RNA FOR CANCER THERAPY

Abstract

The present invention relates to RNA, particularly an immunostimulatory RNA (isRNA), a coding RNA or a combination thereof, for use in the treatment or prophylaxis of a disease, in particular a tumor and/or cancer disease. The present invention also provides pharmaceutical compositions, and a kit comprising the RNA(s). Further, the invention also comprises medical uses of the RNA(s) and compositions comprising the RNA(s).

Claims

1. Immunostimulatory RNA (isRNA) for use in the treatment or prophylaxis of a tumor or cancer disease selected from the group consisting of cutaneous melanoma (cMEL), cutaneous squamous cell carcinoma (cSCC), head and neck squamous cell carcinoma (HNSCC), adenoid cystic carcinoma (ACC), cutaneous T-cell lymphoma, preferably cutaneous T-cell lymphoma of mycosis fungoides subtype, and vulvar squamous cell cancer (VSCC), wherein the tumor or the cancer disease is preferably at an advanced stage and/or refractory to standard therapy.

2. The isRNA for use according to claim 1, wherein the isRNA is administered intratumorally.

3. The isRNA for use according to claim 2, wherein the isRNA is administered by injection.

4. The isRNA for use according to any of the preceding claims, wherein the isRNA is a non-coding RNA.

5. The isRNA for use according to any of the preceding claims, wherein the isRNA comprises a nucleic acid sequence according to
(G.sub.lX.sub.mG.sub.n),formula (I) wherein: G is guanosine (guanine), uridine (uracil) or an analogue of guanosine (guanine) or uridine (uracil); X is guanosine (guanine), (uridine) uracil, adenosine (adenine), thymidine (thymine), cytidine (cytosine) or an analogue of the above-mentioned nucleotides (nucleosides); l is an integer from 1 to 40, wherein when l=1 G is guanosine (guanine) or an analogue thereof, when l>1 at least 50% of the nucleotides (nucleosides) are guanosine (guanine) or an analogue thereof; m is an integer and is at least 3; wherein when m=3 X is uridine (uracil) or an analogue thereof, when m>3 at least 3 successive uridines (uracils) or analogues of uridine (uracil) occur; n is an integer from 1 to 40, wherein when n=1 G is guanosine (guanine) or an analogue thereof, when n>1 at least 50% of the nucleotides (nucleosides) are guanosine (guanine) or an analogue thereof; a nucleic acid sequence according to
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.aformula (III) wherein: G is guanosine (guanine), uridine (uracil) or an analogue of guanosine (guanine) or uridine (uracil), preferably guanosine (guanine) or an analogue thereof; X is guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine), or an analogue of these nucleotides (nucleosides), preferably uridine (uracil) or an analogue thereof; N is a nucleic acid sequence having a length of about 4 to 50, preferably of about 4 to 40, more preferably of about 4 to 30 or 4 to 20 nucleic acids, each N independently being selected from guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine) or an analogue of these nucleotides (nucleosides); a is an integer from 1 to 20, preferably from 1 to 15, most preferably from 1 to 10; l is an integer from 1 to 40, wherein when l=1, G is guanosine (guanine) or an analogue thereof, when l>1, at least 50% of these nucleotides (nucleosides) are guanosine (guanine) or an analogue thereof; m is an integer and is at least 3; wherein when m=3, X is uridine (uracil) or an analogue thereof, and when m>3, at least 3 successive uridines (uracils) or analogues of uridine (uracil) occur; n is an integer from 1 to 40, wherein when n=1, G is guanosine (guanine) or an analogue thereof, when n>1, at least 50% of these nucleotides (nucleosides) are guanosine (guanine) or an analogue thereof; u,v may be independently from each other an integer from 0 to 50, preferably wherein when u=0, v1, or when v=0, u1; wherein the nucleic acid molecule of formula (III) has a length of at least 50 nucleotides, preferably of at least 100 nucleotides, more preferably of at least 150 nucleotides, even more preferably of at least 200 nucleotides and most preferably of at least 250 nucleotides; and/or

6. The isRNA for use according to any of the preceding claims, wherein the isRNA comprises at least one nucleic acid sequence according to any one of SEQ ID NOs: 433 to 437, 1014 to 1016, or a fragment or variant of any of these nucleic acid sequences.

7. The isRNA for use according to any of the preceding claims, wherein the isRNA is complexed with a cationic or polycationic compound, preferably with a cationic or polycationic polymer, a cationic or polycationic peptide or protein, a cationic or polycationic polysaccharide and/or a cationic or polycationic lipid.

8. The isRNA for use according to claim 7, wherein the cationic or polycationic compound is a polymeric carrier.

9. The isRNA for use according to claim 8, wherein the polymeric carrier is formed by a disulfide-crosslinked cationic component, preferably a disulfide-crosslinked cationic peptide, wherein the disulfide-crosslinked cationic peptide preferably comprises a peptide according to formula V
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x,(formula (V), wherein l+m+n+o+x=8-15, and l, m, n or o independently of each other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 50% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3 or 4, provided, that the overall content of Xaa does not exceed 50% of all amino acids of the oligopeptide; a peptide according to formula Va
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x(Cys).sub.y}formula (Va), wherein (Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; and x are as defined for formula V, Xaa is any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His, Orn or Cys and y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80 and 81-90, provided that the overall content of Arg (Arginine), Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least 10% of all amino acids of the oligopeptide; a peptide according to formula Vb
Cys1{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}Cys2formula (Vb) wherein empirical formula {(Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x} is as defined for formula (V) and forms a core of an amino acid sequence according to (semiempirical) formula (V) and wherein Cys1 and Cys2 are cysteines proximal to, or terminal to (Arg).sub.l; (Lys).sub.m; (His).sub.n; (Orn).sub.o; (Xaa).sub.x; and/or

10. The isRNA for use according to claim 8 or 9, wherein the polymeric carrier comprises at least one of the disulfide-crosslinked cationic peptides Cys-Arg.sub.12 or Cys-Arg.sub.12-Cys.

11. The isRNA for use according to any of claims 7 to 10, wherein the N/P ratio of the isRNA to the cationic or polycationic compound, preferably the cationic or polycationic peptide or protein, 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.

12. The isRNA for use according to any of the preceding claims, wherein the isRNA is complexed with one or more lipids, thereby forming liposomes, lipid nanoparticles and/or lipoplexes.

13. The isRNA for use according to any of the preceding claims, wherein the treatment comprises administration of at least one additional pharmaceutically active ingredient.

14. The isRNA for use according to claim 13, wherein the at least one additional pharmaceutically active ingredient is a compound that is used in the treatment of a tumor or cancer disease preferably selected from the group consisting of cutaneous melanoma (cMEL), cutaneous squamous cell carcinoma (cSCC), head and neck squamous cell carcinoma (HNSCC), adenoid cystic carcinoma (ACC), cutaneous T-cell lymphoma, preferably cutaneous T-cell lymphoma of mycosis fungoides subtype, and vulvar squamous cell cancer (VSCC), wherein the tumor or the cancer disease is preferably at an advanced stage and/or refractory to standard therapy.

15. The isRNA for use according to claim 13 or 14, wherein the at least one additional pharmaceutically active ingredient is a checkpoint modulator or a fragment or variant thereof.

16. The isRNA for use according to claim 15, wherein the checkpoint modulator is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, a TIGIT-inhibitor an OX40 stimulator, a 4-1BB stimulator, a CD40L stimulator, a CD28 stimulator and a GITR stimulator, or a fragment or variant of any of these checkpoint modulators.

17. The isRNA for use according to claim 16, wherein the checkpoint modulator is a PD-1 inhibitor or a PD-L1 inhibitor, wherein the PD-1 inhibitor is preferably an antagonistic antibody directed against PD-1 and the PD-L1 inhibitor is preferably an antagonistic antibody directed against PD-L1, or a fragment or variant of said antibody.

18. The isRNA for use according to claim 16, wherein the checkpoint modulator is a CTLA-4 inhibitor, preferably an antagonistic antibody directed against CTLA4, or a fragment or variant thereof.

19. The isRNA for use according to claim 13 or 14, wherein the at least one additional pharmaceutically active ingredient is an interleukin, preferably IL-12, or a fragment or variant thereof.

20. The isRNA for use according to any of claims 1 to 19, wherein the treatment comprises administration of at least one coding RNA, preferably at least one mRNA.

21. The isRNA for use according to claim 20, wherein the at least one coding RNA comprises at least one coding sequence encoding at least one peptide or protein comprising at least one peptide or protein selected from the group consisting of IL-12, CD40L, a decoy PD-1 receptor, and an antagonistic antibody directed against CTLA4, or a fragment or variant of any of these.

22. The isRNA for use according to claim 20 or 21, wherein the at least one coding RNA comprises at least one coding sequence encoding a peptide or protein comprising IL-12 or a fragment or variant thereof.

23. The isRNA for use according to claim 20 or 21, wherein the at least one coding RNA comprises at least one coding sequence encoding a peptide or protein comprising CD40L or a CD40L analogue or a fragment or variant thereof.

24. The isRNA for use according to claim 209 or 21, wherein the at least one coding RNA comprises at least one coding sequence encoding a peptide or protein comprising a decoy PD-1 receptor or a PD-1 decoy receptor analogue or a fragment or variant thereof.

25. The isRNA for use according to claim 20 or 21, wherein the at least one coding RNA comprises at least one coding sequence encoding a peptide or protein comprising an antagonistic antibody directed against CTLA4, or an analogue or a fragment or variant thereof.

