NUCLEIC ACIDS COMPRISING FORMULA (NuGlXmGnNv)a AND DERIVATIVES THEREOF AS IMMUNOSTIMULATING AGENT/ADJUVANT

Abstract

The present invention relates to nucleic acids of the general formula (I): (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a and derivatives thereof as an immunostimulating agent/adjuvant and to compositions containing same, optionally comprising an additional adjuvant. The present invention furthermore relates to a pharmaceutical composition or to a vaccine, each containing nucleic acids of formula (I) above and/or derivatives thereof as an immunostimulating agent, and optionally at least one additional pharmaceutically active component, e.g. an antigenic agent. The present invention relates likewise to the use of the pharmaceutical composition or of the vaccine for the treatment of cancer diseases, infectious diseases, allergies and autoimmune diseases etc. Likewise, the present invention includes the use of nucleic acids of the general formula (I): (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a and/or derivatives thereof for the preparation of a pharmaceutical composition for the treatment of such diseases.

Claims

1-25. (canceled)

26. A pharmaceutical composition comprising at least a first RNA molecule said molecule comprising at least 200 consecutive nucleotides of SEQ ID NO: 118 and a pharmaceutically acceptable carrier.

27. The composition of claim 26, wherein the first RNA molecule comprises at least 250 consecutive nucleotides of SEQ ID NO: 118.

28. The composition of claim 27, wherein the first RNA molecule comprises the sequence of SEQ ID NO: 118.

29. The composition of claim 26, wherein the first RNA is a linear RNA.

30. The composition of claim 29, wherein the first RNA comprises a 5′ Cap.

31. The composition of claim 26, wherein the first RNA lacks a 5′ Cap.

32. The composition of claim 30, wherein first RNA molecule comprises a 5′ cap, a Poly-A sequence and, optionally, a Poly-C sequence.

33. The composition of claim 26, wherein first RNA molecule has been purified by HPLC.

34. The composition of claim 26, wherein the composition comprises at least one additional pharmaceutically active component.

35. The composition of claim 26, wherein the first RNA molecule is provided in complex with lipids and/or cationic compounds.

36. The composition of claim 35, wherein the first RNA molecule is provided in complex with cationic peptides.

37. The composition of claim 35, wherein the first RNA molecule is provided in complex with lipids.

38. The composition of claim 34, wherein the composition comprises at least one additional RNA molecule encoding an antigen.

39. The composition of claim 38, wherein the composition comprises at least one additional RNA molecule encoding a tumor antigen.

40. The composition of claim 38, wherein the composition comprises at least one additional RNA molecule encoding a viral antigen.

41. The composition of claim 38, wherein the first RNA molecule comprises at least 250 consecutive nucleotides of SEQ ID NO: 118.

42. The composition of claim 41, wherein the first RNA molecule comprises the sequence of SEQ ID NO: 118.

43. A method of treating a patient comprising administering an effective amount of a composition according to claim 26.

44. A method of producing a RNA: (a) providing a DNA encoding a RNA molecule comprising at least 200 consecutive nucleotides of SEQ ID NO: 118; and (b) performing in vitro transcription to produce the RNA molecule comprising at least 200 consecutive nucleotides of SEQ ID NO: 118.

Description

FIGURES

[0197] The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0198] FIG. 1: shows the TNFα inducing capacity of DOTAP formulated RNAs according to formula (I). PBMCs were seeded at a density of 2*10.sup.5/well/200 μl Medium and stimulated with RNA (4 μg/ml) formulated with DOTAP (12 μg/ml) for 20 h. A TNFα-ELISA was then performed with cell free supernatants. As can be seen in FIG. 1, secretion of TNFα is significantly induced by the inventive nucleic acids according to formula (I), particularly by mRNA sequences according to SEQ ID NOs: 114 to 119 inventive nucleic acids according to formula (I) as defined above, i.e. mRNA sequences according to SEQ ID NOs: 114 to 119 (SEQ ID NO: 114 (R820/(N100).sub.2), SEQ ID NO: 115 (R719/(N100).sub.5), SEQ ID NO: 116 (R720/(N100).sub.10), SEQ ID NO: 117 (R821/(N40T20N40).sub.2), SEQ ID NO: 118 (R722/(N40T20N40).sub.5), and SEQ ID NO: 119 (R723/(N40T20N40).sub.10)) and controls G.sub.2U.sub.20G.sub.2 (GGUUUUUUUUUUUUUUUUUUUUGG) (SEQ ID NO: 125), Seq. U.sub.21: UUUUUUUUUUUUUUUUUUUUU (SEQ ID NO: 126) (Phosphodiester) and Poly(U) (Sigma, 800-1000 kDa).

