Combination Therapy for Immunostimulation

Abstract

The present invention relates to a method for immunostimulation in a mammal, which comprises a. administration of at least one mRNA containing a region which codes for at least one antigen of a pathogen or at least one tumour antigen, and b. administration of at least one cytokine, at least one cytokine mRNA, at least one CpG DNA or at least one adjuvant RNA. The invention likewise relates to a product and a kit comprising the mRNA and cytokine or cytokine mRNA or CpG DNA or adjuvant RNA of the invention.

Description

FIGURES

FIG. 1 shows the in vivo translation of injected mRNA according to the invention. Injection buffer (150 mM NaCl, 10 mM HEPES (buffer), β-galactosidase-coding β-globin UTR-stabilized mRNA, diluted in injection buffer (lac Z mRNA) or β-galactosidase-coding DNA in PBS (lac Z DNA) were injected into the ear pinna of mice. 16 hours after the injection, the mice were sacrificed and the ears were shaved, removed and frozen in embedding medium. Frozen sections were then prepared, fixed and stained overnight with solution containing X-Gal. Cells which expressed β-galactosidase appeared blue. The number of blue cells detected in each section is shown in the graphs (left half of FIG. 1). The length of the ear section analysed is plotted on the x-axis (0 is arbitrarily assigned to the first section which shows blue cells; in the mice injected with buffer, the region lying 2 mm around the injection site was analysed and the 0 determined arbitrarily): Each section is 50 μm and a few successive sections thus cover a total distance of a few millimetres. In each of the graphs (buffer-injected mice, mRNA-injected mice, DNA-injected mice), the two sections which are identified by an asterisk and a grey column are the sections which are shown in the accompanying microscope images (right half of FIG. 1). Open arrows here indicate an endogenous expression of β-galactosidase activity chiefly in the ear follicles. This endogenous activity is detectable by a very weak and diffuse blue colouration. Arrows filled in black indicate blue cells which result from uptake and translation of an exogenous nucleic acid which codes β-galactosidase. Such cells are located in the dermis at the injection site and show an intense blue colouration. Individual sections were photographed. The sections having the most blue cells are shown (they correspond to the sections marked with an asterisk in the graphs). The number of blue cells in each of the successive sections is shown on the y-axes in the graphs (left half of FIG. 1).

FIG. 2 shows the triggering of an antigen-specific immune response of type Th2 by the injection of mRNA. Mice were vaccinated and boosted with mRNA or DNA which codes for β-galactosidase, or they were injected with injection buffer. Two weeks later, the mice received a boost injection. Two weeks later again, the amount of β-galactosidase-specific antibodies present in the serum was determined by ELISA using isotype-specific reagents. The left half of FIG. 2 shows the IgGl production, the right half of FIG. 2 shows the IgG2a production. (▪) shows the curve for DNA-injected mice, (▴) shows the curve for RNA-injected mice and (♦) shows the curve for mice which were injected with injection buffer.

FIG. 3 shows the polarization of a Th2 immune response into a Th1 immune response caused by the injection of GM-CSF. All the results shown relate to mice of the same group in one experiment. The total number of mice which showed an immune response in four independent experiments is shown in Table 1 (FIG. 4).

FIG. 3 a: Mice were injected either with β-galactosidase, emulsified in Freund's adjuvant, or mRNA which codes for β-galactosidase, or injection buffer (as a negative control). GM-CSF (total amount of 2 μg of recombinant protein: approx. 10⁴ U (units)) were injected once, either 24 hours or 2 hours before injection of the mRNA or 24 hours after injection of the mRNA (corresponds to groups GM-CSF T−1, GM-CSF T−0 and GM-CSF T+1). The amount of β-galactosidase-specific IgG1 or IgG2a antibodies contained in the blood of the injected mice was determined by ELISA (1:10 serum dilution). The background which was chiefly obtained by the serum of buffer-injected mice at the same dilution was subtracted. The left half of FIG. 3a shows β-gal-specific IgG1 antibodies (▪), the right half of FIG. 3a shows β-gal-specific IgG2a antibodies (, grey).

