CONSTRUCT COMPRISING mRNA-STRAND AND RETINOIC ACID-INDUCIBLE GENE I (RIG-I)-LIGAND(S), PHARMACEUTICAL COMPOSITION AND KIT COMPRISING THE SAME

Abstract

The present invention relates to a construct comprising a mRNA-strand, and at least one, preferably at least two, retinoic acid-inducible gene I (RIG-l)-ligand(s) comprising a) a nucleic acid sequence being capable of binding to RIG-1, b) a linker moiety L comprising 3 to 24 main chain atoms, and c) a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand. The present invention further provides said construct for use as a medicament and / or for use as a vaccine, a pharmaceutical composition and kit comprising said construct, said construct for use in a method of treatment or prevention of a disease, the use of said construct as a vaccine and / or an immune adjuvant, said construct for use in a method of inducing an immune response as well as a method of treating and a method of inducing an immune response.

Description

CONSTRUCT COMPRISING mRNA-STRAND AND RETINOIC ACID-INDUCIBLE GENE I (RIG-I)-LIGAND(S), PHARMACEUTICAL COMPOSITION AND KIT COMPRISING THE SAMEFIELD OF THE INVENTION

[0001] The present invention relates to a construct comprising a mRNA-strand, and at least one, preferably at least two, retinoic acid-inducible gene I (RIG-l)-ligand(s) comprising a) a nucleic acid sequence being capable of binding to RIG-I, b) a linker moiety L comprising 3 to 24 main chain atoms, preferably comprising 6 to 18 main chain atoms, and c) a singlestranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand. According to the present invention, the construct may comprise one, two, three, four or more RIG-I ligand(s). This construct may be used as a medicament or a vaccine. The present invention also relates to said construct for use in a method of treatment or prevention of a disease. Additionally, the present invention also relates to a respective pharmaceutical composition and further to a respective kit for use in medicine, each comprising said construct.BACKGROUND OF THE INVENTION

[0002] mRNA-based vaccines are increasingly recognized as offering a lower cost, higher safety alternative to classical vaccination strategies such as inactivated pathogens or toxins. This is in part due to the vital role mRNA vaccines have played in vaccination during the worldwide SARS-CoV2 pandemic. Despite the accelerated research into mRNA vaccines in response to SARS-CoV2, optimization of mRNA-vaccine technology is ongoing. Successful immunization via mRNA vaccines typically requires three elements - mRNA encoding an immunogenic pathogen-associated protein, an adjuvant(s) capable of evoking an innate immune response, and an appropriate delivery vector.

[0003] Two currently approved mRNA vaccines (BioNTech and Moderna) induce innate immune activation by virtue of contaminant RNA molecules present in the vaccine preparation due to the nature of the manufacturing process, in addition to the defined requisite mRNA. The quantity and nature of these additional RNA molecules varies depending on the purification processes involved in manufacturing and other as of yet poorly defined factors. This may lead to variability and lack of predictability of adjuvant activity in such vaccines. It is therefore desirable to produce mRNA vaccines containing adjuvants whose quantity, level of immune stimulation, and purity is well-defined, leading to better health outcomes forindividuals in need of vaccination, in terms of both vaccination success itself and the potential for unwanted immunotoxic side-effects, such as allergic reactions or anaphylaxis.

[0004] The present invention provides a construct, which may be used as a vaccine. Such may enable to generate a consistent, predictable level of innate immune activation during vaccination whilst not interfering with vaccine-associated mRNA expression and preventing potential immunotoxic side-effects that may arise from the presence of poorly defined RNA molecules.

[0005] The present invention offers a construct design that can be readily incorporated into mRNA vaccine constructs, that acts via a defined mechanism by virtue of the defined elements present in the construct, and is capable of reliably inducing innate immune activation.SUMMARY OF THE INVENTION

[0006] The present invention relates to a construct, preferably a nucleic acid-construct that may be used in mRNA vaccine compositions that may allow for a defined, predictable innate immune response via a known mechanism of action. This construct allows for innate immune activation, a requisite for successful vaccination, whilst limiting potential adverse side-effects and maintaining high levels of mRNA translation.

[0007] The present invention provides a construct comprising an mRNA-strand, and at least one, preferably at least two, retinoic acid-inducible gene I (RIG-l)-ligand(s) comprising a) a nucleic acid sequence being capable of binding to RIG-1, b) a linker moiety L comprising 3 to 24 main chain atoms, preferably comprising 6 to 18 main chain atoms, and c) a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand.

[0008] The construct may preferably comprise at least two RIG-I ligands or preferably between two to four RIG-I ligands or preferably four RIG-I ligands.

[0009] The construct preferably comprises that the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s) is complementary to at least one nucleic acid sequence comprised in the mRNA strand. It is also preferred that the linker moiety L connects the nucleic acid sequence being able to bind to RIG-I with the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s).

[0010] In order to be able to fulfil its function as a vaccine, it is preferred that the aforementioned mRNA-strand comprises a coding sequence of a protein, of a peptide, or of an antigen that, once translated, is able to induce an immune response, or preferably of a cancer-associated antigen.

[0011] For the construct of the present invention is also preferred that the nucleic acid sequence being able to bind to RIG-I comprises or consists of a double-stranded RNA. ThisdsRNA may be a blunt-ended dsRNA, but may also further comprise an overhang in some embodiments. In that embodiments, it is preferred that said overhang is or comprises a RNA- overhang or that said overhang has or comprises a fluorophore or a lipophilic substitution.

[0012] In addition, it is preferred that the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at least one triphosphate at at least one strand of said double-stranded RNA, preferably at least one triphosphate at the 5'-end of one strand of said double-stranded RNA.

[0013] The length of the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I may have a length in the range of 8 to 20 nt, preferably a length in the range of 10 to 17 nt, more preferably a length in the range of 10 to 15 nt, more preferably a length in the range of 10 to 13 nt, and even more preferably a length of 11 nt.

[0014] In some embodiments, the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I may comprise at least one modification(s). That modification(s) may be selected from the group consisting of O-methyl-group-modification(s), preferably 2 - O-methyl-group-modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group- modification(s), phosphor-thio-modification(s) and locked nucleic acid (LNA)-modification(s). These modifications may be present on at least one of the strands in the dsRNA of the at least one RIG-l-ligand, preferably on both strands of the at least one dsRNA of the at least one RIG- l-ligand. In embodiments in which the dsRNA of the at least one RIG-l-ligand has undergone one or more of the aforementioned modifications, the at least one modification(s) may be present at the 3’-end and / or the 5’-end of at least one strand of the dsRNA of the at least one RIG-l-ligand, preferably at both strands of the dsRNA of the at least one RIG-l-ligand.

[0015] The dsRNA of the at least one RIG-l-ligand may comprise at least one O-methyl-group- modification(s), preferably at least one 2'-O-methyl-group-modification(s), preferably at its 5’- end and / or at it’s 3’-end, at one or both strands of the dsRNA of the at least one RIG-l-ligand.

[0016] The construct of the present invention may comprise a linker moiety L for connecting the nucleic acid sequence being able to bind to RIG-I with the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s). Preferably, the linker moiety L comprises 6 to 18 main chain atoms, more preferably 8 to 16 main chain atoms, more preferably 10 to 14 main chain atoms, even more preferably 12 main chain atoms. The main chain atoms may be carbon atoms that may be optionally replaced by one or more heteroatoms selected from the group consisting of N, O and S.

[0017] The construct of the present invention may comprise a linker moiety L that is a polyalkylene-glycol linker according to Formula IFormula I, wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7.

[0018] In preferred embodiments, the linker moiety L of the at least one RIG-l-ligand(s) is a polyalkylene-glycol-linker according to Formula IIFormula II, wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7. More preferably, the linker moiety L of the at least one RIG-l-ligand(s) is a polyalkylene-glycol-linker according to Formula II, wherein n is 6.

[0019] In a further aspect, the present invention provides the construct according to the present invention and as described herein for use as a medicament and for use as a vaccine.

[0020] In another aspect, the present invention relates to a pharmaceutical composition comprising the construct of the present invention.

[0021] Additionally, the present invention provides the construct according to the present invention and as described herein for use in a method of treatment or prevention of a disease. The disease may be cancer or an infection, preferably a virus infection. The disease may also be a haematological tumour or a solid tumour.

[0022] In a further aspect of the present invention, the construct according to the present invention and as described herein may be for use in a method of infiltrating immune cells, preferably T cells, or for use in a method of turning cold into hot tumours.

[0023] The present invention further relates to the use of the construct of the present invention as a vaccine or as an immune adjuvant.

[0024] The present invention further relates to the use of said construct according to the present invention in the manufacture of a medicament for the treatment or prevention of adisease. The disease may be cancer or an infection, preferably a virus infection. The disease may also be a haematological tumour or a solid tumour.

[0025] In a further aspect, the present invention relates to a method of treating a disease, comprising the step of administering a therapeutically effective amount of the construct according to the present invention and as described herein. The disease may be cancer or an infection, preferably a virus infection. The disease may also be a haematological tumour or a solid tumour. The therapeutically effective amount of the construct may be administered to the subject in need thereof parenteral, subcutaneous, intramuscular, intravenous, oral, intraperitoneal or intranasal.

[0026] In another aspect, the present invention relates to a method of inducing an immune response, comprising the step of administering a therapeutically effective amount of the construct according to the present invention and as described herein. The immune response may be induced by activating the retinoic acid-inducible gene I (RIG-1). Alternatively or additionally, the immune response may be induced by stimulating retinoic acid-inducible gene I (RIG-1). Said construct may have or comprise an immune adjuvant function. The therapeutically effective amount of the construct may be administered to the subject in need thereof parenteral, subcutaneous, intramuscular, intravenous, oral, intraperitoneal or intranasal.

[0027] In a further aspect, the present invention relates to a kit for use in medicine comprising the construct according to the present invention and as described herein. The kit may be a diagnostic kit for selecting a patient for treatment of a disease. Preferably, the disease is an infection or cancer.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings described in the following.

[0029] Figure 1 shows a schematic representation of the construct according to the present invention, depicting the structure of both the mRNA-strand encoding an immunogen and the dsRNA of the RIG-l-ligand capable of activating RIG-I for use as a vaccine. In the embodiment depicted in Figure 1 , four RIG-l-ligands are hybridized to the mRNA-strand.

[0030] Figure 2 shows a schematic representation of the RIG-l-ligand depicted in Figure 1 . This construct comprises three elements in the embodiment depicted: The nucleic acid sequence being capable of binding to RIG-I comprises in this embodiment a blunt-ended dsRNA with a triphosphate moiety attached to the 5’-end of one strand of the dsRNA, a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand, and a linker moiety that connects the two elements mentioned before.

[0031] Figure 3 shows that transfection of THP-1 Dual wt cells with Lipofectamine 2000 of synthetic double-stranded 5'-triphosphate RNA (3p-dsRNA, 250 ng / ml, SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4), DNA template hybridized with four equimolar 5'- triphosphate complementary RIG-l-ligands (I, ppp-Ribo-CRL, 250 ng / ml) and DNA template hybridized with four equimolar 5'-triphosphate modified complementary RIG-l-ligands (II, ppp- mod-CRL, 250 ng / ml) stimulates type I interferon expression as quantified via luciferase reporter activity. For each construct, n = 2 and the reported activity is shown 18 hours after construct application, as well as a schematic representation of the DNA templates (SEQ ID NO: 5) hybridized with ppp-Ribo-CRL (I, SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 66) and ppp-mod-CRL (II, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34).

[0032] Figure 4 shows the dose-dependent induction of type I interferon responses by RIG- l-ligands conjugated to a DNA template. Levels of ISRE reporter activity are shown following transfection of THP-1 Dual wt cells with Lipofectamine 2000 of a DNA template (SEQ ID NO: 5) with no RIG-l-ligand hybridized (grey), or hybridized with one (grey with black border), two (light grey), three (light grey with black border), or four (black and grey horizontally striped) RIG-l-ligands (SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34) to the template (3.9 ng / mL). Also shown is the response to a classical RIG-l-agonist, 3p-dsRNA (black, 3.9 ng / mL, SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4). For each construct, n = 2 and reported activity is shown 18 hours after construct application.

[0033] Figure 5 shows that responses to construct application are RIG-l-dependent when applied hybridized to mRNA, whilst translation of mRNA is only minimal affected. 3p-dsRNA (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4), mRNA (in vitro transcribed based on SEQ ID NO: 36), or mRNA hybridized to four equimolar ppp-mod-CRL ligands (SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34) were applied to either wild-type or RIG-l< / _)THP1 cells. The response was quantified by measurement of CXCL10 levels in the supernatant via ELISA and levels of mRNA translation as quantified by Firefly Luciferase (Flue) expression. Whilst CXCL10 levels are highly reduced in RIG-l< / _)THP1 cells, translation of mRNA is unaffected by either RIG-I knockout or by hybridization with ppp-mod-CRL-ligands. Cells have been transfected with 1000 ng / mL of the respective construct formulated with Lipofectamine MessengerMax. For each construct, n = 3 and reported activity is shown 18 hours after construct application.

[0034] Figure 6 shows the effect of increasing the number of RIG-l-ligands on IFN-I induction and mRNA translation. In both cases, mRNA (in vitro transcribed based on SEQ ID NO: 35) was applied with no RIG-l-ligand hybridized (grey), or hybridized with one (grey with black border), two (light grey), three (light grey with black border), or four (black and grey horizontallystriped) RIG-l-ligands (see SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34) hybridized to the template. Levels of IFN expression are increased by increasing the number of RIG-l- ligands, whilst levels of translation of the mRNA construct are decreased. Despite this decrease, mRNA translation levels remain high, even when four RIG-l-ligands are conjugated to the mRNA-strand. For each construct, 1000 ng / mL has been transfected with Lipofectamine MessengerMax, n = 3 and all quantification is performed 20 hours post-application of the constructs.

[0035] Figure 7 shows the level of IFN-I expression following application of synthetic doublestranded, blunt-ended, 5'-triphosphate RNA (ppp-dsRNA) with a double-stranded portion of either 11 nt (Figure 7A and 7B) or 24 nt (Figure 7C and 7D) in length. For each length, the constructs were applied alone (Ribo-CRL: SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 66, Ribo-24nt-CRL: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively) or hybridized with a DNA template (SEQ ID NO: 67, 2er DNA-Template + Ribo-CRL, 2er DNA-Template + Ribo-24nt-CRL, respectively). A control ligand, 3p-dsRNA (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4), was also applied. Diagrammatic representations of each construct are also depicted. For each length of the dsRNA, conjugation with DNA increases IFN expression. IFN expression is considerably greater for 24 nt ppp-dsRNA versus 11 nt ppp- dsRNA. For each construct, 1000 ng / mL has been transfected with Lipofectamine 2000 to THP-1 Dual cells, n = 2 and reported activity is shown 18 hours after construct application.

[0036] Figure 8 shows the effect of different linker moieties L on interferon induction by 24 nt long ppp-dsRNA of the nucleic acid sequence being capable of binding to RIG-I. In each RIG- l-ligand, the triphosphate group is joined to one strand of the dsRNA that is not attached to the linker moiety L. THP-1. Dual cells were transfected with Lipofectamine 2000 with 1000 ng / mL with a 24 nt long dsRNA with different linker moieties L (marked in this Figure as X, namely 10 uracil-residues (10 U, SEQ ID NO: 16 + SEQ ID NO: 17), 2 uracil-residues (2 U, SEQ ID NO: 18 + SEQ ID NO:19) and Spacer 18 (Sp18, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22), which has the chemical formulaeither individually(see I) or when hybridized to a DNA template (see II, SEQ ID NO: 5). The IFN-inducing effect is the same for both hybridized and non-hybridized ligands and is not greatly affected by the linker moiety L. For each construct, n = 2 and reported activity is shown 18 hours after construct application.

