COMPOSITION FOR TREATING LUNG CANCER, PARTICULARLY OF NON-SMALL LUNG CANCERS (NSCLC)

Abstract

The present invention relates to an active (immunostimulatory) composition comprising at least one RNA, preferably a mRNA, encoding at least two (preferably different) antigens capable of eliciting an (adaptive) immune response in a mammal. The invention furthermore relates to a vaccine comprising said active (immunostimulatory) composition, and to the use of said active (immunostimulatory) composition (for the preparation of a vaccine) and/or of the vaccine for eliciting an (adaptive) immune response for the treatment of lung cancer, particularly of non-small cell lung cancers (NSCLC), preferably selected from the three main sub-types squamous cell lung carcinoma, adenocarcinoma and large cell lung carcinoma, or of disorders related thereto. Finally, the invention relates to kits, particularly to kits of parts, containing the active (immunostimulatory) composition and/or the vaccine.

Claims

1-24. (canceled)

25. An immunostimulatory composition comprising: i) at least a first RNA comprising a coding region encoding NY-ESO-1; and ii) at least a second RNA comprising a coding region encoding MAGE-A3.

26. The immunostimulatory composition of claim 25, wherein the first RNA and the second RNA comprise a 5′ and/or a 3′ UTR sequence.

27. The immunostimulatory composition of claim 25, wherein the first RNA and the second RNA comprise a 5′ and a 3′ UTR sequence.

28. The immunostimulatory composition of claim 25, wherein the first RNA and the second RNA comprise a 3′ Poly-A sequence of 10 to 200 nucleotides.

29. The immunostimulatory composition of claim 25, wherein the first RNA and the second RNA comprise a 5′ Cap.

30. The immunostimulatory composition of claim 25, wherein the first RNA and the second RNA are in complex with a cationic compound.

31. The immunostimulatory composition of claim 25, wherein the cationic compound comprises cationic peptides.

32. The immunostimulatory composition of claim 25, wherein the cationic compound comprises cationic lipids.

33. The immunostimulatory composition of claim 25, wherein the G/C content of the coding region of the first RNA is increased compared to the G/C content of the coding region of the wild-type RNA encoding NY-ESO-1.

34. The immunostimulatory composition of claim 33, wherein the coding region of the first RNA, encoding NY-ESO-1 is at least about 85% identical to the RNA sequence corresponding to SEQ ID NO: 21.

35. The immunostimulatory composition of claim 34, wherein the coding region of the first RNA, encoding NY-ESO-1 is at least about 90% identical to the RNA sequence corresponding to SEQ ID NO: 21.

36. The immunostimulatory composition of claim 25, wherein the G/C content of the coding region of the second RNA is increased compared to the G/C content of the coding region of the wild-type RNA encoding MAGE-A3.

37. The immunostimulatory composition of claim 36, wherein the G/C content of the coding region of the second RNA is increased compared to the G/C content of the coding region of the wild-type RNA encoding MAGE-A3.

38. The immunostimulatory composition of claim 36, wherein the coding region of the second RNA, encoding MAGE-A3 is at least about 85% identical to the RNA sequence corresponding to SEQ ID NO: 17.

39. The immunostimulatory composition of claim 38, wherein the coding region of the second RNA, encoding MAGE-A3 is at least about 90% identical to the RNA sequence corresponding to SEQ ID NO: 17.

40. The immunostimulatory composition of claim 25, further comprising at least one additional RNA encoding a tumor antigen.

41. The immunostimulatory composition of claim 25, further comprising an adjuvant.

42. A kit comprising: i) at least a first composition comprising a RNA encoding NY-ESO1; and ii) at least a second composition comprising a RNA encoding MAGE-A3.

43. A method of stimulating an anti-tumor immune response in a subject comprising administering an effective amount of an immunostimulatory composition of claim 25 to the subject.

44. The method of claim 43, wherein the subject has a cancer.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0609] The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0610] FIG. 1: depicts a RNA sequence (SEQ ID NO: 1) (starting sequence based on the wildtype) encoding MUC1 (HsMUC1-5×VNTR (The wildtype sequence normally shows 40 tandem repeats. These were—for cloning reasons—reduced to 5 tandem repeats). GC content: 61.27%; length: 1668 bp).

