COMPOSITION COMPRISING A COMPLEXED (m)RNA AND A NAKED mRNA FOR PROVIDING OR ENHANCING AN IMMUNOSTIMULATORY RESPONSE IN A MAMMAL AND USES THEREOF

Abstract

The present invention relates to an immunostimulatory composition comprising a) an adjuvant component, comprising or consisting of at least one (m)RNA, complexed with a cationic or polycationic compound, and b) at least one free mRNA, encoding at least one therapeutically active protein, antigen, allergen and/or antibody, wherein the immunostimulatory composition is capable to elicit or enhance an innate and optionally an adaptive immune response in a mammal. The inventive immunostimulatory composition may be a pharmaceutical composition or a vaccine. The invention furthermore relates to a method of preparation of the inventive immunostimulatory composition. The invention also relates to the use of the inventive immunostimulatory composition or its components (for the preparation of a pharmaceutical composition or a vaccine) for the treatment of various diseases. Finally, the invention relates to kits containing the inventive immunostimulatory composition, its components and/or the pharmaceutical composition or vaccine.

Claims

1. A method of stimulating an immune response in a subject in need thereof comprising administering to the subject an effective amount of a composition comprising (a) an adjuvant component, comprising or consisting of at least one RNA, complexed with protamine; and (b) at least one free mRNA, encoding at least one antigen for the prophylaxis and/or treatment of a disease.

2. The method of claim 1, wherein the at least one RNA of the adjuvant component, is selected from a short RNA oligonucleotide, a coding RNA, an mRNA, an immunostimulatory RNA, an siRNA, an antisense RNA, riboswitches, ribozymes or aptamers.

3. (canceled)

4. The method of claim 1, wherein the at least one free mRNA and the at least one RNA of the adjuvant component are identical to each other.

5. The method of claim 1, wherein the at least one free mRNA and the at least one RNA of the adjuvant component are different from each other.

6. The method of claim 1, wherein the N/P ratio of the RNA to the protamine in the adjuvant component is in the range of 0.1-10.

7. The method of claim 1, wherein the molar ratio of the RNA of the adjuvant component to the at least one free mRNA of the second component is 1:1 to 1:4.

8. The method of claim 1, wherein the at least one free mRNA and/or the at least one RNA of the adjuvant component are GC-stabilized.

9. The method of claim 8, wherein the G/C content of the coding region of the GC-stabilized RNA is increased compared with the G/C content of the coding region of the native RNA, the coded amino acid sequence of the GC-stabilized modified RNA not being altered compared with the encoded amino acid sequence of the native modified RNA.

10. (canceled)

11. The method of claim 1, wherein the at least one free mRNA encodes an antigen, selected from 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha5beta1-Integrin, alpha5beta6-Integrin, alpha-methylacyl-coenzyme A racemase, ART-4, B7H4, BAGE-1, BCL-2, BING-4, CA 15-3/CA 27-29, CA 19-9, CA 72-4, CA125, calreticulin, CAMEL, CASP-8, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA, CLCA2, CML28, Coactosin-like protein, Collagen XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10/MAGE-B1, DAM-6/MAGE-B2, EGFR/Her1, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, EZH2, FGF-5, FN, Fra-1, G250/CAIX, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, HAGE, HAST-2, hepsin, Her2/neu/ErbB2, HERV-K-MEL, HNE, homeobox NKX 3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205, KK-LC-1, KM-HN-1, LAGE-1, Livin, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/Melan-A, MART-2, matrix protein 22, MC1R, M-CSF, Mesothelin, MG50/PXDN, MMP 11, MN/CA IX-antigen, MRP-3, MUC1, MUC2, NA88-A, N-acetylglucos-aminyltransferase-V, Neo-PAP, NGEP, NMP22, NPM/ALK, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT, OS-9, osteocalcin, osteopontin, p15, p15, p190 minor bcr-abl, p53, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1-Kinase, Pin1, POTE, PRAME, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, RAGE-1, RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin, survivin-2B, TA-90, TAG-72, TARP, TGFb, TGFbRII, TGM-4, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP-2/INT2, Trp-p8, Tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, WT1; or is selected from mutant antigens expressed in cancer diseases, including alpha-actinin-4/m, ARTC1/m, bcr/abl, beta-Catenin/m, BRCA1/m, BRCA2/m, CASP-5/m, CASP-8/m, CDC27/m, CDK4/m, CDKN2A/m, CML66, COA-1/m, DEK-CAN, EFTUD2/m, ELF2/m, ETV6-AML1, FN1/m, GPNMB/m, HLA-A*0201-R170I, HLA-A11/m, HLA-A2/m, HSP70-2M, KIAA0205/m, K-Ras/m, LDLR-FUT, MART2/m, ME1/m, MUM-1/m, MUM-2/m, MUM-3/m, Myosin class I/m, neo-PAP/m, NFYC/m, N-Ras/m, OGT/m, OS-9/m, p53/m, Pml/RARa, PRDX5/m, PTPRK/m, RBAF600/m, SIRT2/m, SYT-SSX-1, SYT-SSX-2, TEL-AML1, TGFbRII, and TPI/m.