26. The isRNA for use according to claim 20 or 21, wherein the at least one coding RNA comprises at least one coding sequence encoding a peptide or protein comprising IL-12 or a fragment or variant thereof, the same or a different coding RNA comprises at least one coding sequence encoding a peptide or protein comprising CD40L or a fragment or variant thereof, the same or a different coding RNA comprises at least one coding sequence encoding a peptide or protein comprising an antagonistic antibody directed against CTLA4 or a fragment or variant thereof, and optionally the same or a different coding RNA comprises at least one coding sequence encoding a peptide or protein comprising a decoy PD-1 receptor or a fragment or variant thereof.

27. The isRNA for use according to any of claims 20 to 26, wherein the subject does not receive or has not received a treatment with a PD-1 or PD-L1 antagonist and wherein the use comprises administration of at least one peptide or protein comprising a decoy PD-1 receptor or a fragment or variant thereof, or administration of a nucleic acid comprising a nucleic acid sequence encoding at least one peptide or protein comprising a decoy PD-1 receptor or a fragment or variant thereof.

28. The isRNA for use according to claim 26 or 27, wherein the coding sequence encoding a peptide or protein comprising IL-12 or a fragment or variant thereof, the coding sequence encoding a peptide or protein comprising CD40L or a fragment or variant thereof, the coding sequence encoding a peptide or protein comprising an antagonistic antibody directed against CTLA4 or a fragment or variant thereof, and, optionally, the coding sequence encoding a peptide or protein comprising a decoy PD-1 receptor or a fragment or variant thereof, are located on a separate coding RNA, preferably a separate mRNA.

29. The isRNA for use according to claim 26 or 27, wherein at least two of the coding sequences encoding a peptide or protein comprising IL-12 or a fragment or variant thereof, encoding a peptide or protein comprising CD40L or a fragment or variant thereof, encoding a peptide or protein comprising an antagonistic antibody directed against CTLA4 or a fragment or variant thereof, and, optionally, encoding a peptide or protein comprising a decoy PD-1 receptor or a fragment or variant thereof, are located on the same coding RNA, which is preferably a bi- or multicistronic RNA.

30. The isRNA for use according to any of claims 20 to 29, wherein the at least one coding RNA is administered intratumorally.

31. The isRNA for use according to claim 28, wherein the separate coding RNAs are formulated together and administered intratumorally.

32. The isRNA for use according to any of claims 20 to 31, wherein the isRNA is formulated together with the at least one coding RNA.

33. The isRNA for use according to claim 32, wherein the co-formulation is administered intratumorally.

34. The isRNA for use according to any of claims 20 to 33, wherein the at least one coding RNA comprises at least one coding sequence encoding a peptide or protein comprising IL-12, preferably at least one of IL-12A or IL-12B, or a fragment or variant of any of these proteins.

35. The isRNA for use according to claim 34, wherein the encoded peptide or protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 8, or a fragment or variant of any of these sequences.

36. The isRNA for use according to claim 34 or 35, wherein the at least one coding sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 440 to 445 or a fragment or variant of any of these sequences.

37. The isRNA for use according to any of claims 34 to 36, wherein the encoded peptide or protein comprises IL-12A and IL-12B or a fragment or variant of each of these proteins.

38. The isRNA for use according to claim 37, wherein the encoded peptide or protein comprises an amino acid sequence according to SEQ ID NO: 10 or a fragment or variant thereof.

39. The isRNA for use according to any of claims 34 to 38, wherein the at least one coding sequence comprises a nucleic acid sequence according to SEQ ID NO: 447 or a fragment or variant thereof.

40. The isRNA for use according to any of claims 20 to 39, wherein the at least one coding RNA comprises at least one coding sequence encoding a peptide or protein comprising CD40L or a fragment or variant thereof, wherein the encoded peptide or protein preferably comprises an amino acid sequence according to SEQ ID NO: 11 or a fragment or variant thereof.

41. The isRNA for use according to claim 40, wherein the at least one coding sequence comprises a nucleic acid sequence according to SEQ ID NO: 448 or a fragment or variant thereof.

42. The isRNA for use according to any of claims 20 to 41, wherein the at least one coding RNA comprises at least one coding sequence encoding a peptide or protein comprising a decoy PD-1 receptor or a fragment or variant thereof, preferably the extracellular part of a PD-1 receptor or a fragment or variant thereof.

43. The isRNA for use according to claim 42, wherein the decoy PD-1 receptor is a peptide or protein comprising soluble PD-1 or a fragment or variant thereof.

44. The isRNA for use according to claim 42 or 43, wherein the encoded peptide or protein comprises an amino acid sequence according to SEQ ID NO: 2 or a fragment or variant thereof.

45. The isRNA for use according to any of claims 42 to 44, wherein the at least one coding sequence comprises a nucleic acid sequence according to SEQ ID NO: 439 or a fragment or variant thereof.

46. The isRNA for use according to any of claims 20 to 45, wherein the at least one coding RNA comprises at least one coding sequence encoding a peptide or protein comprising an antagonistic antibody directed against CTLA4 or a fragment or variant thereof.

47. The isRNA for use according to claim 46, wherein the encoded peptide or protein comprises an amino acid sequence according to SEQ ID NO: 645 and/or 677, or a fragment or variant of any of these amino acid sequences.

48. The isRNA for use according to claim 46 or 47, wherein the at least one coding sequence comprises a nucleic acid sequence according to SEQ ID NO: 646 and/or 678, or a fragment or variant of any of these nucleic acid sequences.

49. The isRNA for use according to any of claims 20 to 48, wherein the at least one coding RNA comprises at least one coding sequence encoding a peptide or protein comprising a tumor antigen, or a fragment or variant thereof.

50. The isRNA for use according to claim 49, wherein the tumor antigen is preferably selected from the group consisting of 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; B7H.sub.4; BAGE-1; BASI; BCL-2; bcr/abl; beta-catenin/m; BING-4; BIRC7; BRCA1/m; BY55; calreticulin; CAMEL; CASP-8/m; CASPA; cathepsin_B; cathepsin_L; CD1A; CD1B; CD1C; CD1D; CD1E; CD20; CD22; CD276; CD33; CD3E; CD3Z; CD44_Isoform_1; CD44_Isoform_6; CD4; 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; CT45A1; CT55; CT-_9/BRD6; 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; IL10; IL-13Ra2; IL2-RA; IL2-RB; IL2-RG; IL-5; IMP3; ITA5; ITB1; ITB6; kallikrein-2; kallikrein-3; kallikrein-4; KI20A; KIAA0205; KIF2C; KK-LC-1; LDLR; LGMN; LIRB2; LY6K; MAGA5; MAGA8; MAGAB; MAGE-A10; MAGE-A12; MAGE-A1; MAGE-A2; MAGE-A3; MAGE-A4; MAGE-A6; MAGE-A9; MAGE-B10; MAGE-B16; MAGE-B17; MAGE-_B1; 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-E1_(MAGE1); MAGE-E2; MAGE-F1; MAGE-H.sub.1; 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; MYCN; MYO1A; MYO1B; MYO1C; MYO1D; MYO1E; MYO1F; MYO1G; MYO1H; NA17; NA88-A; Neo-PAP; NFYC/m; NGEP; 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; POTEF; POTE; 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-2B; survivin; SYCP1; SYT-SSX-1; SYT-SSX-2; TARP; TCRg; TF2AA; TGFB1; TGFR2; TGM-4; TIE2; TKTL1; TPI/m; TRGV11; TRGV9; TRPC1; TRP-p8; TSG10; TSPY1; TVC_(TRGV3); TX101; tyrosinase; TYRP1; TYRP2; UPA; VEGFR1; WT1; and XAGE1.

51. The isRNA for use according to claim 49 or 50, wherein the at least one coding RNA is not administered intratumorally.

52. The isRNA for use according to any of claims 49 to 51, wherein the at least one coding RNA is administered intradermally, intramuscularly or subcutaneously.

53. The isRNA for use according to any of claims 20 to 52, wherein the at least one coding RNA comprises at least one coding sequence comprising a nucleic acid sequence that is modified compared to the nucleic acid sequence of the coding sequence of the corresponding wild type RNA, and wherein the amino acid sequence encoded by said coding sequence is preferably not modified compared to the amino acid sequence encoded by the coding sequence of the corresponding wild type RNA.

54. The isRNA for use according to claim 53, wherein the at least one coding sequence comprises a) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 25-30; 36-41; 47-52; 58-63; 69-74; 80-85; 91-96; 102-107; 113-118; 124-129; 135-140; 601-606; 612-617; 623-628; 716-725; 727; 1018-1021 and 1059-1062, or a fragment or variant of any of these sequences, preferably from the group consisting of 32; 43; 54; 65; 76; 87; 98; 109; 120; 131; 142; 608; 619; 630; 632-644; 726 and 1058, or a fragment or variant of any of these sequences; b) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 33; 44; 55; 66; 77; 88; 99; 110; 121; 132; 143; 609; 620; 631; 728-738 and 1025-1028, or a fragment or variant of any of these sequences, c) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 646-660; 662-676; 678-692; 694-705; 707-715 or 1029-1041, or a fragment or variant of any of these sequences, and/or d) optionally, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 23; 34; 45; 56; 67; 78; 89; 100; 111; 122; 133; 599; 610; 621; 1022-1024, or a fragment or variant of any of these sequences, preferably from the group consisting of SEQ ID NOs: 24; 35; 46; 57; 68; 79; 90; 101; 112; 123; 134; 600; 611; 622 and 1043-1054, or a fragment or variant of any of these sequences

55. The isRNA for use according to any of claims 20 to 54, wherein the at least one coding RNA comprises at least one coding sequence having a modified, preferably an increased, G/C content compared to the G/C content of the coding sequence of the corresponding wild type RNA, and wherein the amino acid sequence encoded by said coding sequence is preferably not modified compared to the amino acid sequence encoded by the coding sequence of the corresponding wild type RNA.

