Expression Systems for the Treatment of Cancer

Abstract

The present invention relates to a recombinant expression system for the treatment of cancer, comprising two nucleic acid constructs based on "Stimulator of Interferon Genes" (STING). The present invention further relates to an expression vector encoding said nucleic acid constructs or a combination of two expression vectors, and to a viral particle comprising said expression vector or a combination of two viral particles. The present invention also relates to a pharmaceutical composition comprising said recombinant expression system, said expression vector or said viral particle. The present invention also relates to the use of said recombinant expression system, said expression vector, said viral particle or said pharmaceutical composition for use in immunotherapy, in particular in the immunotherapy of proliferative diseases such as cancer. The present invention finally relates to the combined administration of radiation therapy and expression vectors for the treatment of cancer.

Description

[Technical field]

[0001] The present invention relates to a recombinant expression system for the treatment of cancer, based on "Stimulator of Interferon Genes" (STING), comprising two nucleic acid constructs. The present invention further relates to an expression vector encoding said nucleic acid constructs or a combination of two expression vectors, and to a viral particle (or combinations thereof) comprising said expression vector. The present invention also relates to a pharmaceutical composition comprising said recombinant expression system, said expression vector(s) or said viral particle(s). The present invention finally relates to the use of said recombinant expression system, said expression vector, said viral particle or said pharmaceutical composition for use in immunotherapy, in particular in the immunotherapy of proliferative diseases such as cancer. [Background technology]

[0002] Cancer is a group of diseases involving abnormal cell growth and proliferation, with approximately 19 million new cases each year and responsible for approximately 10 million deaths each year (Non-Patent Document 1). Although there have been significant advances in cancer treatment over the past few decades, annual mortality rates indicate that a huge number of people still die from cancer. One serious obstacle in the treatment of cancer is the existence of different cancers, each with its own unique challenges for treatment: for example, some cancers are easily accessible and can be treated locally (i.e., surgical removal, localized radiation therapy), whereas other cancers can only be treated with a systemic approach (i.e., chemotherapy, hormonal therapy, immunotherapy). Many different treatments are available, such as radiation therapy, chemotherapy, surgical removal, hormone therapy, and immunotherapy. Some of these treatments target the tumor by preventing cell proliferation systemically, thereby halting tumor growth. However, physiologically dividing cells, such as hematopoietic cells, cells of the mouth, digestive tract, and reproductive system, are also unnecessarily targeted with these systemic treatments, resulting in severe side effects for the patient, such as fatigue, nausea and vomiting, anemia, and infections. Localized therapies, such as localized radiation therapy, also result in severe side effects depending on the location of the tumor. In contrast, the concept of immunotherapy aims to stimulate the body's immune system to act against cancer cells, thereby reducing undesirable side effects. Immunotherapies can be classified as active or passive. Active immunotherapy is characterized by immunizing the patient with agents that enhance the immune response against tumor antigens. Examples include therapeutic cancer vaccines, cell-based immunotherapy, and targeted antibody therapy. In contrast, passive immunotherapy does not directly target tumor cells, but enhances the immune system's ability to attack cancer cells. For example, checkpoint inhibitors and cytokines are included. However, immunotherapy has been hitherto cited several drawbacks, such as i) insufficient tumor targeting, ii) weak tumor cell killing function, iii) poor results and high cost, iv) tumor immune escape, and v) overstimulation of the immune system, which may cause severe side effects (Non-Patent Document 2).

[0003] STING (also known as endoplasmic reticulum interferon stimulator (ERIS), mediator of IRF3 activation (MITA) or transmembrane protein 173 (TMEM173)) is a four-pass transmembrane protein present in the endoplasmic reticulum (ER) that acts as a sensor for the presence of double-stranded DNA (dsDNA) in the cytosol. Thus, STING is activated by DNA viruses, by DNA damage such as radiation therapy, and by micronuclei that frequently exist in the cytosol of malignant cells (Non-Patent Document 3). STING activates TANK-binding kinase 1 (TBK1), leading to phosphorylation and activation of interferon-regulatory transcription factor 3 (IRF3) and upregulation of type I interferon (IFN) expression. STING also recruits IκB kinase (IKK) and activates the NF-κB pathway, which promotes the production of proinflammatory molecules (including IL-6 and TNF-α) and cooperates with TBK1-IRF3 in the production of type I IFN (Non-Patent Document 3). Type I IFN stimulates immune effector cells, including dendritic cells (DCs) and natural killer (NK) cells, and promotes the production of chemokines such as CXCL9 and CXCL10, which attract T lymphocytes to tumors, which are important for anti-tumor immunity (Non-Patent Document 4). Indeed, STING agonists such as cyclic dinucleotides (CDNs) exert potent anti-tumor effects in different tumor models (Non-Patent Document 5). The importance of STING in cancer pathology may also derive from the fact that STING expression is usually suppressed or lost in many cancers. Unfortunately, significant STING polymorphisms exist in the human population that affect both the molecular responses induced by activation of the protein and the degree of sensitivity to stimulatory ligands, which may in turn greatly affect the efficacy and safety of molecular entities pursued for clinical purposes. Based on these numerous findings, STING is now considered a master regulator in the cancer-immune cycle.

[0004] A well-described activator of STING is 5,6-dimethylxanthenone-4-acetic acid (DMXAA), disclosed in US Pat. No. 5,629,363, although other STING agonists are also known in the art. Clinical trials with DMXAA have shown that it is well tolerated with only minor side effects. However, while the antitumor effects of STING have been reported in animal models, clinical trials have failed due to the lack of efficacy of DMXAA. Further studies have demonstrated the reason for this lack of therapeutic effect, showing that DMXAA potently activates mouse STING but is unable to activate human STING due to different amino acids in the cyclic dinucleotide (CDN) binding site of STING. Patent Documents 2 and 3 disclose cell-based immunotherapy in which immune-competent cells from a patient, such as T cells, are isolated, cultured, modified ex vivo so that a specific cancer is recognized by the cells, and then reinjected into the patient. However, in addition to the elaborate process for generating the ex vivo modified cells, only the specific cancer is targeted.

[0005] Immunotherapy using interleukin-2 (IL-2), a cytokine to stimulate the immune system in targeting cancer cells, was approved for use in the treatment of melanoma and kidney cancer as early as 1992. However, studies show that only about 7%-16% of patients respond to the treatment, and severe side effects are common (Non-Patent Document 6). The use of autologous cancer cells as a vaccine to enhance antitumor immunity has been explored for some time. However, due to the poor immunogenicity of many cancers, down-regulation of MHC molecules, lack of appropriate co-stimulatory molecule expression, and secretion of immune-inhibitory cytokines by cancer cells, responses to such vaccines have not provided long-term efficacy (e.g., Non-Patent Document 7). A number of cytokines have been shown to function in regulating immune responses to tumors. For example, Patent Document 4 discloses the combination of TNF, IL-2, and IFN-β in synergistically effective amounts to combat existing tumors. Patent Documents 5-7 disclose the use of GM-CSF for tumor treatment. However, direct administration of cytokines for cancer treatment may be impractical due to frequent systemic toxicity (Non-Patent Document 8). Patent Document 8 provides compositions and methods for the treatment of cancer using IL-2 immunotherapy. The method of the present invention includes systemic administration of an interleukin-2 / interleukin-2 receptor alpha fusion protein to a patient, which dose-dependently increases circulating NK cells and CD8+ cells in the patient when circulating immunosuppressive T regulatory (Treg) cells do not increase in a dose-dependent manner. However, while the present invention aims to overcome tumor immune evasion, it also includes known drawbacks of cancer immunotherapy, such as weak tumor cell killing ability, and only a portion of patients respond to the treatment at all (Non-Patent Document 9). Furthermore, cytokine-based immunotherapy usually needs to be performed repeatedly to ensure sufficient immune system stimulation while avoiding overstimulation, which results in additional medical costs and burdens on patients. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2004 / 039363 [Patent Document 2] U.S. Patent No. 5,126,132 [Patent Document 3] US Patent Application Publication No. 2005 / 214274 [Patent Document 4] U.S. Patent No. 5,098,702

Patent document 5

Patent document 6

Patent document 7

Patent document 8

Non-licensed literature

[0007]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

[0008] Thus, there remains a need for efficient and safe treatments of proliferative diseases such as cancer, where many different cancers can be effectively targeted, where the treatment has no or few undesirable side effects, and where a wide variety of patients respond to the treatment. [Means for solving the problem]

[0009] It is therefore an object of the present invention to provide an improvement or alternative to the prior art. The problem is solved by providing a recombinant expression system according to claim 1 or 25, an expression vector according to claim 14 or 32 or the combination of two expression vectors according to claim 14, a viral particle according to claim 15 or 33 or the combination of two viral particles according to claim 15, and a pharmaceutical composition according to claim 16 or 34. Embodiments are the subject of further dependent or independent claims. In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary artery disease comprising the steps (a) a first nucleic acid construct comprising a sequence encoding a target STING, the target STING being responsive to a STING agonist that recognizes the target STING but not human STING and its naturally occurring variants (a human non-responsive STING agonist), and the sequence encoding the target STING is operably linked to elements that allow for the expression and / or translation of the target STING; and / or (b) a second nucleic acid construct comprising a sequence encoding at least one immune modulatory gene or protein, wherein the sequence is operably linked to a STING responsive promoter or a radiation inducible promoter. The expression system according to the invention comprises many of the advantages of cancer immunotherapy and avoids the drawbacks of said treatment. This allows the expression of immunomodulatory peptides, particularly cytokines such as interleukins or chemokines, in the tumor to stimulate the immune system and target cancer cells in the tumor, with the preferred intratumoral administration of the expression system resulting in highly localized cytokine expression, avoiding the drawbacks and toxicities of systemic cytokine therapies known in the art.

[0010] The recombinant expression system comprises two expression cassettes represented by two nucleic acid constructs: (a) one cassette represented by a first nucleic acid construct encoding a target STING that responds to a STING agonist that does not activate endogenous human STING (hereinafter also referred to as a "human non-responsive STING agonist"), and (b) two expression cassettes represented by a second nucleic acid construct. (b) The other cassette represented by a second nucleic acid construct encoding an immune-modulating gene or protein under the control of a promoter that is activated upon STING activation. According to the present invention, the recombinant expression system comprises one or two of these cassettes. The combined expression of both cassettes has the advantage that after intratumoral injection of a vector containing the expression cassettes, systemic administration of the agonist of the target STING selectively activates STING in the tumor without affecting endogenous human STING in the rest of the body, thereby avoiding toxicity. Intratumoral activation of targeted STING induces the production of IFN-type, chemokines and other cytokines in the tumor, either by induction of endogenous genes or by activation of the promoter of a second expression cassette encoding an immune-regulating gene or protein, such as IL-12. In this way, systemic administration of the targeted STING agonist induces the intratumoral production of IFN-type and other immune-stimulating peptides derived from STING activation, along with the production of the immune-regulating gene or protein expressed in the second expression cassette. Localized intratumoral production of the combination of immune-regulating genes or proteins promotes antitumor immunity without toxicity. A long-term expression vector (such as an adeno-associated virus vector) containing the two expression cassettes can be injected into the tumor, followed by repeated systemic administration of the targeted STING agonist to induce the production of the above combination of immune-regulating genes or proteins in the tumor.

[0011] The inventors have found that the first nucleic acid of the recombinant expression system according to the present invention induces upregulation of STING-associated genes or proteins when the target STING is specifically activated by administering a STING agonist that recognizes the target STING but does not recognize human STING and its natural variants. Furthermore, it has been found that upregulation of STING-associated genes or proteins is also induced by low-dose radiation therapy. It has been shown that enrichment of STING (e.g., by transducing the target STING into cells) renders the cells responsive to either a STING agonist such as DMXAA (canonical pathway) or low-dose radiation therapy (non-canonical pathway) separately, but when they are applied together, the synergy between the two stimuli (STING agonist and low-dose radiation therapy) is fully displayed (see Figures 9A-C). Thus, in one aspect of the present invention, it is sufficient for the expression system to include only a cassette encoding a target STING that responds to a STING agonist that does not activate human STING.

[0012] Furthermore, the inventors have found that the second nucleic acid of the recombinant expression system according to the invention induces upregulation of an immunomodulatory transgene through STING activity, and that said activity can be specifically induced by a STING agonist that recognizes said target STING but not human STING and its naturally occurring variants, i.e., expression of an immunomodulatory transgene can be controlled by administration of said STING agonist.

[0013] Furthermore, the present inventors found that the recombinant expression system of the present invention was non-toxic and had minimal side effects in vivo. Notably, the present inventors found that when the recombinant expression system was administered in vivo, it reduced tumor size and eventually led to complete remission. Furthermore, it was shown that when the recombinant expression system of the present invention was administered locally to tumors, it not only reduced the size of the target tumor, but also significantly reduced the size of non-target tumors, also known as the abscopal effect. [Brief description of the drawings]

[0014] [Figure 1]A) IFN-β expression in MC38 cells after lentiviral transduction with either hSTINGwt or hSTINGmut and treatment with DMXAA compared to vehicle-treated cells. Data shown are mean ± SEM (n = 3, **P < 0.01, two-way ANOVA followed by Sidak's multiple comparison test); B) IFN-β expression in Hep2G cells after lentiviral transduction with either hSTINGwt or hSTINGmut and treatment with DMXAA compared to vehicle-treated cells. Data shown are mean ± SEM (n = 3, **P < 0.01, two-way ANOVA followed by Sidak's multiple comparison test); C) IFN-β expression in Hep2G cells after lentiviral transduction with mSTINGwt and treatment with DMXAA compared to vehicle-treated cells. Data shown are mean ± SEM (n=3, **P<0.05, **P<0.01, two-way ANOVA followed by Sidak's multiple comparison test);D) STING-derived cytokine expression in MC38 cells with or without luciferase, hSTINGwt, or hSTINGmut lentiviral transduction and then treated with DMXAA compared to vehicle-treated cells. Data shown are mean ± SEM (n=3, *P<0.05, **P<0.01, Fisher LSD test, one sample per run);E) STING-derived cytokine expression in Hep2G cells with or without luciferase, hSTINGwt, or hSTINGmut lentiviral transduction and then treated with DMXAA compared to vehicle-treated cells. Data are means ± SEM (n = 3, *P < 0.05, **P < 0.01, T-test for one sample per run); F) Time course of STING-derived cytokine expression in MC38 cells following lentiviral transduction with hSTINGmut followed by DMXAA treatment compared to vehicle-treated cells.Data are means ± SEM (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA followed by Fisher LSD test); and G) Time course of STING-derived cytokine expression in MC38 cells treated with DMXAA after lentiviral transduction with hSTINGmut compared to vehicle-treated cells. Data are means ± SEM (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA followed by Fisher LSD test); [Diagram 2] Illustrates IFN-β serum levels in mice intravenously injected with AAV8 vectors encoding either mSTING or GFP and repeatedly treated twice weekly with different doses (5, 10, or 20 mg / kg body weight) of DMXAA. Data shown are mean ± SEM (n = 4-12 mice / group) **P < 0.01, ***P < 0.001, one-way ANOVA followed by Dunnett's multiple comparison test. [Diagram 3]A) Luciferase activity of MC38 cells lentivirally transduced with vectors encoding luciferase under the control of different modified IFN-β promoters in the absence or presence of DMXAA. Data are mean ± SEM (n = 2 wells per condition) **p < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA followed by Sidak's multiple comparison test (vehicle-DMXAA); B) Luciferase activity of MC38 cells lentivirally transduced with vectors encoding luciferase under the control of different chemokine promoters in the absence or presence of DMXAA; Data are mean ± SEM (n = 2 wells per condition) ****P < 0.0001, two-way ANOVA followed by Sidak's multiple comparison test (vehicle-DMXAA). C) Luciferase activity of MC38 cells transfected with plasmids encoding luciferase under the control of different synthetic promoters at different time points after DMXAA treatment, data are means ± SEM (n = 2 wells per condition) *P < 0.05, ***P < 0.001, ****P < 0.0001, two-way ANOVA followed by Sidak's multiple comparison test); D) IL-12 protein levels in the supernatant of MC38 cells lentivirally transduced with vectors encoding IL-12 under the control of either the IFN-β, CXCL9- or CXCL10-promoter at different time points after DMXAA treatment, data are means ± SEM (n = 2–8 wells per condition), **** P< 0.0001, Two-way ANOVA followed by Sidak's multiple comparison test; E) and F) Luciferase activity of MC38 cells transfected with plasmids encoding luciferase under the control of different synthetic promoters in the absence or presence of DMXAA, data are means ± SEM (n = 2 wells per condition), * P< 0.05, **** P< 0.001, **** P< 0.0001, Two-way ANOVA followed by Sidak's multiple comparison test; and G) and H) Luciferase activity of HEK293T cells transfected with plasmids encoding luciferase under the control of different synthetic promoters in the absence or presence of DMXAA, data are mean ± SEM (n=2 wells per condition) *P<0.05, ***P<0.001, ****P<0.0001, Two-way ANOVA followed by Sidak's multiple comparison test. [Figure 4] A) IL-12 protein levels in serum of mice injected intravenously with AAV8 encoding IL-12 under the control of either the IFN-β promoter or the ISRE promoter in the absence of DMXAA. Data are mean ± SEM (n = 2-3 animals / condition), *P < 0.05, two-way ANOVA followed by Tukey's multiple comparison test; B) IL-12 protein levels in serum of mice injected intravenously with AAV8 encoding single-chain (sc) IL-12 operably linked to the miR122-binding site under the control of the ISRE promoter or the miR122-binding site-deficient AAV8 in the absence of DMXAA were reduced compared to the miR122-binding site-deficient AAV8 vector. Data are means ± SEM (n = 4-8 animals / condition), *P < 0.0001, Two-way ANOVA followed by Tukey's multiple comparison test; C) Reduction of IL-12 protein levels in serum of mice intravenously injected with AAV8 encoding hSTINGmut under a constitutively active promoter and AAV8 encoding scIL-12 operably linked to a miR122 binding site under the control of the ISRE promoter, or the AAV8 lacking the miR122 binding site, in the presence or absence of DMXAA. Data are means ± SEM (n = 4-8 animals / condition), *P < 0.05, Two-way ANOVA followed by Tukey's multiple comparison test. [Diagram 5]Figure 1 shows the volume of subcutaneous MC38 tumors implanted in STING KO mice treated by intratumoral co-injection of an AAV8 vector encoding hSTINGmut under the control of a constitutive promoter (AAV8-hSTINGmut) at a dose of 1x10vg / mouse and an AAV8 vector encoding scIL-12 operably linked to a miR122 binding site and placed under the control of the ISRE promoter (AAV8-ISREx4IL12miR122T) at a dose of 0.5x10vg / mouse. The animals received either DMXAA (closed triangles) or vehicle (closed circles) at the indicated time intervals. In a further control group, tumors were injected with an AAV8 vector encoding AAV8-hSTINGmut (1x10vg / mouse) + GFP (0.5x10vg / mouse) followed by vehicle (open circles). Data are means ± SEM (n = 4–6 animals / condition *P < 0.05, **P < 0.01, ****P < 0.0001 mixed-effects analysis followed by Tukey's multiple comparison test). [Figure 6] A) Sections of subcutaneous MC38 tumors immunohistochemically stained with anti-GFP antibody 3 days after AAV8-GFP intratumoral injection. Transduction efficiency of AAV8-GFP injected tumors not pretreated with radiation (left) was significantly lower compared to tumors with 8 Gy radiation pretreatment (right). B) Graph shows the percentage of pixels strongly labeled with GFP associated with the tumor area. Data are mean ± SEM (n = 3–6 animals / condition ***P < 0.001 unpaired Student's t test). [Figure 7] Values ​​at given time points for IFN-β protein levels in the supernatants of MC38 cells transduced with irradiated and / or hSTINGmut-encoding vectors quantified at indicated time points after irradiation were compared, data are means ± SEM (n = 2–6 samples per condition; *P<0.05; #P<0.05, ####P<0.0001 vs. MC38 8 Gy, two-way ANOVA followed by Tukey's multiple comparisons test). [Figure 8]A) Time course of tumor volume progression in mm2 of MC38 subcutaneous tumors in C57BL / 6 wild-type mice after intratumoral injection of either AAV8-GFP, AAV8-hSTINGmut, and / or AAV8-ISREx4-IL12miR122T as indicated, followed by either radiation or no radiation. Data are mean ± SEM (n = 4–5 mice / group) and two-way analysis of variance (ANOVA) tests with Tukey's post-hoc test were used to assess significance. Simple main effects analysis showed that treatment had a statistically significant effect on tumor growth (***p<0.001). B) Serum levels of IL-12 measured by ELISA 17 days after treatment of animals in A), points indicate individual values ​​for each mouse, central horizontal line represents median, error bars indicate standard error of the mean (SEM). There were no statistically significant differences between groups as determined by one-way ANOVA (F(2,7) = 0.4375, p = 6622). C) and D) refer to C57BL / 6 wild-type mice with two subcutaneous MC38 tumors (one in the right flank and the other in the left flank), the former was treated and the contralateral tumor was left untreated to analyze the abscopal effect. Right flank tumors were treated by intratumoral injection of either AAV8-GFP, AAV8-hSTINGmut and / or AAV8-ISREx4-IL12miR122T as indicated after 8 Gy of local irradiation. C) shows the growth of treated tumors and D) shows the growth of untreated tumors. Data are mean ± SEM (n = 6 mice / group). Significance was assessed using a two-way analysis of variance (ANOVA) test with Tukey's post-hoc test analysis. Simple main effects analysis showed that the treatment had a statistically significant effect on the growth of treated tumors (***p<0.001), with a statistically significant antitumor effect in contralateral tumors 15 days after treatment (*p<0.01). [Figure 9]A) mRNA levels of IFN-β in MC38 cells upon stimulation with DMXAA or vehicle in untransduced cells or in cells transduced with AAV8-hSTINGmut after 8 Gy of irradiation or not (0 Gy), data are mean ± SEM (n=3, **P<0.05, ***P<0.001, one-way ANOVA followed by Kruskal-Wallis test); B) mRNA levels of IL-12 in MC38 cells after 8 Gy of irradiation or not (0 Gy) in cells transduced with AAV8-hSTINGmut+AAV8-ISREx4-IL12miR122T after stimulation with DMXAA or vehicle. mRNA levels are shown, data are mean ± SEM (n=3, ***P<0,001, one-way ANOVA followed by Kruskal-Wallis test); (C) and D) IFN-β mRNA levels (C) or IL-12 mRNA levels (D) IL-12 in HEK293 cells upon stimulation with DMXAA or vehicle, in cells transduced with AAV8-hSTINGmut+AAV 8-ISREx, or in cells transduced with AAV8-hSTINGmut+AAV-ISREx4-IL12miR122T, after 8 Gy irradiation or without irradiation (0 Gy). mRNA levels are shown and are the mean ± SEM (n=3, **P<0.01, ***P<0.001, ****P<0.0001, two-way ANOVA followed by Tukey's multiple comparison test); E) IFN-β protein levels (pg / ml) obtained by ELISA in the supernatants of cultured non-transduced MC38 cells treated with different doses of DMXAA (0, 5, 10, 20 and 50 μg / mL) 24 hours after irradiation (0, 2 or 8 Gy) and harvested 6, 24 or 48 hours after DMXAA administration. Data are mean. Data are means ± SEM (n = 2-5 samples / condition, ***P<0.001, ****P<0.0001, two-way ANOVA followed by Tukey's multiple comparison test); F) IFN-β protein levels (pg / ml) obtained by ELISA in the supernatants of cultured non-transduced MC38 cells treated with different doses of DMXAA (50 μg / mL) or vehicle 24 h after irradiation with or without irradiation (8 Gy) or without irradiation (0 Gy) and harvested 24 h after DMXAA administration. Data are means ± SEM (n = 3-4 samples / condition, ***P<0.001, ****P<0.0001, two-way ANOVA followed by Tukey's multiple comparison test);001, ****P<0.0001, Two-way ANOVA followed by Tukey's multiple comparison test. ANOVA followed by Tukey's multiple comparison test). [Figure 10] Results obtained in STING KO mice bearing subcutaneous MC38 tumors treated with 0 or 8 Gy radiation and then injected intratumorally with the indicated AAV vectors encoding GFP (control) or hSTINGmut and / or IL12miR122T. Two days later, they received intraperitoneal DMXAA or vehicle. The figure shows serum levels of A) IFN-β, B) IL-12, C) IFN-β at the indicated time points. D represents IL-12 levels in tumor extracts obtained 6 h after DMXAA or vehicle treatment. Values ​​were obtained by ELISA. Data are mean ± SEM (n = 4–5 mice / group), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-way ANOVA followed by Tukey's multiple comparison test. [Figure 11] Results are shown for mouse cells transduced with either an AAV encoding GFP under the control of a constitutive promoter (CAG) (AAV8.CAG.GFP) or an AAV8 encoding IL-12 coupled to miR122T under the control of the ISREx4 promoter (AAV8-ISREx4-IL12miR122T) (MOI 105 in both cases). A shows that transduction efficiency of MC38 cells treated with AAV8-CAG.GFP was approximately 70%, regardless of prior irradiation. B shows that IL-12 production is significantly enhanced by MC38 cells irradiated prior to AAV8-ISREx4-IL12miR122T transduction. Data are mean ± SEM (n = 3 samples / condition). ***P < 0.005 unpaired T-test. [Figure 12]Results are shown for B16 cells transduced with AAV8 encoding luciferase coupled to miR122T under the control of the ISREx4 promoter (AAV8-ISREx4-LucmiR122T) (MOI 105) treated with radiation (8 Gy) or without radiation (0 Gy) in combination with either IFN-α, IFN-β, IFN-γ or vehicle. The figure shows that luciferase activity was induced upon incubation with either IFN-α (A), IFN-β (B) or IFN-γ (C) even in the absence of irradiation, but further irradiation synergistically increased luciferase activity. Data are mean ± SEM (n=3 samples / condition *p<0.01, **P<0.01, ****P<0.0001, two-way ANOVA followed by Tukey's multiple comparison test). [Figure 13]The antitumor effect of combined treatment with radiotherapy and intratumoral injection of AAV8-ISREx4-IL12miR122T (0.5x1011vg) on ​​a mouse model of melanoma (A+B), hepatocellular carcinoma (C-H) and subcutaneous mouse glioblastoma (I+J) is shown, along with intratumoral and serum levels of IL-12 (K+L) and IFN-γ (M+N), and finally the efficacy of the combined approach compared to treatment with only one component of the combined approach or without any component of the combined approach (O). A) Evaluation of tumor volume at given time points after treatment with either AAV8-ISREx4-IL12miR122T combined with radiotherapy (triangles) or AAV8-ISREx4-LucmiR122T combined with radiotherapy (circles) in a mouse model of melanoma (B16 cells). B) Hematoxylin & Eosin staining showing massive lymphocyte infiltration into tumor tissue 3 months after AAV8-ISREx4-IL12miR122T combined with radiotherapy. C) and D) Evaluation of tumor volume (C) or relative tumor volume (D) at the indicated time points after treatment with either AAV8-ISREx4-IL12miR122T combined with radiotherapy (triangles) and AAV 8-Hepat4-LucmiR 122 T combined with radiotherapy (circles) in a mouse model of subcutaneous hepatocellular carcinoma generated by subcutaneous injection of PM299L ISREx cancer cells. E) Evaluation of survival rate shows complete survival of AAV8-ISREx4-IL12miR122T-treated animals throughout the study period (dashed line) compared to early death of all AAV8-ISREx4-LucmiR122T-treated animals (solid line). F) and G) Evaluation of tumor volume (F) or relative tumor volume (G) at the indicated time points after treatment with either AAV8-ISREx4-IL12miR122T in combination with radiotherapy (triangles, dashed line) and AAV8-ISREx4-LucmiR122T in combination with radiotherapy (circles, continuous line) in a mouse model of hepatocellular carcinoma generated by subcutaneous injection of NM53 G03 hepatocellular carcinoma cells. H) Evaluation of survival percentage shows complete survival of AAV8-ISREx4-IL12miR122T-treated animals throughout the study period (dashed line) compared to early death of all AAV8-ISREx4-LucmiR122T-treated animals (continuous line).I) and J) Evaluation of tumor volume at the indicated time points after treatment with either AAV8-ISREx4-IL12miR122T in combination with radiation therapy (I) or AAV8-ISREx4-LucmiR122T in combination with radiation therapy (J) in a subcutaneous mouse glioblastoma model. Lines indicate the change in tumor volume for individual animals over the study period. K)-N) Intratumoral levels of IL-12 (K), serum concentrations of IL-12 (L), intratumoral levels of IFN-γ (M), and serum concentrations of IFN-γ (N) in mice bearing subcutaneous B16 (melanoma) tumors irradiated with 0 or 8 Gy and injected intratumorally with AAV8-ISREx4-IL12miR122T (0.5×1011vg). O) Assessment of tumor volume at indicated time points after no treatment (closed circles), treatment with AAV8-ISREx4-LucmiR122T in combination with radiotherapy (open circles), treatment with AAV8-ISREx4-IL12miR122T but without radiotherapy (squares), and treatment with AAV8-ISREx4-IL12miR122T in combination with radiotherapy (downward pointing triangles) in a mouse model of melanoma (B16 cells). Arrows indicate the start of each treatment. Data are mean ± SEM (n = 4-6 samples per condition) **P < 0.01, ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test (A and O) or Sidak's multiple comparison test (C, D, F, G). K–N) Data are means ± SEM (two-way ANOVA with 4–8 samples per condition followed by Tukey's multiple comparisons *P<0.05, **P<0.01, ***P<0.001). [Figure 14]Antitumor effects of combined treatment with radiotherapy and intratumoral injection of AAV8-ISREx4-IL12miR122T (AAV-IL12T) on a mouse model of melanoma compared to mice injected with AAV8-ISREx4-LucmiR122T (AAV.Luc) and control mice, where the tumor cells lack either MHC class I expression (B2Mko, A) or IFN-γ receptor (B16-IFNRGR-KO) or JAK1 (B16-JAK1-KO) (B). Data are mean ± SEM (n = 5-6 samples / condition; **P < 0.01, ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test). [Figure 15]The expression of either IL-12 or IFN-β in different tumor cell lines including melanoma (B16, Figures A, B and K-P), glioblastoma (GL261, Figures C and D), pancreatic cancer (KPC Figures E and F), colorectal cancer (MC38, Figures G and H) and breast cancer (TSA, Figures I and J) is shown. Cultured cells (85,000 per well) were irradiated with 0 or 8 Gy and then incubated in the presence or absence of a bicistronic vector containing (A-J) scIL-12 and IFN-β separated by a P2A element under the control of the ISREx4 promoter (AAV8-ISREx4-IL12-IFNbmiR122T), (K-L) scIL-12 and IFN-β separated by GGSG under the control of the ISREx4 promoter (AAV8-ISREx4-IL12-GGSGF2A-IFNbmiR122T), (M-N) scIL-12 and IFN-β separated by a short IRES element under the control of the ISREx4 promoter (IRES Gtx9, SEQ ID NO: 83), or (O-P) scIL-12 and IFN-β separated by an IRES2 element under the control of the ISREx4 promoter (AAV8-ISREx4-IL12-IRES2-IFNbmiR122T). After 48 hours, supernatants were harvested and IL-12 and IFN-β were measured by ELISA. Both proteins were detectable only upon incubation with vector, whereas expression of either protein was significantly increased by radiation treatment. Data are mean ± SEM (n = 3 samples per condition). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 One-way ANOVA followed by Sidak's multiple comparison test. [Figure 16] Serum levels of IL-12 (A) and IFN-β (B) are shown in mice intravenously injected with bicistronic AAV8-ISREx4-IL12-IFNbmiR122T (black circles), monocistronic AAV8-ISREx4-IL12miR122T (gray circles), or the monocistronic vector AAV8-ISREx4-IL12 lacking the miRNA122 target sequence (white circles). Data are mean ± SEM (n = 5–7 samples / condition). *p<0.01, ****P<0.0001, ns not significant, two-way ANOVA followed by Tukey's multiple comparison test. [Figure 17] Antitumor effects of combined treatment with radiotherapy and intratumoral injection of either bicistronic AAV8-ISREx4-IL12-IFN-βmiR122T (squares in A, downward triangles in B), monocistronic AAV8-ISREx4-IL12miR122T (upward triangles in A and B), or control vector AAV8-ISREx4-LucmiR122T (circles in A and B) in mouse models of melanoma in which tumor cells express MHC class I (A) or are deficient in MHC class I expression (B). Data are mean ± SEM (n = 6 samples / condition). ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test. [Figure 18] A shows transduced gene expression in irradiated subcutaneous tumors (NM53 hepatocellular carcinoma cells) when AAV8-ISREx4-LucmiR122T was administered by systemic route (intravenous administration) compared to non-irradiated tumors on the opposite side. B is a repeat of the experiment in A. C shows luciferase activity in four mice with orthotopic NM53 hepatocellular carcinoma. Two mice were irradiated with 20 Gy and the other two were not irradiated, after which all mice were injected intravenously with AAV8-ISREx4-LucmiR122T. Luciferase activity was measured 6 days after vector injection. It can be seen that luciferase activity is only present in the liver area when the tumor was pre-irradiated with 20 Gy. Additionally, D shows the time course of tumor volume progression in mm2 of MC38 subcutaneous tumors following intravenous injection of either AAV8-ISREx4-LucmiR122T (AAV.LucT) or AAV8-ISREx4-IL12miR122T (AAV.IL12T) as indicated, after radiation (20 Gy) or no radiation. Data are mean ± SEM (n = 5-6 mice / group), ****P < 0.0001, two-way ANOVA followed by Tukey's post-hoc test. [Figure 19]Figure 1 shows the effect of different radiation doses on transgene expression from AAV8-ISREx4-LucmiR122T when the vector is administered by intratumoral injection. A shows luciferase activity when the vector was injected into the tumor after 15 Gy irradiation and then subjected to three boost doses of 15 Gy (total dose = 60 Gy). B shows the tumor injected with the vector after four daily doses of 15 Gy (total dose = 60 Gy). In C, the tumor received two daily doses of 20 Gy after intratumoral vector injection after 60 Gy (total dose = 100 Gy). In all cases, luciferase activity was evaluated during the next day after vector injection (as indicated in the figure). Thus, A-C show different irradiation and treatment paradigms, while the data are presented collectively in D. *P<0.05. One-way ANOVA followed by Sidak's multiple comparison test. [Figure 20] Figure 1 shows the time course of tumor volume progression in mm2 of B16 subcutaneous tumors following intratumoral injection of the indicated radiation (8 Gy) and either AAV8-ISREx4-LucmiR122T (AAV.LucT) or AAV8-ISREx4-IL12miR122T (AAV.IL12T), followed by administration of either immune checkpoint inhibitor anti-PD1 (αPD1) or immune checkpoint inhibitor anti-CTLA-4 (αCTLA-4), or no further treatment. Data are mean ± SEM (n=5-6 mice / group), ns: not significant, ****P<0.0001, two-way ANOVA followed by Tukey's post hoc test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Recombinant expression systems generate specific and essential immunological pathways in transfected cancer cells that utilize additional endogenous pathway components. Of note, the term "recombinant expression system" includes any nucleotide, analogs thereof, and polymers thereof. Thus, any of the recombinant expression systems disclosed herein refer to polymeric forms of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). The term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, and double- and single-stranded RNA. A more comprehensive definition is contained in the definitions section of this application.