12. The method of claim 1, wherein the at least one free mRNA encodes: a) at least one, two, three or four different antigens of the following group of antigens: (1) PSA (Prostate-Specific Antigen)=KLK3 (Kallikrein-3), (2) PSMA (Prostate-Specific Membrane Antigen), (3) PSCA (Prostate Stem Cell Antigen), (4) STEAP (Six Transmembrane Epithelial Antigen of the Prostate), or b) at least one, two, three, four, five, six, seven, eight, nine, ten eleven or twelve different antigens of the following group of antigens: hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1, and/or MAGE-C2, or a combination thereof.

13. A pharmaceutical composition, comprising an immunostimulatory composition comprising (a) an adjuvant component, comprising at least one RNA, complexed with protamine; (b) at least one free mRNA, encoding at least one antigen; and (c) a pharmaceutically acceptable carrier, adjuvant, and/or vehicle.

14-15. (canceled)

16. The method of claim 1, wherein the subject has a disease selected from cancers or tumor diseases, autoimmune diseases, infectious diseases, viral, bacterial or protozoological infectious diseases or allergic diseases.

17. A kit comprising the pharmaceutical composition according to claim 13, and technical instructions with information on the administration and dosage of the pharmaceutical composition.

18. The method of claim 1, wherein the weight ratio of the at least one RNA to protamine in the adjuvant component is 2:1 to 3:1.

19. The method of claim 1, wherein the molar ratio of the RNA of the adjuvant component to the at least one free mRNA of the second component b) is between 1:1 and 1:3.

20. The method of claim 1, wherein the molar ratio of the RNA of the adjuvant component to the at least one free mRNA of the second component b) is between 1:2 and 1:4.

21. The method of claim 1, wherein the at least one free mRNA encodes an antigen selected from a cancer cell antigen, an autoimmune antigen, an infectious disease antigen or an allergic antigen.

22. The method of claim 1, wherein the at least one free mRNA encodes an infectious disease antigen selected from a viral antigen, a protozoal antigen, or a bacterial antigen.

23. The method of claim 1, wherein the weight ratio of the at least one RNA to protamine in the adjuvant component is 2:1.

24. The method of claim 1, wherein the N/P ratio of the RNA to the protamine in the adjuvant component is 0.3 to 4.

25. A composition comprising: (a) an adjuvant component, comprising or consisting of at least one RNA complexed with protamine; and (b) at least one free mRNA, encoding at least one therapeutically active protein, antigen and/or antibody.

Description

FIGURES

[0333] The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0334] FIG. 1: depicts the results of the expression of luciferase after intradermal injection of mRNA encoding luciferase in vivo, wherein a composition comprising an amount of 10 g free mRNA coding for luciferase (Pp Luc) with or without combination with LacZ-mRNA (wt LacZ) complexed with protamine (2:1 and 1:1) were prepared and injected intradermally into the ear pinna. As can be seen, LacZ-mRNA:protamine complexes, which have been formulated in a ratio of 1:1 or 2:1, respectively, had no negative influence on the expression of luciferase, i.e. no free protamine was present in the solution or only a very small amount, which had nor negative influence on the translation. Accordingly, no complex formation between protamine and ppLuc mRNA was possible, which indicates the stability of the already formed LacZ-mRNA:protamine complexes.

