RNA CONTAINING COMPOSITION FOR TREATMENT OF TUMOR DISEASES

Abstract

The present invention relates to RNA containing compositions for use in the treatment or prophylaxis of tumor and/or cancer diseases, to a pharmaceutical composition, to a kit and to uses of the RNA containing compositions for the treatment or prophylaxis of tumor and/or cancer diseases.

Claims

1. RNA containing composition comprising at least one RNA for use in the treatment or prophylaxis of tumor and/or cancer diseases.

2. The RNA containing composition of claim 1, wherein the RNA containing composition is to be applied intratumorally, especially by injection into tumor tissue.

3. The RNA containing composition of claim 1 or 2, wherein the at least one RNA is selected from the group consisting of coding RNA and non-coding RNA.

4. The RNA containing composition of claim 3, wherein the coding RNA comprises at least one coding region encoding at least one peptide or protein and is preferably selected from the group consisting of mRNA, viral RNA, retroviral RNA, and replicon RNA.

5. The RNA containing composition of claim 4, wherein the coding RNA is mRNA.

6. The RNA containing composition of claim 4 or 5, wherein the at least one peptide or protein is selected or derived from the group consisting of cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, -catenin inhibitors, activators of the STING pathway, checkpoint modulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs), IDO pathway inhibitors, and proteins or peptides that bind inhibitors of apoptosis.

7. The RNA containing composition of claim 6, wherein the cytokine is an interleukin, preferably chosen from the following list: IL-1, IL-1, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL14, IL-15, IL-16, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A/B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35.

8. The RNA containing composition of claim 6 or 7, wherein the interleukin is interleukin-12 (IL-12).

9. The RNA containing composition of claim 6, wherein the cytokine is a member of the TNF family, preferably chosen from the following list: TNF, especially TNF, LT, LT, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK ligand.

10. The RNA containing composition of claim 6, wherein the cytokine is chosen from the following list: FLT3 ligand, G-CSF, GM-CSF, IFN//, IFN, LIF, M-CSF, MIF, OSM, Stem Cell Factor, TGF1, TGF2, TGF3, TSLP ligand.

11. The RNA containing composition of claim 6, wherein the chemokine is chosen from the following list: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, XCL1, XCL2, CX3CL1.

12. The RNA containing composition of claim 6, wherein the suicide gene product is a suicide enzyme, preferably a nucleotide metabolizing enzyme.

13. The RNA containing composition of claim 12, wherein the nucleotide metabolizing enzyme is chosen from the following list: thymidine kinase, preferably Herpes simplex virus thymidine kinase, cytosine deaminase, preferably bacterial cytosine deaminase or Yeast cytosine deaminase, deoxynucleoside kinase, preferably Drosophila melanogaster deoxynucleoside kinase, deoxycytidine kinase, preferably a mammalian deoxycytidine kinase, purine nucleoside phosphorylase, preferably a bacterial purine nucleoside phosphorylase.

14. The RNA containing composition of one of claims 6, 12 or 13, wherein the at least one RNA encoding at least one suicide gene product is used in combination with a prodrug which is a substrate of the suicide gene product.

15. The RNA containing composition of one of claims 6, 12 to 14, wherein the at least one RNA codes for at least one connexin and at least one suicide gene product.

16. The RNA containing composition of one of claims 6, 12 to 14, wherein the RNA composition comprises at least one RNA encoding at least one suicide gene product and wherein the RNA composition is used in combination with a further RNA coding for at least one connexin and/or with a protein of the connexin family or parts or fragments thereof.

17. The RNA containing composition of claim 6, wherein the immunogenic protein or peptide is a protein or peptide of a pathogen, more preferably of a viral or bacterial pathogen.

18. The RNA containing composition of claim 17, wherein the immunogenic protein or peptide is at least one protein or peptide of one virus or bacterium of the following list: influenza virus type A or B or any other orthomyxovirus (influenza type C), picornaviruses, such as rhinovirus or hepatitis A virus, togaviruses, such as alphavirus or rubivirus, e.g. Sindbis, Semliki-Forest or rubeolavirus, rubella virus, coronaviruses, in particular subtypes HCV-229E or HCV-OC43, rhabdoviruses, such as rabies virus, paramyxoviruses, such as mumps virus, reoviruses, such as group A, B or C rotavirus, hepadnaviruses, such as hepatitis B virus, papoviruses, such as human papillomaviruses of any serotype, adenoviruses, in particular type 1 to 47, herpesviruses, such as Herpes simplex virus 1, 2 or 3, cytomegalovirus, preferably CMVpp65, Epstein Barr virus, vacciniaviruses, the bacterium Chlamydophila pneumoniae, Flaviviruses, such as dengue virus type 1 to 4, yellow fever virus, West Nile virus, Japanese encephalitis virus, hepatitis C virus, caliciviruses, filoviruses, such as Ebola virus, bornaviruses, bunyaviruses, such as Rift Valley fever virus, arenaviruses, such as lymphocytic choriomeningitis virus or hemorrhagic fever viruses, retroviruses, such as HIV, parvoviruses.

19. The RNA containing composition of claim 17 or 18, wherein the immunogenic peptide or protein is derived from influenza nucleoprotein.

20. The RNA containing composition of claim 6, wherein the apoptosis inducer is chosen from the group consisting of the Bcl-2 family, tumor suppressor protein p53, ligands of transmembrane death receptors, especially the TNF receptor gene superfamily, pro-apoptic receptor agonists and Beclin-1.

