WO2011069587A1 - Lyophilization of nucleic acids in lactate-containing solutions - Google Patents

Lyophilization of nucleic acids in lactate-containing solutions Download PDF

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Publication number
WO2011069587A1
WO2011069587A1 PCT/EP2010/006789 EP2010006789W WO2011069587A1 WO 2011069587 A1 WO2011069587 A1 WO 2011069587A1 EP 2010006789 W EP2010006789 W EP 2010006789W WO 2011069587 A1 WO2011069587 A1 WO 2011069587A1
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nucleic acid
mage
lyophilized
sequence
lactate
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PCT/EP2010/006789
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French (fr)
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Thorsten Mutzke
Thomas Ketterer
Florian VON DER MüLBE
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Curevac Gmbh
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Publication of WO2011069587A1 publication Critical patent/WO2011069587A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms

Definitions

  • the present invention is directed to the lyophilization of nucleic acids in a lactate- containing solution or formulation.
  • the present invention is particularly suitable for enhancing and improving storage and shipping capabilities of nucleic acids for multiple purposes.
  • the present invention is furthermore directed to methods of lyophilization suitable to prepare such inventive lyophilized nucleic acids, to the use of a lactate- containing solution or formulation for the preparation of such lyophilized nucleic acids or the use of a solution containing at least one nucleic acid (sequence) and (free) lactate for lyophilization of at least one nucleic acid (sequence), to the use of such lyophilized nucleic acids in the preparation of pharmaceutical compositions, to first and second medical indications using such lyophilized nucleic acids and to kits, particularly to kit of parts, comprising such lyophilized nucleic acids.
  • nucleic acids for therapeutic and diagnostic purposes.
  • rapid progress has occurred in recent years in the field of gene therapy and promising results have been achieved.
  • the final dosage form providing these nucleic acids but also production, transport and storage thereof are of particular interest.
  • nucleic acids e.g., naked DNA
  • nucleic acids introduced into a patient' circulatory system are typically not stable and therefore have little chance of affecting most disease processes (see e.g. Poxon et a/., Pharmaceutical development and Technology, 5(1 ), 1 15- 122 (2000)).
  • This obstacle has led to the development of a number of gene delivery systems, e.g.
  • Lyophilization is a worldwide known and recognized method in the art to enhance storage stability of temperature sensitive biomolecules, such as nucleic acids.
  • lyophilization typically water is removed from a frozen sample containing nucleic acids via sublimation.
  • the process of lyophilization is usually characterized by a primary and a secondary drying step. During the primary drying step, free, i.e. unbound, water surrounding the nucleic acid and optionally further components, escapes from the solution. Subsequent thereto water being bound on a molecular basis by the nucleic acids may be removed in a secondary drying step by adding thermal energy. In both cases the hydration sphere around the nucleic acids is lost.
  • the sample containing nucleic acids is initially cooled below the freezing point of the solution and accordingly of the water contained therein. As a result, the water freezes. Dependent on temperature, rate of cooling down (freezing rate), and the time for freezing, the crystal structure of water is changed. This exhibits physical stress on the nucleic acid and other components of the solution, which may lead to a damage of the nucleic acid, e.g. breakage of strands, loss of supercoiling, etc. Furthermore, due to the decrease of volume and loss of the hydration sphere, autocatalytic degradation processes are favored e.g. by traces of transition metals. Additionally, significant changes of pH are possible by concentration of traces of acids and bases.
  • cryoprotectants are understood as excipients, which allow influencing the structure of the ice and/or the eutectical temperature of the mixture. Lyoprotectants are typically excipients, which partially or totally replace the hydration sphere around a molecule and thus prevent catalytic and hydrolytic processes.
  • carbohydrates such as sugars play a central role as lyoprotectants.
  • cryoprotectants and lyoprotectants no general rule may be applied with respect to their impact on different groups of compounds. Therefore, an optimal formulation has to be found using empirical methods.
  • lyophilization causes the removal of the hydration sphere around the DNA, wherein it appears that there are approximately 20 water molecules per nucleotide pair bound most tightly to DNA. These water molecules do not form an ice-like structure upon low-temperature cooling. Upon DNA dehydration over hygroscopic salts at 0% relative humidity, only five or six water molecules remain (see e.g. Tao et a/., Biopolymers, 28, 101 9-1030 (1 989)). Lyophilization may increase the stability of DNA under long-term storage, but may also cause some damage upon the initial lyophilization process, potentially through changes in the DNA secondary structure, breaks of the nucleic acid chain(s) or the concentration of reactive elements such as contaminating metals.