[0199] FIG. 2: shows the IFNα inducing capacity of DOTAP formulated RNAs according to formula (I). PBMCs were seeded at a density of 2*10.sup.5/well/200 μl Medium and stimulated with RNA (2 μg/ml) formulated with DOTAP (12 μg/ml) for 20 h. An IFNα-ELISA was then performed with cell free supernatants. As can be seen in FIG. 2, secretion of IFNα is significantly induced by the inventive nucleic acids according to formula (I), particularly by mRNA sequences according to SEQ ID NOs: 114 to 119 inventive nucleic acids according to formula (I) as defined above, i.e. mRNA sequences according to SEQ ID NOs: 114 to 119 (SEQ ID NO: 114 (R820/(N100).sub.2), SEQ ID NO: 115 (R719/(N100).sub.5), SEQ ID NO: 116 (R720/(N100).sub.10), SEQ ID NO: 117 (R821/(N40T20N40).sub.2), SEQ ID NO: 118 (R722/(N40T20N40).sub.5), and SEQ ID NO: 119 (R723/(N40T20N40).sub.10)) and controls G.sub.2U.sub.20G.sub.2 (GGUUUUUUUUUUUUUUUUUUUUGG) (SEQ ID NO: 125), Seq. U.sub.21: UUUUUUUUUUUUUUUUUUUUU (SEQ ID NO: 126) (Phosphodiester) and Poly(U) (Sigma, 800-1000 kDa).

EXAMPLES

[0200] The following Examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0201] 1. Synthesis of Exemplary Nucleic Acids of Either Formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) According to the Invention [0202] RNA oligonucleotides, as examples of the nucleic acid of the general formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention, were prepared by automatic solid-phase synthesis by means of phosphoramidite chemistry (including sequences according to SEQ ID NOs: 84-85 (formula (I)), SEQ ID NOs: 86-87 (formula (Ia)), SEQ ID NOs: 88-94 (formulas (II), (IIa) and (IIb)), and SEQ ID NOs: 107-108 (formulas (IIIa) and (IIIb))). In each case the RNA-specific 2′-hydroxyl groups of the nucleotides were protected with TBDMS protecting groups. In the synthesis of phosphorothioates, Beaucage reagent was used for the oxidation. The cleavage of carrier material and of the base-labile protecting groups was carried out with methylamine, and the cleavage of the TBDMS protecting group was effected with triethylamine hydrofluoride. [0203] The crude product was purified by means of HPLC either by ion-pair chromatography, by ion-exchange chromatography or by a combination of the two methods, desalinated and dried. The product was checked for purity and correct base composition by mass spectrometry. [0204] According to an alternative way, the above sequences were prepared by in vitro translation based on DNA vectors or oligonucleotide sequences carrying the inventive sequences.

[0205] 2. In Vitro Immunostimulation with Exemplary Nucleic Acids of Either Formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) According to the Invention [0206] a) For the stimulation of mouse BDMCs (bone marrow derived dendritic cells), 3 μl of oligofectamine were mixed with 30 μl of FCS-free IMDM medium (BioWhittaker, catalogue no. BE12-722F) and incubated at room temperature for 5 minutes. 6 μg of a nucleic acid according to SEQ ID NOs: 84-94 and 107-108 (each type of nucleic acid forming a single experiment), respectively, in the form of RNA, was mixed with 60 μl of FCS-free IMDM and mixed with oligofectamine/IMDM, and incubated for 20 minutes at room temperature. 33 μl of this mixture were then placed for cultivation overnight in a well of a 96-well microtitre culture plate which contained 200,000 mouse BDMCs in 200 μl of FCS-free IMDM medium. After 4 hours, 100 μl of IMDM containing 20% FCS were added and, after 16 hours' co-incubation, the supernatant was removed and tested for interleukin-6 (IL-6) and interleukin-12 (IL-12) by a cytokine ELISA. Comparison tests were carried out analogously to the above sequences using the immunostimulating uncapped wild-type mRNA of beta-galactosidase (lacZ), complexed with protamine. [0207] It was possible to show that the nucleic acids of formulas (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention, present in the form of RNA, in particular the sequences according to the invention of SEQ ID NOs: 84-94 and 107-108, have good immunostimulating properties for stimulation of an innate immune response. [0208] b) Human PBMCs were obtained via a Ficoll density gradient and cultivation overnight in X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q), which contained 1% glutamine and 1% penicillin in the presence of 10 μg/ml of the nucleic acids of either formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention in the form of RNA, in particular of the sequences according to the invention of SEQ ID NOs: 84-94 and 107-108 (each type of nucleic acid forming a single experiment). [0209] For stimulation, 3 μl of oligofectamine were mixed with 30 μl of X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q) and incubated at room temperature for 5 minutes. 6 μg of the nucleic acids of either formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention in the form of RNA, in particular the sequences according to the invention SEQ ID NOs: 84-94 and 107-108 (each type of nucleic acid in a single experiment), respectively, were mixed with 60 μl of X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q) and, mixed with oligofectamine/X-VIVO medium, incubated for 20 minutes at room temperature. 33 μl of this mixture were then placed for cultivation overnight in a well of a 96-well microtitre culture plate which contained 200,000 PBMCs in 200 μl of X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q). After co-incubation for 16 hours, the supernatant was removed and tested for interleukin-6 (IL-6) and interleukin-12 (IL-12) and TNFα by means of a cytokine-ELISA. Comparison tests were carried out analogously to the sequences according to the invention (see above) with the immunostimulating oligo RNA40 (5′-GCCCGUCUGUUGUGUGACUC-3′, SEQ ID NO: 113). [0210] It was possible to show that the inventive nucleic acids in the form of RNA, in particular having the sequences according to the invention either formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention as defined above have good immunostimulating properties.