56. The isRNA for use according to claim 55, wherein the at least one coding sequence comprises a) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 25-30; 80-85; 91-96; 102-107; 113-118; 601-606; 124-129; 135-140; 612-617; 623-628; 716-725 and 72, or a fragment or variant of any of these sequences, preferably from the group consisting of SEQ ID NOs: 32; 87; 98; 109; 120; 131; 142; 608; 619; 630; 632; 636-644 and 726, or a fragment or variant of any of these sequences; b) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 33; 88; 99; 110; 121; 132; 143; 609; 620; 631 and 728-738, or a fragment or variant of any of these sequences, and/or c) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 646; 650-658; 662; 666-674; SEQ ID NO: 678; 682-690; 694; 698-705; 707; 710 and 713, or a fragment or variant of any of these sequences, and/or d) optionally, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 23; 78; 89; 100; 111; 122; 133; 599; 610 and 621, or a fragment or variant of any of these sequences, preferably from the group consisting of SEQ ID NOs: 24; 79; 90; 101; 112; 123; 134; 600; 611 and 622, or a fragment or variant of any of these sequences.

57. The isRNA for use according to any of claims 20 to 58, wherein the at least one coding RNA comprises a 5-CAP structure.

58. The isRNA for use according to any of claims 20 to 57, wherein the at least one coding RNA comprises a 5-UTR element and/or a 3-UTR element.

59. The isRNA for use according to any of claims 20 to 58, wherein the at least one coding RNA comprises a poly(A) and/or a poly(C) sequence.

60. The isRNA for use according to any of claims 20 to 59, wherein the at least one coding RNA comprises a histone stem-loop sequence.

61. The isRNA for use according to any of claims 20 to 60, wherein the at least one coding RNA comprises a) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 146-151; 451-456; 157-162; 168-173; 179-184; 190-195; 201-206; 212-217; 223-228; 234-239; 245-250; 256-261 and 267-272, or a fragment or variant of any of these sequences, preferably from the group consisting of SEQ ID NOs: 153; 458; 164; 175; 186; 197; 208; 219; 230; 241; 252; 263, 274; 992 and 598, or a fragment or variant of any of these sequences, b) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 154; 459; 165; 176; 187; 198; 209; 220; 231; 242; 253; 264, 275 and 596, or a fragment or variant of any of these sequences, c) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 594; 595; 860-925, or a fragment or variant of any of these sequences, and/or d) optionally, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 144; 449; 155; 166; 177; 188; 199; 210; 221; 232; 243; 254 and 265, or a fragment or variant of any of these sequences, preferably from the group consisting of SEQ ID NOs: 145; 450; 156; 167; 178; 189; 200; 211; 222; 233; 244; 255; 266 and 597, or a fragment or variant of any of these sequences.

62. The isRNA for use according to claim any of claims 20 to 61, wherein the treatment comprises administration, preferably intratumoral administration, of at least three coding RNAs, wherein a first coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 153; 164; 175; 186; 197; 208; 219; 230; 241; 252; 263; 274; 992; 458 and 598, or a fragment or variant of any of these sequences, a second coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 154; 165; 176; 187; 198; 209; 220; 231; 242; 253; 264; 275; 459 and 596, or a fragment or variant of any of these sequences, and a third coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 920-922 or 923-925, or a fragment or variant of any of these sequences, and optionally, a fourth coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 145; 156; 167; 178; 189; 200; 211; 222; 233; 244; 255; 266; 450 and 597, or a fragment or variant of any of these sequences.

63. The isRNA for use according to claim any of claims 20 to 62, wherein the treatment comprises administration, preferably intratumoral administration, of at least four coding RNAs, wherein a first coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 153; 164; 175; 186; 197; 208; 219; 230; 241; 252; 263; 274; 992; 458 and 598, or a fragment or variant of any of these sequences, a second coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 154; 165; 176; 187; 198; 209; 220; 231; 242; 253; 264; 275; 459 and 596, or a fragment or variant of any of these sequences, a third coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 594 and 860-874, or a fragment or variant of any of these sequences, a fourth coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 595 and 890-904, or a fragment or variant of any of these sequences, and optionally, a fifth coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 145; 156; 167; 178; 189; 200; 211; 222; 233; 244; 255; 266; 450 and 597, or a fragment or variant of any of these sequences.

64. The isRNA for use according to any of claims 20 to 63, wherein the at least one coding RNA comprises a) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 278-283; 289-294; 300-305; 311-316; 322-327; 333-338; 344-349; 355-360; 366-371; 377-382; 388-393; 399-404 and 462-467, or a fragment or variant of any of these sequences, preferably from the group consisting of SEQ ID NOs: 285; 296; 307; 318; 329; 340; 351; 362; 373; 384; 395; 406; 430; 469 and 992, or a fragment or variant of any of these sequences, b) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 286; 297; 308; 319; 330; 341; 352; 363; 374; 385; 396; 470 and 407, or a fragment or variant of any of these sequences, c) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 926-955, or a fragment or variant of any of these nucleic acid sequences, and a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 956-985, or a fragment or variant of any of these nucleic acid sequences; or a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 986-991, or a fragment or variant of any of these nucleic acid sequences, and/or d) optionally, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 276; 287; 298; 309; 320; 331; 342; 353; 364; 375; 386; 460 and 397, or a fragment or variant of any of these sequences, preferably from the group consisting of SEQ ID NOs: 277; 288; 299; 310; 321; 332; 343; 354; 365; 376; 461; 387 and 398, or a fragment or variant of any of these sequences.

65. The isRNA for use according to claim any of claims 20 to 64, wherein the treatment comprises administration, preferably intratumoral administration, of at least four coding RNAs, wherein a first coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 285; 296; 307; 318; 329; 340; 351; 362; 373; 384; 395; 406; 430; 469 and 992, or a fragment or variant of any of these sequences, a second coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 286; 297; 308; 319; 330; 341; 352; 363; 374; 385; 396; 470 and 407, or a fragment or variant of any of these sequences, a third coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 926-940, or a fragment or variant of any of these sequences, a fourth coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 956-970, or a fragment or variant of any of these sequences, and optionally, a fifth coding RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 277; 288; 299; 310; 321; 332; 343; 354; 365; 376; 387; 461 and 398, or a fragment or variant of any of these sequences.

66. The isRNA for use according to any of claims 20 to 65, wherein the isRNA is administered as RNA complexed with one or more cationic or polycationic compounds, and the at least one coding RNA, preferably an mRNA, is administered as free RNA or is administered a RNA that is complexed with one or more lipids, thereby forming liposomes, lipid nanoparticles and/or lipoplexes.

67. The isRNA for use according to any of claims 1 to 66, wherein the treatment comprises chemotherapy, radiation therapy and/or surgery.

68. Pharmaceutical composition comprising an immunostimulatory RNA (isRNA) and a pharmaceutically acceptable carrier and/or vehicle for use in the treatment of a tumor or cancer disease preferably selected from the group consisting of cutaneous melanoma (cMEL), cutaneous squamous cell carcinoma (cSCC), head and neck squamous cell carcinoma (HNSCC), adenoid cystic carcinoma (ACC), cutaneous T-cell lymphoma, preferably cutaneous T-cell lymphoma of mycosis fungoides subtype, and vulvar squamous cell cancer (VSCC), wherein the tumor or the cancer disease is preferably at an advanced stage and/or refractory to standard therapy.

69. The pharmaceutical composition for use according to claim 68, wherein the isRNA is as defined in claims 1 to 12.

70. The pharmaceutical composition for use according to claim 68 or 69, wherein the treatment comprises administration of at least one additional pharmaceutically active ingredient, preferably as defined in any of claims 13 to 62.

71. Kit or kit of parts comprising an immunostimulatory RNA (isRNA), preferably the isRNA as defined in claims 1 to 12, or the pharmaceutical composition as defined in any of claims 68 to 70, and optionally technical instructions with information on the administration and dosage for administration, for use in the treatment of a tumor or cancer disease preferably selected from the group consisting of cutaneous melanoma (cMEL), cutaneous squamous cell carcinoma (cSCC), head and neck squamous cell carcinoma (HNSCC), adenoid cystic carcinoma (ACC), cutaneous T-cell lymphoma, preferably cutaneous T-cell lymphoma of mycosis fungoides subtype, and vulvar squamous cell cancer (VSCC), wherein the tumor or the cancer disease is preferably at an advanced stage and/or refractory to standard therapy, wherein the pharmaceutical composition is administered intratumorally.

72. Use of an isRNA, preferably the isRNA as defined according to any of claims 1 to 12, the pharmaceutical composition as defined in any of claims 68 to 70, or the kit or kit of parts as defined in claim 71, for use in the manufacture of a medicament for treatment of a tumor or cancer disease preferably selected from the group consisting of cutaneous melanoma (cMEL), cutaneous squamous cell carcinoma (cSCC), head and neck squamous cell carcinoma (HNSCC), adenoid cystic carcinoma (ACC), cutaneous T-cell lymphoma, preferably cutaneous T-cell lymphoma of mycosis fungoides subtype, and vulvar squamous cell cancer (VSCC), wherein the tumor or the cancer disease is preferably at an advanced stage and/or refractory to standard therapy, for intratumoral administration.