FIG. 3 b: The in vitro reactivation of T cells by β-galactosidase was checked with the aid of a cytokine detection on day 4 of the culture. The content of IFNγ (▪) and IL-4 (, grey) in the supernatant of the splenocyte culture used was measured by means of ELISA.

FIG. 3 c: The cytotoxic activity of splenocytes which were cultured in the presence of purified β-galactosidase for six days was checked in a chromium release assay. The target cells were P815 (H2^d) cells, which were either charged (▪) with the synthetic peptide TPHPARIGL, which corresponds to the dominant H2-L^d epitope of β-galactosidase, or were not charged (□).

FIG. 4 shows Table 1, in which the total number of mice injected is shown. The total number of mice whose splenocytes showed a detectable cytokine release or a β-galactosidase-specific cytotoxic activity in vitro in independent experiments is shown. Mice in which at least 10% more TPHPARIGL-charged cells were killed, compared with the average of the cells killed in the negative control group (buffer-injected mice), were classified as mice with an immune response (responding). Splenocyte cultures which contained at least 100 pg/ml of cytokine more than the total content of cytokine in the splenocyte cultures of the negative control mice (buffer-injected mice) were classified as responding cultures (responding mice). The figures in bold indicate groups in which more than half of the mice showed an immune response to the vaccine according to the parameters investigated (cytokine or cytotoxic activity).

FIG. 5: shows the polarization of a Th2 immune response into a Th1 immune response caused by the injection of GM-CSF RNA in addition to the mRNA according to the invention. All the results shown relate to mice of the same group in one experiment. For this, mice were injected with mRNA which codes for β-galactosidase, GM-CSF RNA or injection buffer. GM-CSF RNA (total amount 50 μg) was injected once, either 24 hours or 2 hours before injection of the mRNA or 24 hours after injection of the mRNA (corresponds to groups GM-CSF RNA T−1, GM-CSF RNA T−0 and GM-CSF RNA T+1). The amount of IFN-γ secreted which was contained in the blood of the injected mice was determined by ELISA.

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

EXAMPLES

Example 1

Preparation of the mRNA

The mRNA was obtained by in vitro transcription of suitable template DNA and subsequent extraction and purification of the mRNA. Standard methods which are described in numerous instances in the prior art and with which the person skilled in the art is familiar can be used for this. For example, Maniatis et al. (2001), Molecular Cloning: Laboratory Manual, Cold Spring Harbour Laboratory Press. The same also applies to the sequencing of the mRNA, which followed the purification (described below) of the mRNA. The NBLAST program in particular was used here.

The mRNA according to the invention was generally prepared in accordance with the following procedure:

1. Vector

The genes for which the particular mRNA codes were inserted into the plasmid vector pT7TS. pT7TS contains untranslated regions of the alpha- or beta-globin gene and a polyA tail of 70 nucleotides:

Plasmids of high purity were obtained with the Qiagen Endo-free Maxipreparation Kit or with the Machery-Nagel GigaPrep Kit. The sequence of the vector was checked via a double-strand sequencing from the T7 promoter up to the PstI or XbaI site and documented. Plasmids in which the gene sequence cloned in was correct and without mutations were used for the in vitro transcription.

2. Genes

The genes for which the mRNA according to the invention codes were amplified by means of PCR or extracted from the plasmids (described above). Examples of gene constructs which were employed are

GP100 (accession number M77348): PCR fragment SpeI in T7TS HinDIII blunt/SpeI

MAGE-A1 (accession number M77481): plasmid fragment HinDIII/SpeI in T7TS HinDIII/SpeI

MAGE-A6 (accession number: NM_—005363): PCR fragment SpeI in T7TS HinDIIIblunt/SpeI

Her2/neu (accession number: M11730): PCR fragment HinDIII/SpeI in T7TS HinDIII/SpeI