[0037] Figure 9 shows the comparison of the effect of different RIG-l-ligands on interferon induction, with both 24 nt (Tockary-24mer RNA-Sp18, see SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8) and 11 nt long ppp-dsRNA (Tockary-11mer RNA-Sp18: see SEQ ID NO: 13,SEQ ID NO: 14 and SEQ ID NO: 15; CRL rev-Sp18: SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22) applied. In each ligand, the triphosphate group is joined to the strand of the dsRNA that is not attached to the linker moiety L. As linker moiety L, Spaced 8 as defined above for Figure 8 were used. As with Figure 7, THP-1 Dual cells were transfected with Lipofectamine 2000 with 1000 ng / mL RIG-l-ligands either individually (see I) or when hybridized to a DNA template (see II, SEQ ID NO: 5). Comparable to Figure 8, hybridization of the 24 nt long dsRNA to the DNA template does not alter IFN-induction, whereas hybridization of the 11 nt long dsRNAs to the DNA template results in higher levels of IFN- induction. For each construct, n = 2 and reported activity is shown 18 hours after construct application.

[0038] Figure 10 shows the potency of different 11 nt long RIG-l-ligands hybridized to DNA as quantified by their capacity to induce interferon expression. Except for the ligand with Sp18 (CRL mod), in each ligand the triphosphate group is joined to the strand of the dsRNA that is not attached to the linker moiety L. THP-1 Dual cells were transfected with Lipofectamine 2000 with 1000 ng / mL of different 11 nt ppp-dsRNA either individually (see I) or when hybridized to a DNA template (see II, SEQ ID NO: 5). Once again, DNA-hybridized RIG-l-ligands display a higher potency than the same ligand when applied individually. For each construct, n = 2 and reported activity is shown 18 hours after construct application. The definitions of the expressions and the respective RNA sequences used in Figure 10 as well as the chemical formula of “SP18”, “SP9”and “SpC3” are given herein in Tables 2 and 5 below.

[0039] Figure 11 shows examples of linker moieties L with 18, 9 and 4 main chain atoms (here carbon and oxygen atoms) and the respective numbering of main chain atoms as defined herein.

[0040] Figure 12 shows the surface expression of A) CD86 (see Figure 12A), B) CD69 (see Figure 12B), C) MHC I (see Figure 12C) and D) MHC II on murine BMDCs after transfection with model mRNAs ± CRL (see Figure 12D). BMDCs were transfected with different mRNA constructs and controls (1000 ng / mL) in complex with Lipofectamine MessengerMax. Surface markers were stained 18 h after transfection and detected with flow cytometry on CD11b+ CD11c+ cells. E) The level of secreted IFNa was analyzed in the supernatant (see Figure 12E). The results represent six experiments and mean ± SEM is shown. *p < 0.05, paired t- test. CRL corresponds to CRL-mod (pppCGGCGAGUmUmAmG (see SEQ ID NO: 32) X fGfAfUfUfGfAfGfCfGfGmC (see SEQ ID NO: 33) + mCmUmAACUCGCCG (see SEQ ID NO: 34)). pppCGGCGAGUmUmAmG with comments to modifications corresponds to SEQ ID NO: 62 in the sequence listing (“m” means a 2'-O-methyl-modification and “f” means a 2'-fluoro- modification), fGfAfUfUfGfAfGfCfGfGmC with comments to modifications corresponds to SEQ ID NO: 63 in the sequence listing (“m” means a 2'-O-methyl-modification and “f’ means a 2 - fluoro-modification), pppCGGCGAGUmUmAmGXfGfAfUfUfGfAfGfCfGfGmC with commentsto modifications and spacer corresponds to SEQ ID NO: 64 in the sequence listing (“m” means a 2'-O-methyl-modification, “f” means a 2'-fluoro-modification and “X” means Spacer) and CmllmAmACUCGCCG with comments to modifications corresponds to SEQ ID NO: 65 in the sequence listing (“m” means a 2'-O-methyl-modification) (see Table 4). As controls, the CRL without mRNA, as well as IVT4, as known RIG-l-ligand (SEQ ID NO: 52) has been transfected. mRNAs used in this example corresponds to in vitro transcribed mRNAs based on the EGFP- construct (SEQ ID NO: 45), EGFP-Fluc-construct (SEQ ID NO: 46), mCherry-construct (SEQ ID NO: 47) and mCherry-Fluc construct (SEQ ID NO: 49).

[0041] Figure 13 shows the percentage of A) EGFP expressing (see Figure 13A) and B) mCherry expressing CD11 b+ CD11c+ cells after transfection of murine BMDCs with model mRNAs ± CRL (see Figure 13B). BMDCs were transfected with different mRNA constructs (1000 ng / mL) in complex with Lipofectamine MessengerMax. 18 h after transfection, BMDCs have been analyzed with flow cytometry. The results represent six experiments and mean ± SEM is shown. CRL corresponds to CRL-mod (pppCGGCGAGUmUmAmG (see SEQ ID NO: 32) X fGfAfUfUfGfAfGfCfGfGmC (see SEQ ID NO: 33) + CmUmAm ACUCGCCG (see SEQ ID NO: 34). pppCGGCGAGUmUmAmG with comments to modifications corresponds to SEQ ID NO: 62 in the sequence listing (“m” means a 2'-O-methyl-modification and “f’ means a 2 - fluoro-modification), fGfAfUfUfGfAfGfCfGfGmC with comments to modifications corresponds to SEQ ID NO: 63 in the sequence listing (“m” means a 2'-O-methyl-modification and “f” means a 2'-fluoro-modification), pppCGGCGAGUmUmAmG X fGfAfUfUfGfAfGfCfGfGmC with comments to modifications and spacer corresponds to SEQ ID NO: 64 in the sequence listing (“m” means a 2'-O-methyl-modification and “f” means a 2'-fluoro-modification) and CmUmAmACUCGCCG with comments to modifications corresponds to SEQ ID NO: 65 in the sequence listing (“m” means a 2'-O-methyl-modification and “f” means a 2'-fluoro- modification) (see Table 4). mRNAs used in this example corresponds to in vitro transcribed mRNAs based on the EGFP-construct (SEQ ID NO: 45), EGFP-Fluc-construct (SEQ ID NO: 46), mCherry-construct (SEQ ID NO: 47) and mCherry-Fluc construct (SEQ ID NO: 49).

[0042] Figure 14 shows the surface expression of A) CD86 (see Figure 14A), B) CD69 (see Figure 14B), C) MHC I (see Figure 14C) and D) MHC II on murine BMDCs after transfection with MCMV protein coding mRNAs ± CRL (see Figure 14D). BMDCs were transfected with different mRNA constructs and controls (1000 ng / mL) in complex with Lipofectamine 2000. Surface markers were stained 18 h after transfection and detected with flow cytometry on CD11 b+ CD11c+ cells. E) The level of secreted IFNa was analyzed in the supernatant (see Figure 14E). The results represent four experiments and mean ± SEM is shown. *p < 0.05, paired t-test. CRL corresponds to pppSO-11-3F-hsf (SEQ ID NO: 50) + SO-3f-2xDNA (SEQ ID NO: 51). As controls, HT-DNA, IVT4 (SEQ ID NO: 52), as well as CA21 (SEQ ID NO: 53) has been transfected. mRNAs used in this example corresponds to in vitro transcribed mRNAsbased on the gO-construct (SEQ ID NO: 54), gL-construct (SEQ ID NO: 56), gH-construct (SEQ ID NO: 58) and IE1 construct (SEQ ID NO: 60).DETAILED DESCRIPTION OF THE INVENTION

[0043] As described herein, the present invention relates to a construct comprising a mRNA- strand, and at least one, preferably at least two, retinoic acid-inducible gene I (RIG-l)-ligand(s) comprising a) a nucleic acid sequence being capable of binding to RIG-1, b) a linker moiety L comprising 3 to 24 main chain atoms, preferably comprising 6 to 18 main chain atoms, and c) a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand.

[0044] The RIG-l-ligand may be hybridized to the mRNA-strand and may act as a vaccine adjuvant in an mRNA vaccine. This construct may be used in a method of vaccination that results in the defined and predictable induction of an innate immune response.

[0045] mRNA vaccines

[0046] mRNA vaccines consist of mRNA molecules that encode a desired antigenic protein that is capable of stimulating an immunogenic reaction, initiating a process that ends with the generation of acquired immunity and therefore protection against future infection of the relevant pathogen(s). This is achieved through the local cytoplasmic translation of vaccine mRNA to produce the antigenic protein(s), which is typically then incorporated into the cell membrane and / or secreted.

[0047] In mRNA vaccines, the mRNA may be constructed so as to guarantee optimal translation of the antigen. This requires the insertion of a cap structure (N7-methyl-guanosine 5’-5’-linked to the first nucleotide of the mRNA) at the 5’-end.

[0048] For example, when administered intramuscularly, mRNA vaccines typically lead to adaptive immune system activation via initial transfection of muscle cells, epidermal cells, and tissue-resident antigen presenting cells, e.g. dendritic cells and macrophages, thereby initiating priming of T and B cells. Later, vaccine particles will typically move from the injection site to secondary lymphoid tissue via lymphatic drainage. Transfection of lymphoid tissue antigen presenting cells initiates priming and activation of both T and B cells, contributing to generation of adaptive immunity. This adaptive immunity is mediated by, amongst other cell types, memory B cells, plasma cells, and T helper cells.

[0049] In addition to adaptive immune responses, innate immune activation is key to successful immunization following administration of an mRNA vaccine. Non-self RNA is a potent activator of the innate immune system, with host RNA undergoing extensive intracellular modification to prevent innate immune recognition by clearly differentiating self and non-self RNA. This property means that RNA can serve as both an indirect immunogen by way of protein expression, as described herein, and an adjuvant in the composition of vaccines.

[0050] As used herein, "inducing an immune response" may mean initiating or causing an increase in one or more of B-cell activation, T-cell activation, natural killer cell activation, activation of antigen presenting cells (e.g., B cells, dendritic cells, monocytes and macrophages), cytokine production, chemokine production, specific cell surface marker expression, in particular, expression of co-stimulatory molecules. In one aspect, such an immune response may involve the production of type I IFN, in particular IFN-a, in cells such as PDC (plasmacytoid dendritic cells) and / or monocytes.

[0051] Vaccine adjuvants

[0052] Adjuvant activity requires activation of the innate immune system, acting to enhance vaccine immunogenicity and degree of protection conferred by vaccination. Currently approved mRNA vaccines are believed to activate the innate immune system by virtue of the presence of contaminant RNA particles that are a by-product of the method of production. Although these contaminants are able to activate the innate immune system and thus act as adjuvants, the lack of control over their quantity, quality, and method of action means that mRNA vaccines will benefit from the invention of defined adjuvants that may be coadministered. Such adjuvants may be adjusted in strength and / or mechanism of immune activation to best meet the needs of a given mRNA vaccine construct.

[0053] The present invention provides a construct, which is able to activate RIG-I, an innate immune receptor involved in innate immune recognition of RNA viruses, and can be used as an mRNA vaccine adjuvant.

[0054] A vaccine adjuvant may mean any material, which is incorporated with a vaccine for the purpose of enhancing the immune response of the vaccine. This is typically an innate immune response. This can be a contaminant from the manufacturing process, such as bacterial lipopolysaccharide, inorganic compounds such as aluminium salts, or defined immune stimulants, such as cytokines, e.g. interleukin-1. In many cases, adjuvants function by mimicking molecules that are termed pathogen-associated molecular patterns (PAMPs), evolutionarily conserved molecular motifs that act to indicate that a cell or molecule is non-self and must therefore evoke an immune response.

[0055] mRNA vaccine adjuvants

[0056] Initial activation of the innate immune system is a necessary step in mRNA-vaccine elicited adaptive immunity. Hence, mRNA vaccines are typically dependent upon vaccine adjuvants for their functionality. RNA can elicit innate immune activation via various endosomal and cytosolic immune receptors. For example, non-host RNA binds and activates, amongst others, toll-like receptor s (TLR3) and 7 (TLR7), melanoma differentiation-associated protein 5 (MDA5), and retinoic acid-inducible gene I (RIG-1). Activation of these receptors drives type I interferon (IFN I) responses and plays a key role in innate antiviral responses.

[0057] Due to the potential adverse effects that systemic innate immune activation could drive, many mRNA vaccines have been modified to attempt to evade innate immune recognition of the antigen-encoding mRNA. For example, replacement of uridine with naturally occurring uridine-derivatives, such as pseudo-uridine, which is part of the processing that allows host mRNA to avoid stimulating innate immune receptors, or addition of a 2’-O-methyl-group onto the first nucleotide to prevent binding of RIG-1.

[0058] Despite steps taken to avoid cytoplasmic RNA sensing when preparing mRNA for use in vaccine compositions, it is likely that mRNA-vaccines stimulate the innate immune system, in part, via unintended nucleic acid contaminants generated during the manufacturing process, e.g. RNA by-products. This draws a parallel to the presence of minute quantities of microbial contaminants that act as adjuvants in protein-antigen-based vaccines, a now well- characterized and necessary element of vaccine.

[0059] The exact nature of the adjuvant-contaminants that allow for successful immunization via mRNA vaccines remains unclear, as few studies have studied the activation of the innate immune system in vivo following vaccination with approved mRNA vaccines. However, it is clear that a robust antiviral and IFN-I response is elicited just one day post-vacci nation, providing a clear indication that the mRNA vaccine triggers direct innate immune activation.

[0060] Despite the efficacy of said mRNA vaccines, it is readily apparent that reliance on undefined, non-controlled contaminants to elicit a key step in vaccine-induced immunity is undesirable. Future mRNA-based vaccines would therefore benefit from two elements - improved purification and / or synthesis to limit contaminants from RNA manufacturing processes and the creation of well-defined, controlled adjuvants to fulfil the role of removed contaminants that can be adapted in both strength and nature to best suit the needs of the vaccine in question.

[0061] Construct

[0062] It is to this end that the construct of the present invention has been designed, providing a construct capable of binding and activating retinoic acid-inducible gene I (RIG-I) and may serve as or part of an mRNA vaccine.

[0063] Thus, in a first aspect, the present invention relates to a construct comprising- a mRNA-strand, and- at least one, preferably at least two, retinoic acid-inducible gene I (RIG-l)-ligand(s) comprising a) a nucleic acid sequence being capable of binding to RIG-1, b) a linker moiety L comprising 3 to 24 main chain atoms, preferably comprising 6 to 18 main chain atoms, and c) a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand.

[0064] It is preferred that the construct of the present invention is a nucleic acid-construct. It is more preferred that the construct of the present invention is an engineered nucleic acidconstruct. The term “engineered” as used herein may mean that such a construct was artificially made and does not occur as such in nature. It is further preferred that the construct of the present invention comprises at least two of the retinoic acid-inducible gene I (RIG-I)- ligand(s), more preferred that the construct of the present invention comprises two to four of the retinoic acid-inducible gene I (RIG-l)-ligand(s) and even more preferred that the construct comprises four of the retinoic acid-inducible gene I (RIG-l)-ligand(s) as described herein.

[0065] mRNA strand

[0066] Messenger RNA (mRNA) is a single-stranded RNA molecule that results from transcription of DNA by RNA polymerase. The initial mRNA transcript is then processed to produce a mature mRNA molecule that may be read by the ribosome to produce a polypeptide (protein).

