RNA ENCODING A TUMOR ANTIGEN

Abstract

The present invention relates to an RNA encoding a tumor antigen. In particular, the present invention relates to RNA suitable for treatment and/or prophylaxis of cancer and related diseases. The present invention concerns such novel RNA as well as compositions, vaccines and kits comprising the RNA. Furthermore, the present invention relates to the RNA, compositions, vaccines or kits as disclosed herein for use in the treatment and/or prophylaxis of cancer and related diseases.

Claims

1. An RNA comprising at least one coding sequence, wherein the coding sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 505-4033; 4561-4591, or a fragment or variant of any one of said nucleic acid sequences.

2. The RNA according to claim 1, wherein the at least one coding sequence encodes a tumor antigen, or a fragment or variant of a tumor antigen, 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; B7H4; 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-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; 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.

3. (canceled)

4. The RNA according to claim 1, wherein the RNA is mono-, bi-, or multicistronic.

5. The RNA according to claim 1, wherein the RNA is an mRNA, a viral RNA or a replicon RNA.

6. The RNA according to claim 1, wherein the RNA is a modified RNA, preferably a stabilized RNA.

7. The RNA according to claim 1, wherein the G/C content of the at least one coding sequence of the RNA is increased compared to the G/C content of the corresponding coding sequence of the corresponding wild-type RNA, and/or wherein the C content of the at least one coding sequence of the RNA is increased compared to the C content of the corresponding coding sequence of the corresponding wild-type RNA, and/or wherein the codons in the at least one coding sequence of the RNA are adapted to human codon usage, wherein the codon adaptation index (CAI) is preferably increased or maximised in the at least one coding sequence of the RNA, wherein the amino acid sequence encoded by the RNA is preferably not being modified compared to the amino acid sequence encoded by the corresponding wild-type RNA.

8.-10. (canceled)

11. The RNA according to claim 1, which comprises a 5-CAP structure and/or at least one 3-untranslated region element (3-UTR element).

12. The RNA according to claim 1, which comprises at least one histone stem-loop.

13.-15. (canceled)

16. The RNA according to claim 1, wherein the at least one RNA comprises a poly(A) sequence, preferably comprising 10 to 200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides, and/or a poly(C) sequence, preferably comprising 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides.

17. The RNA according to claim 1, which comprises, preferably in 5 to 3 direction, the following elements: a) a 5-CAP structure, preferably m7GpppN, b) at least one coding sequence as defined in claim 8, c) a poly(A) tail, preferably consisting of 10 to 200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides, d) a poly(C) tail, preferably consisting of 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides, and e) a histone stem-loop, preferably comprising the RNA sequence according to SEQ ID NO: 4556.

18. The RNA according to claim 11, which comprises a 3-UTR element and wherein the 3-UTR element comprises a nucleic acid sequence derived from a 3-UTR of a gene, which preferably encodes a stable mRNA, or from a homolog, a fragment or a variant of said gene.

19.-20. (canceled)

21. The RNA according to claim 1, which comprises, preferably in 5 to 3 direction, the following elements: a) a 5-CAP structure, preferably m7GpppN, b) at least one coding sequence, c) a 3-UTR element comprising a nucleic acid sequence, which is derived from an -globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 4547, or a homolog, a fragment or a variant thereof, d) a poly(A) tail, preferably consisting of 10 to 200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides, e) a poly(C) tail, preferably consisting of 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides, and f) a histone stem-loop, preferably comprising the RNA sequence according to SEQ ID NO: 4556.

22.-23. (canceled)

24. The RNA according to claim 1, wherein the RNA comprises a 5-UTR element.

25.-28. (canceled)

29. The RNA according to claim 1, which comprises, preferably in 5 to 3 direction, the following elements: a) a 5-CAP structure, preferably m7GpppN, b) a 5-UTR element, which comprises or consists of a nucleic acid sequence, which is derived from the 5-UTR of a TOP gene, preferably comprising an RNA sequence corresponding to the nucleic acid sequence according to SEQ ID NO: 4537, or a homolog, a fragment or a variant thereof, c) at least one coding sequence, d) a 3-UTR element comprising a nucleic acid sequence, which is derived from an -globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 4547, or a homolog, a fragment or a variant thereof; and/or a 3-UTR element comprising a nucleic acid sequence, which is derived from an albumin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 4551 or SEQ ID NO: 4553, or a homolog, a fragment or a variant thereof, e) a poly(A) tail, preferably consisting of 10 to 200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides, f) a poly(C) tail, preferably consisting of 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides, and g) a histone stem-loop, preferably comprising the RNA sequence according to SEQ ID NO: 4556

30. A composition comprising the RNA according to claim 1 and a pharmaceutically acceptable carrier.

31.-35. (canceled)

36. The composition according to claim 30, wherein the RNA is complexed with one or more lipids, thereby forming liposomes, lipid nanoparticles and/or lipoplexes.