Tyrosinase (accession number: NM_—000372): plasmid fragment EcoRI blunt in T7TS HinDIII blunt/SpeI blunt

Melan-A (accession number: NM_—005511): plasmid fragment NotI blunt in T7TS HindIII blunt/SpeI blunt

CEA (accession number: NM_—004363): PCR fragment HinDIII/SpeI in T7TS HinDIII/SpeI

Tert (accession number: NM_—003219): PCR fragment HindIII/SpeI in T7TS HinDIII/SpeI

WT1 (accession number: NM_—000378): plasmid fragment EcoRV/KpnI blunt in T7TS HinDIII blunt/SpeI blunt

PR3 (accession number: NM_—002777): plasmid fragment EcoR1 blunt/Xbal in T7TS HinDIII blunt/SpeI

PRAME (accession number: NM_—006115): plasmid fragment BamH1 blunt/XbaI in T7TS HinDIII blunt/SpeI

Survivin (accession number AF077350): PCR fragment HinDIII/SpeI in T7TS HinDIII/SpeI

Mucin1 (accession number NM_—002456): plasmid fragment: SacI blunt/BamHI in T7TS HinDIII blunt/BglII

Tenascin (accession number X78565): PCR fragment BglII blunt/SpeI in T7TS HinDIII blunt/SpeI

EGFR1 (accession number AF288738): PCR fragment HinDIII/Spel in T7TS HinDIII/Spe I

Sox9 (accession number Z46629): PCR fragment HinDIII/Spel in T7TS HinDIII/SpeI

Sec61G (accession number NM_—014302): PCR fragment HinDIII/Spel in T7TS HinDIII/SpeI

PTRZ1 (accession number NM_—002851): PCR fragment EcoRV/SpeI in T7TS HinDIII blunt/SpeI

3. In Vitro Transcription

3.1. Preparation of Protein-free DNA

500 μg of each of the plasmids described above were linearized in a volume of 2.5 ml by digestion with the restriction enzyme PstI or XbaI in a 15 ml Falcon tube. This cleaved DNA construct was transferred into the RNA production unit. 2.5 ml of a mixture of phenol/chloroform/ isoamyl alcohol were added to the linearized DNA. The reaction vessel was vortexed for 2 minutes and centrifuged at 4,000 rpm for 5 minutes. The aqueous phase was removed and mixed with 1.75 ml 2-propanol in a 15 ml Falcon tube. This vessel was centrifuged at 4,000 rpm for 30 minutes, the supernatant was discarded and 5 ml 75% ethanol were added. The reaction vessel was centrifuged at 4,000 rpm for 10 minutes and the ethanol was removed. The vessel was centrifuged for a further 2 minutes and the residues of the ethanol were removed with a microlitre pipette tip. The DNA pellet was then dissolved in 500 μl RNase-free water (1 μg/μl).

3.2. Enzymatic mRNA Synthesis

Materials:

T7 polymerase: purified from an E. coli strain which contains a plasmid with the gene for the polymerase. This RNA polymerase uses as the substrate only T7 phage promoter sequences (Fermentas),

NTPs: synthesized chemically and purified via HPLC. Purity more than 96% (Fermentas),

CAP analogue: synthesized chemically and purified via HPLC. Purity more than 90% (Trilink),

RNase inhibitor: RNasin, injectable grade, prepared by a recombinant method (E. coli) (Fermentas),

DNase: distributed as a medicament via pharmacies as Pulmozym® (dornase alfa) (Roche).

The following reaction mixture was pipetted into a 15 ml Falcon tube:

• • 100 μg linearized protein-free DNA,
  • 400 μl 5× buffer (Tris-HCl pH 7.5, MgCl₂, spermidine, DTT, inorganic pyrophosphotase 25 U),
  • 20 μl ribonuclease inhibitor (recombinant, 40 U/μ;);
  • 80 μl rNTP-mix (ATP, CTP, UTP 100 mM), 29 μl GTP (100 mM);
  • 116 μl cap analogue (100 mM);
  • 50 μl T7 RNA polymerase (200 U/μl);
  • 1,045 μl RNase-free water.