21. The RNA containing composition of claim 6 or 20, wherein the apoptosis inducer is chosen from the following list: Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, Blk, Cytochrome c, Caspases, especially Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Death domain, especially Fas, preferably FasL, TNF, Apo2L/TRAIL, agonist of DR4 and/or DR5, Apo3L, DR4 agonistic antibody, DR5 agonistic antibody, protein kinase R (PKR), Granzyme B.

22. The RNA containing composition of claim 6, wherein the angiogenesis inhibitor is chosen from the following list: IFN-, IFN-, IFN-, CXCL9, CXCL10, IL-12, PF-4, TNF-, sFLT-1, FLK-1, Angiostatin, Endostatin, Vasostatin, Canstatin, Tumstatin, 16 kD prolacin fragment, TIMP-1, TIMP-2, TIMP-3, TSP-1, TSP-2, Maspin, PEX, sTie1, sTie2, Angiopoietin-1, Angiopoietin-2, Anti-VEGFR2 antibody, Anti-VEGF antibody and Anti-VEGFR1 antibody.

23. The RNA containing composition of claim 6, wherein the heat shock protein is chosen from the following list: HSP27, HSP47, HSP60, HSP70, HSC70, GRP78, HSP90, HSP110, GRP94, GRP170, PDI/PDIA, CRT/CALR.

24. The RNA containing composition of claim 6, wherein the tumor antigen is chosen from the following list: 1A01_HLA-A/m; 1A02; 5T4; ACRBP; AFP; AKAP4; alpha-actinin-_4/m; alpha-methylacyl-coenzyme_A_racemase; ANDR; ART-4; ARTC1/m; AURKB; B2MG; B3GN5; B4GN1; B7H4; BAGE-1; BASI; BCL-2; bcr/abl; beta-catenin/m; BING-4; BIRC7; BRCA1/m; BY55; calreticulin; CAMEL; CASPA; Caspase_8; cathepsin_B; cathepsin_L; CD1A; CD1B; CD1C; CD1D; CD1E; CD20; CD22; CD276; CD33; CD3E; CD3Z; CD4; CD44_Isoform_1; CD44_Isoform_6; CD52; CD55; CD56; CD80; CD86; CD8A; CDC27/m; CDE30; CDK4/m; CDKN2A/m; CEA; CEAM6; CH3L2; CLCA2; CML28; CML66; COA-1/m; coactosin-like_protein; collagen_XXIII; COX-2; CP1B1; CSAG2; CT-_9/BRD6; CT45A1; CT55; CTAG2_Isoform_LAGE-1A; CTAG2_Isoform_LAGE-1B; CTCFL; Cten; cyclin_B1; cyclin_D1; cyp-B; DAM-10; DEP1A; E7; EF1A2; EFTUD2/m; EGFR; EGLN3; ELF2/m; EMMPRIN; EpCam; EphA2; EphA3; ErbB3; ERBB4; ERG; ETV6; EWS; EZH2; FABP7; FCGR3A_Version_1; FCGR3A_Version_2; FGF5; FGFR2; fibronectin; FOS; FOXP3; FUT1; G250; GAGE-1; GAGE-2; GAGE-3; GAGE-4; GAGE-5; GAGE-6; GAGE7b; GAGE-8_(GAGE-2D); GASR; GnT-V; GPC3; GPNMB/m; GRM3; HAGE; hepsin; Her2/neu; HLA-A2/m; homeobox_NKX3.1; HOM-TES-85; HPG1; HS71A; HS71B; HST-2; hTERT; iCE; IF2B3; IL-10; IL-13Ra2; IL2-RA; IL2-RB; IL2-RG; IL-5; IMP3; ITA5; ITB1; ITB6; kallikrein-2; kallikrein-4; KI20A; KIAA0205; KIF2C; KK-LC-1; LDLR; LGMN; LIRB2; LY6K; MAGA5; MAGA8; MAGAB; MAGE-_B1; MAGE-_E1; MAGE-A1; MAGE-A10; MAGE-A12; MAGE-A2; MAGE-A3; MAGE-A4; MAGE-A6; MAGE-A9; 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_(MAGE1); MAGE-E2; MAGE-F1; MAGE-H1; MAGEL2; mammaglobin_A; MART-1/melan-A; MART-2; MC1_R; M-CSF; mesothelin; MITF; MMP1_1; MMP7; MUC-1; MUM-1/m; MUM-2/m; MYO1A; MYO1B; MYO1C; MYO1D; MYO1E; MYO1F; MYO1G; MYO1H; NA17; NA88-A; Neo-PAP; NFYC/m; NGEP; N-myc; NPM; NRCAM; NSE; NUF2; NY-ESO-1; OA1; OGT; OS-9; osteocalcin; osteopontin; p53; PAGE-4; PAI-1; PAI-2; PAP; PATE; PAX3; PAX5; PD1L1; PDCD1; PDEF; PECA1; PGCB; PGFRB; Pim-1_-Kinase; Pin-1; PLAC1; PMEL; PML; POTE; POTEF; PRAME; PRDX5/m; PRM2; prostein; proteinase-3; PSA; PSB9; PSCA; PSGR; PSM; PTPRC; RAB8A; RAGE-1; RARA; RASH; RASK; RASN; RGS5; RHAMM/CD168; RHOC; RSSA; RU1; RU2; RUNX1; S-100; SAGE; SART-_1; SART-2; SART-3; SEPR; SERPINB5; SIA7F; SIA8A; SIAT9; SIRT2/m; SOX10; SP17; SPNXA; SPXN3; SSX-1; SSX-2; SSX3; SSX-4; ST1A1; STAG2; STAMP-1; STEAP-1; survivin; Survivin-2B; SYCP1; SYT-SSX-1; SYT-SSX-2; TARP; TCRg; TF2AA; TGFbeta1; TGFR2; TGM-4; TIE2; TKTL1; TPI/m; TRGV11; TRGV9; TRPC1; TRP-p8; TSG10; TSPY1; TVC_(TRGV3); TX101; tyrosinase; TYRP1; TYRP2; UPA; VEGFR1; WT1; XAGE1.