37. The composition according to claim 30, wherein the composition comprises at least one adjuvant.

38.-41. (canceled)

42. A kit, preferably kit of parts, comprising an RNA according to claim 1, and optionally a liquid vehicle for solubilising and optionally technical instructions with information on the administration and dosage of the RNA, the RNA.

43.-45. (canceled)

46. A method of treating or preventing cancer comprising administering to a subject in need thereof an RNA according to claim 1.

47.-55. (canceled)

Description

FIGURES

[0346] The figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.

[0347] FIG. 1: shows the presence of IgG2a antibodies specific for the cancer antigen NY-ESO-1 in mice that were vaccinated with NY-ESO-1 mRNA vaccine. A detailed description of the experiment is provided in the example section (see Example 2).

[0348] FIG. 2: shows the induction of antigen-specific T-lymphocytes directed against the cancer antigen NY-ESO-1 in mice that were vaccinated with NY-ESO-1 mRNA vaccine. A detailed description of the experiment is provided in the example section (see Example 2).

[0349] FIG. 3: shows the presence of IgG2a antibodies specific for the cancer antigen PSA in mice that were vaccinated with PSA mRNA vaccine. A detailed description of the experiment is provided in the example section (see Example 3).

[0350] FIG. 4: shows the induction of antigen-specific T-lymphocytes directed against the cancer antigen PSA in mice that were vaccinated with PSA mRNA vaccine. A detailed description of the experiment is provided in the example section (see Example 3).

[0351] FIG. 5: shows the induction of antigen-specific T-lymphocytes directed against the cancer antigen PSM (PSMA) in mice that were vaccinated with PSM (PSMA) mRNA vaccine. A detailed description of the experiment is provided in the example section (see Example 4).

[0352] FIG. 6: shows the induction of antigen-specific T-lymphocytes directed against the cancer antigen MAGE-A2 in mice that were vaccinated with MAGE-A2 mRNA vaccine. A detailed description of the experiment is provided in the example section (see Example 5).

[0353] FIG. 7: shows the induction of antigen-specific T-lymphocytes directed against the cancer antigen MAGE-C2 in mice that were vaccinated with MAGE-C2 mRNA vaccine. A detailed description of the experiment is provided in the example section (see Example 5).

[0354] FIG. 8: shows the induction of antigen-specific T-lymphocytes directed against the cancer antigen MAGE-A3 in mice that were vaccinated with MAGE-A3 mRNA vaccine. A detailed description of the experiment is provided in the example section (see Example 5).

[0355] FIG. 9: shows the induction of antigen-specific T-lymphocytes directed against the cancer antigen PMEL (human gp100, hgp100) in mice that were vaccinated with PMEL mRNA vaccine. A detailed description of the experiment is provided in the example section (see Example B).

EXAMPLES

[0356] 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.

Example 1: Preparation of mRNA Cancer Vaccine

[0357] 1.1 Preparation of DNA and mRNA Constructs

[0358] For the present examples, DNA sequences encoding cancer antigens, are prepared and used for subsequent RNA in vitro transcription reactions.

[0359] Most DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence. Sequences were introduced into a pUC19 derived vector and modified to comprise stabilizing sequences derived from alpha-globin-3-UTR, a stretch of 30 cytosines, and a stretch of B4 adenosines at the 3-terminal and (poly-A-tail).

[0360] The following constructs, coding for the indicated antigens, are used in the present example:

[0361] NY-ESO-1 (SEQ ID NO: 458B), PSA (SEQ ID NO: 4590), PSM (SEQ ID NO: 4589), MAGE-A2 (SEQ ID NO: 4588), MAGE-A3 (SEQ ID NO: 4587), MAGE-C2 (SEQ ID NO: 4585) and PMEL (SEQ ID NO: 4591).

[0362] The obtained plasmid DNA constructs were transformed and propagated in bacteria (Escherichia coli) using common protocols known in the art.