[0067] It is preferred that the mRNA-strand comprises a coding sequence of a protein, a peptide or an antigen, preferably an antigen that is able to induce an immune response or preferably a cancer antigen. Thus, the mRNA is able to encode (a) relevant antigen(s) via its coding sequence. The coding sequence is defined herein as the sequence of mRNA that encodes a protein or polypeptide.

[0068] The term “protein” is equally used herein with the term "polypeptide". Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids). The term "polypeptide" as used herein describes a group of molecules, which, for example, consist of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homoor heterotrimers etc. The terms "polypeptide" and "protein" may also refer to naturally modified polypeptides / proteins, wherein the modification is affected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.

[0069] As used herein and in the context of the present invention, the term “peptide” means a compound consisting of two or more amino acids linked in a chain, the carboxyl group of each acid being joined to the amino group of the next by a bond of the type -OC-NH-.

[0070] As used herein and in the context of the present invention, the term “antigen” means a substance that can stimulate the immune system to produce a set of specific antibodies and that combines with the antibody through a specific binding site or epitope.

[0071] An antigen that is able to induce an immune response as used herein and in the context of the present invention may mean an antigen capable of inducing an immune response by activating retinoic acid-inducible gene I (RIG-I).

[0072] A cancer antigen as used herein and in the context of the present invention may mean an antigen produced by a tumour cell. Cancer antigens are therefore useable as biomarkers and may be treatment targets for immunotherapy-based cancer treatment.

[0073] Expression of the mRNA may result in expression of said antigen(s) in transfected cells, with subsequent cell surface externalization and / or secretion resulting in recognition of said antigen by appropriate immune cells.

[0074] In some embodiments, the mRNA may be non-replicating and may simply encode the required antigen(s). In other embodiments, the mRNA may also encode viral replication machinery that results in intracellular amplification of said mRNA, thereby amplifying antigen expression.

[0075] In the present invention, the mRNA itself may typically have no intrinsic immune- stimulatory activity in the absence of the conjugated complementary RIG-I ligand(s). The inventors of the present invention demonstrate this in Figure 12 and Figure 14, which both show that an mRNA, when applied in the absence of hybridized RIG-l-ligands, induces little to no expression of markers of immune activation.

[0076] It is preferred that the single-stranded nucleic acid sequence of the at least one RIG- l-ligand(s) is complementary to at least one nucleic acid sequence comprised in the mRNA strand. In some embodiments of the present invention, the mRNA-strand, which may be used synonymously to “(the) mRNA” herein, may contain one or more untranslated regions (UTR). This is a region of the mRNA transcript that is not translated into protein. Such a region may be before the coding region (5’-UTR) or after it (3’-UTR). Both, the 5’-UTR and the 3’-UTR, may serve as the site of hybridization between the mRNA-strand and the single stranded nucleic acid sequence of the RIG-l-ligand. Thus, it is preferred that the single-stranded nucleic acid sequence is capable of hybridizing to at least one nucleic acid sequence located in a non-coding nucleic acid sequence of the mRNA-strand, preferably the 5'-UTR or 3'-UTR region of the mRNA-strand.

[0077] It is further preferred that the non-coding nucleic acid sequence of the mRNA-strand is or comprises a 3'-untranslated region (UTR)-nucleic acid sequence or a 5'-untranslated region (UTR)-nucleic acid sequence or a poly-A-tail.

[0078] As used herein, a poly-A tail refers to a region of mRNA at the 3’-end of an mRNA sequence that exclusively contains adenine ribonucleotides. The region is non-coding, but controls mRNA processing and export.

[0079] As used herein, the term “non-coding nucleic acid sequence” refers to a region of DNA or RNA that is not translated during protein translation.

[0080] Non-coding regions of mRNA are often found between the 5’-end of the mRNA strand and the start codon of the coding sequence, termed the 5’-untranslated region (5’-UTR), or between the stop codon of the coding sequence and the 3’-end of the mRNA, termed the 3’- untranslated region (3’-UTR).

[0081] It is preferred that the mRNA-strand comprises such a 3'-untranslated region (UTR)- nucleic acid sequence and that the single-stranded nucleic acid sequence is capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand comprised in said 3'- untranslated region (UTR)-nucleic acid sequence. Further, it is preferred that the mRNA- strand comprises a 5'-untranslated region (UTR)-nucleic acid sequence and that the singlestranded nucleic acid sequence is capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand comprised in said 5'-untranslated region (UTR)-nucleic acid sequence. More preferably, the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s) is capable of hybridizing to the at least one nucleic acid sequence comprised in the 3'- or 5'- untranslated region (UTR)-nucleic acid sequence of the mRNA-strand via Watson-Crick-base- pairing.

[0082] The inventors of the present invention demonstrate in Figure 6B that the hybridization of RIG-I ligand(s) does not prevent the successful translation of the encoded protein, although expression levels may drop as a function of the number of hybridized ligands. However, even with four ligands hybridized, expression levels of the encoded protein can remain high, at about 50% of levels achieved when mRNA is transfected in isolation.

[0083] Retinoic acid-inducible gene (RIG-I) ligand

[0084] “Retinoic acid-inducible gene (RIG-I)” is an innate immune surveillance protein and member of the pattern recognition receptor family, which detects the intracellular presence of viral genomic RNA, both single- and double-stranded, via common molecular motifs found in viral genomes. Activation of RIG-I is an important step in innate immune activation in response to the presence of certain viruses. RIG-I activation initiates downstream signalling pathwaysthat ultimately trigger expression and release of, amongst other immune mediators, interferon(s). Interferon release triggers further innate immune activation in proximal cells, allowing them to prepare an antiviral response.

[0085] RIG-1 activation is a promising mechanism by which the innate immune system can be stimulated. Thus, the inventors of the present invention have found a construct comprising at least one, preferably at least two, ligand(s) for RIG-1 activation, which can serve as an adjuvant in mRNA vaccine compositions. The innate immune activation, which can be induced by the present invention, is demonstrated to be RIG-l-dependent in Figure 5.

[0086] “RIG-1 activation”, as used herein and in the context of the present invention, may comprise the binding of RIG-1 to a suitable ligand, typically viral genomic RNA, the subsequent displacement of the inhibitory caspase activation and recruitment domains (CARDs) of RIG-1, the oligomerization of multiple RNA-bound RIG-1 monomers, and the interaction between oligomerized RIG-1 CARD domains and mitochondrial antiviral-signalling protein (MAVS), which induces gene expression, most importantly expression of type I IFNs, via kinase-based signalling pathways.

[0087] A “RIG-l-ligand” is hereby defined as a substance that is capable of inducing RIG-I- activation, as defined herein.

[0088] It is intended that the present invention is not limited with regard to defining likely signalling pathways by which the present application will act as an adjuvant via RIG-I- activation.

[0089] Nucleic acid sequence

[0090] For the construct of the present invention, the at least one RIG-1 ligand(s) comprises a nucleic acid sequence being capable of binding to RIG-1. That nucleic acid sequence may be a polynucleotide, however structured. In some embodiments of the present invention, the nucleic acid sequence may be a nucleic acid sequence that is capable of binding to and activating RIG-1.

[0091] In some embodiments, the nucleic acid sequence capable of binding to RIG-I may comprise or may consist of a ribonucleic acid (RNA) sequence. This sequence may, in preferred embodiments, be a double-stranded RNA (dsRNA). Thus, the nucleic acid sequence being able to bind to RIG-I may comprise or consist of a double-stranded RNA.

[0092] Double-stranded RNA

[0093] As used herein, the term “double-stranded RNA” (dsRNA) refers to two strands of ribonucleotides whose bases are joined by hydrogen bonding, typically via Watson-Crick base pairing rules. The double-stranded RNA may be self-complementary.

[0094] The inventors of the present invention have found out that the present invention is independent from the specific sequence of the double-stranded RNA.

[0095] In certain embodiments of the present invention, said dsRNA of the nucleic acid sequence being capable of binding to RIG-1 has a length in the range of 8 to 20 nt. Preferably, the double-stranded RNA may have a length in the range of 10 to 17 nt. More preferably, the double-stranded RNA may have a length in the range of 10 to 15 nt, more preferably a length in the range of 10 to 15 nt, and more preferably a length in the range of 10 to 13 nt, and even more preferably a length of 11 nt.

[0096] Said double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I may be a blunt-ended double-stranded RNA. Alternatively, the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I may comprise an overhang. Such an overhang may be a RNA-overhang or an overhang with a fluorophore or a lipophilic substitution.

[0097] The nucleic acid sequence being capable of binding to RIG-I may further comprise at least one triphosphate. A triphosphate group, also named as “ppp” here within, refers to three phosphate groups (PO43') sequentially bound to the ribose sugar of a nucleotide molecule. In case that nucleic acid sequence comprises or consists of a dsRNA, that dsRNA may comprise at least one triphosphate at at least one strand of said dsRNA, preferably at least one triphosphate at the 5'-end of one strand of said dsRNA.

[0098] In preferred embodiments, the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises one triphosphate at the 5'-end of one strand of said double-stranded RNA.

[0099] Modification(s)

[0100] According to the present invention, the construct as described herein may comprise at least one modification(s). Specifically, the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I may comprise at least one modification(s). The at least one modification(s) may be selected from the group consisting of O-methyl-group-modification(s), preferably 2'-O-methyl-group-modification(s), fluoro-group-modification(s), preferably 2 - fluoro-group-modification(s), phosphoro-thio-modification(s), deoxy-modifications and locked nucleic acid (LNA)-modification(s).

[0101] Preferably, the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at at least one strand of the double-stranded RNA the at least one modification(s), more preferably at both strands of the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I.

[0102] It is also preferred that the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I further comprises at least one triphosphate at one end of at leastone strand of said double-stranded RNA and the at least one modification(s) is / are at the other end of the at least one strand of said double-stranded RNA.

[0103] Preferably, the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at its 3'-end and / or at its 5'-end of at least one strand of the doublestranded RNA the at least one modification(s), preferably at both strands of the doublestranded RNA of the nucleic acid sequence being capable of binding to RIG-I.

[0104] The double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I may comprise at its 3'-end of at least one strand of the double-stranded RNA the at least one modification(s). Alternatively, the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I may comprise at its 5'-end of at least one strand of the double-stranded RNA the at least one modification(s).

[0105] It is also preferred that the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at its 5'-end and / or at its 3'-end at least one O-methyl- group-modification(s), more preferably at least one 2'-O-methyl-group-modification(s). More preferably, the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at both strands of the double-stranded RNA at least one O-methyl-group- modification(s).

[0106] Also, the mRNA-strand of the construct of the present invention may comprise at least one modification(s). That at least one modification(s) of the mRNA-strand may be (a) pseudo- uridine-modification(s).

[0107] For the at least one modification(s), it has to be considered that the construct of the present invention may be a ribonucleic acid (RNA) construct. Each nucleotide in RNA contains a ribose sugar, with carbons numbered T through 5'. A base is attached to the T-position, in general, adenine (A), cytosine (C), guanine (G), or uracil (II). Adenine and guanine are purines, cytosine and uracil are pyrimidines. A phosphate group is attached to the 3'-position of one ribose and the 5'-position of the next. The phosphate groups have a negative charge each, making RNA a charged molecule (polyanion). The bases form hydrogen bonds between cytosine and guanine, between adenine and uracil and between guanine and uracil.

[0108] The construct of the present invention may be built of oligonucleotides which may be short, single- or double-stranded RNA molecules, and may include antisense oligonucleotides (ASO), RNA interference (RNAi), and aptamer RNAs.

[0109] As used herein, the term "oligonucleotide" refers to a polynucleotide formed from a plurality of linked nucleoside units. The terms "oligonucleotide" and "polynucleotide" are used synonymously. Such oligonucleotides can be obtained from existing nucleic acid sources, including genomic or cDNA, but are preferably produced by synthetic methods including chemical synthesis, in vitro and in vivo transcription. The nucleoside residues can be coupled to each other by any of the numerous known internucleoside linkages. Such internucleosidelinkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, pyrophosphate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. The term "oligonucleotide" may also encompass polynucleosides having one or more stereospecific internucleoside linkage (e.g., (Rp)- or (Sp)-phosphorothioate, alkylphosphonate, or phosphotriester linkages).

[0110] The oligonucleotides comprised in the construct of the present invention may include naturally occurring nucleosides, modified nucleosides, or mixtures thereof. As used herein, the term "modified nucleoside" is a nucleoside that includes a modified heterocyclic base, a modified sugar moiety, or a combination thereof. In some embodiments, the modified nucleoside is a non-natural pyrimidine or purine nucleoside. In some embodiments, the modified nucleoside is a 2'-substituted ribonucleoside, an arabino-nucleoside or a 2'-deoxy- 2'-substituted-arabinoside.

[0111] The term “pseudo-uridine-modification(s)”, as used herein, may mean that, for example, uridine is substituted by pseudouridine. Pseudouridine is the most frequently naturally occurring RNA modification, found in all classes of biologically functional RNAs. Compared to uridine, pseudouridine contains an additional hydrogen bond donor group and is therefore widely regarded as a structure stabilizing modification. The effects of replacing specific uridines with pseudouridines on RNA dynamics crucially depend on the exact location of the replacement site and can range from destabilizing to locally or even globally stabilizing. By using a combination of NMR spectroscopy, MD simulations and QM calculations, one can rationalize the observed effects on a structural and dynamical level.

[0112] The term “O-methyl-group-modification(s)”, as used herein, may mean that the construct is modified such that it comprises one or more O-methyl-group(s). Preferably, it may be substituted to comprise a 2'-O-methyl-group. 2'-O-methylation is a common nucleoside modification of RNA, where a methyl group is added to the 2'-hydroxyl of the ribose moiety of a nucleoside, producing a methoxy-group.

[0113] The term “fluoro-group-modification(s)”, as used herein, means that the construct of the present invention is modified such that it comprises one or more fluoro-group(s).

[0114] The term “phosphoro-thio-modification(s)”, as used herein, means that the construct of the present invention is modified such that it comprises one or more phosphoro-thio- group(s). Replacing a non-bridging oxygen in the phosphodiester linkage of RNA by sulfur results in phosphoro-thio-modified RNA. Phosphoro-thio-modification of mRNA enhances translation efficiency and thus provides a further strategy to improve mRNA functionality.

[0115] A locked nucleic acid (LNA), also known as bridged nucleic acid (BNA), and often referred to as inaccessible RNA, is a modified RNA nucleotide in which the ribose moiety ismodified with an extra bridge connecting the 2'-oxygen and 4'-carbon. The bridge "locks" the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. This structure provides for increased stability against enzymatic degradation. LNA also offers improved specificity and affinity in base-pairing as a monomer or a constituent of an oligonucleotide. LNA nucleotides can be mixed with DNA or RNA residues in an oligonucleotide.

[0116] As used herein, the term "2'-substituted ribonucleoside" or "2'-substituted arabinoside" includes ribonucleosides or arabinonucleoside, in which the hydroxyl group at the 2' position of the pentose moiety is substituted to produce a 2'-substituted or 2'-O-substituted ribonucleoside. Preferably, such substitution is with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an aryl group having 6-10 carbon atoms, wherein such alkyl, or aryl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups. Examples of 2'-O-substituted ribonucleosides or 2'-O-substituted-arabinosides include, without limitation, 2'-O-methylribonucleosides or 2'-O-methylarabinosides and 2'-O- methoxyethylribonucleosides or 2'-O-methoxyethylarabinosides.

[0117] The term "2'-substituted ribonucleoside" or "2'-substituted arabinoside" also includes ribonucleosides or arabinonucleosides, in which the 2'-hydroxyl group is replaced with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an amino or halo group. Examples of such 2'-substituted ribonucleosides or 2'-substituted arabinosides include, without limitation, 2'-amino, 2'-fluoro, 2'-allyl, and 2'-propargyl ribonucleosides or arabinosides.