  • Lyophilization can also cause damage upon the initial lyophilization process in other nucleic acid molecules, e.g. RNA.
  • Agents that can substitute for non-freezable water, such as trehalose can demonstrate cryoprotective properties for DNA and other molecules during lyophilization of intact bacteria (see e.g. Israeli et a/, Cryobiology, 30, 51 9-523 (1 993); or Rudolph et a/, Arch. Biochem. Biophys., 245, 1 34-143 (1986)).
  • cryoprotective agents such as polyols, amino acids, sugars, and lyotropic salts
  • Poxon et a/. (2000, supra) investigated the effect of lyophilization on plasmid DNA activity.
  • Poxon et a/. (2000, supra) hypothetized, that a change in the DNA conformation from supercoi led to open circular and linear form would be indicative of damage of the plasmid DNA.
  • the percentage of supercoiled DNA did not change after lyophi lization and subsequent DMED treatment, suggesting that other effects drew responsible for the loss of transfection efficiency.
  • Poxon et a/. (2000, supra) found that a decrease in plasmid DNA activity as measured by an in vitro transfection assay can be ameliorated by the use of carbohydrates during lyophilization of the plasmid DNA.
  • a statistically significant loss of transfection efficiency (p ⁇ 0.05) by lyophilization of pRL-CMV plasmid DNA could completely be restored by using mono- and disaccharides during lyophilization.
  • lyoprotectants glucose (monosaccaride), sucrose and lactose (disaccharides) were used.
  • Poxon eta/. (2000, supra) only carried out investigations with lyophilized plasmid DNA using carbohydrate lyoprotectants. No other nucleic acids, such as RNA or PNA, were discussed. Poxon et a/. (2000, supra), did also not investigate if the addition of sugars to the lyophilization affects the stability of the lyophilized plasmid DNA.
  • Molina et al. see e.g. Molina et al., J. Pharm. Sci. 2004 Sep, 93(9), 2259-73).
  • Molina et al. (2004, supra) lyophilized lipid/DNA complexes in the presence of the carbohydrates glucose, sucrose and trehalose, i.e. mono- or disaccharide sugars.
  • Molina et al. (2004, supra) furthermore used a specific liposome preparation, preferably liposomes containing DOTAP in a 1 :1 (w/w) ratio with the zwitterionic lipid DOPE, wherein a 3:1 lipid:DNA weight ratio was applied.
  • the plasmid product must be of high purity, essentially in its supercoiled form and free of host-cell proteins, chromosomal RNA, RNA, preferably without the use of RNase A, and endotoxins.
  • Quaak et al. (2008, supra) used as excipients sucrose, trehalose, mannitol and polyvinylpyrrolidone (PVP), wherein lyophilization of formulations containing sucrose as a bulking agent in a concentration of (2%) turned out to result in a stable product.
  • PVP polyvinylpyrrolidone
  • RNAs nucleic acids
  • provision of stabilization of RNAs during lyophilization and long-term storage is particularly important.
  • the physico chemical stability of RNAs in solution is extremely low.
  • RNA is typically completely degraded even in the absence of RNases when stored a few days at room temperature. To avoid such degradation and a loss of function, particularly when regarding coding RNAs such as mRNAs the RNA is to be stored at -20°C or even -80°C.
  • Yadava et a/ when using nucleic acids, particularly RNAs, as an active agent in a pharmaceutical composition or a vaccine, Yadava et a/, (see Yadava et a/., AAPS Pharm. Sci. Tech., Vol. 9, No. 2, June 2008, pp. 335- 341 ) discuss the effect of lyophilization and freeze-thawing on the stability of siRNA- liposome complexes.
  • the lipoplexes in Yadava et a/. (2008, supra) when lyophilized in the presence of sugars, such as glucose or sucrose, could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions, they lost 65-75% of their biological functionality.
