cancer is as defined in any one of items 80-91.

[0596] 201. The use according to item 199 or 200, wherein said treatment is a prophylactic and / or a therapeutic treatment.

[0597] 202. The use according to any one of items 199-201, wherein said treatment is a therapeutic treatment.

[0598] 203. A use of the gene therapy system as defined in item 127 or 128, in the manufacture of a medicament.

[0599] 204. A use of the gene therapy system as defined in item 127 or 128, in the manufacture of a medicament for the treatment of cancer, such as wherein said cancer is a malignancy arising from epithelial tissue or a malignancy arising from non-epithelial tissue. 205. The use according to item 204, wherein said cancer is as defined in any one of items 80-91.

[0600] 206. The use according to item 204 or 205, wherein said treatment is a prophylactic and / or a therapeutic treatment. 207. The use according to any one of items 204-206, wherein said treatment is a therapeutic treatment.

Claims

CLAIMS1. A vector comprising(i) a first nucleic acid sequence encoding a galactosyltransferase, an enzymatically active variant thereof, or an enzymatically active truncated form thereof; and(ii) a second nucleic acid sequence encoding a Helicobacter pylori neutrophilactivating protein (NAP), an immunologically equivalent variant thereof, or an immunologically equivalent fragment thereof.

2. The vector according to claim 1, wherein said galactosyltransferase is an N- acetyllactosaminide alpha-l,3-galactosyltransferase.

3. The vector according to claim 2, wherein said N-acetyllactosaminide alpha-1, 3- galactosyltransferase comprises an amino acid sequence selected from a group consisting of an amino acid sequence according to SEQ ID NO:2 and amino acid sequences having at least 70% sequence identity to SEQ ID NO:2.

4. The vector according to any one of claims 1-3, wherein said NAP comprises an amino acid sequence selected from a group consisting of an amino acid sequence according to SEQ ID NO:4 and amino acid sequences having at least 70% sequence identity to SEQ ID NO:4.

5. The vector according to any one of claims 1-4, wherein said vector is a non-viral vector.

6. The vector according to any one of claims 1-4, wherein said vector is a viral vector.

7. The vector according to any one of claims 1-4 and 6, wherein said vector is a viral vector selected from the group consisting of a retrovirus, a lentivirus, a vaccinia virus, an adenovirus and an adeno-associated virus.

8. The vector according to any one of claims 1-4, 6 and 7, wherein said vector is an adenovirus.

9. The vector according to any one of claims 6-8, wherein said viral vector is an oncolytic virus, such as an oncolytic adenovirus.

10. A vector system for simultaneous or subsequent delivery of a first and a second vector to a cell and / or for generating an isolated cell population as defined in claim11, said vector system comprising(a) the first vector comprising said first nucleic acid sequence as defined in any one of claims 1-3, and(b) the second vector comprising said second nucleic acid sequence as defined in any one of claims 1 and 4; wherein said first and said second vectors are separate vectors.

11. An isolated cell population comprising said first nucleic acid sequence as defined in any one of claims 1-3 and / or a galactosyltransferase, an enzymatically active variant thereof, or an enzymatically active truncated form thereof, encoded by said first nucleic acid sequence; and said second nucleic acid sequence as defined in any one of claims 1 and 4 and / or a NAP, an immunologically equivalent variant thereof, or an immunologically equivalent fragment thereof, encoded by said second nucleic acid sequence.

12. A pharmaceutical composition comprising the vector as defined in any one of claims 1-9, the vector system as defined in claim 10, or the isolated cell population as defined in claim 11; and at least one pharmaceutically acceptable salt, carrier and / or excipient.

13. The vector as defined in any one of claims 1-9, the vector system as defined in claim 10, and / or the isolated cell population as defined in claim 11, for use as a medicament.15114. The vector as defined in any one of claims 1-9, the vector system as defined in claim 10, and / or the isolated cell population as defined in claim 11, for use in the treatment of cancer, such as wherein said cancer is a malignancy arising from epithelial tissue or a malignancy arising from non-epithelial tissue.

15. The vector, the vector system or the isolated cell population for use according to claim 14, wherein said cancer is carcinoma.

16. The vector, the vector system or the isolated cell population for use according to any one of claims 14 and 15, wherein said cancer is pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC), such as stage IV PDAC.

17. The vector, the vector system or the isolated cell population for use according to claim 14, wherein said cancer is melanoma.

18. The isolated cell population for use according to any one of claims 14-17, wherein said use comprises(e) obtaining a precursor cell population, such as a malignant cell population, such as a cancer cell population, from a subject suffering from cancer, such as cancer as defined in any one of claims 15- 17,(f) ex vivo transfecting or transducing the precursor cell population obtained in step (e) to obtain the isolated cell population as defined in claim 11,(g) administering the isolated cell population obtained in step (f) to the subject from which the precursor cell population was obtained in step (e).

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