[0118] The term "oligonucleotide" may also include hybrid and chimeric oligonucleotides. A "chimeric oligonucleotide" is an oligonucleotide having more than one type of internucleoside linkage. One preferred example of such a chimeric oligonucleotide is a chimeric oligonucleotide comprising a phosphorothioate, phosphodiester or phosphorodithioate region and non-ionic linkages such as alkylphosphonate or alkylphosphonothioate linkages (see e.g., U.S. Pat. Nos. 5,635,377 and 5,366,878).

[0119] A "hybrid oligonucleotide" is an oligonucleotide having more than one type of nucleoside. One preferred example of such a hybrid oligonucleotide comprises a ribonucleotide or 2'-substituted ribonucleotide region, and a deoxyribonucleotide region (see, e.g., U.S. Pat. Nos. 5,652,355, 6,346,614 and 6,143,881).

[0120] RNA oligonucleotides discussed herein may include otherwise unmodified RNA as well as RNA, which has been modified (e.g., to improve efficacy), and polymers of nucleoside surrogates.

[0121] Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably those which occur naturally in the human body. That may berare or unusual, but naturally occurring, RNAs as modified RNAs. Such rare or unusual RNAs, often termed modified RNAs (apparently, because these are typically, the result of a post- transcriptional modification) are within the term unmodified RNA, as used herein.

[0122] Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as modified "RNAs", they will, of course, because of the modification, include molecules which are not RNAs.

[0123] Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone.

[0124] All nucleic acid sequences listed herein are in the 5'- to 3'-direction unless otherwise indicated.

[0125] A single-stranded RNA oligonucleotide may contain self-complementary sequences and may form a hairpin, for example with the sequence 5'-GACCTAGCCTAAAACTAGGTC- 3' (SEQ ID NO: 38). The self-complementary sequence may be a palindromic sequence. For example, 5'-AAAGATCCGGATCAAAA-3' (SEQ ID NO: 39).

[0126] Linker moiety L

[0127] It is preferred for the construct of the present invention that the linker moiety L connects the nucleic acid sequence being capable of binding to RIG-I with the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s).

[0128] The term “linker moiety”, as used herein, means any component of the aforementioned construct who’s primary function comprises joining two or more constituent components of the construct together without serving any inherent signalling or pharmacological function perse.

[0129] That linker moiety L may be any linker moiety that provides enough flexibility for the construct of the present invention to induce an immune response and / or to induce RIG-I- activity. It may be a flexible linker moiety, which allows the nucleic acid sequence being capable of binding to RIG-I to be connected to the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s) to be able to activate RIG-I.

[0130] The linker moiety L comprises 3 to 24 main chain atoms, preferably 6 to 18 main chain atoms, more preferably 8 to 16 main chain atoms, even more preferably 10 to 14 main chain atoms and even more preferably 12 main chain atoms. The main chain atoms are carbon atoms that may be optionally replaced by one or more heteroatoms selected from the group consisting of N, O and S.

[0131] When used herein, the term “main chain atoms” refers to the atoms present in the backbone of the linker moiety L. That backbone may be build by carbon-atoms, but in case, where the carbon-atoms are replaced by O, N or S, those also count to the total number of main chain atoms.

[0132] In some embodiments, the linker moiety L is a nucleoside-linker. It is preferred when the linker moiety L is a nucleoside-linker that the nucleoside-linker has a length of at least 2 nucleosides. It is also preferred when the linker moiety L is a nucleoside-linker that the nucleoside-linker has a length of 2 to 10 nucleosides. It is further preferred when the linker moiety L is a nucleoside-linker that the nucleoside-linker is an adenosine-linker, preferably with at least 2 adenosines, more preferably with 2 adenosines.

[0133] It is also preferred for the linker moiety L that it is a hydrocarbon-based linker, preferably a hydrocarbon-based linker according to Formula IFormula I, wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7. In the case of Formula I, the carbon-atoms of the (C2-C3)-alkylene as well as the O-atom count to the total number of main chain atoms.

[0134] More preferably, the linker-moiety L is a polyalkylene-glycol-linker according to Formula IIwherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7. In the case of Formula II, the two carbon-atoms and the O-atom in the bracket of Formula II count to the total number of main chain atoms.

[0135] It is further preferred that the linker moiety L is a polyalkylene-glycol-linker according to Formula II and that n is 6. In this case, the number of the main chain atoms is 18.

[0136] It is further preferred that the linker moiety L is a polyethylene-glycol-linker, wherein the number of main chain atoms as defined herein is 18. An example for a linker moiety comprising 18 main chain atoms (here carbon and oxygen atoms) is Spaced 8 as used in the Examples presented herein. The chemical formula thereof and its numbering of main chain atoms as defined herein is displayed in Figure 11.

[0137] It is also preferred that the linker moiety L is a polyethylene-glycol-linker, wherein the number of main chain atoms as defined herein is 9. An example for a linker moiety comprising 9 main chain atoms (here carbon and oxygen atoms) is Spacer9 as used in the Examples presented herein. The chemical formula thereof and its numbering of main chain atoms as defined herein is displayed in Figure 11.

[0138] In one aspect, the linker moiety L is a linker as displayed in Figure 11 as SpacerC3. This linker moiety comprises 4 main chain atoms (here carbon and oxygen atoms) as defined herein and may therefore be also called Spacer4. The chemical formula thereof and its numbering of main chain atoms as defined herein is displayed in Figure 11.

[0139] The inventors of the present invention demonstrate the effect of different linker moieties on the capacity for the construct of the present invention to induce an IFN-I response in Figure 10. This demonstrates the need for linker optimization and indicates that flexibility is a desirable feature for said linkers, as the most flexible linkers correspond to the greatest degree of IFN-I activation.

[0140] Overhang

[0141] In certain embodiments, the nucleic acid capable of binding to RIG-I comprises an overhang. The term “overhang” is used interchangeably herein with the term “single-stranded overhang” and refers to a non-blunt ended sequence, as defined herein, in the construct.

[0142] In the present invention, an overhang may comprise either a single-stranded nucleic acid sequence at either of the termini of either strand of an otherwise double-stranded nucleic acid construct or may comprise a non-nucleic acid based overhang. For example, in certain embodiments this overhang may comprise a fluorophore or lipophilic substitution.

[0143] For example, the fluorophore used in the context of the present invention may be FAM, FITC, or any Atto Dye.

[0144] Further, a preferred lipophilic substitution used in the context of the present invention may be cholesterol, oleat or palmitat.

[0145] In alternative embodiments, the nucleic acid capable of binding to RIG-I does not contain an overhang and is thus a blunt-ended nucleic acid.

[0146] Blunt ended nucleic acid

[0147] A blunt ended nucleic acid, as used herein, refers to a double-stranded nucleic acid whose strands are equal in length. This is in direct contrast to a nucleic acid that contains an overhang, as defined herein.

[0148] In a preferred embodiment of the present invention, the dsRNA of the nucleic acid sequence capable of binding to RIG-I is a blunt ended double-stranded RNA.

[0149] Single-stranded nucleic acid sequence

[0150] As discussed above, the dsRNA of the nucleic acid sequence capable of binding to RIG-I may be joined directly to a mRNA-strand by virtue of a single-stranded nucleic acid sequence that may be joined to the dsRNA moiety via the linker moiety L as defined herein. This single-stranded nucleic acid sequence may be complementary to a nucleic acid sequence present in the mRNA-strand and will thus mediate hybridization between the dsRNA and the mRNA-strand. This may occur via Watson-Crick base pairing, i.e. the formation of hydrogen bonds between nucleoside bases such that adenine pairs with uracil / thymine and guanosine with cytosine. In certain embodiments, the aforementioned site of hybridization may be found in the 5’-UTR or the 3-’UTR of the mRNA-strand.

[0151] Preferably, the single-stranded nucleic acid sequence of the at least one RIG-I- ligand(s) has a length in the range of 5 to 15 nt, more preferably a length in the range of 8 to 13 nt.

[0152] As used herein, a poly-A tail refers to a region of mRNA at the 3’-end of an mRNA sequence that exclusively contains adenine ribonucleotides. The region is non-coding, but controls mRNA processing and export.

[0153] Transfection reagent

[0154] The construct of the present invention may be used without the need to apply a transfection reagent.

[0155] The term “transfection reagent” as used herein may mean a reagent that is used to introduce naked or purified nucleic acids into, e.g. eukaryotic cells. These reagents may include plasmids, reagents, kits, primer sets, and more.

[0156] Medical uses

[0157] In a further aspect of the present invention, the construct according to the present invention is for use as a medicament.

[0158] In a further aspect of the present invention, the construct according to the present invention is for use in a method of treatment or prevention of a disease.

[0159] That disease may be cancer or an infection, preferably a virus infection. Thus, the construct of the present invention may be for use in a method of treatment or prevention of a virus or viral infection.

[0160] The terms “treat” and “prevent” as well as words stemming therefrom, like “treatment” or “prevention”, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods of the present invention can provide any amount of any level of treatment or prevention of cancer in a mammal. Furthermore, the treatment or prevention provided by the method of the present invention can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention can include promoting the regression of a tumour. Also, for purposes herein, “prevention” can encompass delaying the onset of the cancer, or a symptom or condition thereof.

[0161] Construct for use as a vaccine

[0162] In a further aspect of the present invention, the construct according to the invention is for use as a vaccine, preferably an mRNA vaccine, more preferably an mRNA vaccine for use in the treatment or prevention of an infection, preferably a virus or viral infection.

[0163] Viruses or viral infections may include, but are not limited to, infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), hepatitis C, hepatitis B, herpes simplex virus (HSV), HIV-AIDS, poliovirus, encephalomyocarditis virus (EMCV) and small-pox virus. Examples of (+) strand RNA viruses which can be targeted for inhibition include, without limitation, picornaviruses, caliciviruses, nodaviruses, coronaviruses, arteriviruses, flaviviruses, and togaviruses. Examples of picornaviruses include enterovirus (poliovirus 1), rhinovirus (human rhinovirus 1A), hepatovirus (hepatitis A virus), cardiovirus (encephalomyocarditis virus), aphthovirus (foot-and-mouth disease virus O), and parechovirus (human echovirus 22). Examples of caliciviruses include vesiculovirus (swine vesicular exanthema virus), lagovirus (rabbit hemorrhagic disease virus), "Norwalk-like viruses" (Norwalk virus), "Sapporo-like viruses" (Sapporo virus), and "hepatitis E-like viruses" (hepatitis E virus). Betanodavirus (striped jack nervous necrosis virus) is the representative nodavirus. Coronaviruses include coronavirus (avian infections bronchitis virus) and torovirus (Berne virus). Arterivirus (equine arteritis virus) is the representative arteriviridus. Togaviruses include alphavirus (Sindbis virus) and rubivirus (Rubella virus). Finally, the flaviviruses include flavivirus (Yellow fever virus), pestivirus (bovine diarrhea virus), and hepacivirus (hepatitis C virus).

[0164] In case the disease is cancer, the cancer may preferably be selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skincancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof. However, in some embodiments, the disease may be a haematological tumour or a solid tumour.

[0165] Use of the construct as an immune adjuvant

[0166] In a further aspect, the present invention provides the use of the construct according to the present invention as an immune adjuvant. It may also be used as a vaccine adjuvant and / or for use in a method of vaccination. As used herein, “immune adjuvant” means a substance that increases or modulates the immune response of a subject as such. As used herein, “vaccine adjuvant” means a substance that increases or modulates the immune response to a vaccine. Adjuvants are often used to modify or augment the effects of a vaccine by stimulating the immune system to respond to the vaccine more vigorously, and thus providing increased immunity to a particular disease. Some adjuvants accomplish this task by mimicking specific sets of evolutionarily conserved molecules, so called pathogen-associated molecular patterns, which include liposomes, lipopolysaccharide, molecular cages for antigens, components of bacterial cell walls, and endocytosed nucleic acids such as RNA, double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA. The ribonucleic acid construct of the present invention is able to serve as such an immune adjuvant.

[0167] Construct for use in a method of infiltrating immune cells

[0168] In one further aspect of the present invention, the construct according to the present invention is for use in a method of infiltrating immune cells, preferably T cells.

[0169] In another aspect of the present invention, the construct according to the present invention is for use in a method of stimulating immune cells. Thus, the construct may have immunostimulatory activity. As used herein, "immunostimulatory activity" refers to the capability of an agent, such as a molecule or a composition, here of the construct of the present invention, to induce an immune response. Thus, the present invention provides a construct capable of inducing immunostimulatory activity, in particular anti-viral responses, in particular type l-IFN-production.

[0170] In some embodiments, the immunostimulatory activity refers to the type I IFN-inducing activity, in particular, IFN-a inducing activity. As used herein, "IFN-a inducing activity" refersto the capability of an agent, such as a molecule or composition, here of the construct of the present invention, to induce IFN-a production from a cell capable of producing IFN-a. Cells capable of producing IFN-a include, but are not limited to, peripheral blood mononuclear cells (PBMC) (e.g., B cells, dendritic cells (myeloid dendritic cells and plasmacytoid dendritic cells), macrophages, monocytes, natural killer cells, granulocytes), endothelial cells, and cell lines.

[0171] Construct for use in a method of turning cold into hot tumours

[0172] In one further aspect of the present invention, the construct according to the present invention is for use in a method of turning cold into hot tumours. The lymphocyte infiltration and IFN-y status may be key factors for effective therapy by defining a “T cell inflamed” phenotype (“hot tumours”). In contrast, lack of T cells infiltrating the tumour characterizes “noninflamed” or “cold tumours” (in which other immune populations or myeloid cells can however be observed).

[0173] Use of the construct as a vaccine

[0174] In a further aspect, the present invention provides the use of the construct according to the present invention as a vaccine.

[0175] Use of the construct in the manufacture of a medicament

[0176] In a further aspect, the present invention provides the use of the construct according to the present invention and as described herein in the manufacture of a medicament for the treatment or prevention of a disease. The disease may be cancer or an infection, preferably a virus infection. In case, the disease is cancer, the cancer is preferably selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof. However, for example, the disease may be a haematological tumour or a solid tumour.

[0177] Pharmaceutical composition

[0178] The present invention further provides a pharmaceutical composition comprising the construct of the present invention. That composition may further comprise one or more component(s), such as a pharmaceutically acceptable carrier, an excipient or a delivery agent. 1

[0179] In some embodiments, the delivery agent is a complexation agent which forms a complex with the oligonucleotide or the precursor thereof and facilitates the delivery of the oligonucleotide into cells. In some embodiments, the complexation agent is a polymer, preferably a cationic polymer. In a preferred embodiment, the complexation agent is a cationic lipid. In another preferred embodiment, the complexation agent is polyethylenimine (PEI) (K. Wu et al., Brain Research 1008(2):284-287 (May 22, 2004); B. Urban-Klein et al. Gene Therapy 12(5):461-466 (2005)). Additional examples of complexation agents include, but are not limited to, collagen derivatives (Y. Minakuchi et al. Nucleic Acids Research 32(13):e109 (2004)), and biodegradable microspheres such as liposomes (M. Sioud, D. Sorensen, Biochem Biophys Res Commun 312(4):1220-1225 (2003); PY Chien et al. Cancer Gene Therapy 12(3):321-328 (2005)), virosomes (J de Jonge et al. Gene Therapy, 13:400-411 (2006)), SNALPs (J J Rossi, Gene Therapy 13:583-584 (2006)), ISCOMATRIXo (CSL Limited) (I.D. Davis et al. PNAS 101 (29): 10697-10702 (July 20, 2004); MJ Pearse, D. Drane, Adv Drug Deliv Rev 57(3):465-474 (Jan 10, 2005)), and poly (D,L-lactide-co-glycolide) copolymer (PLGA) microspheres (A. Khan et al. J Drug Target 12(6):393-404 (2004)).