25. The RNA containing composition of claim 6, wherein the -catenin inhibitor is chosen from the following list: TAT-NLS-BLBD-6, axin-1, TCF-4, GSK-3b, DKK-1, Dvl-1.

26. The RNA containing composition of claim 6, wherein the activator of the STING (stimulator of interferon genes) pathway is an activating protein or a constitutively active protein of the STING pathway, preferably of DDX41, STING, cGAS, IRF3, TBK1, or STAT6.

27. The RNA containing composition of claim 6, wherein the checkpoint modulator is a modulator of B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7/HHLA2, BTLA, CD28, CD28H/IGPR-1, CTLA-4, ICOS, PD-1, PD-L2/B7-DC, PDCD6, VISTA/B7-H5/PD-1H, BTN1A1/Butyrophilin, BTN2A1, BTN2A2/Butyrophilin 2A2, BTN3A1/2, BTN3A2, BTN3A3, BTNL2/Butyrophilin-like 2, BTNL3, BTNL4, BTNL6, BTNL8, BTNL9, BTNL10, CD277/BTN3A1, LAIR1, LAIR2, CD96, CD155/PVR, CRTAM, DNAM-1/CD226, Nectin-2/CD112, Nectin-3, TIGIT, LILRA3/CD85e, LILRA4/CD85g/ILT7, LILRB1/CD85j/ILT2, LILRB2/CD85d/ILT4, LILRB3/CD85a/ILT5, LILRB4/CD85k/ILT3, 4-1BB/TNFRSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TNF-beta, OX40/TNFRSF4, OX40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNF-alpha, TNF RII/TNFRSF1B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, SLAM/CD150, TIM-1/KIM-1/HAVCR, TIM-3, TIM-4, CD7, CD96, CD160, CD200, CD300a/LMIR1, CRTAM, DAP12, Dectin-1/CLEC7A, DPPIV/CD26, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3, TIM-1/KIM-1/HAVCR, TIM-4, TSLP R, or any combinations thereof.

28. The RNA containing composition of claim 6 or 27, wherein the checkpoint modulator is selected from the group consisting of an agonistic antibody, an antagonistic antibody, a dominant negative receptor, a decoy receptor and a ligand.

29. The RNA containing composition of claim 28, wherein the antagonistic antibody is directed against PD-1, PD-L1 or CTLA-4.

30. The RNA containing composition of claim 28, wherein the agonistic antibody is directed against OX-40.

31. The RNA containing composition of claim 28, wherein the decoy receptor is a soluble PD-1 receptor.

32. The RNA containing composition of claim 6, wherein the antibody, is an agonistic antibody, an antagonistic antibody, or a neutralizing antibody.

33. The RNA containing composition of claim 6 or 32, wherein the antibody is directed against a tumor antigen or a tumor associated antigen.

34. The RNA containing composition of one of claims 3-33, wherein the G/C content of the coding region of the coding RNA, preferably mRNA is increased compared with the G/C content of the coding region of the wild type RNA, and wherein the coded amino acid sequence of said G/C-enriched RNA is preferably not being modified compared with the encoded amino acid sequence of the wild type RNA.

35. The RNA containing composition of one of claims 3-34, wherein the coding RNA, preferably mRNA comprises additionally a 5-UTR element and/or a 3-UTR element.

36. The RNA containing composition of one of claims 3-35, wherein the coding RNA, preferably mRNA comprises additionally at least one histone stem-loop.

37. The RNA containing composition of one of claims 3-36, wherein the coding RNA, preferably mRNA comprises additionally a 5-CAP structure and/or a poly(A) sequence and/or a poly(C) sequence.

38. The RNA containing composition of claim 3, wherein the non-coding RNA is selected from the group consisting of small interfering RNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA), ribozymes, aptamers, riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

39. The RNA containing composition of claim 38, wherein the immunostimulating RNA comprises at least one RNA sequence according to formula (III) (GlXmGn), formula (IV) (ClXmCn), formula (V) (NuGlXmGnNv)a, and/or formula (VI) (NuClXmCnNv)a).

40. The RNA containing composition of claim 38 or 39, wherein the immunostimulating RNA comprises at least one RNA sequence according to SEQ ID NO. 5, 394 and 10072.

41. The RNA containing composition of any of the preceding claims, wherein the at least one RNA is complexed with one or more cationic or polycationic compounds, preferably with cationic or polycationic polymers, cationic or polycationic peptides or proteins, e.g. protamine, cationic or polycationic polysaccharides and/or cationic or polycationic lipids.

42. The RNA containing composition of claim 41, wherein the cationic or polycationic compound is a polymeric carrier.

43. The RNA containing composition of claim 42, wherein the polymeric carrier is formed by disulfide-crosslinked cationic components, preferably disulfide-crosslinked cationic peptides, preferably comprising peptides according to formula VII, VIIa and/or VIIb and/or a compound according to formula (VIII) (L-P1-S[S-P2-S]n-S-P3-L).

44. The RNA containing composition of claims 41-43, wherein the N/P ratio of the at least one RNA to the one or more cationic or polycationic compounds, preferably cationic or polycationic peptides or proteins is in the range of about 0.1 to 10, including a range of about 0.3 to 4, of about 0.5 to 2, of about 0.7 to 2 and of about 0.7 to 1.5.