  • RNA interference is the post-transcriptional gene silencing due to the cleavage of mRNA, triggered by small double stranded RNA molecules (small interfering RNAs, siRNAs), homologous in sequence to the target mRNA, which are typically less sensitive to loss of biological activity due to breakage and (partial) degradation than mRNAs or RNAs in general, as these siRNAs may even exhibit a biological activity, when their (short) sequence is partially degraded. Yadava et a/.
  • RNAvaccination using RNAs RNAs
  • gene therapy using RNAs etc.
  • the problem underlying the present invention is solved by a lyophilized nucleic acid, which has been lyophilized from a lactate containing solution.
  • the inventive nucleic acid (sequence), lyophilized or to be lyophilized is prepared using a method as described herein, particularly a method of lyophilization of a nucleic acid according to the present invention.
  • the present invention also provides the use of a lactate containing solution, preferably as defined herein for lyophilizing a nucleic acid (sequence) preferably as defined herein, or the use of a solution containing at least one nucleic acid (sequence) as defined herein and (free) lactate as defined herein for lyophilization of at least one nucleic acid (sequence).
  • "free” preferably means unbound or unconjugated, e.g.
  • the lactate is not covalently bound to the nucleic acid(sequence) to be lyophilized, or in other words, the lactate is unconjugated, preferably with respect to the nucleic acid (sequence) to be lyophilized).
  • a lactate as defined herein may be any lactate available in the art.
  • a lactate within the context of the present invention is defined as a chemical compound, particularly a salt, derived from (free) lactic acid (lUPAC systematic name: 2- hydroxypropanoic acid), also known as milk acid, including its optical isomers L-(+)-lactic acid, (5)-lactic acid, D-(-)-lactic acid or (A)-lactic acid, more preferably its biologically active optical isomer L-(+)-lactic acid, wherein the salt or an anion thereof, preferably may be selected from sodium-lactate, potassium- lactate, or Al 3 + -lactate, NH 4 + -lactate, Fe-lactate, Li-lactate, Mg-lactate, Ca-lactate, Mn-lactate or Ag-lactate, or selected from Ringer's lactate (RiLa), lactated Ringer's solution (main content sodium lactate, also termed "Hartmann's Solution”
  • Lactic acid is a chemical compound that plays a role in several biochemical processes. It was first isolated in 1 780 by a Swedish chemist, Carl Wilhelm Scheele, and is a carboxylic acid with a chemical formula of C 3 H 6 0 3 . It has a hydroxyl group adjacent to the carboxyl group, making it an alpha hydroxy acid (AHA). In solution, it can lose a proton from the acidic group, producing the lactate ion CH 3 CH(OH)COO " . Lactic acid is chiral and has two optical isomers.
  • L-(+)-lactic acid or (5)-lactic acid is D-(-)-lactic acid or ( ⁇ -lactic acid, wherein L-(+)-lactic acid is the biologically important isomer.
  • L-lactate is constantly produced in animals from pyruvate via the enzyme lactate dehydrogenase (LDH) in a process of fermentation during normal metabolism and exercise.
  • LDH lactate dehydrogenase
  • lactic acid is typically produced via fermentation using among others bacteria such as Lactobacillus bacteria, etc.
  • the present invention preferably uses lactate as a monomer and therefore preferably excludes polymeric forms of lactic acid, in particular poly lactic acid (PLA), or salts therefrom from the scope of the present invention.
  • the lactate containing solution as defined herein which is used for lyophilizing a nucleic acid (sequence) as defined herein, typically comprises a lactate concentration prior to lyophilization in the range of about 3 mM to about 300 mM, preferably in the range of about 5 mM to about 200 mM, more preferably in the range of about 10 mM to about 150 mM, even more preferably about 15 mM to about 35 mM, and most preferably 20 mM to about 31 mM.
  • the lactate containing solution as defined herein which is used for preparation of the inventive lyophilizing a nucleic acid (sequence) as defined herein, typically comprises a Ringer's lactate concentration (or a concentration of any of the afore mentioned lactate containing solutions) prior to lyophilization e.g. in the range of about 10% (w/w) to about 100% (w/w), e.g.
  • Ringer's lactate (100 % (w/w)) is typically defined as a solution comprising 131 mM Na + , 5,36 mM K + , 1 ,84 mM Ca 2+ , and 28,3 mM Lactate).