[0180] Polyethylenimine (PEI) can be linear or branched. In a preferred embodiment, PEI is in vivo-jetPEI, which is a linear PEI developed by Poly Plus-transfection for effective and reproducible delivery of anionic oligonucleotides with low toxicity in vivo. The preferred in vivo routes of administration include, but are not limited to, intravenous, intracerebral and intraperitoneal routes.

[0181] SNALPs stand for Stable-Nucleic-Acid-Lipid Particles and contain a lipid bilayer comprised of a mixture of cationic and fusogenic lipid coated with diffusible polyethylene glycol (PEG). The SNALPs are in the 120 nanometer diameter size range, protect the enclosed nucleic acid from serum nucleases and allow cellular endosomal uptake and subsequent cytoplasmic release of the nucleic acid.

[0182] ISCOMATRIXo is made from saponin, cholesterol and phospholipids under defined conditions and forms cage-like structures typically 40 nm in diameter. ISCOMATRIXo has the duel capability of facilitating cargo (e.g., antigen) delivery and stimulating the immune system, both the cellular and humoral immune response.

[0183] The pharmaceutical composition of the present invention may further comprise another agent such as an agent that stabilizes the oligonucleotide, in particular, RNA oligonucleotide, e.g., a protein that complexes with the oligonucleotide agent to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg2+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

[0184] A formulated composition can assume a variety of states. In some examples, the pharmaceutical composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10 % water). In another example, theoligonucleotide agent is in an aqueous phase, e.g., in a solution that includes water, this form being the preferred form for administration via inhalation.

[0185] The aqueous phase or the crystalline compositions can be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase), or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the oligonucleotide composition is formulated in a manner that is compatible with the intended method of administration.

[0186] In some embodiments, it is preferred that the pharmaceutical composition of the present invention does not comprise any transfection reagent(s) or complex(es) or composition(s) thereof. The transfection reagent mentioned for those embodiments means such a transfection reagent as defined herein above. In some embodiments, administration routes may include administration via intranasal spray.

[0187] The pharmaceutical composition of the invention can be used to generate a large amount of type I IFN, in particular, IFN-, IL-18 and / or IL-1 , in vitro and / or in vivo. The type I IFN, in particular, IFN-, IL-18 and / or IL-1 , can be generated at high quantities from different cellular sources, including both immune and non-immune cells, from different species of vertebrates.

[0188] Construct for use in a method of inducing an immune response

[0189] The present application also provides the construct according to the present invention for use in a method of inducing an immune response.

[0190] The construct may be an antigen recognizing construct or may be part of an antigen recognizing construct or is comprised in an antigen recognizing construct.

[0191] Further, the antigen recognizing construct may be an antibody, or fragment thereof, or a T cell receptor (TCR), or fragment thereof, or a chimeric antigen receptor (CAR), or a fragment thereof.

[0192] The term “antibody” in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. , molecules that contain an antibody combining site or a paratope. Such molecules are also referred to as "antigen binding fragments" of immunoglobulin molecules.

[0193] The term “T cell receptor” in general and as used in the context of the present invention refers to an immunoglobulin superfamily member having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail (see, e.g., Janeway el al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997) capable of specifically binding to an antigen peptide bound to an MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generallyis comprised of a heterodimer having a and p chains (also known as TCRa and TCRp, respectively), or y and 5 chains (also known as TCRy and TCR5, respectively).

[0194] Chimeric antigen receptors (CARs) - also known as chimeric immuno-receptors, chimeric T cell receptors or artificial T cell receptors - are receptor proteins that have been engineered to give T cells the new ability to target a specific antigen. The receptors are chimeric in that they combine both antigen-binding and T cell activating functions into a single receptor. CAR T cell therapy uses T cells engineered with CARs to treat cancer. The premise of CAR T immunotherapy is to modify T cells to recognize cancer cells in order to more effectively target and destroy them.

[0195] Method of treating a disease

[0196] The present application provides in a further aspect a method of treating a disease, comprising the step of administering a therapeutically effective amount of the construct according to the present invention to a subject in need thereof.

[0197] In some embodiments of the method of treating according to the present invention, the disease may be cancer or an infection, preferably a virus infection.

[0198] In case when the disease is cancer, the cancer may be selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof. However, the disease may also be a haematological tumour or a solid tumour.

[0199] In some embodiments of the method of treating according to the present invention, the construct may be administered in a multimerized form. This means that the construct is present in the form of a multimer such that at least two, e.g. three or four of such complexes are present.

[0200] For said method of treating according to the present invention, the construct may be administered by any means known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), ocular, rectal, vaginal, and topical (including buccal and sublingual) administration. In preferred embodiments, the administration may be intravenous or by intra- parenteral infusion or injection. For the method of treating according to the present invention,the construct may also be administered intra-parenchymally, intrathecally, and / or by stereotactic injection. It is preferred for the method of treating according to the present invention that the therapeutically effective amount of the construct may be administered to the subject in need thereof parenteral, subcutaneous, intramuscular, intravenous, oral or intranasal.

[0201] For oral administration, the construct may be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.

[0202] Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents may include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

[0203] Capsules for oral use may include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules, wherein the active ingredient is mixed with water or an oil, such as peanut oil, liquid paraffin or olive oil.

[0204] For intramuscular, intraperitoneal, subcutaneous and intravenous use, the construct of the present invention may be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the present invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0205] The therapeutically effective amount of the construct may also be administered as encapsulated formulations to protect the construct against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, asdescribed in U.S. Patent No. 4,522,811 ; PCT publication WO 91 / 06309; and European patent publication EP-A-43075.

[0206] A suitable dose or therapeutically effective amount of the ribonucleic acid construct may be in the range of 0.001 to 500 milligrams per kilogram body weight of the recipient per day (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 100 milligrams per kilogram, about 1 milligrams per kilogram to about 75 milligrams per kilogram, about 10 micrograms per kilogram to about 50 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram).

[0207] The step of administering a therapeutically effective amount of the construct may be once per day, or the construct may be administered as two, three, four, five, six or more subdoses at appropriate intervals throughout the day. In that case, the construct contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the oligonucleotide agent over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0208] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the infection or disease / disorder, previous treatments, the general health and / or age of the subject, and other diseases / disorders present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual ribonucleic acid construct according to the present invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model.

[0209] Toxicity and therapeutic efficacy of the ribonucleic acid construct and the pharmaceutical composition comprising said construct of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Ribonucleic acid agents that exhibit high therapeutic indices are preferred.

[0210] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosages of the construct or pharmaceutical compositions comprising the construct of the present invention are preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any ribonucleic acid agent used in the method of treating of thepresent invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the ribonucleic acid agent that includes the IC50 (i.e., the concentration of the test ribonucleic acid agent which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0211] The administering physician can adjust the amount and timing of the administration of the construct of the present invention on the basis of results observed using standard measures of efficacy known in the art or described herein.

[0212] Method of inducing an immune response

[0213] In a further aspect, the present invention provides a method of inducing an immune response, comprising the step of administering a therapeutically effective amount of the construct according to the present invention to a subject in need thereof. Preferably, the immune response may be induced by activating retinoic acid-inducible gene I (RIG-I). The immune response may be in addition or alternatively induced by stimulating retinoic acidinducible gene I (RIG-I).

[0214] For the method of inducing an immune response according to the present invention, the construct may have or comprise an immune adjuvant function.

[0215] For the method of inducing an immune response according to the present invention, the construct may comprise at least one, preferably at least two, RIG-l-ligand(s), preferably at least two RIG-l-ligand(s), more preferably at least three RIG-l-ligand(s), and preferably four RIG-l-ligand(s).

[0216] In some embodiments for the method of inducing an immune response, the construct is administered in a multimerized form as described herein above.

[0217] For the method of inducing an immune response of the present invention, it is preferred that the therapeutically effective amount of the ribonucleic acid construct is administered to the subject in need thereof parenteral, subcutaneous, intramuscular, intravenous, oral or intranasal. The definitions given above with regard to these administration routes also apply to the method of inducing an immune response. The embodiments for the method of treating according to the present invention also apply here for the method of inducing an immune response.

[0218] Kit

[0219] In a further aspect, the present invention is directed to a kit for use in medicine comprising the construct according to the present invention.

[0220] It is preferred that the kit is a diagnostic kit for selecting a patient for treatment of a disease, preferably wherein the disease is an infection or cancer.

[0221] It is preferred that - in case the disease is cancer - the cancer may be selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof. However, the disease may also be a haematological tumour or a solid tumour.

[0222] The invention will be further illustrated by the following, non-limiting items:1 . A construct comprising- an mRNA-strand, and- at least one, preferably at least two, retinoic acid-inducible gene I (RIG-l)-ligand(s) comprising, a) a nucleic acid sequence being capable of binding to RIG-I, b) a linker moiety L comprising 3 to 24 main chain atoms, preferably comprising 6 to 18 main chain atoms, and c) a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand.2. The construct according to item 1 , wherein the construct is a nucleic acid-construct.3. The construct according to item 2, wherein the nucleic acid-construct is an engineered nucleic acid-construct.4. The construct according to any one of the preceding items, wherein the construct comprises at least two of the retinoic acid-inducible gene I (RIG-l)-ligand(s).5. The construct according to any one of the preceding items, wherein the construct comprises two to four of the retinoic acid-inducible gene I (RIG-l)-ligand(s).he construct according to any one of the preceding items, wherein the construct comprises four of the retinoic acid-inducible gene I (RIG-l)-ligand(s). he construct according to any one of the preceding items, wherein the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s) is complementary to at least one nucleic acid sequence comprised in the mRNA strand. he construct according to any one of the preceding items, wherein the linker moiety L connects the nucleic acid sequence being able to bind to RIG-1 with the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s). he construct according to any one of the preceding items, wherein the mRNA-strand comprises a coding sequence of a protein, a peptide or an antigen, preferably an antigen that is able to induce an immune response or preferably a cancer antigen. . The construct according to any one of the preceding items, wherein the single-stranded nucleic acid sequence is capable of hybridizing to at least one nucleic acid sequence located in a non-coding nucleic acid sequence of the mRNA-strand. . The construct according to item 10, wherein the non-coding nucleic acid sequence of the mRNA-strand is or comprises a 3'-untranslated region (UTR)-nucleic acid sequence or a 5'-untranslated region (UTR)-nucleic acid sequence or a poly-A-tail. . The construct according to any one of the preceding items, wherein the mRNA-strand comprises a 3'-untranslated region (UTR)-nucleic acid sequence and the single-stranded nucleic acid sequence is capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand comprised in said 3'-untranslated region (UTR)-nucleic acid sequence. . The construct according to any one of the preceding items, wherein the mRNA-strand comprises a 5'-untranslated region (UTR)-nucleic acid sequence and the single-stranded nucleic acid sequence is capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand comprised in said 5'-untranslated region (UTR)-nucleic acid sequence. . The construct according to item 12 or 13, wherein the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s) is capable of hybridizing to the at least one nucleic acid sequence comprised in the 3'- or 5'-untranslated region (UTR)-nucleic acid sequence of the mRNA-strand via Watson-Crick-base-pairing.The construct according to any one of the preceding items, wherein the nucleic acid sequence being capable of binding to RIG-I comprises at least one triphosphate. The construct according to any one of the preceding items, wherein the nucleic acid sequence being able to bind to RIG-I comprises or consists of a double-stranded RNA. The construct according to item 16, wherein the double-stranded RNA of the nucleic acid sequence being able to bind to RIG-I is a blunt-ended double-stranded RNA. The construct according to item 16, wherein the double-stranded RNA of the nucleic acid sequence being able to bind to RIG-I further comprises an overhang. The construct according to item 18, wherein the overhang is a RNA-overhang or an overhang with a fluorophore or a lipophilic substitution. The construct according to any one of items 16 to 19, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at least one triphosphate at at least one strand of said double-stranded RNA, preferably at least one triphosphate at the 5'-end of one strand of said double-stranded RNA. The construct according to any one of items 16 to 20, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I has a length in the range of 8 to 20 nt, preferably wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I has a length in the range of 10 to 17 nt. The construct according to any one of items 16 to 21 , wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I has a length in the range of 10 to 15 nt. The construct according to any one of items 16 to 22, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I has a length in the range of 10 to 13 nt. The construct according to any one of items 16 to 23, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I has a length of 11 nt.he construct according to any one of items 16 to 24, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises one triphosphate at the 5'-end of one strand of said double-stranded RNA. he construct according to any one of items 16 to 25, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at least one modification(s). he construct according to item 26, wherein the at least one modification(s) is / are selected from the group consisting of O-methyl-group-modification(s), preferably 2'-O- methyl-group-modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group- modification(s), phosphor-thio-modification(s) and locked nucleic acid (LNA)- modification(s). he construct according to any one of the preceding items, wherein the mRNA-strand comprises at least one modification(s). he construct according to item 28, wherein the at least one modification(s) of the mRNA- strand is / are pseudo-uridine-modification(s). he construct according to item 26 or 27, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at at least one strand of the double-stranded RNA at least one modification(s), preferably at both strands of the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I. he construct according to item 30, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I further comprises at least one triphosphate at one end of at least one strand of said double-stranded RNA and at least one modification(s) is / are at the other end of the at least one strand of said double-stranded RNA. he construct according to any one of items 26 to 31 , wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at its 3'-end and / or at its 5'-end of at least one strand of the double-stranded RNA at least one modification(s), preferably at both strands of the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I.The construct according to any one of items 26 to 32, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-1 comprises at its 3'-end of at least one strand of the double-stranded RNA at least one modification(s). . The construct according to any one of items 26 to 32, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-1 comprises at its 5'-end of at least one strand of the double-stranded RNA at least one modification(s). . The construct according to any one of items 26 to 34, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-1 comprises at its 5'-end and / or at its 3'-end at least one O-methyl-group-modification(s), preferably at least one 2'-O-methyl-group-modification(s). . The construct according to item 35, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-1 comprises at both strands of the doublestranded RNA at least one O-methyl-group-modification(s). . The construct according to any one of the preceding items, wherein the linker moiety L comprises 6 to 18 main chain atoms. . The construct according to any one of the preceding items, wherein the linker moiety L comprises 8 to 16 main chain atoms. . The construct according to any one of the preceding items, wherein the linker moiety L comprises 10 to 14 main chain atoms. . The construct according to any one of the preceding items, wherein the linker moiety L comprises 12 main chain atoms. . The construct according to any one of the preceding items, wherein the main chain atoms are carbon atoms that are optionally replaced by one or more heteroatoms selected from the group consisting of N, O and S. . The construct according to any one of the preceding items, wherein the linker moiety L of the at least one RIG-l-ligand(s) is a polyalkylene-glycol-linker according to Formula IFormula I, wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7. he construct according to any one of the preceding items, wherein the linker moiety L of the at least one RIG-l-ligand(s) is a polyalkylene-glycol-linker according to Formula IIFormula II, wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7. he construct according to item 43, wherein the linker moiety L of the at least one RIG-I- ligand(s) is a polyalkylene-glycol-linker according to Formula II and wherein n is 6. he construct according to any one of the preceding items, wherein the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s) has a length in the range of 5 to 15 nt. he construct according to item 45, wherein the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s) has a length in the range of 8 to 13 nt. he construct according to any one of the preceding items for use as a medicament. he construct according to any one of the preceding items for use as a vaccine. Pharmaceutical composition comprising the construct according to any one of items 1 to 46.he construct according to any one of items 1 to 46 for use in a method of treatment or prevention of a disease. he construct for use according to item 50, wherein the disease is cancer or an infection, preferably a virus infection. he construct for use according to item 50 or 51 , wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof. he construct for use according to item 50 or 51 , wherein the disease is a haematological tumour or a solid tumour. he construct according to any one of items 1 to 46 for use in a method of infiltrating immune cells, preferably T cells. he construct according to any one of items 1 to 46 for use in a method of turning cold into hot tumours. Use of the construct according to any one of items 1 to 46 as a vaccine. Use of the construct according to any one of items 1 to 46 as an immune adjuvant. Use of the construct according to any one of items 1 to 46 in the manufacture of a medicament for the treatment or prevention of a disease. he use according to item 58, wherein the disease is cancer or an infection, preferably a virus infection.he use according to item 58 or 59, wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof. he use according to item 59 or 60, wherein the disease is a haematological tumour or a solid tumour. he construct according to any one of items 1 to 46 for use in a method of inducing an immune response. he construct according to any one of items 1 to 46, wherein the construct is an antigen recognizing construct or part of an antigen recognizing construct or is comprised in an antigen recognizing construct. method of treating a disease, comprising the step of administering a therapeutically effective amount of the construct according to any one of items 1 to 46 to a subject in need thereof. he method of treating according to item 64, wherein the disease is cancer or an infection, preferably a virus infection. he method of treating according to item 65, wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer,osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof. he method of treating according to item 66, wherein the disease is a haematological tumour or a solid tumour. he method of treating according to any one of items 64 to 67, wherein the therapeutically effective amount of the construct is administered to the subject in need thereof parenteral, subcutaneous, intramuscular, intravenous, oral, intraperitoneal or intranasal. method of inducing an immune response, comprising the step of administering a therapeutically effective amount of the construct according to any one of items 1 to 46 to a subject in need thereof. he method according to item 69, wherein the immune response is induced by activating retinoic acid-inducible gene I (RIG-I). Method according to item 70, wherein the immune response is induced by stimulating retinoic acid-inducible gene I (RIG-I). Method according to any one of items 69 to 71 , wherein the construct has or comprises an immune adjuvant function. Method according to any one of items 69 to 72, wherein the construct comprises at least one, preferably at least two, RIG-l-ligand(s). Method according to any one of items 69 to 73, wherein the construct comprises at least two RIG-l-ligand(s). Method according to any one of items 69 to 74, wherein the construct comprises at least three RIG-l-ligand(s). he method according to any one of items 69 to 75, wherein the construct comprises four RIG-l-ligand(s).77. The method according to any one of items 69 to 76, wherein the therapeutically effective amount of the construct is administered to the subject in need thereof parenteral, subcutaneous, intramuscular, intravenous, oral, intraperitoneal or intranasal.78. A kit for use in medicine comprising the construct according to any one of items 1 to 46.79. The kit according to item 78, wherein the kit is a diagnostic kit for selecting a patient for treatment of a disease, preferably wherein the disease is an infection or cancer.