[0335] FIG. 2: shows the results of a vaccination reaction in the E.G7-OVA-model for therapeutic purposes. For the experiment, 300,000 E.G7-OVA tumor cells were implanted in C57 BL/6 mice and the mice were vaccinated 8 times within 2 weeks with the inventive immunostimulatory composition comprising 20 g GC-enriched mRNA coding for Gallus gallus Ovalbumin. In a first experiment, either an RNA was used, which was entirely complexed with protamine in a ratio of 3:1 or an RNA, wherein the complexed RNA was mixed with free RNA in a ratio of 1:1, 1:4 and 1:8 (w/w). As can be seen in FIG. 2, free RNA as well as protamine alone do not exhibit any effect on tumor growth in comparison to the buffer control (Ringer-lactate solution). Surprisingly, a vaccination with a protamine complexed RNA in a ratio of 3:1 (RNA:protamine) significantly reduces tumor growth. Addition of free RNA enhances the tumor response even further, wherein a ratio of more than 1:8 (complexed RNA: free RNA), e.g. 1: 4 or even 1:1, has been proven to be particularly advantageous.

[0336] FIG. 3: describes the results of a vaccination reaction in the E.G7-OVA-model for therapeutic purposes similar to the Experiment in FIG. 2. For this second experiment either an RNA was used, which was entirely complexed with protamine in a ratio of 3:1 or an RNA according to an improved protocol, wherein the complexed RNA was mixed with free RNA in a ratio of RNA:protamine 3:1+free RNA (1:1)). As can be seen in FIG. 3, the combination of complexed RNA and free RNA, particular in the described ratios, leads to an improved tumor defense.

[0337] FIG. 4: shows the statistical (mathematical) analysis of the experiment according to FIG. 3. FIG. 4 particularly shows that the difference between the groups are significant. The statistical (mathematical) analysis was carried out with the GraphPad Prism Software and the p values were determined using the Mann Whitney test. The analysis underlines the results shown in FIG. 3.

[0338] FIG. 5: depicts the results of the detection of immunostimulatory properties and the stimulation of hPBMC with mRNA (CAP-GgOva(GC)-muag-A70-C30) (A70 and C30 disclosed as SEQ ID NOS 141-142, respectively). For this experiment 210.sup.5 hPBMCs were seeded in 200 l medium per well in 96 well plates and 50 l of the inventive composition, comprising 10 g mRNA coding for Gallus gallus ovalbumin were added to stimulate cytokine release over night at 37 C. The secretion of cytokines (TNFalpha and IL6) in hPBMCs with compositions comprising complexed and free RNA, showed a significant elevation in an ELISA detection indicating good immunostimulatory properties.

[0339] FIG. 6: describes the results of the induction of a humoral immune response in vivo, wherein C57 BL/6 mice were vaccinated 8-times with each 16 g GC-enrichen mRNA (CAP-GgOva(GC)-muag-A70-C30) (A70 and C30 disclosed as SEQ ID NOS 141-142, respectively) coding for Gallus gallus Ovalbumine or an irrelevant control RNA (pB-Luc RNA). As can be seen in FIG. 6, the combination of a complexed RNA with a free RNA, particularly in a ratio of (1:1) leads to an elevated humoral immune response compared to the vaccination with an entirely complexed RNA.

[0340] FIG. 7: shows the mRNA sequence according to SEQ ID NO: 120, which exhibits a length of 1365 nucleotides and was termed CAP-GgOva(GC)-muag-A70-C30 (A70 and C30 disclosed as SEQ ID NOS 141-142, respectively). The mRNA sequence CAP-GgOva(GC)-muag-A70-C30 (A70 and C30 disclosed as SEQ ID NOS 141-142, respectively) contained following sequence elements: [0341] GC-optimized sequence for a better codon usage and stabilization [0342] muag (mutated alpha-globin-3-UTR) [0343] 70adenosine at the 3-terminal end (poly-A-tail) (SEQ ID NO: 141), [0344] 30cytosine at the 3-terminal end (poly-C-tail) (SEQ ID NO: 142).
The ORF is indicated in italic letters, muag (mutated alpha-globin-3-UTR is indicated with a dotted line, the poly-A-tail is underlined with a single line and the poly-C-tail is underlined with a double line.

[0345] FIG. 8: depicts the mRNA sequence according to SEQ ID NO: 121, which exhibits a length of 1816 nucleotides and was termed T7TS-Ppluc(wt)-A70 (A70 disclosed as SEQ ID NO: 141). The coding sequence (CDS) of the entire mRNA sequence is indicated in italic letters, the poly-A-tail is underlined with a single line.

