Expression system for treating cancer

Abstract

The present invention relates to a recombinant expression system for the treatment of cancer, which is based on the "Stimulating Factor of Interferon Gene" (STING) and comprises two nucleic acid constructs. The invention also relates to expression vectors or a combination of two expression vectors encoding one or more of said nucleic acid constructs, and to viral particles or a combination of two viral particles comprising one or more of said expression vectors. The invention also relates to a pharmaceutical composition comprising the recombinant expression system, one or more of the expression vectors or one or more of the virus particles. The invention also relates to the use of the recombinant expression system, one or more of the expression vectors, one or more of the viral particles or the pharmaceutical composition for immunotherapy, especially for immunotherapy of proliferative disorders such as cancer. The invention finally relates to the administration of an expression vector in combination with radiation therapy for the treatment of cancer.

Description

Field of the Invention

[0001] The present invention relates to a recombinant expression system for treating cancer, which is based on "stimulator of interferon genes" (STING) and comprises two nucleic acid constructs. The present invention also relates to an expression vector encoding one or more of the nucleic acid constructs or a combination of two expression vectors, and a viral particle (or a combination thereof) comprising one or more of the expression vectors. The present invention also relates to a pharmaceutical composition comprising the recombinant expression system, one or more of the expression vectors or one or more of the viral particles. Finally, the present invention relates to the use of the recombinant expression system, one or more of the expression vectors, one or more of the viral particles or the pharmaceutical composition for immunotherapy, especially for immunotherapy of proliferative diseases such as cancer. Background of the Invention

[0003] Cancer is a group of diseases involving abnormal cell growth and proliferation, with approximately 19 million new cases each year and is the cause of approximately 10 million deaths each year (Global Cancer Observatory, 2020). Although significant advances have been made in cancer treatment over the past few decades, annual mortality rates show that a large number of people still die from cancer.

[0004] A serious obstacle to cancer treatment is that there are several different types of cancer, each with its own challenges to treat. For example, some cancers are easily accessible and therefore can be treated locally (i.e., surgical resection, localized radiation therapy), while other cancers can only be treated with systemic approaches (i.e., chemotherapy, hormone therapy, immunotherapy).

[0005] There are many different treatment methods, such as radiotherapy, chemotherapy, surgical resection, hormone therapy and immunotherapy. Some of these treatments target tumors by systemically interfering with cell proliferation, thereby stopping tumor growth. However, these treatments cause serious side effects to patients, such as fatigue, nausea and vomiting, anemia and infection, because these systemic treatments also do not expect to target physiological dividing cells such as hematopoietic cells, cells in the oral cavity, digestive tract and reproductive system. In addition, depending on the location of the tumor, local therapies such as local radiotherapy can cause serious side effects. In contrast, the concept of immunotherapy is to stimulate the body's immune system to fight cancer cells, thereby reducing unwanted side effects. Immunotherapy can be divided into active or passive immunotherapy. Active immunotherapy is characterized in that the patient is immunized with an agent that enhances the immune response to 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 ability of the immune system to attack cancer cells. Examples include checkpoint inhibitors and cytokines. However, to date, immunotherapy has shown several disadvantages, such as i) poor tumor targeting, ii) weak tumor cell killing ability, iii) laborious and therefore costly, iv) tumor immune escape, and v) overstimulation of the immune system that can lead to serious side effects (Tan et al. 2020, Biomed Pharmacother. 124: 109821).

[0006] STING (also known as endoplasmic reticulum interferon stimulator (ERIS), mediator of IRF3 activation (MITA), or transmembrane protein 173 (TMEM173) is a four-transmembrane ER protein present in the endoplasmic reticulum (ER) that acts as a sensor for the presence of double-stranded DNA (dsDNA) in the cytosol. As such, STING is activated by DNA viruses, DNA damaging lesions (such as radiation therapy), and micronuclei that are frequently present in the cytosol of malignant cells (Li et al. Journal of Hematology & Oncology 2019, 12:35). STING activates TANK-binding kinase 1 (TBK1), leading to phosphorylation and activation of interferon-regulated 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 TNFα) and cooperates with TBK1-IRF3 to produce type I IFN (Li et al. Journal of Hematology & Oncology 2019,12:35). Type I IFN stimulates immune effector cells including dendritic cells (DC) and natural killer (NK) cells, and promotes the production of chemokines such as CXCL9 and CXCL10, which attract T lymphocytes to tumors, which is crucial for anti-tumor immunity (Mowat, C. et al. J. Exp. Med. 2021, Vol. 218, No. 9, e20210108). In fact, STING agonists, like cyclic dinucleotides (CDNs), exert powerful anti-tumor effects in different tumor models (Sivick Ke et al. Cell Reports 2018; 25, 3074-3085). The importance of STING in cancer pathology can also be derived from the fact that STING expression is often suppressed or lost in many cancers.

[0007] Unfortunately, there are significant STING polymorphisms in the human population, which influence both the molecular responses induced by activation of the protein and the degree of sensitivity to stimulatory ligands. Therefore, this can greatly affect the efficacy and safety of molecular entities pursued for clinical purposes.

[0008] Based on these numerous findings, STING is now considered a master regulator in the cancer immunity circuit.

[0009] A well-described STING activator disclosed in WO 2004 / 039363 A1 is 5,6-dimethylxanthone-4-acetic acid (DMXAA), but other STING agonists are also known in the art. Clinical trials of DMXAA have shown that it is well tolerated and exhibits only mild side effects. However, despite the anti-tumor effects of STING described in animal models, clinical trials have shown that DMXAA failed in trials due to lack of efficacy. Further studies have demonstrated the reason for this treatment failure, showing that DMXAA strongly activates mouse STING, but cannot activate human STING due to amino acid differences in the cyclic dinucleotide (CDN) binding site of STING.

[0010] Documents US 5,126,132 A and US2005 / 214274 A1 disclose cell-based immunotherapy, in which immune active cells such as T cells from a patient are isolated, cultured and modified ex vivo so that these cells recognize a specific type of cancer and then reinjected into the patient. However, in addition to the complex process of generating these ex vivo modified cells, they only target a specific type of cancer.

[0011] Immunotherapy, which uses the cytokine interleukin-2 (IL-2) to stimulate the immune system to target cancer cells, was approved for the treatment of melanoma and kidney cancer as early as 1992. However, studies have shown that only about 7% to 16% of patients respond to the treatment, and serious side effects are common (Marabondo et al. Expert Opin Drug Saf. 2017 Dec; 16(12): 1347-1357.).

[0012] The use of autologous cancer cells as vaccines to enhance anti-tumor immunity has been explored for some time. However, due to the weak immunogenicity of many cancers, the downregulation of MHC molecules, the lack of sufficient co-stimulatory molecule expression and the secretion of immunosuppressive cytokines by cancer cells, the response to such vaccines does not produce long-term efficacy (see, for example, Armstrong TD and Jaffee EM, Surg Oncol Clin N Am. 1 1 (3): 681-96, 2002). Many cytokines have been shown to play a role in regulating the immune response to tumors. For example, U.S. Patent No. 5,098,702 describes the use of a combination of TNF, IL-2 and IFN-β in a synergistically effective amount to combat existing tumors. U.S. Patent Nos. 5,078,996, 5,637,483 and 5,904,920 describe the use of GM-CSF for treating tumors. However, direct administration of cytokines to treat cancer may not be practical because they are often systemically toxic (Asher et al., J. Immunol. 146:3227-3234, 1991).

[0013] Document US2021 / 038684 A1 provides compositions and methods for treating cancer using IL-2 immunotherapy. The method of the invention includes systemic administration of interleukin-2 / interleukin-2-receptor alpha fusion protein to the patient, wherein administration results in a dose-dependent increase in circulating NK cells and CD8+ cells in the patient, while there is no dose-dependent increase in circulating immunosuppressive T regulatory (Treg) cells. However, despite the intention to overcome tumor immune escape, the invention includes known disadvantages of cancer immunotherapy, such as weak tumor cell killing ability, and only some patients respond to treatment (Boni et al., Journal of Clinical Oncology 2021, 39: 15_suppl, 2513-2513). In addition, cytokine-based immunotherapy usually needs to be repeated (avoiding excessive stimulation that brings additional medical expenses and burdens to patients) to ensure sufficient immune system stimulation.

[0014] Therefore, there remains a need for effective and safe treatments for proliferative diseases such as cancer, in which many different types of cancer are efficiently targeted, which treatments show no or mild undesirable side effects, and to which a wide variety of patients respond.

[0015] Therefore, an object of the present invention is to provide an improvement or alternative to the prior art.

[0016] This 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 a combination of two expression vectors according to claim 14, a viral particle according to claim 15 or 33 or a combination of two viral particles according to claim 15 and a pharmaceutical composition according to claim 16 or 34. Specific embodiments are subject matter of further dependent or independent claims. Summary of the invention:

[0018] In a first aspect, the present invention provides a recombinant expression system comprising

[0019] a. a first nucleic acid construct comprising a sequence encoding a target STING that responds to a STING agonist that recognizes the target STING but not human STING and its natural variants (human non-responsive STING agonist), wherein the sequence encoding the target STING is operably linked to an element that allows expression and / or translation of the target STING; and / or

[0020] b. A second nucleic acid construct comprising a sequence encoding at least one immune regulatory gene or protein, said sequence being operably linked to a STING responsive promoter or a radiation inducible promoter.

[0021] The expression system according to the invention combines many of the advantageous aspects of cancer immunotherapy while avoiding the disadvantages of said therapy.

[0022] It allows the expression of immunomodulatory peptides, in particular cytokines such as interleukins or chemokines, in tumors, thereby stimulating the immune system to target cancer cells of the tumor. Due to the preferred intratumoral administration of the expression system, cytokine expression is highly localized, thereby avoiding the adverse aspects and toxicity of systemic cytokine therapies known in the art.

[0023] The recombinant expression system comprises two expression cassettes represented by two nucleic acid constructs: a) one expression 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) another expression cassette represented by a second nucleic acid construct, encoding an immunomodulatory gene or protein under the control of a promoter that is activated when STING is activated. According to the present invention, the recombinant expression system comprises both cassettes or only one of the cassettes. The combined expression of the two expression cassettes has the following advantages: after intratumoral injection of one or more vectors containing the expression cassettes, systemic administration of the agonist of the target STING enables selective activation of STING in the tumor without affecting the endogenous human STING in the rest of the body, thus avoiding toxicity. Intratumoral activation of the target STING induces the production of type I IFN, chemokines and other cytokines in the tumor by inducing endogenous genes or by activating the promoter of the second expression cassette encoding an immunomodulatory gene or protein (such as IL-12). In this way, systemic administration of the agonist of the target STING triggers the intratumoral production of type I IFN and other immunostimulatory peptides produced by STING activation, as well as the production of immunomodulatory genes or proteins expressed by the second expression cassette. The local intratumoral production of this combination of immunomodulatory genes or proteins can promote anti-tumor immunity without toxicity. By injecting the tumor with a long-term expression vector (such as an adeno-associated virus vector) containing these two expression cassettes, it is possible to induce the production of the above combination of immunomodulatory genes or proteins in the tumor by repeated systemic administration of the agonist of the target STING.

[0024] The inventors found that the first nucleic acid of the recombinant expression system according to the present invention induces upregulation of STING-related genes or proteins when the target STING is specifically activated by administering a STING agonist that recognizes the target STING but not human STING and its natural variants. It was further found that low-dose radiotherapy also induces upregulation of STING-related genes or proteins. It can be demonstrated that enrichment of STING (for example, by transducing cells with target STING) makes the cells more sensitive to STING agonists such as DMXAA (classical pathway) or low-dose radiotherapy (non-canonical pathway), respectively, and when the two stimuli (STING agonists and low-dose radiotherapy) are applied together, the synergistic effect between them can be fully demonstrated (see Fig.9A -C). Thus, in one aspect of the invention, it is sufficient that the expression system only comprises a cassette encoding a target STING that is responsive to a STING agonist that does not activate human STING.

[0025] In addition, the inventors found that the second nucleic acid of the recombinant expression system according to the present invention induces upregulation of an immunomodulatory transgene that is dependent on STING activity, and that this activity can be specifically induced by a STING agonist that recognizes the target STING but not human STING and its natural variants. Therefore, the expression of the immunomodulatory transgene can be controlled by administering the STING agonist.

[0026] Furthermore, the inventors have found that the recombinant expression system according to the present invention is non-toxic and shows less side effects in vivo. Notably, the inventors have found that when administered in vivo, the recombinant expression system leads to tumor reduction, ultimately leading to complete remission. Furthermore, by locally administering the recombinant expression system of the present invention in a tumor, not only the size of the target tumor is reduced, but also non-target tumors show a significant size reduction, which is also referred to as the abscopal effect. DETAILED DESCRIPTION OF THE INVENTION

[0028] The recombinant expression system generates specific and basal immune pathways in transfected cancer cells that utilize otherwise endogenous pathway components.

[0029] It is noteworthy that the term "recombinant expression system" includes any nucleotide, its analog and its polymer. Therefore, any recombinant expression system disclosed herein refers to a polymeric form of nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)) of any length. These terms refer to the primary structure of the molecule, and therefore include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. A more comprehensive definition is included in the definition section of this application.

[0030] The first component of this pathway is the so-called "target STING", which is defined by its responsiveness to STING agonists that cannot activate human STING, thereby allowing selective activation of target STING. The first component is given by the first nucleic acid construct of the present invention and can be regarded as the "synergistic effector" and "activator" part of the pathway. It is worth noting that this first component can also be administered alone, so that cells expressing this first component respond to STING agonists and low-dose radiotherapy, so that when these two stimuli (STING agonists and low-dose radiotherapy) are applied together, the synergistic effect between them is fully demonstrated.

[0031] In the first nucleic acid construct according to the present invention, the target STING is operably linked to an element that allows expression and / or translation of the target STING. In the case where the recombinant expression system is a DNA-based expression system, the element is typically a promoter that allows transcription of mRNA encoding the target STING. In the case where the recombinant expression system is an RNA-based expression system, the element is typically an RNA element that allows translation of mRNA encoding the target STING, such as a CAP, a 5′UTR, a 3′UTR, or a poly A site.

[0032] The second component of the pathway is an artificial gene construct, which includes an immunomodulatory gene or protein, the sequence of which is operably linked to a STING responsive promoter or a radiation-inducible promoter. The immunomodulatory gene or protein activates innate and adaptive immune responses to produce a specific cancer immune response. The second component is given by the second nucleic acid construct of the present invention and can be regarded as the "synergistic effector" part of the pathway.

[0033] It is worth noting that target STING differs from native human STING in that it is activated by human non-responsive agonists such as DMXAA. However, after activation, target STING exhibits the biological activity of human STING because target STING is still able to bind and activate downstream effector proteins, which are also activated by human STING (such as iRhom2 and TANK binding kinase 1 (TBK1)). After activation, STING undergoes conformational changes, leading to TBK1 recruitment. In turn, TBK1 phosphorylates transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB), which translocate to the nucleus and stimulate the production of proinflammatory molecules such as type I interferons, IL6, TNF-α or CXCL10. (Ahn, J. et al. Exp Mol Med 51, 1-10 (2019)).

[0034] It can be provided that the target STING responds to an agonist that does not react with human STING. The human non-responsive STING agonist can be selected from the group consisting of: 5,6-dimethylxanthone-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)fluorene-9-one (tiloron) and 10-carboxymethyl-9-acridone (CMA). Those skilled in the art recognize that analogs of the molecules disclosed herein and any stereoisomers thereof can also be used. In addition, one or more atoms of the above-mentioned molecules, their analogs and / or their stereoisomers can be replaced by their respective isotopes.

[0035] When used for human treatment, the use of human non-responsive STING agonists in the description of the present invention has some advantages. By definition, such human non-responsive STING agonists cannot bind to human wild-type STING or its natural variants in a manner that causes STING activation. Therefore, it can be administered systemically without causing a systemic STING response, thereby avoiding unwanted side effects. Since the present invention is intended to express a target STING that responds to a human non-responsive STING agonist, it is possible to specifically activate this transgenic STING, thereby causing local STING activation. Therefore, in the description of the present invention, the human non-responsive STING agonist has two effects: it is used to define STING used as a target STING in gene therapy, and in addition, it is also used in subsequent drug therapy to activate the target STING, thereby achieving a highly selective treatment method.

[0036] Preferably, the target STING is responsive to DMXAA. The use of DMXAA-responsive STING has the advantage that the STING agonist DMXAA has been demonstrated in clinical studies to be a molecule with a favorable safety profile and only mild side effects.

[0037] Furthermore, it could be shown that activation of the target STING by DMXAA showed a dose-dependent induction of downstream STING targets such as IFN-β (see Example 1). This allows the immune response to be controlled by simply adjusting the DMXAA dose during treatment.

[0038] Intratumoral injection of DMXAA has been shown to effectively elicit CD8+ T cell responses in mice and promote the elimination of established tumors in a STING-dependent manner (Corrales et al. Cell Reports, 2015; 11, 1018-1030). Clinical trials demonstrating the anti-tumor properties of DMXAA have shown that it exhibits only mild side effects. However, these trials have shown that DMXAA cannot activate human wild-type STING or its natural variants. However, murine STING and rat STING respond to DMXAA as they display the biological activity of STING.

[0039] It is also envisaged 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 natural 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 a human non-responsive STING agonist as determined in an in vitro agonist assay.

[0040] In vitro agonist assays can be used to identify human non-responsive STING agonists. Several possible methods are known to those skilled in the art for conducting such assays. In a non-limiting example, a vector encoding a reporter gene (e.g., luciferase) under the control of a STING responsive promoter can be constructed. Cells can be transformed, transfected, or transduced with the vector and additionally with a vector that induces expression of human STING or its natural variant, mutated human STING variant, or non-human STING. These cells should then be contacted with a STING agonist (preferably DMXAA), and luciferase activity should be measured. If luciferase activity in cells transformed, transfected, or transduced with a vector that induces expression of a mutated human STING variant or non-human STING is detectable, but luciferase activity in cells transformed, transfected, or transduced with a vector that induces expression of human STING or its natural variant is not significantly different from a mock-treated control group, the agonist is considered to be "human non-responsive". In this case, no significant difference from the mock-treated control group 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 by using hSTING[R]-S162A / G230I / Q266I (SEQ ID NO: 6). Another example of an in vitro agonist assay is to directly measure IFN-β levels when cells transformed, transfected or transduced with a vector that induces a mutation in a human STING variant, a non-human STING, or a human STING or a natural variant thereof are contacted with a STING agonist. In this case, the human non-responsive STING agonist will provide at least 25%, preferably at least 50%, more preferably at least 75%, and even more preferably 90% of the luciferase activity obtained by using hSTING[R]-S162A / G230I / Q266I (SEQ ID NO: 6) The IFN-β level obtained.

[0041] It is also envisaged 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 non-human responsive STING agonist as determined in an in vitro agonist assay, wherein the human non-responsive STING agonist is preferably DMXAA. Non-limiting examples of non-human STING that are sensitive to a human non-responsive STING agonist as determined in an in vitro agonist assay are murine STING or a natural variant thereof, rat STING or a natural variant thereof, or a hybrid STING. In a preferred embodiment, the non-human STING is murine STING according to SEQ ID NO: 7, which encodes a protein of SEQ ID NO: 8.

[0042] Alternatively, a radiation-inducible promoter may be provided. As used herein, a radiation-inducible promoter is a promoter that induces expression of a therapeutic gene sequence when a cell is irradiated. These promoters may be natural, synthetic or artificially produced, selected from recombinant products of natural promoters. For example, these promoters may contain at least one of the following sequences, either individually or in a repetitive manner:

[0043] a. the eukaryotic radiation-inducible sequence CC(A / T)(6)GG (SEQ ID NO:87), also known as a CArG element or [CC(A+T-rich)6GG], such as the sequence of the early growth response-1 (Egr-1) promoter or the pE9 promoter, 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 alternatively the sequence comprises at least two sequences according to SEQ ID NO:87, wherein the at least two sequences may be separated by additional nucleotides;

[0044] b. a low frequency CCAAT box sequence, such as the CCAAT box sequence of the survivin promoter, preferably the sequence has 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 88 or is identical to SEQ ID NO: 88 (CCAAT-rich domain of the survivin promoter);

[0045] c. cyclin-dependent kinase inhibitor 1 (also known as CDKN1A or p21) promoter;

[0046] d. Late herpes simplex virus 1 promoters, such as glycoprotein C promoter or U S 11 promoter;

[0047] e. Combinations of the above or artificial mutations thereof.

[0048] In a preferred embodiment of the present invention, the target STING is DMXAA-sensitive human STING as determined in an in vitro DMXAA assay. As described above, the advantages of using DMXAA for human therapy are that DMXAA has been shown to have a favorable safety profile, exhibits only minor side effects, and because it cannot activate human STING, it can be administered systemically without causing systemic STING activation.

[0049] In vitro DMXAA assays can be used to identify STING that responds to DMXAA. Cells can be transduced with vectors that induce expression of any STING (e.g., human wild-type STING, mouse STING). If, after the addition of DMXAA, cells respond by showing a statistically significant increase in the mRNA levels of IFN-β and other cytokines measured by quantitative PCR (qPCR) compared to mock-treated controls in triplicate assays, the expressed STING is considered to be DMXAA-sensitive (see, e.g., FIG. 1 ). In this case, a statistically significant increase means that the mRNA level is at least 10%, preferably at least 20%, more preferably at least 30%, and in particular at least 50% higher than the mock-treated control. An example of a DMXAA-unresponsive STING is human wild-type STING or a natural variant thereof. Another example of an in vitro DMXAA assay is to directly measure the IFN-β level of cells transformed, transfected, or transduced with a vector encoding STING (e.g., human wild-type STING, mouse STING) after contact with DMXAA. In this case, DMXAA-sensitive human STING will provide at least 25%, preferably at least 50%, more preferably at least 75%, and even more preferably 90% of the IFN-β levels as obtained by using hSTING[R]-S162A / G230I / Q266I (SEQ ID NO:6) treated with DMXAA.

[0050] Advantageously, in the description of the present invention, it may be provided that the target STING encoded by the sequence of the first nucleic acid construct is a DMXAA-sensitive human STING variant selected from the group consisting of:

[0051] a. a human STING[R] variant, further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I;

[0052] b. a human STING[AQ] variant further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of: S162A, A230I, Q266I, E260I, and E260V;

[0053] c. a human STING[H] variant further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I; and

[0054] d. A truncated human STING[H] variant further comprising at least one or at least two amino acid substitutions selected from the group consisting of S162A and G230I.

[0055] The skilled person noted that all of the above mutations are located in the cyclic dinucleotide (CDN) binding site (binding pocket) and / or lid region of the binding pocket of STING. Therefore, in another embodiment, endogenous human STING may contain a mutation in one of the amino acids located in the binding pocket region and / or lid region to make it DMXAA-sensitive human STING.

[0056] For example, several natural variants of human STING have been identified as a result of several single polymorphisms (SNPs) and SNP haplotypes that produce several allelic polymorphic forms of the protein. The amino acid sequence of the most common natural variant or allele of human STING is SEQ ID NO: 1, which is referred to as wild-type human STING. The most common natural allelic variants of human STING include: hSTING [R] as described in SEQ ID NO: 1, usually referred to as hSTING R232, i.e., the amino acid at position 232 is arginine, which STING is also considered to be a wild-type isoform; hSTING [H] as described in SEQ ID NO: 2, usually referred to as hSTING H232, i.e., the amino acid at position 232 is histidine; hSTING [AQ] as described in SEQ ID NO: 3, whose sequence differs from hSTING [R] by the incorporation of amino acid substitutions G230A and R293Q. In addition, a truncated form of hSTING [H] as described in SEQ ID NO: 4 is characterized. Another natural variant is STING[HAQ], which has amino acid substitutions R71H, G230A, and R293Q. Thus, the target STING differs from one of the known haplotypes of human STING (i.e., hSTING[R] as described in SEQ ID NO: 1, hSTING[H] as described in SEQ ID NO: 2, hSTING[AQ] as described in SEQ ID NO: 3, hSTING[H] as described in SEQ ID NO: 4, STING[Q] having amino acid substitution R293Q, or STING[HAQ] having amino acid substitutions R71H, G230A, and R293Q). In addition, the target STING can be responsive to any one of the human non-responsive STING agonists selected from the group consisting of 5,6-dimethylxanthone-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-acridone (CMA). Thus, administration of the agonist results in activation of exogenous STING provided by the first nucleic acid of the recombinant expression system, wherein endogenous STING encoded in the genome of the human subject is not activated.

[0057] In another possibility, a DMXAA-sensitive human STING variant encoded by the sequence of the first nucleic acid construct may be provided 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, hSTING[AQ]-Q266I, hSTING[R]-S162A / Q230I, hSTING[AQ]-S162A, hSTING[ hSTING[AQ]-S162A / R169A, hSTING[AQ]-S162A / T267A, hSTING[AQ]-E260I, 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, and preferably hSTING[R]-S162A / G230I / Q266I.

[0058] Experiments have been performed showing that mutation S162A is critical in allowing DMXAA to bind and activate human STING, and mutations Q266I and G230I or A230I increase sensitivity to DMXAA (Gao et al., 2014, Cell Reports 8, 1668-1676).

[0059] According to another advantageous aspect, it can be provided that the sequence of the first nucleic acid construct encoding the DMXAA-sensitive human STING variant is selected from the group consisting of: SEQ ID NO: 5 (which is 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 relative to SEQ ID NO: 5. The sequence identity of the polynucleotide sequence relative to SEQ ID NO: 5 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%. Notably, the polypeptide encoded by any of the foregoing polynucleotides has biological activity in response to the agonist DMXAA as determined in an in vitro DMXAA assay.

[0060] According to another advantageous aspect, it can be provided that the sequence of the first nucleic acid construct encoding the DMXAA-sensitive human STING variant is selected from the group consisting of: a polynucleotide sequence encoding a polypeptide comprising SEQ ID NO: 6 (which is the sequence of hSTING [R] -S162A / G230I / Q266I), or 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 relative to SEQ ID NO: 6. The sequence identity of the polypeptide encoded by the polynucleotide sequence relative to SEQ ID NO: 6 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%. Notably, a polypeptide encoded by any of the foregoing polynucleotides has biological activity that is responsive to a human non-responsive STING agonist as determined in an in vitro agonist assay.

[0061] 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, in cancer treatment, the use of variants of human STING may be preferred over the use of non-human STING because the former may show better interactions with signaling molecules in human cells. In addition, the use of non-human STING may induce unwanted immune responses.

[0062] It is noteworthy that the polynucleotide sequence from the first nucleic acid construct described herein above may contain at least one modification of the polypeptide, which is an amino acid substitution, an amino acid insertion, an amino acid deletion, a C-terminal truncation or an N-terminal truncation. In addition, according to the present invention, a polynucleotide sequence that hybridizes with the complementary sequence of the above-mentioned polynucleotide sequence under stringent conditions is also included. In addition, according to the present invention, a fragment of at least 50 amino acid residues of a DMXAA-sensitive human STING variant is included. It is noteworthy that the polypeptide encoded by any of the aforementioned polynucleotides has a biological activity that responds to a human non-responsive STING agonist as determined in an in vitro agonist assay.