73. Method of treating or preventing a disorder selected from the group consisting of a tumor or cancer disease preferably selected from the group consisting of cutaneous melanoma (cMEL), cutaneous squamous cell carcinoma (cSCC), head and neck squamous cell carcinoma (HNSCC), adenoid cystic carcinoma (ACC), cutaneous T-cell lymphoma, preferably cutaneous T-cell lymphoma of mycosis fungoides subtype, and vulvar squamous cell cancer (VSCC), wherein the tumor or the cancer disease is preferably at an advanced stage and/or refractory to standard therapy, wherein the method comprises administering, preferably intratumorally, to a subject in need thereof an effective amount of an isRNA, preferably the isRNA as defined according to any of claims 1 to 12, the pharmaceutical composition as defined in any of claims 68 to 70, or the kit or kit of parts as defined in claim 71.

Description

BRIEF DESCRIPTION OF THE FIGURES

[1227] The examples and figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures and examples shall not be construed to limit the present invention thereto.

[1228] FIG. 1: Panel (A) shows an analysis of the median tumor growth of Balb/C mice bearing CT26 tumors after intratumoral treatment with RNAdjuvant, mRNA encoding IL-12 and mRNA-encoded 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 1. [1229] Panel (B) shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with RNAdjuvant, mRNA encoding IL-12 and mRNA encoded 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 1. Kaplan-Meier survival curves are presented.

[1230] FIG. 2: shows survival proportions of Balb/C 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 2. Kaplan-Meier survival curves are presented.

[1231] FIG. 3: shows survival proportions of Balb/C mice bearing CT26 tumors after intratumoral treatment with RNAdjuvant, mRNA encoding IL-12 and mRNA-encoded 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 3. Kaplan-Meier survival curves are presented.

[1232] FIG. 4: shows an analysis of the median tumor growth after re-challenge of Balb/C mice with syngeneic CT26 colon carcinoma cells at day 113 after the first tumor challenge. Mice were previously treated intratumorally with RNAdjuvant alone or in combination with anti-PD1 treatment. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 4.

[1233] FIG. 5: shows an analysis of the median tumor growth after re-challenge of Balb/C mice with syngeneic CT26 colon carcinoma cells at day 113 after the first tumor challenge. Mice were previously treated intratumorally with RNAdjuvant alone or in combination with an mRNA encoding CD40L and an mRNA-encoded IL-12. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 5.

[1234] FIG. 6: shows an analysis of the median tumor growth of Balb/C mice bearing CT26 tumors after intratumoral treatment with immunostimulating RNA (RNAdjuvant), mRNA encoding soluble PD1 (solPD1) and CD40 ligand (CD40L) in combination with a checkpoint inhibitor anti CTLA4 antibody. The experiment was performed as described in Example 7.

[1235] FIG. 7: shows an analysis of the median tumor growth of the untreated lesion of Balb/C mice bearing CT26 tumors in both flanks after intratumoral treatment of one lesion with immunostimulating RNA (RNAdjuvant), mRNA encoding soluble PD1 (solPD1) and CD40 ligand (CD40L) in combination with an anti-CTLA4 checkpoint antibody. The experiment was performed as described in Example 8.

[1236] FIG. 8: Panel (A) shows an analysis of the median tumor growth of Balb/C mice bearing E.G7-OVA tumors after intratumoral treatment with immunostimulatory RNAdjuvant and vaccinated i.d. with OVA (RNActive) or in combination with an anti-PD1 checkpoint inhibitor (administered i.p.) and PpLuc RNActive or buffer as unspecific control. The experiment was performed as described in Example 10. [1237] Panel (B) shows survival proportions of mice bearing E.G7-OVA tumors after intratumoral treatment with immunostimulatory RNAdjuvant and vaccinated i.d. with OVA (RNActive) or in combination with a checkpoint inhibitor anti PD1 (administered i.p.) and PpLuc RNActive or buffer as unspecific control. The experiment was performed as described in Example 10. Kaplan-Meier survival curves are presented.

[1238] FIG. 9: Panel (A) shows an analysis of the median tumor growth of Balb/C mice bearing E.G7-OVA tumors after intratumoral treatment with immunostimulatory RNAdjuvant and an mRNA encoding IL12 vaccinated i.d. with OVA (RNActive) and PpLuc RNActive or buffer as unspecific control. The experiment was performed as described in Example 11. [1239] Panel (B) shows survival proportions of mice bearing E.G7-OVA tumors after intratumoral treatment with immunostimulatory RNAdjuvant and an mRNA encoding IL12 vaccinated i.d. with OVA (RNActive) and PpLuc RNActive or buffer as unspecific control. The experiment was performed as described in Example 11. Kaplan-Meier survival curves are presented.

[1240] FIG. 10: Panel (A) shows translated mRNA products of IL12 in the supernatant of RNA transfected A375 cells after 5 hours. The experiment was performed as described in Example 13. [1241] Panel (B) shows translated mRNA products of IL12 in the supernatant of RNA transfected A375 cells after 24 hours. The experiment was performed as described in Example 13.

[1242] FIG. 11: Panel (A) shows translated mRNA products of solPD1 in the supernatant of RNA transfected A375 cells after 5 hours. The experiment was performed as described in Example 13. [1243] Panel (B) shows translated mRNA products of solPD1 in the supernatant of RNA transfected A375 cells after 24 hours. The experiment was performed as described in Example 13.

[1244] FIG. 12: Panel (A) shows translated mRNA products of anti-CTLA4 antibody in the supernatant of RNA transfected A375 cells after 5 hours. The experiment was performed as described in Example 13. [1245] Panel (B) shows translated mRNA products of anti-CTLA4 antibody in the supernatant of RNA transfected A375 cells after 24 hours. The experiment was performed as described in Example 13.

[1246] FIG. 13: shows membrane bound translated mRNA product of CD40LG on transfected A375 cells after 24 hours analyzed by FACS analysis. The experiment was performed as described in Example 13.

EXAMPLES

[1247] The Examples shown in the following are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.

[1248] Preparation of DNA, mRNA Constructs and Immunostimulatory RNA (RNAdjuvant)

[1249] 1. Preparation of DNA and RNA Constructs

TABLE-US-00004 TABLE 3 RNA constructs SEQ ID RNA Description NO: R2025 R2391 Non-coding immunostimulatory 433 RNA (RNAdjuvant) R2763 mRNA encoding murine 430 IL-12 (IL-12 (GC)) R3971 mRNA encoding murine soluble 431 PD-1 (soluble PD-1 (GC)) R3571 mRNA encoding murine CD40L 432 (CD40L (GC)) R5417 mRNA encoding heavy chain (HC) of 594 anti-CTLA4 antibody (HC anti-CTLA4 Ab (GC)) R5418 mRNA encoding light chain (LC) of 595 anti-CTLA4 antibody (HLC anti-CTLA4 Ab (GC))

[1250] The constructs of IL-12 (GC), soluble PD-1 (GC) (solPD1), CD40L (GC) and two anti-CTLA4 antibody chains were prepared 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. The mRNAs expressing human IL-12, soluble PD-1 receptor, CD40L and anti-CTLA4 antibody are prepared in analogous manner by using the corresponding human coding sequences.

[1251] 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.

[1252] 2. RNA In Vitro Transcription

[1253] 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, CD40L, soluble PD-1 and anti-CTLA4 antibody encoding constructs were performed in the presence of a CAP analog (m7GpppG). The isRNA R2025 was prepared without CAP analog. Subsequently, the RNA was purified using PureMessenger (CureVac, Tbingen, Germany; WO2008077592).

[1254] 3. Preparation of the Polymeric Cargo Complex (RNAdjuvant)

[1255] The following cationic peptide as cationic component of the polymeric carrier was used (Cys-Arg12-Cys or CR12C) according to SEQ ID NO: 579 or SEQ ID NO: 580.

[1256] 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 CR12C 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.

[1257] The mass ratio of peptide:RNA was 1:3,7. The polymeric carrier cargo complex is formed by the disulfide-crosslinked cationic peptide CR12C as carrier and the immunostimulatory R2025 as nucleic acid cargo. This polymeric carrier cargo complex R2025/CR12C (R2391) was used as adjuvant in the following examples (referred to as RNAdjuvant)

[1258] 4. Preparation of the RNA for Administration

[1259] IL-12 mRNA (R1328), soluble PD-1 mRNA (R3971) and 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 Treatment with an Immunostimulating RNA (RNAdjuvant) and an mRNA Encoding Soluble PD-1 and an mRNA Encoding IL-12

[1260] Balb/c mice (see Table 4) 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 50 mm.sup.3. Intratumoral (i.t.) therapy started at day 9 and continued twice a week for three weeks. Mice were injected with a combination of RNAdjuvant (25 g of R2391), mRNA-encoded IL-12 (25 g of R2763) and mRNA-encoded soluble PD-1 (R3971) (group A according to Table 2) or mRNA-encoded IL-12 (25 g of R2763) (group B according to Table 2) alone or RNAdjuvant (25 g of R2391) alone (group C according to Table 2). To control for anti-tumor effects due to injection procedure, mice were injected with buffer (RiLa, group D according to Table 4), respectively.

[1261] 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]$\mathrm{volume}.Math..Math.\left({\mathrm{mm}}^3\right)=\frac{\mathrm{length}.Math..Math.\left(\mathrm{mm}\right){\mathrm{width}}^2\left({\mathrm{mm}}^2\right)}{6}$

[1262] On days 9, 11, 14, 17 and 21 of the experiment mice were injected intratumorally (i.t.) with RNA according to the Table 4 below. The volume for intratumoral injection was 50 l.

