Composition comprising a complexed (M)RNA and a naked mRNA for providing or enhancing an immunostimulatory response in a mammal and uses thereof

1. An immunostimulatory composition comprising:

a) an adjuvant component, comprising at least one mRNA, complexed with protamine wherein the weight ratio of the at least one mRNA to protamine in the adjuvant component is 2:1 to 3:1; and

b) at least one free mRNA, encoding at least one antigen, wherein the molar ratio of the RNA of the adjuvant component to the at least one free mRNA of the second component b) is 1:1 to 1:4.

2. The immunostimulatory composition according to claim 1 , wherein the at least one free mRNA and the at least one mRNA of the adjuvant component are identical to each other.

3. The immunostimulatory composition according to claim 1 , wherein the at least one free mRNA and the at least one mRNA of the adjuvant component are different from each other.

4. The immunostimulatory composition according to claim 1 , wherein the N/P ratio of the mRNA to the protamine in the adjuvant component is in the range of 0.1-10.

5. The immunostimulatory composition according to claim 1 , wherein the at least one free mRNA and/or the at least one mRNA of the adjuvant component are GC-stabilized.

6. The immunostimulatory composition according to claim 5 , 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.

7. The immunostimulatory composition according to 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-acetylglucosaminyltransferase-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, 8400, SAGE, SART-1, SART-2, SART-3, SCC, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin, survivin-213, TA-90, TAG-72, TARP, TGFb, TGFbR11, TGM-4, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP-2/INT2, Trp-p8, Tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, WT1; 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 1/m, neo-PAP/m, NFYC/m, N-Ras/m, OGT/m, OS-9/m, p53/m, Pml/RARa, PRDX5/m, PTPRX/m, RBAF600/m, SIRT2/m, SYT-SSX-1, SYT-SSX-2, TEL-AML1, TGFbRII, and TPI/m.

8. The immunostimulatory composition according to claim 1 , wherein the at least one free mRNA encodes: (i) 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 (ii) 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.

9. A pharmaceutical composition, comprising an immunostimulatory composition according to claim 1 and optionally a pharmaceutically acceptable carrier, adjuvant, and/or vehicle.

10. A pharmaceutical composition according to claim 9 , wherein the pharmaceutical composition is formulated to provide immune stimulation.

11. A method for preparing an immunostimulatory composition as defined according to claim 1 , comprising following steps: (i) preparing the adjuvant component by mixing in a specific ratio the at least one mRNA and the protamine; and (ii) preparing the immunostimulatory composition by adding in a specific ratio of the at least one free mRNA to the adjuvant component prepared according to step (i).

12. A kit comprising the immunostimulatory composition according to claim 1 , and a pharmaceutically acceptable carrier and technical instructions with information on the administration and dosage of the immunostimulatory composition and/or the pharmaceutically acceptable carrier.

13. The immunostimulatory composition according to claim 1 , wherein the molar ratio of the mRNA of the adjuvant component to the at least one free mRNA of the second component b) is 1:1 and 1:3.

14. The immunostimulatory composition according to claim 1 , wherein the molar ratio of the mRNA of the adjuvant component to the at least one free mRNA of the second component b) is 1:2 and 1:4.

15. The immunostimulatory composition according to 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.

16. The immunostimulatory composition according to claim 15 , wherein the at least one free mRNA encodes an infectious disease antigen selected from a viral antigen, a protozoal antigen, or a bacterial antigen.

17. The immunostimulatory composition according to claim 1 , wherein the weight ratio of the at least one mRNA to protamine in the adjuvant component is 2:1.

18. The immunostimulatory composition according to claim 1 , wherein the weight ratio of the at least one mRNA to protamine in the adjuvant component is 3:1.

19. The immunostimulatory composition according to claim 4 , wherein the N/P ratio of the mRNA to the protamine in the adjuvant component is 0.3 to 4.

20. The immunostimulatory composition according to claim 1 , wherein the N/P ratio of the mRNA to the protamine in the adjuvant component is 0.5 to 2.

21. The immunostimulatory composition according to claim 1 , wherein the N/P ratio of the mRNA to the protamine in the adjuvant component is 0.7 to 2.

22. The immunostimulatory composition according to claim 1 , wherein the at least one free mRNA encodes a cancer cell antigen.