45. The RNA containing composition of any of the preceding claims wherein the RNA containing composition comprises at least one RNA, which is complexed with one or more cationic or polycationic compounds, and at least one free RNA, preferably coding RNA, more preferably mRNA.

46. The RNA containing composition of any of the preceding claims, wherein the at least one mRNA is complexed with one or more lipids and thereby forming liposomes, lipid nanoparticles and/or lipoplexes.

47. The RNA containing composition of any of the preceding claims, wherein the RNA containing composition comprises a polymeric carrier cargo complex, formed by a polymeric carrier, preferably comprising disulfide-crosslinked cationic peptides, preferably Cys-Arg.sub.12, and/or Cys-Arg.sub.12-Cys, and an immunostimulating RNA, preferably the RNA sequence according to SEQ ID NO: 5, 394 or 10072.

48. Pharmaceutical composition comprising the RNA containing composition as defined according to claims 1 to 47 and a pharmaceutically acceptable carrier and/or vehicle.

49. The pharmaceutical composition of claim 48, prepared for injection into tumor tissue.

50. Kit or kit of parts comprising the RNA containing composition as defined according to claims 1 to 47, or the pharmaceutical composition as defined according to claim 48 or 49, and optionally technical instructions with information on the administration and dosage for administration.

51. The RNA containing composition as defined according to one of claims 1 to 47, or the pharmaceutical composition as defined according to claim 48 or 49, or the kit or kit of parts as defined according to claim 50 for use as a medicament.

52. The RNA containing composition as defined according to claims 1 to 47, or the pharmaceutical composition as defined according to claim 48 or 49, or the kit or kit of parts as defined according to claim 50 for use in the treatment or prophylaxis of tumor and/or cancer diseases preferably by intratumoral application, especially by injection into tumor tissue.

53. Use of the RNA containing composition as defined according to claims 1 to 47, or the pharmaceutical composition as defined according to claim 48 or 49, or the kit or kit of parts as defined according to claim 50 for the treatment or prophylaxis of tumor and/or cancer diseases, preferably by intratumoral application, especially by injection into tumor tissue.

54. The use of claim 53, wherein the treatment or prophylaxis comprises the administration of at least one additional pharmaceutically active compound.

55. The use of claim 54, wherein the at least one additonal pharmaceutically active compound is selected from the group consisting of cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, -catenin inhibitors, activators of the STING pathway, checkpoint modulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs), IDO pathway inhibitors, proteins or peptides that bind inhibitors of apoptosis, anti-bacterial agents, anti-viral agents, drugs, adjuvants, chemotherapeutic agents and kinase inhibitors.

56. The use of claim 54 or 55, wherein the treatment further comprises radiation therapy and/or surgery.

57. The use of claim 55, wherein the checkpoint modulator is selected from a modulator as defined in claim 27.

58. The use of claim 57, wherein the checkpoint modulator is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, an OX-40 stimulator, a 4-1BB stimulator, a CD40L stimulator, a CD28 stimulator, a GITR stimulator.

59. The use of claim 58, wherein the PD-1 inhibitor is an antagonistic antibody directed against PD-1 and the PD-L1 inhibitor is an antagonistic antibody directed against PD-1.

60. The use of claim 54, wherein the antibody is selected from an antibody directed against CD73 and/or CD137.

61. Use of the RNA containing composition as defined according to one of claims 1 to 47, or the pharmaceutical composition as defined according to claim 48 or 49, or the kit or kit of parts as defined according to claim 50 for preparation of a medicament for treatment of tumor and/or cancer diseases, preferably by intratumoral application, especially by injection into tumor tissue.

62. Method of treatment of tumor and/or cancer diseases with the RNA containing composition as defined according to one of claims 1 to 47, or the pharmaceutical composition as defined according to claim 48 or 49, or the kit or kit of parts as defined according to claim 50, preferably by intratumoral application, especially by injection into tumor tissue.

Description

SHORT DESCRIPTION OF THE FIGURES

[0784] FIG. 1: shows survival proportions of mice bearing E.G7-OVA tumors after intratumoral treatment with mRNA encoding IL-12 (IL-12 mRNA) or with recombinant IL-12 protein (rIL-12 protein). The experiment was performed as described in Example 1. Kaplan-Meier survival curves are presented.

[0785] FIG. 2: shows that intratumoral treatment of mice with a combination of IL-12 mRNA (R2763, SEQ ID NO: 1) and the polymeric carrier cargo complex (R2391, RNAdjuvant*, prepared as described in methods) led to a significantly decreased tumor volume compared to control groups. The experiment was performed as described in Example 2. FIG. 2 shows the mean tumor volume at day 21 after tumor challenge (the last day when all animals were alive). Statistical analysis was performed in GraphPad Prism version 5.04 using Mann Whitney test.

[0786] FIG. 3: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with a combination of IL-12 mRNA and the polymeric carrier cargo complex (RNAdjuvant) as described in Example 2 and in legend of FIG. 2. Kaplan-Meier survival curves are presented. Statistical analysis was performed in GraphPad Prism version 5.04 using Log-rank test.

[0787] FIG. 4: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with mRNA encoding the influenza nucleoprotein. The experiment was performed as described in Example 3. Kaplan-Meier survival curves are presented.

[0788] FIGS. 5A-B: Panel (A) shows an analysis of the median tumor growth of mice bearing CT26 tumors after intratumoral treatment with mRNA encoding IL-12, RNAdjuvant, and mRNA encoding soluble PD-1. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 4.