[1263] Table 4 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-00005 TABLE 4 Groups, treatment and RNA dilution No. of Amount of Vaccination Group mice Constructs RNA (g) schedule A 10 RNAdjuvant + IL-12 + 25 each 2x week soluble PD-1 B 10 IL-12 25 2x week C 10 RNAdjuvant 25 2x week D 10 RiLa 2x week

[1264] Tumor Challenge and Administration of the Inventive Composition:

[1265] Mice were injected according to the indicated scheme shown in Table 4. Median tumor growth was determined according to formula above. The results of the experiment are shown in FIG. 1, wherein FIG. 1A shows the effect of the inventive composition on tumor growth and FIG. 1B shows the effect of the inventive composition on survival.

[1266] Results:

[1267] The results in FIG. 1A 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 4) strongly decreased the median tumor volume compared to the other treatments (groups B-D according to Table 4). In addition, the results in FIG. 1B 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 4) strongly increased the survival of tumor challenged mice compared to the other treatments (groups B-D according to Table 4).

Example 2: Treatment with an Immunostimulating RNA (RNAdjuvant) in Combination with a Checkpoint Inhibitor Anti PD-1 Antibody

[1268] Table 5 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 intraperitoneal (i.p.) in CT26 tumor challenged mice, survival rates were analyzed.

TABLE-US-00006 TABLE 5 Groups, treatment and RNA dilution/antibody dilution Construct Amount Antibody No. of (i.t. of RNA (i.p. Vaccination Group mice treatment) (g) treatment) schedule A 8 RiLa 2x week B 8 RNAdjuvant 25 Control Ab 2x week (100 g) C 7 RNAdjuvant 25 Anti-PD-1 2x week (200 g) D 6 RiLa Anti-PD-1 2x week (200 g)

[1269] Tumor Challenge and Administration of the Inventive Composition:

[1270] The tumor challenge was performed according to the previous experiment (see Example 1). Mice were injected according to the indicated scheme shown in Table 5. The results of the experiment are shown in FIG. 2.

[1271] Results:

[1272] FIG. 2 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 5) 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 5) 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 5).

Example 3: Intratumoral Treatment with an Immunostimulating RNA (RNAdjuvant) and an mRNA Encoding CD40 Ligand (CD40L) and an mRNA Encoding IL-12

[1273] Table 6 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 analysed.

TABLE-US-00007 TABLE 6 Groups, treatment and RNA dilution No. of Constructs Vaccination Group mice (amount of RNA) schedule A 10 RNAdjuvant (50 g) + 2x week IL-12 (75 g) + CD40L (75 g) B 10 RNAdjuvant (100 g) 2x week C 10 RiLa 2x week

[1274] Tumor Challenge and Administration of the Inventive Composition:

[1275] The tumor challenge was performed according to the previous experiments (see Example 1). Mice were injected according to the indicated scheme shown in Table 6. The results of the experiment are shown in FIG. 3.

[1276] Results:

[1277] The results in FIG. 3 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 6) strongly increased the median survival of tumor challenged mice compared to the other treatments (groups B to C according to Table 6).

Example 4: Induction of Systemic Anti-Tumoral Memory Response by Combination of Intratumoral Treatment with an Immunostimulating RNA (RNAdjuvant) and Systemic Anti-PD-1 Treatment

[1278] Table 7 summarizes the treatment as used in the present example. RNAdjuvant (administered i.t.) and systemic treatment with a checkpoint inhibitor anti PD-1 (BioXCell) was evaluated by performing re-challenge of mice completely eradicating the primary CT-26 tumor after first treatment, survival rates were analyzed.

TABLE-US-00008 TABLE 7 Groups, treatment and RNA dilution Constructs of first tumor challenge No. of (amount of RNA/Ab + Group mice route of treatment) A 7 RNAdjuvant (100 g, i.t.) + Anti-PD-1 (200 g, i.p.) B 3 RNAdjuvant (100 g, i.t.) C 7 RiLa

[1279] Tumor Re-Challenge after Administration of the Inventive Composition:

[1280] First tumor challenge was performed by s.c. injection of CT-26 tumor cells on the right flank in Balb/C mice, whereas re-challenge with 110.sup.6 syngeneic CT26 colon carcinoma cells was performed on the left flank at day 113 after first tumor challenge. Challenge of nave animals served as control. Tumor eradication of the primary tumor was noted in a lower percentage of animals with intratumoral RNAdjuvant alone (3 out of 10 mice) as compared to the combination of systemic anti-PD-1 with intratumoral RNAdjuvant (7 out of 9 mice). The results of the experiment are shown in FIG. 4.

[1281] Results:

[1282] The results in FIG. 4 show that all mice which have eradicated the first tumor were completely protected against the second tumor challenge demonstrating the induction of systemic memory response. Systemic memory response was also induced by intratumoral RNAdjuvant treatment alone. The induction of a systemic memory response is remarkable as no vaccine inducing an adaptive immune response was administered. Therefore administration of the inventive isRNA, particularly in combination with systemic anti-PD-1 treatment, was sufficient to induce a systemic immune response which could not have been expected.

Example 5: Induction of Systemic Anti-Tumoral Memory Response by Combination of Intratumoral Treatment with an mRNA Encoding CD40 Ligand (CD40L) and an mRNA Encoding IL-12

[1283] Table 8 summarizes the treatment as used in the present example. RNAdjuvant and the mRNA constructs encoding IL-12 and murine CD40L (administered i.t.) were evaluated by performing re-challenge of mice completely eradicating the primary CT-26 tumor after treatment, survival rates were analyzed.

TABLE-US-00009 TABLE 8 Groups, treatment and RNA dilution Constructs of first No. of tumor challenge Group mice (amount of RNA) A 5 RNAdjuvant (50 g) + CD40L (75 g) + IL-12 (75 g) B 3 RNAdjuvant (100 g) C 7 RiLa

[1284] Tumor Re-Challenge after Administration of the Inventive Composition:

[1285] First tumor challenge was performed by s.c. injection of CT-26 tumor cells on the right flank in Balb/C mice. After intratumorally treatment with RNAdjuvant alone or in combination with an mRNA encoding CD40 ligand (CD40L) and an mRNA encoding IL-12 treatment, re-challenge with 110.sup.6 syngeneic CT26 colon carcinoma cells was performed on the left flank at day 113 after the first tumor challenge. Challenge of nave animals served as control. Tumor eradication of the primary tumor was noted in a lower percentage of animals with intratumoral RNAdjuvant alone (3 out of 10 mice) as compared to the combination of mRNA encoding CD40 ligand (CD40L) and an mRNA-encoded IL-12 with intratumoral RNAdjuvant (5 out of 10 mice). The results of the experiment are shown in FIG. 5.

[1286] Results:

[1287] The results in FIG. 5 show that all mice which have eradicated the first tumor were completely protected against the second tumor challenge demonstrating the induction of systemic memory response. Systemic memory response was also induced by intratumoral RNAdjuvant treatment alone.

Example 6: Phase I/II Study of Intratumoral Application of RNAdjuvant (CV8102) in Patients with Advanced Cutaneous Melanoma (cMEL), Cutaneous Squamous Cell Carcinoma (cSCC), Head and Neck Squamous Cell Carcinoma (hnSCC), or Adenoid Cystic Carcinoma (ACC)

[1288] Part 1:

[1289] Phase I, open label, cohort based dose escalation & expansion study of intratumorally administered RNAdjuvant, with or without systemic anti-PD-1 treatment, in patients having advanced cutaneous melanoma (cMEL), cutaneous squamous cell carcinoma (cSCC), head and neck squamous cell carcinoma (hnSCC), or adenoid cystic carcinoma (ACC).

[1290] Intratumoral injection of RNAdjuvant: CV8102 is administered to cutaneous, subcutaneous, or readily accessible lymph node lesions that can be injected using direct visualization or imaging-guidance (ultrasound) as clinically determined.

[1291] Schedule: Patients receive a maximum of 8 intratumoral administrations of CV8102, unless disease progression requiring initiation of next-line therapy or unacceptable toxicity occurs:

[1292] The first 5 administrations are performed in weekly intervals (Days 1, 8, 15, 22, 29). For patients on anti-PD-1 treatment in Cohorts C and D, CV8102 treatment is initiated on a day of anti-PD-1 treatment and will follow the anti-PD-1 treatment schedule after day 29.

[1293] The subsequent 3 administrations of CV8102 are performed with 2-week intervals (Cohorts A and B). For patients on anti-PD-1 treatment in study Cohorts C and D, the administrations of CV8102 after Day 29 follow the anti-PD-1 treatment schedule and are performed on the day of anti-PD-1 administration; i.e., patients on nivolumab receive CV8102 every 2nd week; patients on pembrolizumab receive CV8102 every 3rd week.

[1294] Part A: Dose Escalation of Single Agent RNAdjuvant

[1295] Part A of the study uses a 2 parameter Bayesian logistic regression model with overdose control for dose escalation. Cohorts of at least 1 (starting dose level) or 2 patients (any other dose level) with advanced cMEL, cSCC, hnSCC, or ACC are treated at escalating doses of intratumorally administered RNAdjuvant until identification of the maximum tolerated dose (MTD) and determination of the recommended dose (RD). A minimum of 7 patients should be enrolled in Part A.

[1296] The starting dose in Part A is 25 g of RNAdjuvant. The further dose levels are listed in Table 9 below.