[0611] FIG. 2: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 2) encoding MUC1 (HsMUC1 GC-5×VNTR, 1. GC maximized, 2. Codon usage) GC content: 73.56%; length 1668 bp. Difference to basic sequence (FIG. 1 (SEQ ID NO: 1)): 398/1668 bases=23.86%.

[0612] FIG. 3: depicts a RNA sequence (SEQ ID NO: 3) (starting sequence based on the wildtype) encoding 5T4 (Hs5T4 (trophoblast glycoprotein TPBG); GC content: 61.60%; length: 1263 bp.

[0613] FIG. 4: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 4) encoding 5T4 (Hs5T4 GC, 1. GC-maximized, 2. Codon usage); GC content: 70.47%; length 1263 bp. Difference to basic sequence (FIG. 3 (SEQ ID NO: 3)): 247/1263 Bases=19.56%.

[0614] FIG. 5: depicts a RNA sequence (SEQ ID NO: 5) (starting sequence based on the wildtype) encoding Her-2/neu (HsHer2/neu (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2)); GC content: 60.78%; length: 3768 bp.

[0615] FIG. 6: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 6) encoding Her-2/neu (HsHer2/neu GC, 1. GC-maximized, 2. Codon usage); GC content: 70.54%; length 3768 bp. Difference to basic sequence (FIG. 5 (SEQ ID NO: 5)): 772/3768 Bases=20.49%.

[0616] FIG. 7: depicts a RNA sequence (SEQ ID NO: 7) (starting sequence based on the wildtype) encoding hTERT (HsTERT (telomerase reverse transcriptase); GC Content: 66.08%; Length: 3399 bp.

[0617] FIG. 8: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 8) encoding hTERT (HsTERT GC, 1. GC-maximized, 2. Codon usage); GC Content: 72.96%; Length 3399 bp, Difference to basic sequence (FIG. 7 (SEQ ID NO: 7)): 566/3399 Bases=16.65%.

[0618] FIG. 9: depicts a RNA sequence (SEQ ID NO: 9) (starting sequence based on the wildtype) encoding WT1 (HsWT1 (Wilms tumor 1)); GC Content: 61.78%; Length: 1554 bp.

[0619] FIGS. 10A-D: FIG. 10 A) depicts a RNA sequence (SEQ ID NO: 10) encoding WT1 (HsWT1 (Wilms tumor 1)) showing a sequence with a reduced GC content in region 325-408 of said sequence compared to the corresponding region of the wildtype sequence.

[0620] FIGS. 10 B), C) and D) show a comparison of the corresponding regions 325-408:

[0621] in B) the wildtype sequence according to FIG. 9 (SEQ ID NO: 53),

[0622] in C) the GC-maximized sequence according to FIG. 11 (SEQ ID NOS: 54 and 55, respectively, in order of appearance),

[0623] and

[0624] in D) the GC-reduced sequence according to FIG. 10 (SEQ ID NOS: 56 and 57, respectively, in order of appearance),

[0625] which all show a different GC-pattern.

[0626] FIG. 11: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 11) encoding WT1 (HsWT1 GC, 1. GC-maximized, 2. Codon usage); GC Content: 72.59%; Length 1554 bp. Difference to basic sequence (FIG. 9 (SEQ ID NO: 9)): 322/1554 Bases=20.72%.

[0627] FIG. 12: depicts a RNA sequence (SEQ ID NO: 12) (starting sequence based on the wildtype) encoding CEA (CEA (carcinoembryonic antigen) HsCEACAM5); GC Content: 52.20%; Length: 2109 bp.

[0628] FIG. 13: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 13) encoding CEA (CEACAM5 GC, 1. GC-maximized, 2. Codon usage, already in place); GC Content: 66.24%; Length 2109 bp. Difference to basic sequence (FIG. 12 (SEQ ID NO: 12)): 495/2109 Bases=23.47%.