[0363] 1.2. RNA In Vitro Transcription

[0364] The DNA plasmids prepared according to paragraph 1 are enzymatically linearized using EcoRI and transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture and CAP analog (m7GpppG) under suitable buffer conditions. The obtained mRNAs purified using PureMessenger (CureVac, Tobingen, Germany; WO 2008/077592 A1) are used for in vitro vaccination experiments (see Examples 2-B).

[0365] 1.3. Preparation of Protamine Complexed mRNA (RNActive Formulation):

[0366] The obtained antigen mRNA constructs were complexed with protamine prior to use in in vivo vaccination experiments. The mRNA complexation consists of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA is complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes are stably generated, free mRNA is added, and the final concentration of the vaccine is adjusted with Ringer's lactate solution.

Example 2: Vaccination Experiment with NY-ESO-1 mRNA Vaccine

[0367] To test the NY-ESO-1 mRNA vaccine obtained according to Example 1 in vivo. C57BL/6 mice were intradermally injected with NY-ESO-1 mRNA vaccine. The immune responses of NY-ESO-1 vaccinated mice were analysed (NY-ESO-1 specific immune response and NY-ESDI specific cellular immune response). A detailed description of the experiment is provided below.

[0368] 2.1. Detection of an NY-ESD-1-Specific Immune Response (B-Cell Immune Response):

[0369] For vaccination 5 mice (C57 BL/6) per group were intradermally injected B times within 3 weeks with NY-ESO-1 mRNA vaccine. As negative control lacZ mRNA was injected. Detection of an antigen-specific immune response (B-cell immune response) was carried out by detecting NY-ESO-1 specific antibodies. Therefore, blood samples were taken from the vaccinated mice one week after the last vaccination and sera were prepared. MaxiSorb plates (Nalgene Nuno International) were coated with the antigenic protein (0.5 g/well). After blocking with 1PBS containing 0.05% Tween-20 and 1% BSA the plates were incubated with diluted mouse serum (1:30, 1:90, 1:270, 1:810). Subsequently a biotin-coupled secondary antibody (anti-mouse-IgG2a, Pharmingen) was added. After washing, the plate was incubated with horseradish peroxidase-streptavidin and subsequently the conversion of the ABTS substrate (2,2-azino-bis(3-ethyl-benzthiazoline-B-sulfonic acid) was measured. The result of the experiment is shown in FIG. 1.

[0370] 2.2. Detection of an NY-ESO-1-Specific Cellular Immune Response (CTL) by ELISPOT

[0371] 2 weeks after the last vaccination mice were sacrificed, the spleens were removed and the splenocytes were isolated. The splenocytes were restimulated for 7 days in the presence of NY-ESO-1 peptides (peptide library); a peptide library of HIV antigen was used as control. To determine an antigen-specific cellular immune response IFNgamma secretion was measured after re-stimulation with peptide. For detection of IFNgamma a coat multiscreen plate (Millipore) was incubated overnight with coating buffer (0.1 M Carbonat-Bicarbonat Buffer pH 9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising antibody against IFNgamma (BD Pharmingen. Heidelberg, Germany). After coating effector cells (510.sup.5/well) were incubated with the antigen-specific peptide library, an unspecific peptide, DMSO or medium as control for 24h in the plate. Subsequently the plate was washed with 1PBS and incubated with a biotin-coupled secondary antibody. After washing with 1PBS/0.05% Tween-20 the substrate (5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium Liquid Substrate System from Sigma Aldrich. Taufkirchen, Germany) was added to the plate and the conversion of the substrate could be detected visually. The results are shown in FIG. 1.

[0372] Results:

[0373] The data shows that we detected antigen specific IgG2a antibodies in serum of mice vaccinated with the NY-ESO-1 mRNA vaccine demonstrating that the mRNAs are functional and immunogenic in vive. The result is shown in FIG. 1. The presence of antigen-specific cytotoxic T-cells in splenocytes from NY-ESO-1 vaccinated mice was investigated using the ELISPOT technique. As shown in FIG. 2, the stimulation with NY-ESO-1 peptide library led to high IFN-gamma secretion in splenocytes from mice vaccinated with NY-ESO-1 mRNA vaccine and not in splenocytes from control mice, vaccinated with mRNA coding for irrelevant protein beta-galactosidase. None of the splenocytes reacted to the HIV-derived control peptide library. The result is shown in FIG. 2.