[0211] 3. In Vivo Immunostimulation with Exemplary Nucleic Acids of Either Formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) According to the Invention—Use as Adjuvant [0212] BALB/c mice (5 per group) were injected with beta-galactosidase protein and with an adjuvant (as defined herein) on days 0 and 10. The mice were sacrificed on day 20 and the blood serum was used for an antibody test against beta-galactosidase protein by means of ELISA, and the IL-6, IL-12 and TNF-alpha values were determined analogously to the above-described in vitro cultures.

[0213] 4. Stimulation of Human Cells with an Adjuvant According to the Invention in the Form of a Nucleic Acid Molecule of Either Formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) [0214] a) In order to determine the immunogenic activity of nucleic acids of either formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention as defined above in the form of adjuvants, particularly of nucleic acids containing a sequence according to SEQ ID NOs: 84-94 and 107-108 (each type of nucleic acid again forming a single experiment) were co-incubated with human cells. To this end, human PBMC cells, for example, were co-incubated for 16 hours in X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q), enriched with 2 mM L-glutamine (BioWhittaker), 10 U/ml penicillin (BioWhittaker) and 10 μg/ml streptomycin, with 10 μg/ml of RNA (mRNA coding for β-galactosidase and optionally with 10 μg/ml protamine. The supernatants were removed and the release of IL-6 and TNFalpha was analysed by means of ELISA. [0215] b) In a further experiment, the release of TNF-alpha by human PBMC cells was determined after stimulation with inventive nucleic acids of either formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention (SEQ ID NOs: 84-94 and 107-108, each type of nucleic acid in a single experiment, see above) and also adjuvants used according to the invention. [0216] To that end, human PBMC cells were co-incubated for 16 hours with 10 μg/ml said inventive nucleic acids in X-VIVO 15 medium (BioWhittaker), enriched with 2 mM L-glutamine (BioWhittaker), 10 U/ml penicillin (BioWhittaker) and 10 μg/ml streptomycin. The supernatants were removed and analysed by means of ELISA.