[0346] FIG. 9: shows the statistical analysis of the expression of luciferase in Balb/c mice. The statistical analysis was carried out with the GraphPad Prism Software and the p values were determined using the Mann Whitney test. The results in FIG. 9 show that the RNA complexed according to the invention (2:1 (50%)+free (50%) exhibits the same expression when compared to naked RNA and RNA which is complexed in the ratio of 4:1 with protamine. As can be seen, differences between the relevant groups are not significant (ns). Thus, an immune stimulation can be efficiently provided with the inventive complexed RNA wherein expression level is maintained compared to naked RNA and RNA which is complexed in the ratio of 4:1 with protamine (see als FIG. 10).

[0347] FIG. 10: depicts the statistical analysis of induction of IL-12 in Balb/c mice. The statistical analysis was carried out with the GraphPad Prism Software and the p values were determined using the Mann Whitney test. The results show that the difference between the inventive complexed RNA (2:1 (50%+free (50%)) and the group 4:1 (100%) which comprises the same amount of RNA and protamine is significant. Thus, an immune stimulation can be efficiently provided with the inventive complexed RNA wherein expression level is maintained compared to naked RNA and RNA which is complexed in the ratio of 4:1 with protamine (see also FIG. 9).

[0348] FIG. 11: shows the induction of IgG2a antibodies against the Influenza antigen hemagglutinin (HA). Therefore mice were vaccinated 2 times with 20 g GC-enriched mRNA coding for hemagglutinin (HA) naked or formulated with protamine hydrochloride (Prot. Val) or protamine sulphate (Prot. Leo) as indicated. As can be seen in FIG. 11, the combination of a complexed RNA with free RNA leads to an elevated humoral immune response compared to the vaccination with an entirely complexed RNA. Compared to naked RNA, complexed RNA leads to higher IgG2a antibody titers, an indicator of the Th1 driven response. Complexation of RNA with protamine hydrochloride (Protamine Valeant) has a stronger effect than complexation with protamine sulphate.

[0349] FIG. 12: illustrates the induction of IgG2a-specific antibodies against the Influenza antigen HA over 15 weeks after immunization. Therefore mice were vaccinated 2 times with 20 g GC-enriched mRNA coding for hemagglutinin (HA) naked or complexed with protamine (RNA:Protamine 2:1+free RNA (1:1) (w/w)) and IgG2a antibodies were measured at the indicated time points. Sera from different time points after immunization were analysed by ELISA. Hemagglutinin-specific antibody titers of the IgG2a subtype are plotted for the groups treated with buffer (80% RiLa), naked or complexed HA mRNA.

[0350] FIG. 13: depicts the induction of antibodies against the influenza antigen HA by HAI assay. Therefore mice were vaccinated 2 times with 20 g GC-enriched mRNA coding for hemagglutinin (HA) naked or formulated with protamine (RNA:Protamine 2:1+free RNA (1:1) (w/w)) and the HAI assay was performed at the indicated time points.

[0351] FIG. 14: shows the mRNA sequence (SEQ ID NO: 124) encoding the pathogenic antigen hemagglutinin (HA) from influenza virus.

EXAMPLES

[0352] The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

Example 1Preparation of mRNA Construct Encoding Pp Luciferase (Photinus pyralis)

[0353] For the following experiments a DNA sequence, encoding Pp luciferase (Photinus pyralis) and corresponding to the respective mRNA encoding Pp luciferase sequences as used herein, was prepared and used for subsequent transfection and vaccination experiments. Thereby, the DNA sequence corresponding to the native Pp Luciferase encoding mRNA was modified with a poly-A-tag (A70) (SEQ ID NO: 141) leading to SEQ ID NO: 121 (see FIG. 8). The final construct had a length of 1816 nucleotides and was termed T7TS-Ppluc(wt)-A70 (A70 disclosed as SEQ ID NO: 141).