[0063] Preferably, it can be specified that the promoter of the first nucleic acid construct is a constitutive promoter, such as SV40, CMV0, UBC, EF1a, PGK, CAG, EFS, CBh, CBA, human beta actin, Ac5, polyhedrin, TEF1, GDS, CaMV35S, Ubi, H1 or U6. Preferably, the promoter is selected from the list consisting of: SV40, CMV0, UBC, EF1a, PGK, CAG, EFS, CBh and CBA. The use of a constitutive promoter in the specification of the present invention has the advantage of ensuring that the "activator" part of the pathway is expressed. Typically, constitutive promoters show higher expression levels compared to inducible or tissue-specific promoters. However, those skilled in the art recognize that in the specification of the present invention, cancer-specific promoters are also feasible promoters for the first nucleic acid construct.

[0064] In an alternative embodiment, the promoter of the first nucleic acid construct is an inducible promoter, preferably selected from the list 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 element (TTF1).

[0065] In addition, it can be advantageous that within the scope of the present invention, at least one immunomodulatory gene or protein encoded by the sequence of the second nucleic acid construct is selected from the group consisting of: interleukin family, chemokine family, colony stimulating factor family, transforming growth factor family, tumor necrosis factor family, any blocking peptide for immunosuppressive factor, any monoclonal antibody or any scFv or any nano antibody reactive with immune checkpoint protein, or shRNA targeting immunosuppressive factor. A non-limiting list of immunomodulatory proteins is 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, CCL19, CCL20, CCL21, CCL22, CCL23, CCL 24, 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-stimulating factor, type I interferon, I FN-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 that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3 and fragments or variants thereof; a non-limiting list of immunomodulatory genes consists of shRNA targeting TGF-β, shRNA targeting IL-10 or shRNA targeting FoxP3.

[0066] In a preferred embodiment of the present invention, at least one immunomodulatory gene or protein encoded by the sequence of the second nucleic acid 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-macrophage colony stimulating factor (GM-CSF), type I interferon (IFN-α and IFN-β), IFN-γ, TNF-α, CXCL10, FLT 3-ligand, blocking peptides targeting TGF-β, blocking peptides targeting IL-10, blocking peptides targeting FoxP3, monoclonal antibodies or scFv or nanobodies that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3 and fragments or variants thereof.

[0067] Those skilled in the art will recognize 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.

[0068] It is known in the art that there are several immunomodulatory proteins for immunotherapy for cancer treatment. For example, IL-2 is a protein that has been used in cancer treatment for more than 20 years for immunotherapy. However, only a very small number of patients respond to IL-2-related immunotherapy, and these therapies are known to cause serious side effects. IL-12 reprograms myeloid-derived suppressor cells (MDSC) into efficient antigen presenting cells (APC). In addition, IL-12 strongly stimulates T cells and NK cells, and increases IFN-γ production, promoting Th1-type responses that can control the growth of treated tumors and the growth of distant metastases (Lasek et al. Cancer Immunol. Immunother. 2014; 63: 419ff.). In addition, IL-12 has an anti-angiogenic effect, which can impair tumor vascularization, thereby limiting tumor growth. IL-12 is a heterodimeric cytokine encoded by two independent genes. In order to promote its expression in a recombinant expression system, it is feasible to use a recombinant single-chain IL-12 transgene encoding two subunits of IL-12. Therefore, in a preferred embodiment, the immunomodulatory protein according to the invention is IL-12.

[0069] In one embodiment, the at least one immunomodulatory protein is a complex or fusion protein of IL-15 and the Sushi domain of the IL-15 receptor α chain to enhance the agonist activity of IL-15 through trans expression such as provided by compound P22339 (Hu, Q., Ye, X., Qu, X. et al., SciRep 2018, 7675).

[0070] Optionally, at least one immunomodulatory protein may be selected from the group consisting of: an α subunit of IL-12, a β subunit of IL-12, a single-chain IL-12 comprising an α subunit and a β subunit of IL-12. In the present specification, single-chain IL-12 (scIL-12) is defined as a fusion protein comprising an α and β subunit, wherein the peptide sequences are either directly connected or separated by a linker peptide, wherein the linker peptide may have a length of 1 to 100 amino acids. Preferably, the length of the linker ranges from 4 to 60 amino acids, more preferably from 8 to 40 amino acids, and particularly preferably from 12 to 25 amino acids. The length of the linker can 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 acids. In addition, the at least one immunomodulatory protein can be a single-chain IL-12 (human or mouse 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 relative to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12. The sequence identity of the polypeptide sequence 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, for a polypeptide to be considered to have interleukin-12 biological activity, it must 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 assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 9 or 10.

[0071] The biological activity of IL-12 can be assessed in vitro by the ability of the peptide to increase IFN-γ production, which is produced by cells expressing the IL-12 receptor and the corresponding signaling pathway, such as peripheral blood mononuclear cells or human macrophage cell lines. Alternatively, a reporter cell line can be used as, for example, a Promega bioluminescent IL-12 bioassay (Catalog No. JA2601, JA2605).

[0072] In vivo IL-12 bioactivity can be assessed by its ability to elevate IFN-γ serum levels.

[0073] In addition, the at least 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% identity to SEQ ID NO: 79, wherein the polypeptide has the biological activity of IFN-β. The sequence identity of the polypeptide sequence 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, for a polypeptide to be considered to have IFN-β biological activity, it must have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of IFN-β assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 79.

[0074] The biological activity of IFN-β can be estimated by the ability of inducing type I IFN response genes. For this purpose, human cell lines (such as HEK 293) can be incubated with polypeptides, and the expression of IFN response genes can be measured by RT-qPCR. Alternatively, IFN reporter cells encoding luciferase under the control of a promoter containing ISRE can be used to assess the IFN-β biological activity of the polypeptide, which is carried out by measuring the expression of luciferase in a luminometer 3h after the polypeptide is added to the cell culture.

[0075] In vivo IFN-β bioactivity can be assessed by induction of IFN response genes in peripheral blood mononuclear cells (RT-qPCR).

[0076] In addition, the at least 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% identity to SEQ ID NO: 80, wherein the polypeptide has the 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, for a polypeptide to be considered to have the biological activity of interleukin-15, it must 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-15 assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 80.

[0077] The biological activity of IL-15 can be assessed in vitro by a CTLL-2 cell proliferation assay.

[0078] In vivo IL-15 bioactivity can be assessed by its ability to induce NK cell and T cell proliferation using FACS analysis.

[0079] In addition, 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% identity to SEQ ID NO: 81, wherein the polypeptide has 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, for a polypeptide to be considered to have the biological activity of CXCL10, it must 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 assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 81. CXCL10 is also known as IP-10.

[0080] The biological activity of CXCL10 can be assessed by its ability to attract T lymphocytes and natural killer cells in vivo or in vitro migration assays.

[0081] In a preferred embodiment, the second nucleic acid construct of the present invention comprises two or more sequences, each sequence 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 relative to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12. The second immunomodulatory protein may be 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 IFN-β; or the second 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 the biological activity of IL-15; or the second immunomodulatory protein may be a chemokine, preferably 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. NO:81. A polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity, wherein the polypeptide has the biological activity of CXCL10.

[0082] In addition, other combinations of immunomodulatory proteins are also feasible. Single-chain IL-12 can be the first immunomodulatory protein, and the second immunomodulatory protein can be selected from the group consisting of: a peptide that blocks TGF-β, a peptide that blocks FoxP3, or a monoclonal antibody against the immune checkpoint inhibitor PD1 or PD-L1, or a nanobody against the immune checkpoint inhibitor PD1 or PD-L1.

[0083] It should be noted that in the context of a second nucleic acid comprising two immunomodulatory proteins, the terms first and second immunomodulatory proteins do not describe the order of the coding sequences on the second nucleic acid, but are only used to illustrate two different immunomodulatory proteins. Therefore, 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. Here, upstream means that the coding sequence of the second immunomodulatory protein is located before the coding sequence of the first immunomodulatory protein in the 5' to 3' direction on the nucleic acid.

[0084] TGF-β blocking peptides are peptides that interfere with TGF-β itself or interfere with the receptors that bind TGF-β, so that at least some TGF-β signaling is reduced or even completely prevented. Examples of TGF-β blocking peptides are P17 and P144.

[0085] A FoxP3 blocking peptide is a peptide that interferes with FoxP3 itself or with a receptor that binds FoxP3, such that at least some FoxP3 signaling is reduced or even completely prevented. An example of a FoxP3 blocking peptide is P60.

[0086] In another preferred embodiment, the second nucleic acid construct is a bicistronic or multicistronic 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 sequence encoding a P2A peptide). The IRES sequence and the 2A peptide coding sequence are translational regulatory elements (TRE).

[0087] In the present specification, a protein or RNA having anti-cancer properties is any such molecule that is capable of delaying, stopping or reversing the progression of cancer.

[0088] A bicistronic or polycistronic expression cassette is a gene construct that contains two or more independent coding regions or genes in a single transcript. Generally, it contains two or more different sequences in a single transcript. In order to allow the transcription of two or more immunomodulatory proteins and / or immunomodulatory genes, a type of TRE that allows the translation of proteins individually is required.

[0089] One possible solution is to use an internal ribosome entry site (IRES). An IRES is a specific RNA sequence in a transcript that allows translation to be initiated at non-AUG codons, bypassing the normal cap-dependent initiation process. This mechanism allows regulation of gene expression and co-expression of multiple proteins from a single mRNA molecule.

[0090] Another possible solution is 2A peptides, also known as 2A self-cleaving peptides. 2A peptides are a class of 18-22aa long peptides that can induce ribosome skipping during intracellular protein translation. These peptides share the core sequence motif of DxExNPGP (SEQ ID NO:32) and are present in a wide range of viral families. They help produce polyproteins by causing ribosomes to be unable to form peptide bonds. Due to its short length, 2A peptides are very suitable for viral vector systems with limited capacity, such as AAV. Another advantage of these peptides is that they allow equimolar expression of the coding sequences separated by these peptides. Members of the 2A peptide family are T2A, P2A, E2A and F2A. The flanks of these peptides can have linker peptides of different lengths, which can lead to improved functionality. For example, the F2A element with a linker domain consisting of the amino acid sequence GGSG (SEQ ID NO:82) in front shows significant expression of proteins separated by the element.

[0091] Table 1: Examples of combinations of two immunomodulatory proteins separated by a translational regulatory element (TRE) in a bicistronic vector

[0092]

[0093]

[0094] A - preferably human single chain IL-12 as described in SEQ ID NO:9

[0095] B—Preferably human IFN-β as described in SEQ ID NO: 79

[0096] C-preferably human IL-15-sushi as described in SEQ ID NO: 80

[0097] D—preferably human CXCL10 as described in SEQ ID NO: 81

[0098] Immunotherapy involving the systemic availability of immunomodulatory proteins generally causes unwanted side effects. However, an advantageous aspect of the present invention is that the recombinant expression system used herein induces local expression of the immunomodulatory protein, 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 a tumor. Without wishing to be bound by theory, it is believed that the release of immunomodulatory proteins in or near a tumor causes T cells to infiltrate near the tumor, and may result in the recognition of tumor cells that usually escape the recognition of the immune system, thereby enabling the immune system to target and kill these cells. Therefore, the progression of the tumor is at least delayed, preferably stopped, and more preferably reversed.

[0099] In a preferred embodiment, the immunomodulatory gene or protein of the present invention is an immunostimulatory gene or protein, i.e., a gene or protein that stimulates the immune system, such as by directly stimulating the immune system (e.g., IL-12) or inhibiting immunosuppressive molecules (e.g., blocking peptides or shRNA targeting TGF-β).

[0100] According to another possibility, it can be provided that the STING-responsive promoter of the second nucleic acid construct is 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 IRSF3 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), a NFκB motif promoter (preferably according to SEQ ID NO: 29 or 30) and a STAT6 motif promoter. In a preferred embodiment, the STING-responsive promoter comprises at least one interferon response element (ISRE).

[0101] By effectively linking the expression of immunomodulatory genes or proteins to the activity of STING using a STING-responsive promoter according to the present invention, several advantages are achieved. First, the expression of immunomodulatory proteins can be controlled by using human non-responsive STING agonists. Therefore, it is possible to adjust the expression of immunomodulatory proteins according to the needs of treatment, rather than causing continuous high-level expression of proteins. Second, targeted and localized expression of immunomodulatory proteins can be achieved, thereby avoiding potential side effects of systemic expression of proteins.

[0102] Optionally, it is possible that the STING responsive promoter is selected from the list consisting of:

[0103] 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 relative to SEQ ID NO: 11 or a sequence identical to SEQ ID NO: 11;

[0104] b. The nucleotide sequence according to a., wherein the copies are spaced up to 40 nucleotides apart, preferably 4 to 35 nucleotides, more preferably 10 to 30 nucleotides, particularly preferably 15 to 25 nucleotides apart;

[0105] c. A nucleotide sequence according to SEQ ID NO: 12, comprising four copies of ISRE separated by 20 nucleotides;

[0106] d. The nucleotide sequence according to SEQ ID NO: 13, comprising four copies of ISREs separated by 6 nucleotides.

[0107] The use of STING-responsive promoters in the present specification ensures controlled and targeted expression of immunomodulatory proteins. In addition, the aforementioned STING-responsive promoters have been shown to have additional advantages. As detailed in the examples of the present application, STING-responsive promoters have been shown to be inducible by administering human non-responsive STING agonists in the presence of target STING. In addition, the induction of these promoters has been shown to be specific, as only limited expression occurs in the absence of a selective STING agonist. In particular, a promoter carrying four copies of an ISRE as described in SEQ ID NO: 12 is highly inducible while also exhibiting high specificity.

[0108] Preferably, the nucleotide sequence of the STING-responsive promoter described in a. and b. 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. does not contain any other 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 the ISRE of SEQ ID NO: 11, wherein the nucleotides are selected in such a way that these additional nucleotides do not form any transcriptional regulatory elements (e.g. randomly). Particularly preferably, the nucleotide sequence of the STING-responsive promoter as described in a. and b. consists of 1-10 copies, preferably 2-7 copies, more preferably 3-5 copies, and particularly preferably 4 copies of a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity relative to SEQ ID NO: 11 or identical to SEQ ID NO: 11.

[0109] Furthermore, the STING-responsive promoters described in a. to d. may already represent functional promoters, and thus these promoters do not require additional elements to induce expression of an operably linked transgene.

[0110] According to a further possibility, it can be provided that the second nucleic acid construct also contains at least one sequence motif, which inhibits the transgenic expression of the immunomodulatory protein in tissues or cells (preferably the liver) that are not intended to express the protein, wherein the sequence motif is preferably a target sequence of a microRNA that is highly abundant in the liver.

[0111] It is known that some viral particles, especially AAV, show tropism for hepatocytes. This means that when the viral particles enter the blood circulation system, they preferentially transduce hepatocytes, ultimately resulting in the expression of the encoded transgene in the liver. Likewise, in the case of intratumoral injection of the viral particles, some of these particles will enter the blood circulation system, since tumors are highly vascularized. In order to prevent the unwanted expression of the transgene encoded by the viral particles in the liver, it is therefore appropriate to control its expression. Several methods are known to those skilled in the art to achieve this goal.

[0112] In general, a suitable method for restricting transgene expression to a specific tissue or a specific cell type is to use a viral particle with a distinct tropism for the tissue or cell type. A method for achieving this is by changing the serotype of the AAV or by changing the capsid of the AAV. Several AAV serotypes are known in the art, each with a different tropism. However, since the present invention is intended to target several different types of cancer, it is necessary to optimize the serotype or capsid for each cancer or tumor type. Nevertheless, this may be a suitable way to achieve tissue or cell type specific expression of a transgene.

[0113] The skilled artisan may also use DNA-binding repressors or RNA-binding repressors to avoid unwanted expression of a transgene encoded by a viral particle in tissues or cells that are not intended to express the transgene. Thus, the second nucleic acid construct may comprise an operator that binds a repressor. The repressor may be operably linked to a promoter that is active in hepatocytes, thereby inducing expression of the repressor in these cells, thereby inhibiting expression of the transgene.

[0114] Another possibility is to use RNA interference (RNAi). RNAi is characterized in that RNA molecules reduce or suppress the expression of nucleic acid sequences with substantial or complete homology to the RNA molecules. Non-limiting examples of RNAi are small interfering RNA, small hairpin RNA or microRNA (miR). In order to use RNAi to reduce or suppress transgenic expression in specific tissues, it is conceivable that, for example, shRNA targeting the transgenic nucleotide sequence can be effectively connected to a tissue-specific promoter. When the shRNA is used, it therefore reduces or suppresses transgenic expression in tissues. Although this method is feasible in the specification of the present invention, it may be disadvantageous because it is necessary to additionally use the shRNA to limit the expression of transgenic in target tissues such as the liver.

[0115] A good way to avoid this shortcoming is to effectively link the miRNA target sequence to the nucleotide sequence of the transgene. Therefore, in the tissue where there is a miRNA associated with the miRNA target sequence, the expression of the transgene will be reduced or inhibited. Therefore, a preferred embodiment of the present invention includes a target sequence of a microRNA whose sequence motif is highly abundant in the liver. Therefore, 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 weakened.

[0116] Optionally, it is conceivable that the microRNA abundant in the liver is selected from the group consisting of: miR-122, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c or let7f. In addition, it may be advantageous that the microRNA abundant in the liver is miR-122. The advantage of miR-122 is that it is highly expressed in the liver, and more importantly, specifically expressed in the liver. Therefore, by selecting the miR-122 target sequence, a significant reduction of the transgene in the liver is ensured, while the expression in other tissues is not weakened. In addition, it has been shown that miR-122 is downregulated in most liver tumors, so the recombinant expression system according to the present invention is also suitable for treating liver cancer.

[0117] According to a preferred embodiment of the present invention, the corresponding miR-122 target sequence is a miR-122 target sequence having at least 90% or at least 95% sequence identity relative to SEQ ID NO: 14 or being identical to SEQ ID NO: 14. Preferably, the target sequence comprises 1 to 12, preferably 3 to 10, more preferably 4 to 8, and particularly preferably 5 copies of the miR-122 target sequence as described in the previous sentence. Increasing the number of copies of the miR-122 target sequence increases the miR-mediated reduction, while no significant improvement is observed for numbers higher than the disclosed 5 copies. In a preferred embodiment, the copies are separated by 1 to 40 nucleotides, preferably 1 to 30 nucleotides, more preferably 2 to 20 nucleotides, and particularly preferably 2 to 8 nucleotides.

[0118] Preferably, the nucleotide sequence comprises 5 copies of the miR-122 target sequence described in SEQ ID NO: 15. As described in the examples of the present application, the nucleotide sequence described in SEQ ID NO: 15 substantially inhibits transgene expression in the liver, thereby resulting in undetectable plasma levels of the transgene by the method described in Example 4.

[0119] According to a further possibility, it can be provided that the second nucleic acid construct further comprises a transgene encoding thymidine kinase (preferably a thymidine kinase according to SEQ ID NO: 16). The nucleotide sequence encoding thymidine kinase is linked to the nucleotide sequence of the immunomodulatory gene or protein and can be separated by a 2A self-cleaving peptide (so-called 2A peptide). The 2A peptide consists of a class of 18-22 amino acid long peptides that can induce ribosome jumping during intracellular protein translation. These peptides share the core sequence motif "DXEXNPGP" and are found in a wide range of viral families. They help produce polyproteins by causing the ribosome to be unable to form peptide bonds. Those skilled in the art recognize that the use of 2A peptides allows the co-expression of immunomodulatory proteins and thymidine kinase. The co-expression of thymidine kinase is advantageous because it allows tumor transduction to be assessed by PET imaging, and in the case of potential IL-12-mediated toxicity, cells expressing cytokines are eliminated by administering thymidine kinase substrates (e.g., acyclovir, penciclovir, famciclovir or ganciclovir).

[0120] The first and / or second nucleic acid constructs may also include a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). WPRE is known to increase transgene expression and is therefore most effective when placed downstream of the transgene (i.e., target STING sequence or immune modulatory gene or protein), close to the polyadenylation signal.

[0121] Conveniently, the first and / or second nucleic acid construct further comprises a polyadenylation signal located at a 3' position relative to the target STING or immune modulatory gene or protein.The length of the Poly(A) tail may be optimized to stabilize the mRNA and subsequently increase protein translation.

[0122] The skilled person is also aware of other methods for increasing protein translation, such as

[0123] a. by sequence modification to optimize G / C content and / or overstabilizing secondary structure codons, or

[0124] b. alteration of the 5′ cap by addition of synthetic or natural 5′ end cap analogs such as m7GpppG or initiator trimers such as “CleanCap”, or stabilization by capping enzymes, or

[0125] c. Adding regulatory elements

[0126] c.1 in the 5′-untranslated region (UTR), such as by optimizing or modifying the length and sequence to avoid overly stable structures through appropriate cis-regulatory sequences, or

[0127] c.2 In the 3′-UTR, stable elements or repetitive motifs or synthetic or natural sequences are introduced by sequence modification, which have been shown to be effective in other mRNAs.

[0128] In a specific embodiment, the second nucleic acid construct of the recombinant expression system consists of the nucleic acid construct given in the table below, wherein the different elements are shown in the 5′ to 3′ direction as a promoter, a first open reading frame (ORF1), a first translation regulatory element (TRE1), a second open reading frame (ORF2), a second translation regulatory element (TRE2), and a target sequence for a microRNA (microRNA element).

[0129] Table 2: Embodiments of the second nucleic acid construct of the expression system

[0130] Promoter ORF1 TRE1 ORF2 TRE2 microRNA elements <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 C ]]> IFN-β minimal promoter <![CDATA[scIL12 B ]]> IFN-β minimal promoter <![CDATA[scIL12 C ]]> <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 C ]]> <![CDATA[miR122 target site E > IFN-β minimal promoter <![CDATA[scIL12 B ]]> <![CDATA[miR122 target site E > IFN-β minimal promoter <![CDATA[scIL12 C ]]> <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 C ]]> WPRE <![CDATA[miR122 target site E > IFN-β minimal promoter <![CDATA[scIL12 B ]]> WPRE <![CDATA[miR122 target site E > IFN-β minimal promoter <![CDATA[scIL12 C ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[HSV-TK D ]]> p2A <![CDATA[scIL12 B ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[HSV-TK D ]]> p2A <![CDATA[scIL12 C ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> p2A <![CDATA[HSV-TK D ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 C ]]> p2A <![CDATA[HSV-TK D ]]> WPRE <![CDATA[miR122 target site E >

[0131] A - preferably 4 copies of ISRE (preferably given by SEQ ID NO: 12)

[0132] B - preferably human single chain IL-12 as described in SEQ ID NO:9

[0133] C - preferably murine single chain IL-12 as described in SEQ ID NO: 10

[0134] D—preferably HSV thymidine kinase as described in SEQ ID NO: 16

[0135] E—preferably 5 copies of the miR-122 target sequence as described in SEQ ID NO: 15

[0136] Another aspect of the present invention is an expression vector encoding the first and second nucleic acid constructs according to the present invention. The use of an expression vector comprising the first and second nucleic acid constructs has certain advantages. First, the expression vector is specifically designed to allow the nucleotide sequence of the encoding to be introduced into a host cell and to control their expression. Second, the expression vector is a very stable plasmid and is therefore protected from degradation. Third, the expression vector can be used for gene delivery using different methods. Non-limiting examples of the expression vector of the present invention and preferred embodiments are viral vectors, more preferably viral vectors selected from the list consisting of: adeno-associated virus (AAV) vectors, adenovirus vectors, lentiviral vectors, vaccinia virus vectors or herpes simplex virus vectors. In particular, viral vectors combine several advantages of expression vectors, such as general safety, low toxicity and stability.

[0137] It is conceivable that the first and second nucleic acid constructs of the recombinant expression system according to the invention are contained in a single expression vector. This has the advantage that a single administration of the expression vector is sufficient to express the recombinant expression system according to the invention in the patient.

[0138] 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 nucleotide sequence is not as limited as combining two nucleic acid constructs in one vector. Size limitation is a common problem for viral vectors. For example, adeno-associated virus (AAV) is limited to a size of about 5 kb, however, it must be taken into account that ITR, promoter and other elements are not included in 5 kb.

[0139] Thus, in an alternative aspect, the recombinant expression system of the present invention provides a combination of a first expression vector encoding a first nucleic acid construct and a second expression vector encoding a second nucleic acid construct.

[0140] It is also advantageous that the first and / or second nucleic acid construct of the one or more expression vectors further comprises 5'ITR and 3'ITR sequences, preferably AAV ITRs as described in SEQ ID NO: 17. It may be advantageous that the one or more expression vectors are AAV vectors, and preferably have serotypes AAV1, AAV3, AAV6, AAV8, AAV9, AAV2, AAV5, AAVrh.10 or any gain-of-function mutant of AAVrh.10, with AAV2 being most preferred. AAV is commonly used in biotechnology and is therefore well characterized. Advantages of using AAV vectors are that they are generally considered safe, exhibit low toxicity and are stable. In addition, AAV has been approved for use in humans by several governmental organizations around the world.

[0141] It is also advantageous that the first and / or second nucleic acid construct of the one or more expression vectors further comprises 5'ITR, ψ packaging signal and 3'ITR sequences, preferably 5'ITR, ψ packaging signal and 3'ITR of adenovirus (Ad). It can be advantageous that the one or more expression vectors are adenoviral vectors, preferably high-capacity adenoviral vectors. Adenoviruses are commonly used in biotechnology and are therefore well characterized. An advantage of adenoviral vectors is that they have a higher size restriction than AAV vectors. In addition, adenoviruses have been approved for use in humans by several government organizations around the world.

[0142] It is also advantageous that the one or more expression vectors are lentiviral vectors, vaccinia virus vectors or herpes simplex virus vectors. These vectors are commonly used in biotechnology and are therefore well characterized.

[0143] It is also contemplated that nanoparticles can be used to deliver the first and / or second nucleic acid of the present invention.Nanoparticles as gene delivery systems are known in the art and have advantages such as adjustable size, shape and / or surface, allowing for modulation of their biological behavior. For effective delivery and cellular uptake, the nucleic acid construct can be packaged into nanoparticles (preferably liposomes) made of a pharmaceutically acceptable carrier, which is composed of physiologically compatible lipids, proteins, polysaccharides or synthetic polymers (such as standard lipids compatible with biological membranes) in different proportions, wherein the standard lipids are selected from the following list: phospholipids, such as 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-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 corresponding enantiomers, among which distearoylphosphatidylglycerol and distearoylphosphatidylcholine are most preferred. Another suitable example is that cardiolipin is preferably used in combination with cholesterol.Alternative nanoparticles are 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; (SM-102) heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (ALC 0159) N,N-dimyristamide of 2-hydroxyacetic acid (O-PEGylated to a PEG chain of about 2 kilodaltons), (ALC 0315) (4-hydroxybutyl) azadiyl] 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, such as DP7-C (cholesterol-modified cationic peptide DP7) (VQWRIRVAVIRK, SEQ ID NO: 85), polymers such as polyethylene glycol preparations composed of polymer molecules of different molecular sizes, which can be non-covalently bound or covalently linked to the lipids in the preparation.

[0144] Another aspect of the present invention is a viral particle comprising the aforementioned viral vector, said viral vector comprising the first and second nucleic acid constructs of the present invention. This has the advantage that a single administration of said viral particle is sufficient to express the recombinant expression system according to the present invention in a patient.

[0145] Another aspect of the present invention is the combination of a first and a second viral particle, the first viral particle comprising a viral vector encoding a first nucleic acid construct, 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 limited as when two nucleic acid constructs are combined in one viral vector of a single viral particle. Size limitation is a problem that often occurs for viral vectors. For example, adeno-associated virus (AAV) is limited to a size of about 5 kb, but it must be taken into account that ITR, promoters and other elements are not included in the 5 kb.