[0223] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0224] As used herein, “a” and “an” refer to not only a single individual, but also a group or species of entities unless otherwise noted.

[0225] All terms used herein bear the meanings that are established in the art, unless otherwise noted. Techniques disclosed herein can be performed by a person skilled in the art following the same present description and / or established protocols such as those disclosed in Molecular cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbour Laboratory, New York), Current Protocols in Molecular Biology (Ausubel et al., 2007, John Wiley & Sons, New York), and Current Protocols in Immunology (Coligan et al., 2007, John Wiley & Sons, New York).

[0226] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0227] As used herein, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an", as used herein, refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include", "have", and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

[0228] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been usedas terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein and herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments of the invention will become apparent from the following claims.EXAMPLES

[0229] Materials and Methods

[0230] Cell lines

[0231] Monocytic leukemia cell line THP-1 (Tsuchiya et al., 1980) was a kind gift from the Clinic of Anaesthesiology (University Hospital Bonn). The inventors of the present invention generated RIG-I deficient THP-1 cells by Crispr-Cas9 gene-editing. The respective monoclonal cell line has been published previously (RIG-I - / - #1 in Ofir-Birin etal., 2021). THP- 1 , THP-1 RIG-l / _and THP-1 Dual wt (InvivoGen) were cultured in RPMI 1640 (Gibco) in a humidified incubator at 37°C and 5% CO2. The medium was supplemented with 10% fetal calf serum (Gibco), 100 U / ml penicillin, and 100 pg / ml streptomycin (Gibco), 1 mM sodium pyruvate (Gibco) and 0,1 mM MEM non-essential amino acids (Gibco). Cell lines were verified to be mycoplasma negative by using a mycoplasma-specific PCR approach at regular intervals.

[0232] Bone marrow was isolated from the femurs of 8-12 week old C57BL / 6 mice. Erythrocytes in the suspension were lysed with 1 X RBC lysis buffer (BioLegend). Remaining cells were sedimented and resuspended in differentiation medium (RPMI 1640 (Gibco) + 13 % GM-CSF). 5X 106cells were seeded in 010 cm culture dishes and cultured in a humidified incubator at 37°C and 5% CO2. After 4 days, two thirds of the medium were exchanged with fresh differentiation medium. Six days after isolation, the bone marrow derived dendritic cells (BMDC) were harvested. The adherent cells were incubated twice with DPBS (Thermo Fisher Scientific) + 2 mM EDTA (Invitrogen) for 30 min at 37°C to detach. For transfection, BMDC were seeded at a density of 2x106cells / mL in culture medium (RPMI 1640 (Gibco) supplemented with 10% fetal calf serum (FCS, Gibco), 100 U / ml penicillin, and 100 pg / ml streptomycin (Gibco), 1 mM sodium pyruvate (Gibco) and 0,1 mM MEM non-essential amino acids (Gibco) in 96-well plates.

[0233] C57BL / 6 mice were obtained from Charles River and kept in-house in the House of Experimental Therapy at the University Hospital Bonn.

[0234] Plasmid Templates

[0235] To generate a plasmid DNA template (pDNA) for mRNA synthesis, T7-promoter sequence, coding sequences and RIG-I ligand hybridization sites were cloned into plasmid pEF-BOS (see Mizushima S., et al., 1990). The template comprises the T7 promoter, a short 5’-UTR containing a Kozak sequence, different coding regions indicated in the respective experiment and sequence listing, such as EGFP, Firefly Luciferase, SIINFEKL Peptid or Flag peptide (see respective constructs in the sequence listing and Table 4); Stop Codon, the RIGI ligand hybridization sequence, a 3’-UTR followed by a poly-A sequence. The respective DNA sequences, which comprise the nucleic acid sequence of the T7 promoter, the 5’-UTR, the coding sequence, the RIG-1 hybridization sides, the 3’-UTR and the poly-A-tail is given herein as SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 and SEQ ID NO: 60.

[0236] Modell mRNAs (see, for example, Example 9):

[0237] pDNA was linearized by either EcoRI (Fast Digest, Thermo Fisher Scientific, SEQ ID NO: 35) or BcoDI (New England Biolabs, see SEQ ID NO: 36, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 49) digestion and purified by ROTI®Phenol / Chloroform / lsoamyl alcohol (Carl Roth). The aqueous phase was two times extracted with pure chloroform (Carl Roth), followed by addition of 2 volumes 100% ethanol and 0.1 volumes 3 M sodium acetate (Thermo Fisher Scientific). Upon incubation at -20°C for at least 30 min, samples were centrifuged at 16,000 ref at 4°C for 30 min. The resulting DNA pellet was air-dried and resuspended in water.

[0238] In vitro transcription

[0239] Linearized DNA was used as template (2000 ng). In-vitro transcribed RNA was performed with Hi-T7 polymerase (high concentration, M0470T, New England Biolabs) in transcription buffer (40 mM Tris / HCI pH 7.5, 1 mM DTT, 2.5 mM spermidine, 50 mM sodium chloride, 16.5 mM magnesium chloride) supplemented with 10 mM ATP, CTP, GTP (Thermo Fisher Scientific), nl m^PTP (Jena Bioscience). Additionally, 4 mM CleanCap AG (Trilink), 20 U murine RNAse Inhibitor (New England Biolabs), 0.05 U Yeast Inorganic Pyrophosphatase (New England Biolabs) were added into the transcription reaction. After incubation at 50°C for 60 min, DNAse I (Thermo Fisher Scientific) was added and the mixture incubated at 37°C for 30 min.

[0240] RNA was purified with ROTI®Phenol / Chloroform / lsoamyl alcohol (Carl Roth), followed by two times extraction with pure chloroform (Carl Roth) and ethanol precipitation. The resulting RNA pellet was air-dried and resuspended in water.

[0241] Concerning Example 10:

[0242] CMV mRNAs: pDNA was linearized by Esp3l (ThermoFisher, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 and SEQ ID NO: 60) digestion and purified by ROTI®Phenol / Chloroform / lsoamyl alcohol (Carl Roth). The aqueous phase was two times extracted with pure chloroform (Carl Roth), followed by addition of 2 volumes 100% ethanol and 0.1 volumes 3 M sodium acetate (Thermo Fisher Scientific). Upon incubation at -20°C forat least 30 min, samples were centrifuged at 16,000 ref at 4°C for 30 min. The resulting DNA pellet was air-dried and resuspended in water.

[0243] In vitro transcription:

[0244] Linearized DNA was used as template (1000 ng). In-vitro transcribed RNA was performed with the TranscriptAid T7 transcription kit (ThermoFisher). The concentration of NTPs was modified to 10 mM ATP, CTP, GTP (Thermo Fisher Scientific), nl m^PTP (Jena Bioscience). Additionally, 4 mM CleanCap AG (Hongene) was added into the transcription reaction for cotranscriptional capping. After incubation at 37°C for 2 h, DNAse I (ThermoFisher) was added and the mixture incubated at 37°C for 30 min.

[0245] RNA was purified by adding one volume of water and one volume of LiCI (7.5 mM) to the reaction. The mRNA was precipitated for 30 min at -20 °C and centrifuged for 30 min at 16,000 ref and 4 °C. The pellet was washed in 70 % Ethanol and resuspended in water.

[0246] ppp-RNA synthesis

[0247] ppp-RNA was synthesized according to US 11142763 B2.

[0248] Lipofection based transfectionRNA transfection was performed in duplicates. Cells were plated at 80.000 cells / well in a 96- well plate in 100 pL RPMI medium. Cells were transfected as indicated with either Lipofectamine 2000 (Thermo Fisher Scientific) or Lipofectamine MessengerMax (Thermo Fisher Scientific) following the manufacturer’s instruction.

[0249] Luciferase AssayLuciferase assay was performed as indicated 18 h or 24 h after stimulation. To measure Lucia luciferase production, as surrogate for type I IFN induction, 25 pL of the supernatant was mixed with the same volume of luciferase substrate (1 pg / mL coelenterazine in 100 mM Tris pH 7.4, 300 mM Sodium Ascorbate). Measurement of firefly luciferase, as translation level indicator for the mRNA, was conducted on lysed cells. Cells were lysed with saponin buffer and 25 pL lysed-cells were mixed with 25 pL D-luciferin-containing buffer. Luminescence was immediately measured with the EnVision 2104 Multilabel reader (PerkinElmer).

[0250] CXCL10 ELISA

[0251] Human CXCL10 was measured according to manufacturer’s instructions with the Human IP10 ELISA Set (BD).

[0252] Transfection and Readout of murine BMDCs

[0253] Murine BMDCs were transfected with controls, mRNA or mRNA+CRL (1 pg / mL) using either Lipofectamine MessengerMax (ThermoFisher, Example 9, model mRNAs) or Lipofectamine 2000 (ThermoFisher, Example 10, CMV mRNAs) according to the manufacturers protocol.

[0254] After 18 h incubation at 37 °C, surface expression of MHC proteins and activation markers was examined by flow cytometry. Secreted IFNa was detected in the supernatant using enzyme-linked immunosorbent assay (ELISA).

[0255] Staining and detection of surface proteins by flow cytometry:

[0256] Transfected BMDCs were incubated twice for 30 minutes at 37°C with DPBS (Thermo Fisher Scientific) + 2 mM EDTA (Invitrogen) to detach from the wells. The cells were sedimented by centrifugation (400 ref, RT, 3 min) and washed with FACS buffer, DPBS (Thermo Fisher Scientific), 5 % FCS (Gibco), 2 mM EDTA (Invitrogen), 0.05 % NaNs (Carl Roth) between all staining steps.

[0257] Cells were incubated with Zombie NIR Fixable Viability Dye (BioLegend) prediluted 1 :4000 in PBS for 20 min at RT in the dark. After washing, Fc receptors were blocked by incubation with 2.5 pg / mL purified Rat Anti-Mouse CD16 / CD32 (Mouse BD Fc Block™, BD Pharmingen™) for 5 min at RT. Antibodies against the surface targets (Table 1) were prediluted 1 :100 in FACS buffer. The antibody mix was applied directly to the cells in Fc block solution at equal volume and incubated for 20 min at 4 °C. After two final washing steps, the stained cells are stored in FACS buffer on ice until measurement in the Attune NxT Acoustic Focusing Cytometer AFC2 (Invitrogen).

[0258] Gating

[0259] For quantification of the stained markers, single cells were selected and dead, Zombie NIR positive cells were excluded. The mean fluorescence of each dye was determined in population of CD11b+ CD11c+ cells.Table 1 : Antibody clones for labeling the indicated markers for flow cytometry:FlowJo 10.8.1 was used for data analysis.

[0260] Murine IFNa ELISA

[0261] ELISA medium-binding 96-well plates (Grainer Bio-One) were coated with 1 pg / mL rat monoclonal antibody against murine IFNa (RMMA-1 , PBL Assay Science) in coating buffer (0.2 M sodium phosphate, pH 6.5) at 4 °C overnight.

[0262] Plates were washed with DPBS (Gibco) + 0.05 % Tween (Carl Roth, PBS-T) and blocked with assay buffer (DPBS (Gibco) + 10 % FCS (Gibco)) for 1 h at RT. After washing again with PBS-T, the supernatant of transfected cells and a dilutional series of recombinant murine IFNa (PBL Assay Science) were loaded and incubated overnight at 4 °C.

[0263] Plates were washed with PBS-T and incubated with anti-mouse IFNa rabbit serum (1x102Neutralizing Units / ml, PBL Assay Systems) in assay buffer for 3 h at RT. After washing with PBS-T, the samples were incubated with goat anti-rabbit IgG (H+L)-HRP conjugate (BioRad) diluted 1 :5000 in assay buffer for 2h at RT. Plates were washed with PBS-T and readout was performed with the TMB substrate reagent set (BD OptEIA™) according to the manufacturer’s instructions. The enzymatic reaction was stopped after 20 min with 1 M sulfuric acid (Carl Roth). Absorbance at 450 nm and 570 nm was measured with an Epoch™ Microplate Spectrophotometer (BioTek Instruments Inc.). The concentration of IFNa in the supernatant was calculated according to the standard curve.

[0264] Statistical analysis

[0265] Graphs show the mean with standard error. Statistical analysis was performed using multiple paired ratio t tests with a false discovery rate (FDR) set to 0.05 and the two-stage step- up (Benjamini, Krieger, and Yekutieli) method.

[0266] Prism 10 software (GraphPad, California, USA) was used for analysis and visualization.