[0016] The first component of this pathway is the so-called "targeted STING", which is defined by its responsiveness to STING agonists that cannot activate human STING and can selectively activate targeted STING. The first component is identified in the first nucleic acid construct of the present invention and can be considered as both the "co-effector" and "activator" parts of the pathway. In particular, the first component can be given alone, rendering cells expressing the first component responsive to STING agonists and low-dose radiation therapy, such that when these two stimuli (STING agonists and low-dose radiation therapy) are co-applied, these two stimuli are fully synergized. In the first nucleic acid construct of the present invention, the target STING is operably linked to an element that allows the expression and / or translation of said target STING. When the recombinant expression system is a DNA-based expression system, this element is usually a promoter that can transcribe the mRNA encoding the target STING. When the recombinant expression system is an RNA-based expression system, this element is usually an RNA element, such as a CAP, 5'UTR, 3'UTR or polyA site, that can translate the mRNA encoding the target STING.

[0017] The second component of the pathway is an artificial gene construct comprising an immunomodulatory gene or protein, said sequence being operably linked to a STING-responsive or radiation-inducible promoter. The immunomodulatory gene or protein activates both the innate and adaptive immune responses to generate a specific cancer immune response. The second component is specified in a second nucleic acid construct of the invention and can be considered as the "co-effector" portion of the pathway.

[0018] Notably, targeted STING differs from native human STING because it is activated by human non-reactive agonists such as DMXAA. However, once activated, targeted STING exhibits the biological activity of human STING in that it can still bind and activate downstream effector proteins that are also activated by human STING, such as iRhom2 and TANK-binding kinase 1 (TBK1). Upon activation, STING undergoes a conformational change that recruits TBK1, which then phosphorylates the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB), translocating them to the nucleus and stimulating the production of pro-inflammatory molecules such as type I interferons, IL6, TNF-α, or CXCL10. (Ahn, J.et al, Exp Mol Med 51, 1-10 (2019)). The target STING may be responsive to an agonist that is not reactive with human STING. The human non-responsive STING agonist may be selected from the group consisting of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), 7-bromo-DMXAA, 7-iodo-DMXAA, 7-hydroxy-DMXAA, 7-formyl-DMXAA, 7-hydroxy-methyl-DMXAA, 7-(2-hydroxy-ethyl)-DMXAA, flavone-8-acetic acid (FAA), 2,7-bis(2-dimethylaminoethoxy)fluoren-9-one (tilorone), and 10-carboxymethyl-9-acridan (CMA). Those skilled in the art will recognize that any of the analogs of the molecules disclosed herein and their stereoisomers may be used. Furthermore, one or more atoms of the above molecules, their analogs, and / or their stereoisomers may be replaced by their respective isotopes.

[0019] There are several advantages to using the human non-responsive STING agonist in the context of the present invention in human therapy. By definition, the human non-responsive STING agonist cannot bind to human wild-type STING or its natural variants to activate STING. It can be administered systemically without systemic STING response, thus avoiding undesirable side effects. The present invention intends to express a target STING that responds to the human non-responsive STING agonist, so that this transgenic STING can be specifically activated, thus resulting in local STING activation. Thus, in the context of the present invention, the human non-responsive STING agonist has two functions. It is used to define the STING to be used as the target STING in gene therapy, and further used in subsequent drug therapy to activate this target STING, thus allowing a highly selective therapeutic approach. Preferably, the target STING is responsive to DMXAA. The use of DMXAA-responsive STING has the advantage that the STING agonist DMXAA has already been proven in clinical trials as a molecule with excellent safety and minimal side effects. Furthermore, targeting STING activation by DMXAA showed dose-dependent induction of downstream STING targets such as IFN-β (see Example 1), which allows immune responses to be controlled by simply adjusting the DMXAA dose during treatment. Intratumoral injection of DMXAA was shown to effectively prime CD8+ T cell responses in a STING-dependent manner in mice, promoting the elimination of established tumors (Corrales et al. Cell Reports, 2015;11,1018-1030). Clinical trials of DMXAA's antitumor properties showed that it exhibited minimal side effects. However, the studies showed that DMXAA was unable to activate human wild-type STING or its natural mutants. Nevertheless, mouse STING and rat STING are responsive to DMXAA in that they exhibit STING biological activity.

[0020] It is contemplated within the scope of the present invention that the target STING encoded by the sequence of the first nucleic acid construct is a mutated form of a naturally occurring variant of human STING that further comprises at least one, at least two, or at least three amino acid substitutions that render the human STING variant sensitive to a human non-responsive STING agonist, as determined in an in vitro agonist determination assay. Human non-responsive STING agonists can be identified using an in vitro agonist determination assay. There are several methods for carrying out such assays known to those skilled in the art. In one non-limiting example, a vector can be constructed that encodes a reporter (e.g., luciferase) under the control of a STING-responsive promoter. Cells can be transformed, transfected or transduced with said vector and also with a vector that induces the expression of human STING or its natural variants, mutated human STING variants or non-human STING. The cells should then be contacted with a STING agonist, preferably DMXAA, and luciferase activity should be measured. Although luciferase activity in cells transformed, transfected or transduced with a vector that induces the expression of mutated human STING variants or non-human STING can be detected, if the luciferase activity in cells transformed, transfected or transduced with a vector that induces the expression of human STING or its natural variants is not significantly different from the mock-treated control group, the agonist is considered to be "human non-responsive". In the present context, not significantly different from mock-treated control means that the luciferase activity is less than 30%, preferably less than 10%, more preferably less than 5%, and particularly preferably less than 1% of the luciferase activity obtained with hSTING[R]-S162A / G230I / Q266I (SEQ ID NO: 6). Another example of an in vitro agonist determination assay is to directly measure IFN-β levels when cells transformed, transfected, or transduced with a mutated human STING variant, non-human STING, or a vector directing the expression of human STING or a naturally occurring variant thereof are contacted with a STING agonist. In the present context, a human non-responsive STING agonist results in at least 25%, preferably at least 50%, more preferably at least 75%, and even more preferably 90% of the IFN-β levels obtained with hSTING[R]-S162A / G230I / Q266I (SEQ ID NO: 6).

[0021] It may be within the scope of the present invention that the target STING encoded by the sequence of the first nucleic acid construct is a non-human STING that is sensitive to a human non-reactive STING agonist as determined by an in vitro agonist determination assay.The human non-reactive STING agonist is preferably DMXAA, and non-limiting examples of the non-human STING that is sensitive to a human non-reactive STING agonist as determined by an in vitro agonist determination assay are mouse STING or its natural variant, rat STING or its natural variant, or fusion STING.In a preferred embodiment, the non-human STING is mouse STING as set forth in SEQ ID NO:7, which encodes the protein of SEQ ID NO:8. Alternatively, a radiation-inducible promoter may be provided. As used herein, a radiation-inducible promoter is a promoter that induces the expression of a therapeutic gene sequence when cells are irradiated. The promoter may be natural, synthetic, or artificially created, selected from recombinant products of natural promoters. For example, the promoter may have the following sequence: (a) a eukaryotic radiation-inducible sequence CC(A / T)(6)GG (SEQ ID NO:87), such as the early growth response 1 (Egr1) promoter or pE9 promoter, also known as the CArG element or [CC(A+Trich)6 GG], preferably the sequence has 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:86, or is identical to SEQ ID NO:86 (Egr1 promoter), or comprises at least two sequences according to SEQ ID NO:87, wherein the at least two sequences may be separated by additional nucleotides; (b) low frequency CCAAT box sequences, e.g., from the survivin promoter, preferably having 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 88 or a sequence identical to SEQ ID NO: 88 (CCAAT-rich domain of the survivin promoter); (c) cyclin-dependent kinase inhibitor 1 (also known as CDKN1A or p21) promoter; (d) a late herpes simplex virus 1 promoter, such as the glycoprotein C promoter or the US11 promoter; (e) a combination or artificial mutation of the above;

[0022] In a preferred embodiment of the present invention, the target STING is DMXAA-sensitive human STING as determined by in vitro DMXAA determination assay.As described above, the use of DMXAA for human treatment has the advantage that DMXAA is safe, has minimal side effects, and cannot activate human STING, so it can be administered systemically without activating systemic STING.

[0023] In vitro DMXAA determination assay can be used to identify STING that is responsive to DMXAA. Cells can be transduced with vectors that induce the expression of any STING (e.g., human wild-type STING, mouse STING). If upon addition of DMXAA, cells respond by showing a statistically significant increase in mRNA levels of IFN-β and other cytokines measured by quantitative PCR (qPCR) in triplicate measurements compared to mock-treated controls, the expressed STING is considered to be DMXAA sensitive. In this context, a statistically significant increase means that the mRNA levels are at least 10%, preferably at least 20%, more preferably at least 30%, particularly at least 50% higher compared to mock-treated controls. An example of DMXAA non-responsive STING is human wild-type STING or a naturally occurring variant thereof. Another example of an in vitro DMXAA determination assay is to directly measure the IFN-β levels of cells transformed, transfected, or transduced with vectors that code for STING (e.g., human wild-type STING, mouse STING) after contact with DMXAA. In the present context, DMXAA-sensitive human STING provides at least 25%, preferably at least 50%, more preferably at least 75%, and even more preferably 90% of the IFN-β levels obtained with hSTING[R]-S162A / G230I / Q266I (SEQ ID NO: 6) treated with DMXAA.

[0024] Advantageously, in the context of the present invention, the target STING encoded by the sequence of the first nucleic acid construct is one of the following: (a) a human STING[R] variant further comprising at least one, at least two, or at least three amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I; (b) a human STING[AQ] mutant further comprising at least one, at least two, or at least three amino acid substitutions selected from the group consisting of S162A, A230I, Q266I, E260I, and E260V; (c) a human STING[H] variant further comprising at least one, at least two, or at least three amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I; and (d) a truncated human STING[H] mutant comprising at least one or at least two amino acid substitutions selected from the group consisting of S162A and G230I;

[0025] Those skilled in the art will appreciate that all of the above mutations are within the cyclic dinucleotide (CDN) binding site (binding pocket) and / or the lid region of the binding pocket of STING. Thus, in another embodiment, a mutation in one of the amino acids present in the binding pocket region and / or the lid region of endogenous human STING may result in DMXAA-sensitive human STING.

[0026] Several naturally occurring variants of human STING have been identified, for example as a result of certain single polymorphisms (SNPs) and SNP haplotypes that produce several allelic polymorphic forms of the protein. The amino acid sequence of the most common naturally occurring variant or allele of human STING is represented in SEQ ID NO: 1, which is referred to as wild-type human STING. The most common naturally occurring allelic variants of human STING include the following: hSTING[R], commonly referred to as hSTING R232, represented by SEQ ID NO:1, i.e., the amino acid at position 232 is arginine, is also considered to be the wild-type isoform of STING; hSTING[H], commonly referred to as hSTING H232, represented by SEQ ID NO:2, i.e., the amino acid at position 232 is histidine; hSTING[AQ], represented by SEQ ID NO:3, the sequence of which differs from hSTING[R] by the incorporation of the amino acid substitutions G230A and R293Q; Further, a truncated form of hSTING[H] as set forth in SEQ ID NO: 4 is characterized. A further naturally occurring variant is STING[HAQ] with amino acid substitutions R71H, G230A and R293Q. Thus, the target STING is not identical to one of the known haplotypes of human STING, i.e., hSTING[R] as set forth in SEQ ID NO: 1, hSTING[H] as set forth in SEQ ID NO: 2, hSTING[AQ] as set forth in SEQ ID NO: 3, hSTING[H] as set forth in SEQ ID NO: 4, amino acid substitution R293QSTING[Q], or STING[HAQ] with amino acid substitutions R71H, G230A and R293Q. Additionally, the targeted STING may be responsive to any of the human non-responsive STING agonists selected from the group consisting of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), 7-bromo-DMXAA, 7-iodo-DMXAA, 7-hydroxy-DMXAA, 7-formyl-DMXAA, 7-hydroxymethyl-DMXAA, 7-(2-hydroxyethyl)-DMXAA, flavone-8-acetic acid (FAA), 2,7-bis(2-dimethylaminoethoxy)fluoren-9-one (tilorone), and 10-carboxylmethyl-9-acridan (CMA). Thus, administration of the agonist activates exogenous STING provided by the first nucleic acid of the recombinant expression system, but not endogenous STING encoded by the genome of the human subject.

[0027] In a further advantage, the DMXAA sensitive human STING mutants encoded by the sequence of the first nucleic acid construct are selected from the group consisting of hSTING[R]-S162A / G230I / Q266I, hSTING[R]-S162A / Q266I, hSTING[R]-G230I, hSTING[R]-S162A, hSTING[R]-Q266I, hSTING[R]-S162A / G230I, hSTING[AQ]-S162A / A230I / Q266I, hSTING[AQ]-S162A, hSTING[AQ]-S162A / R169A, hSTING[AQ]-S162A / T267A, hSTING[AQ]-E260I, hSTING[AQ]-E260V, It may be selected from the group consisting of hSTING[H]-S162A / G230I / Q266I, hSTING[H]-S162A / Q266I, hSTING[H]-G230I, hSTING[H]-S162A, hSTING[H]-S162A / G230I, and hSTING[H]-Q266I, preferably hSTING[R]-S162A / G230I / Q266I. The experiments performed show that the mutation S162A is important in allowing DMXAA to bind and activate human STING, and that the mutations Q266I and G230I or A230I increased sensitivity to DMXAA (Gao et al., 2014, Cell Reports 8, 1668-1676). In a further advantage, the sequence of the first nucleic acid construct encoding the DMXAA sensitive human STING mutant may be selected from the group consisting of SEQ ID NO:5, the sequence of hSTING[R]-S162A / G230I / Q266I, or a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:5. The sequence identity of the polynucleotide sequence to SEQ ID NO:5 may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In particular, a polypeptide encoded by any of the above polynucleotides has biological activity in response to the agonist DMXAA, as determined in an in vitro DMXAA determination assay. In a further advantage, the sequence of the first nucleic acid construct encoding the DMXAA-sensitive human STING mutant is selected from the group consisting of polynucleotide sequences encoding a polypeptide comprising SEQ ID NO:6, wherein SEQ ID NO:6 is hSTING[R]-S162A / G230I / Q266I. The sequence identity of the polypeptide encoded by the polynucleotide sequence with respect to SEQ ID NO:6 can be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In particular, the polypeptide encoded by any of the above polynucleotides is biologically active in response to a human non-responsive STING agonist as determined in an in vitro agonist determination assay.

[0028] Both hSTING[R]-S162A / G230I / Q266I and mouse STING have been extensively studied and are known to be highly activated by DMXAA and CMA. In addition, mouse STING can also be activated by FAA and tilorone. However, using mutants of human STING may be more advantageous than using non-human STING in the treatment of cancer, since the former may exhibit better interactions with signaling molecules in human cells. In addition, using non-human STING may result in undesirable immune responses.

[0029] In particular, the polynucleotide sequence derived from the first nucleic acid construct may include at least one modification of the polypeptide, which is amino acid substitution, amino acid insertion, amino acid deletion, C-terminal truncation or N-terminal truncation.In addition, the polynucleotide sequence that hybridizes to the complement of the polynucleotide sequence under stringent conditions is also included in the present invention.In addition, the fragment of at least 50 amino acid residues of DMXAA-sensitive human STING mutant is included in the present invention.In particular, the polypeptide encoded by any of the above polynucleotides has biological activity that responds to human non-responsive STING agonist as determined by in vitro agonist determination assay. Preferably, the promoter of the first construct may be a constitutive promoter, such as SV40, CMV0, UBC, EF1a, PGK, CAG, EFS, CBh, CBA, human β-actin, Ac5, polyhedrin, TEF1, GDS, CaMV35S, Ubi, H1 or U6. Preferably, the promoter is selected from the group consisting of SV40, CMV0, UBC, EF1a, PGK, CAG, EFS, CBh and CBA. The use of a constitutive promoter in the context of the present invention has the advantage of ensuring expression of the "activator" part of the pathway. In general, constitutive promoters show higher expression levels compared to inducible or tissue-specific promoters. However, the skilled artisan will recognize that cancer-specific promoters are also viable promoters of the first nucleic acid construct in the context of the present invention.

[0030] In another embodiment, the promoter of the first nucleotide construct is an inducible promoter, preferably selected from the group consisting of a telomerase responsive promoter, an IL-6 responsive promoter, a TGF-β inducible promoter, a VEGFR inducible promoter, an epidermal growth factor receptor inducible promoter, a HER2 / Neu inducible promoter, a MUC1 promoter, a BRCA1 promoter, a BRCA2 promoter, a LALBA promoter, a PSA promoter, an ERBB2 promoter, a CEA promoter, a vPAR promoter, and a thyroid transcription factor 1 (TTF1).

[0031] Furthermore, it may be advantageous within the scope of the present invention that the at least one immune-modulating gene or protein encoded by the sequence of the second nucleic acid construct is selected from the group consisting of the interleukin family, the chemokine family, the colony-stimulating factor family, the transforming growth factor family, the tumor necrosis factor family, any blocking peptide against an immune suppressor, any monoclonal antibody or any scFv or any nanobody reactive with an immune checkpoint protein, or an shRNA targeting an immune suppressor.A non-limiting list of immune modulating proteins includes interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-7, interleukin-8, interleukin-9, interleukin-11, single chain interleukin-12, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21, interleukin-22, interleukin-23, interleukin-24, interleukin-25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, interleukin-30, interleukin-31, interleukin-32, interleukin-33, interleukin-34, interleukin-35, interleukin-36, interleukin-37, interleukin-38, interleukin-39, interleukin-40, interleukin-41, interleukin-42, interleukin-43, interleukin-44, interleukin-45, interleukin-46, interleukin-47, interleukin-48, interleukin-49, interleukin-50, interleukin-51, interleukin-52, interleukin-53, interleukin-54, interleukin-55, interleukin-56, interleukin-57, interleukin-58, interleukin-59, interleukin-60, interleukin-61, interleukin-62, interleukin-63, interleukin-64, inter -sushi, Interleukin-16, Interleukin-17, Interleukin-18, Interleukin-19, Interleukin-20, Interleukin-21, Interleukin-22, Interleukin-23, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CC L18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6 , CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, granulocyte macroph phage colony stimulating factor, type I interferon, IFN-γ, TNF-α, FLT3-ligand, blocking peptides targeting TGF-β, blocking peptides targeting IL-10, blocking peptides targeting FoxP3, monoclonal antibodies or single chain variable fragments (scFv) or nanobodies neutralizing PD1, PDL1, CTLA4, CD137, TIM3, LAG3, and fragments or variants thereof. A non-limiting list of immune modulating genes consists of shRNAs targeting TGF-β, shRNAs targeting IL-10, or shRNAs targeting FoxP3.

[0032] In a preferred embodiment of the present invention, the at least one immune-modulating gene or protein encoded by the sequence of the second nucleotide construct is selected from the group consisting of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-12 (IL-12), single-chain interleukin-12 (scIL-12), interleukin-15 (IL-15), interleukin-15-sushi (IL-15-sushi), interleukin-21 (IL-21), interleukin-23 (IL-23), granulocyte colony-stimulating factor (GM-CSF), type I interferons (IFN-α and IFN-β), IFN-γ, TNF-α, CXCL10, FLT 3-ligand, a blocking peptide targeting TGF-β, a blocking peptide targeting IL-10, a blocking peptide targeting FoxP3, a monoclonal antibody or scFv or nanobody neutralizing PD1, PDL1, CTLA4, CD137, TIM3, LAG3, and fragments or variants thereof. One of skill in the art will recognize that any immunomodulating protein, or fragment or variant thereof, that stimulates the immune system to target cancer cells is a suitable immunomodulating protein according to the present invention.

[0033] There are immune-modulating proteins known in the art that are used in immunotherapy for cancer treatment. For example, IL-2 is a protein that has been used in immunotherapy for over 20 years in the treatment of cancer. However, only a small number of patients respond to IL-2-related immunotherapy, and it is known that the therapy causes severe side effects. IL-12 reprograms myeloid-derived suppressor cells (MDSCs) to become efficient antigen-presenting cells (APCs). In addition, IL-12 strongly stimulates T cells and NK cells, enhances the production of IFN-γ, and promotes a Th1-type response that can control not only the growth of the treated tumor, but also the growth of distant metastases (Lasek et al. Cancer Immunol. Immunother. 2014; 63:419 ff.). In addition, IL-12 has an anti-angiogenic effect, which can prevent tumor angiogenesis and limit tumor growth. IL-12 is a heterodimeric cytokine encoded by two separate genes. The use of a recombinant single chain IL-12 transgene encoding both subunits of IL-12 facilitates expression in recombinant expression systems. Thus, in a preferred embodiment, the immunomodulatory protein according to the invention is IL-12.

[0034] In one embodiment, the at least one immunomodulatory protein is a complex or fusion protein of IL-15 with the Sushi domain of the IL-15 receptor alpha chain to enhance the agonist activity of IL-15 via trans-presentation, such as that provided by the compound P22339 (Hu, Q., Ye, X., Qu, X. et al., Sci Rep 2018, 7675). Optionally, the at least one immunomodulatory protein may be selected from the group consisting of the α-subunit of IL-12, the β-subunit of IL-12, single chain IL-12 comprising the α and β subunits of IL-12. In the context of the present invention, single chain IL-12 (scIL-12) is defined as a fusion protein comprising the α and β subunits, wherein the peptide sequences are directly linked or separated by a linker peptide, wherein the linker peptide may have an amino acid length of 1 to 100. Preferably, the linker has an amino acid length of 4 to 60 amino acids, more preferably 8 to 40 amino acids, particularly preferably 12 to 25 amino acids. The amino acid length of the linker may be 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid. Furthermore, the at least one immunomodulatory protein may be a single chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10 (human or murine single chain IL-12), or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9 or 10, and having the biological activity of interleukin-12. The sequence identity of the polypeptide sequence with respect to SEQ ID NO: 9 or 10 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, a polypeptide that is considered to have a biological activity of interleukin-12 should have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of interleukin-12 as assessed in one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 9 or 10.

[0035] The biological activity of IL-12 can be estimated in vitro by the ability of the peptides to enhance IFN-γ production by cells that both express the IL-12 receptor and the corresponding signaling pathway, such as peripheral blood mononuclear cells or human macrophage cell lines. Alternatively, reporter cell lines may be used, such as the Promega bioluminescent IL-12 bioassay (Cat. Nos. JA2601, JA2605). In vivo IL-12 bioactivity can be estimated by its ability to increase IFN-γ serum levels. Additionally, the at least one immunomodulatory protein can be an IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO: 79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 79, and having the biological activity of an IFN-β. The sequence identity of the polypeptide sequence relative to SEQ ID NO: 79 can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, a polypeptide that is considered to have IFN-β biological activity should have at least 10%, preferably at least 20%, more preferably at least 30%, and even more preferably at least 50% of the IFN-β biological activity as assessed in one of the biological activity tests described below, compared to the biological activity of the polypeptide set forth in SEQ ID NO:79. The biological activity of IFN-β can be estimated by its ability to induce IFN type I responsive genes. For this purpose, human cell lines (such as HEK 293) can be incubated with the polypeptide and the expression of IFN responsive genes can be determined by RT-qPCR. Alternatively, IFN-reporter cells encoding luciferase under the control of an ISRE-containing promoter can be used to estimate the IFN-β biological activity of a polypeptide by measuring luciferase expression in a luminometer 3 hours after addition of the polypeptide to the cell culture. In vivo IFN-β bioactivity can be estimated by induction of IFN-responsive genes in peripheral blood mononuclear cells (RT-qPCR).

[0036] Further, the at least one immunomodulatory protein may be IL-15-sushi comprising or consisting of an amino acid sequence of SEQ ID NO: 80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 80, wherein the polypeptide has a biological activity of IL-15. The sequence identity of the polypeptide sequence to SEQ ID NO: 80 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, a polypeptide that is considered to have the biological activity of interleukin-15 should have at least 10%, preferably at least 20%, more preferably at least 30%, and even more preferably at least 50% of the biological activity of interleukin-15 as assessed in one of the biological activity tests described below, compared to the biological activity of the polypeptide set forth in SEQ ID NO:80. The biological activity of IL-15 can be estimated in vitro by a CTLL-2 cell proliferation assay. In vivo IL-15 bioactivity can be estimated by its ability to induce proliferation of NK cells and T cells using FACS analysis.

[0037] Additionally, the at least one immunomodulatory protein may be CXCL10 comprising or consisting of the amino acid sequence of SEQ ID NO: 81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 81, and having the biological activity of CXCL10. The sequence identity of the polypeptide sequence to SEQ ID NO: 81 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, a polypeptide that is considered to have the biological activity of CXCL10 should have at least 10%, preferably at least 20%, more preferably at least 30%, and even more preferably at least 50% of the biological activity of CXCL10 as assessed in one of the biological activity tests described below, compared to the biological activity of the polypeptide represented by SEQ ID NO: 81. CXCL10 is also known as IP-10. The biological activity of CXCL10 can be assessed by its ability to attract T lymphocytes and natural killer cells, either in vivo or in vitro migration assays.

[0038] In a preferred embodiment, the second nucleic acid construct of the invention comprises two or more sequences each encoding a different immunomodulatory protein, and the first immunomodulatory protein is a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9 or 10, and the polypeptide has the biological activity of interleukin-12. The second immunomodulatory protein may be an IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO:79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:79, and having the biological activity of IFN-β; alternatively, the second immunomodulatory protein may be a chemokine comprising or consisting of the amino acid sequence of SEQ ID NO:81, preferably CXCL10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:81, and having the biological activity of CXCL10.

[0039] Also, immunomodulatory proteins may be used in combination with others. Single chain IL-12 may be the first immunomodulatory protein and the second immunomodulatory protein may be selected from the group consisting of a peptide blocking TGF-β, a peptide blocking FoxP3, or a monoclonal antibody against the immune checkpoint inhibitors PD1 or PD-L1, or a nanobody against the immune checkpoint inhibitors PD1 or PD-L1.

[0040] It should be noted that the term "first and second immunomodulatory proteins" in the context of a second nucleic acid comprising two immunomodulatory proteins is not used to describe the order of the coding sequences on the second nucleic acid, but simply to illustrate two different immunomodulatory proteins. Thus, the coding sequence of the second immunomodulatory protein can be located upstream of the coding sequence of the first immunomodulatory protein on the nucleic acid, where upstream means that the coding sequence of the second immunomodulatory protein precedes the coding sequence of the first immunomodulatory protein in the 5' to 3' direction on the nucleic acid. A TGF-β blocking peptide is one that interferes with either TGF-β itself or the receptor that binds to TGF-β, such that at least a portion of TGF-β signaling is reduced or completely prevented. Examples of TGF-β blocking peptides are P17 and P144. A FoxP3 blocking peptide is one that interferes with either FoxP3 itself or a receptor that binds to FoxP3, such that at least a portion of FoxP3 signaling is reduced or completely prevented. An example of a FoxP3 blocking peptide is P60. In another preferred embodiment, the second nucleic acid construct is a bi- or polycistronic expression cassette, wherein the sequence encoding the immunomodulatory gene or protein is separated by a sequence encoding an IRES and / or the sequence encoding the immunomodulatory gene or protein is separated by a sequence encoding a 2A peptide, preferably a P2A peptide. The IRES sequence and the 2A peptide coding sequence are translational regulatory elements (TREs). In the present context, a protein or RNA with anti-cancer properties is any of the above molecules that are capable of slowing, stopping or reversing the progression of cancer. A bicistronic expression cassette is a genetic construct that contains two or more additional coding regions or genes within a single transcript. Typically, it contains two or more different sequences within a single transcript. To allow separate translation of two or more immunomodulatory proteins and / or transcription of immunomodulatory genes, a TRE is required to allow the proteins to be translated individually. One possible solution is to use an internal ribosome entry site (IRES), a specific sequence of RNA within a transcript that initiates translation at a non-AUG codon, bypassing the normal cap-dependent initiation process. This mechanism allows for the regulation of gene expression and the co-expression of multiple proteins from a single mRNA molecule. Another possible solution is 2A peptides, also known as 2A self-cleaving peptides. 2A peptides are a class of 18-22 amino acid peptides that can induce ribosome skipping during translation of proteins in cells. They share the core sequence motif of DxExNPGP (SEQ ID NO: 32) and are found in a wide range of viral families. They contribute to the production of polyproteins by preventing ribosomes from forming peptide bonds. Due to their short length, 2A peptides are ideal for use in viral vector systems with limited functionality, such as AAV. Another advantage of the peptides is that they allow equimolar expression of coding sequences separated by the peptide. Members of the 2A peptide family are T2A, P2A, E2A and F2A. The peptides can be flanked by linker peptides of different lengths, which can improve function. For example, proteins separated by an F2A element preceded by a linker domain consisting of the amino acid sequence GGSG (SEQ ID NO: 82) showed significant expression. Table 1: Examples of combinations of two immunomodulatory proteins separated by a translational regulatory element (TRE) in a bicistronic vector.

[0041] [Table 1] A - preferably a human single chain IL-12 as shown in SEQ ID NO:9. B - preferably human IFN-β as represented by SEQ ID NO:79. C- is preferably human IL-15-sushi represented by SEQ ID NO:80. D- is preferably human CXCL10 represented by SEQ ID NO:81.