[0789] Panel (B) shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with mRNA encoding IL-12, RNAdjuvant, and mRNA encoding soluble PD-1. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 4. Kaplan-Meier survival curves are presented.

[0790] FIG. 6: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with mRNA encoding IL-12, RNAdjuvant, mRNA encoding soluble PD-1 and intraperitoneal treatment of an anti-CD73 antibody. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 5. Kaplan-Meier survival curves are presented.

[0791] FIG. 7: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with mRNA encoding IL-12, RNAdjuvant, mRNA encoding soluble PD-1 and intraperitoneal treatment of an anti-CD173 antibody. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 6. Kaplan-Meier survival curves are presented.

[0792] FIG. 8: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with RNAdjuvant and intraperitoneal treatment of an anti-PD-1 antibody. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 7. Kaplan-Meier survival curves are presented.

[0793] FIG. 9: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with mRNA encoding IL-12, RNAdjuvant, mRNA encoding CD40L compared to intratumoral treatment with mRNA encoding IL-12 alone. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 8. Kaplan-Meier survival curves are presented.

[0794] FIG. 10: shows the mRNA sequence R3571 encoding murine CD40L (MmCD40L) according to SEQ ID NO. 10.073

EXAMPLES

Methods: Preparation of the RNA

1. Preparation of DNA and RNA Constructs

[0795] For the present examples DNA sequences encoding the indicated RNAs (see Table 17) were prepared and used for subsequent RNA in vitro transcription reactions.

TABLE-US-00019 TABLE 17 RNA constructs RNA Description 5-UTR 3-UTR SEQ ID NO. R1328 Murine IL-12 encoding mRNA Muag (3-UTR of SEQ ID NO: (MmIL-12(GC))-sc-Flag) alpha globin)-A64- 1 C30 R491 mRNA encoding Photinus Muag (3-UTR of SEQ ID NO: pyralis luciferase (pPLuc (GC)) alpha globin)-A64- 2 (irrelevant mRNA) C30 R2763 Murine IL-12 encoding mRNA 5-TOP-UTR albumin-3-UTR-A64- SEQ ID NO: (MmIL-12 (GC)) derived from C30-histone stem- 3 the ribosomal loop protein 32L R2244 Luciferase encoding mRNA 5-TOP-UTR albumin-3-UTR-A64- SEQ ID NO: (PpLuc(GC)) derived from C30-histone stem- 4 the ribosomal loop protein 32L R2025 Non-coding SEQ ID NO: R2391 immunostimulatory RNA 5 (RNAdjuvant) (SEQ ID NO. 118 of WO2009095226) R2650 mRNA coding for the 5-TOP-UTR albumin-3-UTR-A64- SEQ ID NO: R2651 influenza nucleoprotein derived from C30-histone stem- 6 (H1N1(PR8)-NP(GC)) the ribosomal loop protein 32L R3971 mRNA encoding solPD-1 5-TOP-UTR albumin-3-UTR-A64- SEQ ID NO: derived from C30-histone stem- 389 the ribosomal loop protein 32L R3571 mRNA encoding murine 5-TOP-UTR albumin-3-UTR-A64- SEQ ID NO: CD40L (MmCD40L) derived from C30-histone stem- 10.073 the ribosomal loop protein 32L

[0796] The constructs of MmIL-12(GC), Influenza NP (GC), solPD-1 and PpLuc(GC)) were prepared by introducing a 5-TOP-UTR derived from the ribosomal protein 32L, modifying the wild type coding sequence by introducing a GC-optimized sequence for stabilization, followed by a stabilizing sequence derived from the albumin-3-UTR, a stretch of 64 adenosines (poly(A)-sequence), a stretch of 30 cytosines (poly(C)-sequence), and a histone stem loop. Most DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization, using an in silico algorithm that increase the GC content of the respective coding sequence compared to the wild type coding sequence (in Table 12 indicated as GC).

[0797] For the present example a DNA sequence encoding the non-coding immunostimulatory RNA (isRNA) R2025 was prepared and used for subsequent RNA in vitro transcription reactions.

2. RNA In Vitro Transcription

[0798] The respective DNA plasmids prepared according to section 1 above were transcribed in vitro using T7 polymerase. The RNA in vitro transcription reactions of the IL-12, the NP, PpLuc, CD40L and soluble PD-1 encoding constructs were performed in the presence of a CAP analog (m.sup.7GpppG). The isRNA R2025 was prepared without CAP analog. Subsequently, the RNA was purified using PureMessenger (CureVac, Tubingen, Germany; WO2008/077592A1).

3. Preparation of the Polymeric Cargo Complex (RNAdjuvant)

[0799] The following cationic peptide as cationic component of the polymeric carrier was used (Cys-Arg.sub.12-Cys or CR.sub.12C) according to SEQ ID NO: 7.

[0800] For synthesis of the polymeric carrier cargo complexes an RNA molecule having the RNA sequence R2025 as defined in section 1 above was mixed with the cationic CR.sub.12C peptide component as defined above. The specified amount of the RNA was mixed with the respective cationic component in mass ratios as indicated below, thereby forming a complex. If polymerizing cationic components were used according to the present invention, polymerization of the cationic components took place simultaneously to complexation of the nucleic acid cargo. Afterwards, the resulting solution was adjusted with water to a final volume of 50 l and incubated for 30 minutes at room temperature. Further details are described in WO2012013326.