[0146] The one or more viral particles are preferably AAV or one or more adenoviral particles; more preferably one or more AAV viral particles. As described above, Ad and AAV exhibit several advantages over other viral particles, such as they are well characterized, frequently used, and approved for human treatment. These viruses exhibit other advantages that make them particularly useful in gene therapy, such as they are able to transduce dividing as well as non-dividing cells, and they do not integrate into the host genome.

[0147] In a specific embodiment, the combination of the first expression vector and the second expression vector, or the combination of the first viral particle and the second viral particle is provided as a kit of parts.

[0148] Another aspect of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a recombinant expression system, one or more expression vectors or one or more viral particles according to the present invention. The advantage of pharmaceutical compositions is that they are "ready-to-use" because they can be directly administered to the patient to be treated. In addition, the pharmaceutical composition is customized according to the treatment needs to optimize the stability and availability of the ingredients while avoiding unwanted side effects to the patient.

[0149] In another aspect of the invention, the recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical compositions according to the invention can each be used in therapy, preferably immunotherapy, or are provided for use in therapy, preferably immunotherapy.

[0150] In a preferred embodiment, the immunotherapy is an immunotherapy for a proliferative disorder, more preferably an immunotherapy for cancer, and even more preferably an immunotherapy for a solid cancer. Although it is conceivable that non-solid cancers can also be treated by the immunotherapy of the present invention, the treatment for solid cancers has the advantage that these cancers are confined to the local area and can therefore be treated by topical administration. Therefore, the recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical compositions according to the present invention can be administered topically to the tumor by intratumoral administration. This has the advantage of inducing a local and targeted immune response, thereby causing a delay, stoppage or reversal of tumor progression. In addition, although the present invention takes measures, such as by using miR target sequences to avoid systemic expression, local administration even further reduces unwanted total irradiation expression.

[0151] As mentioned above, cancer immunotherapy has several limitations, such as i) poor tumor targeting, ii) weak tumor cell killing ability, iii) laborious and therefore costly, iv) tumor immune escape, v) overstimulation of the immune system that can lead to serious side effects (Tan et al. 2020, Biomed Pharmacother. 124: 109821). The immunotherapy according to the present invention is an extended immunotherapy because it is also combined with gene therapy. Therefore, the immunotherapy according to the present invention is not just immunotherapy itself, but a combination of immunotherapy and gene therapy, hereinafter referred to as "immunogene therapy", to effectively treat proliferative diseases such as cancer.

[0152] The cancer may also be selected from the list consisting of: cancers 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, gastric cancer, gallbladder cancer, cervical cancer, thyroid cancer and skin cancer (including squamous cell carcinoma); lymphoid hematopoietic tumors including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, histiocytic lymphoma and Burkitt's lymphoma; central and peripheral nervous system tumors including astrocytoma, neuroblastoma, glioma and schwannoma; fibrosarcoma, Mesenchymal tumors including rhabdomyosarcoma and osteosarcoma; Other tumors including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratoma; 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 cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, gastric cancer, bladder cancer, urothelial carcinoma, hepatocellular carcinoma, hepatoblastoma, liver cancer, breast cancer, colon cancer and head and neck cancer, gastric cancer, germ cell tumor, bone cancer, bone tumor, malignant fibrous histiocytoma of bone in adults; malignant fibrous histiocytoma of bone in children, sarcoma, pediatric sarcoma, natural killer cells of the nasal cavity and paranasal sinuses, tumors, plasma cell tumors, carcinoid tumors, neuroendocrine tumors, bile duct cancer and Merkel cell carcinoma.

[0153] In another preferred embodiment, the recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical compositions of the present invention are used in combination therapy with at least one other therapy selected from the group consisting of: a therapy comprising at least one check point 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.

[0154] The inhibitor of the therapy including at least one checkpoint inhibitor can be selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR inhibitors, more preferably selected from the group consisting of: PD-1, PD-L1, PD-L2 or CTLA-4 inhibitors. Immune checkpoints are molecules expressed on T cells or cells that interact with T cells, which help regulate immune responses and prevent excessive or prolonged immune activation. However, cancer cells can use these checkpoint proteins to escape detection by the immune system. By blocking these checkpoint proteins, immune checkpoint inhibitors can release the brakes on the immune response and allow the immune system to attack and destroy cancer cells. In the specification of the present invention, it is particularly advantageous to use immune checkpoint inhibitors as an additional therapy because the expression system of the present invention causes T cells to infiltrate the tumor environment. Since the function of these T cells may be impaired due to immune checkpoints, methods to overcome this obstacle can greatly benefit the treatment results.

[0155] The therapy including at least one adoptive cell therapy can be adoptive NK cell therapy, adoptive dendritic cell therapy or adoptive T cell therapy, wherein adoptive T cell therapy is preferably CAR-T cell therapy or a therapy using tumor infiltrating leukocytes (TIL). In some cases, tumor silences the expression of MHC class I molecules and / or IFN-γ signaling molecules as a means of escaping T cell immunity. These problems can be avoided using adoptive cell therapy because these adoptive cells are generally able to identify and target tumor cells. Therefore, a synergistic effect can be expected in a combined approach.

[0156] Furthermore, the combination of the expression system of the present invention and a cancer vaccine or exogenous cytokine can cause a synergistic effect because the recruited T cells are directed toward cancer cells by the cancer vaccine or exogenous cytokine.

[0157] In a preferred embodiment of the invention, the cancer is selected from the list consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymus cancer, cervical cancer, colon cancer, esophageal cancer, stomach 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 carcinoma, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasmacytoma, bile duct cancer and neuroendocrine tumors or carcinoid tumors.

[0158] In another aspect, the present invention relates to a recombinant expression system, one or more expression vectors, one or more viral particles or a pharmaceutical composition of the present invention for use in immunotherapy, wherein the immunotherapy comprises the following steps:

[0159] a. administering a recombinant expression system according to the invention, one or more expression vectors 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 produce a therapeutically effective transgenic expression of the target STING and an immunomodulatory protein, wherein the administration is preferably intratumoral or systemic;

[0160] b. Optionally, prior to and / or simultaneously with the administration according to step a), treating the patient with radiation therapy (preferably radiation therapy directed to tissues or organs intended to express one or more of said transgenes from a); and / or;

[0161] c. After administration according to step a), treating the patient with a STING agonist that recognizes the transgenic expressed target STING other than human STING and its natural variants and / or with radiation therapy (preferably radiation therapy directed to tissues or organs intended to express one or more of said transgenes from a).

[0162] It is worth noting that step c) provides three possible different treatment options. For example, only STING agonists that recognize the transgenic expressed target STING but not human STING and its natural variants (human non-responsive STING agonists) can be applied. Another possibility is to treat the patient with radiation therapy alone. Finally, these two options can also be combined.

[0163] In another embodiment, before, simultaneously and / or after administration according to step a), the patient is treated 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, treatment with at least one other therapy occurs after administration according to step a).

[0164] In a similar aspect, the invention relates to a method of treating a proliferative disorder, preferably cancer, in a patient, the method comprising:

[0165] a. administering a recombinant expression system according to the invention, one or more expression vectors according to the invention, one or more viral particles according to the invention or a pharmaceutical composition according to the invention to a patient suffering from a proliferative disorder, preferably cancer, to produce a therapeutically effective transgenic expression of a target protein and an immunomodulatory protein, wherein the administration is preferably intratumoral or systemic;

[0166] b. Optionally, prior to and / or simultaneously with the administration according to step a), treating the patient with radiation therapy (preferably radiation therapy directed to tissues or organs intended to express one or more of said transgenes from a); and / or;

[0167] c. After administration according to step a), treating the patient with a STING agonist that recognizes the transgenic expressed target STING other than human STING and its natural variants and / or with radiation therapy (preferably radiation therapy directed to tissues or organs intended to express one or more of said transgenes from a).

[0168] In another embodiment, before, simultaneously and / or after administration according to step a), the patient is treated 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, treatment with at least one other therapy occurs after administration according to step a).

[0169] Notably, the embodiments described below are applicable to the recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical compositions of the present invention for use in immunotherapy and methods of treating proliferative disorders.

[0170] It is envisaged that the use of the immunotherapy or method of treating a proliferative disorder according to the invention may be combined with other treatments as anticancer therapies. Several anticancer therapies are known in the art, such as chemotherapy, hormone therapy, radiotherapy, photodynamic therapy, stem cell transplantation, surgery, targeted therapy, hyperthermia or other immunotherapies. It may be advantageous to direct the radiotherapy to the tumor that has received intratumoral administration according to step a).

[0171] It is also advantageous if in step c), the STING agonist is selected from the group consisting of 5,6-dimethylxanthone-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 (tiloron) and 10-carboxymethyl-9-acridone (CMA). Preferably, the STING agonist is DMXAA. DMXAA has been shown in clinical studies to be a molecule with a beneficial safety profile and no serious side effects. In addition, according to the present invention, DMXAA activates the target STING from step a without activating human wild-type STING or its natural variants, thereby making the treatment generally safe and avoiding systemic STING activation.

[0172] It is also optionally contemplated that in step b) and / or step c), a radiotherapy of 100 Gy or less is used. Any ionizing radiation dose of 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 in step b) and / or step c), a low-dose radiotherapy of 40 Gy or less, preferably 10 Gy or less, more preferably 8 Gy or less is used. Low dose radiation therapy can be any radiation therapy with 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 can be better tolerated by patients because they experience fewer or no side effects that are otherwise common in radiotherapy. As used herein, the indicated dose refers to the total dose, meaning that the total dose used for treatment in step b) or step c) does not exceed the required dose. Thus, the dose of 40 Gy applied in step b) can be achieved by a single dose of 40 Gy or by divided doses (e.g., four doses of 10 Gy, given over a period of weeks or even months).

[0173] In another embodiment, the radiation therapy in step c) comprises several individual radiation therapy treatments, wherein the cumulative dose of the individual radiation therapy treatments totals less than 100 Gy, preferably less than 40 Gy, more preferably less than 25 Gy. The cumulative dose of the individual radiation therapy treatments may be 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, 44 Gy, 40 Gy, 38 Gy, 36 Gy, 34 Gy, 32 Gy, 30 Gy, 28 Gy, 26 Gy, 24 Gy, 22 Gy, 25 Gy, 26 Gy, 24 Gy, 25 Gy, 26 ... 0Gy, 19Gy, 18Gy, 17Gy, 16Gy, 15Gy, 14Gy, 13Gy, 12Gy, 11Gy, 10Gy, 9Gy, 8Gy, 7Gy, 6Gy, 5 Gy, 4Gy, 3Gy, 2.5Gy, 2.0Gy, 1.5Gy, 1Gy, 0.5Gy, 0.25Gy, 0.2Gy, 0.15Gy, 0.1Gy or 0.05Gy.

[0174] It is noteworthy that radiotherapy can be applied externally or internally. Those skilled in the art know several methods for providing external radiotherapy. For example, external radiotherapy can be provided by gamma rays, electron beams or proton accelerators sufficient for therapeutic purposes. However, for some tumors, it may be advantageous to use internal radiotherapy. For therapeutic purposes, internal radiotherapy can be provided by administering radioisotopes in pharmacologically acceptable forms and dosages. The embodiment of internal radiotherapy can be brachytherapy or non-localized radiotherapy. Brachytherapy physically limits radioactive deposits so that they can not be moved from the position where they have been deposited. Radioactive deposits can be placed in the gap (interstitial radiotherapy), which is used for example for a considerable percentage of prostate tumors and some head and neck tumors, or radioactive deposits can be placed in the cavity (intracavitary radiotherapy), which is used for example for some uterine, bronchial or esophageal tumors. Nonlocalized radiation therapy can be a localized administration of partial diffusion, such as selective internal radiation therapy (SIRT), in which a suspension of material is injected into a specific artery or vein with the goal of keeping it close to the injection site rather than being widely distributed in the body, or it can be a completely systemic approach, such as the use of synthetic soluble chemicals with radioisotopes that find their site of action by affinity or biologics such as radiolabeled antibodies to better deliver radiation to the target tumor. A typical example of the latter is iodine-131. While different isotopes may be used for different therapeutic 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.

[0175] As discovered by the present inventors, there is a synergistic effect between the canonical STING pathway activated by DMXAA and the atypical STING pathway activated by radiotherapy in vitro (see Example 9) and in vivo (see Example 10), which supports the beneficial combination of STING agonist treatment and radiotherapy in step c.

[0176] In addition, the second nucleic acid construct has the advantage that it can be applied systemically and that the expression of at least one immunomodulatory gene or protein can be induced by radiation. Thus, radiation has a dual effect, since it not only promotes the transduction of the virus into the irradiated cells, but also induces the expression of at least one immunomodulatory gene or protein.

[0177] The first effect described above relates to an important concept of the present invention, namely that radiotherapy marks the tumor for transgene expression from a gene vector such as an AAV vector. This fact has very important clinical applications because the data of the present application show that when the AAV vector is administered parenterally (e.g., intravenously), tumor irradiation converts the tumor into a target for transgene (e.g., IL-12) expression. Using this method, the inventors were even able to cure subcutaneous tumors when the vector was administered intravenously. After intravenous administration of AAV.ISREx4.IL12miR122T to animals with subcutaneous tumors, the tumor was eliminated if the tumor had been previously irradiated, but not if it had not received any irradiation. In these experiments, tumor irradiation alone (by intravenous administration of a control virus encoding luciferase) could not induce tumor regression.

[0178] In another aspect, the present invention includes a recombinant expression system consisting of a nucleic acid construct comprising sequences encoding at least two immunomodulatory proteins, which 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 protein in tissues or cells that are not intended to express the protein.

[0179] As discovered by the present inventors, an expression system consisting of nucleic acids comprising at least two immunomodulatory proteins under the control of a STING-responsive promoter induces upregulation of immunomodulatory proteins upon irradiation even in the absence of the target STING (Example 15). In addition, the expression system alone induces significant anti-tumor effects (Example 17) without causing off-site expression (Example 16).

[0180] This expression system combines many of the favorable aspects of cancer immunotherapy while avoiding the disadvantages of said therapy.

[0181] It allows the expression of immunomodulatory peptides, in particular cytokines such as interleukins or chemokines, in tumors, thereby stimulating the immune system to target cancer cells of the tumor. Due to the preferred intratumoral administration of the expression system, cytokine expression is highly localized, thereby avoiding the adverse aspects and toxicity of systemic cytokine therapies known in the art.

[0182] Furthermore, the inventors have found that the nucleic acid of the recombinant expression system induces upregulation of the immunomodulatory transgene even when irradiated alone. Thus, the expression of the immunomodulatory transgene can be controlled by administering radiation therapy.

[0183] Furthermore, the inventors have found that the recombinant expression system is non-toxic and exhibits minor side effects in vivo. Notably, the inventors have found that when administered in vivo, the recombinant expression system results in tumor reduction, ultimately leading to complete remission.

[0184] This recombinant expression system generates a specific basal immune pathway in transfected cancer cells that utilizes other endogenous pathway components.

[0185] The nucleic acid construct of the expression system is an artificial gene 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. The immunomodulatory proteins activate innate and adaptive immune responses to generate specific cancer immune responses.

[0186] Alternatively, a radiation-inducible promoter may be provided. As used herein, a radiation-inducible promoter is a promoter that induces expression of a therapeutic gene sequence when a cell is irradiated. These promoters may be natural, synthetic or artificially produced, selected from recombinant products of natural promoters. For example, these promoters may contain at least one of the following sequences, either individually or in a repetitive manner:

[0187] a. the eukaryotic radiation-inducible sequence CC(A / T)(6)GG (SEQ ID NO:87), also known as a CArG element or [CC(A+T-rich)6GG], such as the sequence of the early growth response-1 (Egr-1) promoter or the pE9 promoter, 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 alternatively the sequence comprises at least two sequences according to SEQ ID NO:87, wherein the at least two sequences may be separated by additional nucleotides;

[0188] b. a low frequency CCAAT box sequence, such as the CCAAT box sequence of the survivin promoter, preferably the sequence has 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 88 or is identical to SEQ ID NO: 88 (CCAAT-rich domain of the survivin promoter);

[0189] c. cyclin-dependent kinase inhibitor 1 (also known as CDKN1A or p21) promoter;

[0190] d. Late herpes simplex virus 1 promoters, such as glycoprotein C promoter or U S 11 promoter;

[0191] e. Combinations of the above or artificial mutations thereof.

[0192] By using sequence motifs that inhibit transgenic expression of an immunomodulatory protein in tissues or cells not intended to express the protein, protein expression can be restricted to different tissues, tissue regions, or cell populations.

[0193] Furthermore, it is advantageous within the scope of the present invention that at least two immunomodulatory proteins encoded by the sequence of the nucleic acid construct are selected from the group consisting of: interleukin family, chemokine family, colony stimulating factor family, transforming growth factor family, tumor necrosis factor family, any blocking peptide for immunosuppressive factor, any monoclonal antibody or any scFv or any nano antibody reactive with immune checkpoint protein, or shRNA targeting immunosuppressive factor. A non-limiting list of immunomodulatory proteins is 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, 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-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, CCL1 9. 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 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 that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3, and fragments or variants thereof;

[0194] In a preferred embodiment of the present invention, the at least two immunomodulatory proteins encoded by the nucleic acid construct sequence 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-macrophage colony-stimulating factor (GM-CSF), type I interferon (IFN-α and IFN-β), IFN-γ, TNF-α, CXCL10, FLT3-ligand, blocking peptides targeting TGF-β, blocking peptides targeting IL-10, blocking peptides targeting FoxP3, monoclonal antibodies or scFvs or nanobodies that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3 and fragments or variants thereof.

[0195] Those skilled in the art will recognize 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.

[0196] It is known in the art that there are several immunomodulatory proteins for immunotherapy for cancer treatment. For example, IL-2 is a protein that has been used in cancer treatment for more than 20 years for immunotherapy. However, only a very small number of patients respond to IL-2-related immunotherapy, and these therapies are known to cause serious side effects. IL-12 reprograms myeloid-derived suppressor cells (MDSC) into efficient antigen presenting cells (APC). In addition, IL-12 strongly stimulates T cells and NK cells, and increases IFN-γ production, promoting Th1-type responses that can control the growth of treated tumors and the growth of distant metastases (Lasek et al. Cancer Immunol. Immunother. 2014; 63: 419ff.). In addition, IL-12 has an anti-angiogenic effect, which can impair tumor vascularization, thereby limiting tumor growth. IL-12 is a heterodimeric cytokine encoded by two independent genes. In order to promote its expression in a recombinant expression system, it is feasible to use a recombinant single-chain IL-12 transgene encoding two subunits of IL-12. Thus, in a preferred embodiment, one of the immunomodulatory proteins according to this aspect of the invention is IL-12.

[0197] 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 α chain to enhance the agonist activity of IL-15 through trans expression such as provided by compound P22339 (Hu, Q., Ye, X., Qu, X. et al., Sci Rep 2018, 7675).

[0198] One of the immunomodulatory proteins encoded by the nucleic acid construct sequence can be optionally selected from the group consisting of: IL-12 α subunit, IL-12 β subunit, single-chain IL-12 comprising IL-12 α subunit and β subunit. It is worth noting that single-chain IL-12 (scIL-12) is a fusion protein comprising α and β subunits, wherein the peptide sequences are either directly connected or separated by a linker peptide, wherein the linker peptide can have a length of 1 to 100 amino acids. Preferably, the length of the linker ranges from 4 to 60 amino acids, more preferably from 8 to 40 amino acids, and particularly preferably from 12 to 25 amino acids. The length of the linker can 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 acids. In addition, one of the immunomodulatory proteins encoded by the nucleic acid construct sequence can be a single-chain IL-12 (human or mouse 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 relative to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12. The sequence identity of the polypeptide sequence 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, for a polypeptide to be considered to have interleukin-12 biological activity, it must 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 assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 9 or 10.

[0199] The biological activity of IL-12 can be assessed in vitro by the ability of the peptide to increase IFN-γ production, which is produced by cells expressing the IL-12 receptor and the corresponding signaling pathway, such as peripheral blood mononuclear cells or human macrophage cell lines. Alternatively, a reporter cell line can be used as, for example, a Promega bioluminescent IL-12 bioassay (Catalog No. JA2601, JA2605).

[0200] In vivo IL-12 bioactivity can be assessed by its ability to elevate IFN-γ serum levels.

[0201] In addition, an 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% identity to SEQ ID NO: 79, wherein the polypeptide has the biological activity of IFN-β. The sequence identity of the polypeptide sequence 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, for a polypeptide to be considered to have IFN-β biological activity, it must have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of IFN-β assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 79.

[0202] The biological activity of IFN-β can be estimated by the ability of inducing type I IFN response genes. For this purpose, human cell lines (such as HEK 293) can be incubated with polypeptides, and the expression of IFN response genes can be measured by RT-qPCR. Alternatively, IFN reporter cells encoding luciferase under the control of a promoter containing ISRE can be used to assess the IFN-β biological activity of the polypeptide, which is carried out by measuring the expression of luciferase in a luminometer 3h after the polypeptide is added to the cell culture.

[0203] In vivo IFN-β bioactivity can be assessed by induction of IFN response genes in peripheral blood mononuclear cells (RT-qPCR).

[0204] In addition, an immunomodulatory protein may be IL-15-sushi, which comprises or consists 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 the 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, for a polypeptide to be regarded as having the biological activity of interleukin-15, it must 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-15 as assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 80.

[0205] The biological activity of IL-15 can be assessed in vitro by a CTLL-2 cell proliferation assay.

[0206] The in vivo biological activity of IL-15 can be assessed by its ability to induce proliferation of NK cells and T cells using FACS analysis.

[0207] In addition, 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, wherein the polypeptide has 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, for a polypeptide to be considered to have the biological activity of CXCL10, it must 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-15 as assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 81. CXCL10 is also known as IP-10.

[0208] The biological activity of CXCL10 can be assessed by its ability to attract T lymphocytes and natural killer cells in vivo or in vitro migration assays.

[0209] 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 relative to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12. The second immunomodulatory protein may be 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 IFN-β; or the second 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 the biological activity of IL-15; or the second immunomodulatory protein may be a chemokine, preferably 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. NO:81. A polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity, wherein the polypeptide has the biological activity of CXCL10.

[0210] In addition, other combinations of immunomodulatory proteins are also feasible. Single-chain IL-12 can be the first immunomodulatory protein, and the second immunomodulatory protein can be selected from the group consisting of: a peptide that blocks TGF-β, a peptide that blocks FoxP3, or a monoclonal antibody against the immune checkpoint inhibitor PD1 or PD-L1, or a nanobody against the immune checkpoint inhibitor PD1 or PD-L1.

[0211] It should be noted that in the context of a nucleic acid comprising two immunomodulatory proteins, the terms first and second immunomodulatory proteins do not describe the order of the coding sequences on the nucleic acid, but are only used to illustrate two different immunomodulatory proteins. Therefore, 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. Here, upstream means that the coding sequence of the second immunomodulatory protein is located before the coding sequence of the first immunomodulatory protein in the 5' to 3' direction on the nucleic acid.

[0212] TGF-β blocking peptides are peptides that interfere with TGF-β itself or interfere with the receptors that bind TGF-β, so that at least some TGF-β signaling is reduced or even completely prevented. Examples of TGF-β blocking peptides are P17 and P144.

[0213] A FoxP3 blocking peptide is a peptide that interferes with FoxP3 itself or with a receptor that binds FoxP3, such that at least some FoxP3 signaling is reduced or even completely prevented. An example of a FoxP3 blocking peptide is P60.

[0214] In another preferred embodiment, the nucleic acid construct is a bicistronic or multicistronic 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 sequence encoding a P2A peptide). The IRES sequence and the 2A peptide coding sequence are translational regulatory elements (TRE).

[0215] In the present specification, a protein or RNA having anti-cancer properties is any such molecule that is capable of delaying, stopping or reversing the progression of cancer.

[0216] A bicistronic or polycistronic expression cassette is a gene construct that contains two or more independent coding regions or genes in a single transcript. Generally, it contains two or more different sequences in a single transcript. In order to allow the transcription of two or more immunomodulatory proteins and / or immunomodulatory genes, a type of TRE that allows the translation of proteins individually is required.

[0217] One possible solution is to use an internal ribosome entry site (IRES). An IRES is a specific RNA sequence in a transcript that allows translation to be initiated at non-AUG codons, bypassing the normal cap-dependent initiation process. This mechanism allows regulation of gene expression and co-expression of multiple proteins from a single mRNA molecule.

[0218] Another possible solution is 2A peptides, also known as 2A self-cleaving peptides. 2A peptides are a class of 18-22aa long peptides that can induce ribosome skipping during intracellular protein translation. These peptides share the core sequence motif of DxExNPGP (SEQ ID NO:32) and are present in a wide range of viral families. They help produce polyproteins by causing ribosomes to be unable to form peptide bonds. Due to its short length, 2A peptides are very suitable for viral vector systems with limited capacity, such as AAV. Another advantage of these peptides is that they allow equimolar expression of the coding sequences separated by these peptides. Members of the 2A peptide family are T2A, P2A, E2A and F2A. The flanks of these peptides can have linker peptides of different lengths, which can lead to improved functionality. For example, the F2A element with a linker domain consisting of the amino acid sequence GGSG (SEQ ID NO:82) before it shows significant expression of the protein separated by the element.

[0219] Table 3: Examples of combinations of two immunomodulatory proteins separated by a translational regulatory element (TRE) in a bicistronic vector

[0220] First immunomodulatory protein TRE Second immunomodulatory protein <![CDATA[Single-chain IL-12 A > IRES <![CDATA[Interferon-β B > <![CDATA[Single-chain IL-12 A > IRES <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > IRES <![CDATA[CXCL10 D ]]> <![CDATA[Single-chain IL-12 A > IRES Peptides that block FoxP3 <![CDATA[Single-chain IL-12 A > IRES Peptides that block TGF-β <![CDATA[Single-chain IL-12 A > IRES Anti-PD1 monoclonal antibody <![CDATA[Single-chain IL-12 A > IRES Anti-PD-L1 monoclonal antibodies <![CDATA[Single-chain IL-12 A > IRES Anti-PD1 Nanobody <![CDATA[Single-chain IL-12 A > IRES Anti-PD-L1 Nanobody <![CDATA[IFN-β B ]]> IRES <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > P2A <![CDATA[IFN-β B ]]> <![CDATA[Single-chain IL-12 A > P2A <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > P2A <![CDATA[CXCL10 D ]]> <![CDATA[Single-chain IL-12 A > P2A Peptides that block FoxP3 <![CDATA[Single-chain IL-12 A > P2A Peptides that block TGF-β <![CDATA[Single-chain IL-12 A > P2A Anti-PD1 monoclonal antibody <![CDATA[Single-chain IL-12 A > P2A Anti-PD-L1 monoclonal antibodies <![CDATA[Single-chain IL-12 A > P2A Anti-PD1 Nanobody <![CDATA[Single-chain IL-12 A > P2A Anti-PD-L1 Nanobody <![CDATA[IFN-β B ]]> P2A <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > F2A <![CDATA[IFN-β B ]]> <![CDATA[Single-chain IL-12 A > F2A <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > F2A <![CDATA[CXCL10 D ]]> <![CDATA[Single-chain IL-12 A > F2A Peptides that block FoxP3 <![CDATA[Single-chain IL-12 A > F2A Peptides that block TGF-β <![CDATA[Single-chain IL-12 A > F2A Anti-PD1 monoclonal antibody <![CDATA[Single-chain IL-12 A > F2A Anti-PD-L1 monoclonal antibodies <![CDATA[Single-chain IL-12 A > F2A Anti-PD1 Nanobody <![CDATA[Single-chain IL-12 A > F2A Anti-PD-L1 Nanobody <![CDATA[IFN-β B ]]> F2A <![CDATA[IL-15-sushi C ]]>

[0221] A - preferably human single chain IL-12 as described in SEQ ID NO:9

[0222] B—Preferably human IFN-β as described in SEQ ID NO: 79

[0223] C-preferably human IL-15-sushi as described in SEQ ID NO: 80

[0224] D—preferably human CXCL10 as described in SEQ ID NO: 81

[0225] Immunotherapy involving the systemic availability of immunomodulatory proteins generally causes unwanted side effects. However, the advantageous aspect of this aspect of the present invention is that the recombinant expression system used herein induces local expression of the immunomodulatory protein, thereby inducing a highly localized and targeted immune response. For example, the intratumoral administration of the recombinant expression system according to this aspect of the present invention induces an immune response in or near a tumor. Without wishing to be bound by theory, it is believed that the release of immunomodulatory proteins in or near a tumor causes T cells to infiltrate near the tumor, and may result in the recognition of tumor cells that usually escape recognition by the immune system, thereby enabling the immune system to target and kill these cells. Therefore, the progression of the tumor is at least delayed, preferably stopped, and more preferably reversed.