[0267] Results

[0268] In the following examples, the construct according to the present invention, which has been used, is a construct that comprises one or more RIG-l-ligand(s), which comprise a blunt- ended dsRNA as nucleic acid sequence being capable of binding to RIG-I. The blunt-ended dsRNA comprises a triphosphate moiety at one of the strands of the dsRNA. This construct comprises a linker moiety L (or in the Examples alternatively named X). The one or more RIG- l-ligand comprises a single-stranded nucleic acid sequence that may hybridize with a certain region of mRNA-strand. The mRNA-strand encodes the immunogen.

[0269] The inventors of the present invention have demonstrated the immunostimulatory capacity of the construct of the present invention via application of the construct, in the variousexamples described herein, e.g. by application of the construct(s) to THP1 Dual cells (InvivoGen). THP1 Dual cells are cells that are derived from the human THP-1 monocyte cell line and into which a luciferase reporter gene has been stably integrated. The translation of the reporter gene is activated via an interferon (IFN) promoter. In the presence of type I IFN, luciferase is thus expressed which, after addition of the substrate, is quantified by reading out the luciferase activity in a Perkin Elmer multi-label reader.

[0270] Example 1

[0271] By Figure 3, the inventors demonstrated that the construct according to the present invention is capable of inducing IFN-I expression, as quantified via levels of luminescence resulting from an interferon-stimulated response element (ISRE)-reporter cell line (THP-1 Dual wt, invivogen) capable of secreting Lucia luciferase after activating the IRF pathway. This is an important step in validating the construct as a viable immune adjuvant for use in mRNA vaccination, as adjuvancy requires innate immune activity, with the expression and release of interferons comprising an important element of innate immune responses. In this example, RIG-l-ligands were hybridized to a DNA template and were applied to THP-1 Dual cells at a concentration of 250 ng / mL. The RIG-l-ligand comprises a blunt-ended dsRNA with a 5’- triphosphate present at one strand of the dsRNA, a single-stranded RNA region that can hybridize to a DNA template, and a linker moiety to connect them. Two versions of the RIG-l- ligand were applied - an unmodified (I, ppp-Ribo-CRL, SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 66) and modified (II, ppp-mod-CRL) version (the modifications are according to SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34), both were applied and hybridized to the DNA template (SEQ ID NO: 5) at a ratio of 4 RIG-l-ligands per DNA template. A schematic depiction of both constructs can be found on the right side of Figure 3. For each construct, the construct was applied for 18 hours before resultant luminescence was assayed. Assaying was performed by extracting 25 pL cell supernatant, mixing it with 25 pL coelentarazine (1 pg / mL) and recording the resultant luminescence emitted. The two RIG-l-ligands were compared with triphosphate dsRNA (3p-dsRNA, 250 ng / ml, SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4), a known potent activator of RIG-I that may induce interferon expression. It can be seen that ppp-Ribo-CRL (I) is a less potent immune activator than either 3p-dsRNA or ppp-mod-CRL (II), which outperforms both construct I and the 3p-dsRNA control. For each construct, n = 2 and each bar represents mean ± standard error of the mean (SEM).

[0272] Example 2

[0273] The inventors of the present invention have demonstrated that the construct according to the present invention is an innate immune activator that is capable of inducing IFN- expression. The inventors have further demonstrated that this innate immune activation iscontrollable by the number of constructs hybridized to the DNA template. This is important, as different mRNA vaccines may require different intensity adjuvant activity for optimal vaccination. Thus, in Figure 4 as shown herein, a DNA template (SEQ ID NO: 5) is applied either alone (grey), with one (grey with black border), two (light grey), three (light grey with black border), or four (black and grey horizontally striped) ppp-mod-CRL (SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34) ligands being hybridized. For each construct, the construct was applied at a concentration of 3.9 ng / mL for 18 hours before resultant luminescence was assayed. Assaying was performed by extracting 25 pL cell supernatant, mixing it with 25 pL coelentarazine (1 pg / mL) and recording the resultant luminescence emitted. ISRE reporter activity increases as the number of ppp-mod-CRL ligands per DNA template is increased. Results were once again contrasted with 3p-dsRNA (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4). For each construct, n = 2 and each bar represents mean ± standard error of the mean (SEM).

[0274] Example 3

[0275] Figure 5 expands on the demonstrated ability of the present invention to induce interferon-expression by confirming that it is a) mechanistically dependent on RIG-I immunostimulatory activity and b) hybridization of the dsRNA construct does not interfere with the translation of the mRNA to which it is hybridized. The RIG-I dependent nature of the present invention's immune activation was demonstrated through the use of RIG-I homozygous knockout THP-1 dual cells (RIG-I^). 3p-dsRNA (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4), mRNA alone (in vitro transcribed based on SEQ ID NO: 36), and mRNA with 4 ppp-mod-CRL ligands (SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34) hybridized were applied at a concentration of 1000 ng / mL and formulated in Lipofectamine MessengerMax. 18 h after transfection, RIG-I activation was quantified by measuring the CXCL10 levels in the supernatant by ELISA. When supernatant CXCL10 was compared between WT (Figure 5A) and RIG-l< / _)(Figure 5B) cells, it could be seen that expression of CXCL10 is orders of magnitude lower in RIG-l( / )cells (y-axis values in Figure 5A range from 0 to 1 .5 x 104pg / mL and in Figure 5C from 0 to 40 pg / mL). Quantification of successful mRNA translation was performed by transfecting cells with mRNA (with or without hybridized ppp-mod-CRL) encoding non-secreted firefly luciferase (Flue). Thus, resultant luminescence was proportionate to the level of translation of the encoded luciferase. No difference in translation could be seen between mRNA and mRNA with hybridized RIG-I ligand in either WT (Figure 5B) or RIG-l< / _)(Figure 5D) cells. The success of translation was not influenced by RIG-I, as no difference could be seen between translation levels between WT (Figure 5B) and RIG-l< / _)(Figure 5D) cells. For each experiment, n = 3 and each bar represents mean ± standard error of the mean (SEM).

[0276] Example 4

[0277] The utility of the present invention derives, in part, from it’s modularity, as the number of RIG-l-ligand(s) per mRNA-strand can be altered to best suit a given vaccine construct. By Figure 6A, the inventors have demonstrated that the strength of interferon induction is proportionate to the number of ppp-mod-CRL ligands hybridized to an mRNA-strand, rather than the DNA template used in Figure 4 to better replicate the intended use of the present invention. ISRE reporter activity was quantified in THP-1 Dual cells, following the transfection of 1000 ng / mL of the cells with Lipofectamine MessengerMax with mRNA alone (grey, in vitro transcribed based on SEQ ID NO:35), or mRNA plus one (grey with black border), two (light grey), three (light grey with black border), or four (black and grey horizontally striped) ppp- mod-CRL ligands (SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34) hybridized to the mRNA-strand {in vitro transcribed based on SEQ ID NO: 35). Strength of interferon induction was increased by the addition of ppp-mod-CRL ligands. For each construct, interferon response was quantified at 20 hours post-transfection. For each experiment, n = 3 and each bar represents mean ± standard error of the mean (SEM).

[0278] Equally important in mRNA vaccine efficacy is the ability of the mRNA construct to transfect and induce translation of the immunogenic protein within the transfected cell. In Figure 6B, the inventors of the present invention repeated the experimental protocol employed in Figure 5B and 5D, quantifying levels of mRNA translation by quantifying the intensity of luminescence produced by a non-secreted Firefly luciferase (Flue) encoded by the mRNA. As with Figure 6A, cells were transfected with Lipofectamine MessengerMax in a concentration of 1000 ng / mL with either mRNA alone in vitro transcribed based on SEQ ID NO: 35), or mRNA plus one (grey with black border), two (light grey), three (light grey with black border), or four (black and grey horizontally striped) ppp-mod-CRL ligands (SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34) hybridized to the mRNA-strand {in vitro transcribed based on SEQ ID NO: 35). mRNA translation was decreased by increasing the number of hybridized ligands indicating that there is an adjustable and as part of the present invention controllable range between protein translation and immune stimulation.

[0279] Example 5

[0280] By the creation of Figure 7, the inventors of the present invention compared the efficacy of an 11 nt ribonucleotide RIG-I ligand (Ribo-CRL, SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 66) to a 24 nt ribonucleotide RIG-I ligand (Ribo-24nt-CRL, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8), applied either alone or when hybridized to a DNA template (SEQ ID NO: 67). THP-1 Dual cells were transfected with the two aforementioned constructs and with 3p-dsRNA. All constructs were applied at a concentration of 1000 ng / ml and transfectedwith Lipofectamine 2000. After 18 hours, 25 pL of cell supernatant was mixed with 25 pL coelenterazine (1 pg / ml), and the resultant luciferase activity was quantified. Figure 7A shows the constructs quantified in Figure 7B, by which the inventors demonstrated that DNA- hybridized Ribo-CRL induces stronger IFN-expression than Ribo-CRL alone. Similarly, Figure 7C shows the constructs quantified in Figure 7D, by which the inventors demonstrated that, in contrast to Ribo-CRL, Ribo-24nt-CRL potency is unaltered by hybridization to DNA. The ratio of ISRE reporter activity between either ligand alone and the ligand when hybridized to a DNA template is shown in Figure 7E, confirming the potentiation of Ribo-CRL by DNA hybridization and the lack of change in Ribo-24nt-CRL when similarly hybridized.

[0281] Example 6

[0282] By Figure 8, the inventors of the present invention compared various synthetic 5’- triphosphate complementary RIG-I ligands with 24 nt long dsRNA elements and different spacers / linker moieties L when transfected either individually (see I) or when hybridized to a 4-strand DNA template (see II, SEQ ID NO: 5), at a concentration of 1000 ng / mL. THP-1 Dual WT cells were transfected via Lipofectamine 2000 and luciferase activity, indicative of IFN expression levels, quantified after 18 hours. With this experiment, the present inventors sought to quantify a recently described 24 nt triphosphate RIG-I ligand described by Tockary et al. For each spacer (X) (namely 10 uracil-residues (10 U, SEQ ID NO: 16 + SEQ ID NO: 17), 2 uracil-residues (2 U, SEQ ID NO: 18 + SEQ ID NO:19) and Spacer 18 (Sp18, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22), ISRE reporter activity could be seen to be within the same range for the ligand when applied alone (see I) or when hybridized with the DNA template (see II). IFN expression range does not differ between different spacers.

[0283] Example 7

[0284] With Figure 9, the inventors sought to expand the data demonstrated in Figure 8 by performing similar experiments, but comparing a 24 nt long ppp-dsRNA to a 11 nt long dsRNA. Following the same protocol as employed in Figure 8, the inventors of the present invention demonstrated that unlike the 24 nt version described similar in Tockary et al. (Tockary 24mer RNA - Sp18, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8), a 11 nt ppp-dsRNA ligand based on the same RNA sequence (Tockary 11mer RNA - Sp18: SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15) demonstrated a template-effect, with application of the ligand in isolation (see I) inducing substantially lower IFN-expression, when compared with the ligand applied and hybridized to an appropriate DNA template (see II, SEQ ID NO: 5). The same effect could be seen when the construct of the present invention (CRL rev - Sp18: SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22) was applied in isolation (I) versus whenhybridized (II). For each experiment, n = 2 and each bar represents mean ± standard error of the mean (SEM).

[0285] Example 8

[0286] A key element of the construct according to the present invention is a flexible linker moiety that joins the ppp-dsRNA of the RIG-l-ligand to the ssRNA that enables hybridization to an appropriate nucleic acid strand. With Figure 10, the inventors sought to quantify the effects of different linkers of varying flexibility on immunogenicity of the construct according to the present invention, as quantified by interferon expression-related luminescence, described above. To make each construct comparable to the RIG-l-ligand described in Tockary et al., each construct is the construct of the present invention in a “reversed” conformation with respect to the positioning of the triphosphate moiety. The triphosphate was located on the opposite RNA-strand of the dsRNA of the RIG-l-ligand than as compared to the construct of the present invention. Each construct was applied at a concentration of 1000 ng / mL either individually (see I) or when hybridized to a 4-strand DNA template (see II) to THP-1 Dual cells by transfection with Lipofectamine 2000. Luciferase activity after 18 hours of application was shown along with a schematic representation of each construct, above. For each linker moiety L (in this Example marked as X), the immunogenicity of the construct was increased by hybridization to the aforementioned DNA template. The inventors also noted that conformational flexibility is positively correlated with immunogenicity, with high flexibility linkers (e.g. 10 II, 2 x Sp18) achieving strong IFN expression levels. This suggested that RIG- I activation is optimized when the linker moiety can move relative to the single-strand nucleic acid sequence that is able to bind to a sequence of the mRNA-strand. The strongest IFN- induction was achieved by the modified version of the construct according to the present invention (CRL mod) in the non-reversed conformation, with high ISRE reporter activity for both the ligand applied in isolation and when being hybridized. The definitions of the expressions and the respective RNA sequences used in Figure 10 as well as the chemical formula of “SP18”, “SP9”and “SpC3” are given in Tables 2 and 5.

[0287] Example 9

[0288] Example 9 (see Figure 12) shows the surface expression of A) CD86 (see Figure 12A), B) CD69 (see Figure 12B), C) MHC I (see Figure 12C) and D) MHC II (see Figure 12D), on murine BMDCs after transfection with model mRNAs ± CRL, as well as E) the level of secreted IFNa in the supernatant of the transfected cells (see Figure 12E). The results show that CD11b+ CD11c+ BMDCs transfected with mRNA + CRL upregulate the expression of activation marker CD86 and CD69, and enhance presentation on MHC I and MHC II compared to non-adjuvanted mRNA. BMDCs were transfected with different mRNA constructsand controls (1000 ng / mL) in complex with Lipofectamine MessengerMax. 18 h after transfection, level of secreted IFNa was analyzed in the supernatant, as well as surface markers were stained and detected with flow cytometry on CD11b+ CD11c+ cells. The results represent six experiments and mean ± SEM is shown. *p < 0.05, paired t-test.

[0289] Example 10

[0290] Example 10 (see Figure 13) shows the percentage of A) EGFP expressing (see Figure 13A) and B) mCherry expressing CD11 b+ CD11c+ cells (see Figure 13B) after transfection of murine BMDCs with model mRNAs ± CRL. BMDCs were transfected with different mRNA constructs (1000 ng / mL) in complex with Lipofectamine MessengerMax. Protein expression has been quantified 18 h after transfection via flow cytometry. CD11 b+ CD11c+ positive fluorescent cells have been found at comparable levels for the mRNA and the RIG-I ligand (CRL)-adjuvanted mRNA transfected cells. The results represent six experiments and mean ± SEM is shown.

[0291] Example 11

[0292] Example 11 (see Figure 14) shows the surface expression of A) CD86 (see Figure 14A), B) CD69 (see Figure 14B), C) MHC I (see Figure 14C) and D) MHC II (see Figure 14D), of murine BMDCs after transfection with MCMV protein coding mRNAs ± CRL, as well as E) the level of secreted IFNa in the supernatant of the transfected cells (see Figure 14E). The results show that CD11b+ CD11c+ BMDCs transfected with mRNA + CRL upregulate the expression of activation marker CD69 and CD86, and enhance presentation on MHC I and MHC II compared to non-adjuvanted mRNA. BMDCs were transfected with different mRNA constructs and controls (1000 ng / mL) in complex with Lipofectamine 2000. Surface markers were stained 18 h after transfection and detected with flow cytometry on CD11 b+ CD11c+ cells. The level of secreted IFNa was analyzed in the supernatant 18 h after transfection. The results represent four experiments and mean ± SEM is shown. *p < 0.05, paired t-test.