The total volume was 2 ml and was incubated at 37° C. for 2 hours in a heating block. Thereafter, 300 μl DNase: Pulmozyme™ (1 U/μl) were added and the mixture was incubated at 37° C. for a further 30 minutes. The DNA template was enzymatically degraded by this procedure.

5. Purification of the mRNAs

5.1. LiCl Precipitation (Lithium Chloride/Ethanol Precipitation)

Based on 20-40 μg RNA, this was carried out as follows:

LiCl precipitation 25 μl LiCl solution [8 M]

30 μl WFI (water for injection) were added to the transcription batch (20 μl) and the components were mixed carefully. 25 μl LiCl solution were added to the reaction vessel and the solutions were vortexed for at least 10 seconds. The batch was incubated at −20° C. for at least 1 hour. The closed vessel was then centrifuged at 4,000 rpm for 30 minutes at 4° C. The supernatant was discarded.

Washing

5 μl 75% ethanol were added to each pellet (under a safety workbench). The closed vessels were centrifuged at 4,000 rpm for 20 minutes at 4° C. The supernatant was discarded (under a safety workbench) and centrifugation was carried out again at 4,000 rpm for 2 minutes at 4° C. The supernatant was carefully removed with a pipette (under a safety workbench). Thereafter, the pellet was dried for approx. 1 hour (under a safety workbench).

Resuspension

In each case 10 μl WFI were added to the thoroughly dried pellets (under a safety workbench). The particular pellet was then dissolved in a shaking apparatus overnight at 4° C.

5.2. Final Purification

The final purification was carried out by phenol/chloroform extraction. However, it can likewise be carried out by means of anion exchange chromatography (e.g. MEGAclear™ from Ambion or Rneasy from Qiagen). After this purification of the mRNA, the RNA was precipitated against isopropanol and NaCl (1 M NaCl 1:10, isopropanol 1:1), vortexed, and centrifuged at 4,000 rpm for 30 min at 4° C,, and the pellet was washed with 75% ethanol. The RNA purified by means of phenol/chloroform extraction was dissolved in RNase-free water and incubated at 4° C. for at least 12 hours. The concentration of each mRNA was measured at OD₂₆₀ absorption. (The chloroform/phenol extraction was carried out in accordance with Sambrook J., Fritsch E. F., and Maniatis T., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, vol. 1, 2, 3 (1989)).

Example 2

Stabilizing of the mRNA

An example of an embodiment of the stabilized mRNA according to the invention relates to a β-globin UTR-stabilized mRNA. An mRNA stabilized in this manner had the following structure: cap-β-globin UTR (80 bases)-β-galactosidase coding sequence-β-globin 3′-UTR (approx. 180 bases)-poly A tail (A₃₀C₃₀). Instead of the β-galactosidase coding sequence, constructs which had a sequence which codes for an antigen from a pathogen or tumour already described above were likewise produced.

As a further example of an embodiment of the stabilized mRNA according to the invention, the nucleic acid sequence of the coding region of the mRNA was optimized in respect of its G/C content. To determine the sequence of a modified mRNA according to the invention, the computer program described in WO 02/098443 was used, which, with the aid of the genetic code or the degenerative nature thereof, modifies the nucleotide sequence of any desired mRNA such that a maximum G/C content results, in combination with the use of codons which code for tRNAs occurring as frequently as possible in the cell, the amino acid sequence coded by the modified mRNA preferably being identical to the non-modified sequence. Alternatively, it is also possible to modify only the G/C content or only the codon usage compared with the original sequence. The source code in Visual Basic 6.0 (development environment used: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3) is likewise described in WO 02/098443, the disclosure of which is subject matter of the present invention.