Example 2Preparation of mRNA Construct Encoding Gallus gallus Ovalbumin

[0354] For the following experiments a further DNA sequence, encoding Gallus gallus Ovalbumin and corresponding to the respective mRNA sequences, was prepared and used for subsequent transfection and vaccination experiments. Thereby, the DNA sequence corresponding to the native Gallus gallus Ovalbumin encoding mRNA was GC-optimized for a better codon-usage and stabilization. Then, the DNA sequence corresponding to the coding Gallus gallus Ovalbumin mRNA sequence was transferred into an RNActive construct, which has been modified with a poly-A-tag and a poly-C-tag (A70-C30) (A70 and C30 disclosed as SEQ ID NOS 141-142, respectively). The final construct had a length of 1365 nucleotides and was termed CAP-GgOva(GC)-muag-A70-C30 (A70 and C30 disclosed as SEQ ID NOS 141-142, respectively). It contained following sequence elements: [0355] GC-optimized sequence for a better codon usage and stabilization [0356] muag (mutated alpha-globin-3-UTR) [0357] 70adenosine at the 3-terminal end (poly-A-tail) (SEQ ID NO: 141), [0358] 30cytosine at the 3-terminal end (poly-C-tail) (SEQ ID NO: 142).

[0359] The corresponding mRNA sequence is shown in FIG. 7 (see SEQ ID NO: 120).

Example 3In Vitro-Transcription Experiments

[0360] The recombinant plasmid DNA was linearized and subsequently in vitro transcribed using the T7 RNA polymerase. The DNA template was then degraded by DNAseI digestion. The RNA was recovered by LiCl precipitation and further cleaned by HPLC extraction (PUREMessenger, CureVac GmbH, Tbingen, Germany).

Example 4Making of the Inventive Composition

[0361] The mRNA used in the experiments below was complexed with protamine by addition of protamine to the mRNA in the indicated ratios (1:1-1:4) (w/w). After incubation for 10 min, the free RNA was added.

Example 5Expression of Luciferase after Intradermal Injection of mRNA Encoding Luciferase In Vivo

[0362] In this experiment the influence of compositions comprising readily prepared mRNA:protamine complexes and/or free mRNA on the translation was investigated. Therefore, a composition comprising an amount of 10 g free mRNA coding for luciferase (Pp Luc) with or without combination with LacZ-mRNA complexed with protamine (2:1 and 1:1) were prepared and injected intradermally into the ear pinna.

[0363] The mRNA encoding Pp Luciferase was prepared as described above. The composition to be administered contained either no further complexed RNA, lacZ-mRNA:protamine in a concentration of 2:1 or lacZ-mRNA:protamine in a concentration of 1:1. As a control, a composition was administered containing no mRNA encoding Pp Luciferase (Photinus pyralis). Protamine was used as a control. In order to prepare these compositions, lacZ-mRNA was formulated in a first step with protamine in different amounts and ratios (2:1 und 1:1 (w/w)) and added as a first component. Then, free Pp Luc mRNA was added 10 minutes later to the composition.

[0364] After 24 h the ear pinna were removed and frozen in liquid nitrogen. For homogenization, the samples were placed in a TissueLyser for 3 min at 30 s.sup.1. Then 800 l lysis-buffer (25 mM Tris-HCl pH (7.5-7.8); 2 mM EDTA; 10% (w/v) Glycerol; 1% (w/v) Triton-X-100; 2 mM DTT; 1 mM PMSF) is added and samples are placed again in the TissueLyser for 6 min at 30 s.sup.1. Samples are centrifuged for 10 min at 13500 rpm and 4 C. The supernatant is removed and stored at 80 C. until luciferase measurement. Supernatants were mixed with Luciferin Buffer (25 mM Glycylglycin, 15 mM MgSO.sub.4, 5 mM ATP, 62.5 M Luciferin) and the luminescence was measured with a luminometer (Lumat LB 9507; Berthold Technologies, Bad Wildbad, Germany).

[0365] As a result (see FIG. 1), LacZ-mRNA:protamine complexes, which have been formulated in a ratio of 1:1 or 2:1, respectively, had no negative influence on the expression of luciferase, i.e. no free protamine was present in the solution or only a very small amount, which had no negative influence on the translation. Accordingly, no complex formation between protamine and ppLuc mRNA was possible, which indicates the stability of the already formed LacZ-mRNA:protamine complexes.

Example 6Vaccination in the e.g7-OVA-Model for Therapeutic Purposes

General Method:

[0366] 300000 E.G7-OVA tumor cells were implanted in C57 BL/6 mice. In the following 3 weeks the mice were vaccinated 8 times within 3 weeks with the inventive composition comprising 20 pg GC-enriched mRNA coding for Gallus gallus Ovalbumine. The tumor size was measured 18 days after the implantation of the tumor cells.