[0226] In preferred embodiments, at least one immunomodulatory protein of this aspect of the invention is an immunostimulatory protein. This 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 targeting TGF-β).

[0227] According to a further possibility, it can be provided that the STING-responsive promoter of the nucleic acid construct of this aspect of the invention is 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 IRSF3 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), a NFκB motif promoter (preferably according to SEQ ID NO: 29 or 30) and a STAT6 motif promoter. In a preferred embodiment, the STING-responsive promoter comprises at least one interferon response element (ISRE).

[0228] By operatively linking the expression of immunomodulatory proteins to a STING-responsive promoter according to the present invention, targeted and localized expression of immunomodulatory proteins can be achieved, thereby avoiding potential side effects resulting from systemic expression of the proteins.

[0229] Optionally, the STING responsive promoter of this aspect of the invention may be selected from the list consisting of:

[0230] 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 relative to SEQ ID NO: 11 or a sequence identical to SEQ ID NO: 11;

[0231] b. The nucleotide sequence according to a., wherein the copies are spaced up to 40 nucleotides apart, preferably 4 to 35 nucleotides, more preferably 10 to 30 nucleotides, particularly preferably 15 to 25 nucleotides apart;

[0232] c. A nucleotide sequence according to SEQ ID NO: 12, comprising four copies of ISRE separated by 20 nucleotides;

[0233] d. The nucleotide sequence according to SEQ ID NO: 13, comprising four copies of ISREs separated by 6 nucleotides.

[0234] The use of STING-responsive promoters in the present specification ensures controlled and targeted expression of immunomodulatory proteins. In addition, the aforementioned STING-responsive promoters have been shown to have additional advantages. As detailed in the examples of the present application, STING-responsive promoters have been shown to be inducible by administering human non-responsive STING agonists in the presence of target STING. In addition, the induction of these promoters has been shown to be specific, as only limited expression occurs in the absence of a selective STING agonist. In particular, a promoter carrying four copies of an ISRE as described in SEQ ID NO: 12 is highly inducible while also exhibiting high specificity. Notably, expression can be achieved even in the absence of STING when radiotherapy is applied.

[0235] Preferably, the nucleotide sequence of the STING-responsive promoter described in a. and b. 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. does not contain any other 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 the ISRE of SEQ ID NO: 11, wherein the nucleotides are selected in such a way that these additional nucleotides do not form any transcriptional regulatory elements (e.g. randomly). Particularly preferably, the nucleotide sequence of the STING-responsive promoter as described in a. and b. consists of 1-10 copies, preferably 2-7 copies, more preferably 3-5 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 relative to SEQ ID NO: 11 or a sequence identical to SEQ ID NO: 11.

[0236] Furthermore, the nucleotide sequences of the STING-responsive promoters described in a. to d. may already represent functional promoters, and thus these promoters do not require additional elements to induce expression of an operably linked transgene.

[0237] According to another possibility, it can be provided that the nucleic acid construct of this aspect of the invention also comprises at least one sequence motif which inhibits the transgenic expression of the immunomodulatory protein in tissues or cells (preferably the liver) that are not intended to express the protein, wherein the sequence motif is preferably a target sequence of a microRNA that is highly abundant in the liver.

[0238] It is known that some viral particles, especially AAV, show tropism for hepatocytes. This means that when the viral particles enter the blood circulation system, they preferentially transduce hepatocytes, ultimately resulting in the expression of the encoded transgene in the liver. In addition, in the case of intratumoral injection of the viral particles, some of these particles will enter the blood circulation system, because tumors are highly vascularized. In order to prevent the unwanted expression of the transgene encoded by the viral particles in the liver, it is therefore appropriate to control their expression. Several methods are known to those skilled in the art to achieve this goal.

[0239] In general, a suitable method for restricting transgene expression to a specific tissue or a specific cell type is to use a viral particle with a distinct tropism for the tissue or cell type. A method for achieving this is by changing the serotype of the AAV or by changing the capsid of the AAV. Several AAV serotypes are known in the art, each with a different tropism. However, since the present invention is intended to target several different types of cancer, it is necessary to optimize the serotype or capsid for each cancer or tumor type. Nevertheless, this may be a suitable way to achieve tissue or cell type specific expression of a transgene.

[0240] The skilled artisan may also use DNA binding repressors or RNA binding repressors to avoid unwanted expression of a transgene encoded by a viral particle in tissues or cells that are not intended to express the transgene. Thus, the nucleic acid construct of this aspect of the invention may comprise an operator that binds a repressor. The repressor may be operably linked to a promoter that is activated in tissues or cells that are not intended to express the transgene, thereby inducing expression of the repressor in these tissues or cells, thereby inhibiting expression of the transgene.

[0241] Another possibility is to use RNA interference (RNAi). RNAi is characterized in that RNA molecules reduce or suppress the expression of nucleic acid sequences with substantial or complete homology to the RNA molecules. Non-limiting examples of RNAi are small interfering RNA, small hairpin RNA or microRNA (miR). In order to use RNAi to reduce or suppress transgenic expression in specific tissues, it is conceivable that, for example, shRNA targeting the transgenic nucleotide sequence can be effectively connected to a tissue-specific promoter. When the shRNA is used, it therefore reduces or suppresses transgenic expression in tissues. Although this method is feasible in the specification of the present invention, it may be disadvantageous because it is necessary to additionally use the shRNA to limit the expression of transgenic in target tissues such as the liver.

[0242] A good way to avoid this shortcoming is to effectively link the miRNA target sequence to the nucleotide sequence of the transgene. Therefore, in the tissue where there is a miRNA associated with the miRNA target sequence, the expression of the transgene will be reduced or inhibited. Therefore, a preferred embodiment of the present invention includes a target sequence of a microRNA whose sequence motif is highly abundant in the liver. Therefore, 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 weakened.

[0243] Optionally, it is conceivable that the microRNA abundant in the liver is selected from the group consisting of: miR-122, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c or let7f. In addition, it may be advantageous that the microRNA abundant in the liver is miR-122. The advantage of miR-122 is that it is highly expressed in the liver, and more importantly, specifically expressed in the liver. Therefore, by selecting the miR-122 target sequence, a significant reduction of the transgene in the liver is ensured, while the expression in other tissues is not weakened. In addition, it has been shown that miR-122 is downregulated in most liver tumors, so the recombinant expression system according to this aspect of the invention is also suitable for treating liver cancer.

[0244] According to a preferred embodiment of this aspect of the invention, the corresponding miR-122 target sequence is a miR-122 target sequence that has at least 90% or 95% sequence identity relative to SEQ ID NO: 14 or is identical to SEQ ID NO: 14. Preferably, the target sequence comprises 1 to 12, preferably 3 to 10, more preferably 4 to 8, and particularly preferably 5 copies of the miR-122 target sequence as described in the previous sentence. Increasing the number of copies of the miR-122 target sequence increases miR-mediated reduction, while no significant improvement is observed for numbers higher than the disclosed 5 copies. In a preferred embodiment, the copies are separated by 1 to 40 nucleotides, preferably 1 to 30 nucleotides, more preferably 2 to 20 nucleotides, and particularly preferably 2 to 8 nucleotides.

[0245] Preferably, the nucleotide sequence comprises 5 copies of the miR-122 target sequence as described in SEQ ID NO: 15. As described in the examples of the present application, the nucleotide sequence described in SEQ ID NO: 15 has been shown to substantially inhibit the expression of the transgene in the liver, thereby resulting in undetectable plasma levels of the transgene by the method described in Example 16.

[0246] According to a further possibility, it can be provided that the nucleic acid construct of this aspect of the invention further comprises a transgene encoding a thymidine kinase (preferably a thymidine kinase according to SEQ ID NO: 16). The nucleotide sequence encoding the thymidine kinase is linked to the nucleotide sequence of the immunomodulatory protein and can be separated by a 2A self-cleaving peptide (so-called 2A peptide). The 2A peptide constitutes a class of 18-22 amino acid long peptides that can induce ribosome skipping during intracellular protein translation. These peptides share the core sequence motif "DXEXNPGP" and are found in a wide range of viral families. They help produce polyproteins by causing the ribosome to be unable to form peptide bonds. Those skilled in the art recognize that the use of 2A peptides allows the co-expression of immunomodulatory proteins and thymidine kinase. The co-expression of thymidine kinase is advantageous because it allows the assessment of tumor transduction by PET imaging, and in the case of potential IL-12-mediated toxicity, the cells expressing cytokines are eliminated by administering thymidine kinase substrates (e.g., acyclovir, penciclovir, famciclovir or ganciclovir).

[0247] The nucleic acid constructs of this aspect of the invention may also comprise a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). WPRE is known to increase transgene expression and is therefore most effective when placed downstream of the transgene (ie, immunomodulatory protein), close to the polyadenylation signal.

[0248] Conveniently, the nucleic acid construct of this aspect of the invention further comprises a polyadenylation signal located at a 3' position relative to the immunomodulatory protein.The length of the poly(A) tail may be optimized to stabilize the mRNA and subsequently increase protein translation.

[0249] The skilled person is also aware of other methods for increasing protein translation, such as

[0250] a. by sequence modification to optimize G / C content and / or overstabilizing secondary structure codons, or

[0251] b. alteration of the 5′ cap by addition of synthetic or natural 5′ end cap analogs such as m7GpppG or initiator trimers such as “CleanCap”, or stabilization by capping enzymes, or

[0252] c. Adding regulatory elements

[0253] c.1 in the 5′-untranslated region (UTR), such as by optimizing or modifying the length and sequence to avoid overly stable structures through appropriate cis-regulatory sequences, or

[0254] c.2 In the 3′-UTR, stable elements or repetitive motifs or synthetic or natural sequences are introduced by sequence modification, which have been shown to be effective in other mRNAs.

[0255] In a specific embodiment, the nucleic acid construct of the recombinant expression system of this aspect of the invention consists of the nucleic acid construct given in the table below, wherein the different elements are shown in the 5′ to 3′ direction as a promoter, a first open reading frame (ORF1), a first translation regulatory element (TRE1), a second open reading frame (ORF2), a second translation regulatory element (TRE2) and a target sequence of a microRNA (microRNA element).

[0256] Table 4: Embodiments of nucleic acid constructs of expression systems

[0257]

[0258] A - preferably 4 copies of an ISRE preferably given by SEQ ID NO: 12

[0259] B - preferably human single chain IL-12 as described in SEQ ID NO:9

[0260] C—preferably human IFN-β as described in SEQ ID NO: 79

[0261] D-preferably human IL-15-sushi as described in SEQ ID NO: 80

[0262] E—preferably 5 copies of the miR-122 target sequence as described in SEQ ID NO: 15

[0263] Another aspect of the present invention is an expression vector encoding a nucleic acid construct comprising a sequence encoding at least two immunomodulatory proteins. Using an expression vector comprising this nucleic acid construct has certain advantages. First, the expression vector is specifically designed to allow the nucleotide sequence of the encoding to be introduced into a host cell and to control their expression. Secondly, the expression vector is a very stable plasmid, so it is protected and not degraded. The third, the expression vector can be used for gene delivery using different methods. The non-limiting examples of expression vectors and the preferred embodiments of this aspect of the present invention are viral vectors, more preferably viral vectors selected from adeno-associated virus (AAV) vectors, adenovirus vectors, lentiviral vectors, vaccinia virus vectors or herpes simplex virus vectors. In particular, viral vectors combine several advantages of expression vectors, such as general safety, low toxicity and stability.

[0264] It is also advantageous that the nucleic acid construct comprising sequences encoding at least two immunomodulatory proteins of the expression vector further comprises 5'ITR and 3'ITR sequences, preferably the AAV ITRs described in SEQ ID NO: 17. It may be advantageous that the expression vector is an AAV vector, and preferably has 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. An advantage of using AAV vectors is that they are generally considered safe, exhibit low toxicity and are stable. In addition, AAV has been approved for use in humans by several governmental organizations around the world.

[0265] It is also advantageous that the nucleic acid construct comprising the sequence of at least two immunomodulatory proteins encoding the expression vector also comprises 5'ITR, ψ packaging signal and 3'ITR sequence, preferably 5'ITR, ψ packaging signal and 3'ITR of adenovirus (Ad). It may be advantageous that the expression vector is an adenoviral vector, preferably a high-capacity adenoviral vector. Adenoviruses are commonly used in biotechnology and are therefore well characterized. An advantage of adenoviral vectors is that they have a higher size restriction compared to AAV vectors. In addition, adenoviruses have been approved for use in humans by several government organizations in the world.

[0266] It may also be advantageous that the expression vector is a lentiviral vector, a vaccinia virus vector or a herpes simplex virus vector. These vectors are commonly used in biotechnology and are therefore well characterized.

[0267] It is also contemplated that nanoparticles may be used to deliver nucleic acids comprising sequences encoding at least two immunomodulatory proteins.Nanoparticles are known in the art as gene delivery systems and have advantages such as adjustable size, shape and / or surface, allowing for modulation of their biological behavior. For effective delivery and cellular uptake, the nucleic acid construct can be packaged into nanoparticles (preferably liposomes) made of a pharmaceutically acceptable carrier, which is composed of physiologically compatible lipids, proteins, polysaccharides or synthetic polymers (such as standard lipids compatible with biological membranes) in different proportions, wherein the standard lipids are selected from the following list: phospholipids, such as 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-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 corresponding enantiomers, among which distearoylphosphatidylglycerol and distearoylphosphatidylcholine are most preferred. Another suitable example is that cardiolipin is preferably used in combination with cholesterol.Alternative nanoparticles are 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-Oi 10; (SM-102) heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (ALC0159) N,N-dimyristamide of 2-hydroxyacetic acid (O-PEGylated to about 2 kilodaltons PEG chain), (ALC 0315) (4-hydroxybutyl) azadiyl] di(hexane-6,1-diyl) bis(2-hexyldecanoate), Lipofectamine, Trans-IT, Ins tantFECT, (DOTAP) 1,2-dioleoyl-3-trimethylammonium. Other components may include peptides and polypeptides, such as DP7-C (cholesterol-modified cationic peptide DP7) (VQWRIRVAVIRK, SEQ ID NO: 85), polymers such as polyethylene glycol preparations composed of polymer molecules of different molecular sizes, which can be non-covalently bound or covalently linked to the lipids in the preparation.

[0268] Another aspect of the present invention is a viral particle comprising the aforementioned viral vector, wherein the viral vector comprises a nucleic acid construct comprising a sequence encoding at least two immunomodulatory proteins. This has the advantage that a single administration of the viral particle is sufficient to express the recombinant expression system in the patient.

[0269] The aforementioned viral particles are preferably AAV or adenoviral particles; more preferably AAV viral particles. As described above, Ad and AAV exhibit several advantages over other viral particles, such as they are well characterized, frequently used, and approved for human treatment. These viruses exhibit other advantages that make them particularly useful in gene therapy, such as they are able to transduce dividing and non-dividing cells, and they do not integrate into the host genome.

[0270] Another aspect of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a recombinant expression system, wherein the recombinant expression system comprises a sequence encoding at least two immunomodulatory proteins, the expression vector comprises a sequence encoding at least two immunomodulatory proteins, or the viral particle comprises a sequence encoding at least two immunomodulatory proteins. The advantage of pharmaceutical compositions is that they are "ready-to-use" because they can be directly administered to the patient to be treated. In addition, pharmaceutical compositions are customized according to treatment needs to optimize the stability and availability of the ingredients while avoiding unwanted side effects to the patient.

[0271] In another aspect of the present invention, a recombinant expression system, an expression vector, a viral particle or a pharmaceutical composition each comprising sequences encoding at least two immunomodulatory proteins can be used in therapy (preferably immunotherapy), or is provided for therapy (preferably immunotherapy).

[0272] In a preferred embodiment, immunotherapy is an immunotherapy for proliferative disorders, more preferably an immunotherapy for cancer, and even more preferably an immunotherapy for solid cancers. Although it is conceivable that non-solid cancers can also be treated by the immunotherapy of the present invention, the therapy for solid cancers has such an advantage that these cancers are confined to local areas and can therefore be treated by topical administration. Therefore, a recombinant expression system, expression vector, viral particle or pharmaceutical composition each comprising a sequence encoding at least two immunomodulatory proteins can be administered locally to a tumor by intratumoral administration. This has the advantage of being able to induce local and targeted immune responses, thereby causing a delay, stop or reversal of tumor progression. In addition, although the present invention has taken measures to avoid systemic expression, local administration even further reduces unwanted systemic expression.

[0273] As described above, cancer immunotherapy has several limitations, such as i) poor tumor targeting, ii) weak tumor cell killing ability, iii) laborious and therefore costly, iv) tumor immune escape, v) overstimulation of the immune system that can lead to serious side effects (Tan et al. 2020, Biomed Pharmacother. 124: 109821). Immunotherapy as described above is an extended immunotherapy because it is also combined with gene therapy. Therefore, this immunotherapy is not only immunotherapy itself, but also a combination of immunotherapy and gene therapy, hereinafter referred to as "immunogene therapy", to effectively treat proliferative disorders such as cancer.

[0274] The cancer may also be selected from the list consisting of: cancers 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, gastric cancer, gallbladder cancer, cervical cancer, thyroid cancer and skin cancer (including squamous cell carcinoma); lymphoid hematopoietic tumors including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, histiocytic lymphoma and Burkitt's lymphoma; central and peripheral nervous system tumors including astrocytoma, neuroblastoma, glioma and schwannoma; fibrosarcoma, Mesenchymal tumors including rhabdomyosarcoma and osteosarcoma; Other tumors including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratoma; 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 cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, gastric cancer, bladder cancer, urothelial carcinoma, hepatocellular carcinoma, hepatoblastoma, liver cancer, breast cancer, colon cancer and head and neck cancer, gastric cancer, germ cell tumor, bone cancer, bone tumor, malignant fibrous histiocytoma of bone in adults; malignant fibrous histiocytoma of bone in children, sarcoma, pediatric sarcoma, natural killer cells of the nasal cavity and paranasal sinuses, tumors, plasma cell tumors, carcinoid tumors, neuroendocrine tumors, bile duct cancer and Merkel cell carcinoma.

[0275] 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 combination therapy with at least one other therapy, wherein the other therapy is selected from the group consisting of: a therapy comprising at least one check point 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.

[0276] The inhibitor of the therapy including at least one checkpoint inhibitor can be selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR inhibitors, more preferably selected from the group consisting of: PD-1, PD-L1, PD-L2 or CTLA-4 inhibitors. Immune checkpoints are molecules expressed on T cells or cells that interact with T cells, which help regulate immune responses and prevent excessive or prolonged immune activation. However, cancer cells can use these checkpoint proteins to escape detection by the immune system. By blocking these checkpoint proteins, immune checkpoint inhibitors can release the brakes on the immune response and allow the immune system to attack and destroy cancer cells. In the description of this aspect of the invention, it is particularly advantageous to use immune checkpoint inhibitors as additional therapy because the expression system containing sequences encoding at least two immunomodulatory proteins causes T cells to infiltrate the tumor environment. Since the function of these T cells may be impaired due to immune checkpoints, methods to overcome this obstacle can greatly benefit treatment outcomes.

[0277] The therapy including at least one adoptive cell therapy can be adoptive NK cell therapy, adoptive dendritic cell therapy or adoptive T cell therapy, wherein adoptive T cell therapy is preferably CAR-T cell therapy or a therapy using tumor infiltrating leukocytes (TIL). In some cases, tumor silences the expression of MHC class I molecules and / or IFN-γ signaling molecules as a means of escaping T cell immunity. These problems can be avoided using adoptive cell therapy because these adoptive cells are generally able to identify and target tumor cells. Therefore, a synergistic effect can be expected in a combined approach.

[0278] Furthermore, the combination of the expression system of the present invention and a cancer vaccine or exogenous cytokine can cause a synergistic effect because the recruited T cells are directed toward cancer cells by the cancer vaccine or exogenous cytokine.

[0279] In a preferred embodiment of this aspect of the invention, the cancer is selected from the list consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymic cancer, cervical cancer, colon cancer, esophageal cancer, stomach 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 carcinoma, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasmacytoma, bile duct cancer and neuroendocrine tumors or carcinoid tumors.

[0280] In another aspect, the present invention relates to a recombinant expression system, an expression vector, a viral particle or a pharmaceutical composition for immunotherapy, each of which comprises a sequence encoding at least two immunomodulatory proteins, wherein the immunotherapy comprises the following steps:

[0281] a. administering to a patient suffering from a proliferative disorder 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 produce therapeutically effective transgenic expression of the immunomodulatory proteins, wherein the administration is preferably intratumoral administration or systemic administration;

[0282] b. Optionally, prior to and / or simultaneously with the administration according to step a), treating the patient with radiation therapy (preferably radiation therapy directed to the tissue or organ intended to express the transgene from a); and / or;

[0283] c. After administration according to step a), treating the patient with radiation therapy, preferably radiation therapy directed to the tissue or organ intended to express the transgene from a).

[0284] In a similar aspect, the invention relates to a method of treating a proliferative disorder, preferably cancer, in a patient, the method comprising:

[0285] a. administering to a patient suffering from a proliferative disorder (preferably cancer) 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 produce therapeutically effective transgenic expression of the immunomodulatory proteins, wherein the administration is preferably intratumoral administration or systemic administration;

[0286] b. Optionally, prior to and / or simultaneously with the administration according to step a), treating the patient with radiation therapy (preferably radiation therapy directed to the tissue or organ intended to express the transgene from a); and / or;

[0287] c. After administration according to step a), treating the patient with radiation therapy, preferably radiation therapy directed to the tissue or organ intended to express the transgene from a).

[0288] In another embodiment, before, simultaneously with and / or after administration according to step a), the patient is treated with at least one other therapy, the other therapy treating the patient, the other therapy being 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, treatment with at least one other therapy occurs after administration according to step a).

[0289] It is noteworthy that the embodiments described below are applicable to recombinant expression systems, expression vectors, viral particles or pharmaceutical compositions, each of which comprises sequences encoding at least two immunomodulatory proteins, for use in immunotherapy and methods of treating proliferative disorders.

[0290] It is contemplated that the use of immunotherapy or the method for treating a proliferative disease may be combined with other treatments as anticancer therapies. Several anticancer therapies are known in the art, such as chemotherapy, hormone therapy, radiotherapy, photodynamic therapy, stem cell transplantation, surgery, targeted therapy, hyperthermia or other immunotherapies. It may be advantageous to direct the radiotherapy to the tumor that has received intratumoral administration according to step a).

[0291] It is also optionally contemplated that in step b) and / or step c), a radiotherapy of 100 Gy or less is used. Any ionizing radiation dose of 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 in step b) and / or step c), a low-dose radiotherapy of 40 Gy or less, preferably 10 Gy or less, more preferably 8 Gy or less is used. Low dose radiation therapy can be any radiation therapy with 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 can be better tolerated by patients because they experience fewer or no side effects that are common in radiotherapy. As used herein, the indicated dose means that the total dose used for step b) or step c) does not exceed the required dose. Thus, the dose of 40 Gy applied in step b) can be achieved by a single dose of 40 Gy or by fractionated doses (e.g., four doses of 10 Gy) given over a period of weeks or even months.

[0292] In another embodiment, the radiation therapy in step c) comprises several individual radiation therapy treatments, wherein the cumulative dose of the individual radiation therapy treatments totals less than 100 Gy, preferably less than 40 Gy, more preferably less than 25 Gy. The cumulative dose of the individual radiation therapy treatments may be 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, 44 Gy, 40 Gy, 38 Gy, 36 Gy, 34 Gy, 32 Gy, 30 Gy, 28 Gy, 26 Gy, 24 Gy, 22 Gy, 25 Gy, 26 Gy, 24 Gy, 25 Gy, 26 ... 0Gy, 19Gy, 18Gy, 17Gy, 16Gy, 15Gy, 14Gy, 13Gy, 12Gy, 11Gy, 10Gy, 9Gy, 8Gy, 7Gy, 6Gy, 5 Gy, 4Gy, 3Gy, 2.5Gy, 2.0Gy, 1.5Gy, 1Gy, 0.5Gy, 0.25Gy, 0.2Gy, 0.15Gy, 0.1Gy or 0.05Gy.

[0293] It is noteworthy that radiotherapy can be applied externally or internally. Those skilled in the art know several methods for providing external radiotherapy. For example, external radiotherapy can be provided by gamma rays, electron beams or proton accelerators sufficient for therapeutic purposes. However, for some tumors, it may be advantageous to use internal radiotherapy. For therapeutic purposes, internal radiotherapy can be provided by administering radioisotopes in pharmacologically acceptable forms and dosages. The embodiment of internal radiotherapy can be brachytherapy or non-localized radiotherapy. Brachytherapy physically limits radioactive deposits so that they can not be moved from the position where they have been deposited. Radioactive deposits can be placed in the gap (interstitial radiotherapy), which is used for example for a considerable percentage of prostate tumors and some head and neck tumors, or radioactive deposits can be placed in the cavity (intracavitary radiotherapy), which is used for example for some uterine, bronchial or esophageal tumors. Nonlocalized radiation therapy can be a localized administration of partial diffusion, such as selective internal radiation therapy (SIRT), in which a suspension of material is injected into a specific artery or vein with the goal of keeping it close to the injection site rather than being widely distributed in the body, or it can be a completely systemic approach, such as the use of synthetic soluble chemicals with radioisotopes that find their site of action by affinity or biologics such as radiolabeled antibodies to better deliver radiation to the target tumor. A typical example of the latter is iodine-131. While different isotopes may be used for different therapeutic 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.

[0294] In another aspect, the present invention relates to embodiments 1 to 18 as described below:

[0295] Embodiment 1: A recombinant expression system comprising

[0296] a. a first nucleic acid construct comprising a sequence encoding a target STING that responds to a STING agonist that recognizes the target STING but not human STING and its natural variants, the sequence encoding the target STING being operably linked to an element that allows expression and / or translation of the target STING; and / or

[0297] b. A second nucleic acid construct comprising a sequence encoding at least one immunomodulatory gene or protein, said sequence being operably linked to a STING-responsive promoter.

[0298] Embodiment 2: The recombinant expression system of embodiment 1, wherein the target STING responds to a human non-responsive STING agonist selected from the group consisting of: 5,6-dimethylxanthone-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-acridone (CMA), and wherein preferably the target STING responds to DMXAA.