[0374] Overall, the results shown in FIG. 1 and FIG. 2 demonstrate that the inventive NY-ESO-1 mRNA vaccine induces antigen specific B-cell and T-cell responses in vive.

Example 3: Vaccination Experiment with PSA mRNA Vaccine

[0375] To test the PSA mRNA vaccine obtained according to Example 1 in vivo, C57BL/6 mice were intradermally injected with PSA mRNA vaccine. The immune responses of PSA vaccinated mice were analysed (PSA specific immune response and PSA specific cellular immune response). The results of the experiment are shown in FIGS. 3 and 4. A detailed description of the experiment is provided below.

[0376] 3.1. Detection of an PSA-Specific Immune Response (B-Cell Immune Response):

[0377] For vaccination 4 mice (C57 BL/6) per group were intradermally injected 4 times within 3 weeks with 5 g PSA mRNA vaccine. As negative control, an injection buffer was applied. Detection of an antigen-specific immune response (B-cell immune response) was carried out by detecting PSA specific antibodies. The detection of antigen-specific immune response (B-cell immune response) was performed according to Example 2.2. The results are shown in FIG. 3.

[0378] 3.2. Detection of an PSA-Specific Cellular Immune Response by ELISPOT:

[0379] 2 months after the last vaccination mice were sacrificed, the spleens were removed and the splenocytes were isolated. The splenocytes were incubated for 7 days in presence of IL-4 to select dendritic cells. To determine an antigen-specific cellular immune response INFgamma secretion was measured after re-stimulation. The detection of an antigen-specific cellular immune response by ELISPOT was essentially performed according to Example 2.3. The results are shown in FIG. 4.

[0380] Results:

[0381] The data shows that we detected antigen specific IgG2a antibodies in serum of mice vaccinated with the PSA mRNA vaccine demonstrating that the mRNAs are functional and immunogenic in vivo. The result is shown in FIG. 3. The presence of antigen-specific cytotoxic T-cells in splenocytes from PSA vaccinated mice was investigated using the ELISPOT technique. As shown in FIG. 4, the stimulation with PSA peptide library led to high IFN-gamma secretion in splenocytes from mice vaccinated with PSA mRNA vaccine and not in splenocytes from control mice, vaccinated with buffer. None of the splenocytes reacted to the control peptide library. The result is shown in FIG. 4.

[0382] Overall, the results shown in FIG. 3 and FIG. 4 demonstrate that the inventive PSA mRNA vaccine induces antigen specific B-cell and T-cell responses in vive.

Example 4: Vaccination Experiment with PSM mRNA Vaccine

[0383] To test the PSM mRNA vaccine obtained according to Example 1 in vivo. C57BL/6 mice were intradermally injected with PSM mRNA vaccine. The immune responses of PSM vaccinated mice were analysed (PSM specific cellular immune response). The result of the experiment is shown in FIG. 5. A detailed description of the experiment is provided below.

[0384] 4.1. Detection of an PSM-Specific Cellular Immune Response by ELISPOT:

[0385] For vaccination B mice (C57 BL/6) per group were intradermally injected 5 times with 32 g PSA mRNA vaccine. As negative control, ringer lactate (RiLa) buffer was applied. After the last vaccination mice were sacrificed, the spleens were removed and the splenocytes were isolated. For re-stimulation 310.sup.7 splenocytes were incubated for 5 days with a PSM peptide library (1 g/ml per peptide) in presence of IL-2 (30 U/ml). The detection of an antigen-specific cellular immune response by ELISPOT was essentially performed according to Example 2.3. The result is shown in FIG. 5.

[0386] Results:

[0387] The presence of antigen-specific cytotoxic T-cells in splenocytes from PSM vaccinated mice was investigated using the ELISPOT technique. As shown in FIG. 5, the stimulation with PSM peptide library led to high IFN-gamma secretion in splenocytes from mice vaccinated with PSM mRNA vaccine and not in splenocytes from control mice, vaccinated with RiLa buffer. The result is shown in FIG. 5.

[0388] Overall, the results shown in FIG. 5 demonstrate that the inventive PSM mRNA vaccine induces antigen specific T-cell responses in vivo.