Example 3

Cell Culture

P815 cells were supplemented with 10% heat-inactivated foetal calf serum (PAN systems, Germany), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and cultured in an RPMI 1640 (Bio-Whittaker, Verviers, Belgium). The CTL culture was carried out in RPMI 1640 medium, supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM β-mercaptoethanol, 50 μg/ml gentamycin, 1× MEM non-essential amino acids and 1 mM sodium pyruvate. The CTLs were restimulated for one week with 1 μg/ml β-galactosidase (Sigma, Taufkirchen, Germany). On day 4, the supernatants were carefully collected and replaced by fresh medium containing 10 U/ml rIL-2 (final concentration).

In parallel experimental set-ups, the restimulation was carried out with in each case 1.3 μg/ml survivin, 1 μg MAGE-3 and 0.8 μg Muc-1. All the other conditions in these experimental set-ups were identical to the conditions described above.

Example 4

Immunization of Mice

Female BALB/c AnNCrlBR (H-2d) mice 6 to 12 weeks old were obtained from Charles River (Sulzfeld, Germany). Approval for the genetic (DNA and mRNA) vaccination of the mice was granted by the Committee for Animal Ethics in Tübingen (number IM/200). The BALB mice were anaesthetized with 20 mg pentobarbital intraperitoneally. The mice were then injected intradermally in both ear pinnae with 25 μg β-globin UTR-stabilized mRNA coding for β-galactosidase, which was diluted with injection buffer (150 mM NaCl, 10 mM HEPES). 5·10³ units (1 μg) of GM-CSF (Peprotech, Inc., Rocky Hill, N.Y., USA), diluted with 25 μl PBS, were subsequently injected. This corresponded to a total amount of 2 μg (approx. 10⁴ units), which was injected only once. Such a dosage lies in the lowest range of the dosages normally chosen in mice (26). Two weeks after the first injection, the mice were treated under the same conditions (as with the first injection).

In parallel experimental set-ups I, II+III, which were carried out under the same conditions described above, mice were injected with, instead of 25 μg β-globin UTR-stabilized mRNA which coded for β-galactosidase and 1 μg pg GM-CSF, in

• • Experimental set-up I: 30 μg β-globin UTR-stabilized mRNA coding for survivin and 1.2 μg IL-2, in
  • Experimental set-up II: 23 μg β-globin UTR-stabilized mRNA coding for MAGE-3 and 2 μg IL-12, and in
  • Experimental set-up III: 18 μg β-globin UTR-stabilized mRNA coding for Muc-1 and 1 μg IFN-α.

GM-CSF (total amount of 2 μg of recombinant protein: approx. 10⁴ U (units)) were injected once, either 24 hours or 2 hours before injection of the mRNA or 24 hours after injection of the mRNA (corresponds to groups GM-CSF T−1, GM-CSF T−0 and GM-CSF T+1). The amount of β-galactosidase-specific IgG1 or IgG2a antibodies contained in the blood of the injected mice was determined by ELISA (1:10 serum dilution). The background, which was chiefly obtained by the serum of buffer-injected mice at the same dilution, was subtracted.

Example 5

Chromium Release Assay

Splenocytes were stimulated in vitro with purified β-galactosidase (1 mg/ml) and the CTL activity was determined after 6 days using a standard ⁵¹Cr release assay (as described, for example, by Rammensee et al. (1989), Immunogenetics 30: 296-302). The death rate of the cells was determined with the aid of the amount of ⁵¹Cr released into the medium (A) compared with the amount of spontaneous ⁵¹Cr release of the target cells (B) and the total content of ⁵¹Cr of target cells lysed with 1% Triton-X-100 (C) by means of the formula

% cell lysis=(A−B)÷(C−B)×100

Stimulation of the splenocytes with survivin, MAGE-3 and Muc-1 (concentration in each case 1 mg/ml) was carried out in parallel experimental set-ups. All the other conditions in these experimental set-ups were identical to the conditions described above.