[0042] Immunotherapy involving systemic availability of immunomodulatory proteins often results in undesirable side effects. However, an advantage of the present invention is that the recombinant expression system used herein induces local expression of said immunomodulatory proteins, thereby inducing a highly localized and targeted immune response. For example, intratumoral administration of the recombinant expression system according to the present invention induces an immune response in or near the tumor. Without intending to be bound by theory, it is believed that release of the immunomodulatory protein in or near the tumor allows T cells to infiltrate the tumor vicinity, recognizing tumor cells that normally evade immune system recognition, allowing the immune system to target and kill said cells. Thus, tumor progression is at least delayed, preferably stopped, and more preferably reversed. In a preferred embodiment, the immunomodulatory gene or protein of the invention is an immune stimulatory gene or protein, which is a gene or protein that stimulates the immune system, such as by directly stimulating the immune system (e.g., IL-12) or by inhibiting immune suppressive molecules (e.g., blocking peptides or shRNAs targeting TGF-β).

[0043] In a further possibility, the second STING responsive promoter may be selected from the group consisting of an IFN-β responsive promoter (preferably represented by SEQ ID NO: 25), an IFN-β minimal promoter (preferably represented by SEQ ID NO: 31), a CXCL9 responsive promoter (preferably represented by SEQ ID NO: 23), a CXCL10 responsive promoter (preferably represented by SEQ ID NO: 24), a STAT motif promoter (preferably represented by SEQ ID NO: 26), an IR SF 3 motif promoter (preferably represented by SEQ ID NO: 27 or SEQ ID NO: 28), an ISRE motif promoter (preferably represented by SEQ ID NO: 12 or SEQ ID NO: 13), an NFβ motif promoter (preferably represented by SEQ ID NO: 29 or 30) and a STAT 6 motif promoter. In a preferred embodiment, the STING responsive promoter comprises at least one interferon response element (ISRE). By using the STING-responsive promoter of the present invention to operably link the expression of immunomodulatory genes or proteins to the activity of STING, several advantages are achieved. First, the expression of immunomodulatory proteins can be controlled using human non-responsive STING agonists. Thus, rather than the protein being continuously expressed at high levels, the expression of immunomodulatory proteins can be tailored when treatment is required. Second, targeted local expression of immunomodulatory proteins can be achieved, avoiding potential side effects from systemic expression of proteins. The STING responsive promoter optionally comprises: (a) a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 11, or containing 1 to 10 copies, preferably 2 to 7 copies, more preferably 3 to 5 copies, and particularly preferably 4 copies of a sequence identical to SEQ ID NO: 11; (b) the nucleotide sequence according to (a), wherein the copies are separated by up to 40 nucleotides, preferably by 4 to 35 nucleotides, more preferably by 10 to 30 nucleotides, particularly preferably by 15 to 25 nucleotides; (c) a nucleotide sequence set forth in SEQ ID NO: 12, comprising four copies of an ISRE separated by 20 nucleotides; (d) a nucleotide sequence set forth in SEQ ID NO: 13, comprising 4 copies of the ISRE separated by 6 nucleotides; may be selected from the group consisting of:

[0044] The use of STING-responsive promoters in the context of the present invention ensures the targeted expression of immunomodulatory proteins is controlled. Moreover, the STING-responsive promoters have been shown to have additional advantages. As described in the examples of the present application, it has been shown that STING-responsive promoters can be induced by administering a human non-responsive STING agonist in the presence of target STING. Furthermore, it has been shown that the induction of the promoter is specific in that expression is limited in the absence of a selective STING agonist. In particular, a promoter with four copies of ISRE, such as that represented by SEQ ID NO: 12, is both highly inducible and highly specific.

[0045] Preferably, the nucleotide sequence of the STING-responsive promoter described in (a) and (b) is devoid of AP-1 or NFκB sites. Even more preferably, the nucleotide sequence of the STING-responsive promoter described in (a) and (b) does not contain further motifs that contribute to the inducibility, leakiness or expression efficiency of the promoter. In other words, the nucleotide sequence of the promoter described in (a) or (b) may comprise several nucleotides before, between and / or after SEQ ID NO: 11, such further nucleotides being selected (e.g. randomly) such that they do not form any transcriptional regulatory element. Particularly preferably, the nucleotide sequence of the STING-responsive promoter described in (a) and (b) consists of 1 to 10 copies, preferably 2 to 7 copies, more preferably 3 to 5 copies, particularly preferably 4 copies of a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO: 11 or is identical to SEQ ID NO: 11. Furthermore, the STING-responsive promoters described in (a)-(d) may already represent functional promoters, such that no other elements are required for the promoter to induce expression of a transduced gene to which it is operably linked.

[0046] In a further possibility, the second nucleic acid construct further comprises at least one sequence motif that inhibits transgenic expression of the immunomodulatory protein in tissues or cells not intended to express the immunomodulatory protein, preferably in the liver, and the sequence motif may preferably be a target sequence of a microRNA that is significantly enriched in the liver. It is known that some viral particles, especially AAV, are directed to hepatocytes. This means that when viral particles approach the blood circulation system, they preferentially transduce hepatocytes, which ultimately results in the expression of the transgene encoded in the liver. In addition, in the case of intratumoral injection of said viral particles, a portion of said particles has access to the blood circulation system, since tumors are highly vascularized. Therefore, in order to prevent undesired expression of the transgene encoded by the viral particles in the liver, it is appropriate to control its expression. Methods for achieving this purpose are known to those skilled in the art.

[0047] In general, a suitable method for limiting transgene expression to a specific tissue or a specific cell type is to use viral particles with different tropism for said tissue or cell type. A way to achieve this is by changing the serotype of AAV or by changing the capsid of AAV. Certain AAV serotypes are known in the art, each with different tropism. However, since the present invention is intended to target several different cancers, it will be necessary to optimize the serotype or capsid for each cancer or tumor. Nevertheless, this may be a suitable method for achieving tissue or cell type specific expression of transgene.

[0048] One skilled in the art may also use a DNA or RNA binding repressor to avoid undesired expression of the transgene encoded by the viral particle in tissues or cells that are not intended to express the transgene. Thus, the second nucleic acid construct may contain an operon that binds to the repressor. The repressor may be operably linked to a promoter that is active in hepatocytes, thus inducing expression of the repressor in said cells, thereby inhibiting expression of the transgene. Another possibility is to use RNA interference (RNAi). RNAi is characterized in that an RNA molecule reduces or inhibits the expression of a nucleic acid sequence with which it shares substantial or complete homology. Non-limiting examples of RNAi are small interfering RNA, small hairpin RNA, or microRNA (miR). To use RNAi to reduce or inhibit transgene expression in a specific tissue, it may be envisaged that, for example, an shRNA targeting the nucleotide sequence of said transgene can be operably linked to a tissue-specific promoter. When said shRNA is administered, it reduces or inhibits transgene expression in the tissue. Although this method is feasible in the context of the present invention, it may be disadvantageous in that an additional administration of said shRNA is required to limit transgene expression in the target tissue, for example the liver. An elegant way to circumvent this drawback is to operably link a miRNA target sequence to the nucleotide sequence of the transgene, so that expression of the transgene is reduced or inhibited in tissues in which the miRNA associated with said miRNA target sequence is present. Thus, a preferred embodiment of the present invention comprises that the sequence motif is a target sequence of a microRNA that is significantly enriched in the liver, so that a single administration of the nucleic acid construct is sufficient to avoid transgene expression in the liver, while transcription in other tissues or organs is not impaired. In some cases, it is considered that the liver-enriched microRNA is selected from the group consisting of: miR-122, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c, let7f. Furthermore, it may be advantageous that the liver-enriched microRNA is miR-122. The advantage of miR-122 is that it is highly expressed in the liver, and importantly, it is specifically expressed in the liver. Therefore, the selection of miR-122 target sequence ensures a significant reduction in transduced gene in the liver, while not compromising expression in other tissues. Furthermore, miR-122 is downregulated in most liver tumors, and as a result, it has been shown that the recombinant expression system according to the present invention is also suitable for treating liver cancer.

[0049] According to a preferred embodiment of the invention, each miR-122 target sequence is a miR-122 target sequence having 90% or at least 95% sequence identity with SEQ ID NO: 14 or is identical to SEQ ID NO: 14. Preferably, the target sequence comprises 1-12, preferably 3-10, more preferably 4-8, particularly preferably 5 copies of the miR-122 target sequence as expressed in the previous sentence. With increasing copy number of the miR-122 target sequence, miR-mediated activity was lessened, but no significant improvement was observed beyond the disclosed number of 5 copies. In a preferred embodiment, the copies are spaced apart by 1-40 nucleotides, preferably 1-30 nucleotides, more preferably 2-20 nucleotides, particularly preferably 2-8 nucleotides. The nucleotide sequence preferably comprises five copies of the miR-122 target sequence represented by SEQ ID NO: 15. As described in the Examples of the present application, it has been shown that the nucleotide sequence represented by SEQ ID NO: 15 substantially inhibits transgene expression in the liver, such that the transgene was undetectable at plasma levels using the method described in Example 4.

[0050] In a further possibility, the second nucleic acid construct may further comprise a transduced gene encoding a thymidine kinase, preferably the thymidine kinase set forth in SEQ ID NO: 16. The nucleotide sequence encoding the thymidine kinase may be linked to the nucleotide sequence of an immunomodulatory gene or protein and separated by a 2A self-cleaving peptide (so-called 2A peptide). 2A peptides constitute a class of peptides of 18-22 amino acids in length that can induce ribosome skipping during translation of proteins in cells. The peptides share a core sequence motif of "DXEXNPGP" and are found in a wide range of virus families. They facilitate the production of polyproteins without forming ribosomal peptide bonds. The skilled artisan will recognize that the use of 2A peptides allows for simultaneous expression of an immunomodulatory protein and a thymidine kinase. Co-expression of thymidine kinase is advantageous in that it allows assessment of tumor transduction by PET imaging and, in the case of potential IL-12-mediated toxicity, the administration of thymidine kinase substrates (e.g., acyclovir, penciclovir, famciclovir or ganciclovir) to eliminate cells expressing the cytokine.

[0051] The first and / or second nucleic acid constructs may further comprise a Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE), which is known to enhance transgene expression and is therefore most effective when placed downstream of the transgene (i.e., the target STING or immune-modulating gene or protein) proximal to the polyadenylation signal. Conveniently, the first and / or second nucleic acid construct further comprises a polyadenylation signal located 3' to the target STING or immune modulatory gene or protein. Optimizing the length of the poly(A) tail can stabilize the mRNA and enhance subsequent protein translation.

[0052] Those skilled in the art will also be aware of other means for enhancing protein translation, e.g. (a) sequence modification to optimize G / C content and / or over-stabilize secondary structure codons, or (b) alternating 5' caps by adding synthetic or natural 5' cap analogs, such as m7GpppG or starter trimers, such as "CleanCap", or stabilization by capping enzymes; or (c) Addition of regulatory elements in the following cases: (c1) optimization in the 5' untranslated region (UTR), for example via appropriate cis-regulatory sequences, or modification of the length and sequence to avoid overly stable structures, or (c2) Addition, via sequence modification, of stable elements or repeated motifs in the 3'-UTR that have proven effective in other mRNAs or synthetic or naturally occurring sequences are known.

[0053] In a particular embodiment, the second nucleic acid construct of the recombinant expression system consists of a nucleic acid construct represented in the table below, where the different elements are indicated in the 5' to 3' direction as: promoter, first open reading frame (ORF1), first translational regulatory element (TRE1), second open reading frame (ORF2), second translational regulatory element (TRE2) and target sequence of microRNA (microRNA element). Table 2: Second nucleic acid construct embodiments of the expression system

[0054] [Table 2] A - an ISRE, preferably four copies, preferably represented by SEQ ID NO:12. B - preferably a human single chain IL-12 as shown in SEQ ID NO:9. C- is preferably a murine single chain IL-12 as shown in SEQ ID NO:10. D- is preferably an HSV thymidine kinase represented by SEQ ID NO:16. E - preferably 5 copies of the miR-122 target sequence represented by SEQ ID NO:15.

[0055] Another aspect of the present invention is an expression vector encoding the first and second nucleic acid constructs according to the present invention. Using an expression vector comprising the first and second nucleic acid constructs has certain advantages. First, the expression vector is specifically designed to transduce and express the encoded nucleotide sequence into a host cell. Second, the expression vector is a plasmid that is highly stable and protected from degradation. Third, the expression vector can be considered for gene delivery using different methods. Non-limiting examples of the expression vector and preferred embodiments of the present invention are viral vectors, more preferably viral vectors selected from the group consisting of adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, vaccinia virus vectors, or herpes simplex virus vectors. In particular, viral vectors have the advantages of expression vectors, such as general safety, low toxicity, and stability.

[0056] It is envisaged that the first and second nucleic acid constructs of the recombinant expression system according to the invention are comprised within a single expression vector, which has the advantage that a single administration of said expression vector is sufficient to express the recombinant expression system according to the invention in a patient.

[0057] Another aspect of the present invention is the combination of a first and a second expression vector, the first vector encoding a first nucleic acid construct and the second vector encoding a second nucleic acid construct according to the present invention. This has the advantage that the size of each nucleic acid construct is not as limited as when both nucleic acid constructs are combined in one vector. Size limitation is a frequent problem with viral vectors. For example, the size of adeno-associated virus (AAV) is limited to about 5 kb, but it must be taken into account that ITRs, promoters and other elements are not included in the 5 kb. Thus, in another aspect, the recombinant expression system of the present invention relates to a combination of a first expression vector encoding a first nucleic acid construct and a second expression vector encoding a second nucleic acid construct. It is also advantageous that the first and / or second nucleic acid construct of the expression vector further comprises 5'ITR and 3'ITR sequences, preferably the AAV ITRs as represented by SEQ ID NO: 17. It may be advantageous that the expression vector is an AAV vector, preferably of serotype AAV1, AAV3, AAV6, AAV8, AAV9, AAV2, AAV5, AAVrh.10, or any gain-of-function mutant of AAVrh.10, with AAV2 being the most preferred. AAV is commonly used in biotechnology and is therefore well characterized. The advantage of using AAV vectors is that they are generally considered safe, have low toxicity, and are stable. Moreover, AAV has already been approved by certain government agencies around the world for use in humans.

[0058] It is also advantageous that the first and / or second nucleic acid construct of the expression vector further comprises 5'ITR, psi packaging signal and 3'ITR sequences, preferably 5'ITR, psi packaging signal and 3'ITR of adenovirus (Ad). The expression vector may be advantageously an adenovirus vector, preferably a high-capacity adenovirus vector. Adenoviruses are commonly used in biotechnology and therefore well characterized. The advantage of adenovirus vectors is that they have a higher size limit compared to AAV vectors. Moreover, adenoviruses have already been approved by certain government agencies around the world for use in humans. It is also advantageous if the expression vector is a lentiviral, vaccinia virus or herpes simplex virus vector, which are commonly used in biotechnology and therefore well characterized.

[0059] It is also contemplated that the first and / or second nucleic acid of the invention can be delivered using nanoparticles. Nanoparticles as gene delivery systems are known in the art and have the advantage that they can be tailored in terms of size, shape and / or surface, etc., allowing for modulation of their biological behavior. For effective delivery and uptake by cells, the nucleic acid constructs may be prepared using a method comprising administering to the subject a nucleic acid molecule selected from the group consisting of distearoylphosphatidylglycerol, distearoylphosphatidylcholine, (DLPC) 1,2-dilauroyl-sn-glycero-3-phosphocholine, (DMPC) 1,2-dimyristoyl-sn-glycero-3-phosphocholine, (DPPC) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, (DSPC) 1,2-distearoyl-sn-glycero-3-phosphocholine, (DOPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine, (DMPE) 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, (DPPE) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, (DOPE) 1,2-dioleoyl-sn-glycero-3 Nanoparticles, preferably packaged in liposomes, made of pharma- ceutically acceptable carriers composed of physiologically compatible lipids, proteins, polysaccharides or synthetic polymers in various ratios, such as standard lipids compatible with biological membranes selected from the group consisting of phospholipids such as phosphoethanolamine, (DOPS) 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, (DPPS) 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, (DMPS) 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, (DOPG) 2,3-dimyristoyl-sn-glycero-1-phosphocholine, and their respective enantiomers, with distearoylphosphatidylglycerol and distearoylphosphatidylcholine being the most preferred. Another suitable example is the combination of preferably cholesterol and cardiolipin.Alternatively, the nanoparticles may be dendrimer nanoparticles, protamine liposomes, cationic polymers, cationic polymer liposomes, polysaccharide particles, cationic lipid particles, cationic lipid cholesterol nanoparticles, cationic lipid cholesterol PEG nanoparticles, ionizable lipids, preferably lipids positively charged under physiological conditions, including amino lipids such as MC3 (DLin-MC3-DMA), C12-200, cKK-E12, ZA3-Ep10, 200-Oi10; (SM-102) heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid, (ALC 0159) N,N-dimyristylamide of 2-hydroxyacetic acid, O-pegylated to PEG chains of approximately 2 kilodaltons, (ALC 0315)(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate), lipofectamine, Trans-IT, InstantFECT, (DOTAP)1,2-dioleoyl-3-trimethylammonium. Other components may include peptides and polypeptides (e.g., DP7-C, cholesterol-modified cationic peptide DP7 (VQWRIRVAVIRK, SEQ ID NO:85)), polymers (e.g., polyethylene glycol preparations composed of polymer molecules of various molecular sizes that may be non-covalently bound or covalently linked to lipids in the formulation).

[0060] Another aspect of the invention is a viral particle comprising the above-mentioned viral vector comprising the first and second nucleic acid constructs of the invention, which has the advantage that a single administration of said viral particle is sufficient to express the recombinant expression system according to the invention in a patient.

[0061] Yet another aspect of the present invention is the combination of a first viral particle with a second viral particle, the first viral particle comprising a viral vector encoding a first nucleic acid construct according to the present invention, and the second viral particle comprising a viral vector encoding a second nucleic acid construct according to the present invention. This has the advantage that the size of each nucleotide sequence is not as limited as when both nucleic acid constructs are combined in one viral vector in a single viral particle. Size limitations are a frequent problem with viral vectors. For example, the size of adeno-associated virus (AAV) is limited to about 5 kb, but it must be taken into account that ITRs, promoters and other elements are not included in the 5 kb. The viral particle is preferably an AAV or adenoviral particle, more preferably an AAV viral particle. As described hereinabove, Ad and AAV exhibit several advantages over other viral particles, such as that they are well characterized, frequently used, and approved for treatment in humans. The viruses exhibit additional advantages that make them particularly useful in gene therapy, such as the ability to transduce dividing as well as non-dividing cells and not integrating into the host genome. In certain embodiments, the combination of the first and second expression vectors, or the combination of the first and second viral particles, is provided as a kit-of-parts.

[0062] Another aspect of the invention is a pharmaceutical composition comprising a pharma- ceutical acceptable excipient together with a recombinant expression system, expression vector(s) or viral particle(s) according to the invention. The pharmaceutical composition has the advantage of being "ready to use", in that it can be administered directly to a patient for treatment. Moreover, the pharmaceutical composition is tailored to optimize the stability and availability of the components according to the therapeutic needs, while avoiding undesirable side effects for the patient.

[0063] In another aspect of the invention, the recombinant expression system, the expression vector, the viral particle or the pharmaceutical composition, respectively, according to the invention may be or relates to use in therapy, preferably immunotherapy. In a preferred embodiment, the immunotherapy is an immunotherapy of a proliferative disorder, more preferably an immunotherapy of a cancer, even more preferably an immunotherapy of a solid cancer. Although it is considered that non-solid cancers can also be treated by the immunotherapy of the present invention, the use of solid cancer therapy has the advantage that the cancer is locally delimited and therefore accessible by local administration. Thus, the recombinant expression system, expression vector, viral particle or pharmaceutical composition according to the present invention can be administered locally to the tumor by intratumoral administration. This has the advantage that a local targeted immune response is induced to slow, stop or reverse tumor progression. Furthermore, the present invention takes measures to avoid systemic expression, for example by using miR targeting sequences, whereas local administration further reduces undesired systemic expression. As mentioned above, cancer immunotherapy has several limitations, such as i) insufficient tumor targeting, ii) weak tumor cell killing function, iii) laborious and expensive, iv) tumor immune evasion, v) overstimulation of the immune system (<1 μg / mL) which may cause severe side effects, and v) reduced immunogenicity (Non-Patent Document 2). The immunotherapy according to the present invention is an extended immunotherapy that also combines gene therapy. Thus, the immunotherapy according to the present invention is not simply immunotherapy per se, but a combination of immunotherapy and gene therapy (hereinafter referred to as "immunogenic therapy") for effectively treating proliferative disorders such as cancer. Cancer further includes hematopoietic tumors of the lymphatic system, including bladder cancer (including accelerated and metastatic bladder cancer), breast cancer, colon cancer (including colorectal cancer), kidney cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer and lung adenocarcinoma), ovarian cancer, prostate cancer, testicular cancer, genitourinary tract cancer, lymphatic system cancer, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), esophageal cancer, stomach cancer, gallbladder cancer, cervical cancer, thyroid cancer and skin cancer (including squamous cell carcinoma); tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors, including melanoma, xeroderma pigmentosum, keratinizing acanthoma, dermoid carcinoma, follicular thyroid carcinoma, and teratocarcinoma; The cancer may be selected from the group consisting of melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian carcinoma, liver cancer, colorectal cancer, endometrial cancer, renal cancer, prostate carcinoma, thyroid carcinoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, gastric cancer, bladder cancer, urothelial carcinoma, hepatocellular carcinoma, embryonal tumor, liver cancer, breast carcinoma, colon carcinoma, and head and neck cancer, gastric carcinoma, germ cell tumor, osteosarcoma, bone tumor, adult malignant fibrous histiocytoma of bone; childhood, malignant fibrous histiocytoma of bone, sarcoma, pediatric sarcoma, Sino-nasal natural killer, neoplasm, plasmacytoma, carcinoid tumor, neuroendocrine tumor, cholangiocarcinoma, and Merkel cell tumor.

[0064] In another preferred embodiment, the recombinant expression system, expression vector, viral particle or pharmaceutical composition of the invention is used in a combination therapy with at least one other therapy selected from the group consisting of a therapy comprising at least one checkpoint inhibitor, a therapy comprising at least one adoptive cell therapy, a therapy comprising at least one cancer vaccine, a therapy comprising at least one exogenous cytokine and / or a therapy comprising radiation therapy. The inhibitor of the treatment comprising at least one checkpoint inhibitor may be selected from the group consisting of inhibitors of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR, more preferably from the group consisting of inhibitors of PD-1, PD-L1, PD-L2, or CTLA-4. Immune checkpoints are molecules expressed on T cells or cells that interact with T cells that regulate the immune response and help prevent excessive or prolonged immune activation. However, by utilizing the checkpoint proteins, cancer cells can avoid detection by the immune system. By blocking the checkpoint proteins, immune checkpoint inhibitors release the brakes on the immune response, allowing the immune system to attack and destroy the cancer cells. The use of immune checkpoint inhibitors as an additional therapy is particularly advantageous in the context of the present invention, since the expression system of the present invention results in the infiltration of T cells into the tumor environment. As such T cells may be functionally compromised due to immune checkpoints, measures to overcome this hurdle could greatly benefit therapeutic outcome.

[0065] The therapy comprising at least one adoptive cell therapy can be adoptive NK cell therapy, adoptive dendritic cell therapy, or adoptive T cell therapy, and the adoptive T cell therapy is preferably CAR-T cell therapy or tumor infiltrating leukocyte (TIL) therapy. In some cases, tumors silence the expression of MHC class I molecules and / or IFN-γ signaling molecules as a means of avoiding T cell immunity. Using adoptive cell therapy can avoid this problem because the adoptive cells can normally recognize and target tumor cells. Therefore, synergistic effects can be expected in combination approaches. Additionally, the use of the expression system of the present invention in combination with a cancer vaccine or exogenous cytokines may produce a synergistic effect, as the recruited T cells are primed against cancer cells by the cancer vaccine or exogenous cytokines.

[0066] In a preferred embodiment of the invention, the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymic cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, urothelial cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasma cell neoplasms, cholangiocarcinoma, and neuroendocrine or carcinoid tumors. In yet another aspect the present invention relates to a recombinant expression system, an expression vector, a viral particle or a pharmaceutical composition of the invention for use in immunotherapy, said immunotherapy comprising the steps of: (a) administering a recombinant expression system according to the invention, an expression vector according to the invention, a viral particle according to the invention, or a pharmaceutical composition according to the invention to a patient suffering from a proliferative disorder, preferably cancer, to obtain therapeutically effective transgenic expression of target STING and immunomodulatory proteins, preferably wherein the administration is intratumoral or systemic; (b) optionally, prior to and / or simultaneously with administration according to (a), treating the patient with radiation therapy, preferably radiation therapy directed to the tissue or organ intended to express the transduced gene(s) from (a); and / or (c) following administration according to (a), treating the patient with a STING agonist that recognizes the transgenically expressed target STING, but does not recognize human STING and its naturally occurring variants, and / or with radiation therapy, preferably radiation therapy directed to the tissue or organ intended to express the transduced gene(s) from (a). It is worth noting that (c) above provides three possible different treatment options. For example, only STING agonists that recognize transgenically expressed target STING but do not recognize human STING and its natural variants (human non-reactive STING agonists) may be applied. Another possibility is to treat the patient with radiation therapy alone. Finally, these can also be used in combination. In other embodiments, the patient is treated prior to, concurrently with, and / or after administration with (a) with at least one other therapy selected from the group consisting of a therapy comprising at least one checkpoint inhibitor, a therapy comprising at least one adoptive cell therapy, a therapy comprising at least one cancer vaccine, and / or a therapy comprising at least one exogenous cytokine. In a preferred embodiment, the treatment with at least one other therapy is administered after administration with (a).

[0067] In a similar aspect, the present invention provides a method of treating a proliferative disorder, preferably cancer, in a patient, comprising administering to the patient: (a) administering a recombinant expression system according to the invention, an expression vector according to the invention, a viral particle according to the invention, or a pharmaceutical composition according to the invention to a patient suffering from a proliferative disorder, preferably cancer, to obtain therapeutically effective transgenic expression of target STING and immunomodulatory proteins, preferably wherein the administration is intratumoral or systemic; (b) optionally treating the patient with radiation therapy prior to and / or simultaneously with administration of (a), preferably radiation therapy directed to the tissue or organ intended to express the transduced gene(s) from (a); and / or (c) following administration according to (a), treating the patient with a STING agonist that recognizes the transgenically expressed target STING, but does not recognize human STING and its naturally occurring variants, and / or with radiation therapy, preferably radiation therapy directed to the tissue or organ intended to express the transduced gene(s) from (a). In other embodiments, the patient is treated prior to, concurrently with, and / or after administration with (a) with at least one other therapy selected from the group consisting of a therapy comprising at least one checkpoint inhibitor, a therapy comprising at least one adoptive cell therapy, a therapy comprising at least one cancer vaccine, and / or a therapy comprising at least one exogenous cytokine. In a preferred embodiment, the treatment with at least one other therapy is administered after administration with (a). In particular, the embodiments described below apply to both the recombinant expression systems, expression vectors, viral particles, or pharmaceutical compositions of the invention for use in immunotherapy and in methods of treating proliferative disorders.

[0068] The use for immunotherapy or immune cell therapy is considered. The compounds according to the present invention for treating proliferative disorders can be used in combination with other anti-cancer therapies, such as chemotherapy, hormonal therapy, radiation therapy, photodynamic therapy, stem cell transplantation, surgery, targeted therapy, hyperthermia, or other immunotherapy, which are known in the art. It may be advantageous to direct radiation therapy to the tumor that has been administered intratumorally in (a) above. It is also advantageous if in c) the STING agonist is selected from the group consisting of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), 7-bromo-DMXAA, 7-iodo-DMXAA, 7-hydroxy-DMXAA, 7-formyl-DMXAA, 7-hydroxymethyl-DMXAA, 7-(2-hydroxyethyl)-DMXAA, flavone-8-acetic acid (FAA), 2,7-bis(2-dimethylamino ethoxy)fluoren-9-one (tilorone), and 10-carboxymethyl-9-acridan (CMA). Preferably, the STING agonist is DMXAA. DMXAA has already been proven in clinical trials to be a molecule with excellent safety and no serious side effects. Furthermore, in the present invention, DMXAA activates the target STING from (a) above without activating human wild-type STING or its natural mutants, making the treatment generally safe and avoiding systemic STING activation.

[0069] It is also contemplated that radiation therapy of 100 Gy or less may be used in (b) and / or (c) in some cases. Any dose of ionizing radiation less than 100 Gy, 96 Gy, 92 Gy, 88 Gy, 84 Gy, 80 Gy, 76 Gy, 72 Gy, 68 Gy, 64 Gy, 60 Gy, 56 Gy, 52 Gy, 48 Gy, or 44 Gy may be used. It is also contemplated that low dose radiation therapy of 40 Gy or less, preferably 10 Gy or less, more preferably 8 Gy or less may be used in (b) and / or (c). Low dose radiation therapy can be any radiation therapy using a dose of ionizing radiation less than 40 Gy, 38 Gy, 36 Gy, 34 Gy, 32 Gy, 30 Gy, 28 Gy, 26 Gy, 24 Gy, 22 Gy, 20 Gy, 19 Gy, 18 Gy, 17 Gy, 16 Gy, 15 Gy, 14 Gy, 13 Gy, 12 Gy, 11 Gy, 10 Gy, 9 Gy, 8 Gy, 7 Gy, 6 Gy, 5 Gy, 4 Gy, 3 Gy, 2.5 Gy, 2.0 Gy, 1.5 Gy, 1 Gy, 0.5 Gy, 0.25 Gy, 0.2 Gy, 0.15 Gy, 0.1 Gy, 0.05 Gy, or 0.01 Gy. The advantage of using low-dose radiotherapy is that it is better tolerated by patients, in that they experience little or no side effects that are common with radiotherapy.Dose as used herein refers to total dose, meaning that the total dose for treatment in (b) or (c) above does not exceed the dose claimed.Thus, the dose of 40Gy applied in (b) above can be achieved by a single dose or fractionated dose of 40Gy, for example, 4 fractions of 10Gy given over a period of several weeks or even months. In another embodiment, the radiation therapy in (c) comprises several further radiation treatments, the cumulative dose of the further radiation treatments being added up to less than 100 Gy, preferably less than 40 Gy, more preferably less than 25 Gy. The cumulative dose of the further radiation treatments is 100 Gy, 96 Gy, 92 Gy, 88 Gy, 84 Gy, 80 Gy, 76 Gy, 72 Gy, 68 Gy, 64 Gy, 60 Gy, 56 Gy, 52 Gy, 48 Gy, 44 Gy, 40 Gy, 38 Gy, 36 Gy, 34 Gy, 32 Gy, 30 Gy, 28 Gy, 26 Gy, 24 Gy, 22 Gy, 20 Gy, It may be less than 19 Gy, 18 Gy, 17 Gy, 16 Gy, 15 Gy, 14 Gy, 13 Gy, 12 Gy, 11 Gy, 10 Gy, 9 Gy, 8 Gy, 7 Gy, 6 Gy, 5 Gy, 4 Gy, 3 Gy, 2.5 Gy, 2.0 Gy, 1.5 Gy, 1 Gy, 0.5 Gy, 0.25 Gy, 0.2 Gy, 0.15 Gy, 0.1 Gy or 0.05 Gy.

[0070] In particular, radiotherapy can be applied externally or internally. Those skilled in the art are aware of several means for providing external radiotherapy. For example, external radiotherapy can be provided by gamma rays, electron beams or proton accelerators suitable for therapeutic purposes. However, for some tumors, it may be advantageous to use internal radiotherapy. Internal radiotherapy can be provided by administration of a radioisotope in a pharmacologically acceptable form and dose for therapeutic purposes. An embodiment of internal radiotherapy can be brachytherapy or non-localized radiotherapy. Brachytherapy physically restrains the radioactive deposits so that they essentially cannot move from the deposited location. Radioactive deposits can be placed in the stroma (interstitial radiotherapy), for example used in many prostate tumors and some head and neck tumors, or they can be placed in the cavity (intracavity radiotherapy), for example used in some uterine, bronchial or esophageal tumors. Non-localized radiation therapy may be a partially diffuse local administration, such as selective internal radiation therapy (SIRT), in which a suspension of material is injected into a specific artery or vein with the purpose of settling near the injection site rather than undergoing widespread distribution in the body, or it may be a fully systemic approach, such as the use of biological agents, such as soluble chemicals synthesized with radioisotopes that find their point of action by affinity, or radiolabeled antibodies, to better deliver radiation to the target tumor. A classic example of the latter is iodine-131. Although different isotopes may be used for different treatment paradigms, isotopes useful in the present invention may preferably be selected from the list consisting of yttrium-90, iodine-131, lutetium-177, iridium-192, astatine-211, bismuth-213, actinium-225, samarium-153, radium-223, phosphorus-32, strontium-89, erbium-169, holmium-166, terbium-161, gallium-68, rhenium-188 or rhenium-186.