TABLE-US-00010 TABLE 9 Dose levels of RNAdjuvant Dose Levels evaluated in Part A and C Dose Level RNAdjuvant No of Patients.sup. Level 1 25 g 1 Level 2* 50 g 2 Level 3 100 g 2 Level 4 150 g 2 *Starting dose level Part A; potential starting dose level Part C, based on review of Part A data .sup.A minimum of 3 patients will be treated per cohort

[1297] Inclusion Criteria:

[1298] Cohort A:

[1299] Patients with histologically confirmed advanced (unresectable or metastatic) cMEL or cSCC who failed approved standard therapy or for whom no standard therapy is indicated

[1300] Not amenable to surgical resection or locoregional radiation therapy with curative intent

[1301] Progression on/after at least one line of therapy

or

[1302] Histologically confirmed recurrent or metastatic hnSCC

[1303] Not amenable to surgical resection or locoregional radiation therapy with curative intent

[1304] Progression documented radiologically (documentation of a new or progressive lesion on/after at least one line of therapy.

or

[1305] Histologically or cytologically confirmed ACC

[1306] Not amenable to surgical resection or locoregional radiation therapy with curative intent

[1307] Progression documented radiologically; on/after at least one line of therapy

[1308] Part B: Expansion Cohort

[1309] After completion of Part A, patients with advanced cSCC or ACCare enrolled to separate expansion cohorts at the previously defined recommended dose to further characterize the tolerability and safety profile of intratumorally administered RNAdjuvant in these patient populations and to collect preliminary evidence of anti-tumor activity. Part B should enroll up to 10 patients per expansion cohort. Inclusion criteria:

[1310] Cohort B:

[1311] Expansion cohorts B1 and B2

[1312] Patients with histologically confirmed advanced (unresectable/metastatic) cSCC (B1) or ACC (B2) who failed standard therapy or for whom no standard therapy is indicated

[1313] Not amenable to curative locoregional treatments

[1314] Progression on/after at least one line of therapy (radiological documentation required for ACC)

[1315] Part C: Dose Escalation of RNAdjuvant in Combination with Anti-PD-1 Therapy in Patients with Advanced cMEL or HNSCC

[1316] Part C enrolls patients with advanced cMEL or HNSCC currently receiving anti-PD-1 therapy. Patients must have stable disease or slowly progressive disease after at least 12 weeks of anti-PD-1 therapy prior to application of RNAdjuvant. Cohorts of at least 2 patients are treated sequentially at escalating doses of RNAdjuvant. The dose escalation and the determination of the MTD and RCD (recommended combination dose) are guided by a 5 parameter bayesian logistic regression model with overdose control.

[1317] Dose escalation in Part C starts as soon as at least 3 doses of RNAdjuvant have been evaluated in Part A. Starting dose in Part C is one dose level below the highest dose level considered tolerable from part A at the time Part C is commenced (see Table 9).

[1318] Inclusion Criteria:

[1319] Cohorts C and D:

[1320] Histologically confirmed advanced (unresectable or metastatic) cMEL in Cohorts C and D1

[1321] Currently receiving standard anti-PD-1 therapy according to the Summary of Product Characteristics (SPC) Stable or slowly progressing disease after at least 12 weeks of anti-PD-1 therapy according to investigator assessment

[1322] Intention to continue current anti-PD-1 therapy due to an assumed clinical benefit by continued anti-PD-1 therapy according to investigators judgement

[1323] Histologically confirmed recurrent or metastatic hnSCC in Cohorts C and D2

[1324] Currently receiving standard anti-PD-1 therapy according to the SPC

[1325] Stable or slowly progressing disease after at least 12 weeks of anti-PD-1 therapy, defined as follows:

[1326] Stable disease according to irRECIST

[1327] Intention to continue current anti-PD-1 therapy due to an assumed clinical benefit according to investigators judgement

[1328] Part D: Expansion Cohort of RNAdjuvant in Combination with Anti-PD-1 Therapy in Patients with Advanced cMEL or HNSCC.

[1329] Once the RCD (recommended combination dose) has been established in Part C, the expansion Part D enrolls additional patients with advanced cMEL or HNSCC on treatment with a PD-1 antagonist (refer to eligibility requirements Part C) to further characterize the tolerability and the safety profile and to evaluate the anti-tumor activity of the combination therapy. Part D should enroll about 21 patients.

[1330] Part 2 (May Alternatively be Performed as Separate Clinical Trial)

[1331] Phase I, open label, cohort based dose escalation & expansion study of intratumorally administered RNAdjuvant and RNArt with or without systemic anti-PD-1 treatment in patients advanced malignant melanoma, squamous cell carcinoma of the skin (SCCs), adenocystic carcinoma (ACC), cutaneous T-cell lymphoma, or squamous cell carcinoma of the head and neck (HNSCC).

[1332] In Part 2 of the phase I clinical trial, Part 1 is repeated, but a fixed dose combination of RNArt and RNAdjuvant is investigated. Dose escalation methodology and cohort definitions including clinical indications are similar to Part 1. Please refer to the previous section for details.

[1333] However, for Part 2, information gained from Part 1 is considered that may lead to a change of the study design of Part 1 and/or 2. RNArt comprises 3 compounds based on optimized RNA that encodes IL-12, PD-1 decoy receptor and CD-40L.

Example 7: Intratumoral Treatment with an Immunostimulating RNA (RNAdjuvant) and an mRNA Encoding CD40 Ligand (CD40L) and mRNA Encoding Soluble PD1 (solPD1) in Combination with a Checkpoint Inhibitor Anti CTLA-4 Antibody

[1334] Table 10 summarizes the treatment as used in the present example. RNAdjuvant and an mRNA encoding soluble PD1 and CD40L in combination with a checkpoint inhibitor anti-CTLA4 antibody (BioXcell, clone 9H10) were administered intratumorally (i.t.) in CT26 tumor challenged mice, median tumor growth were analyzed.

TABLE-US-00011 TABLE 10 Groups, treatment and RNA dilution/antibody dilution RNAdjuvant RNArt anti-CTLA4 (i.t.) (i.t.) (i.t) No. of Group (25 g) (25g) (50 g) mice A RNAdjuvant solPD1 + CD40L anti-CTLA4 10 B RNAdjuvant IL12 + solPD1 + CD40L 10 C Buffer 9

[1335] Tumor Challenge and Administration of the Inventive Composition:

[1336] The tumor challenge was performed according to the previous experiments (see Example 1). Mice were injected according to the indicated scheme shown in Table 10. The results of the experiment are shown in FIG. 6.

[1337] Results:

[1338] The results in FIG. 6 show that the inventive composition comprising RNAdjuvant and an mRNA encoding soluble PD1 and CD40L in combination with a checkpoint inhibitor anti-CTLA4 antibody (group A according to Table 10) strongly decreased the median tumor volume.

Example 8: Abscopal Effect of Intratumoral Treatment with an Immunostimulating RNA (RNAdjuvant) and an mRNA Encoding CD40 Ligand (CD40L) and mRNA Encoding Soluble PD1 (solPD1) in Combination with a Checkpoint Inhibitor Anti CTLA-4 Antibody

[1339] Tumor Challenge and Administration of the Inventive Composition:

[1340] Balb/c mice 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. On day 5 of the experiment mice were injected subcutaneously (s.c.) with 110.sup.6 CT26 cells (in a volume of 100 l PBS) on the left flank to observe an abscopal effect (effect on the untreated tumor) of the inventive composition. Table 10 of Example 7 summarizes the treatment as used in the present example. RNAdjuvant and an mRNA encoding soluble PD1 and CD40L in combination with an anti-CTLA4 checkpoint inhibitor were administered intratumorally (i.t.) in CT26 tumor bearing mice (right flank), median tumor growth of the untreated tumor (left flank) were analyzed.

[1341] Mice were injected according to the indicated scheme shown in Table 10 of Example 7. Median tumor growth of the untreated tumor (left flank) was analyzed. Results of the experiment are shown in FIG. 7.

[1342] Results:

[1343] Intratumoral treatment of one lesion with anti-CTLA4 antibody in combination with RNAdjuvant, mRNA encoding soluble PD1 and mRNA encoding CD40 ligand induces a systemic effect and inhibits tumor growth of the untreated tumor.

Example 9: Intratumoral Treatment with an Immunostimulating RNA (RNAdjuvant) and mRNA Encoding CD40 Ligand (CD40L), mRNA Encoding IL12, mRNA Encoding Soluble PD1 (solPD1) and Anti-CTLA4 Antibody in Combination with Anti-PD1 Antibody (Administered i.p.)

[1344] Tumor Challenge and Administration of the Inventive Composition:

[1345] The tumor challenge is performed according to the previous experiments (see Example 1). Mice are injected according to the indicated scheme shown in Table 11.

[1346] Table 11 summarizes the treatment as use in the present example. RNAdjuvant and an mRNA encoding CD40L, mRNA encoding soluble PD1, mRNA encoding IL12, mRNA encoding CD40L and mRNA encoding the checkpoint inhibitor anti-CTLA4 antibody are administered intratumorally (i.t.) in combination with checkpoint inhibitor anti PD1 antibody in CT26 tumor challenged mice, median tumor growth and survival rates are analyzed.

TABLE-US-00012 TABLE 11 Groups, treatment and RNA dilution/antibody dilution Group RNAdjuvant (i.t.) RNArt (i.t.) anti-CTLA4 (i.t.) anti-PD1 (i.p.) No. of mice 1 RNAdjuvant IL12 + CD40L anti-CTLA4 anti-PD1 10 3 RNAdjuvant IL12 + CD40L + solPD1 anti-CTLA4 10 2 RNAdjuvant IL12 + CD40L anti-CTLA4 10 4 RNAdjuvant IL12 + CD40L anti-PD1 10 5 RNAdjuvant IL12 + CD40L + solPD1 10 6 RNAdjuvant IL12 + CD40L 10 7 anti-CTLA4 anti-PD1 10 8 anti-CTLA4 10 9 anti-PD1 10 10 Puffer 8

Example 10: Intratumoral Treatment with an Immunostimulating RNA (RNAdjuvant) and an mRNA Encoding for an Antigen (RNActive) Administered Intradermally (i.d.) in Combination with Anti-PD1 Antibody (Administered i.p.)