[0299] Embodiment 3: The recombinant expression system of embodiment 1 or 2, wherein the target sequence encoded by the sequence of the first nucleic acid construct is

[0300] a. a mutant form of a naturally occurring variant of human STING further comprising 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 assay; or

[0301] b. a non-human STING that is sensitive to a human non-responsive STING agonist as determined in an in vitro agonist assay, preferably a murine STING or a hybrid STING, even more preferably a murine STING according to SEQ ID NO: 7;

[0302] Wherein the target STING is preferably DMXAA-sensitive human STING as determined in an in vitro DMXAA assay.

[0303] Embodiment 4: The recombinant expression system of embodiment 3, wherein the target STING encoded by the sequence of the first nucleic acid construct is a DMXAA-sensitive human STING variant selected from the group consisting of:

[0304] a. a human STING[R] variant, further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I;

[0305] b. a human STING[AQ] variant further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of S162A, A230I, Q266I, E260I, and E260V;

[0306] c. a human STING[H] variant further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I; and

[0307] d. A truncated human STING[H] variant further comprising at least one or at least two amino acid substitutions selected from the group consisting of S162A and G230I.

[0308] Embodiment 5: The recombinant expression system of embodiment 4, wherein the DMXAA-sensitive human STING variant 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[R]-S162A / Q266I, hSTING[R]-S162A / Q230I hSTING[AQ]-S162A / R169A, hSTING[AQ]-S162A / T267A, hSTING[AQ]-E260I, 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, and preferably hSTING[R]-S162A / G230I / Q266I.

[0309] Embodiment 6: The recombinant expression system of embodiment 4, wherein the sequence of the first nucleic acid construct encoding the DMXAA-sensitive human STING variant is selected from the group consisting of:

[0310] a. SEQ ID NO: 5;

[0311] b. a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity relative to SEQ ID NO: 5;

[0312] c. a polynucleotide sequence encoding a polypeptide comprising SEQ ID NO: 6;

[0313] 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 relative to SEQ ID NO: 6;

[0314] e. a polynucleotide sequence encoding a polypeptide of c) or d) having at least one modification, wherein the modification is an amino acid substitution, an amino acid insertion, an amino acid deletion, a C-terminal truncation or an N-terminal truncation;

[0315] f. a polynucleotide sequence that hybridizes under at least moderately stringent conditions to the complement of a) to e) in any one of the polynucleotide sequences;

[0316] A region of the polynucleotide sequence of ga) to f), which encodes a fragment of at least 50 amino acid residues of a DMXAA-sensitive human STING variant;

[0317] Wherein the polypeptide encoded by the polynucleotide of any one of a) to g) has a biological activity responsive to the agonist DMXAA as determined in an in vitro DMXAA assay.

[0318] Embodiment 7: The recombinant expression system of any one of the above embodiments 1 to 6, wherein the promoter of the first nucleic acid 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;

[0319] or

[0320] The promoter of the first nucleic acid construct is an inducible promoter, preferably selected from the list consisting of: telomerase responsive promoter, IL-6 responsive promoter, TGF-β inducible promoter, VEGFR inducible promoter, epidermal growth factor receptor inducible promoter, HER2 / Neu inducible promoter, MUC1 promoter, BRCA1 promoter, BRCA2 promoter, LALBA promoter, PSA promoter, ERBB2 promoter, CEA promoter, uPAR promoter and thyroid transcription factor 1 (TTF1).

[0321] Embodiment 8: The recombinant expression system of any one of embodiments 1 to 7 above, wherein the at least one immunomodulatory gene or protein encoded by the sequence of the second nucleic acid 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, 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-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, C CL21, 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 Macrophage 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 that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3 and fragments or variants thereof; or shRNA targeting TGF-β, shRNA targeting IL-10, or shRNA targeting FoxP3.

[0322] Embodiment 9: The recombinant expression system of any one of embodiments 1 to 8 above, wherein the at least one immunomodulatory protein is selected from the group consisting of:

[0323] a. α subunit of IL-12;

[0324] b. β subunit of IL-12;

[0325] c. Single chain IL-12 containing α and β subunits of IL-12;

[0326] d. a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10;

[0327] e. 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.

[0328] Embodiment 10: The recombinant expression system of any one of embodiments 1 to 9 above, wherein the STING response promoter of the second nucleic acid construct is selected from the group consisting of: IFN-β response promoter, IFN-β minimal promoter, CXCL9 response promoter, CXCL10 response promoter, STAT motif promoter, IRSF3 motif promoter, ISRE motif promoter, NFκB motif promoter and STAT6 motif promoter, wherein the STING response promoter preferably comprises at least one interferon-stimulated response element (ISRE), more preferably selected from the list consisting of:

[0329] a. A nucleotide sequence comprising 1 to 4 copies of an ISRE as described in SEQ ID NO: 11, preferably comprising four copies of the ISRE;

[0330] b. according to the nucleotide sequence of SEQ ID NO:12;

[0331] c. The nucleotide sequence according to SEQ ID NO:13.

[0332] Embodiment 11: The recombinant expression system of any one of embodiments 1 to 10 above, wherein the second nucleic acid construct further comprises at least one sequence motif that inhibits transgenic expression of an immunomodulatory protein in the liver, wherein the sequence motif is preferably a target sequence of a microRNA that is highly abundant in the liver, more preferably selected from the group consisting of miR1-22, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c, let7f, and even more preferably a miR-122 target sequence selected from the list consisting of:

[0333] a. miR-122 target sequence as described in SEQ ID NO: 14;

[0334] 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;

[0335] c. A nucleotide sequence comprising 5 copies of the miR-122 target sequence as described in SEQ ID NO: 15.

[0336] Embodiment 12: An expression vector encoding the first and / or second nucleic acid construct according to any one of embodiments 1 to 11 above, or a combination of the first and second expression vectors, wherein the first vector encodes the first nucleic acid construct according to any one of embodiments 1 to 11 above, and the second vector encodes the second nucleic acid construct according to any one of embodiments 1 to 11 above, wherein one or more of the expression vectors are preferably viral vectors, more preferably viral vectors selected from the list consisting of: adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, vaccinia virus vectors or herpes simplex virus vectors, and even more preferably adeno-associated vectors having one or more of the following characteristics:

[0337] a. The first and / or second nucleic acid construct sequence further comprises a 5′ITR and a 3′ITR sequence, preferably the AAV ITR described in SEQ ID NO: 17;

[0338] b. The AAV vector is preferably AAV8, AAV1, AAV3, AAV6, AAV9, AAV2, AAV5, AAVrh.10 or any gain-of-function mutant of AAVrh.10.

[0339] Embodiment 13: A viral particle comprising a viral vector containing the first and / or second nucleic acid construct of Embodiment 12, or a combination of the first and second viral particles, wherein the first viral particle comprises a viral vector encoding the first nucleic acid construct of Embodiment 12, and the second viral particle comprises a viral vector encoding the second nucleic acid construct of Embodiment 12, wherein one or more of the viral particles are preferably AAV or one or more Ad viral particles; more preferably one or more AAV viral particles.

[0340] Embodiment 14: A pharmaceutical composition comprising a pharmaceutically acceptable excipient and:

[0341] a. the recombinant expression system of any one of embodiments 1 to 11; or

[0342] b. one or more expression vectors of embodiment 12; or

[0343] c. One or more viral particles of embodiment 13.

[0344] Embodiment 15: The recombinant expression system of any one of embodiments 1 to 11; one or more expression vectors of embodiment 12; one or more viral particles of embodiment 13 or the pharmaceutical composition according to embodiment 14, for use in immunotherapy, preferably for immunotherapy of proliferative disorders, more preferably for immunotherapy of cancer, and even more preferably for immunotherapy of solid cancers.

[0345] Embodiment 16: A recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical composition for use according to embodiment 15, wherein the cancer is selected from the list 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 lymphoma, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasmacytoma, bile duct cancer and neuroendocrine tumors or carcinoid tumors.

[0346] Embodiment 17: The recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical composition according to embodiment 15 or 16, wherein the use in immunotherapy is a combination of:

[0347] a. administering the recombinant expression system of any one of embodiments 1 to 11, one or more expression vectors of embodiment 12, one or more viral particles of embodiment 13 or the pharmaceutical composition according to embodiment 14 to a patient suffering from a proliferative disorder, preferably cancer, to produce a therapeutically effective transgenic expression of a target STING and / or an immunomodulatory protein, wherein the administration is preferably intratumoral administration;

[0348] b. Optionally, prior to administration according to step a), treating the patient with radiation therapy (preferably radiation therapy directed to tissues or organs intended to express one or more of said transgenes from a); and

[0349] c. After administration according to step a), treating the patient with a STING agonist that recognizes the transgenic expressed target STING other than human STING and its natural variants and / or with radiation therapy (preferably radiation therapy directed to tissues or organs intended to express one or more of said transgenes from a).

[0350] Embodiment 18: A recombinant expression system, one or more expression vectors, one or more viral particles or a pharmaceutical composition for use according to embodiment 17, wherein in step c), a STING agonist according to embodiment 2 is used, preferably DMXAA.

[0351] Embodiment 19: A recombinant expression system, one or more expression vectors, one or more viral particles or a pharmaceutical composition for use according to Embodiment 17 or 18, wherein a medium or low dose radiotherapy of 40 Gy or less, preferably 8 Gy or less, is used in step b).

[0352] It should also be explicitly noted that in the context of the present patent application, indefinite articles and numerical indications such as "a", "an", "one", "two", etc. are generally understood as "at least"-indications, i.e., "at least one... ", "at least two... ", etc., unless it is clearly inferred from the respective context or it is obvious or technically mandatory for a person skilled in the art to indicate that there is only "exactly one... ", "exactly two... ", etc.

[0353] Other advantages, features and details of the invention will become apparent from the following description, in which embodiments of the invention are described in detail with reference to the accompanying drawings. In this regard, the features mentioned in the claims and in the description may each be essential to the invention individually or in any combination.

[0354] The foregoing explanation of the embodiments specifically describes the invention in the context of examples. Of course, without departing from the scope of the invention, the individual features of the embodiments can be freely combined with each other as long as it is technically reasonable.

[0355] definition

[0356] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena are rarely, if ever, completed and / or carried out to perfection or to achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0357] Approximately: As used herein, the term "approximately" may be used to modify any quantitative comparison, value, measurement, or other representation that could permissibly vary without resulting in a change in the basic function to which it is related.

[0358] A / an: As used in the specification, "a" or "an" may mean one or more. As used herein in one or more claims, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more.

[0359] Or / and / or: Use of the term "or" in the claims is intended to mean "and / or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, although this disclosure supports a definition referring only to alternatives and "and / or."

[0360] The term "about" generally refers to within the standard deviation of the stated value as determined using standard analytical techniques for measuring the stated value. These terms can also mean plus or minus 5% of the stated value.

[0361] Substantially: The term "substantially" should be understood to mean that the methods or compositions include only the specified steps or materials, as well as those steps or materials that do not materially affect the basic and novel characteristics of such methods and compositions.

[0362] Stereoisomers: In addition, the compounds used in the present invention may contain one or more asymmetric carbon atoms and therefore exist in two or more stereoisomeric forms. When the compound contains an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur. The present invention includes the use of individual stereoisomers of the compound and (where appropriate) its individual tautomeric forms as well as mixtures thereof.

[0363] Analog: As used herein, the term "analog" refers to a compound that is similar in structure to another compound but has a slightly different composition, such as the replacement of one atom by an atom of a different element or a functional group.

[0364] Isotopes in compounds: The present invention also includes the use of all suitable isotopic variations of the compounds described herein or their pharmaceutically acceptable salts. An isotopic variation of a compound of the present invention or its pharmaceutically acceptable salt is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into compounds and their pharmaceutically 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 compounds and their pharmaceutically acceptable salts, such as those in which radioactive isotopes such as 3H or 14C are incorporated, can be used for drug and / or substrate tissue distribution studies. Tritiated isotopes (i.e., 3H) and carbon-14 isotopes (i.e., 14C) are particularly preferred because of their ease of preparation and detectability. Further substitution with isotopes such as deuterium (i.e. 2H) may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., prolonged in vivo half-life or reduced dosage requirements) and may therefore be preferred in some circumstances. Isotopic variations of the compounds of the invention and pharmaceutically acceptable salts thereof can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.

[0365] Cytokine: The term "cytokine" refers to a general term for biomolecules that affect / influence cells of the immune system. This definition is intended to include, but is not limited to, those biomolecules that act locally or can circulate in the blood, and when used in the compositions or methods of the present invention, are used to regulate or modulate an individual's immune response to cancer. Cytokines can be many different substances, such as interferons, interleukins, chemokines, and growth factors. Exemplary cytokines for practicing the present invention include, but are not limited to, interferons (e.g., IFN-α, IFN-β, and IFN-γ), interleukins (e.g., IL-1 to 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-macrophage colony stimulating factor (GM-CSF), TGF-β, FLT3-ligand, and fragments or variants thereof. It is worth noting that exogenously provided cytokines are a type of immunotherapy.

[0366] Immunomodulation: As used herein, the term "immunomodulation" refers to the property of initiating or altering (e.g., enhancing or attenuating) the activity of cells involved in an immune response. An immunomodulatory composition or method can enhance the activity of cells involved in an immune response, for example, by increasing pro-inflammatory or anti-inflammatory markers, and / or can reduce the activity of cells involved in an immune response, for example, by reducing pro-inflammatory or anti-inflammatory markers.

[0367] 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 its natural variants, but binds to mutated human STING or STING from other species, such as STING from a mouse, rat, or monkey. Optionally, the term "human non-responsive STING agonist" refers to an agonist that binds to human wild-type STING or its natural variants without inducing STING activity, while the STING activity of mutated human STING or STING from other species (such as STING from a mouse, rat, or monkey) is induced when the agonist is bound. 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] as described in SEQ ID NO:1, hSTING[H] as described in SEQ ID NO:2, hSTING[AQ] as described in SEQ ID NO:3, hSTING[H] as described in SEQ ID NO:4, STING[Q] having the amino acid substitution R293Q, or STING[HAQ] having the amino acid substitutions R71H, G230A, and R293Q, wherein the exogenously provided DMXAA-sensitive human STING responds to the "human non-responsive" agonist.

[0368] Protein / polypeptide: The terms "protein", "peptide" and "polypeptide" are used interchangeably herein to 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 in length. A protein, peptide or polypeptide may refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide or polypeptide may be modified, for example, by the addition of chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization or other modifications, and the like. A protein, peptide or polypeptide may also be a single molecule or a multimolecular complex. A protein, peptide or polypeptide may be simply a fragment of a naturally occurring protein or peptide. A protein, peptide or polypeptide may be naturally occurring, recombinant or synthetic, or any combination thereof.

[0369] Antibody / monoclonal antibody: As used herein, the term "antibody" refers collectively to immunoglobulins or immunoglobulin-like molecules, including, for example, but not limited to, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, such as mammals such as humans, goats, rabbits, camels and mice, and 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 cells into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, such as by producing cells that form hybrid antibodies from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0370] Single-chain variable fragment / scFv: The terms "single-chain variable region" and "scFv" refer to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are continuously connected by a short flexible polypeptide linker and can be expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0371] As used herein, the term "nanobody" refers to the smallest antigen-binding fragment or single variable domain (VHH) derived from naturally occurring heavy chain antibodies, and is known to those skilled in the art.

[0372] The term "blocking peptide" as used herein refers to peptides that can interfere with the molecules they target in such a way that the interaction of the molecule with its target is weakened. For example, a blocking peptide targeting TGF-β would weaken its immunosuppressive function.

[0373] Nucleic acid: The term "nucleic acid" includes any nucleotide, its analogs and polymers thereof. The terms "polynucleotide", "nucleic acid construct" or "nucleotide sequence" as used herein refer to a polymeric form of nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)) of any length. These terms refer to the primary structure of the molecule and thus include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. These terms include, as equivalents, analogs of RNA or DNA prepared from nucleotide analogs and modified polynucleotides (such as but not limited to methylated, protected and / or capped nucleotides or polynucleotides). These terms include polyribonucleotides or oligoribonucleotides (RNA) and polydeoxyribonucleotides or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides 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 ribose moieties, nucleic acids containing deoxyribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. Technicians know that he can replace a variable number of nucleosides in nucleic acids with modified nucleosides, which may include derivatives of purine and pyrimidine bases, such as pseudouridine, N1-methyl pseudouridine, 5-methyluridine, 5-methylcytosine, 6-methyladenosine, 2-thiouridine to improve translation efficiency, and in some embodiments, the prefix poly-refers to nucleic acids containing 2 to about 10,000, 2 to about 50,000, or 2 to about 100,000 nucleotide monomer units. In some embodiments, "oligonucleotide" refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

[0374] Gene: As used herein, the term "gene" means a DNA segment that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression. In addition, shRNA is also included in this definition.

[0375] Hybridization: As used herein, the term "hybridizing" or "hybridizes" will be understood as the formation of hydrogen bonds between base pairs on opposing strands, thereby forming two nucleic acid strands (eg, an oligonucleotide and a target nucleic acid) of a duplex.

[0376] Promoter: The term "promoter" is used herein in its ordinary sense to refer to a nucleotide region containing a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene and is capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. It may contain genetic elements to which regulatory proteins and molecules (such as RNA polymerase and other transcription factors) can bind to initiate specific transcription of a nucleic acid sequence. The phrases "operably positioned", "operably linked", "under control" and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation relative to a nucleic acid sequence to control transcription initiation and / or expression of the sequence.

[0377] 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 the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0378] 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 the gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

[0379] 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, enables the gene product to be produced in a cell substantially only when 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-α.

[0380] 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 the position of the residue in the sequence and the identity of the newly replaced residue. Various methods for generating amino acid substitutions (mutations) provided herein are well known in the art and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0381] Identity: As used herein, the term "identity" refers to the overall correlation between polymer molecules, such as polypeptide molecules and / or nucleic acid molecules (e.g., DNA molecules and / or RNA molecules). In some embodiments, if the sequence of the polymer molecule is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical, then they are considered to be "substantially identical" to each other. For example, the calculation of the identity percentage of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for the purpose of optimal comparison (e.g., for optimal alignment, a gap can be introduced in one or both of the first and second sequences, and non-identical sequences can be ignored for the purpose of comparison). In certain embodiments, the length of the sequence 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. Then compare the nucleotide or amino acid at the corresponding position.When the position in the first sequence is occupied by the residue (for example, nucleotide or amino acid) identical to the corresponding position in the second sequence, then the molecule is identical at this position.Considering the number of spaces and the length of each space that need to be introduced for the optimal comparison of two sequences, the identity percentage between the two sequences is a function of the number of identical positions shared by the sequences.The determination of the identity percentage between the comparison of sequences and two sequences can be completed using a mathematical algorithm.For example, the algorithm (CABIOS, 1989, 4:11-17) of Meyers and Miller (which has been incorporated into the ALIGN program (version 2.0)) can be used to determine the identity percentage between two nucleotide sequences.In some exemplary embodiments, the nucleic acid sequence comparison performed with the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.Or, the GAP program in the GCG software package can be used to determine the identity percentage between two nucleotide sequences using the NWSgapdna.CMP matrix.

[0382] Hybridization under stringent conditions: In the present description, hybridization under stringent conditions refers to hybridization conditions in which only long sequences with almost perfect complementary matches are guaranteed to anneal. This term is well known in the field of genetic engineering and has been used for many years in patent practice as a quasi-structural feature for defining DNA claims. It is noteworthy that the corresponding claims define their subject matter not only by the ability to hybridize under stringent conditions, but also by a further functional feature, namely that the polypeptide encoded by the claimed DNA sequence has at least the functional properties of STING.

[0383] Connected: As used herein, the term "connected", when used for two or more parts, means that the parts are physically bound or connected to each other, forming a sufficiently stable molecular structure so that the parts remain bound under conditions in which the linkage is formed, preferably under conditions in which the new molecular structure is used (e.g., physiological conditions). In certain preferred embodiments of the present invention, the linkage is a covalent linkage. In other embodiments, the linkage is non-covalent. The parts can be directly or indirectly connected. When two parts are directly connected, they are either covalently bonded to each other, or close enough so that the intermolecular forces between the two parts maintain their association. When two parts are indirectly connected, they are each covalently or non-covalently connected to a third part, and the third part maintains the association between the two parts. Generally, when two parts are referred to as being connected by a "linker" or "linking moiety" or "linking port", the linkage between the two connected parts is indirect, and generally each linking moiety is covalently bonded to the linker. The linker can be any suitable moiety that reacts with the two moieties to be joined in sufficient quantity to produce a reasonable yield, within a reasonable period of time, under conditions consistent with the stability of the moiety (which can be appropriately protected as appropriate).

[0384] Operably linked / effectively linked: As used herein, the term "operably linked" or "effectively linked" is a juxtaposition in which the components are in a relationship that allows them to function in their intended manner. A control element such as a promoter that is "operably linked" to a functional element is associated in such a way 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 target coding element; in some embodiments, the control element acts in trans on the target functional element.

[0385] 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 that shares substantial or complete homology with the RNA molecule. 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 called Dicer, which cuts long double-stranded RNA (dsRNA) into double-stranded fragments of 3' overhangs of typically 21-23 base pairs and 2 bases (although variations in length and overhangs are also contemplated), referred to as "short interfering RNA" ("siRNA"). Such siRNAs comprise two single-stranded RNAs (ssRNAs): an "antisense strand" or "guide strand" comprising a region that is substantially complementary to the target sequence and a "sense strand" or "passenger strand" comprising a region that is substantially complementary to the antisense strand. It will be appreciated by those of ordinary skill in the art that the guide strand may be fully complementary to the target region of the target RNA, or may be incompletely complementary to the target region of the target RNA. As used herein, the term "microRNA" or "miRNA" or "miR" refers to a specific type of siRNA that is physiologically produced in an organism, rather than synthetically produced. The miRNA binds to the corresponding miRNA target sequence and reduces or inhibits the expression of a nucleic acid sequence that shares basic or complete homology with the miRNA. As used herein, the term "shRNA" or "short hairpin RNA" refers to a single transcript that adopts a stem-loop structure, which is processed into siRNA by the RNAi mechanism. A typical shRNA molecule comprises two inverted repeat sequences, which contain sense and antisense target sequences separated by a loop sequence. The base-paired segment can be any suitable length that allows in vivo target gene inactivation, wherein one strand of the base-paired stem is complementary to the mRNA of the target gene. The loop of the shRNA stem-loop structure can be any suitable length that allows in vivo target gene inactivation. The base-paired stem can be completely base-paired, or can have 1 or 2 mismatched base pairs.

[0386] Target gene / RNA: As used herein, "target gene" refers to a gene whose expression will be regulated (e.g., upregulated or inhibited). As used herein, the term "target RNA" refers to an RNA to be degraded or translationally inhibited or otherwise inhibited using one or more agents (e.g., one or more miRNAs or siRNAs). Target RNA may also be referred to as a target sequence or target transcript. RNA may be a primary RNA transcript (e.g., pre-mRNA) or a processed transcript transcribed from a target gene, such as an mRNA encoding a polypeptide. As used herein, the term "target portion" or "target region" refers to a continuous portion of the nucleotide sequence of a target RNA. In some embodiments, the target portion of an mRNA is at least long enough to serve as a substrate for RNA interference (RNAi)-mediated cleavage in the presence of a suitable miRNA or siRNA. The length of the target portion may be about 8-36 nucleotides, e.g., about 10-20 or about 15-30 nucleotides. The target portion length may have a specific value or sub-range within the above range. For example, in certain embodiments, the length of the target portion can be 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, 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 nucleotides.

[0387] Recombinant: As used herein, the term "recombinant" refers to a nucleic acid or polypeptide designed, modified, prepared, expressed, produced, manufactured and / or isolated by recombinant means, such as 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 has been otherwise manipulated to express one or more genes or gene components that encode and / or direct the expression of a polypeptide or one or more components, parts, elements or domains thereof; and / or a polypeptide prepared, expressed, produced or isolated by any other means, including splicing or connecting selected nucleic acid sequence elements to each other, chemically synthesizing selected sequence elements, and / or otherwise generating nucleic acids that encode and / or direct the expression of a polypeptide or one or more components, parts, elements or domains thereof. In some embodiments, one or more of such selected sequence elements exist in nature. In some embodiments, one or more of such selected sequence elements are designed on a computer. In some embodiments, one or more such selected sequence elements are generated by 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.)).

[0388] Natural variant: The term "natural variant" refers to a variant of a gene or transcript that is derived from the same locus as the target nucleic acid, but may differ, for example, due to the degeneracy of the genetic code (which results in a diversity of codons encoding 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. The natural variants exist in nature and are not artificially produced. In contrast, when the term "variant" is used without the word "natural" associated therewith, the variant may include artificially produced variants, such as mutations. With respect to human STING, some known natural variants are truncated forms of STING[R], STING[AQ], STING[H], STING[HAQ], and STING[H].

[0389] Wild type: As used herein, "wild type" refers to a naturally occurring nucleic acid sequence on a genetic locus in an organism's genome, as well as a sequence transcribed or translated from such a nucleic acid. Therefore, the term "wild type" may also refer to an amino acid sequence encoded by a nucleic acid. Since a locus may have more than one sequence or allele in an individual population, the term "wild type" includes 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) on a genetic locus of an individual in a population. As used herein, "mutant" refers to a change in the sequence of a nucleic acid or a protein, polypeptide or peptide encoded therein, which is the result of recombinant DNA technology.

[0390] 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 still make them human, while hybridization of a portion of a human protein with a portion of a homologous protein of another species makes it non-human.

[0391] Hybrid STING: As used herein, the term "hybrid STING" refers to STING in which a portion of the protein sequence is derived from human STING and another portion is derived from a homologous STING of another species.

[0392] Target STING: As used herein, a target STING is a STING that responds to a STING agonist that recognizes the target STING but not human STING and its natural variants. Thus, the target STING may be a mutated human STING, but not the endogenous human STING encoded in the genome of a human subject (according to one of the known haplotypes of human STING), i.e., hSTING[R] as described in SEQ ID NO: 1, hSTING[H] as described in SEQ ID NO: 2, hSTING[R] as described in SEQ ID NO: 3, hSTING[H] as described in SEQ ID NO: 4, STING[Q] with the amino acid substitution R293Q, or STING[HAQ] with the amino acid substitutions R71H, G230A, and R293Q. Thus, the responsiveness to a STING agonist is determined in an "in vitro-DMXAA assay". Non-limiting examples of target STING are murine STING or a mutated human STING hSTING[R]-S162A / G230I / Q266I.

[0393] Cistron / monocistron / bicistron / polycistronic: As used herein, the term "cistron" refers to a segment of a DNA or RNA molecule that encodes a single protein or a single functional RNA molecule. The terms "monocistron", "bicistron" or "polycistronic" refer to the number of cistrons on a DNA or RNA molecule. For example, a bicistronic vector comprising two cistrons under the control of one promoter results in the translation of two different proteins.

[0394] Exogenous / foreign: The term "exogenous" or "foreign" when applied 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, the term refers to a cell that has been isolated and subsequently introduced into other cells or organisms by artificial or natural means. An exogenous nucleic acid can be from a different organism or cell, or can be one or more additional copies of a nucleic acid that occurs naturally in an organism or cell. An exogenous cell can be from a different organism or from the same organism. As non-limiting examples, an exogenous nucleic acid is a nucleic acid that is located in a chromosomal location different from the chromosomal location in which it is located in a natural cell, or alternatively is a nucleic acid that is flanked by a nucleic acid sequence that is different from a naturally occurring nucleic acid sequence.