[0801] The mass ratio of peptide:RNA was 1:3.7. The polymeric carrier cargo complex is formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the immunostimulatory R2025 as nucleic acid cargo. This polymeric carrier cargo complex R2025/CR.sub.12C (designated R2391) was used as adjuvant in the following examples (referred to as RNAdjuvant)

4. Preparation of the Vaccine Formulation Coding for the Influenza Nucleoprotein (H1N1(PR8)-NP(GC)) (R2651)

[0802] The mRNA (R2650) was complexed with protamine by addition of protamine to the mRNA in the ratio (1:2) (w/w) (adjuvant component). After incubation for 10 min, the same amount of free mRNA used as antigen-providing mRNA was added. This vaccine formulation is termed herein R2651 (according to WO2010037539). The vaccine was administered in Ringer's Lactate solution.

5. Preparation of the RNA for Administration

[0803] The naked (that is, non-formulated) PpLuc mRNA (R2244, R491)), IL-12 mRNA (R2763, R1328), soluble PD-1 mRNA (R3971), CD40L mRNA (R3571) were administered in Ringer's Lactate (RiLa) solution. The co-formulation of naked mRNAs and the polymeric carrier cargo complex RNAdjuvant (R2391) were also administered in Ringer's Lactate (RiLa) after mixing of both components directly before injection.

Example 1: Intratumoral Application of mRNA Coding for IL-12

[0804] 5 female C57BL/6 mice per treatment group were inoculated with 10 cells E.G7-OVA cells 5 days before the first treatment. For each treatment group 5 established (about 100 mm.sup.3) subcutaneously implanted EG.7-OVA tumors were treated. Tumors were treated with 16 g mRNA coding for MmIL-12 (MmIL-12(GC))-sc-Flag) (R1328) or 0.5 g MmIL-12 protein on d 0, 2, 4, 21, 23 and 25 with 50 g (1 g/l). As control mice were treated with an irrelevant mRNA (pPLuc) (R491).

[0805] Study day 0 is defined as the first day of treatment. Tumor growth was monitored frequently (every 2-3 days). Mice with a volume of >3 cm.sup.3 were killed.

Results of Example 1

[0806] FIG. 1 shows that the intratumoral treatment with the mRNA-encoded IL-12 (IL-12 mRNA) resulted in a significant increase in survival compared to the control group. Furthermore an increased survival could be observed compared to the intratumoral application of recombinant IL-12 protein (rIL-12 protein).

Example 2: Intratumoral Treatment with mRNA Encoding IL-12 in Combination with an Immunostimulating RNA (RNAdjuvant)

[0807] The following table 18 summarizes the RNA constructs used for the example 2.

TABLE-US-00020 TABLE 18 RNA constructs for example 2 RNA Description FIG. SEQ ID NO. R2763 Murine IL-12 encoding mRNA 1 SEQ ID NO. 1 R2244 Luciferase encoding mRNA (PpLuc) 2 SEQ ID NO. 2 R2025 Non-coding immunostimulatory RNA 3 SEQ ID NO. 3

[0808] Balb/c mice (n=6 or 7, see table 14) were injected subcutaneously (s.c.) with 110.sup.6 CT26 cells (colon carcinoma cell line) per mouse (in a volume of 100 l PBS) on the right flank on day 0 of the experiment. At day 9 after tumor challenge, mice were sorted according to the tumor size to obtain groups with a mean tumor volume of approximately 60 mm.sup.3. Intratumoral (i.t.) therapy started at day 9 and continued for additional 4 injections every 3-4 days. Mice were injected with a combination of mRNA-encoded IL-12 (25 g of R2763)+RNAdjuvant (25 g of R2391) (group A). To control for local inflammation due to RNA application or the injection procedure, mice were injected with control mRNA coding for luciferase (PpLuc, R2244, group B) or buffer (RiLa, group C), respectively. Untreated mice served as additional control (group D).

[0809] Tumor growth was monitored by measuring the tumor size in three dimensions using a calliper. Tumor volume was calculated according to the following formula:

[00001]$\mathrm{volume}\left({\mathrm{mm}}^3\right)=\frac{\mathrm{length}\left(\mathrm{mm}\right){\mathrm{width}}^2\left({\mathrm{mm}}^2\right)}{6}$

[0810] On day 9, 11, 14, 17 and 21 of the experiment mice were injected intratumorally (i.t.) with RNA according to the table 19 below. The volume for intratumoral injection was 50 l.

TABLE-US-00021 TABLE 19 Animal groups Strain Number Dose per Route, Group sex of mice RNA mouse volume A BALB/c 7 R2763, 25 g of i.t., Female R2391 each RNA 50 l B BALB/c 7 R2244 50 g i.t., Female 50 l C BALB/c 6 RiLa i.t., Female 50 l D BALB/c 6 Female

Results of Example 2

[0811] FIG. 2 shows that the intratumoral treatment with the combination of mRNA-encoded IL-12 (R2763) and RNAdjuvant (R2391) resulted in a statistically significant decrease in tumor volume at day 21 after tumor challenge compared to all control groups.

[0812] FIG. 3 shows that the intratumoral treatment with the combination of mRNA-encoded IL-12 (R2763) and RNAdjuvant (R2391) resulted in a statistically significant increase in survival compared to all three control groups (group A vs. group B * p=0.0104, group A vs. group C ** p=0.0035, group A vs. Group D * p==0.0263).

Example 3: Vaccination of Mice with mRNA Encoding the Influenza Nucleoprotein (NP) and Subsequent Intratumoral Treatment with NP-Encoding mRNA

[0813] The objective of this experiment was to test whether a pre-existing immune response can be harnessed against an established tumor. To this end, mice were first vaccinated with RNActive (vaccine formulation complexed with protamine) encoding the influenza nucleoprotein (NP) (R2651) which induces a high level of anti-NP CD8 T cell responses, then challenged with CT26 tumor cells followed by intratumoral treatment with naked RNA encoding NP (R2650).