[0629] FIG. 14: depicts a RNA sequence (SEQ ID NO: 14) (starting sequence based on the wildtype) encoding MAGE-A2 (HsMAGE-A2 (melanoma antigen family A, 2) HsMAGE-A2B). GC Content: 55.87%; Length: 945 bp.

[0630] FIG. 15: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 15) encoding MAGE-A2 (HsMAGE-A2B GC, 1. GC-maximized, 2. Codon usage); GC Content: 68.57%; Length 945 bp. Difference to basic sequence (FIG. 14 (SEQ ID NO: 14)): 187/945 Bases=19.79%.

[0631] FIG. 16: depicts a RNA sequence (SEQ ID NO: 16) (starting sequence based on the wildtype) encoding MAGE-A3 (MAGE-A3 (melanoma antigen family A, 3) MAGE-A3) GC Content: 56.30%; Length: 945 bp.

[0632] FIG. 17: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 17) encoding MAGE-A3 (MAGE-A3 GC, 1.GC-maximized, 2. Codon usage, already known GC-Enrichment); GC Content: 69.00%; Length 945 bp. Difference to basic sequence (FIG. 16 (SEQ ID NO: 16)): 190/945 Bases=20.11%.

[0633] FIG. 18: depicts a RNA sequence (SEQ ID NO: 18) (starting sequence based on the wildtype) encoding Survivin (Survivin (baculoviral IAP repeat-containing 5, BRC5) HsSurvivin(wt)); GC Content: 52.68%; Length: 429 bp.

[0634] FIG. 19: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 19) encoding Survivin (HsSurvivin(GC), 1. GC-maximized, 2. Codon Usage, already known GC-Enrichment); GC Content: 65.27%; Length: 429 bp. Difference to basic sequence (FIG. 18 (SEQ ID NO: 18)): 72/429 Bases=16.78%.

[0635] FIG. 20: depicts a RNA sequence (SEQ ID NO: 20) (starting sequence based on the wildtype) encoding NY-ESO-1 (Homo sapiens NY-ESO-1 (NY-ESO-1(wt)); GC-Content 67.4%.

[0636] FIG. 21: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 21) encoding NY-ESO-1 (NY-ESO-1(GC), GC-Content 79.56%, (already known GC-Enrichment); Difference to wt (FIG. 20 (SEQ ID NO: 20)): 112/543 Bases, 20.63%.

[0637] FIG. 22: depicts a RNA sequence (SEQ ID NO: 22) (starting sequence based on the wildtype) encoding MAGE-C1 (HsMAGEC1 (melanoma antigen family C, 1) HsMAGEC1(wt)) GC Content: 51.86%; Length: 3429 bp.

[0638] FIG. 23: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 23) encoding MAGE-C1 (HsMAGEC1(GC), 1. GC-maximized, 2. Codon usage). GC Content: 68.73%; Length 3429 bp. Difference to basic sequence (FIG. 22 (SEQ ID NO: 22)): 964/3429 Bases=28.11%

[0639] FIG. 24: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 24) encoding a truncated MAGE-C1 (HsMAGEC1(GC), 1. GC-maximized, 2. Codon usage). In comparison to the basic sequence (FIG. 22 (SEQ ID NO: 22)) the repeat regions were deleted and the sequence according to FIG. 24, following an initial start codon (ATG), starts at aa 613 of the GC-maximized wildtype sequence (FIG. 23 (SEQ ID NO: 23)).

[0640] FIG. 25: depicts a RNA sequence (SEQ ID NO: 25) (starting sequence based on the wildtype) encoding MAGE-C2 (HsMAGE-C2 (melanoma antigen family C, 2)HsMAGE-C2); GC Content: 50.81%; Length: 1122 bp.

[0641] FIG. 26: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 26) encoding MAGE-C2 (HsMAGE-C2 GC, 1. GC-maximized, 2. Codon usage); GC Content: 66.58%; Length 1122 bp, Difference to basic sequence (FIG. 25 (SEQ ID NO: 25)): 264/1122 Bases=23.53%.

[0642] FIG. 27 shows the presence of IgG1 antibodies specific for the tumour antigen NY-ESO-1 in mice which were vaccinated with the mRNA vaccine consisting of 5 components, each containing mRNA coding for one NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4) formulated with protamine at a mass ratio of 4:1.