A) First Experiment

[0367] 300000 E.G7-OVA tumor cells were implanted into C57 BL/6-mice. Within the following two weeks the mice were vaccinated 8-times with each 20 g GC-enriched mRNA encoding Gallus gallus Ovalbumin. For this experiment, either an RNA was used, which was entirely complexed with protamine in a ratio of 3:1 or an RNA, wherein the complexed RNA was mixed with free RNA in a ratio of 1:1, 1:4 and 1:8 (w/w). [0368] The results are shown in FIG. 2. As can be seen in FIG. 2, free RNA as well as protamine alone does not exhibit any little effect on tumor growth in comparison to the buffer control (Ringer-lactate solution). Surprisingly, a vaccination with a protamine complexed RNA in a ratio of 3:1 (RNA:protamine) significantly reduces tumor growth. Addition of free RNA enhances the tumor response even further, wherein a ratio of more than 1:8 (complexed RNA: free RNA), e.g. 1:4 or even 1:1, has been proven to be particularly advantageous.

B) Second Experiment

[0369] 300000 E.G7-OVA tumor cells were implanted into C57 BL/6-mice. Within the following three weeks the mice were vaccinated 8-times with each 20 g GC-enriched mRNA encoding Gallus gallus Ovalbumin. For this experiment, either an RNA was used, which was entirely complexed with protamine in a ratio of 3:1 or an RNA according to an improved protocol, wherein the complexed RNA was mixed with free RNA in a ratio of RNA:Protamin 3:1+free RNA (1:1)). [0370] The results of this experiment are depicted in FIG. 3. As can be seen in FIG. 3, the combination of complexed RNA and free RNA, particular in the described ratios, leads to an improved tumor defense. [0371] The statistical (mathematical) analysis was carried out with the GraphPad Prism Software. [0372] The p values were determined using the Mann Whitney test (see FIG. 4).

Example 7Detection of Immunostimulatory Properties, and Stimulation of hPBMC with mRNA

[0373] For this experiment hPBMC were isolated by centrifugation on Ficoll (20 min at 2000 rpm) and subsequently washed two times in PBS. hPBMC were then resuspended in FCS, 10% DMSO at a density of 510.sup.7/ml. 1 ml aliquots were frozen and stored at 80 C.

[0374] Prior to the experiment, hPBMc were thawed by resuspending in PBS, followed by two washes in PBS. hPBMC were then suspended in X-Vivo 15, 1% glutamine, 1% Pen/Strep at a density of 110.sup.6/ml. After seeding hPBMCs at 210.sup.5 per well in 96 well plates, 50 l of the inventive composition, comprising 8 g mRNA coding for Gallus gallus ovalbumin were added to stimulate cytokine release over night at 37 C.

[0375] Therefore, human PBMCs were incubated for 20 hours with RNA, encoding Gallus gallus Ovalbumin (OVA), complexed with protamine (3:1) plus 50% free RNA (formulated as follows: RNA:protamine 3:1+free RNA (1:1) (w/w)). The secretion of cytokines (TNFalpha and IL6) was measured and detected in the supernatant using Standard ELISA.

[0376] For the TNFalpha and IL6 quantification (ELISA) Maxisorb plates were coated over night (4 C.) with capture antibody (1 g/ml) and subsequently blocked with 1% BSA for 1 hour at room temperature (RT). After three washes with 0.05% Tween, 50 l (TNF) or 50 l (IL-6) hPBMC supernatant, adjusted with blocking buffer 15 to 100 l, were added to the wells. Binding was allowed to proceed for two hours (RT). The plate was then washed and 100 l streptavidin-conjugated horseradish peroxidise was added. After incubation for 30 minutes and washing a colorimetric substrate (TMB, Perbio Science) was added. Optical densities were measured at 450 nm using a Tecan ELISA plate reader. All incubations were performed at room temperature and washing steps include at least 3 steps using PBS/Tween20 (0.05% v/v).

[0377] 100 l/well of a mixture of Strept-HRP (diluted 1/1000) and biotinylated detection antibody (0.5 g/ml) were added. Incubation for one hour at RT was followed by three washes with 0.05% Tween. Finally, 100 l/well of Amplex Red HRP substrate (50 M), 0.014% H.sub.2O.sub.2 were added. Fluorescence was measured in a Spectramax Gemini plate reader (Ex 540 nm, Em 590 nm, cutoff 590 nm).