Example 6

ELISA

MaxiSorb plates from Nalgene Nunc International (Nalge, Denmark) were coated overnight at 4° C. with 100 μl β-galactosidase at a concentration of 100 μg/ml (antibody ELISA) or with 50 μl of anti-mouse anti-IFN-γ or -IL-4 (cytokine ELISA) capture antibodies (Becton Dickinson, Heidelberg, Germany) at a concentration of 1 μg/ml in coating buffer (0.02% NaN₃, 15 mM Na₂CO₃, 15 mM NaHCO₃, pH 9.6). The plates were then saturated for 2 hours at 37° C. with 200 μl of blocking buffer (PBS-0.05% Tween 20-1% BSA). They were subsequently incubated at 37° C. for 4 to 5 days with sera (antibody ELISA) at 1:10, 1:30 and 1:90 dilutions in washing buffer or 100 μl of the cell culture supernatant (cytokine ELISA). 100 μl of 1:1,000 dilutions of goat anti-mouse IgG1 or IgG2a antibodies (antibody ELISA) from Caltag (Burlington, Calif., USA) or 100 μl/well of biotinylated anti-mouse anti-IFN-γ or -IL-4 (cytokine ELISA) detection antibodies (Becton Dickinson, Heidelberg, Germany) at a concentration of 0.5 μg/ml in blocking buffer were then added and the plates were incubated at room temperature for 1 hour.

For the cytokine ELISA, after 3 washing steps with washing buffer, 100 μl of a 1:1,000 dilution of streptavidin-HRP (BD Biosciences, Heidelberg, Germany) were added per well. After 30 minutes at room temperature, 100 μl ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) concentrate at a concentration of 300 mg/l in 0.1 M citric acid, pH 4.35) were added per well. After a further 15 to 30 min at room temperature, the extinction at OD₄₀₅ was measured with a Sunrise ELISA Reader from Tecan (Crailsheim, Germany). The amounts of the cytokines were calculated with the aid of a standard curve plotted by titration of certain amounts of recombinant cytokines (BD Pharmingen, Heidelberg, Germany).

In parallel experimental set-ups, the MaxiSorb plates were coated with survivin, MAGE-3 and Muc-1 (in each case 100 μl). All the other conditions in these parallel experimental set-ups were identical to the conditions described above.

Example 7

Immunization of Mice with GM-CSF RNA (cf. FIG. 5)

Female BALB/c AnNCrlBR (H-2d) mice 6 to 12 weeks old (Charles River, Sulzfeld, Germany) BALB mice were anaesthetized with 20 mg pentobarbital intraperitoneally analogously to Example 4 (see above). The mice were then injected intradermally in both ear pinnae with 25 μg of β-globin UTR-stabilized mRNA coding for β-galactosidase, which was diluted with injection buffer (150 mM NaCl, 10 mM HEPES). 50 μg GM-CSF RNA were subsequently injected once into the ear pinnae. Two weeks after the first injection, the mice were treated under the same conditions (as with the first injection).

In parallel experimental set-ups I, II, III, IV and V, which were carried out under the same conditions described above, mice were, in

• • Experimental set-up I: injected only with injection buffer (control);
  • Experimental set-up II: injected with 50 μg GM-CSF RNA alone (control);
  • Experimental set-up III: injected with 25 μg β-globin UTR-stabilized mRNA which coded for β-galactosidase, and 50 μg GM-CSF RNA, the GM-CSF RNA being administered 24 hours before the β-globin UTR-stabilized mRNA coding for β-galactosidase (corresponding to t−1);
  • Experimental set-up IV: injected with 25 μg β-globin UTR-stabilized mRNA which coded for β-galactosidase, and 50 μg GM-CSF RNA, the GM-CSF RNA being administered 2 hours before the β-globin UTR-stabilized mRNA coding for β-galactosidase (corresponding to t−0);
  • Experimental set-up V: injected with 25 μg β-globin UTR-stabilized mRNA which coded for β-galactosidase, and 50 μg GM-CSF RNA, the GM-CSF RNA being administered 24 hours after the β-globin UTR-stabilized mRNA coding for β-galactosidase (corresponding to t+1).