[1347] C57BL/6 mice were inoculated s.c. with 310.sup.5 E.G7-OVA tumor cells in the right flank. Treatment was start at a mean tumor size of 30 mm.sup.3. Mice were treated i.t. with immunostimulatory RNAdjuvant and vaccinated i.d. with OVA RNActive (mRNA encoding ovalbumine) in combination with an anti PD1 antibody (administered i.p.). mRNA encoding Photinus pyralis Luciferase (PpLuc) or buffer were used as unspecific control. On days 7, 11, 14, 17, and 20 of the experiment mice were treated according to Table 12 below. Median tumor growth and survival rates were analyzed.

TABLE-US-00013 TABLE 12 Groups, treatment and RNA dilution/antibody dilution Anti-PD1 RNAdjuvant RNActive No. of (i.p.) (i.t.) (i.d.) Group mice (200 g) (25 g) (32 g) A 10 Anti-PD1 RNAdjuvant OVA B 10 Anti-PD1 RNAdjuvant C 10 OVA D 10 PpLuc PpLuc E 10 Buffer Buffer

[1348] Tumor Challenge and Administration of the Inventive Composition:

[1349] Mice were injected according to the indicated scheme shown in Table 12. The results of the experiment are shown in FIG. 8.

[1350] Results:

[1351] The results in FIG. 8 show that the immunostimulatory RNA (RNAdjuvant) administered i.t. in combination with mRNA encoding the tumor antigen ovalbumine (OVA) and in combination with an anti PD1 antibody strongly decreased the tumor growth compared to the other treatments (groups B-E according to Table 12). Remarkably, the results in FIG. 8 show that the immunostimulatory RNAdjuvant in combination with a checkpoint inhibitor PD1 antibody and mRNA vaccination (OVA, administered i.d.) induced complete tumor remission and significantly increased the survival of tumor challenged mice compared to the other treatments (groups B-E according to Table 13).

Example 11: Intratumoral Treatment with an Immunostimulating RNA (RNAdjuvant) and an mRNA Encoding IL12 Administered i.t. in Combination with an mRNA Encoding an Antigen (RNActive) Administered i.d.

[1352] C57BL/6 mice were inoculated s.c. with 3105 E.G7-OVA tumor cells in the right flank. Treatment was started at a mean tumor size of 30 mm.sup.3. Mice were treated i.t. with immunostimulatory RNAdjuvant and vaccinated i.d. with mRNA encoding the tumor antigen ovalbumine (OVA RNActive) in combination with an mRNA encoding IL12 (administered i.t.). mRNA encoding Photinus pyralis luciferase (PpLuc RNActive) or buffer were used as unspecific control. On days 7, 11, 14, 17, and 20 of the experiment mice were treated according to Table 13 below. Median tumor growth and survival rates were analyzed.

TABLE-US-00014 TABLE 13 Groups, treatment and RNA dilution RNAdjuvant RNArt RNActive No. of (i.t.) (i.t.) (i.d.) Group mice 25 g 25 g 32 g A 10 RNAdjuvant IL12 OVA B 10 RNAdjuvant IL12 C 10 OVA D 10 PpLuc PpLuc E 10 Buffer Buffer

[1353] Tumor Challenge and Administration of the Inventive Composition:

[1354] Mice were injected according to the indicated scheme shown in Table 13. The results of the experiment are shown in FIG. 9.

[1355] Results:

[1356] The results in FIG. 9 show that intratumoral treatment with immunostimulatory RNA (RNAdjuvant) and an mRNA encoding IL12 in combination with intradermal vaccination with OVA RNActive strongly decreased the median tumor volume compared to the other treatments (groups B-E according to Table 13). In addition, the results in FIG. 9 show that the inventive composition comprising an immunostimulatory RNA (RNAdjuvant) and an mRNA encoding IL12 combined with mRNA vaccination (OVA RNActive) strongly increased the survival of tumor challenged mice compared to the other treatments (groups B-E according to Table 13).

Example 12: Phase I/II Study of Intratumoral Application of RNAdjuvant (CV8102) in Patients with Advanced Cutaneous Melanoma (cMEL), Cutaneous Squamous Cell Carcinoma (cSCC), Head and Neck Squamous Cell Carcinoma (hnSCC), Adenoid Cystic Carcinoma (ACC), Vulvar Squamous Cell Carcinoma (VSCC), or Cutaneous T-Cell Lymphoma, Mycosis Fungoides Subtype (CTCL-MF)

[1357] Part 1:

[1358] Phase I, open label, cohort based dose escalation & expansion study of intratumorally administered RNAdjuvant, with or without systemic anti-PD-1 treatment, in patients having advanced cutaneous melanoma (cMEL), cutaneous squamous cell carcinoma (cSCC), head and neck squamous cell carcinoma (hnSCC), adenoid cystic carcinoma (ACC), vulvar squamous cell carcinoma (VSCC), or cutaneous T-cell lymphoma, mycosis fungoides subtype (CTCL-MF).

[1359] Intratumoral injection of RNAdjuvant: CV8102 is administered to cutaneous, subcutaneous, or readily accessible lymph node lesions that can be injected using direct visualization or imaging-guidance (ultrasound) as clinically determined.

[1360] Schedule: Patients receive a maximum of 8 intratumoral administrations of CV8102, unless disease progression requiring initiation of next-line therapy or unacceptable toxicity occurs:

[1361] The first 5 administrations are performed in weekly intervals (Days 1, 8, 15, 22, 29). For patients on anti-PD-1 treatment in Cohorts C and D, CV8102 treatment is initiated on a day of anti-PD-1 treatment and will follow the anti-PD-1 treatment schedule after day 29.

[1362] The subsequent 3 administrations of CV8102 are performed with 2-week intervals (Cohorts A and B). For patients on anti-PD-1 treatment in study Cohorts C and D, the administrations of CV8102 after Day 29 follow the anti-PD-1 treatment schedule and are performed on the day of anti-PD-1 administration; i.e., patients on nivolumab receive CV8102 every 2nd week; patients on pembrolizumab receive CV8102 every 3rd week.

[1363] Part A: Dose Escalation of Single Agent RNAdjuvant

[1364] Part A of the study uses a 2 parameter Bayesian logistic regression model with overdose control for dose escalation. Cohorts of at least 1 (starting dose level) or 2 patients (any other dose level) with advanced cMEL, cSCC, hnSCC, or ACC are treated at escalating doses of intratumorally administered RNAdjuvant until identification of the maximum tolerated dose (MTD) and determination of the recommended dose (RD). A minimum of 7 patients should be enrolled in Part A.

[1365] The starting dose in Part A is 25 g of RNAdjuvant. The further dose levels are listed in Table 14 below.

TABLE-US-00015 TABLE 14 Dose levels of RNAdjuvant Dose Levels evaluated in Part A and C Dose Level RNAdjuvant No of Patients.sup. Level 1 25 g 1 Level 2 50 g 2 Level 3 100 g 2 Level 4 150 g 2 .sup.A minimum of 2 patients will be treated per cohort

[1366] Inclusion Criteria:

[1367] Cohort A:

[1368] Patients with histologically confirmed advanced (unresectable or metastatic) cMEL or cSCC who failed approved standard therapy or for whom no standard therapy is indicated

[1369] Not amenable to surgical resection or locoregional radiation therapy with curative intent

[1370] Progression on/after at least one line of therapy

or

[1371] Histologically confirmed recurrent or metastatic hnSCC

[1372] Not amenable to surgical resection or locoregional radiation therapy with curative intent

[1373] Progression documented radiologically (documentation of a new or progressive lesion on/after at least one line of therapy.

or

[1374] Histologically or cytologically confirmed ACC

[1375] Not amenable to surgical resection or locoregional radiation therapy with curative intent

[1376] Progression documented radiologically; on/after at least one line of therapy

or

[1377] Histologically confirmed recurrent or metastatic VSCC

[1378] Not amenable to surgery, radio- or radiochemotherapy with curative intent

[1379] Not a candidate for standard systemic therapies

or

[1380] Relapsed or refractory CTCL, mycosis fungoides (MF) subtype

[1381] Diagnosis based upon standard staging classification system

[1382] MF without CD30.sup.+ large cell transformation and no evidence of visceral involvement

[1383] Relapsed, refractory or progressed after at least one prior treatment

[1384] Part B: Expansion Cohort

[1385] After completion of Part A, patients with advanced cSCC, ACC, VSCC, or CTCL-MF are enrolled to separate expansion cohorts at the previously defined recommended dose to further characterize the tolerability and safety profile of intratumorally administered RNAdjuvant in these patient populations and to collect preliminary evidence of anti-tumor activity. Part B should enroll up to 10 patients per expansion cohort. Inclusion criteria:

[1386] Cohort B:

[1387] Expansion cohorts B1, B2

[1388] Patients with histologically confirmed advanced (unresectable/metastatic) cSCC (B1) or ACC (B2) who failed standard therapy or for whom no standard therapy is indicated

[1389] Not amenable to curative locoregional treatments

[1390] Progression on/after at least one line of therapy (radiological documentation required for ACC)

[1391] Expansion cohort B3

[1392] Histologically confirmed recurrent or metastatic VSCC

[1393] Not amenable to surgery, radio- or radiochemotherapy with curative intent

[1394] Not a candidate for standard systemic therapies

[1395] Expansion cohort B4

[1396] Relapsed or refractory CTCL, mycosis fungoides (MF) subtype

[1397] Diagnosis based upon standard staging classification system

[1398] MF without CD30.sup.+ large cell transformation and no evidence of visceral involvement

[1399] Relapsed, refractory or progressed after at least one prior treatment

[1400] Part C: Dose Escalation of RNAdjuvant in Combination with Anti-PD-1 Therapy in Patients with Advanced cMEL or HNSCC

[1401] Part C enrolls patients with advanced cMEL or HNSCC currently receiving anti-PD-1 therapy. Patients must have stable disease or slowly progressive disease after at least 12 weeks of anti-PD-1 therapy prior to application of RNAdjuvant. Cohorts of at least 2 patients are treated sequentially at escalating doses of RNAdjuvant. The dose escalation and the determination of the MTD and RCD (recommended combination dose) are guided by a 5 parameter bayesian logistic regression model with overdose control.