[0071] As the inventors have found, there is synergy between the canonical STING pathway activated by DMXAA and the non-canonical STING pathway activated by radiation therapy in vitro (see Example 9) and in vivo (see Example 10), supporting the beneficial combination of STING agonist therapy and radiation therapy in (c) above. A further advantage of the second nucleic acid construct is that it can be applied systemically and the expression of at least one immunomodulatory gene or protein can be induced by radiation, thus providing a synergistic effect in that irradiation promotes viral transduction in irradiated cells and also induces the expression of at least one immunomodulatory gene or protein. The first effect above relates to a key concept of the present invention, namely, that radiation therapy marks tumors for transgene expression from genetic vectors such as AAV vectors. This fact has significant clinical application, as the data in this application show that tumor irradiation converts tumors into targets for transgene (e.g., IL-12) expression upon parenteral (e.g., intravenous) administration of AAV vectors. Using this approach, the inventors were able to cure subcutaneous tumors even when the vector was administered intravenously. After intravenous administration of AAV.ISREx4.IL12miR122T to mice bearing subcutaneous tumors, the tumors were eliminated when irradiated but not when not irradiated. In the present experiment, tumor irradiation alone (with intravenous administration of a control virus encoding luciferase) could not induce tumor regression.

[0072] In another aspect, the invention includes a recombinant expression system consisting of a nucleic acid construct comprising sequences encoding at least two immunomodulatory proteins, said sequences being operably linked to a STING-responsive or radiation-inducible promoter and at least one sequence motif that inhibits transgenic expression of the immunomodulatory proteins in tissues or cells not intended to express the proteins. As the present inventors found, an expression system consisting of a nucleic acid containing at least two immunomodulatory proteins under the control of a STING-responsive promoter induces upregulation of the immunomodulatory proteins upon radiation even in the absence of targeted STING (Example 15). Moreover, this expression system alone was not expressed outside the site (Example 16) and had a significant antitumor effect (Example 17). This expression system offers many advantages for cancer immunotherapy while avoiding some of the drawbacks of said therapy. Thus, expression of immunomodulatory peptides, particularly cytokines such as interleukins or chemokines, in tumors can stimulate the immune system to target cancer cells in the tumor. The preferred intratumoral administration of the expression system results in highly localized cytokine expression, thus avoiding the drawbacks and toxicities of systemic cytokine therapies known in the art.

[0073] Furthermore, the present inventors have found that the nucleic acid of this recombinant expression system upregulates the immunomodulatory transgene even in the presence of radiation alone, thus allowing the expression of the immunomodulatory transgene to be regulated by the administration of radiation therapy. Furthermore, the inventors found that this recombinant expression system is non-toxic and has minimal side effects in vivo. Remarkably, the inventors found that this recombinant expression system, when administered in vivo, reduced tumors and ultimately led to complete remission. This recombinant expression system generates a specific and essential immunological pathway in transfected cancer cells that utilizes additional endogenous pathway components. The nucleic acid construct of the expression system is an artificial gene construct that includes sequences encoding at least two immunomodulatory proteins, which are operably linked to a STING-responsive promoter or a radiation-inducible promoter. The immunomodulatory proteins activate both innate and adaptive immune responses to specifically initiate a cancer immune response. Alternatively, a radiation-inducible promoter may be provided. As used herein, a radiation-inducible promoter is a promoter that induces the expression of a therapeutic gene sequence upon irradiation of a cell. The promoter may be natural, synthetic, or artificially created, selected from recombinant products of natural promoters. For example, the promoter may have the following sequence: (a) a eukaryotic radiation-inducible sequence CC(A / T)(6)GG (SEQ ID NO:87), such as that of the early growth response 1 (Egr1) promoter or pE9 promoter, also known as the CArG element or [CC(A+Trich)6 GG], preferably the sequence has 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:86 or is identical to SEQ ID NO:86 (Egr1 promoter), or the sequence contains at least two sequences according to SEQ ID NO:87, which may be separated by additional nucleotides; (b) low frequency CCAAT box sequences, such as those of the survivin promoter, preferably having 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 88 or a sequence identical to SEQ ID NO: 88 (CCAAT-rich domain of the survivin promoter); (c) cyclin-dependent kinase inhibitor 1 (also known as CDKN1A or p21) promoter; (d) a late herpes simplex virus 1 promoter, such as the glycoprotein C promoter or the US11 promoter; (e) Combinations or artificial variants of the above may include at least one of, individually or repeatedly.

[0074] Sequence motifs that inhibit transgenic expression of immunomodulatory proteins in tissues or cells not intended to express the protein may be used to restrict expression of the protein from different tissues, tissue regions or cell populations. Furthermore, it may be advantageous within the scope of the present invention that the at least two immunomodulatory proteins encoded by the sequence of the nucleic acid construct are selected from the group consisting of the interleukin family, the chemokine family, the colony stimulating factor family, the transforming growth factor family, the tumor necrosis factor family, any blocking peptide against an immunosuppressant, any monoclonal antibody or any scFv or any nanobody reactive with an immune checkpoint protein, or an shRNA targeting an immunosuppressant. A non-limiting list of immune modulating proteins includes interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-7, interleukin-8, interleukin-9, interleukin-11, single chain interleukin-12, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-15-sushi, interleukin-16, interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21, interleukin-22, interleukin-23, CCL1, CCL2, CCL3, CCL4, CCCL5, CCCL6, CCCL7, CCCL8, CCCL9, CCCL10, CCCL11, CCCL12, CCCL13, CCCL14, CCCL15, CCCL16, CCCL17, CCCL18, CCCL19, CCCL19, CCCL110, CCCL1111, CCCL121, CCCL132, CCCL14, CCCL15, CCCL162, CCCL17, CCCL18, CCCL19, CCCL19, CCCL19, CCCL19, CCCL19, CCCL102, CCCL103, CCCL104, CCCL105, CCCL106, CCCL107, CCCL108, CCCL109, CCCL1109, CCCL1111, CCCL112, CCCL1133, CCCL114, CCCL115, CCCL115, CCCL1163, CCCL117, CCCL118, CCCL119, CCCL119, CCCL124, CCCL125, CCCL126, CCCL127, CCCL1 L5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, TIM3, LAG3, and fragments or variants thereof. In a preferred embodiment of the invention, the at least two immunomodulatory proteins encoded by the sequences are selected from the group consisting of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-12 (IL-12), single chain interleukin-12 (scIL-12), interleukin-15 (IL-15), interleukin-15-sushi (IL-15-sushi), interleukin-21 (IL-21), interleukin-23 (IL-23), granulocyte colony stimulating factor (GM-CSF), type I interferons (IFN-α and IFN-β), IFN-γ, TNF-α, CXCL10, FLT 3-ligand, a blocking peptide targeting TGF-β, a blocking peptide targeting IL-10, a blocking peptide targeting FoxP3, a monoclonal antibody or scFv or nanobody neutralizing PD1, PDL1, CTLA4, CD137, TIM3, LAG3, and fragments or variants thereof. One of skill in the art will appreciate that any immunomodulatory protein, or fragment or variant thereof, that stimulates the immune system to target cancer cells is a suitable immunomodulatory protein according to the present invention. There are several immune-modulating proteins known in the art that are used in immunotherapy for cancer treatment. For example, IL-2 is a protein that has been used in immunotherapy for over 20 years in the treatment of cancer. However, only a small number of patients respond to IL-2-related immunotherapy, and the therapy is known to have severe side effects. IL-12 reprograms myeloid-derived suppressor cells (MDSCs) to become efficient antigen-presenting cells (APCs). In addition, IL-12 strongly stimulates T cells and NK cells, increases IFN-γ production, and promotes a Th1-type response that can control not only the growth of the treated tumor, but also the growth of distant metastases (Lasek et al. Cancer Immunol. Immunother. 2014; 63:419 ff.). In addition, IL-12 has antiangiogenic effects, which can impair tumor vascularization and limit tumor growth. IL-12 is a heterodimeric cytokine encoded by two separate genes. To facilitate expression in recombinant expression systems, a recombinant single chain IL-12 transgene encoding both subunits of IL-12 may be used. Thus, in a preferred embodiment, one of the immunomodulatory proteins according to this aspect of the invention is IL-12.

[0075] In one embodiment, one of the immunomodulatory proteins is a complex or fusion protein of IL-15 with the Sushi domain of the IL-15 receptor alpha chain to enhance the agonist activity of IL-15 via transpresentation as provided by compound P22339 (Hu, Q., Ye, X., Qu, X. et al., Sci Rep 2018, 7675). In some cases, it is possible to encode one of the immunomodulatory proteins selected from the group consisting of the α-subunit of IL-12, the β-subunit of IL-12, and single-chain IL-12 comprising the α- and β-subunits. Of note, single-chain IL-12 (scIL-12) is a fusion protein comprising the α- and β-subunits, where the peptide sequences are directly linked or separated by a linker peptide, where the amino acid length of the linker peptide can be 1-100. Preferably, the amino acid length of the linker ranges from 4-60 amino acids, more preferably 8-40 amino acids, and particularly preferably 12-25 amino acids. The amino acid length of the linker may be 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid. Furthermore, one of the immunomodulatory proteins encoded by the sequence of the nucleic acid construct may be a single chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10 (human or mouse single chain IL-12), or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9 or 10, and having the biological activity of interleukin-12. The sequence identity of the polypeptide sequence with respect to SEQ ID NO: 9 or 10 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, a polypeptide that is considered to have a biological activity of interleukin-12 should have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of interleukin-12 as assessed in one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 9 or 10. The biological activity of IL-12 can be estimated in vitro by the ability of the peptides to increase IFN-γ production by cells expressing both the IL-12 receptor and the corresponding signaling pathway, such as peripheral blood mononuclear cells or human macrophage cell lines. Alternatively, reporter cell lines can be used, such as the Promega bioluminescent IL-12 bioassay (catalog no. JA2601, JA2605). In vivo IL-12 bioactivity can be estimated by its ability to increase IFN-γ serum levels. Additionally, one immunomodulatory protein may be an IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO: 79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 79, wherein the polypeptide has the biological activity of an IFN-β. The sequence identity of the polypeptide sequence with respect to SEQ ID NO: 79 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, a polypeptide that is considered to have IFN-β biological activity should have at least 10%, preferably at least 20%, more preferably at least 30%, and even more preferably at least 50% of the IFN-β biological activity as assessed in one of the biological activity tests described below, compared to the biological activity of the polypeptide set forth in SEQ ID NO:79. The biological activity of IFN-β can be estimated by its ability to induce IFN type I responsive genes. For this purpose, human cell lines (such as HEK 293) can be incubated with the polypeptide and the expression of IFN responsive genes can be determined by RT-qPCR. Alternatively, IFN-reporter cells encoding luciferase under the control of an ISRE-containing promoter can be used to estimate the IFN-β biological activity of a polypeptide by measuring luciferase expression in a luminometer 3 hours after addition of the polypeptide to the cell culture. In vivo IFN-β bioactivity can be estimated by induction of IFN-responsive genes in peripheral blood mononuclear cells (RT-qPCR). Further, one immunomodulatory protein may be IL-15-sushi comprising or consisting of the amino acid sequence of SEQ ID NO: 80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 80, wherein the polypeptide has a biological activity of IL-15. The sequence identity of the polypeptide sequence to SEQ ID NO: 80 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, a polypeptide that is considered to have the biological activity of interleukin-15 should have at least 10%, preferably at least 20%, more preferably at least 30%, and even more preferably at least 50% of the biological activity of interleukin-15 as assessed in one of the biological activity tests described below, compared to the biological activity of the polypeptide set forth in SEQ ID NO:80. The biological activity of IL-15 can be estimated in vitro by a CTLL-2 cell proliferation assay. In vivo IL-15 bioactivity can be estimated by its ability to induce proliferation of NK cells and T cells using FACS analysis.

[0076] Additionally, the at least one immunomodulatory protein may be CXCL10 comprising or consisting of the amino acid sequence of SEQ ID NO: 81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 81, and having the biological activity of CXCL10. The sequence identity of the polypeptide sequence to SEQ ID NO: 81 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, a polypeptide to be considered as having a biological activity of CXCL10 should have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of CXCL10 as assessed in one of the biological activity tests described below, compared to the biological activity of a polypeptide according to SEQ ID NO: 81. CXCL10 is also known as IP-10. The biological activity of CXCL10 can be estimated by its ability to attract T lymphocytes and natural killer cells, either in vivo or in vitro migration assays. In a preferred embodiment, the first immunomodulatory protein is a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO:9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:9 or 10, wherein the polypeptide has the biological activity of interleukin-12. The second immunomodulatory protein may be an IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO:79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:79, and having the biological activity of IFN-β; alternatively, the second immunomodulatory protein may be a chemokine comprising or consisting of the amino acid sequence of SEQ ID NO:81, preferably CXCL10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:81, and having the biological activity of CXCL10.

[0077] Other combinations of immunomodulatory proteins are also possible: single chain IL-12 may be the first immunomodulatory protein and the second immunomodulatory protein may be selected from the group consisting of a peptide blocking TGF-β, a peptide blocking FoxP3, or a monoclonal antibody against the immune checkpoint inhibitors PD1 or PD-L1, or a nanobody against the immune checkpoint inhibitors PD1 or PD-L1. It should be noted that the term "first and second immunomodulatory proteins" in the context of a nucleic acid comprising two immunomodulatory proteins does not describe the order of the coding sequences on the nucleic acid, but is used simply to illustrate two different immunomodulatory proteins. Thus, the coding sequence of the second immunomodulatory protein may be located upstream of the coding sequence of the first immunomodulatory protein on the nucleic acid, where upstream means that the coding sequence of the second immunomodulatory protein precedes the coding sequence of the first immunomodulatory protein in the 5' to 3' direction on the nucleic acid.

[0078] A TGF-β blocking peptide is one that interferes with either TGF-β itself or the receptor that binds to TGF-β, such that at least a portion of TGF-β signaling is reduced or completely prevented. Examples of TGF-β blocking peptides are P17 and P144. A FoxP3 blocking peptide is one that interferes with either FoxP3 itself or a receptor that binds to FoxP3, such that at least a portion of FoxP3 signaling is reduced or completely prevented. An example of a FoxP3 blocking peptide is P60.

[0079] In another preferred embodiment, the nucleic acid construct is a bi- or polycistronic expression cassette, wherein the sequence encoding the immunomodulatory protein is separated by a sequence encoding an IRES and / or the sequence encoding the immunomodulatory protein is separated by a sequence encoding a 2A peptide, preferably a P2A peptide, and the IRES sequence and the 2A peptide coding sequence are translational regulatory elements (TREs). In the present context, a protein or RNA with anti-cancer properties is any molecule that is capable of slowing, halting or reversing the progression of cancer. A bicistronic expression cassette is a genetic construct that contains two or more additional coding regions or genes in a single transcript. Generally, it contains two or more different sequences in a single transcript. To allow separate translation of two or more immunomodulatory proteins and / or transcription of immunomodulatory genes, a type of TRE is required to allow the proteins to be translated individually. One possible solution is to use an internal ribosome entry site (IRES). IRES are specific sequences of RNA within a transcript that allow translation to begin at a non-AUG codon, bypassing the normal cap-dependent initiation process. This mechanism allows for the regulation of gene expression and the simultaneous expression of multiple proteins from a single mRNA molecule. Another possible solution is the 2A peptides, also known as 2A self-cleaving peptides. 2A peptides are a class of peptides, 18-22 amino acids long, that can induce ribosome skipping during translation of proteins in cells. The peptides share the core sequence motif of DxExNPGP (SEQ ID NO: 32) and are found in a wide range of viral families. They aid in the production of polyproteins by preventing ribosomes from forming peptide bonds. Their short length makes 2A peptides ideal for use in viral vector systems with limited functionality, such as AAV. Another advantage of the peptides is that they allow equimolar expression of coding sequences separated by the peptide. Members of the 2A peptide family are T2A, P2A, E2A and F2A. The peptides can be flanked by linker peptides of different lengths, which may result in improved functionality. For example, an F2A element preceded by a linker domain consisting of the amino acid sequence GGSG (SEQ ID NO: 82) showed significant expression of proteins separated by the element. Table 3: Example of combination of two immunomodulatory proteins separated by a translational regulatory element (TRE) in a bicistronic vector

[0080] [Table 3] A - preferably a human single chain IL-12 as shown in SEQ ID NO:9. B - preferably human IFN-β as represented by SEQ ID NO:79. C- is preferably human IL-15-sushi represented by SEQ ID NO:80. D- is preferably human CXCL10 represented by SEQ ID NO:81.

[0081] Immunotherapy involving systemic availability of immunomodulatory proteins often results in undesirable side effects. However, an advantage of this aspect of the invention is that the recombinant expression system used herein induces local expression of said immunomodulatory proteins, thereby inducing a highly localized and targeted immune response. For example, intratumoral administration of a recombinant expression system according to this aspect of the invention induces an immune response in or near the tumor. Without wishing to be bound by theory, it is believed that release of the immunomodulatory protein in or near the tumor allows T cells to infiltrate the tumor vicinity and recognize tumor cells that normally evade immune system recognition, which allows the immune system to target and kill the cells. Thus, tumor progression is at least slowed, preferably stopped, and more preferably reversed. In a preferred embodiment, at least one of the immunomodulatory proteins of this aspect of the invention is an immunostimulatory protein, that is, a protein that stimulates the immune system, such as by directly stimulating the immune system (e.g., IL-12) or by inhibiting immunosuppressive molecules (e.g., blocking peptides that target TGF-β).

[0082] In a further possibility, the STING responsive promoter of this aspect of the invention may be selected from the group consisting of an IFN-β responsive promoter (preferably according to SEQ ID NO: 25), an IFN-β minimal promoter (preferably according to SEQ ID NO: 31), a CXCL9 responsive promoter (preferably according to SEQ ID NO: 23), a CXCL10 responsive promoter (preferably according to SEQ ID NO: 24), a STAT motif promoter (preferably according to SEQ ID NO: 26), an IR SF 3 motif promoter (preferably according to SEQ ID NO: 27 or SEQ ID NO: 28), an ISRE motif promoter (preferably according to SEQ ID NO: 12 or SEQ ID NO: 13), an NFβ motif promoter (preferably according to SEQ ID NO: 29 or 30) and a STAT 6 motif promoter. In a preferred embodiment, the STING responsive promoter comprises at least one interferon response element (ISRE). By operably linking the expression of an immunomodulatory protein to a STING-responsive promoter according to the present invention, targeted, localized expression of the immunomodulatory protein can be achieved, thereby avoiding potential side effects from systemic expression of the protein.

[0083] The STING responsive promoter of this aspect of the invention comprises: (a) a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 11, or containing 1 to 10 copies, preferably 2 to 7 copies, more preferably 3 to 5 copies, and particularly preferably 4 copies of a sequence identical to SEQ ID NO: 11; (b) the nucleotide sequence according to (a), wherein the copies are separated by up to 40 nucleotides, preferably by 4 to 35 nucleotides, more preferably by 10 to 30 nucleotides, particularly preferably by 15 to 25 nucleotides; (c) a nucleotide sequence set forth in SEQ ID NO: 12, comprising four copies of an ISRE spaced 20 nucleotides apart; (d) a nucleotide sequence as set forth in SEQ ID NO: 13 comprising 4 copies of an ISRE spaced 6 nucleotides apart;

[0084] The use of STING-responsive promoters in the context of the present invention ensures the controlled targeted expression of immunomodulatory proteins. Moreover, the STING-responsive promoters are shown to have additional advantages. As detailed in the examples of the present application, the STING-responsive promoters are shown to be inducible by administering human non-responsive STING agonists in the presence of targeted STING. Furthermore, the induction of the promoters is shown to be specific in that in the absence of selective STING agonists, only limited expression occurs. In particular, the promoter carrying four copies of ISRE, as represented by SEQ ID NO: 12, is highly inducible while also being highly specific. Notably, expression can be achieved even in the absence of STING when applying radiation therapy. Preferably, the nucleotide sequence of the STING-responsive promoter described in (a) and (b) above does not contain an AP-1 site or an NFκB site. Even more preferably, the nucleotide sequence of the STING-responsive promoter described in (a) and (b) above does not contain any further motifs that contribute to the inducibility, leakage or expression efficiency of the promoter, in other words, the nucleotide sequence of the promoter described in (a) and (b) above may contain some nucleotides before, between and / or after the ISRE of SEQ ID NO: 11, where said further nucleotides are selected in such a manner (e.g. randomly) that they do not form any transcriptional regulatory element. Particularly preferred is the nucleotide sequence of the STING-responsive promoter described in (a) and (b) above. It consists of 1 to 10 copies, preferably 2 to 7 copies, more preferably 3 to 5 copies, particularly preferably 4 copies of a sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 11, or is identical to SEQ ID NO: 11. Furthermore, the nucleotide sequences of the STING-responsive promoters described in (a)-(d) above may already represent functional promoters, such that no other elements are required for said promoter to induce expression of an operably linked transduced gene.

[0085] In a further possibility, the nucleic acid construct of this aspect of the invention further comprises at least one sequence motif that inhibits transgenic expression of the immunomodulatory protein in tissues or cells not intended to express the protein, preferably in the liver, which sequence motif may preferably be a target sequence of a microRNA that is significantly enriched in the liver. It is known that some viral particles, especially AAV, show tropism for hepatocytes. This means that when viral particles access the blood circulation system, they preferentially transduce hepatocytes, which ultimately results in the expression of the transgene encoded in the liver. In addition, in the case of intratumoral injection of the viral particles, a portion of the particles accesses the blood circulation system, since tumors are highly vascularized. Therefore, in order to prevent the undesired expression of the transgene encoded by the viral particles in the liver, it is appropriate to control their expression. Several methods for achieving this purpose are known to those skilled in the art. In general, a suitable method for limiting transgene expression to a specific tissue or a specific cell type is to use viral particles with different tropism for said tissue or cell type. A way to achieve this is by changing the serotype of AAV or by changing the capsid of AAV. Several AAV serotypes are known in the art, each with different tropism. However, since the present invention is intended to target several different cancers, it will be necessary to optimize the serotype or capsid for each cancer or tumor. Nevertheless, this may be a suitable method for achieving tissue- or cell type-specific expression of transgenes.

[0086] One skilled in the art may also use a DNA or RNA binding repressor to avoid undesired expression of the transgene encoded by the viral particle in tissues or cells where it is not intended to express the transgene. Thus, the nucleic acid construct of this aspect of the invention may comprise an operon that binds to the repressor. The repressor may be operably linked to a promoter that is activated in tissues or cells where it is not intended to express the transgene, thus inducing expression of the repressor in said tissues or cells, thereby inhibiting expression of the transgene.

[0087] Another possibility is to use RNA interference (RNAi). RNAi is characterized in that an RNA molecule reduces or inhibits the expression of a nucleic acid sequence with which it shares substantial or complete homology. Non-limiting examples of RNAi are small interfering RNA, small hairpin RNA, or microRNA (miR). To reduce or inhibit transgene expression in a specific tissue using RNAi, it may be considered that, for example, an shRNA targeting the nucleotide sequence of said transgene can be operably linked to a tissue-specific promoter. When said shRNA is administered, it reduces or inhibits transgene expression in the tissue. Although this method is feasible in the context of the present invention, it may be disadvantageous in that an additional administration of said shRNA is required to limit transgene expression in the target tissue, for example the liver.

[0088] An elegant way to circumvent this drawback is to operably link a miRNA target sequence to the nucleotide sequence of the transgene, so that expression of the transgene is reduced or inhibited in tissues in which the miRNA associated with said miRNA target sequence is present. Thus, a preferred embodiment of the present invention comprises that the sequence motif is a target sequence of a microRNA that is significantly enriched in the liver, so that a single administration of the nucleic acid construct is sufficient to avoid transgene expression in the liver, while transcription in other tissues or organs is not impaired. In some cases, the liver-enriched microRNA may be selected from the group consisting of miR-122, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c or let7f. Furthermore, it may be advantageous that the liver-enriched microRNA is miR-122. The advantage of miR-122 is that it is highly expressed in the liver, and importantly, it is specifically expressed in the liver. Therefore, by selecting the miR-122 target sequence, it is ensured that the transduced gene in the liver is significantly reduced, while the expression in other tissues is not impaired. Furthermore, miR-122 is downregulated in most liver tumors, and as a result, it has been shown that the recombinant expression system according to this aspect of the present invention is also suitable for treating liver cancer.

[0089] According to a preferred embodiment of this aspect of the invention, each miR-122 target sequence is a miR-122 target sequence having 90% or at least 95% sequence identity with SEQ ID NO: 14 or is identical to SEQ ID NO: 14. Preferably, the target sequence comprises 1-12, preferably 3-10, more preferably 4-8, particularly preferably 5 copies of the miR-122 target sequence represented in the previous sentence. Increasing the copy number of the miR-122 target sequence increased the miR-mediated reduction, but no significant improvement was observed beyond the disclosed number of 5 copies. In a preferred embodiment, the copies are spaced 1-40 nucleotides, preferably 1-30 nucleotides, more preferably 2-20 nucleotides, particularly preferably 2-8 nucleotides apart. Preferably, the nucleotide sequence comprises five copies of the miR-122 target sequence represented by SEQ ID NO: 15. As detailed in the Examples of the present application, the nucleotide sequence represented by SEQ ID NO: 15 substantially inhibits transgene expression in the liver, such that plasma levels of the transgene were undetectable by the method described in Example 16.

[0090] In a further possibility, it may be provided that the nucleic acid construct of this aspect of the invention further comprises a transgene encoding a thymidine kinase, preferably the thymidine kinase according to SEQ ID NO: 16. The nucleotide sequence encoding the thymidine kinase may be linked to the nucleotide sequence of the immunomodulatory protein and spaced apart by a 2A self-cleaving peptide (so-called 2A peptide). 2A peptides constitute a class of peptides of 18-22 amino acids in length that can induce ribosome skipping during translation of proteins in cells. The peptides share a core sequence motif of "DXEXNPGP" and are found in a wide range of viral families. They aid in the production of polyproteins by preventing the ribosome from forming peptide bonds. The skilled artisan will appreciate that the use of 2A peptides allows for simultaneous expression of the immunomodulatory protein and the thymidine kinase. Co-expression of thymidine kinase is advantageous in that it allows assessment of tumor transduction by PET imaging and, in the case of potential IL-12-mediated toxicity, elimination of cells expressing the cytokine by administration of thymidine kinase substrates (e.g., acyclovir, penciclovir, famciclovir or ganciclovir). The nucleic acid construct of this aspect of the invention may further comprise a Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE), which is known to increase transgene expression and is therefore most effective when placed downstream of the transgene (i.e., an immunomodulatory protein) proximal to the polyadenylation signal. Conveniently, the nucleic acid construct of this aspect of the invention further comprises a polyadenylation signal located 3' to the immunomodulatory protein. Optimizing the length of the poly(A) tail can stabilize the mRNA and enhance subsequent protein translation. The skilled artisan will also be familiar with other means for increasing protein translation, e.g. (a) by sequence modification to optimize G / C content and / or over-stabilizing secondary structure codons, or (b) alternating 5' caps by adding synthetic or natural 5' cap analogs, such as m7GpppG or starter trimers, such as "CleanCap", or stabilization with a capping enzyme; or (c) Addition of regulatory elements (c1) in the 5' untranslated region (UTR), for example, by optimization via appropriate cis-regulatory sequences or by modification of the length and sequence to avoid overly stable structures; or (c2) in the 3'-UTR through sequence modification to introduce stable elements or repetitive motifs or synthetic or naturally occurring sequences that have proven effective in other mRNAs;

[0091] In a particular embodiment, the nucleic acid construct of the recombinant expression system of this aspect of the invention consists of the nucleic acid construct represented in the table below, where the different elements are indicated in the 5' to 3' direction as: promoter, first open reading frame (ORF1), first translational regulatory element (TRE1), second open reading frame (ORF2), second translational regulatory element (TRE2) and target sequence of microRNA (microRNA element). Table 4: Nucleic acid construct embodiments of the expression system

[0092] [Table 4] A - preferably 4 copies of an ISRE, preferably as given by SEQ ID NO:12. B - preferably a human single chain IL-12 as shown in SEQ ID NO:9. C- is preferably human IFN-β represented by SEQ ID NO:79. D- is preferably human IL-15-sushi represented by SEQ ID NO:80. E - preferably 5 copies of the miR-122 target sequence represented by SEQ ID NO:15.

[0093] Another aspect of the invention is an expression vector encoding a nucleic acid construct comprising sequences encoding at least two immunomodulatory proteins. Using an expression vector comprising the nucleic acid construct has certain advantages. First, the expression vector is specifically designed to transduce and express the encoded nucleotide sequence into a host cell. Second, the expression vector is a plasmid that is extremely stable and protected from degradation. Third, the expression vector is contemplated for gene delivery using different methods. Non-limiting examples of expression vectors and preferred embodiments of this aspect of the invention are viral vectors, more preferably viral vectors selected from the group consisting of adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, vaccinia virus vectors, or herpes simplex virus vectors. In particular, viral vectors combine some advantages of expression vectors, such as general safety, low toxicity, and stability.

[0094] It is also advantageous that the nucleic acid construct comprising the sequence encoding at least two immunomodulatory proteins of the expression vector further comprises 5'ITR and 3'ITR sequences, preferably the AAV ITRs represented by SEQ ID NO: 17. It may be advantageous that the expression vector is an AAV vector, preferably of serotype AAV1, AAV3, AAV6, AAV8, AAV9, AAV2, AAV5, AAVrh.10, or any gain-of-function mutant of AAVrh.10, but AAV2 is most preferred. AAV is commonly used in biotechnology and is therefore well characterized. The advantage of using AAV vectors is that they are generally considered safe, exhibit low toxicity, and are stable. Moreover, AAV has already been approved by several government agencies around the world for use in humans.

[0095] It is also advantageous that the nucleic acid construct comprising the sequence encoding at least two immunomodulatory proteins of the expression vector further comprises 5'ITR, psi packaging signal and 3'ITR sequences, preferably 5'ITR, psi packaging signal and 3'ITR of adenovirus (Ad). It may be advantageous that the expression vector is an adenovirus vector, preferably a high-capacity adenovirus vector. Adenoviruses are commonly used in biotechnology and therefore well characterized. The advantage of adenovirus vectors is that they have a higher size limit compared to AAV vectors. Moreover, adenoviruses have already been approved by several government agencies around the world for use in humans. It is also advantageous if the expression vector is a lentiviral, vaccinia virus or herpes simplex virus vector, which are commonly used in biotechnology and are therefore well characterized.

[0096] It is also contemplated that nanoparticles can be used to deliver nucleic acids comprising sequences encoding at least two immunomodulatory proteins. Nanoparticles as gene delivery systems are known in the art and offer advantages such as tunable size, shape and / or surface, which allow for tailoring of their biological behavior. For effective delivery and uptake by cells, the nucleic acid constructs may be prepared using a method comprising administering to the subject a nucleic acid molecule selected from the group consisting of distearoylphosphatidylglycerol, distearoylphosphatidylcholine, (DLPC) 1,2-dilauroyl-sn-glycero-3-phosphocholine, (DMPC) 1,2-dimyristoyl-sn-glycero-3-phosphocholine, (DPPC) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, (DSPC) 1,2-distearoyl-sn-glycero-3-phosphocholine, (DOPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine, (DMPE) 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, (DPPE) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, (DOPE) 1,2-dioleoyl-sn-glycero-3 Nanoparticles, preferably packaged in liposomes, made of pharma- ceutically acceptable carriers composed of physiologically compatible lipids, proteins, polysaccharides or synthetic polymers in various ratios, such as standard lipids compatible with biological membranes selected from the group consisting of phospholipids such as phosphoethanolamine, (DOPS) 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, (DPPS) 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, (DMPS) 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, (DOPG) 2,3-dimyristoyl-sn-glycero-1-phosphocholine, and their respective enantiomers, where distearoylphosphatidylglycerol and distearoylphosphatidylcholine are most preferred. Another suitable example is the use of cardiolipin, preferably in combination with cholesterol.Alternative nanoparticles include dendrimer nanoparticles, protamine liposomes, cationic polymers, cationic polymer liposomes, polysaccharide particles, cationic lipid particles, cationic lipid cholesterol nanoparticles, cationic lipid cholesterol PEG nanoparticles, ionizable lipids, preferably lipids that are positively charged under physiological conditions, including amino lipids such as MC3 (DLin-MC3-DMA), C12-200, cKK-E12, ZA3-Ep10, 200-Oi10, etc.; (SM-102) heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid, (ALC 0159), N,N-dimyristylamide of 2-hydroxyacetic acid O-PEGylated to PEG chains of approximately 2 kilodaltons; (ALC 0315)(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate), Lipofectamine, Trans-IT, InstantFECT, (DOTAP)1,2-dioleoyl-3-trimethylammonium. Other components may include peptides and polypeptides (e.g., DP7-C, cholesterol-modified cationic peptide DP7 (VQWRIRVAVIRK, SEQ ID NO:85)), polymers (e.g., polyethylene glycol preparations composed of polymer molecules of various molecular sizes that may be non-covalently bound or covalently linked to lipids in the formulation).