[0814] 27 Balb/c mice were vaccinated intradermally (i.d.) with 40 g of H1N1(PR8)-NP(GC) RNActive (R2651) (250 l) or Ringer-Lactate buffer (RiLa) as control on day 0, day 7 and day 16 of the experiment. On day 14 all mice were challenged subcutaneously (s.c.) with 110.sup.6 CT26 cells per mouse (in a volume of 100 l PBS) on the right flank. On day 22, mice were assigned to the different groups as shown in Table 20.

[0815] On day 23, seven days after the second boost, intratumoral (i.t.) application of 50 g naked H1N1(PR8)-NP(GC) mRNA (R2650) started (only group C) and continued for additional four injections (at day 25, day 28, day 31 and day 35). The volume for intratumoral injection was 50 l. A detailed treatment schedule is shown in Table 21.

[0816] Tumor growth was monitored by measuring the tumor size in three dimensions using a calliper. Tumor volume was calculated according to the following formula:

[00002]$\mathrm{volume}\left({\mathrm{mm}}^3\right)=\frac{\mathrm{length}\left(\mathrm{mm}\right){\mathrm{width}}^2\left({\mathrm{mm}}^2\right)}{6}$

TABLE-US-00022 TABLE 20 Animal groups Strain Number mRNA mRNA Group sex of mice i.d. i.t. A BALB/c 9 RiLa Female B BALB/c 9 R2651 Female (40 g) C BALB/c 9 R2651 R2650 Female (40 g) (50 g)

TABLE-US-00023 TABLE 21 Vaccination schedule Day Treatment 0 i.d. vaccination all groups 7 i.d. vaccination all groups 14 Tumor challenge of all groups (1 10.sup.6 CT26 cells/mouse) 16 i.d. vaccination all groups 23 i.t. vaccination group C 25, 28, 31, 35 i.t. vaccination group C

Results of Example 3

[0817] FIG. 4 shows that pre-existent immunity (induced in this model by the NP vaccination) increased the median survival time (MST) of mice which received intratumoral application of NP-encoded mRNA compared to mice which were treated with buffer only (MST=28 vs. MST=21, respectively).

Example 4: Intratumoral Treatment with an Immunostimulating RNA (RNAdjuvant) and an mRNA Encoding Soluble PD-1 and and an mRNA Encoding IL-12

[0818] Table 22 summarizes the treatment as used in the present example. RNAdjuvant and the mRNA constructs encoding IL-12 and soluble PD-1 were administered intratumorally (i.t.). In CT26 tumor challenged mice, survival rates and median tumor growth were analyzed.

TABLE-US-00024 TABLE 22 Groups, treatment and RNA dilution Nr. of i.t. treatment Group mice (25 g for each component) Vaccination schedule A 10 IL-12 + RNAdjuvant + 2X week soluble PD-1 B 10 IL-12 2X week C 10 RNAdjuvant 2X week D 10 RiLa 2X week

Tumor Challenge and Administration of the Inventive Composition:

[0819] 60 Balb/c mice were challenged subcutaneously with 110.sup.6 CT26 cells per mouse (volume in 100 l PBS) on the right flank on day 0 of the experiment. On day 8 mice were sorted according to tumor size. According to tumor size, the first vaccination took place on day 8 or 9 (tumors should have a size of about 40-50 mm.sup.3). Mice were vaccinated with different combinations of mRNAs and RNAdjuvant according to the table above. Six vaccinations took place. Volume for intratumoral injection was 50 l.

[0820] Mice were injected according to the indicated scheme shown in Table 22. Median tumor growth was determined according to example 3. The results of the experiment are shown in FIG. 5, wherein FIG. 5A shows the effect of the inventive composition on tumor growth, and FIG. 5B shows the effect of the inventive composition on survival.

Results

[0821] The results in FIG. 5A show that the inventive composition comprising an mRNA encoding IL-12 and mRNA encoding soluble PD-1 in combination with RNAdjuvant (group A according to Table 22) strongly decreased the median tumor volume compared to the other treatments (groups B-D according to Table 22). In addition, the results in FIG. 5B show that the inventive composition comprising an mRNA encoding IL-12 and mRNA encoding soluble PD-1 in combination with RNAdjuvant (group A according to Table 22) strongly increased the survival of tumor challenged mice compared to the other treatments (groups B-D according to Table 22).

Example 5: Intratumoral Treatment with mRNA Encoding IL-12 in Combination with an Immunostimulating RNA (RNAdjuvant) and mRNA Encoding Sol PD-1 and Anti-CD73 Antibody

[0822] Table 23 summarizes the treatment as used in the present example. In addition to RNAdjuvant and mRNA constructs encoding IL-12 and soluble PD-1 (administered intratumorally (i.t.)), an anti CD73 antibody (BioXCell) was co-administered intraperitoreally (i.p.). In CT26 tumor challenged mice, survival rates were analyzed.

TABLE-US-00025 TABLE 23 Groups, treatment and RNA dilution Nr. of i.t. treatment i.p. Vaccination Group mice (25 g for each component) treatment schedule A 10 IL-12 + RNAdjuvant + a-CD73 2X week soluble PD-1 B 10 IL-12 + RNAdjuvant + Rat IgG2a 2X week soluble PD-1 C 10 RiLa a-CD73 2X week D 10 RiLa Rat IgG2a 2X week

Tumor Challenge and Administration of the Inventive Composition:

[0823] The tumor challenge was performed according to the previous experiments (see Example 4).