[0395] Plasmid: A "plasmid" is a common type of vector, an extrachromosomal DNA molecule that is capable of replication independently of chromosomal DNA. In some cases, it is circular and double-stranded.

[0396] Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA or RNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell after introduction into the host cell, thereby replicating with the host genome. In addition, certain vectors are capable of directing the expression of genes operatively connected thereto. Such vectors are referred to herein as "expression vectors". It is understood by those of ordinary skill in the art that the "viral vectors" described herein include viral components, such as capsid proteins, in addition to the transgenes described herein.

[0397] Expression system: "Expression system" or "expression construct" means one or more nucleic acid molecules capable of directing transcription. An expression system includes at least one or more transcription control elements (such as promoters, enhancers or their structural and functional equivalents) 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. Therefore, the terms "expression system" and "recombinant expression system" encompass DNA or RNA nucleic acid molecules capable of directing transcription. In the specification of the present invention, the corresponding DNA or RNA nucleic acid molecules are also referred to as "nucleic acid constructs".

[0398] AAV: The term "AAV" refers to adeno-associated virus and can be used to refer to the naturally occurring wild-type virus itself or its derivatives. Unless otherwise required, the term covers all subtypes, serotypes and pseudotypes, as well as naturally occurring and recombinant forms. The AAV genome consists of single-stranded DNA and contains inverted terminal repeats (ITRs) at both ends of the DNA chain and two open reading frames: rep and cap, encoding replication proteins and capsid proteins, respectively. Foreign polynucleotides can replace the natural rep and cap genes. AAV can be made with a variety of different serotypes of capsids, which have different transduction spectra, or as used herein, have "tropism" for different tissue types. As used herein, the term "serotype" refers to an AAV that is recognized and distinguished from other AAVs based on the reactivity of the capsid protein with a determined antiserum, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAVrh10. For example, serotype AAV2 is used to refer to an AAV containing a capsid protein encoded by the cap gene of AAV2 and a genome containing 5' and 3' ITR sequences from the same AAV2 serotype. Pseudotyped AAV refers to an AAV containing a capsid protein from one serotype and a viral genome including 5'-3' ITRs of a second serotype. Pseudotyped adenoviruses are expected to have the cell surface binding properties of the capsid serotype and genetic properties consistent with the ITR serotype. Pseudotyped rAAVs are produced using standard techniques described in the art.

[0399] Adenovirus: The term "adenovirus" refers to any virus in the genus Adenovirus (including but not limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenus). Typically, adenoviral vectors are generated by introducing one or more mutations (e.g., deletions, insertions or substitutions) into the adenoviral genome of an adenovirus to accommodate the insertion of non-native nucleic acid sequences (e.g., for gene transfer) into the adenovirus.

[0400] Lentivirus: As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Vesna-Medivirus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0401] Virus particle: As used herein, the term "virus particle" refers to all or part of a virion. For example, a virus particle comprises a recombinant genome and may further comprise a capsid. The virus particle may be a gene therapy vector. In this article, the terms "virus particle" and "vector" are used interchangeably. For the purposes of this application, a "gene therapy" vector is a virus particle that can be used for gene therapy, i.e., a virus particle that contains all the required functional elements for expressing a transgene (such as a CFI nucleotide sequence) in a host cell after administration. Suitable virus particles of the present invention include parvoviruses, retroviruses, lentiviruses, or herpes simplex viruses. The parvovirus may be an adeno-associated virus (AAV). Optionally, the virus particle is an AAV or adenovirus particle.

[0402] Host cell: As used herein, the term "host cell" refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Those skilled in the art who read this disclosure will understand that such terms refer not only to the specific subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be the same as 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 some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI 38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, MC38 cells, myeloma cells, tumor cells, and cell lines derived from the above cells. In some embodiments, the cell comprises one or more viral genes.

[0403] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a person at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, animals can be transgenic animals, genetically engineered animals, and / or clones.

[0404] Treatment / treating: "Treatment" or "treating" includes (1) inhibiting a disease, disorder or condition in a subject or patient who is experiencing or exhibiting pathology or symptomology of the disease (e.g., arresting further development of the pathology and / or symptomology), (2) ameliorating a disease, disorder or condition in a subject or patient who is experiencing or exhibiting pathology or symptomology of the disease (e.g., reversing the pathology and / or symptomology), and / or (3) achieving any measurable reduction in a disease, disorder or condition in a subject or patient who is experiencing or exhibiting the disease, disorder or condition.

[0405] Prophylactic treatment: "Prophylactic treatment" includes: (1) reducing or alleviating the risk of disease in a subject or patient who may be at risk and / or susceptible to the disease but does not yet experience or display any or all pathology or symptoms of the disease, and / or (2) slowing the onset of disease pathology or symptoms in a subject or patient who may be at risk and / or susceptible to the disease but does not yet experience or display any or all pathology or symptoms of the disease.

[0406] Local administration / local delivery: As used herein, the term "local administration" or "local delivery" refers to delivery that does not rely on the transport of the product to its intended target tissue or site through the vascular system when it refers to the delivery of the recombinant expression system, expression vector, viral particle or pharmaceutical composition described herein. The product described herein can be delivered directly to its intended target tissue or site, or near it, for example, in close proximity to the intended target tissue or site. For example, the product can be delivered by injection or implantation of a composition or compound or by injection or implantation of a device containing a composition or compound. After local administration near the target tissue or site, the product described herein or one or more components thereof can diffuse to the intended target tissue or site. It should be understood that once it has been locally delivered, a portion of the product described herein (usually only a small portion of the administered dose) can enter the vascular system and be transported to another location, including back to its intended target tissue or site. As used herein, the term "local administration" or "local delivery" refers to delivery that can rely on the transport of the viral vector or pharmaceutical composition to its intended target tissue or site through the vascular system when it refers to the delivery of the viral particle or pharmaceutical composition described herein.

[0407] Simultaneous administration: As used herein, the term "simultaneous administration" with respect to two or more compounds (e.g., therapeutic agents) is an administration performed using a dosage and a time interval, which allows the administered compounds to be present together in the body in a non-negligible amount over a period of time, such as at one or more sites of action in the body. The time interval may be a few minutes (e.g., at least 1 minute, 1-30 minutes, 30-60 minutes), a few hours (e.g., at least 1 hour, 1-2 hours, 2-6 hours, 6-12 hours, 12-24 hours), a few days (e.g., at least 1 day, 1-2 days, 2-4 days, 4-7 days, etc.), a few weeks (e.g., at least 1 week, 2 weeks, or 3 weeks, etc.). Therefore, the compounds may, but need not, be administered together as part of a single composition. In addition, the compounds may, but need not, be administered substantially simultaneously (e.g., less than 5 minutes apart, or less than 1 minute apart) or within a short time of each other (e.g., less than 1 hour apart, less than 30 minutes apart, less than 10 minutes apart, about 5 minutes apart). According to various embodiments of the present disclosure, the compounds administered within such a time interval may be considered to be administered substantially simultaneously. In certain embodiments of the present disclosure, the compounds administered simultaneously are present in vivo (e.g., in the blood and / or at sites of local complement activation) at effective concentrations over a period of time. When administered simultaneously, the effective concentration of each compound required to elicit a specific biological response may be lower than the effective concentration of each compound when administered alone, thereby reducing the dose required for one or more of the compounds relative to the compound when administered as a single compound. The effects of multiple compounds may be, but not necessarily, 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 elicit a specific biological response (e.g., a desired biological response).

[0408] Sequential administration: As used herein, the term "sequential administration" of two or more compounds refers to administering two or more compounds to a subject so that the compounds are not present together in the subject's body or at relevant active sites in the body at concentrations greater than non-negligible. Administration of the compounds may be alternating, but need not be. Each compound may be administered multiple times.

[0409] Systemic administration: As used herein, the term "systemic administration" and similar terms are used herein in accordance with their usage in the art and refer to the 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 widely distributed in the body in significant amounts and has a biological effect, such as its desired effect, in the blood, and / or reaches its desired site of action through the vascular system. Typical systemic administration routes include (i) administration by direct introduction of the compound into the vascular system, or (ii) subcutaneous, oral, pulmonary, or intramuscular administration, wherein the compound is absorbed, enters the vascular system, and is transported to one or more desired sites of action through the blood.

[0410] Intratumoral administration: The term "intratumoral administration" refers to the 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 after administration. One of ordinary skill in the art will appreciate that administration can occur directly within a tumor or in close proximity to a tumor.

[0411] Subject: As used herein, the term "subject," "test subject," or "patient" refers to any organism to which a provided compound or composition is administered according to the invention, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject may suffer from and / or be susceptible to a disease, disorder, and / or condition.

[0412] Effective / Therapeutically Effective Amount: As used herein, the term "effective amount" or "therapeutically effective amount" means at least the minimum amount of a compound (e.g., a therapeutic agent, expression vector, viral particle, pharmaceutical composition, and / or formulation) that causes a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to a disease, condition, and / or disorder. As will be understood by one of ordinary skill in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and the like. For example, an effective amount of a compound for treating a disease, condition, and / or condition in a formulation 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 a disease, condition, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; 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 deleterious 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 the size of a tumor, inhibiting (i.e., slowing down or ideally preventing to some extent) the infiltration of cancer cells into peripheral organs, inhibiting (i.e., slowing down and ideally 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 disease.

[0413] Combination of expression vectors / combination of viral particles: The phrase "combination of expression vectors" or "combination of viral particles" means herein that a first expression vector or a first viral particle is administered to a patient before, during (including simultaneously with - preferably co-formulated with) 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 to administer any expression vector and viral particle mentioned herein in a pharmaceutically effective amount, i.e., administration involving the total amount of each component of the combination sufficient to show a meaningful patient benefit. The formulations may be conveniently provided in unit dosage form by methods known to those skilled in the art.

[0414] Kit: As used herein, a "kit" 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 combined administration. Preferably, the kit may 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 so that the desired effect can be obtained when administered to a subject. Thus, preferably the medical package contains a certain amount of dosage units corresponding to the relevant dosage regimen. Preferably, the kit contains instructions for using the dosage form to achieve the desired effect and the amount of the dosage form to be taken within a specified time period.

[0415] Immunotherapy: The term "immunotherapy" as used herein refers to a therapy by which the regulation or stimulation of the immune system is used to treat a disease, preferably a proliferative disease such as cancer, using immunomodulatory genes or proteins. Immunotherapy can cause stimulation of the immune system, which is commonly used for immunotherapy against cancer. However, other diseases such as autoimmune diseases, diabetes, cardiovascular disease, 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 regulate the balance of regulatory T cells and effector T cells, thereby maintaining the function of β cells. Immunotherapy can be used to treat non-cancer diseases such as lupus, rheumatoid arthritis, and diabetes.

[0416] Immune checkpoint inhibitors: 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 the checkpoint signaling pathway include, for example, CD27, CD28, CD40, CD 122, 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" means a composition having a molecular weight of less than about 5 kDa to 50 Da, such as 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 about 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, peptide mimetics, carbohydrates, lipids, or other organic or inorganic molecules. Alternatively, immune checkpoint inhibitors are antibodies or fragments thereof. For example, the antibody or fragment thereof is specific for a protein in a checkpoint signaling pathway, such as CD27, CD28, CD40, CD 122, 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 relalizumab (anti-LAG-3).

[0417] 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. Various cell types can be used for this purpose, such as dendritic cells, natural killer (NK) cells, or T cells.

[0418] 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 separate tumor infiltrating leukocytes (TILs) from a tumor, optionally expand these cells outside the patient's body and infuse them back into the patient. Another adoptive T cell therapy is chimeric antigen receptor (CAR) T cell therapy. CAR-T cells can be designed in a variety of ways to enhance tumor cell toxicity and specificity, avoid tumor immunosuppression, avoid host rejection, and extend their therapeutic half-life.

[0419] Cancer vaccine: As used herein, the term "cancer vaccine" refers to a vaccine that treats an existing cancer or prevents cancer from developing.

[0420] Combination therapy: As used herein, the term "combination therapy" refers to the situation where two or more different compounds (e.g., therapeutic agents, expression vectors, viral particles, pharmaceutical compositions, and / or preparations) are administered in overlapping schemes to expose the subject to two compounds at the same time. When used in combination therapy, two or more different compounds can be administered simultaneously or separately. This combination 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, wherein the compounds are present in separate preparations. In another alternative, a first compound can be administered, followed by one or more additional compounds. In a separate administration scheme, two or more compounds can be administered at intervals of minutes, hours, days, or weeks. In some embodiments, two or more compounds can be administered at intervals of 1-2 weeks.

[0421] Radiotherapy: The term "radiotherapy" refers hereinafter to the medical use of ionizing radiation, usually as part of cancer treatment to control or kill malignant cells. Ionizing radiation can be based on photon rays, such as X-rays or gamma rays, or particle rays, wherein the particles of the particle rays are preferably selected from the group consisting of beta particles, alpha particles, neutrons, muons, pions, protons or other heavier positive ions. It can also be used as part of adjuvant therapy to prevent tumor recurrence after surgery to remove primary malignant tumors. Radiotherapy can work synergistically with other anticancer therapies and can be used before, during and after other anticancer therapies for susceptible cancers. The term "low-dose radiotherapy" refers hereinafter to radiotherapy with an ionizing radiation dose of less than 40Gy, preferably 10Gy or less, more preferably 8Gy or less. Low dose radiation therapy can be any radiation therapy with 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.

[0422] Proliferative disease: As used herein, the term "proliferative disease" refers to a condition in which the unregulated or abnormal growth of cells, or both, can lead to the development of an unwanted condition or disease, which may or may not be cancerous. Exemplary cell proliferative disorders of the present invention encompass a variety of conditions in which cell division is dysregulated. Exemplary cell proliferative disorders include, but are not limited to, tumors, benign tumors, malignant tumors, precancerous conditions, tumors in situ, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immune tumors, blood tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing cells.

[0423] Cancer / tumor: The term "cancer" or "tumor" refers to the presence of cells with typical characteristics of cancer cells (such as uncontrolled proliferation, immortality, invasion or metastasis potential, rapid growth, and certain characteristic morphological features). In some embodiments, such cells partially or completely exhibit such characteristics due to the expression and activity of immune checkpoint proteins such as PD-1, PD-L1, PD-L2 and / or CTLA-4. Cancer cells usually exist in the form of tumors, but such cells can exist alone in animals, or can be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term "cancer" includes pre-malignant as well as malignant cancers.Cancer includes, but is not limited to, various cancers, 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, gastric cancer, gallbladder cancer, cervical cancer, thyroid cancer and skin cancer (including squamous cell carcinoma); including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, Hematopoietic neoplasms of the lymphoid lineage including myeloid leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, histiocytic lymphoma and Burkitt's lymphoma; myeloid hematopoietic neoplasms including acute and chronic myeloid leukemia, myelodysplastic syndrome, myeloid leukemia and promyelocytic leukemia; central and peripheral nervous system tumors including astrocytoma, neuroblastoma, glioma and schwannoma; mesenchymal tumors including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; melanoma Other tumors include: melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, liver cancer, bile duct cancer, breast cancer, colon cancer and head and neck cancer, stomach cancer, germ cell tumor , bone cancer, bone tumors, adult malignant fibrous histiocytoma of bone; childhood malignant fibrous histiocytoma of bone, sarcoma, pediatric sarcoma, sinonasal natural killer cells, tumors, plasma cell neoplasms; myelodysplastic syndrome; neuroblastoma; testicular germ cell tumors, intraocular melanoma, myelodysplastic syndrome; myelodysplastic / myeloproliferative disorders, synovial sarcoma, chronic myeloid leukemia, acute lymphocytic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL), multiple myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, mastocytosis and any symptoms associated with mastocytosis and any metastases thereof.In addition, conditions include urticaria pigmentosa, mastocytosis such as diffuse cutaneous mastocytosis, solitary mastocytoma in humans, and some rare subtypes such as bullous, erythrodermic, and telangiectatic mastocytosis, mastocytosis with associated blood disorders such as myeloproliferative or myelodysplastic syndrome, or acute leukemia, myeloproliferative disorders associated with mastocytosis, mast cell leukemia, and other cancers. Other cancers are also included within the scope of the disorder, including, but not limited to: cancers including bladder cancer, urothelial cancer, 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, particularly testicular seminoma, and skin cancer; including squamous cell carcinoma; gastrointestinal stromal tumor ("GIST"); lymphoid hematopoietic tumors including leukemias, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma, and Burkitt lymphoma; myeloid hematopoietic tumors, These include acute and chronic myeloid leukemias and promyelocytic leukemias; mesenchymal tumors, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratoma, neuroblastoma and glioma; central and peripheral nervous system tumors, including astrocytoma, neuroblastoma, glioma and schwannoma; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, teratoma, chemotherapy-refractory non-seminomatous germ cell tumors and Kaposi's sarcoma and any metastases thereof.Other non-limiting examples of cancer types suitable for the methods contemplated by the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, 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, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bone cancer, brain tumor, lung cancer (including lung adenocarcinoma), small cell lung cancer, bladder cancer, upper melanoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; 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); as well as polycythemia vera, lymphomas (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 and includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney 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.

[0424] Anti-cancer: An "anti-cancer" agent is capable of negatively affecting cancer cells / tumors in a subject, for example, by promoting the killing of cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or prolonging the lifespan of a subject suffering from cancer.

[0425] Pharmaceutical composition: The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the preparation is to be administered. Such preparations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient used.

[0426] Synergy / Synergistic effect: The term "synergy" or "synergistic effect" refers to the combined effect of two or more compounds that is greater than the sum of the individual effects of the individual cancer compounds / therapies.

[0427] In vitro agonist assay / in vitro DMXAA assay: The term "in vitro agonist assay" refers to an assay that allows identification of human non-responsive STING agonists, wherein the agonist activates human STING or a variant of non-human STING, but does not activate human wild-type STING or a naturally occurring variant thereof. The term "in vitro DMXAA assay" refers to an assay that allows identification of STING that responds to DMXAA.

[0428] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reaction and purification techniques can be performed according to the manufacturer's instructions or as usually completed in this area or as described herein. The aforementioned techniques and procedures can be performed according to conventional methods known in the art, and are described in various general and more specific references as quoted and discussed in this specification in full. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose. BRIEF DESCRIPTION OF THE DRAWINGS

[0429] Figure 1, including Figures AG, illustrates A) IFN-β expression in MC38 cells after lentiviral transduction with hSTINGwt or hSTINGmut and subsequent DMXAA treatment relative 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 hSTINGwt or hSTINGmut and subsequent DMXAA treatment relative 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 subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 9, **P < 0.01, two-way ANOVA followed by Sidak's multiple comparison test); D) STING-derived cytokine expression in MC38 cells after lentiviral transduction with luciferase, hSTINGwt or hSTINGmut or no transduction and subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 3, *P < 0,05, **P < 0.01, one sample at a time by one-sample T test E) STING-derived cytokine expression in Hep2G cells after lentiviral transduction with luciferase, hSTINGwt or hSTINGmut or without transduction and subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 3, *P < 0,05, **P < 0.01, one sample at a time by one-sample T test); F) Time course of STING-derived cytokine expression in MC38 cells after lentiviral transduction with hSTINGmut and subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 3, *P < 0,05, **P < 0.01, ***P < 0,001, two-way ANOVA followed by Fisher's LSD test); and G) Time course of STING-derived cytokine expression in MC38 cells after lentiviral transduction with hSTINGmut and subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA followed by Fisher's LSD test).

[0430] Figure 2 Depicted are IFN-β serum levels in mice injected intravenously with AAV8 vectors encoding mSTING or GFP and treated twice weekly with different doses of DMXAA (5, 10 or 20 mg / kg body weight). 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.

[0431] Figure 3, including panels AH, illustrates A) luciferase activity of MC38 cells transduced by lentivirus 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 / 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 transduced by lentivirus with vectors encoding luciferase under the control of different modified chemokine promoters in the absence or presence of DMXAA. Luciferase activity of MC38 cells transduced with vectors encoding IL-12 under the control of IFN-β promoter, CXCL9 promoter or CXCL10 promoter by lentiviral transduction at different time points after DMXAA treatment; data shown are mean ± SEM (n = 3, *P < 0.05, two-way ANOVA, followed by Kruskal-K ... kal-Wallis test; D) IL-12 protein levels in the supernatants of MC38 cells transduced by lentiviral transduction with vectors encoding IL-12 under the control of the IFN-β promoter, CXCL9 promoter, or CXCL10 promoter at different time points after DMXAA treatment; data are mean ± SEM (n = 2-8 wells / condition), ****P < 0.0001, two-way ANOVA followed by Sidak's multiple comparison test; E) and F) MC38 cells transfected with plasmids encoding luciferase under the control of different synthetic promoters in the absence or presence of DMXAA. Luciferase activity of C38 cells; data are mean ± SEM (n = 2 wells / 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 / condition), ****P < 0.0001, two-way ANOVA followed by Sidak's multiple comparison test.

[0432] Figure 4, including panels AC, illustrates A) IL-12 protein levels in mouse serum after intravenous injection with AAV8 encoding IL-12 under the control of 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 mouse serum after intravenous injection with AAV8 encoding single-chain (sc) IL-12 operably linked to a miR122 binding site under the control of the ISRE promoter in the absence of DMXAA compared to the AAV8 vector lacking the miR122 binding site. -12 protein levels were reduced; data are mean ± SEM (n = 4 animals / condition), ****P < 0.0001, Student's t-test; and C) IL-12 protein levels were reduced in mouse serum after intravenous injection with AAV8 encoding hSTINGmut under the control of a constitutively active promoter and AAV8 encoding scIL-12 operably linked to a miR122 binding site under the control of an ISRE promoter or without the miR122 binding site in the presence or absence of DMXAA; data are mean ± SEM (n = 4-8 animals / condition), *P < 0.05, two-way ANOVA followed by Tukey's multiple comparison test.

[0433] Figure 5 Figure 2 shows the volume of subcutaneous MC38 tumors implanted in STING KO mice that had been co-injected intratumorally with an AAV8 vector encoding hSTINGmut under the control of a constitutive promoter (AAV8-hSTINGmut) (dose of 1×10 11 vg / mouse) + AAV8 vector encoding scIL-12 operably linked to the miR122 binding site and placed under the control of the ISRE promoter (AAV8-ISREx4IL12miR122T) (dose 0.5x10 11 vg / mouse). These animals received DMXAA (solid triangles) or vehicle (solid circles) at the indicated time intervals. In an additional control group, tumors were injected with AAV8-hSTINGmut (1x10 11 vg / mouse) + AAV8 vector encoding GFP (0.5x10 11 vg / mouse) followed by vehicle injection (open circles). Data are mean ± 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.

[0434] Figure 6, including Fig. 6A and Figure 6B , illustrates A) subcutaneous MC38 tumor sections immunohistochemically stained with anti-GFP antibody 3 days after intratumoral injection of AAV8-GFP. The transduction efficiency of tumors injected with AAV8-GFP without radiation pretreatment (left) was significantly reduced compared with tumors pretreated with 8Gy radiation (right). B) The figure shows the percentage of strongly GFP-labeled pixels associated with tumor area. Data are mean ± SEM (n = 3-6 animals / condition) ***P < 0.001, unpaired Student's t-test.

[0435] Figure 7 Shown are quantified IFN-β protein levels in supernatants of MC38 cells irradiated and / or transduced with vectors encoding hSTINGmut at the indicated time points after irradiation; data are mean ± SEM (n = 2-6 samples / condition), *P < 0.05, mixed effects followed by Tukey's multiple comparison test (individual effects). #P < 0.05, ####P < 0.0001 vs. MC38 8Gy, two-way ANOVA followed by Tukey's multiple comparison test (comparison of values ​​at given time points).

[0436] Figure 8, including Figures AD, illustrates A) the time course of tumor volume progression in mm3 of MC38 subcutaneous tumors in C57BL / 6 wild-type mice after irradiation or non-irradiation as indicated, followed by intratumoral injection of AAV8-GFP, AAV8-hSTINGmut and / or AAV8-ISREx4-IL12miR122T. Data are mean ± SEM (n = 4-5 mice / group), and significance was assessed using a two-way analysis of variance (ANOVA) test and Tukey post hoc test analysis. 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 on day 17 after treatment of animals in A). The dots represent individual values ​​for each mouse, the middle horizontal line represents the median, and the error bars represent the standard error of the mean (SEM). There were no statistically significant differences between the groups as determined by one-way ANOVA (F(2,7)=0.4375, p=0.6622). 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, while the contralateral tumor was not treated in order to analyze the abscopal effect. The right flank tumors were treated with 8Gy of local irradiation, followed by intratumoral injection of AAV8-GFP, AAV8-hSTINGmut and / or AAV8-ISREx4-IL12miR122T as indicated. 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 and Tukey post hoc test analysis. Simple main effects analysis showed that treatment had a statistically significant effect on the growth of treated tumors (***p<0.001) and a statistically significant anti-tumor effect on contralateral tumors (*p<0.01) on day 15 after treatment.

[0437] Figure 9, including Figures AF, illustrates A) IFN-β mRNA levels in MC38 cells upon stimulation with DMXAA or vehicle in non-transduced cells or cells transduced with AAV8-hSTINGmut after 8 Gy of irradiation or without irradiation (0 Gy); data shown are mean ± SEM (n = 3, *P < 0.05, **P < 0.01, one-way ANOVA followed by Kruskal-Wallis test); B) IFN-β mRNA levels in MC38 cells upon stimulation with DMXAA or vehicle in non-transduced cells or cells transduced with AAV8-hSTINGmut after 8 Gy of irradiation or without irradiation (0 Gy); data shown are mean ± SEM (n = 3, *P < 0.05, **P < 0.01, one-way ANOVA followed by Kruskal-Wallis test); A) IL-12 mRNA levels in MC38 cells upon stimulation with AAV8-hSTINGmut+AAV8-GFP, AAV8-GFP+AAV-ISREx4-IL12miR122T, or AAV8-hSTINGmut+AAV-ISREx4-IL12miR122; data shown are mean ± SEM (n = 3, ***P < 0.001, one-way ANOVA followed by Kruskal-Wallis test); C) and D) IL-12 mRNA levels in MC38 cells upon stimulation with DMXAA or vehicle after 8 Gy of irradiation or without irradiation (0 Gy). IFN-β mRNA levels (in C) or IL-12 mRNA levels (in D) in HEK293 cells upon stimulation; data shown are 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 collected 6, 24 or 48 hours after DMXAA administration; data Mean ± SEM (n = 2-5 samples / condition), ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test; and F) IFN-β protein levels (pg / ml) obtained by ELISA in supernatants of cultured non-transduced MC38 cells treated with DMXAA (50 μg / mL) or vehicle 24 hours after irradiation (8 Gy) or without irradiation (0 Gy) and collected 24 h after DMXAA administration; Data are mean ± SEM (n = 3-4 samples / condition), ***P < 0.001, ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test.

[0438] Figure 10, including Figures AD, shows the results obtained in STING KO mice with subcutaneous MC38 tumors that had been treated with 0 or 8 Gy irradiation as indicated, followed by intratumoral injection of AAV vectors encoding GFP (control) or hSTINGmut and / or IL12miR122T. Two days later, they received intraperitoneal DMXAA or vehicle. The figure illustrates serum levels of A) IFN-β, B) IL-12, C) IFN-γ at the indicated time points. Figure D represents IL-12 levels in tumor extracts obtained 6 h after DMXAA or vehicle treatment values ​​obtained by ELISA. Data are mean ± SEM (n = 4-5 mice per group), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test.