[0217] 5. Secretion of TNFα and IFN-α in Human PBMCs [0218] For this experiments, several inventive nucleic acids according to formula (I) as defined above, i.e. mRNA sequences according to SEQ ID NOs: 114 to 119, were formulated with DOTAP (Roche). [0219] The inventive nucleic acid sequences used in the experiment were [0220] SEQ ID NO: 114 (R820/(N100).sub.2); [0221] SEQ ID NO: 115 (R719/(N100).sub.5); [0222] SEQ ID NO: 116 (R720/(N100).sub.10); [0223] SEQ ID NO: 117 (R821/(N40T20N40).sub.2); [0224] SEQ ID NO: 118 (R722/(N40T20N40).sub.5); and [0225] SEQ ID NO: 119 (R723/(N40T20N40).sub.10). [0226] Human PBMCs were then stimulated with the formulated RNAs at a concentration of 8 μg/ml and 12 μg/ml DOTAP for 20 hours. The Supernatants were then investigated for the secretion of TNFα and IFN-α using a matched-paired ELISA. [0227] For the experiment, human PBMCs were obtained via a Ficoll density gradient and cultivation for 20 hours in X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q), which contained 1% glutamine and 1% penicillin in the presence of 2 or 4 μg/ml of the above nucleic acids for IFNα or TNFα stimulation respectively. For formulation and stimulation, 3 or 6 μg RNA in HBS buffer were transferred to a vial containing 18 μg N-[1-(2,3-Dioleoyloxy)propyl]-N,N,Ntrimethylammonium methylsulfate (DOTAP) (Roche Diagnostics, catalogue no. 11 811 177 001) in HBS buffer and carefully mixed by gently pipetting the mixture several times. The transfection mixture was incubated for 15 min at 15-25° C. 1 volume of the DOTAP/nucleic acid mixture was then gently diluted with 7.3 volumes of X-Vivo medium. 100 μl of this mixture were then placed for cultivation overnight in a well of a 96-well microtitre culture plate which contained 2*10.sup.5 PBMCs in 100 μl of X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q). After coincubation for 20 hours, the supernatant was removed and tested for IFNα and TNFα□ by means of a cytokine-ELISA. Comparison tests were carried out analogously to the sequences according to the invention (see above) with the immunostimulating oligo G.sub.2U.sub.20G.sub.2 (Phosphothioat-modifed), Poly(U) (Sigma, Taufkirchen, Germany) and the oligo U.sub.21 (Phophodiester). [0228] The results are shown in FIGS. 1 and 2. FIG. 1 shows the TNFα inducing capacity of DOTAP formulated RNAs. PBMCs were seeded at a density of 2*10.sup.5/well/200 μl Medium and stimulated with RNA (4 μg/ml) formulated with DOTAP (12 μg/ml) for 20 h. A TNFα-ELISA was then performed with cell free supernatants. FIG. 2 shows the IFNα inducing capacity of DOTAP formulated RNAs. PBMCs were seeded at a density of 2*10.sup.5/well/200 μl Medium and stimulated with RNA (2 μg/ml) formulated with DOTAP (12 μg/ml) for 20 h. An IFNα-ELISA was then performed with cell free supernatants. [0229] As can be seen in FIG. 1 and FIG. 2, both secretion of TNFα and IFNα is significantly induced by the inventive nucleic acids according to formula (I), particularly by mRNA sequences according to SEQ ID NOs: 114 to 119 inventive nucleic acids according to formula (I) as defined above, i.e. mRNA sequences according to SEQ ID NOs: 114 to 119 (SEQ ID NO: 114 (R820/(N100).sub.2), SEQ ID NO: 115 (R719/(N100).sub.5), SEQ ID NO: 116 (R720/(N100).sub.10), SEQ ID NO: 117 (R821/(N40T20N40).sub.2), SEQ ID NO: 118 (R722/(N40T20N40).sub.5), and SEQ ID NO: 119 (R723/(N40T20N40).sub.10)) versus control sequences G.sub.2U.sub.20G.sub.2(Phosphothioat-modifed), Poly(U) (Sigma, Taufkirchen, Germany) and the oligo U.sub.21 (Phophodiester).

Advantages of the Invention

[0230] A nucleic acid of the general formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) according to the invention may be used as immunostimulating agent as such for stimulating the innate immune system of a patient to be treated. This immunostimulating property may well be enhanced by the addition of other compounds known in the art as actively stimulating the innate immune response to the inventive nucleic acids, e.g. by lipid modification or addition of additional adjuvants. The inventive nucleic acids as defined herein, particularly those according to formula (I) comprising the structure (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a, or of derivatives thereof, exhibit a significant better amplification in bacteria, e.g. E. coli. It is furthermore particularly advantageous, if the inventive nucleic acid (N.sub.vG.sub.lX.sub.mG.sub.nN.sub.v).sub.a of formula (I), or of derivatives thereof, is a partially double-stranded nucleic acid molecule or a mixture of a single-stranded and a double-stranded nucleic acid molecule, since such a (partially double-stranded) inventive nucleic acid molecule according to formula (I) (or of formula (Ia), (II) (IIa), (IIb), (IIIa) and/or (IIIb)), can positively stimulate the innate immune response in a patient to be treated by addressing the PAMP—(pathogen associated molecular pattern) receptors for single-stranded RNA (TLR-7 and TLR-8) as well as the PAMP-receptors for double-stranded RNA (TLR-3, RIG-I and MDA-5). Receptors TLR-3, TLR-7 and TLR-8 are located in the endosome and are activated by RNA taken up by the endosome. In contrast, RIG-I and MDA-5 are cytoplasmic receptors, which are activated by RNA which was directly taken up into the cytoplasm or which has been released from the endosomes (endosomal release or endosomal escape). Accordingly, a partially double-stranded inventive nucleic acid (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a of formula (I) (or of derivatives thereof, e.g. (a partially double-stranded) inventive nucleic acid molecule according to formula (Ia), (II) (IIa), (IIb), (IIIa) and (IIIb) as defined herein)) is capable of activating different signal cascades of immunostimulation and thus leads to an increased innate immune response or enhances such a response significantly. A further advantage of the invention is the high induction of the antiviral cytokine IFNalpha which is preferred in stimulation of the innate immune system. An often underestimated limitation of generally accepted immunostimulating nucleic acids (e.g. poly A:U and poly I:C) is the undefined structure of them which results in regulatory restrictions.