[0643] FIG. 28: shows the presence of IgG2a antibodies specific for the tumour antigen NY-ESO-1 in mice which were vaccinated with the mRNA vaccine consisting of 5 components, each containing mRNA coding for one NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4) formulated with protamine at a mass ratio of 4:1.

[0644] FIG. 29: shows the presence of IgG1 antibodies specific for the tumour antigen MAGE-C1 in mice which were vaccinated with the mRNA vaccine consisting of 5 components, each containing mRNA coding for one NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4) formulated with protamine at a mass ratio of 4:1.

[0645] FIG. 30: shows the presence of IgG2a antibodies specific for the tumour antigen MAGE-C1 in mice which were vaccinated with the mRNA vaccine consisting of 5 components, each containing mRNA coding for one NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4) formulated with protamine at a mass ratio of 4:1.

[0646] FIG. 31 shows the presence of IgG1 antibodies specific for the tumour antigen MAGE-C2 in mice which were vaccinated with the mRNA vaccine consisting of 5 components, each containing mRNA coding for one NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4) formulated with protamine at a mass ratio of 4:1.

[0647] FIG. 32: shows the presence of IgG2a antibodies specific for the tumour antigen MAGE-C2 in mice which were vaccinated with the mRNA vaccine consisting of 5 components, each containing mRNA coding for one NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4) formulated with protamine at a mass ratio of 4:1.

[0648] FIG. 33: shows the induction of antigen-specific T-lymphocytes directed against the tumour antigen 5T4 in mice which were vaccinated with the mRNA vaccine consisting of 5 components, each containing mRNA coding for one NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4) formulated with protamine at a mass ratio of 4:1.

[0649] FIG. 34: shows the induction of antigen-specific T-lymphocytes directed against the tumour antigen NY-ESO-1 in mice which were vaccinated with the mRNA vaccine consisting of 5 components, each containing mRNA coding for one NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4) formulated with protamine at a mass ratio of 4:1.

EXAMPLES

[0650] The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

1. Preparation of Encoding Plasmids:

[0651] In the following experiment DNA sequences, corresponding to the respective mRNA sequences end encoding the antigens [0652] hTERT, [0653] WT1, [0654] MAGE-A2, [0655] 5T4, [0656] MAGE-A3, [0657] MUC1, [0658] Her-2/neu, [0659] NY-ESO-1, [0660] CEA, [0661] Survivin, [0662] MAGE-C1, or [0663] MAGE-C2. [0664] respectively, were prepared and used for in vitro transcription and transfection experiments. Thereby, the DNA sequence corresponding to the native antigen encoding mRNA was increased in GC-content and codon-optimized. Then, the coding sequence was transferred into an RNActive construct (CureVac GmbH, Tubingen, Germany), which has been modified with a poly-A-tag and a poly-C-tag (A70-C30).

2. In Vitro Transcription:

[0665] Based on the recombinant plasmid DNA obtained in Example 1 the RNA sequences were prepared by in vitro transcription. Therefore, the recombinant plasmid DNA was linearized and subsequently in vitro transcribed using the T7 RNA polymerase. The DNA template was then degraded by DNase I digestion, and the RNA was recovered by LiCl precipitation and further cleaned by HPLC extraction (PUREMessenger®, CureVac GmbH, Tubingen, Germany).
3. Complexation with Protamine [0666] For transfection of the RNA into cells and organisms the RNA obtained by in vitro transcription was preferably complexed, more preferably with protamine upon mixing the RNA with protamine.

4. Vaccination Experiments

[0667] For vaccination the RNA obtained by the in vitro transcription experiment as shown above (see Experiment 2) was transfected into mice (Mice: C57 BL/6), preferably when complexed with protamine (see Experiment 3). Transfection occurred in different groups, wherein 5 mice (C57 BL/6) per group were immunized intradermally 8 times within 3 weeks with the inventive mRNA cocktail, i.e. a mixture of mRNA complexed with protamine, wherein the RNA codes for at least two of the antigens hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1, or MAGE-C2.