[0097] Another aspect of the invention is a viral particle comprising the above-mentioned viral vector comprising a nucleic acid construct comprising sequences encoding at least two immunomodulatory proteins, which has the advantage that a single administration of said viral particle is sufficient to express the recombinant expression system in the patient. The viral particle is preferably an AAV or adenoviral particle, more preferably an AAV viral particle. As described hereinabove, Ad and AAV exhibit advantages over other viral particles, such as that they are well characterized, widely used, and approved for human therapy. The viruses exhibit further advantages that make them particularly useful in gene therapy, such as the ability to transduce dividing as well as non-dividing cells and not integrating into the host genome.

[0098] Another aspect of the invention is a pharmaceutical composition comprising a recombinant expression system comprising a sequence encoding at least two immunomodulatory proteins, an expression vector comprising a sequence encoding at least two immunomodulatory proteins, or a viral particle comprising a sequence encoding at least two immunomodulatory proteins, together with a pharma- ceutically acceptable excipient. The pharmaceutical composition has the advantage of being "ready to use", in that it can be administered directly to a patient for treatment. Furthermore, the pharmaceutical composition is tailored to optimize the stability and availability of the components according to the needs of the treatment, while avoiding undesirable side effects for the patient.

[0099] In other aspects of the invention, recombinant expression systems, expression vectors, viral particles or pharmaceutical compositions each comprising sequences encoding at least two immunomodulatory proteins may be used or are provided for use in therapy, preferably immunotherapy. In a preferred embodiment, the immunotherapy is an immunotherapy of a proliferative disorder, more preferably an immunotherapy of a cancer, even more preferably an immunotherapy of a solid cancer. Although it is considered that non-solid cancers can also be treated by the immunotherapy of the present invention, the use of the immunotherapy of solid cancer has the advantage that the cancer is locally delimited and therefore accessible by local administration. Thus, recombinant expression systems, expression vectors, viral particles or pharmaceutical compositions each comprising sequences encoding at least two immunomodulatory proteins can be administered locally to the tumor by intratumoral administration. This has the advantage that a locally targeted immune response is induced, which can slow, stop or reverse tumor progression. Furthermore, local administration leads to less undesirable systemic expression, whereas the present invention takes measures to avoid systemic expression.

[0100] As mentioned above, cancer immunotherapy has the following problems: i) insufficient tumor targeting, ii) weak tumor cell killing function, iii) high labor and associated high cost, iv) tumor immune evasion, and v) overstimulation of the immune system, which may cause severe side effects (Non-Patent Document 2). The above immunotherapy is an extended immunotherapy since it also incorporates gene therapy. Therefore, this immunotherapy is not simply immunotherapy per se, but a combination of immunotherapy and gene therapy for effectively treating proliferative disorders such as cancer, hereinafter referred to as "immunogen therapy". Cancers further include hematopoietic tumors of the lymphatic system, including bladder cancer (including accelerated and metastatic bladder cancer), breast cancer, colon cancer (including colorectal cancer), kidney cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer and lung adenocarcinoma), ovarian cancer, prostate cancer, testicular cancer, genitourinary tract cancer, lymphatic system cancer, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), esophageal cancer, stomach cancer, gallbladder cancer, cervical cancer, thyroid cancer and skin cancer (including squamous cell carcinoma); tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors, including melanoma, xeroderma pigmentosum, keratinizing acanthoma, dermoid carcinoma, follicular thyroid carcinoma, and teratocarcinoma; The cancer may be selected from the group consisting of malignant melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian carcinoma, liver cancer, colorectal cancer, endometrial cancer, renal cancer, prostate carcinoma, thyroid carcinoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, gastric carcinoma, bladder carcinoma, urothelial carcinoma, hepatocellular carcinoma, embryonal tumor, liver cancer, breast carcinoma, colon carcinoma, and head and neck cancer, gastric carcinoma, germ cell tumor, osteosarcoma, bone tumor, adult malignant fibrous histiocytoma of bone; childhood, malignant fibrous histiocytoma of bone, sarcoma, pediatric sarcoma, Sino-nasal natural killer, neoplasm, plasmacytoma, carcinoid tumor, neuroendocrine tumor, cholangiocarcinoma, and Merkel cell tumor.

[0101] In another preferred embodiment, the recombinant expression system, expression vector, viral particle or pharmaceutical composition each comprising a sequence encoding at least two immunomodulatory proteins is used in a combination therapy with at least one other therapy selected from the group consisting of a therapy comprising at least one checkpoint inhibitor, a therapy comprising at least one adoptive cell therapy, a therapy comprising at least one cancer vaccine, a therapy comprising at least one exogenous cytokine and / or a therapy comprising radiation therapy.

[0102] The inhibitor of the treatment comprising at least one checkpoint inhibitor may be selected from the group consisting of inhibitors of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR, more preferably from the group consisting of inhibitors of PD-1, PD-L1, PD-L2, or CTLA-4. Immune checkpoints are molecules expressed on T cells or cells that interact with T cells, which regulate the immune response and contribute to preventing excessive or prolonged immune activation. However, cancer cells can use the checkpoint proteins to avoid detection by the immune system. By blocking the checkpoint proteins, immune checkpoint inhibitors can release the brakes on the immune response, allowing the immune system to attack and destroy the cancer cells. An expression system comprising sequences encoding at least two immune-modulating proteins is particularly advantageous in the context of this aspect of the invention, as it results in the infiltration of T cells into the tumor environment, with immune checkpoint inhibitors as an additional therapy. Such T cells may be compromised in function due to immune checkpoints, but measures to overcome this could greatly benefit therapeutic outcome.

[0103] The therapy comprising at least one adoptive cell therapy can be adoptive NK cell therapy, adoptive dendritic cell therapy, or adoptive T cell therapy, and the adoptive T cell therapy is preferably CAR-T cell therapy or tumor infiltrating leukocyte (TIL) therapy. In some cases, tumors silence the expression of MHC class I molecules and / or IFN-γ signaling molecules as a means of avoiding T cell immunity. Using adoptive cell therapy, this problem can be avoided because the adoptive cells can normally recognize and target tumor cells. Therefore, synergistic effects can be expected in combination approaches. Additionally, the use of the expression system of the present invention in combination with a cancer vaccine or exogenous cytokines may result in synergistic effects, as the recruited T cells are primed against cancer cells by the cancer vaccine or exogenous cytokines. In preferred embodiments of this aspect of the invention, the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymic cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, urothelial cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasma cell neoplasms, cholangiocarcinoma, and neuroendocrine or carcinoid tumors.

[0104] In yet another aspect, the present invention relates to a recombinant expression system, expression vector, viral particle or pharmaceutical composition, each comprising sequences encoding at least two immunomodulatory proteins, for use in immunotherapy comprising the following: (a) administering a recombinant expression system comprising a sequence encoding at least two immunomodulatory proteins; an expression vector comprising a sequence encoding at least two immunomodulatory proteins, a viral particle comprising a sequence encoding at least two immunomodulatory proteins, or a pharmaceutical composition comprising a sequence encoding at least two immunomodulatory proteins to a patient suffering from a proliferative disorder, preferably cancer, to obtain therapeutically effective transgenic expression of the immunomodulatory proteins, wherein the administration is preferably intratumoral or systemic; (b) optionally treating the patient with radiation therapy prior to and / or simultaneously with administration according to (a), preferably radiation therapy directed to the tissue or organ intended to express the transduced gene from (a); and / or (c) treating the patient following administration with (a) above with radiation therapy, preferably radiation therapy directed to tissues or organs intended to express the transduced gene from (a) above.

[0105] In a similar aspect, the present invention provides a method of treating a proliferative disorder, preferably cancer, in a patient, comprising administering to the patient: (a) administering a recombinant expression system comprising a sequence encoding at least two immunomodulatory proteins; an expression vector comprising a sequence encoding at least two immunomodulatory proteins, a viral particle comprising a sequence encoding at least two immunomodulatory proteins, or a pharmaceutical composition comprising a sequence encoding at least two immunomodulatory proteins to a patient suffering from a proliferative disorder, preferably cancer, to obtain therapeutically effective transgenic expression of the immunomodulatory proteins, wherein the administration is preferably intratumoral or systemic; (b) optionally treating the patient with radiation therapy prior to and / or simultaneously with administration according to (a), preferably radiation therapy directed to the tissue or organ intended to express the transduced gene from (a); and / or (c) treating the patient following administration with (a) above with radiation therapy, preferably radiation therapy directed to tissues or organs intended to express the transduced gene from (a) above. In other embodiments, the patient is treated prior to, concurrently with, and / or after administration with (a) with at least one other therapy selected from the group consisting of a therapy comprising at least one checkpoint inhibitor, a therapy comprising at least one adoptive cell therapy, a therapy comprising at least one cancer vaccine, and / or a therapy comprising at least one exogenous cytokine. In a preferred embodiment, the treatment with at least one other therapy is administered after administration with (a). In particular, the embodiments described below apply to both recombinant expression systems, expression vectors, viral particles, or pharmaceutical compositions, each comprising sequences encoding at least two immunomodulatory proteins for use in immunotherapy and methods of treating proliferative disorders.

[0106] The use for immunotherapy or the method of treating proliferative disorders may be combined with other treatments, which are anti-cancer therapies. Several anti-cancer therapies are known in the art, such as chemotherapy, hormonal therapy, radiation therapy, photodynamic therapy, stem cell transplantation, surgery, targeted therapy, hyperthermia, or other immunotherapies. It may be advantageous to direct radiation therapy to the tumor administered intratumorally by (a) above. It is also contemplated that radiation therapy of 100 Gy or less may be used in (b) and / or (c) above, depending on the circumstances. Any dose of ionizing radiation less than 100 Gy, 96 Gy, 92 Gy, 88 Gy, 84 Gy, 80 Gy, 76 Gy, 72 Gy, 68 Gy, 64 Gy, 60 Gy, 56 Gy, 52 Gy, 48 Gy, or 44 Gy may be used. It is also contemplated that low dose radiation therapy of 40 Gy or less, preferably 10 Gy or less, more preferably 8 Gy or less may be used in (b) and / or (c) above. Low dose radiation therapy can be any radiation therapy using a dose of ionizing radiation less than 40 Gy, 38 Gy, 36 Gy, 34 Gy, 32 Gy, 30 Gy, 28 Gy, 26 Gy, 24 Gy, 22 Gy, 20 Gy, 19 Gy, 18 Gy, 17 Gy, 16 Gy, 15 Gy, 14 Gy, 13 Gy, 12 Gy, 11 Gy, 10 Gy, 9 Gy, 8 Gy, 7 Gy, 6 Gy, 5 Gy, 4 Gy, 3 Gy, 2.5 Gy, 2.0 Gy, 1.5 Gy, 1 Gy, 0.5 Gy, 0.25 Gy, 0.2 Gy, 0.15 Gy, 0.1 Gy, 0.05 Gy, or 0.01 Gy. The advantage of using low-dose radiotherapy is that it is better tolerated by patients, in that they experience little or no side effects that are otherwise common with radiotherapy.As used herein, the dose indicated refers to total dose, meaning that the total dose for treatment in (b) and / or (c) above does not exceed the dose claimed.Therefore, the dose of 40Gy applied in (b) above can be achieved by a single dose or fractionated dose of 40Gy, for example, 4 fractions of 10Gy given over a period of several weeks or even months. In another embodiment, the radiation therapy in (c) comprises several further radiation treatments, the cumulative dose of the further radiation treatments being added up to less than 100 Gy, preferably less than 40 Gy, more preferably less than 25 Gy. The cumulative dose of the further radiation treatments is 100 Gy, 96 Gy, 92 Gy, 88 Gy, 84 Gy, 80 Gy, 76 Gy, 72 Gy, 68 Gy, 64 Gy, 60 Gy, 56 Gy, 52 Gy, 48 Gy, 44 Gy, 40 Gy, 38 Gy, 36 Gy, 34 Gy, 32 Gy, 30 Gy, 28 Gy, 26 Gy, 24 Gy, 22 Gy, 20 Gy, It may be less than 19 Gy, 18 Gy, 17 Gy, 16 Gy, 15 Gy, 14 Gy, 13 Gy, 12 Gy, 11 Gy, 10 Gy, 9 Gy, 8 Gy, 7 Gy, 6 Gy, 5 Gy, 4 Gy, 3 Gy, 2.5 Gy, 2.0 Gy, 1.5 Gy, 1 Gy, 0.5 Gy, 0.25 Gy, 0.2 Gy, 0.15 Gy, 0.1 Gy or 0.05 Gy.

[0107] In particular, radiotherapy can be applied externally or internally. The means for providing external radiotherapy are known to those skilled in the art. For example, external radiotherapy can be provided by gamma rays, electron beams or proton accelerators suitable for therapeutic purposes. However, for some tumors, it may be advantageous to use internal radiotherapy. Internal radiotherapy can be provided by administration of a radioisotope in a pharmacologically acceptable form and dose for therapeutic purposes. An embodiment of internal radiotherapy can be brachytherapy or non-localized radiotherapy. Brachytherapy physically restrains the radioactive deposits so that they essentially cannot move from the location where they are deposited. The radioactive deposits can be placed interstitially (interstitial radiotherapy), which is used, for example, in a significant proportion of prostate tumors and some head and neck tumors, or the radioactive deposits can be placed intracavitary (intracavity radiotherapy), which is used, for example, in some uterine, bronchial or esophageal tumors. Non-localized radiation therapy may be a partially diffuse local administration, such as selective internal radiation therapy (SIRT), in which a suspension of material is injected into a specific artery or vein with the purpose of settling near the injection site rather than undergoing widespread distribution in the body, or it may be a fully systemic approach, such as the use of biological agents, such as soluble chemicals synthesized with radioisotopes that find their point of action by affinity, or radiolabeled antibodies, to better deliver radiation to the target tumor. A typical example of the latter is iodine-131. Although different isotopes may be used for different treatment paradigms, isotopes useful in the present invention may preferably be selected from the list consisting of yttrium-90, iodine-131, lutetium-177, iridium-192, astatine-211, bismuth-213, actinium-225, samarium-153, radium-223, phosphorus-32, strontium-89, erbium-169, holmium-166, terbium-161, gallium-68, rhenium-188 or rhenium-186.

[0108] In further aspects, the present invention relates to embodiments 1 to 18 described below. Embodiment 1: A recombinant expression system comprising: (a) a first nucleic acid construct comprising a sequence encoding a target STING that recognizes a target STING but does not recognize human STING and its naturally occurring variants, the sequence encoding the target STING being operably linked to elements allowing expression and / or translation of the target STING; and / or (b) a second nucleic acid construct comprising a sequence encoding at least one immune modulatory gene or protein, wherein the sequence is operably linked to a STING responsive promoter. Embodiment 2: The recombinant expression system of embodiment 1, including 1, wherein the targeted STING responds to a human non-responsive STING agonist selected from the group consisting of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), 7-bromo-DMXAA, 7-iodo-DMXAA, 7-hydroxy-DMXAA, 7-formyl-DMXAA, 7-hydroxymethyl-DMXAA, 7-(2-hydroxyethyl)-DMXAA, flavone-8-acetic acid (FAA), 2,7-bis(2-dimethylamino ethoxy)fluoren-9-one (tilorone), and 10-carboxymethyl-9-acridan (CMA), preferably, the targeted STING responds to DMXAA.

[0036] Embodiment 3: The target STING encoded by the sequence of the first nucleic acid construct is one of the following: (a) a mutated form of a naturally occurring variant of human STING, which further comprises at least one, at least two, or at least three amino acid substitutions that render said human STING variant sensitive to said human non-responsive STING agonist as determined in an in vitro agonist determination assay; or (b) a non-human STING, preferably mouse STING or fusion STING, even more preferably mouse STING as set forth in SEQ ID NO: 7, that is sensitive to a human non-responsive STING agonist as determined in an in vitro agonist determination assay; wherein the target STING is preferably a DMXAA-sensitive human STING as determined in an in vitro DMXAA determination assay.

[0036] Embodiment 4: The target STING encoded by the sequence of the first nucleic acid construct is one of the following: (a) a human STING[R] variant further comprising at least one, at least two, or at least three amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I; (b) a human STING[AQ] variant further comprising at least one, at least two, or at least three amino acid substitutions selected from the group consisting of S162A, A230I, Q266I, E260I, and E260V; (c) a human STING[H] mutant further comprising at least one, at least two, or at least three amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I. (d) a truncated human STING[H] mutant further comprising at least one or at least two amino acid substitutions selected from the group consisting of S162A and G230I.

[0023] Embodiment 5: The DMXAA sensitive human STING mutant encoded by the sequence of the first nucleic acid construct is selected from the group consisting of hSTING[R]-S162A / G230I / Q266I, hSTING[R]-S162A / Q266I, hSTING[R]-G230I, hSTING[R]-S162A, hSTING[R]-Q266I, hSTING[R]-S162A / G230I, hSTING[AQ]-S162A / A230I / Q266I, hSTING[AQ]-S162A, hSTING[AQ]-S162A / R169A, hSTING[AQ]-S162A / T267A, hSTING[AQ]-E260I, hSTING[AQ]-E260V, 5. The recombinant expression system of embodiment 4, wherein the recombinant expression system is selected from the group consisting of hSTING[H]-S162A / G230I / Q266I, hSTING[H]-S162A / Q266I, hSTING[H]-G230I, hSTING[H]-S162A, hSTING[H]-S162A / G230I, and hSTING[H]-Q266I, preferably hSTING[R]-S162A / G230I / Q266I. Embodiment 6: The sequence of the first nucleic acid construct encoding a DMXAA sensitive human STING mutant is as follows: (a) SEQ ID NO:5; (b) a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:5; (c) a polynucleotide sequence encoding a polypeptide comprising SEQ ID NO:6; (d) a polynucleotide sequence encoding a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:6; (e) a polynucleotide sequence encoding a polypeptide of (c) or (d), wherein the polypeptide has at least one modification which is an amino acid substitution, an amino acid insertion, an amino acid deletion, a C-terminal truncation, or an N-terminal truncation; (f) a polynucleotide sequence that hybridizes under at least moderately stringent conditions to the complement of any of the polynucleotide sequences (a) to (e); (g) a region of the polynucleotide sequence of any of (a)-(f) encoding a fragment of at least 50 amino acid residues of the DMXAA-sensitive human STING mutant; wherein a polypeptide encoded by any of the polynucleotides of (a)-(g) has biological activity that is responsive to the agonist DMXAA as determined in an in vitro DMXAA determination assay. Embodiment 7: The promoter of the first nucleotide construct is a constitutive promoter, preferably selected from the group consisting of SV40, CMV0, UBC, EF1a, PGK, CAG, EFS, CBh, CBA human beta actin, Ac5, polyhedrin, TEF1, GDS, CaMV35S, Ubi, H1 and U6, or The recombinant expression system of any of the above embodiments 1 to 6, wherein the promoter of the first construct is an inducible promoter, preferably selected from the group consisting of a telomerase responsive promoter, an IL-6 responsive promoter, a TGF-β inducible promoter, a VEGFR inducible promoter, an epidermal growth factor receptor inducible promoter, a HER2 / Neu inducible promoter, a MUC1 promoter, a BRCA1 promoter, a BRCA2 promoter, a LALBA promoter, a PSA promoter, an ERBB2 promoter, a CEA promoter, a uPAR promoter and a thyroid transcription factor 1 (TTF1). Embodiment 8: The at least one immunomodulatory gene or protein encoded by the sequence of the second genetic construct is selected from the group consisting of interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-7, interleukin-8, interleukin-9, interleukin-11, single chain interleukin-12, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21, interleukin-22, interleukin-23, interleukin-24, interleukin-25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, interleukin-30, interleukin-31, interleukin-32, interleukin-33, interleukin-34, interleukin-35, interleukin-36, interleukin-37, interleukin-38, interleukin-39, interleukin-40, interleukin-41, interleukin-42, interleukin-43, interleukin-44, interleukin-45, interleukin-46, interleukin-47, interleukin-48, interleukin-49, interleukin-50, interleukin-51, interleukin-52, interleukin-53, interleukin-54, interleukin-55, interleukin-56, interleukin-57, interleukin-58, interleukin-59, interleukin-60, interleukin-61, interleukin-6 Interleukin-15, Interleukin-15-sushi, Interleukin-16, Interleukin-17, Interleukin-18, Interleukin-19, Interleukin-20, Interleukin-21, Interleukin-22, Interleukin-23, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL 7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, granulocyte-macrophage colony-stimulating factor, I 8. The recombinant expression system of any of the above embodiments 1 to 7, wherein the blocking peptide is selected from the group consisting of a blocking peptide targeting type I interferon, IFN-γ, TNF-α, FLT3-ligand, TGF-β, a blocking peptide targeting IL-10, a blocking peptide targeting FoxP3, a monoclonal antibody or single chain variable fragment (scFv) or nanobody neutralizing PD1, PDL1, CTLA4, CD137, TIM3, LAG3, and fragments or variants thereof. Embodiment 9: The at least one immunomodulatory protein is selected from the group consisting of: (a) α subunit of IL-12; (b) the β subunit of IL-12; (c) single-chain IL-12, which contains the α and β subunits of IL-12; (d) a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10; (e) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9 or 10 and having the biological activity of interleukin-12. Embodiment 10: The second said STING responsive promoter comprises an IFN-β responsive promoter, an IFN-β minimal promoter, a CXCL9 responsive promoter, a CXCL10 responsive promoter, a STAT motif promoter, an IRSF3 motif promoter, an ISRE motif promoter, an NFκB motif promoter, and a STAT6 motif promoter, wherein said STING responsive promoter preferably comprises at least one interferon stimulated response element (ISRE), and more preferably comprises one of the following: (a) a nucleotide sequence comprising 1 to 4 copies of the ISRE represented by SEQ ID NO: 11, preferably a nucleotide sequence comprising 4 copies of the ISRE; (b) the nucleotide sequence set forth in SEQ ID NO:12; (c) the nucleotide sequence as set forth in SEQ ID NO: 13. Embodiment 11: The second nucleic acid construct further comprises at least one sequence motif inhibiting transgenic expression of an immunomodulatory protein in the liver, the sequence motif being preferably a target sequence of a microRNA highly abundant in the liver, more preferably said target sequence is selected from the group consisting of miR-122 target sequence, miR-192 target sequence, miR-199a target sequence, miR-101 target sequence, miR-99a target sequence, let7a target sequence, let7b target sequence, let7c target sequence, let7f target sequence, and even more preferably the following: (a) a miR-122 target sequence represented by SEQ ID NO: 14; (b) 1 to 5 copies of the miR-122 target sequence of SEQ ID NO: 14, preferably 5 copies of the miR-122 target sequence; (c) a nucleotide sequence comprising five copies of the miR-122 target sequence represented by SEQ ID NO: 15. Embodiment 12: An expression vector encoding the first and / or second nucleic acid construct according to any of the above embodiments 1-11, or a combination of the first and second expression vectors, wherein said first vector encodes the first nucleic acid construct according to any of the above embodiments 1-11, and said second vector encodes the second nucleic acid construct according to any of the above embodiments 1-11, wherein said expression vector is preferably a viral vector, more preferably a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, a vaccinia viral vector, and a herpes simplex viral vector, and even more preferably has the following characteristics: (a) the sequence of the first and / or second nucleic acid construct further comprises a 5'ITR sequence and a 3'ITR sequence, preferably the AAV ITRs set forth in SEQ ID NO: 17; and (b) The AAV vector is preferably an adeno-associated virus vector having one or more of the following serotypes: AAV8, AAV1, AAV3, AAV6, AAV9, AAV2, AAV5, AAVrh.10, or any gain-of-function mutant of AAVrh.10; more preferably, the AAV vector has serotype AAV2; Embodiment 13: A viral particle, wherein the first viral particle comprises a viral vector encoding the first nucleic acid construct, and the second viral particle comprises a viral vector encoding the second nucleic acid construct of embodiment 12, and the viral particle is preferably an AAV or AAV-like vector. A viral particle comprising a viral vector comprising the first and / or second nucleic acid construct of embodiment 12, or a combination of the first and second viral particles, which is an Ad viral particle; more preferably an AAV viral particle. Embodiment 14: A pharmaceutical composition comprising a pharma- ceutically acceptable excipient and (a) the recombinant expression system of any of embodiments 1 to 11; or (b) an expression vector according to embodiment 12; or (c) a viral particle according to embodiment 13; Embodiment 15: A recombinant expression system of any of embodiments 1 to 11 for use in immunotherapy, preferably the immunotherapy of proliferative disorders, more preferably the immunotherapy of cancer, particularly preferably the immunotherapy of solid cancers; an expression vector(s) of embodiment 12; a viral particle(s) of embodiment 13 or a pharmaceutical composition according to embodiment 14. Embodiment 16: The recombinant expression system, expression vector, viral particle or pharmaceutical composition according to embodiment 15, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymic cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasma cell neoplasms, cholangiocarcinoma, and neuroendocrine or carcinoid tumors. Embodiment 17: Use in immunotherapy (a) administering a recombinant expression system according to any one of embodiments 1 to 11; an expression vector according to embodiment 12, a viral particle according to embodiment 13, or a pharmaceutical composition according to embodiment 14 to a patient suffering from a proliferative disorder, preferably cancer, in order to obtain a therapeutically effective transgenic expression of said target STING and / or said immunomodulatory protein, said administration being preferably intratumoral or systemic; (b) optionally treating the patient with radiation therapy prior to administration of (a), preferably radiation therapy directed to a tissue or organ intended to express the transduced gene from (a); and (c) treating the patient after administration with a STING agonist that recognizes the transgenically expressed target STING but does not recognize human STING and its naturally occurring variants, and / or radiation therapy, preferably radiation therapy directed to the tissue or organ intended to express the transduced gene from (a); Embodiment 18: The recombinant expression system, expression vector, viral particle, or pharmaceutical composition of embodiment 17, wherein in (c), a STING agonist according to embodiment 2 is used, preferably DMXAA. Embodiment 19: The recombinant expression system, expression vector, viral particle, or pharmaceutical composition of embodiment 17 or 18, wherein in (b) above, medium or low dose radiation therapy of 40 Gy or less, preferably 8 Gy or less is used.

[0109] It should also be clearly noted that indefinite articles and numerical indications such as "a", "an", "one", "two" and the like in the context of this application are generally to be understood as indications of "at least", i.e. "at least one", "at least two", etc., unless otherwise clearly understood from the respective context or it is obvious to a person skilled in the art or technically unavoidable that only "exactly one", "exactly two", etc. can be meant. Further advantages, features and details of the invention will become apparent from the following description, in which an embodiment of the invention is described in detail with reference to the drawings. In this regard, the features recited in the claims and in the specification may each be essential to the invention, individually or in any combination. The above description of the embodiments describes the invention only in the context of examples. Of course, individual features of the embodiments can be freely used with one another, as far as is technically appropriate, without departing from the scope of the invention.

[0110] definition "Substantially": As used herein, the term "substantially" refers to a qualitative condition that indicates the entire or nearly entire extent or degree of a feature or characteristic of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness that is inherent in many biological and / or chemical phenomena. As used approximately herein, the term "about" may be applied to modify any quantitative comparison, value, measurement, or other expression that can vary within acceptable limits without resulting in a change in the fundamental function with which it is associated. A / an: As used herein, the terms "a" or "an" may mean one or more than one. When used in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. Or / "And / or": The term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports the definition referring to alternatives only and "and / or." The term "about" generally means within the standard deviation of the stated value as determined using standard analytical techniques to measure the stated value. The term may also be used to refer to ±5% of the stated value. Essentially: It is to be understood that the term "essentially" includes only those methods or compositions that do not materially affect the specified steps or materials, and the basic and novel characteristics of those methods and compositions. Stereoisomers: Additionally, the compounds used in the present invention may contain one or more asymmetric carbon atoms and therefore exist in two or more stereoisomeric forms. Where the compounds contain an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur. The present invention includes the use of the individual stereoisomers of the compounds and, where appropriate, the individual tautomeric forms thereof, as well as mixtures thereof. Analog: As used herein, the term "analog" refers to a compound that is structurally similar to another, but differs slightly in composition (e.g., one atom is replaced by an atom of a different element or by a functional group).

[0111] Isotopes in compounds: The present invention also includes the use of all suitable isotopic variations of the compounds described herein or their pharma- ceutically acceptable salts. An isotopic variation of the compounds of the present invention or its pharma- ceutically acceptable salts is defined as one in which at least one atom is replaced by an atom with the same atomic number but with an atomic mass different from that usually found in nature. Examples of isotopes that can be incorporated into the compounds and their pharma- ceutical acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F and 36Cl, respectively. Certain isotopic variations of the compounds and their pharma- ceutical acceptable salts, such as those incorporating radioactive isotopes such as 3H or 14C, are useful in drug and / or substrate tissue distribution studies. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred due to their ease of preparation and detectability. Further substitution with isotopes such as deuterium, i.e., 2H, can provide certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances. Isotopic variants of the compounds of the present invention and pharma-ceutically acceptable salts thereof can generally be prepared by conventional procedures using the appropriate isotopic variants of suitable reagents.

[0112] Cytokine: The term "cytokine" refers to a general class of biological molecules that affect / have an effect on cells of the immune system. This definition is meant to include, but is not limited to, biological molecules that may act locally or circulate in the blood and, when used in the compositions or methods of the invention, function to regulate or modulate an individual's immune response to cancer. Cytokines can be a number of different substances, such as interferons, interleukins, chemokines, and growth factors. Exemplary cytokines for use in the practice of the invention include, but are not limited to, interferons (e.g., IFN-α, IFN-β, and IFN-γ), interleukins (e.g., IL-1 through IL-29, particularly IL-2, IL-7, IL-12, IL-15, and IL-18), tumor necrosis factors (e.g., TNF-α and TNF-β), chemokines (e.g., CXCL9, CXCL10), granulocyte colony stimulating factor (GM-CSF), TGF-β, FLT 3-ligand, and fragments or variants thereof. In particular, exogenously provided cytokines are a form of immunotherapy. Immunomodulator: As used herein, the term "immunomodulation" refers to the property of initiating or altering (e.g., increasing or decreasing) the activity of cells involved in an immune response. An immunomodulatory composition or method may increase the activity of cells involved in an immune response, e.g., by increasing pro- or anti-inflammatory markers, and / or may decrease the activity of cells involved in an immune response, e.g., by decreasing pro- or anti-inflammatory markers.

[0113] Agonist / human non-responsive STING agonist: As used herein, the term "agonist" refers to a ligand that stimulates the activity of a protein when bound to it. The term "human non-responsive STING agonist" refers to an agonist that does not substantially bind to human wild-type STING or naturally occurring variants thereof, but binds to mutant human STING or STING from other species, such as STING from mouse, rat or monkey. In some cases, the term "human non-responsive STING agonist" refers to an agonist that binds to human wild-type STING or naturally occurring variants thereof without inducing STING activity, but the STING activity of mutant human STING or STING from other species, such as STING from mouse, rat or monkey, is induced upon binding of the agonist. Thus, the term "human non-responsive" refers to an agonist that does not activate one of the endogenous human STING proteins encoded in the genome of a human subject (according to one of the known haplotypes of human STING), i.e., hSTING[R] represented by SEQ ID NO:1, hSTING[H] represented by SEQ ID NO:2, hSTING[AQ] represented by SEQ ID NO:3, hSTING[H] represented by SEQ ID NO:4, STING[Q] with the amino acid substitution R293Q, or STING[HAQ] with the amino acid substitutions R71H, G230A, and R293Q, and exogenously provided DMXAA-sensitive human STING reacts with the "human non-responsive" agonist.