[0824] Mice were injected according to the indicated scheme shown in Table 23.

[0825] The results of the experiment are shown in FIG. 6.

Results

[0826] FIG. 6 shows that the intratumoral treatment with mRNA-encoded IL-12 (R2763), mRNA encoded sol-PD-1 (R3971) and RNAdjuvant (R2391) in combination with an i.p. administration of anti CD73 antibody (Group A according to Table 23) resulted in a statistically significant increase in survival compared to the relevant control group that only received an anti CD73 antibody (Group C according to Table 23) and in an increase in survival rates compared to the the treatment with IL-12+RNAdjuvant+soluble PD-1 and a control antibody (Rat IgG2a, BioXCell) (Group B according to Table 23).

Example 6: Intratumoral Treatment with mRNA Encoding IL-12 in Combination with an Immunostimulating RNA (RNAdjuvant) and an Anti-CD137 Antibody

[0827] Table 24 summarizes the treatment as used in the present example. In addition to RNAdjuvant and the mRNA constructs encoding IL-12 and soluble PD-1 (administered intratumorally (i.t.)), an anti CD137 antibody (BioXCell) was co-administered intraperitoreally (i.p.). In CT26 tumor challenged mice, survival rates were analyzed.

TABLE-US-00026 TABLE 24 Groups, treatment and RNA dilution Nr. of i.p. Vaccination Group mice i.t. treatment (25 g) treatment schedule A 10 IL-12 + RNAdjuvant + a-CD137 2X week soluble PD-1 B 10 IL-12 + RNAdjuvant + Rat IgG2a 2X week soluble PD-1 C 10 RiLa a-CD137 2X week D 10 RiLa Rat IgG2a 2X week

Tumor Challenge and Administration of the Inventive Composition:

[0828] The tumor challenge was performed according to the previous experiments (see Example 4).

[0829] Mice were injected according to the indicated scheme shown in Table 24.

[0830] The results of the experiment are shown in FIG. 7.

Results

[0831] FIG. 7 shows that the intratumoral treatment with mRNA-encoded IL-12 (R2763) sol-PD-1 (R3971) and RNAdjuvant (R2391) in combination with an i.p. administration of anti CD-137 antibody (Group A according to Table 24) resulted in a significant increase in survival compared to the relevant control group that only received the antibody anti CD-137 (Group C according to Table 24) and in an increase in survival rates compared to the the treatment with IL-12+RNAdjuvant+soluble PD-1 and a control antibody (Rat IgG2a, BioXCell) (Group B according to Table 24).

Example 7: Treatment with with an Immunostimulating RNA (RNAdjuvant) in Combination with a Checkpoint Inhibitor Anti PD-1 Antibody

[0832] Table 25 summarizes the treatment as used in the present example. In addition to RNAdjuvant (administered i.t.), a checkpoint inhibitor anti PD-1 (BioXCell) was administered i.p. In CT26 tumor challenged mice, survival rates were analyzed.

TABLE-US-00027 TABLE 25 Groups, treatment and RNA dilution/antibody dilution Amount of RNA Vaccination Group Construct Antibody (g) schedule A RiLa (i.t.) 2X week B RNAdjuvant Control Ab (i.p.)(100 g) 25 2X week (i.t.) C RNAdjuvant Anti-PD-1 (i.p.) (200 g) 25 2X week (i.t.) D RiLa (i.t.) Anti-PD-1 (i.p.) (200 g) 2X week

Tumor Challenge and Administration of the Inventive Composition:

[0833] The tumor challenge was performed according to the previous experiments (see Example 4).

[0834] Mice were injected according to the indicated scheme shown in Table 25.

[0835] The results of the experiment are shown in FIG. 8.

Results

[0836] FIG. 8 shows that the intratumoral (i.t.) treatment with RNAdjuvant (R2391) in combination with an i.p. administration of anti PD-1 antibody (Group C according to Table 25) resulted in an increase in survival compared to the relevant control group that only received the checkpoint inhibitor anti PD-1 antibody (Group D according to Table 25) and in an increase in survival rates compared to the treatment with RNAdjuvant and a control antibody (anti hamster IgG, BioXCell) (Group B according to Table 25).

Example 8: Intratumoral Treatment with an Immunostimulating RNA (RNAdjuvant) and an mRNA Encoding CD40 Ligand (CD40L) and an mRNA Encoding IL-12

[0837] Table 26 summarizes the treatment as used in the present example. RNAdjuvant and the mRNA constructs encoding IL-12 and murine CD40L were administered intratumorally (i.t.). In CT26 tumor challenged mice, survival rates were analyzed.

TABLE-US-00028 TABLE 26 Groups, treatment and RNA dilution Nr. of Vaccination Group mice i.t. treatment (25 g per RNA) schedule A 8 IL-12 + RNAdjuvant + CD40L 2X week B 8 IL-12 2X week C 8 RiLa 2X week

Tumor Challenge and Administration of the Inventive Composition:

[0838] The tumor challenge was performed according to the previous experiments (see Example 4).

[0839] Mice were injected according to the indicated scheme shown in Table 26.

[0840] The results of the experiment are shown in FIG. 9.

Results

[0841] The results in FIG. 9 show that the inventive composition comprising an mRNA encoding IL-12 and an mRNA encoding CD40L in combination with RNAdjuvant (group A according to table 26) strongly increased the median survival of tumor challenged mice compared to the other treatments (groups B-C according to table 26).