5. Detection of an Antigen-Specific Immune Response (B-Cell Immune Response):

[0668] Detection of an antigen-specific immune response (B-cell immune response) was carried out by detecting antigen-specific antibodies. Therefore, blood samples were taken from the vaccinated mice one week after the last vaccination and sera were prepared. MaxiSorb plates (Nalgene Nunc International) were coated with the antigenic protein as encoded by the mRNA-Cocktail (0.5 μg/well). After blocking with 1×PBS 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-6-sulfonic acid) was measured.

6. Detection of an Antigen-Specific Cellular Immune Response (T Cell Immune Response) by ELISPOT:

[0669] 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 peptides from the above antigens (peptide library) or coincubated with dendritic cells generated from bone marrow cells of native syngeneic mice, which are electroporated with RNA coding for the antigen. To determine an antigen-specific cellular immune response INFgamma secretion was measured after re-stimulation. For detection of INFgamma a coat multiscreen plate (Millipore) was incubated overnight with coating buffer 0.1 M carbonate-bicarbonate buffer pH 9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising antibody against INFγ (BD Pharmingen, Heidelberg, Germany). Stimulators and effector cells were incubated together in the plate in the ratio of 1:20 for 24 h. The plate was washed with 1×PBS and incubated with a biotin-coupled secondary antibody. After washing with 1×PBS/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.

7. Tumor Challenge:

[0670] Immunization: [0671] One week after the last immunization 1 Mio B16 melanoma cells or TRAMP-C1 cells were injected subcutaneously in the mice. Within 2 weeks (B16) or 7 weeks (TRAMP-C1), respectively, tumour volume was determined
8. Preparation of a mRNA Vaccine [0672] A particular example of the inventive active (immunostimulatory) composition, comprising a combination of several antigens for the use as a vaccine for the treatment of non-small cell lung cancer (NSCLC) was prepared in the following according to the above disclosure. The exemplary inventive active (immunostimulatory) composition consisted of 5 components, each containing mRNA coding for one NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4, according to SEQ ID NOs: 4, 19, 21, 24 and 26 (GC-enriched sequences)) formulated with protamine at a mass ratio of 4:1.

[0673] Vaccination [0674] C57BL/6 mice were vaccinated intradermally with the mRNA vaccine consisting of 5 components, each containing mRNA coding for one NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4, according to SEQ ID NOs: 4, 19, 21, 24 and 26 (GC-enriched sequences)) formulated with protamine (64 μg per antigen per cycle, divided into 4 injections/cycle). Control vaccination was performed using the corresponding total doses of RNA coding for LacZ (control mRNA lacZ). The vaccination comprised three immunization cycles (week 1, 3, and 5). The groups, number of mice and mouse strains are indicated in the following table:

TABLE-US-00001 Groups Mouse strain Number of mice mRNA vaccine C57BL/6 10 5 for Elispot and 5 for antibody detection in serum by ELISA Control mRNA lacZ C57BL/6 5 3 for Elispot and all 5 for antibody detection in serum by ELISA

[0675] Detection of Antigen-Specific Antibodies [0676] 6 days after last vaccination blood samples (200 μl) were taken retro-orbitally and serum was analyzed for the presence of antigen specific antibody subtypes IgG1 and IgG2a using ELISA. 96-well ELISA plates were coated with recombinant protein (10 μg/ml in coating buffer, incubation at 37° C. for 4 h) and blocked with 200 μl blocking buffer per well over night at 4° C. Subsequently, the samples were incubated with serum pooled from each group of mice and titrated in dilutions ranging from 1:3 to 1:48 for 4 hours at room temperature. After incubation with a specific antibody (1:300 in blocking buffer) against mouse IgG1 or IgG2a and incubation with a HRP-coupled secondary antibody (1:500 in blocking buffer), TMB-substrate was added. The colorimetric reaction was measured at 450 nm using an ELISA reader (Tecan Deutschland GmbH, Crailsheim, Germany).