Maxi Sorb plates from Nalgene Nunc International (Nalge Denmark) were plated out overnight at 4° C. with 50 ml of an anti-mouse anti-interferon-γ(IFN-γ) antibody with 1 mg/ml in a coating buffer (0.02% NaN₃, 15 mM Na₂CO₃, 15 mM NaHCO₃, pH 6.6). The plates were then saturated with 200 ml of the blocking buffer (PBS-0.05% Tween 20-1% BSA) for 2 hours at 37° C. and then incubated at 37° C. for 4-5 h with 100 ml of the cell culture supernatant (cytokine ELISA). 100 μl of 1:1,000 dilutions of 100 μl per well of the biotinylated anti-mouse anti-IFN-γ detection antibody (Becton Dickinson) were added at 0.5 mg/ml in a blocking buffer and incubation was carried out at room temperature for one hour. After 3 washing steps with washing buffer, 100 ml of a 1 to 1,000 dilution of streptavidin-HRP (horseradish peroxidase, BD Biosciences, Heidelberg, Germany) were added per well. After 30 minutes at room temperature, 100 ml per well of ABTS (300 mg/l 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) in 0.1 M citrate, pH 4.35) substrate were added. After 15 to 30 minutes at room temperature, the extinction at OD405 was measured with a Sunrise ELISA reading apparatus from Tecan (Crailsheim, Germany) and the amounts of the cytokine were calculated from a standard curve which was obtained by titration with certain amounts of recombinant cytokines (BD Pharmingen, Heidelberg, Germany). It can be clearly seen that the immunostimulation is significantly increased by administration of GM-CSF mRNA before, at about the same time as and after injection of β-galactosidase mRNA.

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Claims

1. A method for immunostimulation in a mammal, comprising the following steps: (a) administration of at least one mRNA containing a region which codes for at least one antigen of a pathogen or at least one tumour antigen and(b) administration of at least one component of at least one of the following categories chosen from the group consisting of a cytokine, a cytokine mRNA, an adjuvo-viral mRNA, a CpG DNA and an adjuvant RNA.

2. A method according to claim 1, wherein step b. is carried out 1 minute to 48 hours, preferably 20 minutes to 36 hours, equally preferably 30 minutes to 24 hours, more preferably 10 hours to 30 hours, most preferably 12 hours to 28 hours, especially preferably 20 hours to 26 hours after step (a).

3. A method according to claim 1, wherein in step (a) at least one RNase inhibitor, preferably RNasin or aurintricarboxylic acid, is additionally administered.

4. A method according to claim 1, wherein an immune response is intensified or modulated, preferably is modified from a Th2 immune response into a Th1 immune response.

5. A method according to claim 1, wherein the at least one mRNA from step (a) contains a region which codes for at least one antigen from a tumour chosen from the group consisting of: 707-AP, AFP, ART-4, BAGE, β-catenine/m, Bcr-abl, CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, CMV pp65, CT, Cyp-B, DAM, EGFR1, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HBS, HER-2/neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HAST-2, hTERT (or hTRT), influenza matrix protein, in particular influenza A matrix M1 protein or influenza B matrix M1 protein, iCE, KIAA0205, LAGE, e.g. LAGE-1, LDLR/FUT, MAGE, e.g. MAGE-A, MAGE-B, MAGE-C, MAGE-A1, MAGE-2, MAGE-3, MAGE-6, MAGE-10, MART-1/melan-A, MC1R, myosine/m, MUC1, MUM-1, -2, -3, NA88-A, NY-ESO-1, p190 minorbcr-abl, Pml/RARα, PRAME, proteinase 3, PSA, PSM, PTPRZ1, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SEC61G, SOX9, SPC1, SSX, survivin, TEL/AML1, TERT, TNC, TPI/m, TRP-1, TRP-2, TRP-2/INT2, tyrosinase and WT1.