[0439] FIG. 11 , including panels AB, shows the expression of AAV8 encoding GFP under the control of a constitutive promoter (CAG) (AAV8.CAG.GFP) or AAV8 encoding IL-12 coupled to miR122T under the control of the ISRex4 promoter (AAV8-ISREx4-IL12miR122T) (in both cases, MOI was 10 5 ) transduced mouse cells. Panel A shows a transduction efficiency of approximately 70% for AAV8-CAG. GFP-treated MC38 cells were independent of prior irradiation. Panel B illustrates that MC38 cells irradiated prior to AAV8-ISREx4-IL12miR122T transduction significantly enhanced IL-12 production. Data are mean ± SEM (n = 3 samples / condition). ***P < 0.005, unpaired t-test.

[0440] FIG. 12 , comprising panels AC, shows the effects of AAV8 encoding luciferase coupled to miR122T under the control of the ISREX4 promoter (AAV8-ISREx4-LucmiR122T) (MOI 10 10 μg / ml) in 4% paraformaldehyde (P < 0.05) mice treated with irradiation (8 Gy) or without irradiation (0 Gy) in combination with IFN-α, IFN-β, IFN-γ, or vehicle. 5 ) transduced B16 cells. The figure illustrates that luciferase activity was induced when incubated with IFN-α (A), IFN-β (B) or IFN-γ (C) even in the absence of irradiation, while additional irradiation resulted in a synergistic increase in 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.

[0441] FIG. 13 , including FIGS. AO , shows radiation therapy and intratumoral injection of AAV8-ISREx4-IL12miR122T (0.5×10 11vg) antitumor effect of the combined treatment on melanoma mouse model (A+B), hepatocellular carcinoma mouse model (CH) and subcutaneous glioblastoma mouse model (I+J), as well as the intratumoral and serum levels of IL-12 (K+L) and IFN-γ (M+N), and finally the efficiency of the combined method compared with the treatment with only one component of the combined method or without its component. A) In the melanoma mouse model (B16 cells), tumor volume evaluation at the indicated time points after treatment with AAV8-ISREx4-IL12miR122T combined with radiotherapy (triangles) or AAV8-ISREx4-LucmiR122T combined with radiotherapy (circles). B) Hematoxylin-eosin staining shows a large number of lymphocytes infiltrating into tumor tissues three 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 AAV8-ISREx4-IL12miR122T combined with radiotherapy (triangles) and AAV8-ISREx4-LucmiR122T combined with radiotherapy (circles) in a subcutaneous hepatocellular carcinoma mouse model generated by subcutaneous injection of PM299L hepatoma cells. E) Evaluation of survival showed complete survival of AAV8-ISREx4-IL12miR122T treated animals (dashed line) throughout the study period, compared with 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 AAV8-ISREx4-IL12miR122T combined with radiation therapy (triangles, dashed line) and AAV8-ISREx4-LucmiR122T combined with radiation therapy (circles, solid line) in a murine model of hepatocellular carcinoma generated by subcutaneous injection of NM53 G03 hepatoma cells. H) Evaluation of survival rate shows that AAV8-ISREx4-IL12miR122T treated animals survived completely throughout the study period (dashed line) compared to the early death of all AAV8-ISREx4-LucmiR122T treated animals (solid line). I) and J) Evaluation of tumor volume at the indicated time points after treatment with AAV8-ISREx4-IL12miR122T combined with radiation therapy (I) and AAV8-ISREx4-LucmiR122T combined with radiation therapy (J) in a subcutaneous murine glioblastoma model. Lines represent changes in tumor volume in individual animals during the study period. K)-N) Irradiation with 0 or 8 Gy followed by intratumoral injection of AAV8-ISREx4-IL12miR122T (0.5x10 11vg) Intratumoral values ​​of IL-12 (K), serum concentrations of IL-12 (L), intratumoral values ​​of IFN-γ (M), and serum concentrations of IFN-γ (N) in mice with subcutaneous B16 (melanoma) tumors. O) Evaluation of tumor volume at indicated time points after no treatment (black circles), treatment with AAV8-ISREx4-LucmiR122T combined with radiotherapy (white circles), treatment with AAV8-ISREx4-IL12miR122T but without radiotherapy (squares), and treatment with AAV8-ISREx4-IL12miR122T combined with radiotherapy (downward triangles) in a murine model of melanoma (B16 cells). Arrows indicate the start of the corresponding treatment. A), C), D), F), G) and O) data are mean ± SEM (n = 4-6 samples / 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). KN) data are mean ± SEM (4-8 samples per condition). Two-way ANOVA followed by Tukey's multiple comparison *P < 0.05, **P < 0.01, ***P < 0.001.

[0442] Figure 14, including Figures AB, shows the anti-tumor effect of combined treatment with radiation therapy and intratumoral injection of AAV8-ISREx4-IL12miR122T (AAV-IL12T) compared to corresponding controls injected with AAV8-ISREx4-LucmiR122T (AAV.Luc) in a murine melanoma model, in which some tumor cells lack 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 / sample). **P < 0.01, ****P < 0.0001, two-way ANOVA, followed by Tukey's multiple comparison test.

[0443] Figure 15, including Figures AP, shows the expression of IL-12 or IFN-β in different tumor cell lines, including melanoma (B16, Figures A, B, and KP), 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). Cultured cells (85,000 / well) received 0 or 8 Gy and were then incubated in the presence or absence of bicistronic vectors 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 a F2A element preceded by a linker domain consisting of the amino acid sequence GGSG (SEQ ID NO: 82) 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 (IRES Gtx9, SEQ ID NO: 83) under the control of the ISREx4 promoter. Figure 2. ScIL-12 and IFN-β separated by IRES2 element (SEQ ID NO:83) (AAV8-ISREx4-IRESGtx9-IL12-IFNbmiR122T) or (O–P) scIL-12 and IFN-β separated by IRES2 element (SEQ ID NO:84) under the control of ISRex4 promoter (AAV8-ISREx4-IL12-IRES2-IFNbmiR122T). After 48 h, supernatants were collected and IL-12 and IFN-β were measured by ELISA. Both proteins were detectable only when incubated with vector, while the expression of both proteins was greatly increased after irradiation treatment. Data are mean ± SEM (n = 3 samples / condition). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA followed by Sidak's multiple comparison test.

[0444] Figure 16, including Figures AB, shows serum levels of IL-12 (A) and IFN-β (B) in mice injected intravenously with bicistronic AAV8-ISREx4-IL12-IFNbmiR122T (black circles), monocistronic AAV8-ISREx4-IL12miR122T (grey circles), or 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.

[0445] Figure 17, including panels AB, shows the anti-tumor effects of combined treatment with radiation therapy and intratumoral injection of bicistronic AAV8-ISREx4-IL12-IFNbmiR122T (rectangles in A, downward triangles in B), monocistronic AAV8-ISREx4-IL12miR122T (upward triangles in A and B), and control vector AAV8-ISREx4-LucmiR122T (circles in A and B) on a melanoma mouse model in which tumor cells express MHC class I (A) or lack 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.

[0446] Figure 18, including Figures AD, shows transgene expression in irradiated subcutaneous tumors (NM53 liver cancer cells) compared to non-irradiated tumors at the opposite site when AAV8-ISREx4-LucmiR122T is administered by systemic route (intravenous administration). Figure B is a repeat experiment of the experiment in Figure A. Figure C shows luciferase activity in four mice with orthotopic NM53 liver cancer. Two mice were irradiated with 20Gy, and the other two were not irradiated, and then 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 only appears in the liver area where those tumors have been previously irradiated with 20Gy. In addition, Figure D shows the size of MC38 subcutaneous tumors in mm after irradiation (20 Gy) or without irradiation as indicated, followed by intravenous injection of AAV8-ISREx4-LucmiR122T (AAV.LucT) or AAV8-ISREx4-IL12miR122T (AAV.IL12T). 3 Data are mean ± SEM (n = 5-6 mice per group), ****P < 0.0001, two-way ANOVA followed by Tukey post hoc test.

[0447] Figure 19, including Figures AD, shows the effects of different radiation doses on transgene expression from AAV8-ISREx4-LucmiR122T when the vector is administered by intratumoral injection. Figure A shows luciferase activity when the vector is injected into the tumor after irradiation with 15Gy, and then receives 3 additional doses of 15Gy (total dose = 60Gy). Figure B shows a tumor that receives 4 daily doses of 15Gy and then injects the vector (total dose = 60Gy). In Figure C, the tumor receives 60Gy, followed by intratumoral vehicle injection and two subsequent daily doses of 20Gy (total dose = 100Gy). In all cases, luciferase activity was assessed in the following days after vector injection (as shown). Therefore, Figures AC show different irradiation and treatment paradigms, and the data set is shown in Figure D. *P<0.05. After one-way ANOVA, it was followed by Sidak's multiple comparison test.

[0448] Fig. 20 Figure 2 shows the growth of B16 subcutaneous tumors in mm after irradiation (8 Gy) and intratumoral injection of AAV8-ISREx4-LucmiR122T (AAV.LucT) or AAV8-ISREx4-IL12miR122T (AAV.IL12T) as indicated, followed by administration of the immune checkpoint inhibitor anti-PD1 (αPD1) or the immune checkpoint inhibitor anti-CTLA-4 (αCTLA-4) or no further treatment. 3 Data are mean ± SEM (n = 5-6 mice per group), ns: not significant, ****P < 0.0001, two-way ANOVA followed by Tukey post hoc test.

[0449] Specific implementation form

[0450] Example 1: STING response to DMXAA in vitro

[0451] In the first set of three experiments, the responses of different STINGs to DMXAA were investigated.

[0452] Experiment 1: DMXAA dose response to different STING

[0453] The aim of this experiment was to examine which STINGs were sensitive to the STING agonist DMXAA and whether there was a dose-dependency to DMXAA. As a readout, the expression of the downstream target IFN-β was measured by quantitative real-time PCR (qPCR).

[0454] Materials and methods

[0455] To this end, cells from the murine cell line MC38 or cells from the human cell line Hep2G were transduced in vitro with lentivirus encoding wild-type murine STING (mSTINGwt), wild-type human STING (hSTINGwt) or human triple mutant STING-S162A / G230I / Q266I (hSTINGmut). Transduced cells were sorted by flow cytometry based on the expression of the reporter protein to obtain stable cell lines. Individual stable cells were cultured at 750 cells / mm 2 The cells were seeded at a density of 100 μg / mL in growth medium in a 24-well plate. 24 h after seeding, the cells were incubated with different doses of DMXAA for another 3 h (MC38 cells) or 6 h (Hep2G cells). Thereafter, the cells were harvested and homogenized and used RSC s implyRNA Tissue kit protocol (Promega, Madrid, Spain) extracts mRNA. According to the manufacturer's instructions, RNA is reverse transcribed into cDNA using M-MLV reverse transcriptase (Promega, Madrid, Spain). Quantitative real-time PCR is performed using CFX-Connect real-time PCR detection system (BioRad, Hercules, Cal if) to quantify expression levels. Each analysis is performed in a 10 μL total volume, which contains 5 μL 1x IQ SYBR Green Supermix (Bio-Rad Life Science, Hercules, Cal if.), 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 annealed to each gene in the study are listed in detail in Table 5. The expression of IFN-β in any experimental group is measured, and the expression level of cells treated with the vehicle is quantified.

[0456] Results and discussion

[0457] like Figure 1A -C, in human or mouse cells transduced with DMXAA-sensitive STINGs hSTINGmut or mSTINGwt, IFN-β expression increased in a dose-dependent manner after DMXAA treatment. In contrast, no significant changes in IFN-β expression were observed in non-DMXAA-sensitive hSTINGwt. Since IFN-β expression is upregulated upon STING activation, this experiment suggests that i) DMXAA can activate DMXAA-sensitive STING and that these STINGs have the biological activity of STING, and ii) the dose of DMXAA is related to STING activity. Therefore, DMXAA activates DMXAA-sensitive STING but not hSTINGwt.

[0458] Experiment 2: DMXAA-mediated cytokine expression

[0459] The aim of this experiment was to examine whether different STINGs exhibited STING biological activity after DMXAA treatment. As a readout, the expression of STING-derived cytokines was measured by qPCR.

[0460] Materials and methods

[0461] To this end, MC38 and Hep2G cells have been transduced with lentivirus encoding luciferase, hSTINGwt, hSTINGmut to obtain stable cell lines as described above, or have not been transduced with any lentivirus (no vector). 2 The density of 100 μg / mL DMXAA was inoculated in the growth medium of 24-well plates. 24h after inoculation, the cells were incubated with 50 μg / mL DMXAA for another 3h (MC38 cells) or 6h (Hep2G cells). The cells were harvested as described above and prepared for qPCR measurement. The expression of cytokines IFN-β, TNF-α, IL-6, CXCL9 and CXCL10 in any experimental group was measured and the expression level relative to the control (no vector) was quantified.

[0462] Results and discussion

[0463] like Figure 1D -E, the expression of the above cytokines was increased only in cells transduced with hSTINGmut, and no significant changes in expression levels were observed in any other groups. This further confirms the findings of the first experiment, i.e., i) DMXAA can activate DMXAA-sensitive STING, and these STINGs have the biological activity of STING. In addition, this experiment shows that hSTINGmut exhibits the biological activity of STING because it causes upregulation of known STING downstream proteins (Liu et al., 2020117 (24)).

[0464] Experiment 3: Time course expression of STING-derived cytokines after DMXAA treatment

[0465] The purpose of this experiment was to examine the time course of STING-derived cytokine expression following DMXAA treatment. As a readout, the expression of STING-derived cytokines was measured by qPCR.

[0466] Materials and methods

[0467] To this end, stable cell lines of MC38 and Hep2G cells encoding hSTINGmut were obtained as described above. 24 h after inoculation, cells were incubated with 50 μg / mL DMXAA for the indicated time points. Cells were harvested as described above and prepared for qPCR measurements. The expression of cytokines IFN-β, TNF-α, IL-6, CXCL9 and CXCL10 was measured in either experimental group and quantified relative to the expression level of the control group (0 h after DMXAA treatment).

[0468] Results and discussion

[0469] like Figure 1F As shown in Figures 1-G, although the temporal expression profiles of the different cytokines differed for each cytokine and between MC38 cells and Hep2G cells, overall, at any given time point, an increase in expression levels was observed after DMXAA treatment. Thus, the expression of the studied cytokines was upregulated in a DMXAA-dependent manner and was therefore dependent on STING activity, while the different temporal expression patterns indicated that this expression was also dependent on the cell line used and the respective cytokine studied.

[0470] Example 2: STING response to DMXAA in vivo

[0471] Since hSTINGmut has been shown to be generally sensitive to DMXAA in vitro, the aim of this experiment was to examine the response of hSTINGmut to DMXAA in vivo.

[0472] Materials and methods

[0473] C57 wild-type mice aged 6-10 weeks were intravenously injected with AAV8 vector encoding hSTINGmut (AAV8-hSTINGmut, 1x10 11 viral genomes) or AAV8 vector encoding GFP as a control (AAV8-GFP, 1.5x10 11 Two weeks after transduction, mice were injected intraperitoneally repeatedly (twice a week) with different DMXAA doses (5 mg DMXAA / kg body weight, 10 mg DMXAA / kg body weight, and 20 mg DMXAA / kg body weight). Three hours after injection, blood was collected from each mouse, and the IFN-β protein level in serum was determined using VeriKine-HSTM (High Sensitivity) Mouse IFN-β Serum ELISA (pbl Assay Science, Piscataway NJ USA).

[0474] Results and discussion

[0475] like Figure 2As shown, after the first injection, the administration of 20 mg DMXAA / kg body weight already caused an increase in IFN-β levels in AAV8-GFP transduced control animals, and IFN-β levels increased even further after repeated injections. This is expected, because mice express murine STING (mSTINGwt), which is already sensitive to DMXAA, and show IFN-β upregulation in vitro (see Example 1, Experiment 1). It is noteworthy that IFN-β levels in mice transduced with AAV8-hSTINgmut were more than 10 times higher than AAV8-GFP controls. In both cases, systemic STING stimulation led to high toxicity and high mortality (which can be seen from the decrease in the number of remaining animals with continuous DMXAA injections). However, doses of 5 or 10 mg DMXAA / kg body weight showed only very low IFN-β levels in AAV8-hSTINGmut-transduced mice, and, moreover, neither caused significant toxicity nor mortality. These data demonstrate that: i) exogenous STING expression dose-dependently enhances IFN responses to STING agonists, ii) systemic STING activation is associated with unacceptable toxicity, and iii) IFN-β induction in transduced tissues can be triggered on demand by injection of the STING agonist DMXAA.

[0476] Example 3: Promoters dependent on STING activity

[0477] Since DMXAA-induced STING-activity leads to increased cytokine expression levels in cells after transduction with hSTINGmut or mSTINGwt, the aim of the following experiments was to develop a system in which target genes can be expressed dependent on STING activity in transduced cells. Therefore, the aim of the following 4 experiments was to construct vectors with a STING activity-responsive promoter.

[0478] Experiment 1: IFN- β Promoter

[0479] One of the major cytokines upregulated following DMXAA-mediated STING activation is IFN-β, making the IFN-β promoter a viable target to examine its function as a STING activity-dependent promoter. Therefore, the aim of this experiment was to examine the specificity of the differently modified IFN-β promoter in its response to DMXAA-induced STING activity.

[0480] Materials and methods

[0481] To this end, five different modifications of the IFN-β promoter were designed (short IFN-βA according to SEQ ID NO: 18, short IFN-βB according to SEQ ID NO: 19, short IFN-βC according to SEQ ID NO: 20, short IFN-βD according to SEQ ID NO: 21, short IFN-βE according to SEQ ID NO: 22). Lentiviral vectors encoding luciferase under the control of five different promoters were constructed. These vectors were used to lentivirally transduce the MC38 cell line stably expressing hSTINGmut as described above (Example 1), while the control group did not receive further lentiviral transduction (None). 24h after inoculation, cells were treated with DMXAA (50μg / mL) or vehicle. In another 3h ( Figure 3A ) or 3h and 6h( Figure 3B , Figure 3C , Figure 3D ) or 6h( Figure 3E , Figure 3F , Figure 3G and Figure 3H ) after, the cells were homogenized in 50 μL passive lysis buffer (Promega) and frozen in dry ice. After 3 cycles of thawing-vortexing-freezing to ensure lysis, the cells were centrifuged at 12000g for 2 min at 4°C and 20 μL were added to a luminometer 96-well plate, and luciferase activity was determined using respective substrates D-luciferin and coelenterazine.

[0482] Results and discussion

[0483] like Figure 3A As shown in Figure 5, each of the aforementioned promoters showed luciferase activity upon administration of DMXAA, whereas cells transduced with hSTINGmut alone (i.e., cells lacking a vector encoding luciferase) showed no luciferase activity upon administration of DMXAA. Figure 3A denoted as "none" in the figure) showed no luciferase activity, regardless of DMXAA administration. It is noteworthy that the short IFN-βA promoter showed the highest luciferase activity after DMXAA treatment compared to the other promoters. However, significant luciferase activity was observed even in the absence of DMXAA. This suggests that although the short IFN-βA promoter is associated with the activation of hSTINGmut, the specificity of this promoter is somewhat low, because basal luciferase activity (leaky expression) was observed even when hSTINGmut was not activated by DMXAA. Similarly, other promoters also showed significant leaky expression. In addition, the overall responsiveness of other promoters to STING activated by DMXAA is quite low.

[0484] Experiment 2: Chemokine Promoter

[0485] In addition to IFN-β, chemokines CXCL9 and CXCL10 also showed significant upregulation in DMXAA-mediated STING activation, making the CXCL9 promoter and CXCL10 promoter viable targets to examine their function as STING activity-dependent promoters. Therefore, the purpose of this experiment was to examine the specificity of these promoters in response to DMXAA-induced STING activity.

[0486] Materials and methods

[0487] The CXCL9 promoter (CXCL9 according to SEQ ID NO: 23) and the CXCL10 promoter (CXCL10 according to SEQ ID NO: 24) were used in the following experiments. To this end, lentiviral vectors encoding luciferase under the control of these two different promoters were constructed. These vectors were used to stably express hSTINGmut in the MC38 cell line by lentiviral transduction as described above (Example 1), while the control group did not receive further lentiviral transduction (control). In addition, the lentiviral vectors encoding the short IFN-βA promoter, the short IFN-βB promoter, and the short IFN-βC promoter described in Experiment 1 were used as controls. 24h after inoculation, cells were treated with DMXAA (50μg / mL) or vehicle. After another 3 or 6h, cells were harvested and luciferase activity was measured as described in Experiment 1.

[0488] Results and discussion

[0489] like Figure 3B As shown, each of the aforementioned promoters showed luciferase activity when DMXAA was applied, while cells transduced with hSTINGmut alone (control), regardless of whether DMXAA was applied, did not show luciferase activity. It is worth noting that the CXCL9 promoter and the CXCL10 promoter showed significantly higher luciferase activity compared to the IFN-β promoter. However, even in the absence of DMXAA, significant luciferase activity was observed for both chemokine promoters. This suggests that although these promoters cause high luciferase expression when DMXAA is applied, the specificity of these promoters is somewhat low, because even when hSTINGmut is not activated by DMXAA, basal luciferase activity (leakage expression) is observed.

[0490] Experiment 3: Functionality of promoters dependent on STING activity

[0491] Experiments 1 and 2 demonstrated that the IFN-β promoter, CXCL9 promoter, and CXCL10 promoter, when operably linked to a luciferase transgene, caused its upregulation upon DMXAA-induced STING activation. The purpose of the following experiments was to further demonstrate the functionality of these promoters by linking them to the expression of the immunomodulatory single-chain interleukin-12 (scIL-12).

[0492] Materials and methods

[0493] The CXCL9 promoter and CXCL10 promoter from Experiment 2, as well as the full-length IFN-β promoter (IFN-β according to SEQ ID NO: 25) were used in the following experiments. To this end, a lentiviral vector encoding mouse scIL-12 under the control of these three different promoters was constructed. These vectors are used to stably express hSTINGmut MC38 cell lines by lentiviral transduction as described above (Example 1), while the control group does not receive further lentiviral transduction (no vector). 24h after inoculation, cells were treated with DMXAA (50 μg / mL) or vehicle. After the specified time point, cells were harvested. As previously described (Example 1, Experiment 1), cell lysates were used to measure scIL-12 mRNA expression by qPCR, and supernatants were used to measure secreted scIL-12 protein in NUNCMaxiSorp 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.

[0494] Results and discussion

[0495] like Figure 3C -D, cells transduced with hSTINGmut alone showed low expression of scIL-12 mRNA in the cells and did not secrete detectable levels of scIL-12, regardless of whether DMXAA was applied. The IFN-β promoter and the CXCL9-promoter were poorly functional in terms of scIL-12 mRNA or protein expression, even after DMXAA treatment. In contrast, scIL-12 expression was highly upregulated 6 hours after DMXAA treatment and the CXCL10 promoter control, and a large amount of scIL-12 protein was secreted into the supernatant. However, according to previous experiments, the CXCL10 promoter showed significant leakage, as scIL-12 was also produced in the absence of DMXAA. Although this shows that the general concept of promoters that rely on STING activity is feasible, the large leakage of the promoters used in these experiments suggests that it is necessary to test other promoters that are highly inducible when DMXAA is applied and highly specific for STING activity.

[0496] Experiment 4: Synthetic promoter

[0497] As mentioned above, the specificity of the promoters used in previous experiments was somewhat poor. Therefore, the aim of the following experiments was to find other promoters that combined high inducibility after administration of DMXAA with high specificity for STING activity. To achieve this, synthetic promoters have been generated.

[0498] Materials and methods

[0499] Seven different promoters were generated, namely short STATx4 (4 STAT motifs separated by 6 nucleotides according to SEQ ID NO: 26), IRSF3x4 (4 IRSF3 motifs separated by 20 nucleotides according to SEQ ID NO: 27), short IRSF3x4 (4 IRSF3 motifs separated by 6 nucleotides according to SEQ ID NO: 28), ISREX4 (4 ISRE motifs separated by 20 nucleotides according to SEQ ID NO: 12), short ISREX4 (4 ISRE motifs separated by 6 nucleotides according to SEQ ID NO: 13), NFκBx4 (4 NFκB motifs separated by 20 nucleotides according to SEQ ID NO: 29) and short NFκBx4 (4 NFκB motifs separated by 6 nucleotides according to SEQ ID NO: 30). All of the above promoter sequences given by SEQ ID NOs: 12, 13 and 26 to 30 do not contain additional transcriptional regulatory elements that affect the transcription efficiency of the transgene. These promoters are functional promoters themselves and do not require additional elements to provide transcription of the operably linked transgene. A pGL3-basic plasmid containing a luciferase sequence under the control of the respective promoter was designed. For clarity, the pGL3-basic plasmid does not contain any promoter-like sequence, but the promoter is specifically incorporated into the plasmid via the sequences SEQ ID NOs: 12, 13 and 26 to 30 listed above. In addition, a plasmid containing a luciferase sequence under the control of the mouse IFN-β promoter (mIFN-β) and a plasmid containing a luciferase sequence under the control of the CXCL10 promoter were used as controls. MC38 cells or human HEK293T cells (both stably expressing hSTINGmut as described above (Example 1)) were transfected with the indicated plasmids using Lipofectamine 3000 according to the manufacturer's protocol, whereas a mock transfection (basic) was performed on the negative control. 24 h after transfection, cells were treated with DMXAA (50 μg / mL) or vehicle as described in Experiment 1, and luciferase activity was measured after an additional 48 h.

[0500] Results and discussion

[0501] like Figure 3E -F, after DMXAA-mediated STING activation, the ISRE promoter as well as the NFκB promoter led to enhanced luciferase activity in MC38 cells. However, in the absence of DMXAA treatment, the NFκB promoter also exhibited fairly strong luciferase activity. In contrast, the ISRE promoter exhibited high inducibility and low leakiness upon DMXAA stimulation. The short STAT promoter and the two IRSF promoters showed poor inducibility. Regardless of DMXAA treatment, no luciferase activity was observed in the mock-transfected control (basal), a fact that confirms the validity of these assays.

[0502] like Figure 3G -H, after DMXAA-mediated STING activation, the ISRE promoter as well as the NFκB promoter led to enhanced luciferase activity in HEK293T cells. In addition, the leakage of either promoter in HEK293T cells was significantly reduced compared to mouse MC38 cells. However, although no detectable leakage was observed for the ISRE promoter, the NFκB promoter still showed some leakage in the absence of DMXAA treatment. The highest inducibility was observed for the CXCL10 promoter, however, it showed considerable leakage. Other promoters showed fairly low inducibility, but also limited leakage. Therefore, the ISREx4 promoter showed the highest inducibility while showing undetectable leakage.

[0503] In summary, it should be noted that the ISREX4 promoter exhibits leakiness in MC38 cells but not in 239T cells. The former is a tumor cell line, while the latter is a non-malignant cell line of embryonic origin. Tumor cell lines often have chromosomal abnormalities and micronuclei in the cytoplasm, which maintain smoldering STING activation, resulting in low levels of IFN-β production and low levels of activation of STING-responsive promoters.

[0504] Example 4: STING activity-dependent IL-12 expression in vivo

[0505] The ISREx4 promoter exhibited the highest inducibility while maintaining very low leakiness, making it the most promising promoter for in vivo applications. Therefore, the aim of the following experiments was to analyze the translatability of STING activity-mediated IL-12 expression in vivo.