[0114] Protein / Polypeptide: The terms "protein", "peptide" and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids long. A protein, peptide, or polypeptide can refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide can be modified, for example, by addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification. A protein, peptide, or polypeptide can be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof.

[0115] Antibody / Monoclonal Antibody: As used herein, the term "antibody" refers collectively to immunoglobulins or immunoglobulin-like molecules, including, but not limited to, IgA, IgD, IgE, IgG, and IgM and combinations thereof, and similar molecules produced during the immune response in any vertebrate, e.g., mammals such as humans, goats, rabbits, llamas, and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those of skill in the art, e.g., by fusing myeloma cells with immune spleen cells to create fusion antibody-forming cells. Monoclonal antibodies include humanized monoclonal antibodies. Single-chain variable fragment / scFv: The terms "single-chain variable fragment" and "scFv" refer to a fusion protein comprising at least one antibody fragment comprising the variable region of a light chain and at least one antibody fragment comprising the variable region of a heavy chain, where the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker and expressed as a single polypeptide chain, and where the scFv retains the specificity of the intact antibody from which it is derived. The term "nanobody" as used herein refers to the smallest antigen-binding fragment or single variable domain (VHH) derived from a naturally occurring heavy chain antibody, as known to those skilled in the art.

[0116] The term "blocking peptide" as used herein refers to a peptide that can interfere with the molecule and impair its interaction with the target molecule. For example, a blocking peptide that targets TGF-β impairs its immunosuppressive function.

[0117] Nucleic Acid: The term "nucleic acid" includes any nucleotide, its analogs, and polymers thereof. As used herein, the term "polynucleotide", "nucleic acid construct", or "nucleotide sequence" refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). The term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA and double- and single-stranded RNA. The term includes, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides, such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides. The term includes poly- or oligoribonucleotides (RNA) and poly- or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N- or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus atom bridges. The term encompasses nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges. Examples include, but are not limited to, nucleic acids containing a ribose moiety, nucleic acids containing a deoxyribose moiety, nucleic acids containing both a ribose moiety and a deoxyribose moiety, and nucleic acids containing a ribose moiety and a modified ribose moiety. Those skilled in the art will recognize that a variable number of nucleosides in a nucleic acid may be replaced with modified nucleosides, which may include derivatives of purine and pyrimidine bases, such as pseudouridine, N1-methylpseudouridine, 5-methyluridine, and 5-methylcytosine. In certain embodiments, the prefix "poly" is applied to sequences containing 2 to about 10,000, 2 to about 50,000, or 2 to about 100,000 nucleotide monomer units. In certain embodiments, "oligonucleotide" refers to a nucleic acid containing 2 to about 200 nucleotide monomer units. Gene: As used herein, the term "gene" refers to a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression. Also included in this definition are shRNAs. Hybridize: As used herein, the term "hybridizing" or "hybridizing" should be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands, thereby forming a duplex. Promoter: As used herein, the term "promoter" is used in its ordinary sense to refer to a nucleotide region that contains a DNA regulatory sequence, which is derived from a gene and is capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. It may include genetic elements to which regulatory proteins and molecules (e.g., RNA polymerase and other transcription factors) may bind to initiate specific transcription of a nucleic acid sequence. The terms "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence to control transcription initiation and / or expression of that sequence. Constitutive promoter: The term "constitutive promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes a gene product to be produced in a cell under most or all physiological conditions of the cell. Inducible promoter: The term "inducible promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a gene product to be produced in a cell only if an inducer that substantially corresponds to the promoter is present in the cell. STING-responsive promoter: As used herein, the term "STING-responsive promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a gene product to be produced in a cell substantially only if a polypeptide regulated by STING activity is present in the cell. Non-limiting examples of such polypeptides are cytokines, particularly interferons such as IFN-β, chemokines such as CXCL9 or CXCL10, transcription factors such as NFκB or STAT, interleukins such as IL-6, and TNF-β. Mutation: As used herein, the term "mutation" refers to the substitution of a residue in a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or the deletion or insertion of one or more residues in a sequence. Mutations are generally described herein by identifying the original residue, followed by identifying the position of the residue in the sequence, and identifying the newly substituted residue. Various methods for making amino acid substitutions (mutations) provided herein are well known in the art and are described, for example, in Green and Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012).

[0118] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between polypeptide molecules and / or between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules). In some embodiments, polymer molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides or amino acids at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of the percentage of identity between two sequences can be achieved using a mathematical algorithm. For example, the percentage of identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated in the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons performed using the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. Hybridization under stringent conditions: In the context of the present invention, hybridization under stringent conditions refers to hybridization conditions under which only long sequences with near perfect complementary matching will anneal reliably. This term is well known in the field of genetic engineering and has been used in patent practice for many years as a quasi-structural feature for defining DNA claims. In particular, each claim defines its subject matter not only by the function of hybridizing under stringent conditions, but also by a further functional feature, namely, that the polypeptides encoded by the claimed DNA sequences have at least the functional properties of STING.

[0119] Linked: As used herein, the term "linked," when used in reference to two or more moieties, means that the moieties are physically associated or connected to one another to form a molecular structure that is sufficiently stable so that the moieties remain associated under the conditions under which the linkage is formed, preferably under the conditions under which the new molecular structure is used, e.g., physiological conditions. In certain preferred embodiments of the invention, the linkage is a covalent bond. In other embodiments, the linkage is a non-covalent bond. Moieties can be linked either directly or indirectly. When two moieties are directly linked, they are either covalently bonded to one another or are in sufficient proximity that intermolecular forces between the two moieties maintain their association. When two moieties are indirectly linked, they are each linked, either covalently or non-covalently, to a third moiety, which maintains the association between the two moieties. In general, when two moieties are said to be linked by a "linker" or "linking moiety" or "linking portion," the linkage between the two linked moieties is indirect, and usually each of the linked moieties is covalently bonded to the linker. The linker can be any suitable moiety that reacts with the two moieties to be joined in a reasonable amount to provide a reasonable yield, within a reasonable period of time, under conditions consistent with the stability of the moieties (which can be protected as necessary depending on the conditions). Operably linked: As used herein, the terms "operably linked" or "operably linked" refer to a juxtaposition in a relationship permitting the described components to function in their intended manner. A control element, such as a promoter, "operably linked" to a functional element is associated such that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a coding element of interest; in some embodiments, the control element acts in trans with the functional element of interest.

[0120] RNA interference / microRNA / shRNA: As used herein, the term "RNA interference" generally refers to a process in which an RNA molecule reduces or inhibits the expression of a nucleic acid sequence with which it shares substantial or complete homology. Without wishing to be bound by any theory, it is believed that in nature, the RNA interference pathway is initiated by a type III endonuclease known as Dicer, which cleaves long double-stranded RNAs (dsRNAs) into "small interfering RNAs" ("siRNAs"), double-stranded fragments of 21-23 base pairs, usually with two-base 3' overhangs (variations in length and overhangs are contemplated). The siRNAs comprise two single-stranded RNAs (ssRNAs), an "antisense strand" or "guide strand" that includes a region that is substantially complementary to the target sequence, and a "sense strand" or "passenger strand" that includes a region that is substantially complementary to the region of the antisense strand. One of skill in the art will understand that the guide strand may be fully complementary to the target region of the target RNA, or may have less than perfect complementarity to the target region of the target RNA. The term "microRNA" or "miRNA" or "miR" as used herein refers to a specific type of siRNA that is not produced synthetically but is produced physiologically in an organism. The miRNA binds to the respective miRNA target sequence and reduces or inhibits the expression of a nucleic acid sequence with which the miRNA shares substantial or complete homology. The term "shRNA" or "small hairpin RNA" as used herein refers to an individual transcript that adopts a stem-loop structure that is processed into siRNA by the RNAi machinery. A typical shRNA molecule contains two inverted repeats that contain sense and antisense target sequences separated by a loop sequence. The base-paired segment can be of any suitable length that allows inactivation of a target gene in vivo, where one strand of the base-paired stem is complementary to the mRNA of said target gene. The loop of the shRNA stem-loop structure can be of any suitable length that allows inactivation of a target gene in vivo. The base-paired stem can be perfectly base-paired or can have one or two mismatched base pairs.

[0121] Target Gene / RNA: As used herein, a "target gene" refers to a gene whose expression is modulated, e.g., upregulated or inhibited. As used herein, the term "target RNA" refers to an RNA that is degraded or translationally repressed or otherwise inhibited using one or more agents, e.g., one or more miRNAs or siRNAs. A target RNA is also referred to as a target sequence or target transcript. The RNA can be a primary RNA transcript (e.g., pre-mRNA) transcribed from a target gene or a processed transcript, e.g., an mRNA that encodes a polypeptide. As used herein, the term "target portion" or "target region" refers to a contiguous portion of the nucleotide sequence of a target RNA. In certain embodiments, a target portion of an mRNA is at least sufficiently long to serve as a substrate for RNA interference (RNAi)-mediated cleavage within that portion in the presence of an appropriate miRNA or siRNA. The nucleotide length of the target portion can be about 8-36, e.g., about 10-20 or about 15-30. The length of the target portion can be a particular value or subrange within the ranges above. For example, in some embodiments, the nucleotide length of the target portion is about 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19- 28, 19-27, 19-26, 19-25, 19- 24, 19-23, 19-22, It can be 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22.

[0122] Recombinant: As used herein, the term "recombinant" refers to a nucleic acid or polypeptide that is designed, engineered, prepared, expressed, made, manufactured, and / or isolated by recombinant means (e.g., a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or otherwise engineered to express a gene or genetic component that encodes and / or directs the expression of a polypeptide or one or more components, portions, elements, or domains thereof; and / or a process that involves splicing or joining selected nucleic acid sequence elements together, It is intended to refer to a polypeptide that is prepared, expressed, produced, or isolated by any other means, including chemically synthesizing a selected sequence element and / or otherwise generating a nucleic acid that encodes and / or directs the expression of a polypeptide or one or more components, portions, elements, or domains thereof. In some embodiments, one or more of the selected sequence elements are found in nature. In some embodiments, one or more of the selected sequence elements are designed in silico. In some embodiments, one or more of the selected sequence elements result from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source, e.g., in the germline of a source organism of interest (e.g., human, mouse, etc.).

[0123] Naturally occurring variants: The term "naturally occurring variants" refers to variants of genes or transcripts that originate from the same genetic locus as the target nucleic acid, but may differ, for example, due to the degeneracy of the genetic code resulting in a diversity of codons that code for the same amino acid, or due to alternative splicing of pre-mRNA, or the presence of polymorphisms, such as single nucleotide polymorphisms (SNPs), and allelic variants. Such naturally occurring variants are naturally occurring and have not been artificially generated. In contrast, when the term "variant" is used without the associated word "naturally occurring", variants may include artificially generated variants, such as mutations. In the case of human STING, some known naturally occurring variants are STING[R], STING[AQ], STING[H], STING[HAQ], and truncated forms of STING[H]. Wild-type: As used herein, the term "wild-type" refers to the naturally occurring sequence of a nucleic acid at a locus in the genome of an organism and the sequence transcribed or translated from that nucleic acid. Thus, the term "wild-type" can be the amino acid sequence encoded by a nucleic acid. A locus can have more than one sequence or allele in a population of individuals, so the term "wild-type" encompasses all such naturally occurring alleles. As used herein, the term "polymorphism" means that there is a variation (i.e., there are two or more alleles) at a locus in individuals of a population, and the term "mutant" refers to a change in the sequence of a nucleic acid or its encoded protein, polypeptide, or peptide that is the result of recombinant DNA technology.

[0124] Non-human: As used herein, the term "non-human" refers to a gene or protein that is not substantially derived from a wild-type human gene or protein. In the context of the present invention, targeted mutations of human proteins are human, but hybridization of portions of human proteins with portions of homologous proteins of other species results in non-human.

[0125] Fusion STING: As used herein, the term "fusion STING" refers to STING in which parts of the protein sequence are derived from human STING and other parts are derived from a homologous STING from another species. Targeted STING: As used herein, targeted STING is STING that responds to a STING agonist that recognizes the targeted STING but does not recognize human STING and its natural variants. Thus, targeted STING may be a mutant human STING, but is not endogenous human STING (according to one of the known haplotypes of human STING) encoded in the genome of a human subject, i.e., hSTING[R] represented by SEQ ID NO: 1, hSTING[H] represented by SEQ ID NO: 2, hSTING[AQ] represented by SEQ ID NO: 3, hSTING[H] represented by SEQ ID NO: 4, STING[Q] with amino acid substitution R293Q, or STING[HAQ] with amino acid substitutions R71H, G230A, and R293Q. Thus, responsiveness to STING agonists is determined by "in vitro-DMXAA determination assay". Non-limiting examples of targeted STING are mouse STING or mutant human STING hSTING[R]-S162A / G230I / Q266I.

[0126] Cistron / monocistronic / bicistronic / polycistronic: As used herein, the term "cistron" refers to a portion of a DNA or RNA molecule that encodes a single protein or a single functional RNA molecule. The terms "monocistronic", "bicistronic" or "polycistronic" refer to the number of cistrons on a DNA or RNA molecule. For example, a bicistronic vector containing two cistrons under the control of one promoter results in the translation of two different proteins.

[0127] Exogenous / Xenogeneic: The term "exogenous" or "exogenous" when used with respect to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into a cell or organism by artificial or natural means, or with respect to a cell, refers to a cell that has been isolated and then introduced into another cell or organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or may be one or more additional copies of a nucleic acid that is naturally present in the organism or cell. An exogenous cell may be from a different organism, or may be from the same organism. As a non-limiting example, an exogenous nucleic acid is a nucleic acid that is present in a different chromosomal location than present in a natural cell, or that is adjacent to a different nucleic acid sequence than is otherwise found in nature.

[0128] Plasmid: A "plasmid" is a general type of vector, which is an extrachromosomal DNA molecule that can replicate independently of the chromosomal DNA. In certain cases, it is circular and double-stranded. Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting other nucleic acids to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA or RNA segments can be ligated to the viral genome. Certain vectors can replicate autonomously in the host cell into which they are transduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon transduction into the host cell, and are thereby replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". Those skilled in the art will understand that the "viral vectors" described herein include viral components, such as capsid proteins, in addition to the transduced genes described herein.

[0129] Expression system: "Expression system" or "expression construct" refers to one or more nucleic acid molecules capable of directing transcription. An expression system minimally contains one or more transcription control elements (such as promoters, enhancers or functional equivalents thereof) that direct gene expression in one or more desired cell types, tissues or organs. Additional elements such as transcription termination signals may also be included. Thus, the terms "expression system" and "recombinant expression system" encompass DNA or RNA nucleic acid molecules capable of directing transcription. In the context of the present invention, each DNA or RNA nucleic acid molecule is also referred to as a "nucleic acid construct."

[0130] AAV: The term "AAV" refers to adeno-associated virus and may be used to refer to the naturally occurring wild-type virus itself or its derivatives. The term encompasses all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, unless otherwise required. The AAV genome is constructed from a single strand of DNA and contains inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames: rep and cap, which code for replication and capsid proteins, respectively. The foreign polynucleotide may replace the native rep and cap genes. AAV can be produced with a variety of different serotype capsids with different transduction profiles, or "tropisms" as used herein, for different tissue types. The term "serotype" as used herein refers to an AAV, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrhlO, that is identified by and distinguished from other AAVs based on capsid protein reactivity with a defined antiserum. For example, serotype AAV2 is used to refer to an AAV that contains a capsid protein encoded from the cap gene of AAV2 and a genome that contains 5' and 3' ITR sequences from the same AAV2 serotype. A pseudotyped AAV refers to an AAV that contains a capsid protein from one serotype and a viral genome that contains the 5'-3' ITR of a second serotype. A pseudotyped rAAV is expected to have genetic properties that match the cell surface binding properties of the capsid serotype and the ITR serotype. A pseudotyped rAAV is produced using standard techniques described in the art. Adenovirus: The term "adenovirus" refers to any virus of the genus Adenovirus, including, but not limited to, the human, bovine, ovine, equine, canine, porcine, murine, and simian Adenovirus subgenera. Typically, adenoviral vectors are generated by transducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenoviral genome of an adenovirus to accommodate, for example, the insertion of a non-native nucleic acid sequence into the adenovirus for gene transduction.

[0131] Lentivirus: As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV-1 and HIV type 2); Visna-Maedi virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV).

[0132] Viral particle: As used herein, the term "viral particle" refers to all or a portion of a virion. For example, the viral particle may further comprise a recombinant genome and / or a capsid. The viral particle may be a gene therapy vector. As used herein, the terms "viral particle" and "vector" are used interchangeably. For purposes of this application, a "gene therapy" vector is a viral particle that may be used in gene therapy, i.e., a viral particle that contains all of the functional elements necessary to express a transduced gene, such as a CFI nucleotide sequence, in a host cell after administration. Suitable viral particles of the present invention include parvoviruses, retroviruses, lentiviruses, or herpes simplex viruses. The parvovirus may be an adeno-associated virus (AAV). In some cases, the viral particle is an AAV or adenovirus particle.

[0133] Host cell: As used herein, the term "host cell" refers to a cell that has been transduced with exogenous DNA (recombinant or otherwise). One of skill in the art will understand from this disclosure that the term refers not only to the particular subject cell, but also to the progeny of that cell. Because certain modifications may occur in subsequent generations, either by mutation or environmental influences, the progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In certain embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHOK1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT060562, Sertoli cells, BRL3A cells, HT1080 cells, MC38 cells, myeloma cells, tumor cells, and cell lines derived from the above cells. In certain embodiments, the cell contains one or more viral genes.

[0134] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In certain embodiments, "animal" refers to humans at any stage of development. In certain embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In certain embodiments, the animal includes, but is not limited to, a mammal, a bird, a reptile, an amphibian, a fish, and / or a worm. In certain embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0135] Treatment / Treating: "Treatment" or "treating" includes (1) inhibiting a disease, disorder, or condition in a subject or patient experiencing or exhibiting a pathology or symptom of the disease (e.g., arresting further development of the pathology and / or symptom); (2) ameliorating a disease, disorder, or condition in a subject or patient experiencing or exhibiting a pathology or symptom of the disease (e.g., reversing the pathology and / or symptom); and / or (3) causing any measurable decrease in a disease, disorder, or condition in a subject or patient experiencing or exhibiting a pathology or symptom of the disease. Prophylactically Treating: "Prophylactically Treating" includes (1) reducing or reducing the risk of developing a disease in a subject or patient who may be at risk for and / or susceptible to a disease, but who has not yet experienced or displayed any or all of the pathology or symptoms of the disease, and / or (2) delaying the onset of a disease pathology or symptom in a subject or patient who may be at risk for and / or susceptible to a disease, but who has not yet experienced or displayed any or all of the pathology or symptom of the disease.

[0136] Local administration / local delivery: As used herein, with respect to the delivery of a recombinant expression system, expression vector, viral particle, or pharmaceutical composition, the term "local administration" or "local delivery" refers to delivery that does not depend on the transport of the product to its intended target tissue or site via the vascular system. The product disclosed herein may be delivered directly to its intended target tissue or site, or in the vicinity thereof, e.g., in close proximity to the intended target tissue or site. For example, the product may be delivered by injection or implantation of a composition or compound, or by injection or implantation of a device containing the composition or compound. After local administration in the vicinity of a target tissue or site, the product described herein, or one or more components thereof, may diffuse to the intended target tissue or site. It is understood that a portion of the product described herein (usually only a small portion of the administered dose) when delivered locally may enter the vascular system and be transported elsewhere, including back to its intended target tissue or site. The term "local administration" or "local delivery," in the context of delivery of a viral particle or pharmaceutical composition described herein, refers to delivery that may rely on transport of the viral vector or pharmaceutical composition via the vascular system to its intended target tissue or site.

[0137] Co-administration: The term "co-administration" as used herein with respect to two or more compounds, e.g., therapeutic agents, is administration that is performed using doses and time intervals such that the administered compounds are present together in the body, e.g., at one or more sites of action in the body, in non-negligible amounts, over a time interval. The time interval can be minutes (e.g., at least 1 minute, 1-30 minutes, 30-60 minutes), hours (e.g., at least 1 hour, 1-2 hours, 2-6 hours, 6-12 hours, 12-24 hours), days (e.g., at least 1 day, 1-2 days, 2-4 days, 4-7 days, etc.), weeks (e.g., at least 1, 2, or 3 weeks, etc.). Thus, the compounds may, but need not, be administered together as part of a single composition. Additionally, the compounds may, but need not, be administered essentially simultaneously (e.g., within less than 5 minutes, or less than 1 minute apart) or within a short time of each other (e.g., less than 1 hour, less than 30 minutes, less than 10 minutes, about 5 minutes apart). In various embodiments of the present disclosure, compounds administered within the time interval may be considered to be administered substantially simultaneously. In certain embodiments of the present disclosure, the co-administered compounds are present in the body (e.g., in the blood and / or at the site of local complement activation) at effective concentrations over the time interval. When co-administered, the effective concentration of each of the compounds required to induce a particular biological response may be lower than the effective concentration of each compound when administered alone, thereby reducing the dosage of one or more of the compounds compared to the dosage required when the compounds are administered as a single compound. The effect of multiple compounds may be, but is not limited to, additive or synergistic. The compounds may be administered multiple times. A non-negligible concentration of a compound may be, for example, less than about 5% of the concentration required to induce a particular biological response, e.g., a desired biological response.

[0138] Sequential administration: As used herein, the term "sequential administration" of two or more compounds refers to administering two or more compounds to a subject such that the compounds are not both present in the subject's body or at related sites of activity in the body at concentrations greater than negligible. The administration of the compounds may or may not be staggered. Each compound may be administered multiple times.

[0139] Systemic Administration: As used herein, the term "systemic administration" and similar terms are used herein consistently with their usage in the art to refer to administration of a compound (e.g., a therapeutic agent, expression vector, viral particle, pharmaceutical composition, and / or formulation) such that the compound is distributed widely throughout the body in significant quantities, has its biological effect, e.g., its desired effect, in the blood, and / or reaches its desired site of action via the vascular system. Exemplary systemic administration routes include (i) administration by direct transduction of the compound into the vascular system, or (ii) subcutaneous, oral, pulmonary, or intramuscular administration, where the compound is absorbed, enters the vascular system, and is carried via the blood to one or more desired sites of action.

[0140] Intratumoral administration: The term "intratumoral administration" refers to administration of a compound (e.g., a therapeutic agent, an expression vector, a viral particle, a pharmaceutical composition, and / or a formulation) such that the compound is present within a tumor following administration. One of skill in the art will appreciate that administration thereby can occur directly within the tumor or in close proximity to the tumor.

[0141] As used herein, the terms "subject," "test subject," or "patient" refer to any organism to which a provided compound or composition is administered in accordance with the present invention, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Exemplary subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms, etc.) and plants. In certain embodiments, a subject may be suffering from and / or susceptible to a disease, disorder, and / or condition.

[0142] Effective / therapeutically effective amount: As used herein, the term "effective" or "therapeutically effective amount" refers to at least a minimal amount of a compound (e.g., a therapeutic agent, expression vector, viral particle, pharmaceutical composition, and / or formulation) that induces a desired biological response when administered as part of a treatment regimen. In certain embodiments, a therapeutically effective amount of a substance is an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of skill in the art, an effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, improves, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or signs of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose, and in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. In the case of cancer or tumors, an effective amount of a compound may have the effect of reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., slowing or preferably stopping to some extent) cancer cell invasion into peripheral organs; inhibiting (i.e., slowing or preferably stopping to some extent) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating to some extent one or more symptoms associated with the disorder.

[0143] Combination of expression vectors / combination of viral particles: As used herein, the term "combination of expression vectors" or "combination of viral particles" means that a first expression vector or a first viral particle is administered to a patient before, during (including simultaneously - preferably formulated simultaneously) and / or after treatment of the individual with a second expression vector or a second viral particle, respectively. The combined expression vectors or viral particles may be used for simultaneous, sequential or separate administration. In all cases, it is preferred that any of the expression vectors and viral particles described herein are administered in a pharmacologic effective amount, i.e., an administration that includes a total amount of each component of the combination sufficient to show a meaningful patient benefit. The formulations can be conveniently provided in unit dosage form by methods known to those skilled in the art.

[0144] Kit of parts: As used herein, a "kit of parts" is a kit that provides a combination of a first and a second expression vector or a combination of a first and a second viral particle for co-administration. The kit may preferably contain the vectors / particles, for example, in a dosage form for administration. The dosage form contains a sufficient amount of one or more vectors / particles to obtain a desired effect when administered to a subject. Thus, the medical packaging preferably contains dosage units in an amount corresponding to the relevant dosing regimen. Preferably, the kit of parts includes instructions indicating the use of the dosage form to achieve a desired effect and the amount of the dosage form to be taken over a particular period of time.

[0145] Immunotherapy: As used herein, the term "immunotherapy" refers to a therapy that utilizes immune-modulating genes or proteins to modulate or stimulate the immune system to treat a disease, preferably a proliferative disease, such as cancer. Immunotherapy can result in the stimulation of the immune system, which is often used in immunotherapy to target cancer. However, other diseases, such as autoimmune diseases, diabetes, cardiovascular diseases, and inflammation, can also be treated with immunotherapy. For example, IFN-β is used to treat multiple sclerosis. In addition, IL-2 has been shown to modulate the balance between regulatory and effector T cells, thereby preserving β-cell function. Immunotherapy can be used to treat non-cancer diseases, such as lupus, rheumatoid arthritis, and diabetes.

[0146] Immune checkpoint inhibitor: As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a compound that inhibits a protein in a checkpoint signaling pathway. Proteins in a checkpoint signaling pathway include, for example, CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM-3, TIGIT, Lair1, CD244, HAVCR2, CD200, CD200R1, CD200R2, CD200R4, LILRB4, PILRA, ICOSL, 4-1BB, or VISTA. Immune checkpoint inhibitors are known in the art. For example, an immune checkpoint inhibitor can be a small molecule. As used herein, "small molecule" refers to a composition having a molecular weight in the range of about 5 kDa to less than 50 Da, e.g., less than about 4 kDa, less than about 3.5 kDa, less than about 3 kDa, less than about 2.5 kDa, less than about 2 kDa, less than about 1.5 kDa, less than about 1 kDa, less than 750 Da, less than 500 Da, less than about 450 Da, less than about 400 Da, less than about 350 Da, less than 300 Da, less than 250 Da, less than about 200 Da, less than about 150 Da, less than about 100 Da. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic or inorganic molecules. Alternatively, the immune checkpoint inhibitor is an antibody or a fragment thereof. For example, the antibody or fragment thereof is specific for a protein in a checkpoint signaling pathway, such as CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM-3, TIGIT, Lair1, CD244, HAVCR2, CD200, CD200R1, CD200R2, CD200R4, LILRB4, PILRA, ICOSL, 4-1BB, or VISTA.Exemplary anti-immune checkpoint antibodies include, for example, ipilimumab (anti-CTLA-4), pembrolizumab (anti-PD-L1), nivolumab (anti-PD-L1), atezolizumab (anti-PD-L1), duralumab (anti-PD-L1), or relatolimab (anti-LAG-3).

[0147] Adoptive Cell Therapy: As used herein, the term "adoptive cell therapy" refers to a therapy in which autologous or allogeneic immune cells are infused into a patient to treat certain diseases, such as cancer. For this purpose, various cell types can be used, such as dendritic cells, natural killer (NK) cells, or T cells. Adoptive T cell therapy: As used herein, the term "adoptive T cell therapy" refers to a therapy in which autologous or allogeneic T cells are infused into a patient to treat certain diseases, such as cancer. One type of adoptive T cell therapy is to isolate tumor infiltrating leukocytes (TILs) from a tumor, optionally expanding the cells outside the patient, and infusing them back into the patient. Another type of adoptive T cell therapy is chimeric antigen receptor (CAR) T cell therapy. CAR-T cells can be engineered in several ways to enhance tumor cytotoxicity and specificity, circumvent tumor immune suppression, circumvent host rejection, and extend their therapeutic half-life.

[0148] Cancer vaccine: As used herein, the term "cancer vaccine" refers to a vaccine that either treats existing cancer or prevents the development of cancer.

[0149] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which two or more different compounds (e.g., therapeutic agents, expression vectors, viral particles, pharmaceutical compositions, and / or formulations) are administered in an overlapping regimen such that the subject is exposed to both compounds simultaneously. When used in combination therapy, the two or more different compounds may be administered simultaneously or separately. The combination administration can include simultaneous administration of two or more compounds in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, two or more compounds can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more compounds can be administered simultaneously, where the compounds are in separate formulations. In another alternative, one or more additional compounds can be administered after administration of a first compound. In additional administration protocols, the two or more compounds may be administered minutes or hours, days, or weeks apart. In some embodiments, the two or more compounds may be administered 1-2 weeks apart.

[0150] Radiotherapy: The term "radiotherapy" hereinafter generally refers to the medical use of ionizing radiation as part of cancer treatment to control or kill malignant cells. Ionizing radiation may be based on photon radiation such as X-rays or gamma rays or particle radiation, the particles of the particle radiation being preferably selected from the group consisting of beta particles, alpha particles, neutrons, muons, pions, protons or other heavier cations. It may also be used as part of adjuvant therapy to prevent tumor recurrence after surgery to remove the primary malignant tumor. Radiotherapy may be synergistic with other anti-cancer therapies and may be used before, during and after other anti-cancer therapies in susceptible cancers. The term "low dose radiotherapy" hereinafter refers to radiotherapy using a dose of ionizing radiation less than 40 Gy, preferably less than or equal to 10 Gy, more preferably less than or equal to 8 Gy. Low dose radiation therapy can be any radiation therapy using a dose of ionizing radiation less than 40 Gy, 38 Gy, 36 Gy, 34 Gy, 32 Gy, 30 Gy, 28 Gy, 26 Gy, 24 Gy, 22 Gy, 20 Gy, 19 Gy, 18 Gy, 17 Gy, 16 Gy, 15 Gy, 14 Gy, 13 Gy, 12 Gy, 11 Gy, 10 Gy, 9 Gy, 8 Gy, 7 Gy, 6 Gy, 5 Gy, 4 Gy, 3 Gy, 2.5 Gy, 2.0 Gy, 1.5 Gy, 1 Gy, 0.5 Gy, 0.25 Gy, 0.2 Gy, 0.15 Gy, 0.1 Gy, 0.05 Gy, or 0.01 Gy.

