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.

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.