[0293] Table 2: Sequences used herein in the Examples 1 to 8 (the SEQ ID NOs 1 to 34 are marked herein in Table 1 in bold, this means the respective SEQ ID NO is without the triphosphate and without the spacer X)Those sequences according to SEQ ID NOs: 1 - 34 are given in the respective sequence listing of the present application with “t” / ”T” instead of “u” / ”U” (where uracil is present) due to sequence listing program requirements. SEQ ID NO: 32 is displayed in the sequence listing of this application as CGG CGA GTT AG, without the respective modifications mentioned in Table 1 above, wherein “m” means a 2'-O-methyl-modification and “f” means a 2'-fluoro- modification (with modification see SEQ ID NO: 62). SEQ ID NO: 33 is displayed in the sequence listing of this application as GAT TGA GCG GC, without the respective modifications mentioned in Table 1 above, wherein “m” means a 2'-O-methyl-modification and “f’ means a 2'-fluoro-modification (with modification see SEQ ID NO: 63). SEQ ID NO: 34 is displayed in the sequence listing of this application as CTA ACT CGC CG, without the respective modifications mentioned in Table 1 above, wherein “m” means a 2'-O-methyl- modification (with modification see SEQ ID NO: 64). pppCGGCGAGUmllmAmG with comments to modifications corresponds to SEQ ID NO: 62 in the sequence listing (“m” means a 2'-O-methyl-modification and “f” means a 2'-fluoro-modification, see also Table 4),fGfAfUfllfGfAfGfCfGfGmC with comments to modifications corresponds to SEQ ID NO: 63 in the sequence listing (“m” means a 2'-O-methyl-modification and “f” means a 2'-fluoro- modification, see Table 4), pppCGGCGAGUmllmAmG X fGfAfllfllfGfA fGfCfGfGmC with comments to modifications and spacer corresponds to SEQ ID NO: 64 in the sequence listing (“m” means a 2'-O-methyl-modification and “f” means a 2'-fluoro-modification) and mC mil mA ACU CGC CG with comments to modifications corresponds to SEQ ID NO: 65 in the sequence listing. Also in those sequences t” / ”T” is shown as “u” / ”U” (where uracil is present) in the sequence listing due to sequence listing program requirements.

[0294] Table 3: Depiction of further sequences as used in the Examples. When sequences according to this Table 3 correspond to RNA (see sequence listing), t” / ”T” is shown in the sequence listing due to sequence listing program requirements. The mRNA of those sequences has been fully substituted with n1-methyl-pseudouridine (nlm^P) as explained in the Materials and Methods-section. When more than one sequence is cited in brackets in this Table, the respective RNA and DNA sequences are considered.

[0295] Table 4: Depiction of further sequences as used in the Examples 9 to 11. When sequences according to this Table 4 correspond to RNA (see sequence listing), t” / ”T” is shown in the sequence listing due to sequence listing program requirements. The mRNA of those sequences has been fully substituted with n1-methyl-pseudouridine (nlm^P) as explained in the Materials and Methods-section. When more than one sequence is cited in brackets in this Table, the respective RNA and DNA sequences are considered.

[0296] Table 5: Spacers X used as linker moieties L in the Examples (numbering of main chain atoms is given herein in Figure 11)REFERENCES:Mizushima Seiichi, Nagata Shigekazu, pEF-BOS, A powerful mammalian expression vector, Nucleic Acids Research, vol. 18, no. 17, 1990, page5322, https: / / doi.org / 10.1093 / nar / 18.17.5322.Tsuchiya S., Yamabe M., Yamaguchi Y., Kobayashi Y., Konno, T., Tada K., Establishment and characterization of a human acute monocytic leukemia cell line (THP-1), Int J Cancer, 1980, 26(2):171-6, doi: 10.1002 / ijc.2910260208.Ofir-Birin Yifat, Hila Ben Ami Pilo, Abel Cruz Camacho, Ariel Rudik, Anna Rivkin, Or- Yam Revach, Netta Nir, Tai Block Tamin, Paula Abou Karam, Edo Kiper, Yoav Peleg, Reinat Nevo, Aryeh Solomon, Tai Havkin-Solomon, Alicia Rojas, Ron Rotkopf, Ziv Porat, Dror Avni, Eli Schwartz, Thomas Zillinger, Gunther Hartmann, Antonella Di Pizio, Neils Ben Quashie, Rivka Dikstein, Motti Gerlic, Ana Claudia Torrecilhas, Carmit Levy, Esther N. M. Nolte-‘t Hoen, Andrew G. Bowie, and Neta Regev-Rudzki, Malaria parasites both repress host CXCL10 and use it as a cue for growth Acceleration, Nat Commun. 2021 ; 12: 4851, doi: 10.1038 / s41467- 021-24997-7.Tockary T. A., Abbasi S., Masai M., Yoshinaga N., Fukushima S., Kataoka K., llchida, S., Tethering designer short double-stranded RNA to mRNA for co-delivery of molecularly- targeted adjuvants and antigens towards cancer vaccination, Cold Spring Harbor Laboratory, 2022, doi: 10.1101 / 2022.01.18.476829.

Claims

CLAIMS:

1. A construct comprising- an mRNA-strand, and- at least one, preferably at least two, retinoic acid-inducible gene I (RIG-l)-ligand(s) comprising, a) a nucleic acid sequence being capable of binding to RIG-I, b) a linker moiety L comprising 3 to 24 main chain atoms, preferably comprising 6 to 18 main chain atoms, and c) a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand.

2. The construct according to claim 1, wherein the construct is a nucleic acid-construct.

3. The construct according to claim 2, wherein the nucleic acid-construct is an engineered nucleic acid-construct.

4. The construct according to any one of the preceding claims, wherein the construct comprises at least two of the retinoic acid-inducible gene I (RIG-l)-ligand(s).

5. The construct according to any one of the preceding claims, wherein the construct comprises two to four of the retinoic acid-inducible gene I (RIG-l)-ligand(s).

6. The construct according to any one of the preceding claims, wherein the construct comprises four of the retinoic acid-inducible gene I (RIG-l)-ligand(s).

7. The construct according to any one of the preceding claims, wherein the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s) is complementary to at least one nucleic acid sequence comprised in the mRNA strand.

8. The construct according to any one of the preceding claims, wherein the linker moiety L connects the nucleic acid sequence being able to bind to RIG-I with the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s).

9. The construct according to any one of the preceding claims, wherein the mRNA-strand comprises a coding sequence of a protein, a peptide or an antigen, preferably an antigen that is able to induce an immune response or preferably a cancer antigen.

10. The construct according to any one of the preceding claims, wherein the single-stranded nucleic acid sequence is capable of hybridizing to at least one nucleic acid sequence located in a non-coding nucleic acid sequence of the mRNA-strand.11 . The construct according to claim 10, wherein the non-coding nucleic acid sequence of the mRNA-strand is or comprises a 3'-untranslated region (UTR)-nucleic acid sequence or a 5'-untranslated region (UTR)-nucleic acid sequence or a poly-A-tail.

12. The construct according to any one of the preceding claims, wherein the mRNA-strand comprises a 3'-untranslated region (UTR)-nucleic acid sequence and the single-stranded nucleic acid sequence is capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand comprised in said 3'-untranslated region (UTR)-nucleic acid sequence.

13. The construct according to any one of the preceding claims, wherein the mRNA-strand comprises a 5'-untranslated region (UTR)-nucleic acid sequence and the single-stranded nucleic acid sequence is capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand comprised in said 5'-untranslated region (UTR)-nucleic acid sequence.

14. The construct according to claim 12 or 13, wherein the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s) is capable of hybridizing to the at least one nucleic acid sequence comprised in the 3'- or 5'-untranslated region (UTR)-nucleic acid sequence of the mRNA-strand via Watson-Crick-base-pairing.

15. The construct according to any one of the preceding claims, wherein the nucleic acid sequence being capable of binding to RIG-I comprises at least one triphosphate.

16. The construct according to any one of the preceding claims, wherein the nucleic acid sequence being able to bind to RIG-I comprises or consists of a double-stranded RNA.

17. The construct according to claim 16, wherein the double-stranded RNA of the nucleic acid sequence being able to bind to RIG-I is a blunt-ended double-stranded RNA.

18. The construct according to claim 16, wherein the double-stranded RNA of the nucleic acid sequence being able to bind to RIG-I further comprises an overhang.

19. The construct according to claim 18, wherein the overhang is a RNA-overhang or an overhang with a fluorophore or a lipophilic substitution.

20. The construct according to any one of claims 16 to 19, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at least one triphosphate at at least one strand of said double-stranded RNA, preferably at least one triphosphate at the 5'-end of one strand of said double-stranded RNA.21 . The construct according to any one of claims 16 to 20, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I has a length in the range of 8 to 20 nt.

22. The construct according to any one of claims 16 to 21 , wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I has a length in the range of 10 to 17 nt.

23. The construct according to any one of claims 16 to 22, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I has a length in the range of 10 to 15 nt.

24. The construct according to any one of claims 16 to 23, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I has a length in the range of 10 to 13 nt.

25. The construct according to any one of claims 16 to 24, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I has a length of 11 nt.

26. The construct according to any one of claims 16 to 25, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises one triphosphate at the 5'-end of one strand of said double-stranded RNA.

27. The construct according to any one of claims 16 to 26, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at least one modification(s).

28. The construct according to claim 27, wherein the at least one modification(s) is / are selected from the group consisting of O-methyl-group-modification(s), preferably 2'-O-methyl-group-modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group- modification(s), phosphor-thio-modification(s) and locked nucleic acid (LNA)- modification(s).

29. The construct according to any one of the preceding claims, wherein the mRNA-strand comprises at least one modification(s).

30. The construct according to claim 29, wherein the at least one modification(s) of the mRNA- strand is / are pseudo-uridine-modification(s).31 . The construct according to claim 27 or 28, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at at least one strand of the double-stranded RNA at least one modification(s), preferably at both strands of the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I.

32. The construct according to claim 31 , wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I further comprises at least one triphosphate at one end of at least one strand of said double-stranded RNA and at least one modification(s) is / are at the other end of the at least one strand of said double-stranded RNA.

33. The construct according to any one of claims 27 to 32, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at its 3'-end and / or at its 5'-end of at least one strand of the double-stranded RNA at least one modification(s), preferably at both strands of the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I.

34. The construct according to any one of claims 27 to 33, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at its 3'-end of at least one strand of the double-stranded RNA at least one modification(s).

35. The construct according to any one of claims 27 to 33, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at its 5'-end of at least one strand of the double-stranded RNA at least one modification(s).

36. The construct according to any one of claims 27 to 35, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-I comprises at its 5'-endand / or at its 3'-end at least one O-methyl-group-modification(s), preferably at least one 2'-O-methyl-group-modification(s).

37. The construct according to claim 36, wherein the double-stranded RNA of the nucleic acid sequence being capable of binding to RIG-1 comprises at both strands of the doublestranded RNA at least one O-methyl-group-modification(s).

38. The construct according to any one of the preceding claims, wherein the linker moiety L comprises 6 to 18 main chain atoms.

39. The construct according to any one of the preceding claims, wherein the linker moiety L comprises 8 to 16 main chain atoms.

40. The construct according to any one of the preceding claims, wherein the linker moiety L comprises 10 to 14 main chain atoms.

41. The construct according to any one of the preceding claims, wherein the linker moiety L comprises 12 main chain atoms.

42. The construct according to any one of the preceding claims, wherein the main chain atoms are carbon atoms that are optionally replaced by one or more heteroatoms selected from the group consisting of N, O and S.

43. The construct according to any one of the preceding claims, wherein the linker moiety L of the at least one RIG-l-ligand(s) is a polyalkylene-glycol-linker according to Formula IFormula I, wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7.

44. The construct according to any one of the preceding claims, wherein the linker moiety L of the at least one RIG-l-ligand(s) is a polyalkylene-glycol-linker according to Formula IIFormula II, wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7.

45. The construct according to claim 44, wherein the linker moiety L of the at least one RIG-I- ligand(s) is a polyalkylene-glycol-linker according to Formula II and wherein n is 6.

46. The construct according to any one of the preceding claims, wherein the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s) has a length in the range of 5 to 15 nt.

47. The construct according to claim 46, wherein the single-stranded nucleic acid sequence of the at least one RIG-l-ligand(s) has a length in the range of 8 to 13 nt.

48. The construct according to any one of the preceding claims for use as a medicament.

49. The construct according to any one of the preceding claims for use as a vaccine.

50. Pharmaceutical composition comprising the construct according to any one of claims 1 to 47.51 . The construct according to any one of claims 1 to 47 for use in a method of treatment or prevention of a disease.

52. The construct for use according to claim 50, wherein the disease is cancer or an infection, preferably a virus infection.

53. The construct for use according to claim 51 or 52, wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bonecancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof.

54. The construct for use according to claim 51 or 52, wherein the disease is a haematological tumour or a solid tumour.

55. The construct according to any one of claims 1 to 47 for use in a method of infiltrating immune cells, preferably T cells.

56. The construct according to any one of claims 1 to 47 for use in a method of turning cold into hot tumours.

57. Use of the construct according to any one of claims 1 to 47 as a vaccine.

58. Use of the construct according to any one of claims 1 to 47 as an immune adjuvant.

59. Use of the construct according to any one of claims 1 to 47 in the manufacture of a medicament for the treatment or prevention of a disease.

60. The use according to claim 59, wherein the disease is cancer or an infection, preferably a virus infection.

61. The use according to claim 59 or 60, wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof.

62. The use according to claim 60 or 61 , wherein the disease is a haematological tumour or a solid tumour.

63. The construct according to any one of claims 1 to 47 for use in a method of inducing an immune response.

64. The construct according to any one of claims 1 to 47, wherein the construct is an antigen recognizing construct or part of an antigen recognizing construct or is comprised in an antigen recognizing construct.

65. A method of treating a disease, comprising the step of administering a therapeutically effective amount of the construct according to any one of claims 1 to 47 to a subject in need thereof.

66. The method of treating according to claim 65, wherein the disease is cancer or an infection, preferably a virus infection.

67. The method of treating according to claim 66, wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof.

68. The method of treating according to claim 67, wherein the disease is a haematological tumour or a solid tumour.

69. The method of treating according to any one of claims 65 to 68, wherein the therapeutically effective amount of the construct is administered to the subject in need thereof parenteral, subcutaneous, intramuscular, intravenous, oral, intraperitoneal or intranasal.

70. A method of inducing an immune response, comprising the step of administering a therapeutically effective amount of the construct according to any one of claims 1 to 47 to a subject in need thereof.

71. The method according to claim 70, wherein the immune response is induced by activating retinoic acid-inducible gene I (RIG-I).

72. Method according to claim 71 , wherein the immune response is induced by stimulating retinoic acid-inducible gene I (RIG-I).

73. Method according to any one of claims 70 to 72, wherein the construct has or comprises an immune adjuvant function.

74. Method according to any one of claims 70 to 73, wherein the construct comprises at least one, preferably at least two, RIG-l-ligand(s).

75. Method according to any one of claims 70 to 74, wherein the construct comprises at least two RIG-l-ligand(s).

76. Method according to any one of claims 70 to 75, wherein the construct comprises at least three RIG-l-ligand(s).

77. The method according to any one of claims 70 to 76, wherein the construct comprises four RIG-l-ligand(s).

78. The method according to any one of claims 70 to 77, wherein the therapeutically effective amount of the construct is administered to the subject in need thereof parenteral, subcutaneous, intramuscular, intravenous, oral, intraperitoneal or intranasal.

79. A kit for use in medicine comprising the construct according to any one of claims 1 to 47.

80. The kit according to claim 79, wherein the kit is a diagnostic kit for selecting a patient for treatment of a disease, preferably wherein the disease is an infection or cancer.

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