[0151] Proliferative disorder: As used herein, the term "proliferative disorder" refers to a condition in which unregulated or abnormal proliferation of cells, or both, can lead to the development of an undesirable condition or disease, which may or may not be cancerous. Exemplary cell proliferative disorders of the present disclosure encompass a variety of conditions in which cell division is deregulated. Exemplary cell proliferative disorders include, but are not limited to, neoplasms, benign tumors, malignant tumors, precancerous conditions, in situ tumors, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immunological tumors, hematological tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing cells. Cancer / Tumor: The term "cancer" or "tumor" refers to the presence of cells that exhibit characteristics typical of cells from which cancer arises, such as uncontrolled proliferation, immortality, invasive or metastatic potential, rapid growth, and certain characteristic morphological features. In certain embodiments, the cells exhibit such characteristics due in part or in whole to the expression and activity of immune checkpoint proteins, such as PD-1, PD-L1, PD-L2, and / or CTLA-4. Cancer cells are often in the form of tumors, although the cells may exist alone in an animal or may be non-tumorigenic cancer cells (e.g., leukemia cells). As used herein, the term "cancer" includes pre-malignant as well as malignant cancers.Cancers include: various cancers, bladder cancer (including accelerated and metastatic bladder cancer), breast cancer, colon cancer (including colorectal cancer), kidney cancer, liver cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer and lung adenocarcinoma), ovarian cancer, prostate cancer, testicular cancer, genitourinary tract cancer, lymphatic system cancer, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), esophageal cancer, stomach cancer, gallbladder cancer, cervical cancer, thyroid cancer, and skin cancer (including squamous cell carcinoma); leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia hematopoietic malignancies of the lymphoid lineage, including B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphosarcoma, non-Hodgkin's lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burkitt's lymphoma; hematopoietic malignancies of the myeloid lineage, including acute and chronic lymphocytic leukemia, myelodysplastic syndrome, myeloid leukemia, and promyelocytic leukemia; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma, tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; melanoma, xeroderma pigmentosum, keratoacanthoma, and seminiferous epithelium tumors including thyroid cancer, follicular carcinoma of the thyroid, and teratocarcinoma; melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioblastoma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colon cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, bile duct cancer, breast cancer, colon cancer, head and neck cancer, gastric cancer, germ cell tumors, bone cancer, bone tumors, adult malignant fibrous histiocytoma of bone, childhood cancer, malignant fibrous histiocytoma of bone, sarcoma, childhood sarcoma, adenocarcinoma These include, but are not limited to, various cancers such as nasal natural killer, neoplasms, plasma cell neoplasms myelodysplastic syndromes, neuroblastoma, testicular germ cell tumors, intraocular melanoma, myelodysplastic syndromes, myelodysplastic / myeloproliferative disorders, synovial sarcoma, chronic myelogenous leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL), multiple myeloma, acute myelogenous leukemia, chronic lymphocytic leukemia, mastocytosis and any condition related to mastocytosis, and any metastasis thereof.Additionally, the disorders include mastocytosis, such as pigmented urticaria, diffuse cutaneous mastocytosis, human solitary mastocytoma, several rare subtypes such as bullous mastocytosis, erythrodermic mastocytosis, and telangiectatic mastocytosis, mastocytosis with associated hematological disorders such as myeloproliferative syndromes and myelodysplastic syndromes, acute leukemia, myeloproliferative disorders associated with mastocytosis, mast cell leukemia, and other cancers. Other cancers are also included within the scope of disorders, including: bladder cancer, urothelial carcinoma, hepatocellular carcinoma, hepatoblastoma, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, testicular cancer, especially testicular seminoma, skin cancer, carcinomas including squamous cell carcinoma, gastrointestinal stromal tumors (GIST), leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T hematopoietic tumors of the lymphatic system, including acute and chronic myeloid leukemia, promyelocytic leukemia, tumors of mesenchymal origin, such as fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, tetrasarcoma, neuroblastoma and glioma, tumors of the central and peripheral nervous system, such as astrocytoma, neuroblastoma, glioma and schwannoma; and other tumors, including, but not limited to, melanoma, xeroderma pigmentosum, keratinizing acanthoma, seminoma, thyroid follicular carcinoma, teratocarcinoma, chemotherapy-resistant non-seminomatous germ cell tumor, Kaposi's sarcoma, and metastases thereof.Other non-limiting examples of cancer types amenable to the methods of the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bone cancer, brain tumor, lung cancer (lung adenocarcinoma) leukemias, such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is epithelial in nature, including, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, neuroendocrine tumors, carcinoid tumors, Merkel cell carcinoma, or skin cancer.

[0152] Anti-cancer: An "anti-cancer" agent can negatively affect cancer cells / tumors in a subject, for example, by promoting the death of cancer cells, inducing apoptosis in cancer cells, reducing the rate of proliferation of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the progression of cancer, or extending the lifespan of a subject with cancer.

[0153] Pharmaceutical composition: The term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of an active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. The formulation is sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can be reasonably administered to a mammalian subject to provide an effective dosage of the active ingredient employed.

[0154] Synergy / Synergistic Effect: The term "synergism" or "synergistic effect" refers to a combined effect of two or more compounds that is greater than the sum of the separate effects of the cancer compounds / therapies alone.

[0155] In vitro agonist determination assay / in vitro DMXAA determination assay: The term "in vitro agonist determination assay" refers to an assay that can identify human non-responsive STING agonists, which activate human STING or mutants of non-human STING, but do not activate human wild-type STING or its naturally occurring mutants. The term "in vitro-DMXAA determination assay" refers to an assay that can identify STING that responds to DMXAA. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above may generally be performed by conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the specification. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose. EXAMPLES

[0156] STING response to DMXAA in vitro In the first set of three experiments, we investigated differential STING responses to DMXAA. Experiment 1: Dose-response of DMXAA on different STING The aim of this experiment was to test which STING is sensitive to the STING agonist DMXAA and whether there is a dose-dependence to DMXAA. As a readout, the expression of the downstream target IFN-β was measured by quantitative real-time PCR (qPCR). Materials and Methods: For this purpose, cells derived from either the mouse cell line MC38 or the human cell line Hep2G were transduced in vitro with lentiviruses encoding either wild-type mouse STING (mSTINGwt), wild-type human STING (hSTINGwt) or the human triple mutant STING-S162A / G230I / Q266I (hSTINGmut). Transduced cells were sorted by flow cytometry based on reporter protein expression, thereby obtaining stable cell lines. Each stable cell was grown at 750 cells / mm in growth medium. 2Cells were seeded in 24-well plates at a density of 1000 ng / ml. 24 hours after seeding, cells were incubated with different doses of DMXAA for an additional 3 hours (MC38 cells) or 6 hours (Hep2G cells). Cells were harvested, then homogenized, and mRNA was extracted using the Maxwell® RSC simplyRNA Tissue Kit protocol (Promega, Madrid, Spain). RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase (Promega, Madrid, Spain) according to the manufacturer's instructions. Quantitative real-time PCR was performed using a CFX-Connect real-time PCR detection system (BioRad, Hercules, CA) to quantify expression levels. All analyses were performed in a total volume of 10 μL containing 5 μL of 1×IQ SYBR Green Supermix (Bio-Rad Life Science, Hercules, CA), 0.2 μL of each primer (15 μM, 300 nM final concentration), 2.6 μL of distilled water, and 2 μL of template cDNA. The primers annealing to each gene in the study are detailed in Table 5. Expression of IFN-β was measured for either experimental group, and expression levels were quantified relative to vehicle-treated cells.

[0157] [Results and Discussion] As shown in Figure 1A-C, IFN-β expression upon DMXAA treatment was elevated in either human or mouse cells transduced with DMXAA-sensitive STING hSTINGmut or mSTINGwt in a dose-dependent manner. In contrast, no significant change in IFN-β expression was observed in DMXAA-insensitive hSTINGwt. Since IFN-β expression is upregulated upon STING activation, this experiment indicates that i) DMXAA can activate DMXAA-sensitive STING, which has STING biological activity, and ii) the dose of DMXAA correlates with STING activity. Thus, DMXAA activates DMXAA-sensitive STING, but not hSTINGwt.

[0158] Experiment 2: DMXAA-mediated cytokine expression. The aim of this experiment was to investigate whether different STINGs exhibited STING biological activity upon DMXAA treatment. As a readout, the expression of STING-derived cytokines was measured by qPCR. Materials and Methods: For this purpose, MC38 and Hep2G were transduced with lentiviruses encoding luciferase, hSTINGwt, hSTINGmut, or were not transduced with any lentivirus (no vector) to obtain stable cell lines as described above. Each cell was grown at 750 cells / mm in growth medium. 2 Cells were seeded in 24-well plates at a density of 100 μg / mL. 24 hours after seeding, cells were incubated with 50 μg / mL DMXAA for an additional 3 hours (MC38 cells) or 6 hours (Hep2G cells). Cells were harvested and prepared for qPCR measurements as described above. Expression of cytokines IFN-β, TNF-α, IL-6, CXCL9 and CXCL10 was measured for either experimental group, and expression levels were quantified relative to the control (no vector). [Results and Discussion] As shown in Figure 1D-E, the expression of the above cytokines was elevated only in hSTINGmut transduced cells, while no significant changes in expression levels were observed in any of the other groups. This further supports the findings of the first experiment that i) DMXAA can activate DMXAA-sensitive STING, which has STING biological activity. Furthermore, this experiment shows that hSTINGmut exhibits STING biological activity in that it results in upregulation of known STING downstream proteins (Liu et al., 2020 PNAS 117(24)).

[0159] Experiment 3: Time-dependent expression of STING-derived cytokines by DMXAA treatment The aim of this experiment was to investigate the time course of STING-derived cytokine expression upon DMXAA treatment. As a readout, STING-derived cytokine expression was measured by qPCR. Materials and Methods: For this purpose, MC38 and Hep2G stable cell lines encoding hSTINGmut were obtained as described above. 24 hours after seeding, cells were incubated with 50 μg / mL DMXAA for the indicated time points. Cells were harvested and prepared for qPCR measurements as described above. Expression of cytokines IFN-β, TNF-α, IL-6, CXCL9 and CXCL10 was measured for either experimental group, and expression levels were quantified relative to the control (0 hours after DMXAA treatment). [Results and Discussion] As shown in Figure 1F-G, the temporal expression profiles of the different cytokines differed for each cytokine and between MC38 and Hep2G cells, but overall, increased expression levels were observed upon DMXAA treatment for any given time point. Thus, the expression of the investigated cytokines was upregulated in a DMXAA-dependent manner and thus dependent on STING activity, but the different temporal expression patterns indicate that this expression also depends on the cell line used and each cytokine investigated. EXAMPLES

[0160] STING response to DMXAA in vivo Having demonstrated the general sensitivity of hSTINGmut to DMXAA in vitro, the aim of the present experiment was to examine the response of hSTINGmut to DMXAA in vivo. Materials and Methods: Six to 10-week-old C57 wild-type mice were transfected with AAV8-hSTINGmut (1 × 10 11 viral genome) or control GFP (AAV8-GFP, 1.5 × 10 11The study involved intravenously administering an AAV8 vector encoding either the IFN-β or IFN-β viral genome. Two weeks after transduction, mice were injected with different DMXAA doses (5mg DMXAA / kg body weight, 10mg DMXAA / kg body weight, and 20mg DMXAA / kg body weight). Three hours after injection, blood was collected from each mouse, and IFN-β protein levels in serum were measured by VeriKine-HSTM (high sensitivity). Mouse IFN-β serum P ELISA (pbl Assay Science, Piscataway NJ USA). Results and Discussion As shown in Figure 2, administration of 20 mg DMXAA / kg body weight already caused an increase in IFN-β levels in AAV8-GFP transduced control animals after the first injection, but IFN-β levels increased further after repeated injections. This was expected, since the mice express mouse STING (mSTINGwt), which is already sensitive to DMXAA and shows IFN-β upregulation in vitro (s. Example 1, Experiment 1). Notably, IFN-β levels were more than 10-fold higher in AAV8-hSTINGmut transduced mice at any given time point compared to AAV8-GFP controls. In both cases, systemic STING stimulation caused high toxicity and mortality (as can be seen by the reduced number of surviving animals with successive DMXAA injections). However, doses of 5 or 10 mg DMXAA / kg body weight showed significantly lower levels of IFN-β in AAV8-hSTINGmut transduced mice, and furthermore, did not cause significant toxicity or mortality. The data show that i) exogenous STING expression dose-dependently enhances IFN responses to STING agonists, ii) systemic STING activation is associated with unacceptable toxicity, and iii) induction of IFN-β in transduced tissues can be triggered on demand by injection of the STING agonist DMXAA. EXAMPLES

[0161] STING activity-dependent promoters Since DM XAA-induced STING activity caused enhanced cytokine expression levels upon transduction with either hSTINGmut or mSTINGwt, the aim of the following experiments was to develop a system in which target genes could be expressed in transduced cell lines dependent on STING activity. Therefore, the aim of the following four experiments was to construct vectors with STING activity-responsive promoters. Test 1: IFN-β promoter One of the major cytokines upregulated upon DMXAA-mediated STING activation is IFN-β, making the IFN-β promoter a feasible target to test its function as a STING activity-dependent promoter. Therefore, the aim of this experiment was to investigate the specificity of different modified IFN-β promoters in response to DMXAA-induced STING activity. Materials and Methods: For this purpose, five different modifications of the IFN-β promoter (short IFN-βA as set forth in SEQ ID NO: 18, short IFN-βB as set forth in SEQ ID NO: 19, short IFN-βC as set forth in SEQ ID NO: 20, short IFN-βD as set forth in SEQ ID NO: 21, short IFN-βE as set forth in SEQ ID NO: 22) were designed. Lentiviral vectors were constructed that code for luciferase under the control of the five different promoters. The vectors were used to lentivirally transduce MC38 cell lines stably expressing hSTINGmut as described above (Example 1), whereas the control group received no further lentiviral transduction (none). 24 hours after seeding, cells were treated with DMXAA (50 μg / mL) or vehicle. After an additional 3 hours (Figure 3A) or 3 and 6 hours (Figures 3B, 3C, 3D) or 6 hours (Figures 3E, 3F, 3G, and 3h), cells were homogenized in 50 μL of Passive Lysis Buffer (Promega) and frozen in dry ice. After three cycles of thaw-vortex-freeze to ensure lysis, cells were spun at 12000 g for 2 min at 4° C. 20 μL was added to a luminometer 96-well plate and luciferase activity was determined using the respective substrates D-luciferin and coelenterazine. [Results and Discussion] As shown in Figure 3A, each of the above promoters showed luciferase activity upon DMXAA administration, but cells transduced with only hSTINGmut (i.e., cells lacking a vector encoding luciferase, indicated as "none" in Figure 3A) showed no luciferase activity regardless of DMXAA administration. In particular, the short IFN-βA promoter showed the highest luciferase activity upon DMXAA treatment compared to the other promoters. However, significant luciferase activity was observed even in the absence of DMXAA. This indicates that although the short IFN-βA promoter correlates with activation of hSTINGmut, the specificity of this promoter is somewhat low, since basal luciferase activity is observed even when hSTINGmut is not activated by DMXAA (leaky expression). Similarly, the other promoters also showed significant leaky expression. Furthermore, the overall responsiveness of the other promoters to DMXAA-activated STING was rather low.

[0162] Experiment 2: Chemokine promoters Next to IFN-β, the chemokines CXCL9 and CXCL10 also showed clear upregulation upon DMXAA-mediated STING activation, making the CXCL9- and CXCL10-promoters feasible targets to test their function as STING activity-dependent promoters. Therefore, the aim of this experiment was to examine the specificity of the promoters in their response to DMXAA-induced STING activity. Materials and Methods: The CXCL9 promoter (CXCL9 as set forth in SEQ ID NO: 23) and the CXCL10 promoter (CXCL10 as set forth in SEQ ID NO: 24) were used in the following experiments. For this purpose, lentiviral vectors were constructed that encode luciferase under the control of these two different promoters. The vectors were used to lentivirally transduce MC38 cell lines stably expressing hSTINGmut as described above (Example 1), but the control group did not receive any further lentiviral transduction (control). In addition, lentiviral vectors encoding the short IFN-βA, short IFN-βB and short IFN-βC promoters as described in Example 1 were used as comparisons. 24 hours after seeding, cells were treated with DMXAA (50 μg / mL) or vehicle. After a further 3 or 6 hours, cells were harvested and luciferase activity was measured as described in Experiment 1. [Results and Discussion] As shown in Figure 3B, each of the above promoters showed luciferase activity upon DMXAA administration, but cells transduced with hSTINGmut alone (control) showed no luciferase activity regardless of DMXAA administration. In particular, the CXCL9-promoter and CXCL10-promoter showed significantly higher luciferase activity compared to the IFN-β promoter. However, significant luciferase activity was observed for both chemokine promoters even in the absence of DMXAA. This indicates that although the promoters produce high luciferase expression upon DMXAA administration, the specificity of the promoters is somewhat low, since basal luciferase activity is observed even when hSTINGmut is not activated by DMXAA (leaky expression).

[0163] Experiment 3: Functionality of STING activity-dependent promoters Experiments 1 and 2 demonstrate that the IFN-β, CXCL9- and CXCL10-promoters, when operably linked to a luciferase transgene, are upregulated upon DMXAA-induced STING activation. The purpose of the following experiments was to further demonstrate the functionality of the promoters by operably linking them to the expression of the immunomodulatory single chain interleukin-12 (scIL-12). Materials and Methods: The CXCL9 and CXCL10 promoters from Example 2 and the full-length IFN-β promoter (IFN-β as set forth in SEQ ID NO: 25) were used in the following experiments. For this purpose, a lentiviral vector was constructed encoding mouse scIL-12 under the control of the three different promoters. The vector was used to lentivirally transduce an MC38 cell line stably expressing hSTINGmut as described above (Example 1), while a control group did not receive further lentiviral transduction (no vector). 24 hours after seeding, cells were treated with DMXAA (50 μg / mL) or vehicle. After the indicated time points, cells were harvested. Cell lysates were used to measure scIL-12 mRNA expression by qPCR as previously described (Example 1, Experiment 1), while supernatants were used to measure secreted scIL-12 protein in NUNC MaxiSorp flat-bottom 96-well plates using an ELISA assay kit (OptEIA, mouse IL-12 (p70), BD Biosciences, San Diego, CA USA) according to the manufacturer's instructions. [Results and Discussion] As shown in Figure 3C-D, the cell line transduced with hSTINGmut alone showed low expression of scIL-12 mRNA in the cell line, regardless of DMXAA administration, and did not secrete scIL-12 at detectable levels. The function of the IFN-β promoter and CXCL9-promoter for scIL-12 mRNA or protein expression was poor even upon DMXAA treatment. In contrast, scIL-12 expression was highly upregulated 6 hours after DMXAA treatment and control of the CXCL10 promoter, and significant amounts of scIL-12 protein were secreted into the supernatant. However, according to previous experiments, the CXCL10 promoter showed significant leakage in that scIL-12 was produced even in the absence of DMXAA. This indicates that the general concept of a STING activity-dependent promoter is feasible, but the considerable leakage of the promoter used in the experiment makes it necessary to test other promoters that are highly inducible upon DMXAA administration with high specificity for STING activity.

[0164] Experiment 4: Synthetic promoters As mentioned above, the specificity of the promoters used in the previous experiments was somewhat low. Therefore, the aim of the following experiments was to find other promoters that combine high inducibility upon DMXAA administration with high specificity for STING activity. To achieve this, synthetic promoters have been created. Materials and Methods: Seven different promoters were generated, namely short STATx4 (four STAT motifs separated by six nucleotides according to SEQ ID NO:26), IRSF3x4 (four IRSF3 motifs separated by 20 nucleotides according to SEQ ID NO:27), short IRSF3x4 (four IRSF3 motifs separated by six nucleotides according to SEQ ID NO:28), ISREx4 (four ISRE motifs separated by 20 nucleotides according to SEQ ID NO:12), short ISREx4 (four ISRE motifs separated by six nucleotides according to SEQ ID NO:13), NFκBx4 (four NFκB motifs separated by 20 nucleotides according to SEQ ID NO:29) and short NFκBx4 (four NFκB motifs separated by six nucleotides according to SEQ ID NO:30). All the above promoter sequences given by SEQ ID NOs:12, 13 and 26-30 do not contain any further transcriptional regulatory elements that affect the transcription efficiency of the transduced gene. The promoters are themselves functional promoters and do not require any further elements to bring about the transcription of the operably linked transduced gene. pGL3-basic plasmids were designed containing luciferase sequences under the control of the respective promoters. For clarity, pGL 3-basic plasmids do not contain promoter-like sequences, but promoters are exclusively incorporated into the plasmids by SEQ ID NOs: 12, 13 and 26-30 above. In addition, a plasmid containing luciferase sequences under the control of mouse IFN-β promoter (mIFN-β) and a plasmid containing luciferase sequences under the control of CXCL10-promoter were used as controls. Either MC38 cells or human HEK293T cells (both stably expressing hSTINGmut as above (Example 1)) were transfected with the indicated plasmids using Lipofectamine3000 according to the manufacturer's protocol, whereas the negative control was mock transfected (Basic). 24 hours after transfection, cells were treated with DMXAA (50 μg / mL) or vehicle, and luciferase activity was measured after a further 48 hours as described in Experiment 1. Results and Discussion: As shown in Figure 3E-F, both ISRE promoters as well as both NFκB promoters caused an increase in luciferase activity in MC38 cells upon DMXAA-mediated STING activation. However, the NFκB-promoter also showed considerable luciferase activity in the absence of DMXAA treatment. In contrast, the ISRE-promoter showed high inducibility with low leakage upon DMXAA stimulation. The short STAT-promoter and both IR SF-promoters showed poor inducibility. The validity of the assay is supported by the fact that no luciferase activity was observed in mock-transfected controls (basic), regardless of DMXAA treatment. As shown in Figure 3G-H, both ISRE promoters and both NFκB promoters increased luciferase activity in HEK293T cells upon DMXAA-mediated STING activation. Furthermore, compared to mouse MC38 cells, leakage of either promoter was significantly reduced in HEK293T cells. However, no detectable leakage could be observed for the ISRE promoter, whereas the NFκB promoter still leaked some in the absence of DMXAA treatment. The highest inducibility was observed for the CXCL10 promoter, which showed considerable leakage. The inducibility of the other promoters was much lower, but the leakage was also limited. Thus, the ISREx4 promoter showed the highest inducibility, but at the same time the leakage was undetectable. In summary, it should be noted that the ISREx4 promoter exhibits leakage in MC38 cells but not in 293T cells, the former being a tumor cell line and the latter a non-malignant cell line of embryonic origin. The tumor cell lines often harbor chromosomal abnormalities and micronuclei in the cytoplasm, which sustain smoldering STING activation, low levels of IFN-β production, and low-grade activation of STING-responsive promoters. EXAMPLES

[0165] STING activity-dependent IL-12 expression in vivo The ISREx4-promoter was the most highly inducible while maintaining a significantly low leakiness, making it the most promising promoter for in vivo applications. Therefore, the aim of the following experiments was to analyze the translational potential of STING activity-mediated IL-12 expression in vivo. Experiment 1: IL-12 production in the absence of DMXAA-mediated STING induction in vivo The aim of the first experiment was to examine whether IL-12 is produced in vivo in transduced cells even in the absence of DMXAA-mediated STING activation. Materials and Methods An adeno-associated virus (AAV) of serotype 8 (AAV8) encoding scIL-12 under the control of the ISREx4 promoter (AAV8-ISREx4-IL12) was constructed, as well as an AAV8 control vector encoding scIL-12 under the control of the murine IFN-β promoter (AAV8-mIFN-β-IL12). For this purpose, three different doses were administered, namely 1 × 10 10 Viral genome, 5 × 10 10 Viral genome and 1×10 11 The viral genomes AAV8-ISREx4-IL12 or AAV8-pIFN-β-IL12 were intravenously injected into different groups of 6-10 week old C57 wild type mice (n=3 per group) and IL-12 serum levels were measured 7 days later as described in Example 2. [Results and Discussion] As shown in Figure 4A, IL-12 expression was dose-dependent, as higher viral doses caused increased IL-12 production for both AAVs. In particular, expression was obviously higher upon intravenous administration of AAV8-ISREx4-IL12 compared to AAV8-pIFN-β-IL12. Thus, although expression levels were higher for AAV8-ISREx4-IL12, both AAVs caused the production of IL-12 in vivo, even when DMXAA was not administered. Therefore, measures must be taken to reduce leaky IL-12 production in vivo.

[0166] Experiment 2: Inhibition of IL-12 leakage by microRNA binding sites in vivo As shown in the above experiments, upon intravenous administration of AAV8-ISREx4-IL12, IL-12 is produced regardless of the presence of DMXAA, which may cause adverse side effects. Many AAV serotypes, including AAV8, target primarily the liver when present in the blood circulation. Even in the case of intratumoral injection, tumors are usually highly vascularized, so that a portion of AAV may enter the blood circulation and thereby transduce hepatocytes to express IL-12 under the control of the ISRE promoter. The liver is exposed to multiple pathogen-derived molecules of intestinal origin that can induce IFN type I production, which results in the production of IL-12. Since IL-12 is a potent immunostimulatory, but cytotoxic, cytokine, it is important to restrict IL-12 expression to target tissues and prevent IL-12 production in the liver. One way to limit the expression of a protein is to intervene at the post-transcriptional level, for example by miRNA binding and subsequent mRNA degradation. Therefore, to control IL-12 expression, we selected a less leaky STING-responsive promoter and operably linked the IL-12 sequence to a binding site for microRNA122 (miR122), which is highly abundant in hepatocytes, in an attempt to suppress hepatic IL-12 production. [Materials and Methods] A nucleotide sequence was constructed in which five repeats of the miR122 target sequence were placed at the 3' end of the IL-12 sequence. An AAV vector containing this construct under the control of the ISREx4 promoter was constructed, and each viral particle was produced (AAV8-ISREx4-IL12miR122T). Two groups of 6- to 10-week-old C57 wild-type mice (n=4 each) were injected with AAV8-ISREx4-IL12 or AAV8-ISREx4-IL12miR122T (0.5×10 11 Viral genome) were intravenously injected into mice, and IL-12 serum levels were measured 7 days after vector injection as described above. [Results and Discussion] As shown in Figure 4B, animals treated with a vector containing the miR122 binding site (AAV8-ISREx4-IL12miR122T) had undetectable serum levels of IL-12, whereas values ​​above 10 ng / ml were found in mice receiving AAV8-ISREx4-IL12. Thus, undesirable basal hepatic production of IL-12 can be avoided by controlling its expression via the miR122 binding site.

[0167] Experiment 3: Inhibition of IL-12 leakage by microRNA binding sites in the presence of DMXAA in vivo Since the unwanted basal hepatic production of IL-12 can be avoided by controlling its expression via the miR122 binding site, the aim of the following experiments was to elucidate whether the unwanted hepatic production of IL-12 could be achieved even in the presence of the STING agonist DMXAA. Materials and Methods: For this purpose, 6- to 10-week-old C57 wild-type mice were transfected with an AAV8 vector encoding hSTINGmut under the control of the constitutive CAG promoter (AAV8-CAG-hSTINGmut, 1 × 10 11 viral genome) and an ISRE promoter and five miR122 binding sites at the 3' end (AAV8-ISREx4-IL12miR122T, 0.5 × 10 11 viral genome) or without (AAV8-ISREx4-IL12, 0.5 × 10 11 The animals were intravenously injected with the above AAV8 vector containing the viral genome (IL-12) and single chain IL-12 (n=4 animals / group). One week later, the animals were challenged intravenously with DMXAA (20 mg DMXAA / kg body weight), vehicle, or not challenged at all (basal). After 4 hours, 150 μL of blood was collected and IL-12 serum levels were quantified by ELISA as described above. [Results and Discussion] As shown in Figure 4C, mice injected with an IL-12 vector without miR122 binding site (AAV8-ISREx4-IL12) had increased basal serum levels of IL-12, which were further increased upon DMXAA administration, whereas animals injected with an IL-12 vector containing miR122 binding site (AAV8-ISREx4-IL12miR122T) showed almost undetectable IL-12 serum levels at basal status and extremely low levels after DMXAA challenge. Thus, AAV8-ISREx4-IL12miR122T shows little leakage even in the presence of DMXAA. EXAMPLES

[0168] Therapeutic efficacy of intratumoral coinjection of AAV8-hSTINGmut+AAV8-ISREx4-IL12miR122T followed by repeated administration of DMXAA The aim of the following experiments was to analyze whether co-injection of AAV8-hSTINGmut+AAV8-ISREx4-IL12miR122T into tumors followed by repeated DMXAA administration would show a therapeutic effect as measured by a reduction in tumor size. Materials and Methods: For this purpose, we used a mouse model of colon cancer generated by subcutaneous injection of MC38 cells in syngeneic STING-deficient C57 mice (C57BL / 6J-Tmem173gt / J, STING KO). In these animals, DMXAA does not activate STING systemically, but only in tissues transduced with a vector encoding hSTINGmut, thus mimicking the human situation. In this experiment, MC38 subcutaneous tumors were generated in STING KO mice by subcutaneous injection of 500,000 MC38 cells. Tumor nodules were approximately 70 mm 3 When they reached the size of 11 Viral genome) + AAV8-ISREx4-IL12miR122T (0.5 × 10 11 In other mouse control groups, tumors were injected with AAV8-hSTINGmut (1 × 10 11 Viral genome) + AAV8-GFP (0.5 × 1011 Viral genome) and then received vehicle twice weekly (n=3 animals). Results and Discussion As shown in Figure 5, clear antitumor effects were observed in the two groups treated with AAV8-hSTINGmut+AAV8-ISREx4-IL12miR122T, but the antitumor effects were significantly higher in mice additionally treated with DMXAA. Notably, complete tumor elimination was observed in one of six mice treated with DMXAA, but not in mice given vehicle. In contrast, the tumor volume of mice receiving AAV8-GFP+vehicle treatment continued to grow throughout the time course of the experiment. The antitumor effects in mice treated with AAV8-hSTINGmut+AAV8-ISREx4-IL12miR122T given vehicle are attributed to the low level of intratumoral production of IL-12 resulting from the low basal activation of the ISREx4 promoter in MC38 cells, as described above. Importantly, the treatment was well tolerated without any symptoms of toxicity in any of the animals. The strategy described herein was designed to exert a therapeutic effect without toxicity. To avoid toxicity, the AAV vector expressing IL-12 was constructed in such a way that IL-12 is synthesized in the tumor mass but not in the liver, thus eliminating the risk of persistent basal hepatic production of IL-12 by AAV particles that access the blood and liver after their intratumoral injection. On the other hand, an agonist is used that interacts with transgenic STING but not with endogenous human STING. Thus, DMXAA given by intravenous or oral route activates STING in the tumor but does not make the treatment acceptable systemically. The slight activation of transgenic STING expressed by the small fraction of AAV vectors that were able to reach the liver after intratumoral injection caused only a slight increase in type I IFN and IL-12 in serum, less than 0.2 pg / mL (data not shown), which is remarkably well tolerated by experimental animals. EXAMPLES

[0169] Intratumoral transduction efficiency during radiotherapy It is known that different DNA damaging agents, including topoisomerase inhibitors and radiation, have been shown to enhance AAV transduction, possibly by inducing DNA repair synthesis (Russell DW, et al. PNAS 1995;92,5719-5723; Alexander IE et al J Virol 1994;68,8282-8287). AAV is a single-stranded DNA vector that needs to be converted to double-stranded DNA to form a transcription template. The DNA damage response induced by radiation therapy helps convert single-stranded DNA to double-stranded DNA (Peng D. et al. J Hepatol.2000 Jun;32(6):975-85). Using radiation therapy to enhance tumor transduction by AAV vectors is an attractive option, since radiation is frequently given to treat solid tumors. Published data on AAV transduction and radiation refer to in vitro studies using different radiation doses and in vivo studies using high radiation doses. However, high radiation doses significantly alter vascular damage and tissue DNA structure. The aim of the following experiments was to investigate whether low-dose radiation could also enhance intratumoral transduction efficiency. Lower radiation doses preserve tumor vasculature and thus allow lymphocyte trafficking to the tumor. Materials and Methods: MC38 subcutaneous tumors were generated in 6-10 week old C57 mice by subcutaneous injection of 500,000 MC38 cells. Tumor nodules were approximately 70 mm 3 When the tumors reached a size of 1 × 10, they were injected with AAV8 encoding GFP under a constitutively active CAG promoter (AAV8-GFP, 1 × 10 11In a group of mice (N=3), tumors were irradiated with focal medium / low dose radiation (8 Gy) using SARRP (Small Animal Radiation Research Platform, Xstrahl Inc. Suwanee, GA, USA) immediately prior to intratumoral AAV injection, whereas in a control group (N=6), tumors were not irradiated. Two days after injection, immunohistochemical sections of tumors were prepared. Sections were incubated overnight at 4°C with anti-GFP (1:2000, Abcam, ab6556). After rinsing with TBS-T, sections were incubated with goat anti-rabbit labeled polymer EnVisionTM+System (Dako, K400311-2). After treatment with 1000 mM NaCl (Glostrup, Denmark) for 30 min, peroxidase activity was revealed using DAB+ (Dako, K346811-2). Finally, sections were lightly counterstained with Harris hematoxylin, dehydrated, and coverslipped with Eukitt (Labolan, Arizona, 28500). Sections were examined by a pathologist to obtain a subjective assessment of tissue transduction, and they were also scanned using a CS2 Aperio scanner, and the proportion of pixels with strong GFP staining was quantified with Aperio software. [Results and Discussion] As shown in Figure 6A, tumors that did not receive radiation before intratumoral AAV injection showed a transduction efficiency of approximately 10-15%. However, tumors that received 8 Gy radiation therapy immediately before AAV injection showed a transduction efficiency of approximately 25-40% of the tumor area. As shown in Figure 6B, when tumor sections were analyzed using Aperio software, a statistically significant increase in the amount of strong GFP signal relative to the total area was observed in tumors that received 8 Gy compared to non-irradiated tumors. The data show that pretreatment of tumors with low-dose radiation resulted in a marked enhancement of tumor transduction by AAV vectors. EXAMPLES

[0170] Radiotherapy-induced STING activation in vitro Radiation has been shown to increase IFN-β production by tumor cells, an effect mediated by STING. The aim of this experiment was to investigate whether IFN-β production could be further increased by exogenous STING. Materials and Methods: For this purpose, a lentiviral vector encoding hSTINGmut was used to generate MC38 cells stably expressing hSTINGmut as described above (Example 1), except that in the control group, cells were not transduced. Cells were trypsinized, counted, and diluted to 500.000 cells / mL in complete growth medium. The cell suspension was divided into equal volumes and incubated at 750 cells / mm 2 Immediately prior to seeding at a density of 100 μg / ml, cells were either non-irradiated or irradiated for 90 s (8 Gy) in a Gammacell Elan 3000 (Nordion, Canada). At designated time points after irradiation, supernatants were harvested and IFN-β protein levels were quantified by ELISA as described above. Results and Discussion: As shown in Figure 7, IFN-β production remained at low levels throughout the time course of the experiment for cells that were not irradiated and did not express exogenous hSTINGmut. The same was true for cells that expressed exogenous hSTINGmut but were not irradiated. However, with radiation alone, upregulation of IFN-β expression began 24 hours after radiation and was further elevated when hSTINGmut was exogenously expressed. Thus, the effect of radiation on IFN-β production by tumor cells is enhanced by increasing the abundance of STING cells. EXAMPLES

[0171] Local irradiation followed by intratumoral injection of vectors encoding the expression system induces tumor regression and the abscopal effect. Radiation therapy has been shown to induce STING-mediated IFN-β production in tumor cells (Deng L. et al Immunity, 2014;41,843-852). The previous examples show that enrichment of STING increases the production of IFN-β by irradiated cells. By linking IFN-β expression with IL-12 production, the following experiments analyzed whether a strong antitumor effect could be achieved by irradiating and transducing the expression system into tumors. Experiment 1: Antitumor activity of combination treatments without DMXAA. The aim of the first experiment was to investigate whether the synergistic effects observed in vitro are applicable to the in vivo situation and whether combined treatment with radiotherapy and immuno-gene-therapy exerts a strong antitumor effect. Therefore, MC38 tumors were subcutaneously implanted in wild-type C57 mice and then treated with radiotherapy alone, combined immuno-gene therapy and radiotherapy, or no treatment at all. Materials and Methods: Group 1 (n = 4) had MC38 tumors that had not been irradiated or transduced with any vector; Group 2 (n = 5) had tumors irradiated with 8 Gy as above and immediately transduced with AAV8-hSTINGmut (1.0 × 10 11 Viral genome) + AAV8-ISREx4-IL12miR122T (0.5 × 10 11 Group 3 (n = 4) were irradiated with 8 Gy as above and immediately thereafter injected with AAV8-GFP (1.5 × 10 11 The mice were treated with MC38 tumor-bearing IL-12 ELISA kits, which received intratumoral injections of the IL-12 virus genome. Tumor size was measured twice weekly using electronic calipers and volume was calculated ((length x width2) / 2). Additionally, in the second part of the experiment, blood was collected 17 days after transduction and IL-12 serum levels were quantified by ELISA as described above (Example 4). Mice were euthanized when tumors reached 17 mm on the longest axis. [Results and Discussion] As shown in Figure 8A, in the control groups (groups 1 and 3), tumors continued to grow throughout the time course of the experiment. However, due to radiation therapy, tumor growth in group 3 progressed at a slower rhythm than in group 1. In contrast, animals in group 2 eliminated tumors remarkably efficiently. As shown in Figure 8B, serum IL-12 remained undetectable at the time of tumor elimination in four out of five mice in group 2. Of note, the IL-12 serum level in this one mouse was clearly lower than in the previous experiment (Example 4, Experiment 1) that did not use the miRNA122 target site. Serum levels in groups 1 and 3 were undetectable in all mice. The findings point to the concept that radiotherapy-induced DNA damage activates STING, which in turn activates the promoter driving IL-12 expression, leading to increased intratumoral levels of type I IFN and IL-12, resulting in tumor rejection. Furthermore, IL-12 was contained in the tumor, thereby preventing undesirable toxicity. In summary, by combining radiotherapy and immunogen therapy according to the present invention, a synergistic effect is also observed in vivo.