6. A method according to claim 1, wherein the at least one cytokine is chosen from the group consisting of IL-1 (α/β), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-21, IL-22, IL-23, IFN-α, IFN-β, IFN-γ, LT-α, MCAF, RANTES, TGFα, TGFβ1, TGFβ2, TNFα, TNFβ and particularly preferably G-CSF or GM-CSF or M-CSF.

7. A method according to claim 1, wherein the at least one mRNA from step (a) and/or from step (b) is in the form of naked or complexed mRNA.

8. A method according to claim 1, wherein the at least one mRNA from step (a) and/or from step (b) is in the form of globin UTR (untranslated regions)-stabilized mRNA, in particular β-globin UTR-stabilized mRNA.

9. A method according to claim 1, wherein the at least one mRNA from step (a) and/or from step (b) is in the form of modified mRNA, in particular stabilized mRNA.

10. A method according to claim 1, wherein the G/C content of the coding region of the modified mRNA from step (a) and/or from step (b) is increased compared with the G/C content of the coding region of the wild-type RNA, the coded amino acid sequence of the modified mRNA preferably not being modified compared with the coded amino acid sequence of the wild-type mRNA.

11. A method according claim 1, wherein the A/U content in the environment of the ribosome binding site of the modified mRNA from step (a) and/or from step (b) is increased compared with the A/U content in the environment of the ribosome binding site of the wild-type mRNA.

12. A method according to claim 1, wherein the coding region and/or the 5′ and/or 3′ untranslated region of the modified mRNA from step (a) and/or from step (b) is modified compared with the wild-type mRNA such that it contains no destabilizing sequence elements, the coded amino acid sequence of the modified mRNA preferably not being modified compared with the wild-type mRNA.

13. A method according to claim 1, wherein the modified mRNA from step (a) and/or from step (b) has a 5′ cap structure and/or a poly(A) tail, preferably of at least 25 nucleotides, more preferably of at least 50 nucleotides, even more preferably of at least 70 nucleotides, equally more preferably of at least 100 nucleotides, most preferably of at least 200 nucleotides, and/or at least one IRES and/or at least one 5′ and/or 3′ stabilizing sequence.

14. A method according to claim 1, wherein the modified mRNA from step (a) and/or from step (b) or the adjuvant RNA from step (b.) contains at least one analogue of naturally occurring nucleotides.

15. A method according to claim 1, wherein the modified mRNA from step (a) and/or from step (b) or the adjuvant RNA from step (b) is complexed or condensed with at least one cationic or polycationic agent.

16. A method according to claim 1, wherein the cationic or polycationic agent is chosen from the group consisting of: protamine, poly-L-lysine, poly-L-arginine and histones.

17. A method according to claim 1, for treatment of tumour diseases, allergies, autoimmune diseases, such as multiple sclerosis, and protozoological, viral and/or bacterial infections.

18. A product comprising at least one mRNA containing a region which codes for at least one antigen of a pathogen or at least one tumour antigen, and at least one component from at least one of the following categories chosen from the group consisting of: a cytokine, a CpG DNA, a cytokine mRNA, an adjuvo-viral mRNA and an adjuvant RNA, as a combination preparation for simultaneous, separate or time-staggered use in the treatment and/or prophylaxis of tumour diseases, allergies, autoimmune diseases, such as multiple sclerosis, and viral and/or bacterial infections.

19. A kit comprising at least one mRNA containing a region which codes for at least one antigen of a pathogen or at least one tumour antigen, and at least one component of at least one category chosen from the group consisting of: a cytokine, a cytokine mRNA, an adjuvo-viral mRNA, a CpG DNA and an adjuvant RNA, the at least one mRNA containing a region which codes for at least one antigen of a pathogen or at least one tumour antigen, and the at least one cytokine or the at least one cytokine mRNA or the at least one CpG DNA or the at least one adjuvant RNA or the at least one adjuvo-viral mRNA being separate from one another.

20. A use of the kit according to claim 19 for treatment and/or prophylaxis of tumour diseases, allergies, autoimmune diseases, such as multiple sclerosis, and protozoological, viral and/or bacterial infections.