[0506] Experiment 1: IL-12 production in the absence of DMXAA-mediated STING induction in vivo

[0507] The aim of the first experiment was to examine whether IL-12 is produced in transduced cells in vivo even in the absence of DMXAA-mediated STING activation.

[0508] Materials and methods

[0509] Adeno-associated virus (AAV) serotype 8 (AAV8) encoding scIL-12 under the control of the ISRex4 promoter (AAV8-ISREx4-IL12) and an AAV8 control vector encoding scIL-12 under the control of the murine IFN-β promoter (AAV8-mIFN-β-IL12) were constructed. 10 Viral genomes, 5x10 10 viral genomes and 1x10 11 AAV8-ISREx4-IL12 or AAV8-pIFN-β-IL12 containing 10 viral genomes (100 μg / mL) was intravenously injected into different groups of 6- to 10-week-old C57 wild-type mice (n=3 / group), and IL-12 serum levels were measured 7 days later as described in Example 2.

[0510] Results and discussion

[0511] like Figure 4A As shown, IL-12 expression was dose-dependent, as higher viral doses resulted in increased IL-12 production with both AAVs. Notably, expression was significantly higher with intravenous injection of AAV8-ISREx4-IL12 compared to AAV8-pIFN-β-IL12. Thus, despite the higher expression levels of AAV8-ISREx4-IL12, both AAVs were able to result in IL-12 production in vivo even in the absence of DMXAA administration. Therefore, measures must be taken to reduce leaky IL-12 production in vivo.

[0512] Experiment 2: Inhibition of IL-12 leakage by microRNA binding sites in vivo

[0513] As shown in the above experiments, IL-12 is produced after intravenous injection of AAV8-ISREx4-IL12, regardless of the presence or absence of DMXAA, the presence of which can cause adverse side effects. Many AAV serotypes, including AAV8, target the liver primarily when present in the blood circulation. Even in the case of intratumoral injection, some AAV may enter the blood circulation because tumors are often highly vascularized, thereby transducing hepatocytes to express IL-12 under the control of the ISRE promoter. The liver is exposed to a variety of intestinal pathogen-derived molecules that are able to induce the production of type I IFNs, leading to the production of IL-12. Since IL-12 is a potent immunostimulatory but cytotoxic cytokine, it is critical to limit the expression of IL-12 in target tissues and prevent the production of IL-12 in the liver. One way to limit the expression of a certain protein is to intervene at the post-transcriptional level, for example, by miRNA binding and subsequent mRNA degradation. Therefore, to control IL-12 expression, a STING-responsive promoter that is rarely leaky was selected, and in an attempt to inhibit hepatic IL-12 production, the IL-12 sequence was operably linked to the binding site of microRNA 122 (miR122), which is highly abundant in hepatocytes.

[0514] Materials and methods

[0515] A nucleotide sequence was constructed in which five repeats of the target sequence of miR122 were located 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 the corresponding viral particles (AAV8-ISREx4-IL12miR122T) were produced. Two groups of 6-10 week old C57 wild-type mice (n=4 / group) were intravenously injected with AAV8-ISREx4-IL12 or AAV8-ISREx4-IL12miR122T (0.5×10 11 viral genomes), and IL-12 serum levels were measured on day 7 after vector injection as described above.

[0516] Results and discussion

[0517] like Figure 4B As shown, animals treated with a vector containing a miR122 binding site (AAV8-ISREx4-IL12miR122T) had undetectable serum IL-12 levels, whereas values ​​exceeding 10 ng / ml were found in those mice receiving AAV8-ISREx4-IL12. Thus, by controlling the expression of IL-12 via the miR122 binding site, undesirable basal IL-12 production in the liver can be avoided.

[0518] Experiment 3: Inhibition of IL-12 leakage through microRNA binding sites in the presence of DMXAA in vivo

[0519] Since controlling IL-12 expression via miR122 binding sites can avoid undesired hepatic basal IL-12 production, the aim of the following experiments was to elucidate whether undesired hepatic production of IL-12 can be achieved even in the presence of the STING agonist DMXAA.

[0520] Materials and methods

[0521] To this end, 6-10 week old C57 wild-type mice were injected intravenously with an AAV8 vector encoding hSTINGmut under the control of the constitutive CAG promoter (AAV8-CAG-hSTINGmut, 1×10 11 5×10 viral genomes) + the aforementioned AAV 8 vector (n=4 animals / group), wherein the aforementioned AAV 8 vector contains an ISRE promoter and 5 miR122 binding sites at the 3′ end (AAV8-ISREx4-IL12miR122T, 0.5×10 11 viral genomes) or without 5 miR122 binding sites (AAV8-ISREx4-IL12, 0.5x10 11 One week later, the animals were challenged intravenously with DMXAA (20 mg DMXAA / kg body weight), vehicle, or not challenged intravenously at all (basal). After 4 hours, 150 μL of blood was collected and IL-12 serum levels were quantified by ELISA as described above.

[0522] Results and discussion

[0523] like Figure 4C As shown, while mice injected with an IL-12 vector lacking a miR122 binding site (AAV8-ISREx4-IL12) showed elevated basal serum levels of IL-12 that were further elevated after DMXAA administration, those animals injected with an IL-12 vector containing a miR122 binding site (AAV8-ISREx4-IL12miR122T) showed almost undetectable IL-12 serum levels at baseline and very low levels after DMXAA challenge. Thus, AAV8-ISREx4-IL12miR122T showed almost no leakage even in the presence of DMXAA.

[0524] Example 5: Therapeutic effect of repeated administration of DMXAA after intratumoral co-injection of AAV8-hSTINGmut+AAV8-ISREx4-IL12miR122T

[0525] The purpose of the following experiment was to analyze whether co-injection of AAV8-hSTINGmut+AAV8-ISREx4-IL12miR122T into tumors followed by repeated administration of DMXAA would show a therapeutic effect as measured by a reduction in tumor size.

[0526] Materials and methods

[0527] To this end, 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) was used. In these animals, DMXAA does not activate STING systemically, but only in those tissues that have been 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. When the tumor nodules reached about 70 mm 3 When the size of 11 viral genomes)+AAV8-ISREx4-IL12miR122T (0.5x10 11 1x10 viral genomes), and 48 hours later, animals were treated with DMXAA (20 mg / kg body weight) (n=6 animals) or vehicle twice a week (n=5 animals). In another control group of mice, AAV8-hSTINGmut (1x10 11 viral genomes)+AAV8-GFP (0.5x10 11 Tumors were co-injected with 50% paraformaldehyde (4 viral genomes) and then received vehicle twice weekly (n=3 animals).

[0528] Results and discussion

[0529] like Figure 5As shown, a significant anti-tumor effect was observed in both groups treated with AAV8-hSTINGmut+AAV8-ISREx4-IL12miR122T, while the anti-tumor effect was significantly higher in mice treated with DMXAA in addition. It is noteworthy that complete tumor elimination was observed in 1 of 6 mice treated with DMXAA, but no complete tumor elimination was observed in mice treated with the given vehicle. In contrast, the tumor volume of mice treated with AAV8-GFP+vehicle continued to grow throughout the experiment. As described above, the anti-tumor effect in mice treated with the given vehicle of AAV8-hSTINGmut+AAV8-ISREx4-IL12miR122T can be attributed to the low level of intratumoral production of IL-12 caused by the low level of basal activation of the ISREX4 promoter in MC38 cells. Importantly, the therapy was well tolerated in any animal without toxic symptoms. The strategy described here is 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 would be synthesized within the tumor mass rather than in the liver, thereby eliminating the risk of AAV particles that enter the blood and liver after their intratumoral injection continuing to produce IL-12 on a liver basis. On the other hand, an agonist that interacts with transgenic STING but not with endogenous human STING was used. In this way, DMXAA administered by intravenous or oral routes will activate STING in the tumor rather than systemically, making the treatment tolerable. Marginal activation of transgenic STING expressed by a small portion of AAV vectors that can reach the liver after intratumoral injection only caused a slight increase in type I IFN and IL-12 in serum below 0.2 pg / mL (data not shown), which was very well tolerated by experimental animals.

[0530] Example 6: Intratumoral transduction efficiency after radiotherapy

[0531] It is known that different DNA damaging agents including topoisomerase inhibitors and radiation have been shown to enhance AAV transduction, which may be attributed to the induction of 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 into double-stranded DNA to form a transcription template. The DNA damage response caused by radiotherapy helps to convert single-stranded DNA into double-strand...

Claims

1. A recombinant expression system comprising c. a first nucleic acid construct comprising a sequence encoding a target STING that responds to a STING agonist that recognizes the target STING but not human STING and its natural variants (human non-responsive STING agonist), the sequence encoding the target STING being operably linked to an element that allows expression and / or translation of the target STING; and / or d. A second nucleic acid construct comprising a sequence encoding at least one immunomodulatory gene or protein, said sequence being operably linked to a STING-responsive promoter or a radiation-inducible promoter.

2. The recombinant expression system of claim 1, wherein the target STING is responsive to a human non-responsive STING agonist selected from the group consisting of 5,6-dimethylxanthone-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-acridone (CMA), and wherein preferably the target STING is responsive to DMXAA.

3. The recombinant expression system of claim 1 or 2, wherein the target STING encoded by the sequence of the first nucleic acid construct is a. a mutant form of a naturally occurring variant of human STING further comprising 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 assay; or b. a non-human STING that is sensitive to said human non-responsive STING agonist as determined in an in vitro agonist assay, preferably a murine STING or a hybrid STING, even more preferably a murine STING according to SEQ ID NO: 7; Wherein the target STING is preferably DMXAA-sensitive human STING as determined in an in vitro DMXAA assay.

4. The recombinant expression system of claim 3, wherein the target STING encoded by the sequence of the first nucleic acid construct is a DMXAA-sensitive human STING variant selected from the group consisting of: a. a human STING[R] variant, further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of S162A, G230I, and Q266 I; b. a human STING[AQ] variant further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of S162A, A230I, Q266 I, E260I, and E260V; c. a human STING[H] variant further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of S162A, G230I, and Q266 I; and d. A truncated human STING[H] variant further comprising at least one or at least two amino acid substitutions selected from the group consisting of S162A and G230I.

5. The recombinant expression system of claim 4, wherein the DMXAA-sensitive human STING variant encoded by the sequence of the first nucleic acid construct is selected from the group consisting of: hSTING[R]-S162A / G230I / Q266 I, hSTING[R]-S162A / Q266 I, hSTING[R]-G230I, hSTING[R]-S162A, hSTING[R]-Q266 I, hSTING[R]-S162A / G230I, hSTING[AQ]-S162A / A230I / Q266 I. hSTING[AQ]-S162A, hSTING[AQ]-S162A / R169A, hSTING[AQ]-S162A / T267A, hSTING[AQ]-E260I, hSTING[AQ]-E260V, hSTING[H]-S162A / G230I / Q266 I, hSTING[H]-S162A / Q266 I, hSTING[H]-G230I, hSTING[H]-S162A, hSTING[H]-S162A / G230I and hSTING[H]-Q266I, and preferably hSTING[R]-S162A / G230I / Q266I.

6. The recombinant expression system of claim 4, wherein the sequence of the first nucleic acid construct encoding the DMXAA-sensitive human STING variant is selected from the group consisting of: 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 relative 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 relative to SEQ ID NO: 61; e. a polynucleotide sequence encoding a polypeptide of c) or d), wherein the polypeptide has at least one modification, wherein the modification 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 at least under stringent conditions to the complementary sequence of a) to e) in any one of the polynucleotide sequences; A region of the polynucleotide sequence of ga) to f), which encodes a fragment of at least 50 amino acid residues of the DMXAA-sensitive human STING variant; Wherein the polypeptide encoded by the polynucleotide of any one of a) to g) has biological activity in response to the agonist DMXAA as determined in an in vitro DMXAA assay.

7. The recombinant expression system according to any one of the above claims, wherein the promoter of the first nucleic acid construct is a constitutive promoter, preferably selected from the list 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 promoter of the first nucleic acid construct is an inducible promoter, preferably selected from the list consisting of: telomerase responsive promoter, IL-6 responsive promoter, TGF-β inducible promoter, VEGFR inducible promoter, epidermal growth factor receptor inducible promoter, HER2 / Neu inducible promoter, MUC1 promoter, BRCA1 promoter, BRCA2 promoter, LALBA promoter, PSA promoter, ERBB2 promoter, CEA promoter, uPAR promoter and thyroid transcription factor 1 (TTF1).

8. The recombinant expression system of any one of the above claims, wherein the at least one immunomodulatory gene or protein encoded by the sequence of the second nucleic acid 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-1 4. 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, CCL L15, 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, CXCL 15. CXCL16, CXCL17, XCL1, XCL2, CX3CL1, granulocyte-macrophage 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 that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3, and fragments or variants thereof; or shRNA targeting TGF-β, shRNA targeting IL-10, or shRNA targeting FoxP3.

9. The recombinant expression system of any one of the above claims, wherein the at least one immunomodulatory protein is selected from the group consisting of: a. α subunit of IL-12; b. β-subunit of IL-12; c. Single chain IL-12 comprising 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, wherein the polypeptide has the biological activity of interleukin-12; 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, wherein the polypeptide has the biological activity of IFN-β; h. IL-15-sushi, which comprises or consists 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 the biological activity of IL-15; j. CXCL10, which comprises or consists of the amino acid sequence of SEQ ID NO: 81; k. A polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 81, wherein the polypeptide has the biological activity of CXCL10.

10. The recombinant expression system of any one of the above claims, wherein the second nucleic acid construct comprises at least two sequences, each of which encodes 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 relative to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12, and The second immunomodulatory protein is 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 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, wherein the polypeptide has the biological activity of IL-15; or 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, wherein the polypeptide has the biological activity of CXCL10.

11. The recombinant expression system of any one of the above claims, wherein the second nucleic acid construct is a bicistronic or polycistronic expression cassette comprising two or more sequences, each of which encodes a different immunomodulatory protein or a protein or RNA with anticancer properties, wherein a. said sequence is separated by a sequence encoding an IRES; and / or b. The sequence is separated by a sequence encoding a 2A peptide, preferably a sequence encoding a P2A peptide.

12. The recombinant expression system of any one of the above claims, wherein the STING responsive promoter of the second nucleic acid construct is selected from the group consisting of: 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 is more preferably selected from the list consisting of: a. a nucleotide sequence comprising 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%, 80%, 85%, 90% or at least 95% sequence identity with respect to SEQ ID NO: 11 or a sequence identical to SEQ ID NO: 11; b. The nucleotide sequence according to a., wherein the copies are spaced up to 40 nucleotides apart, preferably 4 to 35 nucleotides apart, more preferably 10 to 30 nucleotides apart, particularly preferably 15 to 25 nucleotides apart; c. the nucleotide sequence according to SEQ ID NO: 12; d. The nucleotide sequence according to SEQ ID NO:

13.

13. The recombinant expression system of any one of the above claims, wherein the second nucleic acid construct further comprises at least one sequence motif, which inhibits the transgenic expression of the immunomodulatory protein in tissues or cells that are not intended to express the protein, the tissue being preferably the liver, wherein the sequence motif is preferably a target sequence of a microRNA that is highly abundant in the liver, more preferably the 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, let7b target sequence, even more preferably a miR-122 target sequence selected from the list consisting of: a. a miR-122 target sequence having at least 90% or at least 95% sequence identity to or 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., wherein the copies are spaced 1-40 nucleotides apart, preferably 1-30 nucleotides apart, more preferably 2-20 nucleotides apart, and particularly preferably 2-8 nucleotides apart; d. A nucleotide sequence comprising 5 copies of the miR-122 target sequence as described in SEQ ID NO:

15.

14. An expression vector encoding the first and / or second nucleic acid construct according to any one of the preceding claims, or a combination of the first and second expression vectors, the first vector encoding the first nucleic acid construct according to any one of the preceding claims, and the second vector encoding the second nucleic acid construct according to any one of the preceding claims, wherein the one or more expression vectors are preferably viral vectors, more preferably viral vectors selected from the list consisting of: adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, vaccinia virus vectors or herpes simplex virus vectors, even more preferably adeno-associated virus vectors having one or more of the following characteristics: a. The first nucleic acid construct sequence and / or the second nucleic acid construct sequence further comprises 5′ITR and 3′ITR sequences, preferably the AAV ITR described in SEQ ID NO: 17; b. The AAV vector preferably has serotype AAV8, AAV1, AAV3, AAV6, AAV9, AAV2, AAV5, AAVrh.10 or any gain-of-function mutant of AAVrh.10, and more preferably the AAV vector has serotype AAV2.

15. A viral particle comprising a viral vector containing the first and / or second nucleic acid construct of claim 14 or a combination of a first and a second viral particle, wherein the first viral particle comprises a viral vector encoding the first nucleic acid construct of claim 14, and the second viral particle comprises a viral vector encoding the second nucleic acid construct of claim 14, wherein the one or more viral particles are preferably one or more AAV particles or one or more adenovirus particles; more preferably one or more AAV viral particles.

16. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and: a. The recombinant expression system of any one of claims 1 to 13; or b. one or more expression vectors of claim 14; or c. One or more viral particles according to claim 15.

17. The recombinant expression system of any one of claims 1 to 13; the one or more expression vectors of claim 14; the one or more viral particles of claim 15 or the pharmaceutical composition of claim 16, for use in therapy, preferably in immunotherapy, more preferably in immunotherapy of proliferative disorders, even more preferably in immunotherapy of cancer, particularly preferably in immunotherapy of solid cancers.

18. The recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical composition for use according to claim 17, wherein the recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical composition are used for combination therapy with at least one other therapy selected from the group consisting of: a. a therapy comprising at least one checkpoint inhibitor, preferably 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 selected from the group consisting of inhibitors of PD-1, PD-L1, PD-L2 or CTLA-4; and / or b. a therapy comprising at least one adoptive cell therapy, preferably selected from adoptive NK cell therapy, adoptive dendritic cell therapy or adoptive T cell therapy, wherein the adoptive T cell therapy is preferably CAR-T cell therapy or a therapy using TIL; and / or c. a therapy comprising at least one cancer vaccine; and / or d. A therapy comprising at least one exogenous cytokine; and / or e. Radiation therapy.

19. The recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical composition for use according to claim 17 or 18, wherein the cancer is selected from the list 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 lymphoma, primary liver cancer, metastatic liver cancer, gallbladder cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasmacytoma, bile duct cancer and neuroendocrine tumors or carcinoid tumors.

20. The recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical composition for use according to any one of claims 17 to 19, wherein the use in immunotherapy is a combination of: a. administering the recombinant expression system of any one of claims 1 to 13, the one or more expression vectors of claim 14, the one or more viral particles of claim 15 or the pharmaceutical composition of claim 16 to a patient suffering from a proliferative disorder, to produce a therapeutically effective transgenic expression of the target STING and / or the immunomodulatory protein, wherein the proliferative disorder is preferably cancer, wherein the administration is preferably intratumoral administration or systemic administration; b. Optionally, prior to and / or simultaneously with the administration according to step a), treating the patient with radiation therapy, preferably, the radiation therapy is directed to tissues or organs that are intended to express one or more of the transgenes from a); and c. After administration according to step a), treating the patient with a STING agonist that recognizes the target STING expressed by the transgene other than human STING and its natural variants and / or with radiation therapy, preferably radiation therapy directed to tissues or organs intended to express one or more of the transgenes from a).

21. The recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical composition for use according to claim 20, wherein the patient is treated with at least one other therapy according to claim 18 before, simultaneously with and / or after the administration according to step a), preferably after the administration according to step a).

22. The recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical composition for use according to claim 20 or 21, wherein in step c) a STING agonist according to claim 2 is used, which is preferably DMXAA.

23. The recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical composition for use according to any one of claims 20 to 22, wherein in step b), a radiation therapy with a dose of less than 100 Gy or a low-dose radiation therapy of 40 Gy or less, preferably 8 Gy or less is used.

24. The recombinant expression system, expression vector, viral particle or pharmaceutical composition for use according to any one of claims 20 to 23, wherein step c) comprises several separate radiotherapy treatments, wherein the cumulative dose of the separate radiotherapy treatments totals less than 100 Gy, preferably less than 40 Gy, and more preferably less than 25 Gy.

25. A recombinant expression system consisting of a nucleic acid construct comprising sequences encoding at least two immunomodulatory proteins, the sequences being 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 that are not intended to express the proteins.

26. The recombinant expression system of claim 25, wherein the at least two immunomodulatory proteins are 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-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, C XCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CX CL16, CXCL17, XCL1, XCL2, CX3CL1, granulocyte-macrophage 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 that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3 and fragments or variants thereof.

27. The recombinant expression system according to claim 25 or 26, wherein the at least two immunomodulatory proteins are selected from the group consisting of: a. α subunit of IL-12; b. β-subunit of IL-12; c. Single chain IL-12 comprising 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, wherein the polypeptide has the biological activity of interleukin-12; 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, wherein the polypeptide has the biological activity of IFN-β; h. IL-15-sushi, which comprises or consists 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 the biological activity of IL-15; j. CXCL10, which comprises or consists of the amino acid sequence of SEQ ID NO: 81; k. A polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 81, wherein the polypeptide has the biological activity of CXCL10.

28. The recombinant expression system according to any one of claims 25 to 27, 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 relative to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12, and The second immunomodulatory protein is 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 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, wherein the polypeptide has the biological activity of IL-15; or 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, wherein the polypeptide has the biological activity of CXCL10.

29. The recombinant expression system according to any one of claims 25 to 28, wherein the nucleic acid construct is a bicistronic or polycistronic expression cassette comprising two or more sequences each encoding a different immunomodulatory protein or a protein or RNA with anticancer properties, wherein a. said sequence is separated by a sequence encoding an IRES; and / or b. The sequence is separated by a sequence encoding a 2A peptide, preferably a sequence encoding a P2A peptide.

30. The recombinant expression system according to any one of claims 25 to 29, wherein the STING responsive promoter of the nucleic acid construct is selected from the group consisting of: 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 is more preferably selected from the list consisting of: a. a nucleotide sequence comprising 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%, 90% or at least 95% sequence identity with respect to SEQ ID NO: 11 or a sequence identical to SEQ ID NO: 11; b. The nucleotide sequence according to a., wherein the copies are spaced up to 40 nucleotides apart, preferably 4 to 35 nucleotides apart, more preferably 10 to 30 nucleotides apart, particularly preferably 15 to 25 nucleotides apart; c. the nucleotide sequence according to SEQ ID NO: 12; d. The nucleotide sequence according to SEQ ID NO:

13.

31. The recombinant expression system according to any one of claims 25 to 30, wherein the sequence motif inhibits transgenic expression of the immunomodulatory protein in tissues or cells that are not intended to express the protein, the tissue being preferably the liver, wherein the sequence motif is preferably a target sequence of a microRNA that is highly abundant in the liver, more preferably the 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, even more preferably a miR-122 target sequence selected from the list consisting of: a. a miR-122 target sequence having at least 90% or at least 95% sequence identity to or 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., wherein the copies are spaced 1-40 nucleotides apart, preferably 1-30 nucleotides apart, more preferably 2-20 nucleotides apart, and particularly preferably 2-8 nucleotides apart; d. A nucleotide sequence comprising 5 copies of the miR-122 target sequence as described in SEQ ID NO:

15.

32. The expression vector encoding the nucleic acid construct according to any one of claims 25 to 31, wherein the expression vector is preferably a viral vector, more preferably a viral vector selected from the list consisting of an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, a vaccinia virus vector or a herpes simplex virus vector, even more preferably an adeno-associated virus vector having one or more of the following characteristics: a. The nucleic acid construct sequence further comprises 5′ITR and 3′ITR sequences, preferably AAV ITR as described in SEQ ID NO: 17; b. The AAV vector preferably has serotype AAV2, AAV1, AAV3, AAV6, AAV9, AAV8, AAV5, AAVrh.10 or any gain-of-function mutant of AAVrh.10, and more preferably the AAV vector has serotype AAV2.

33. A viral particle comprising a viral vector containing a nucleic acid construct according to claim 32, wherein the viral particle is preferably an AAV or adenovirus particle; more preferably an AAV viral particle.

34. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and: a. The recombinant expression system of any one of claims 25 to 31; or b. the expression vector of claim 32; or c. The viral particle of claim 33.

35. The recombinant expression system of any one of claims 25 to 31; the expression vector of claim 32; the viral particle of claim 33 or the pharmaceutical composition of claim 34, for use in therapy, preferably in immunotherapy, more preferably in immunotherapy of proliferative disorders, even more preferably in immunotherapy of cancer, particularly preferably in immunotherapy of solid cancer.

36. The recombinant expression system, expression vector, viral particle or pharmaceutical composition for use according to claim 35, wherein the recombinant expression system, the expression vector, the viral particle or the pharmaceutical composition is used in a combination therapy with at least one other therapy selected from the group consisting of a. a therapy comprising at least one checkpoint inhibitor, preferably 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 selected from the group consisting of inhibitors of PD-1, PD-L1, PD-L2 or CTLA-4; and / or b. a therapy comprising at least one adoptive cell therapy, preferably selected from adoptive NK cell therapy, adoptive dendritic cell therapy or adoptive T cell therapy, wherein the adoptive T cell therapy is preferably CAR-T cell therapy or a therapy using TIL; and / or c. a therapy comprising at least one cancer vaccine; and / or d. A therapy comprising at least one exogenous cytokine; and / or e. Treatment including radiation therapy.

37. The recombinant expression system, expression vector, viral particle or pharmaceutical composition for use according to claim 35 or 36, wherein the cancer is selected from the list 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 lymphoma, primary liver cancer, metastatic liver cancer, gallbladder cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, urothelial carcinoma, gallbladder cancer, Merkel cell carcinoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasmacytoma, bile duct cancer and neuroendocrine tumors or carcinoid tumors.

38. The recombinant expression system, expression vector, viral particle or pharmaceutical composition for use according to any one of claims 35 to 37, wherein the use in immunotherapy is a combination of: a. administering the recombinant expression system of any one of claims 25 to 31; the expression vector of claim 32; the viral particle according to claim 33 or the pharmaceutical composition according to claim 34 to produce therapeutically effective transgenic expression of the immunomodulatory protein, wherein the administration is preferably intratumoral administration or systemic administration; b. Optionally, prior to and / or simultaneously with the administration according to step a), treating the patient with radiation therapy, preferably, the radiation therapy is radiation therapy directed to tissues or organs intended to express one or more of the transgenes from a); and c. After administration according to step a), treating the patient with radiation therapy, preferably, the radiation therapy is radiation therapy directed to tissues or organs intended to express one or more of the transgenes from a).

39. The recombinant expression system, one or more expression vectors, one or more viral particles or pharmaceutical composition for use according to claim 38, wherein the patient is treated with at least one other therapy according to claim 36 before, simultaneously with and / or after the administration according to step a), preferably after the administration according to step a).

40. The recombinant expression system, expression vector, viral particle or pharmaceutical composition for use according to claim 38 or 39, wherein in step b), a radiation therapy with a dose of less than 100 Gy is used, or a low-dose radiation therapy of 40 Gy or less, preferably 8 Gy or less is used.

41. The recombinant expression system, expression vector, viral particle or pharmaceutical composition for use according to any one of claims 38 to 40, wherein step c) comprises several separate radiotherapy treatments, wherein the cumulative dose of the separate radiotherapy treatments totals less than 100 Gy, preferably less than 40 Gy, and more preferably less than 25 Gy.

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