[0172] Experiment 2: Abscopal effect of combined treatment without DMXAA As mentioned above, the combination therapy including radiation therapy and immune gene therapy resulted in the disappearance of tumors in all mice. Without wishing to be bound by the treatment, it is believed that immune-modulating molecules such as IL-12 stimulate the immune system to target tumor cells that normally manage to evade the immune system (tumor escape). Thus, even when applied intratumorally, the treatment can stimulate the immune system to target tumors outside the injection site. This is known as the abscopal effect, where local treatment also affects untreated tumors (e.g., metastases). Materials and Methods: In this study, MC38 tumors were implanted into the right flank of 6-10 week old C57BL / 6 wild type mice by subcutaneous injection of tumor cells as described above (Example 6). 5 MC38 tumor cells were inoculated simultaneously into the left flank. Right flank tumors were 70–100 mm 2When tumor volume reached 1.5×10, mice were randomized into different treatment groups (n=6 mice per group). All groups received right flank irradiation with 8 Gy and subsequent intratumoral injection of the respective AAV 11 days after tumor inoculation as above, while the tumor in the left flank was left untreated. Group 1 received AAV8-GFP (1.5×10 11 Group 2 received intratumoral injection of AAV8-hSTINGmut (1.0 × 10 viral genome), and Group 3 received intratumoral injection of AAV8-hSTINGmut (1.0 × 10 11 Viral genome) + AAV8-GFP (0.5 × 10 11 Group 3 received intratumoral injection of AAV8-hSTINGmut (1.0 × 10 viral genome), and Group 4 received intratumoral injection of AAV8-hSTINGmut (1.0 × 10 11 Viral genome) + AAV8-ISREx4-IL12miR122T (0.5 × 10 11 Group 4 received intratumoral injection of AAV8-GFP (1.0 × 10 viral genome), and Group 5 received intratumoral injection of AAV8-GFP (1.0 × 10 11 Viral genome) + AAV8-ISREx4-IL12miR122T (0.5 × 10 11 Mice received intratumoral injections of 1000 μg / mL of 1000 μg / mL of the viral genome. Tumor size was measured twice weekly using electronic calipers and volume was calculated ((length × width2) / 2). Mice were euthanized when tumors reached 17 mm on their longest axis. Results and Discussion: As shown in Figure 8C, the tumor progression of treated tumors (right flank) in groups 1 and 3 was similar to previous experiments. Notably, intratumoral injection with AAV8-hSTINGmut but not AAV8-ISREx4-IL12miR122T (group 2) did not significantly affect tumor progression compared to the AAV8-GFP control group. However, intratumoral injection with AAV8-ISREx4-IL12miR122T but not AAV8-hSTINGmut (group 4) caused tumor elimination with a similar course to the combined treatment (group 3). Notably, the course of tumor progression of untreated tumors (each left flank) was similar, though less pronounced, than for treated tumors, as shown in Figure 8D. This study confirms the potent antitumor effect of therapy on treated tumors, resulting in complete tumor rejection in animals given low-dose radiation therapy together with intratumoral injection of AAV8-hSTINGmut+AAV8-ISREx4-IL12miR122T, and further shows that the combination therapy is also accompanied by a significant abscopal effect. EXAMPLES

[0173] Synergy between the canonical STING pathway activated by DMXAA and the non-canonical STING pathway activated by radiation therapy in vitro As previously reported, radiotherapy-induced DNA damage is known to activate STING. However, this activation is limited due to low expression of cGAS / STING in many tumors (Dunphy et al., 2018, Molecular Cell 71, 745-760). Recently, it has been shown that DNA damaging agents activate STING by a non-canonical cGAMP-independent pathway that favors the NFκB pathway over IFN type I production. Since DMXAA is a can...

Claims

1. below: (c) a first nucleic acid construct comprising a sequence encoding a target STING, the target STING being responsive to a STING agonist that recognizes the target STING but not human STING and its naturally occurring variants (a human non-responsive STING agonist), and the sequence encoding the target STING being operably linked to elements that allow the expression and / or translation of the target STING; and / or (d) a second nucleic acid construct comprising a sequence encoding at least one immune modulatory gene or protein, the sequence being operably linked to a STING-responsive promoter or a radiation-inducible promoter; A recombinant expression system comprising:

2. 2. The recombinant expression system of claim 1, wherein the targeted STING is responsive to a human non-responsive STING agonist selected from the group consisting of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), 7-bromo-DMXAA, 7-iodo-DMXAA, 7-hydroxy-DMXAA, 7-formyl-DMXAA, 7-hydroxymethyl-DMXAA, 7-(2-hydroxyethyl)-DMXAA, flavone-8-acetic acid (FAA), 2,7-bis(2-dimethylaminoethoxy)fluoren-9-one (tilorone) and 10-carboxymethyl-9-acridan (CMA), wherein preferably the targeted STING is responsive to DMXAA.

3. The target STING encoded by the sequence of the first nucleic acid construct is selected from the group consisting of: (a) a mutated form of the naturally occurring variant of human STING, which further comprises at least one, at least two, or at least three amino acid substitutions that render the human STING variant sensitive to the human non-responsive STING agonist as determined in an in vitro agonist determination assay; or (b) a non-human STING, preferably a mouse STING or a fusion STING, even more preferably a mouse STING represented by SEQ ID NO: 7, that is sensitive to said human non-responsive STING agonist as determined in an in vitro agonist determination assay; wherein the target STING is preferably DMXAA-sensitive human STING as determined by an in vitro DMXAA determination assay.

2. The recombinant expression system of claim 1.

4. The target STING encoded by the sequence of the first nucleic acid construct is selected from the group consisting of: (a) a human STING[R] variant further comprising at least one, at least two, or at least three amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I; (b) a human STING[AQ] mutant further comprising at least one, at least two, or at least three amino acid substitutions selected from the group consisting of S162A, A230I, Q266I, E260I, and E260V; (c) a human STING[H] mutant further comprising at least one, at least two, or at least three amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I; and (d) a truncated human STING[H] mutant further comprising at least one or at least two amino acid substitutions selected from the group consisting of S162A and G230I; 4. The recombinant expression system of claim 3, wherein the DMXAA-sensitive human STING mutant is selected from the group consisting of:

5. The DMXAA-sensitive human STING mutant encoded by the sequence of the first nucleic acid construct has the following structure: hSTING[R]-S162A / G230I / Q266I, hSTING[R]-S162A / Q266I, hSTING[R]-G230I, hSTING[R]-S162A, hSTING[R]-Q266I, hSTING[R]-S162A / G2 30I, hSTING[AQ]-S162A / A230I / Q266I, hSTING[AQ]-S162A, hSTING[AQ]-S162A / R169A, hSTING[AQ]-S162A / T267A, hSTING[AQ]-E260I, hST 5. The recombinant expression system of claim 4, wherein the DMXAA-sensitive human STING mutant is selected from the group consisting of hSTING[AQ]-E260V, hSTING[H]-S162A / G230I / Q266I, hSTING[H]-S162A / Q266I, hSTING[H]-G230I, hSTING[H]-S162A, hSTING[H]-S162A / G230I and hSTING[H]-Q266I, wherein the DMXAA-sensitive human STING mutant is preferably hSTING[R]-S162A / G230I / Q266I.

6. The sequence of the first nucleic acid construct encoding the DMXAA-sensitive human STING mutant is as follows: (a) SEQ ID NO:5; (b) a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:5; (c) a polynucleotide sequence encoding a polypeptide comprising SEQ ID NO:6; (d) a polynucleotide sequence encoding a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:6; (e) a polynucleotide sequence encoding a polypeptide of (c) or (d) above, which is modified by at least one of the following: an amino acid substitution, an amino acid insertion, an amino acid deletion, a C-terminal truncation, or an N-terminal truncation; (f) a polynucleotide sequence that hybridizes under at least stringent conditions to the complement of any of the polynucleotide sequences of (a) to (e); and (g) a region of the polynucleotide sequence of (a) to (f) encoding a fragment of at least 50 amino acid residues of the DMXAA-sensitive human STING mutant. and wherein the polypeptide encoded by any of the polynucleotides (a)-(g) has a biological activity that is responsive to the agonist DMXAA as determined in an in vitro DMXAA determination assay.

7. the promoter of the first construct is a constitutive promoter, preferably selected from the group consisting of SV40, CMV0, UBC, EF1a, PGK, CAG, EFS, CBh, CBA, human β-actin, Ac5, polyhedrin, TEF1, GDS, CaMV35S, Ubi, H1 and U6; Or, the promoter of the first construct is an inducible promoter, preferably selected from the group consisting of a telomerase responsive promoter, an IL-6 responsive promoter, a TGF-β inducible promoter, a VEGFR inducible promoter, an epidermal growth factor receptor inducible promoter, a HER2 / Neu inducible promoter, a MUC1 promoter, a BRCA1 promoter, a BRCA2 promoter, a LALBA promoter, a PSA promoter, an ERBB2 promoter, a CEA promoter, a uPAR promoter and a thyroid transcription factor 1 (TTF1); 2. The expression system of claim 1.

8. The at least one immunomodulatory gene or protein encoded by the sequence of the second genetic construct is selected from the group consisting of interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-7, interleukin-8, interleukin-9, interleukin-11, single chain interleukin-12, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21, interleukin-22, interleukin-23, interleukin-24, interleukin-25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, interleukin-30, interleukin-31, interleukin-32, interleukin-33, interleukin-34, interleukin-35, interleukin-36, interleukin-37, interleukin-38, interleukin-39, interleukin-40, interleukin-41, interleukin-42, interleukin-43, interleukin-44, interleukin-45, interleukin-46, interleukin-47, interleukin-48, interleukin-49, interleukin-50, interleukin-51, interleukin-52, interleukin-53, interleukin-54, interleukin-55, interleukin-56, interleukin-57, interleukin-58, interleukin-59, interleukin-60, interleukin-61, interleukin-62, interle Interleukin-15, Interleukin-15-sushi, Interleukin-16, Interleukin-17, Interleukin-18, Interleukin-19, Interleukin-20, Interleukin-21, Interleukin-22, Interleukin-23, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16 , CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, granulocyte macrophage a blocking peptide targeting phage colony stimulating factor, type I interferon, IFN-γ, TNF-α, FLT3-ligand, TGF-β, a blocking peptide targeting IL-10, a blocking peptide targeting FoxP3, a monoclonal antibody or single chain variable fragment (scFv) or nanobody that neutralizes PD1, PDL1, CTLA4, CD137, TIM3, LAG3, and fragments or variants thereof; or shRNA targeting TGF-β, shRNA targeting IL-10, or shRNA targeting FoxP3; 2. The recombinant expression system of claim 1.

9. At least one immunomodulatory protein is selected from the group consisting of: (a) the α subunit of IL-12; (b) the β subunit of IL-12; (c) single-chain IL-12 containing the α and β subunits of IL-12; (d) a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10; (e) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9 or 10 and having interleukin-12 biological activity; (f) IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO: 79; (g) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:79 and having IFN-β biological activity; (h) IL-15-sushi comprising or consisting of the amino acid sequence of SEQ ID NO: 80; (i) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 80, wherein the polypeptide has IL-15 biological activity; (j) CXCL10 comprising or consisting of the amino acid sequence of SEQ ID NO: 81; and (k) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 81 and having CXCL10 biological activity; 2. The recombinant expression system of claim 1, selected from the group consisting of:

10. The second nucleic acid construct comprises at least two sequences each encoding a different immunomodulatory protein, wherein the first immunomodulatory protein is a single chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO:9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:9 or 10 and having the biological activity of interleukin-12; and the second immunomodulatory protein is an IFN-β that comprises or consists of the amino acid sequence of SEQ ID NO:79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:79 and that has the biological activity of an IFN-β; or IL-15-sushi comprising or consisting of the amino acid sequence of SEQ ID NO:80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:80 and having the biological activity of IL-15; or a CXCL10 comprising or consisting of the amino acid sequence of SEQ ID NO:81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:81 and having the biological activity of CXCL10; 2. The recombinant expression system of claim 1.

11. The second nucleic acid construct is a bicistronic or polycistronic expression cassette comprising two or more sequences each encoding a different immunomodulatory protein or anti-cancer protein or RNA, wherein: (a) said sequence is separated by a sequence encoding an IRES; and / or (b) said sequence is separated by a sequence encoding a 2A peptide, preferably a sequence encoding a P2A peptide; 2. The recombinant expression system of claim 1.

12. The second said STING responsive promoter comprises an IFN-β responsive promoter, an IFN-β minimal promoter, a CXCL9 responsive promoter, a CXCL10 responsive promoter, a STAT motif promoter, an IRSF3 motif promoter, an ISRE motif promoter, an NFκB motif promoter, and a STAT6 motif promoter, wherein the STING responsive promoter preferably comprises at least one interferon stimulated response element (ISRE), and more preferably comprises one of the following: (a) a nucleotide sequence comprising 1 to 10 copies, preferably 2 to 7 copies, more preferably 3 to 5 copies, and particularly preferably 4 copies, of a sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to SEQ ID NO: 11; (b) a nucleotide sequence according to (a), wherein the copies are separated by up to 40 nucleotides, preferably from 4 to 35 nucleotides, more preferably from 10 to 30 nucleotides, particularly preferably from 15 to 25 nucleotides; (c) the nucleotide sequence set forth in SEQ ID NO: 12; and (d) the nucleotide sequence set forth in SEQ ID NO:13; Selected from the group consisting of:

2. The recombinant expression system of claim 1.

13. The second nucleic acid construct further comprises at least one sequence motif that inhibits the transgenic expression of an immunomodulatory protein in tissues or cells not intended to express said protein, said tissue being preferably the liver, said sequence motif being a target sequence of a microRNA that is preferably highly abundant in the liver, more preferably said target sequence being selected from the group consisting of miR-122 target sequence, miR-192 target sequence, miR-199a target sequence, miR-101 target sequence, miR-99a target sequence, let7a target sequence, let7b target sequence, let7c target sequence, let7f target sequence, and even more preferably the following: (a) a miR-122 target sequence that has at least 90% or at least 95% sequence identity to, or is identical to, SEQ ID NO:14; (b) a nucleotide sequence comprising 1 to 12, preferably 3 to 10, more preferably 4 to 8, and particularly preferably 5 copies of the miR-122 target sequence of (a); (c) the nucleotide sequence according to (b) above, in which the copies are separated by 1 to 40 nucleotides, preferably by 1 to 30 nucleotides, more preferably by 2 to 20 nucleotides, particularly preferably by 2 to 8 nucleotides; and (d) a nucleotide sequence containing five copies of the miR-122 target sequence shown in SEQ ID NO: 15; A miR-122 target sequence selected from the group consisting of:

2. The recombinant expression system of claim 1.

14. An expression vector encoding the first and / or second nucleic acid construct of claim 1, or a combination of a first and a second expression vector, wherein the first vector encodes the first nucleic acid construct of claim 1 and the second vector encodes the second nucleic acid construct of claim 1, Wherein, the expression vector is preferably a viral vector, more preferably a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, a vaccinia viral vector, and a herpes simplex viral vector, and even more preferably has the following characteristics: (a) the sequence of the first and / or second nucleic acid construct further comprises a 5' ITR sequence and a 3' ITR sequence, preferably the AAV ITRs set forth in SEQ ID NO: 17; and (b) the AAV vector preferably has the serotype AAV8, AAV1, AAV3, AAV6, AAV9, AAV2, AAV5, AAVrh.10, or any gain-of-function mutant of AAVrh.10, more preferably the AAV vector has the serotype AAV2; is an adeno-associated virus vector having one or more of An expression vector or a combination of expression vectors.

15. 15. A viral particle comprising a viral vector or a combination of viral vectors according to claim 14, wherein a first viral particle comprises a viral vector encoding the first nucleic acid construct according to claim 14, and a second viral particle comprises a viral vector encoding the second nucleic acid construct according to claim 14, wherein the viral particle is preferably an AAV or adenoviral particle, more preferably an AAV viral particle.

16. Pharmaceutically acceptable excipients and (a) the recombinant expression system of claim 1; or (b) the expression vector of claim 14; or (c) the virus particle according to claim 15 13. A pharmaceutical composition comprising:

17. 17. A recombinant expression system according to claim 1, an expression vector according to claim 14, a viral particle according to claim 15 or a pharmaceutical composition according to claim 16 for use in therapy, preferably in immunotherapy, more preferably in immunotherapy of a proliferative disorder, even more preferably in immunotherapy of cancer, especially preferably in immunotherapy of solid cancers.

18. below: (a) a therapy comprising at least one checkpoint inhibitor, preferably selected from the group consisting of an inhibitor of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR, more preferably selected from the group consisting of an inhibitor of PD-1, PD-L1, PD-L2, or CTLA-4; and / or (b) a therapy comprising at least one adoptive cell therapy, preferably at least one adoptive cell therapy selected from adoptive NK cell therapy, adoptive dendritic cell therapy and adoptive T cell therapy, wherein said adoptive T cell therapy is preferably CAR-T cell therapy or a therapy using TILs; and / or (c) a therapy comprising at least one cancer vaccine; and / or (d) treatment comprising at least one exogenous cytokine; and / or (e) Radiation therapy For use in combination therapy with at least one other therapy selected from the group consisting of:

18. A recombinant expression system, expression vector, viral particle or pharmaceutical composition according to claim 17.

19. The cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymus cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, primary liver cancer, metastatic liver cancer, gallbladder cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasma cell neoplasms, bile duct cancer, and neuroendocrine or carcinoid tumors; 18. A recombinant expression system, expression vector, viral particle or pharmaceutical composition according to claim 17.

20. The immunotherapy combination includes the following: (a) administering a recombinant expression system according to claim 1; an expression vector according to claim 14; a viral particle according to claim 15 or a pharmaceutical composition according to claim 16 to a patient suffering from a proliferative disorder, preferably cancer, in order to obtain a therapeutically effective transgenic expression of said target STING and / or said immunomodulatory protein, said administration being preferably intratumoral or systemic; (b) optionally, prior to and / or simultaneously with administration of (a), treating the patient with radiation therapy, preferably radiation therapy directed to tissues or organs intended to express the transduced gene from (a); and (c) treating the patient after administration with (a) with a STING agonist that recognizes the transgenically expressed target STING, but does not recognize human STING and its naturally occurring variants, and / or radiation therapy, preferably directed to the tissue or organ intended to express the transgene from (a); It is used in combination with 18. A recombinant expression system, expression vector, viral particle or pharmaceutical composition according to claim 17.

21. 21. The recombinant expression system, expression vector, viral particle or pharmaceutical composition of claim 20, wherein the patient is treated with at least one other therapy of claim 18 prior to, simultaneously with and / or after the administration of claim (a), preferably the patient is treated after the administration of claim (a).

22. 21. The recombinant expression system, expression vector, viral particle or pharmaceutical composition according to claim 20, wherein in (c) a STING agonist according to claim 2, preferably DMXAA, is used.

23. 21. The recombinant expression system, expression vector, viral particle or pharmaceutical composition of claim 20, wherein in (b), radiation therapy is used at a dose of less than 100 Gy, or low-dose radiation therapy is used at 40 Gy or less, preferably 8 Gy or less.

24. 21. The recombinant expression system, expression vector, viral particle or pharmaceutical composition of claim 20, wherein (c) comprises several further radiation treatments, wherein the cumulative dose of said further radiation treatments is added up to less than 100 Gy, preferably less than 40 Gy, more preferably less than 25 Gy.

25. A recombinant expression system comprising a nucleic acid construct comprising sequences encoding at least two immunomodulatory proteins, wherein the sequences are operably linked to a STING-responsive promoter or a radiation-inducible promoter and at least one sequence motif that inhibits transgenic expression of the immunomodulatory proteins in tissues or cells not intended to express the immunomodulatory proteins.

26. At least two of the immunomodulatory proteins are interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-7, interleukin-8, interleukin-9, interleukin-11, single chain interleukin-12, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-15-sushi , Interleukin-16, Interleukin-17, Interleukin-18, Interleukin-19, Interleukin-20, Interleukin-21, Interleukin-22, Interleukin-23, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CC L19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, granulocyte macrophage colony a blocking peptide targeting a stimulatory factor, type I interferon, IFN-γ, TNF-α, FLT3-ligand, TGF-β, a blocking peptide targeting IL-10, a blocking peptide targeting FoxP3, a monoclonal antibody or single chain variable fragment (scFv) or nanobody that neutralizes PD1, PDL1, CTLA4, CD137, TIM3, LAG3, and fragments or variants thereof; 26. The recombinant expression system of claim 25.

27. At least two immunomodulatory proteins are selected from the group consisting of: (a) the α subunit of IL-12; (b) the β subunit of IL-12; (c) single-chain IL-12 containing the α and β subunits of IL-12; (d) a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10; (e) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9 or 10 and having interleukin-12 biological activity; (f) IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO: 79; (g) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:79 and having IFN-β biological activity; (h) IL-15-sushi comprising or consisting of the amino acid sequence of SEQ ID NO: 80; (i) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 80, wherein the polypeptide has IL-15 biological activity; (j) CXCL10 comprising or consisting of the amino acid sequence of SEQ ID NO: 81; and (k) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 81 and having CXCL10 biological activity; 26. The recombinant expression system of claim 25, selected from the group consisting of:

28. the first immunomodulatory protein is a single chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO:9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:9 or 10 and having the biological activity of interleukin-12; and the second immunomodulatory protein is an IFN-β that comprises or consists of the amino acid sequence of SEQ ID NO:79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:79 and that has the biological activity of an IFN-β; or IL-15-sushi comprising or consisting of the amino acid sequence of SEQ ID NO:80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:80 and having the biological activity of IL-15; or a CXCL10 comprising or consisting of the amino acid sequence of SEQ ID NO:81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:81 and having the biological activity of CXCL10; 26. The recombinant expression system of claim 25.

29. The nucleic acid construct is a bicistronic or polycistronic expression cassette comprising two or more sequences each encoding a different immunomodulatory protein or anti-cancer protein or RNA, wherein: (a) said sequence is separated by a sequence encoding an IRES; and / or (b) said sequence is separated by a sequence encoding a 2A peptide, preferably a sequence encoding a P2A peptide; 26. The recombinant expression system of claim 25.

30. The STING responsive promoter comprises an IFN-β responsive promoter, an IFN-β minimal promoter, a CXCL9 responsive promoter, a CXCL10 responsive promoter, a STAT motif promoter, an IRSF3 motif promoter, an ISRE motif promoter, an NFκB motif promoter, and a STAT6 motif promoter, wherein the STING responsive promoter preferably comprises at least one interferon stimulated response element (ISRE), and more preferably comprises the following: (a) a nucleotide sequence comprising 1 to 10 copies, preferably 2 to 7 copies, more preferably 3 to 5 copies, and particularly preferably 4 copies, of a sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to SEQ ID NO: 11; (b) a nucleotide sequence according to (a), wherein the copies are separated by up to 40 nucleotides, preferably from 4 to 35 nucleotides, more preferably from 10 to 30 nucleotides, particularly preferably from 15 to 25 nucleotides; (c) the nucleotide sequence set forth in SEQ ID NO: 12; and (d) the nucleotide sequence set forth in SEQ ID NO:13; Selected from the group consisting of:

26. The recombinant expression system of claim 25.

31. The sequence motif inhibits transgenic expression of said immunomodulatory protein in tissues or cells not intended to express said immunomodulatory protein, said tissue being preferably the liver, said sequence motif being a target sequence of a microRNA that is preferably highly abundant in the liver, more preferably said target sequence being selected from the group consisting of miR-122 target sequence, miR-192 target sequence, miR-199a target sequence, miR-101 target sequence, miR-99a target sequence, let7a target sequence, let7b target sequence, let7c target sequence, let7f target sequence, and even more preferably the following: (a) a miR-122 target sequence that has at least 90% or at least 95% sequence identity to, or is identical to, SEQ ID NO:14; (b) a nucleotide sequence comprising 1 to 12, preferably 3 to 10, more preferably 4 to 8, and particularly preferably 5 copies of the miR-122 target sequence of (a); (c) the nucleotide sequence according to (b) above, in which the copies are separated by 1 to 40 nucleotides, preferably by 1 to 30 nucleotides, more preferably by 2 to 20 nucleotides, particularly preferably by 2 to 8 nucleotides; and (d) a nucleotide sequence containing five copies of the miR-122 target sequence shown in SEQ ID NO: 15; A miR-122 target sequence selected from the group consisting of:

26. The recombinant expression system of claim 25.

32. 26. An expression vector encoding the nucleic acid construct of claim 25, preferably a viral vector, more preferably a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, a vaccinia viral vector, and a herpes simplex viral vector, and even more preferably having the following characteristics: (a) the sequence of the first and / or second nucleic acid construct further comprises a 5'ITR sequence and a 3'ITR sequence, preferably an AAV ITR as set forth in SEQ ID NO: 17; and (b) the AAV vector preferably has the serotype AAV8, AAV1, AAV3, AAV6, AAV9, AAV2, AAV5, AAVrh.10, or any gain-of-function mutant of AAVrh.10, more preferably the AAV vector has the serotype AAV2; is an adeno-associated virus vector having one or more of Expression vector.

33. 33. A viral particle comprising the viral vector according to claim 32, which is preferably an AAV or adenovirus particle, more preferably an AAV viral particle.

34. Pharmaceutically acceptable excipients and (a) a recombinant expression system according to claim 25; or (b) the expression vector of claim 32; or (c) the virus particle according to claim 33 13. A pharmaceutical composition comprising:

35. 35. A recombinant expression system according to claim 25, an expression vector according to claim 32, a viral particle according to claim 33 or a pharmaceutical composition according to claim 34 for use in therapy, preferably in immunotherapy, more preferably in immunotherapy of a proliferative disorder, even more preferably in immunotherapy of cancer, especially preferably in immunotherapy of solid cancers.

36. below: (a) a therapy comprising at least one checkpoint inhibitor, preferably selected from the group consisting of an inhibitor of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR, more preferably selected from the group consisting of an inhibitor of PD-1, PD-L1, PD-L2, or CTLA-4; and / or (b) a therapy comprising at least one adoptive cell therapy, preferably at least one adoptive cell therapy selected from adoptive NK cell therapy, adoptive dendritic cell therapy and adoptive T cell therapy, wherein said adoptive T cell therapy is preferably CAR-T cell therapy or a therapy using TILs; and / or (c) a therapy comprising at least one cancer vaccine; and / or (d) treatment comprising at least one exogenous cytokine; and / or (e) Radiation therapy For use in combination therapy with at least one other therapy selected from the group consisting of:

36. A recombinant expression system, expression vector, viral particle or pharmaceutical composition according to claim 35.

37. The cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymus cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, primary liver cancer, metastatic liver cancer, gallbladder cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasma cell neoplasms, bile duct cancer, and neuroendocrine or carcinoid tumors; 36. A recombinant expression system, expression vector, viral particle or pharmaceutical composition according to claim 35.

38. The immunotherapy combination includes the following: (a) administering a recombinant expression system according to claim 25, an expression vector according to claim 32, a viral particle according to claim 33 or a pharmaceutical composition according to claim 34 to a patient suffering from a proliferative disorder, preferably cancer, in order to obtain a therapeutically effective transgenic expression of said target STING and / or said immunomodulatory protein, said administration being preferably intratumoral or systemic; (b) optionally, prior to and / or simultaneously with administration of (a), treating the patient with radiation therapy, preferably radiation therapy directed to tissues or organs intended to express the transduced gene from (a); and (c) treating the patient after administration with (a) with a STING agonist that recognizes the transgenically expressed target STING, but does not recognize human STING and its naturally occurring variants, and / or radiation therapy, preferably directed to the tissue or organ intended to express the transgene from (a); It is used in combination with 36. A recombinant expression system, expression vector, viral particle or pharmaceutical composition according to claim 35.

39. 39. The recombinant expression system, expression vector, viral particle or pharmaceutical composition of claim 38, wherein the patient is treated with at least one other therapy of claim 36 prior to, simultaneously with and / or after the administration of claim (a), preferably the patient is treated after the administration of claim (a).

40. 39. The recombinant expression system, expression vector, viral particle or pharmaceutical composition of claim 38, wherein in (b), radiation therapy is used at a dose of less than 100 Gy, or low dose radiation therapy is used at 40 Gy or less, preferably 8 Gy or less.

41. 39. The recombinant expression system, expression vector, viral particle or pharmaceutical composition of claim 38, wherein (c) comprises several further radiation treatments, wherein the cumulative dose of said further radiation treatments adds up to less than 100 Gy, preferably less than 40 Gy, more preferably less than 25 Gy.

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