[0378] The results are shown in FIG. 5. As can be seen in FIG. 5, the compositions comprising complexed and free RNA exhibit an immunostimulatory property, which is reflected by a significant secretion of TNFalpha and IL-6 in hPBMCs.

Example 8Induction of a Humoral Immune Response

[0379] C57 BL/6 mice were vacinated 8-times with each 16 g GC-enriched mRNA coding for Gallus gallus Ovalbumine or an irrelevant control RNA (pB-Luc RNA). Thereby, the RNA was either formulated entirely with protamine in a ratio of 2:1 or the ratio was according to an improved protocol RNA:Protamin 2:1+freie RNA (1:1) (w/w))). 2 weeks after the last vaccination blood samples were collected and expression of Ovalbumin-specific antibodies was determined.

[0380] For the detection of antigen-specific antibodies MaxiSorb plates (Nalgene Nunc International) were coated with Antigen (Ovalbumine, recombinant protein). After blocking with 1PBS, 0.05% Tween und 1% BSA the plates were incubated with sera of the mice for 4 hours at room temperature. Subsequently the biotin-coupled secondary antibody was added. After washing the plate was incubated with horseradish peroxidise and the enzyme activity was determined by measuring the conversion of the substrate (2,2-azino-bis(3-ethyl-benzthiazoline-6-sulfonsure) (OD 450 nm). Optical densities were measured at 450 nm using a Tecan ELISA plate reader.

[0381] The results are shown in FIG. 6. As can be seen in FIG. 6, the combination of a complexed RNA with a free RNA leads to an elevated humoral immune response compared to the vaccination with an entirely complexed RNA.

Example 9Statistical Analysis of the Expression of Luciferase in Balb/c Mice

[0382] In this experiment the influence of different formulation strategies with protamine on the translation of luciferase was investigated. Per group 2 mices were injected on 4 different sites intradermally with [0383] (1) a composition comprising 50% protamine-complexed (2:1) Luc-RNA in combination with 50% free RNA, [0384] (2) a composition comprising Luc-RNA complexed with protamine in the ration 4:1, [0385] (3) 100% free Luc-RNA, or [0386] (4) Ringer-Lactate buffer as control.

[0387] Each sample comprised 10 g mRNA coding for luciferase (Luc-RNA, i.e. the above described construct T7TS-Ppluc(wt)-A70 according to SEQ ID NO: 121) (A70 disclosed as SEQ ID NO: 141) in 50 l Ringer-Lactate buffer. The first and the second group comprised also the same amount of protamine, but they were different formulated. The immunostimulatory composition of group (1) was prepared according to the invention.

[0388] The results are shown in FIG. 9. FIG. 9 shows the statistical analysis of the expression of luciferase in Balb/c mice. The statistical analysis was carried out with the GraphPad Prism Software and the p values were determined using the Mann Whitney test. The results in FIG. 9 show that the RNA complexed according to the invention (2:1 (50%)+free (50%), group (1)) exhibits the same expression when compared to naked RNA and RNA which is complexed in the ratio of 4:1 with protamine (groups (2) and (3)). As can be seen, differences between the relevant groups are not significant (ns). Thus, an immune stimulation can be efficiently provided with the inventive immunostimulatory composition wherein expression level is maintained compared to naked RNA and RNA which is complexed in the ratio of 4:1 with protamine (see also FIG. 10).

Example 10Statistical Analysis of Induction of IL-12 in Balb/c Mice

[0389] For this experiment 40 g mRNA coding for Luciferase (Luc-RNA, i.e. the above described construct T7TS-Ppluc(wt)-A70 according to SEQ ID NO: 121) (A70 disclosed as SEQ ID NO: 141) in the following compositions: [0390] (1) 2:1 (50%)+free (50%) comprised 20 ig Luc-RNA complexed with protamine (2:1) (w/w) and 20 g free Luc-RNA (i.e. an inventive immunostimulatory composition), [0391] (2) 4:1 (100%) comprised 40 g Luc-RNA complexed with protamin (4:1) (w/w), [0392] (3) 40 g free Luc-RNA, [0393] (4) 10 g protamine, and [0394] (5) 800 l RiLa (all samples were dissolved in Ringer-Lactate buffer to a final volume of 800 l) were intravenously injected into the tail vein of Balb/c mice (4 mice per group). After 4 hours, blood was taken by puncture of the retro-orbital veins and serum was used for cytokine (IL-12) ELISA. The ELISA was carried out as described for Example 7.