[1402] Dose escalation in Part C starts as soon as at least 3 doses of RNAdjuvant have been evaluated in Part A. Starting dose in Part C is 25 g (see Table 14).

[1403] Inclusion Criteria:

[1404] Cohorts C and D:

[1405] Histologically confirmed advanced (unresectable or metastatic) cMEL in Cohorts C and D1

[1406] Currently receiving standard anti-PD-1 therapy according to the Summary of Product Characteristics (SPC)

[1407] Stable or slowly progressing disease after at least 12 weeks of anti-PD-1 therapy according to investigator assessment

[1408] Intention to continue current anti-PD-1 therapy due to an assumed clinical benefit by continued anti-PD-1 therapy according to investigators judgement

[1409] Histologically confirmed recurrent or metastatic hnSCC in Cohorts C and D2

[1410] Currently receiving standard anti-PD-1 therapy according to the SPC

[1411] Stable or slowly progressing disease after at least 12 weeks of anti-PD-1 therapy, defined as follows:

[1412] Stable disease according to irRECIST

[1413] Stable disease requires a less than or equal 5% decrease in disease (defined as 5% regression in measurable dimension of disease) during an interval of at least 12 weeks prior to Day 1

[1414] Intention to continue current anti-PD-1 therapy due to an assumed clinical benefit according to investigators judgement

[1415] Part D: Expansion Cohort of RNAdjuvant in Combination with Anti-PD-1 Therapy in Patients with Advanced cMEL or HNSCC.

[1416] Once the RCD (recommended combination dose) has been established in Part C, the expansion Part D enrolls additional patients with advanced cMEL or HNSCC on treatment with a PD-1 antagonist (refer to eligibility requirements Part C) to further characterize the tolerability and the safety profile and to evaluate the anti-tumor activity of the combination therapy. Part D should enroll about 21 patients.

[1417] Part 2 (Will be Performed as Separate Clinical Trial)

[1418] Phase I, open label, cohort based dose escalation & expansion study of intratumorally administered RNAdjuvant and RNArt with or without systemic anti-PD-1 treatment in patients percutaneously accessible solid tumors or lymphoma.

[1419] Basically, the design of the phase I clinical trial of RNArt+RNAdjuvant corresponds to the design of the Phase I study of RNAdjuvant (Part 1). The range of tolerated dose combinations will first be investigated using a dose-escalation procedure (cohort A). RNart, comprising 5 RNA compounds encoding IL-12, CD40L, soluble PD-1 an the two chains of anti-CTLA-4, will be increased stepwise while RNAdjuvant will be kept at fixed dose of 25 g. RNArt and RNAdjuvant will be given simultaneously to the same tumor lesion. Dose escalation methodology is similar to Part 1, Cohort A (Bayesian dose-escalation approach). The administration schedule corresponds to the schedule of RNAdjuvant described above (i.e 5 weekly followed by 3 addition injections Q2W).

[1420] The dose escalation part (Cohort A) includes the indications listed in Part 1, plus the following additional indications:

[1421] Patients with

[1422] Human Papilloma virus related advanced tumors, including advanced, recurrent or metastatic vulvar squamous cell carcinoma, cervical cancer, or vaginal cancer

[1423] Not amenable to surgery, radio- or radiochemotherapy with curative intent

[1424] Not a candidate for standard systemic therapies

or

[1425] Follicular low-grade Non-Hodgkin's lymphoma

[1426] Either treatment nave or relapsed or refractory following at least one prior treatment

[1427] Not requiring active therapy (asymptomatic, watchful waiting patients)

or

[1428] Nodal marginal zone B cell lymphoma

[1429] Either treatment nave or relapsed or refractory following at least one prior treatment

[1430] Not requiring active therapy (asymptomatic, watchful waiting patients)

or

[1431] Primary cutaneous anaplastic large-cell lymphoma

[1432] Relapsed, refractory or progressed after at least one prior treatment or for whom no other therapy options are available

or

[1433] Histologically confirmed advanced, recurrent or metastatic adult soft tissue sarcoma

[1434] Not amenable to surgery or other treatment options with curative intent

[1435] At least one line of prior systemic therapy or sarcoma histological subtypes for which there is no known standard systemic therapy

or

[1436] Histologically confirmed advanced (unresectable or metastatic) basal cell carcinoma of the skin

[1437] Not amendable to surgery of any other treatment options with curative intent

[1438] Not a candidate for systemic therapy

[1439] Cohort B: Expansion Cohort

[1440] For indications please refer to Part 1, Expansion cohort B

[1441] Cohort C: Dose Escalation of RNart+RNAdjuvant in Combination with Anti-PD-1 Therapy in Patients with Advanced cMEL, HNSCC or cSCC

[1442] As in the Phase I study with RNAdjuvant, Part C enrolls patients with advanced cMEL or HNSCC. Depending on whether an anti-PD-1 antibody will be approved for treatment of advanced cSCC at the time of start of Cohort C, patients with cSCC will be also enrolled into this cohort. Patients must have stable disease or slowly progressive disease after at least 12 weeks of anti-PD-1 therapy prior to application of RNArt+RNAdjuvant. RNart, comprising 4 RNA compounds encoding IL-12, CD40L and the two chains of anti-CTLA-4, will be increased stepwise while RNAdjuvant will be kept at fixed dose of 25 g. For the dose escalation methodology please refer to Part 1, Cohort C.

[1443] Inclusion Criteria:

[1444] Cohorts C and D:

[1445] Include the indications listed in Part 1, Cohorts C and D. Depending on the approval status of anti-PD-1 antibodies, the following patients will be enrolled to these cohorts:

[1446] Histologically confirmed advanced (unresectable or metastatic) cSCC

[1447] Currently receiving standard anti-PD-1 therapy according to the Summary of Product Characteristics (SPC)

[1448] Stable or slowly progressing disease after at least 12 weeks of anti-PD-1 therapy according to investigator assessment

[1449] Intention to continue current anti-PD-1 therapy due to an assumed clinical benefit by continued anti-PD-1 therapy according to investigators judgement

Experiment 13: Expression of Single RNArt Constructs in A375 Human Melanoma Cells

[1450] A375 cells were seeded in 24-well plates at a density of 50 000 cells/well. After 24 hours cells were transfected with the indicated amounts of RNA and lipofectamine 2000 (Invitrogen) in a constant RNA:lipofectamine ratio of 1:2. After 5 hours the supernatants were collected and 1 ml fresh medium was added. After further 24 hours of incubation supernatants were again collected. Translated mRNA Products of IL12, solPD1 and anti-CTLA4 antibody were measured in the supernatant.

[1451] Human IL12 content in the supernatants was measured with the Human IL-12p70 DuoSet Kit, R&D Systems, Cat: DY1270 according to the manufacturer's protocol using 1:10, 1:100 and 1:1000 dilutions of the supernatants (see FIG. 10).

[1452] Human PD1 ELISA is done in duplicates with the Human PD1 DuoSet Kit, R&D Systems, Cat: DY1086, according to the manufacturer's protocol using 1:10, 1:100 and 1:1000 dilutions of the supernatants (see FIG. 11).

[1453] Human anti-CTLA4 IgG1 ELISA is done in duplicates with Rituximab Antibody for standard (Goat Anti-Human IgG (SouthernBiotech Cat.No: 2044-01, c=1 mg/ml, 1:1000) and Goat Anti-Human IgG Biotin (Dianova Cat.No: 109065088-01, 1:20000) using 1:10, 1:100 and 1:1000 dilutions of the supernatants (see FIG. 12).

[1454] For detection of membrane bound CD40LG protein A375 cells were seeded in 6-well plates at a density of 130 000 cells/well. After 24 hours cells were transfected with the indicated amounts of RNA and lipofectamine 2000 (Invitrogen) in a constant RNA:lipofectamine ratio of 1:2. After 24 hours of incubation cells were collected and cell surface staining was done with CD154 anti-human APC (BD Pharmingen). Finally cell surface expression of CD40LG was analyzed by flow cytometry (see FIG. 13).

TABLE-US-00016 TABLE 15 RNA constructs SEQ ID RNA Description NO: R2025 Non-coding immunostimulatory RNA 433 (RNAdjuvant) R5448/R5939 mRNA encoding human IL-12 598 (hIL-12 (GC)) R5447/R5938 mRNA encoding human soluble PD-1 597 (human solPD1 (GC)) R5446/R5990 mRNA encoding human CD40LG 596 (CD40LG (GC)) R5417 mRNA encoding heavy chain (HC) of 594 anti-CTLA4 antibody (HC anit-CTLA4 Ab (GC)) R5418 mRNA encoding light chain (LC) of 595 anti-CTLA4 antibody (HLC anit-CTLA4 Ab (GC))