[0677] ELISPOT [0678] For the detection of cytotoxic T-lymphocyte (CTL) responses the analysis of the secretion of the effector cytokine IFN-γ in response to a specific stimulus can be visualized at a single cell level using the ELISPOT technique. [0679] Splenocytes from antigen-vaccinated and control mice were isolated 6 days after last vaccination and then transferred into 96-well ELISPOT plates coated with an anti-IFN-γ capture antibody (10 μg/ml). The cells were then stimulated for 24 hours at 37° C. either with relevant antigen-derived peptide library or with the HIV-derived library or the solvent of the peptides, DMSO, or incubated in pure medium as a control. All libraries were used at a concentration of 1 μg/peptide/ml. After the incubation period the cells were washed out of the plate and the IFN-γ secreted by the cells was detected using a biotinylated secondary antibody against murine IFN-γ (1 μg/ml), followed by streptavidin-AKP. Spots were visualized using BCIP/NBT substrate and counted using an automated ELISPOT reader (Immunospot Analyzer, CTL Analyzers LLC).

[0680] Statistical Analysis [0681] Statistical analysis was performed using Graph Pad Prism 5.01 (GraphPad Software, Inc.). All results were expressed as the mean (or median)±standard error of means. For Elispot assays, due to the fact that the basal activation is strongly individual dependent, a background correction was performed individually per mouse by subtraction of the number of spots in medium wells from all other values. Two-tailed Mann-Whitney tests were used to analyze difference between the test groups with a significance level of 5%.

[0682] Results and Discussion [0683] Mice were vaccinated with the mRNA vaccine containing five components as defined above, particularly GC-enriched mRNAs coding for the NSCLC-associated antigens NY-ESO-1, MAGE-C2, MAGE-C1, Survivin and 5T4, (according to SEQ ID NOs: 4, 19, 21, 24 and 26 (GC-enriched sequences)) each formulated separately with the cationic peptide protamine at a mass ratio of 4:1. Control mice were treated with irrelevant RNA coding for LacZ formulated with protamine at the same ratio as the mRNA vaccine. [0684] Using serum isolated from blood drawn from the antigen-vaccinated and control mice, we tested the induction of specific antibodies against the antigens. For three of the five analyzed proteins, MAGE-C1, MAGE-C2 and NY-ESO-1, we detected antigen specific antibodies in serum of mice vaccinated with the mRNA vaccine demonstrating that the mRNAs are functional and immunogenic in vivo. Proteins required for detection of antibodies were produced in E. coli. As production of proteins in E. coli can influence post-translational modifications and these are not well described for the used antigens, this could account for the lack of response seen for the remaining proteins. [0685] Next the activation of cytotoxic T-cells in response to the administration of the mRNA vaccine was analyzed. IFN-γ is the main mediator of Th1 responses and secreted by activated CTLs. Therefore the presence of antigen-specific cytotoxic T-cells in splenocytes from vaccinated mice was investigated using the ELISPOT technique. As an antigenic stimulus for splenocytes restricted peptide libraries were used. Because distinct epitopes of the used human antigens for mouse MHC (H-2K.sup.b and H-2D.sup.b in C57BL/6 mice) are not known, we had to use a hypothetical selection of peptides selected due to potential binding affinity by search of the SYFPEITHI database. Out of peptide libraries (15mers with 11 amino acids overlap) spanning the whole sequences of the proteins, those 15mers containing the hypothetically best epitopes were selected and pooled up to a maximum of 18 peptides. However, these selections might not necessarily contain the correct epitopes so that the detection of immune responses with the help of these tools can easily yield false negative results. Nevertheless, the stimulation with two of these libraries, originating from NY-ESO-1 and 5T4, led to high IFN-γ secretion in splenocytes from mice vaccinated with the mRNA vaccine and not in splenocytes from control mice, vaccinated with mRNA coding for irrelevant protein β-galactosidase. None of the splenocytes reacted to the HIV-derived control peptide library. The number of IFN-γ spots by splenocytes incubated in medium alone represents the basal activation of the freshly isolated cells. Due to the fact that the basal activation is strongly individual dependent, the background correction was performed individually by subtraction of the number of spots in medium wells from all other values. [0686] The results of these experiments are shown in FIGS. 27 to 34.