Example 5: Vaccination Experiment with MAGE-A2, MAGE-A3 and MAGE-C2 mRNA Vaccines

[0389] To test the MAGE-A2, MAGE-A3 and MAGE-C2 mRNA vaccine obtained according to Example 1 in vivo, C57BL/6 mice were intradermally injected with MAGE-A2, MAGE-A3 and MAGE-C2 mRNA vaccines. The immune responses of vaccinated mice were analysed (antigen specific cellular immune response). The results of the experiments are shown in FIGS. 6-8. A detailed description of the experiment is provided below.

[0390] 5.1. Detection of an Antigen-Specific Cellular Immune Response by ELISPOT:

[0391] For the present example, MAGE-A2, MAGE-A3 and MAGE-C2 mRNA vaccines were applied with application regimens as provided below: [0392] MAGE-A2: C57BL/6 mice were vaccinated intradermally with mRNA encoding human MAGE-A2. (20 g/mouse/vaccination day, 5 mice per group). Ringer lactate was used as control. After B vaccinations splenocytes were isolated and analyzed by ELISpot assay. [0393] MAGE-A3: C57BL/6 mice were vaccinated intradermally with mRNA encoding human MAGE-A2. (32 g/mouse/vaccination day. 8 mice per group). Ringer lactate was used as control. After 5 vaccinations splenocytes were isolated and analyzed by ELISpot assay. [0394] MAGE-C2: C57BL/6 mice were vaccinated intradermally with mRNA encoding human MAGE-A2. (20 g/mouse/vaccination day, 5 mice per group). Ringer lactate was used as control. After B vaccinations splenocytes were isolated and analyzed by ELISpot assay.

[0395] After the last vaccination the mice were sacrificed, the spleens were removed and the splenocytes were isolated. For re-stimulation 310.sup.7 splenocytes were incubated for 5 days with the respective antigen-specific peptide or peptide library (1 g/ml per peptide) in presence of IL-2 (30 U/ml). To determine an antigen-specific cellular immune response IFNy secretion was measured after re-stimulation with peptide. The detection of an antigen-specific cellular immune response by ELISPOT was performed according to Example 2.3. The result is shown in FIGS. 6-8.

[0396] Results:

[0397] The presence of antigen-specific cytotoxic T-cells in splenocytes from MAGE-A2, MAGE-A3 and MAGE-C2 vaccinated mice was investigated using the ELISPOT technique. As shown in FIG. 6, the stimulation with MAGE-A2 peptide library led to high IFN-gamma secretion in splenocytes from mice vaccinated with MAGE-A2 mRNA vaccine. Stimulation with a control peptide library did not lead to IFN-gamma secretion. As shown in FIG. 7, the stimulation with MAGE-C2 peptide library led to high IFN-gamma secretion in splenocytes from mice vaccinated with MAGE-C2 mRNA vaccine. Stimulation with a control peptide library did not lead to IFN-gamma secretion. As shown in FIG. 8, the stimulation with MAGE-A3 peptide library led to high IFN-gamma secretion in splenocytes from mice vaccinated with MAGE-A3 mRNA vaccine and not in splenocytes from control mice, vaccinated with RiLa buffer.

[0398] Overall, the results shown in FIGS. 6-8 demonstrate that the inventive MAGE-A2, MAGE-A3 and MAGE-C2 mRNA vaccine induce antigen specific T-cell responses in vivo.

Example B: Vaccination Experiment with PMEL mRNA Vaccine

[0399] To test the PMEL mRNA vaccine obtained according to Example 1 in vivo, C57BL/6 mice were intradermally injected with PMEL mRNA vaccines. The immune response of vaccinated mice was analysed (PMEL specific cellular immune response). The result of the experiment is shown in FIG. 9. A detailed description of the experiment is provided below.

[0400] 6.1. Detection of a PMEL-Specific Cellular Immune Response by ELISPOT:

[0401] 8 mice (C57 BL/6) per group were intradermally injected 4 times within 3 weeks with 64 g PMEL mRNA vaccine. As negative control, RiLa injection buffer was injected. After the last vaccination the mice were sacrificed, the spleens were removed and the splenocytes were isolated. The detection of an antigen-specific cellular immune response by ELISPOT was performed according to Example 2.3. The result is shown in FIG. 9.

[0402] Results:

[0403] The presence of PMEL-specific cytotoxic T-cells in splenocytes from PMEL vaccinated mice was investigated using the ELISPOT technique. As shown in FIG. 9, the stimulation with PMEL peptide library led to high IFN-gamma secretion in splenocytes from mice vaccinated with PMEL mRNA vaccine. Stimulation with a control peptide library did not lead to IFN-gamma secretion.