[0395] The results are shown in FIG. 10. FIG. 10 depicts the statistical analysis of induction of IL-12 in Balb/c mice according to Example 10. The statistical analysis was carried out with the GraphPad Prism Software and the p values were determined using the Mann Whitney test. The results show that the difference between the inventive immunostimulatory composition (2:1 (50%)+free (50%)) and the group 4:1 (100%), which comprises the same amount of RNA and protamine, is in fact significant. Thus, an immune stimulation can be efficiently provided with the inventive immunostimulatory composition wherein expression level is maintained compared to naked RNA and RNA, which is complexed in the ratio of 4:1 with protamine (see also FIG. 9).

Example 11Induction of a Humoral Immune Response Against a Viral Antigen

Vaccination:

[0396] BALB/c mice were vaccinated twice with 20 g GC-enriched mRNA coding for hemagglutinin (HA) of Influenza A/Puerto Rico/8/34 (PR8) or injection buffer (80% Ringer Lactate). Thereby, the RNA was either formulated entirely with protamine in a ratio of 2:1 or the ratio was according to the invention RNA:Protamin 2:1+free RNA (1:1) (w/w). For formulations, two different protamines were tested, protamine hydrochloride (Protamin Valeant) and protamine sulphate (Protamin LEO).

Detection of Specific Antibodies:

[0397] At different time points after last vaccination blood samples were collected and expression of hemagglutinin-specific antibodies was determined by ELISA (FIGS. 11 and 12) or hemagglutination inhibition assay (HAI) (FIG. 13).

Detection of Antigen-Specific Antibodies by ELISA:

[0398] For the detection of antigen-specific antibodies by ELISA, MaxiSorb plates (Nalgene Nunc International) were coated with antigen (inactivated PR8). After blocking with 1PBS, 0.05% Tween und 1% BSA the plates were incubated with sera of the mice for 4 hours at room temperature. Subsequently the biotin-coupled secondary antibody was added. After washing the plate was incubated with horseradish peroxidise and the enzyme activity was determined by measuring the conversion of the substrate (2,2-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (OD 405 nm). Optical densities were measured at 405 nm using a Tecan ELISA plate reader. The results of analysis of sera obtained two weeks after immunization are shown in FIG. 11. As can be seen in FIG. 11, the combination of a complexed RNA with free RNA leads to an elevated humoral immune response compared to the vaccination with an entirely complexed RNA. Compared to naked RNA, complexed RNA leads to higher IgG2a antibody titers, an indicator of the Th1 driven response. Complexation of RNA with protamine hydrochloride (Protamine Valeant) has a stronger effect than complexation with protamine sulphate.

[0399] In FIG. 12, sera from different time points after immunization were analysed by ELISA. Hemagglutinin-specific antibody titers of the IgG2a subtype are plotted for the groups treated with buffer (80% RiLa), naked or complexed HA mRNA. The complexation was done with Protamin Valeant following improved protocol RNA:Protamin 2:1+free RNA (1:1) (w/w).

Detection of Antigen-Specific Antibodies by HAI Assay:

[0400] Sera of immunized mice were also analysed by HAI assay. In an HAI assay, antibodies that neutralize the virus by blocking the interaction of the viral hemagglutinin and the sialic acid on the host cell are detected.

[0401] Sera were inactivated at 56 C. for 10 min to destroy complement and HAI inhibitors. Sera were further incubated with kaolin for 20 min and preadsorbed to chicken red blood cells for 30 min to remove unspecific factors that influence hemagglutination. Pre-treated serum samples were added to a 96 well, U-bottom plate in serial dilution and duplicates. 25 l containing 4 hemagglutinating units of inactivated PR8 in PBS and 50 l of 0.5% chicken red blood cells were then added and incubated at room temperature for 45 min. Endpoint HAI titers were defined as the reciprocal of the highest serum dilution that completely inhibited hemagglutination of the red blood cells. Titers of sera from different time points after immunization are plotted for the groups treated with buffer (80% RiLa), naked or complexed HA mRNA. The complexation was done with Protamin Valeant and the improved protocol RNA:Protamin 2:1+free RNA (1:1) (w/w). A titre of 40 is assumed to be protective in case of influenza infection. Mice immunized with complexed HA RNA show an enduring HAI titer of more than 40, whereas naked HA mRNA led to titers in average lower than the protective titer of 40.