  • the present invention is directed to a nucleic acid, which has been lyophilized or may be lyophilized from a lactate containing solution.
  • a nucleic acid sequence
  • lyophilized or to be lyophilized may be any suitable nucleic acid, selected e.g. from any (double-stranded or single-stranded) DNA, preferably, without being limited thereto, e.g.
  • genomic DNA single-stranded DNA molecules, double-stranded DNA molecules, coding DNA, DNA primers, DNA probes, immunostimulatory DNA, a (short) DNA oligonucleotide ((short) oligodesoxyribonucleotides), or may be selected e.g. from any PNA (peptide nucleic acid) or may be selected e.g.
  • RNA from any (double-stranded or single-stranded) RNA, preferably, without being limited thereto, a (short) RNA oligonucleotide ((short) oligoribonucleotide), a coding RNA, a messenger RNA (mRNA), an immunostimulatory RNA, a siRNA, an antisense RNA, a micro RNA, or riboswitches, ribozymes or aptamers; etc.
  • the nucleic acid (sequence), lyophilized or to be lyophilized may also be a ribosomal RNA (rRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), or a viral RNA (vRNA).
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • mRNA messenger RNA
  • vRNA viral RNA
  • the nucleic acid (sequence), lyophilized or to be lyophilized is an RNA. More preferably, the nucleic acid (sequence), lyophilized or to be lyophilized, may be a (linear) single-stranded RNA, even more preferably an mRNA.
  • an mRNA is typically an RNA, which is composed of several structural elements, e.g. an optional 5'-UTR region, an upstream positioned ribosomal binding site followed by a coding region, an optional 3'-UTR region, which may be followed by a poly-A tail (and/or a poly-C-tail).
  • An mRNA may occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA which carries the coding sequences of one, two or more proteins or peptides.
  • Such coding sequences in di-, or even multicistronic mRNA may be separated by at least one IRES sequence, e.g. as defined herein.
  • the nucleic acid may be a single- or a double-stranded nucleic acid (molecule) (which may also be regarded as a nucleic acid (molecule) due to non-covalent association of two single-stranded nucleic acid(s) (molecules)) or a partially double-stranded or partially single stranded nucleic acid, which are at least partially self complementary (both of these partially double-stranded or partially single stranded nucleic acid molecules are typically formed by a longer and a shorter single- stranded nucleic acid molecule or by two single stranded nucleic acid molecules, which are about equal in length, wherein one single-stranded nucleic acid molecule is in part complementary to the other single-stranded nucleic acid molecules molecule and both thus form a double-stranded nucleic acid molecules molecule in this region, i.e.
  • nucleic acid (sequence), lyophilized or to be lyophilized may be a single-stranded nucleic acid molecule.
  • nucleic acid (sequence), lyophilized or to be lyophilized may be a circular or linear nucleic acid molecule, preferably a linear nucleic acid molecule.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be a coding nucleic acid, e.g. a DNA or RNA.
  • a coding DNA or RNA may be any DNA or RNA as defined above.
  • such a coding DNA or RNA may be a single- or a double-stranded DNA or RNA, more preferably a single-stranded DNA or RNA, and/or a circular or linear DNA or RNA, more preferably a linear DNA or RNA.
  • the coding DNA or RNA may be a (linear) single-stranded DNA or RNA.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be a ((linear) single-stranded) messenger RNA (mRNA).
  • mRNA messenger RNA
  • Such an mRNA may occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA which carries the coding sequences of one, two or more proteins or peptides.
  • Such coding sequences in di-, or even multicistronic mRNA may be separated by at least one IRES sequence, e.g. as defined herein.
  • the lyophilized nucleic acid which has been lyophilized or which is to be lyophilized from a lactate containing solution, is an mRNA (sequence), preferably as defined herein.
  • an inventive lyophilized mRNA (sequence) may be prepared, preferably using a method as described herein, e.g. by the use of a lactate containing solution as defined herein and an mRNA (sequence) as defined herein or a solution containing at least one mRNA (sequence) as defined herein and (free) lactate as defined herein, for lyophilization of at least one mRNA (sequence).
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may encode a protein or a peptide, which may be selected, without being restricted thereto, e.g. from therapeutically active proteins or peptides, from antigens, e.g. tumor antigens, pathogenic antigens (e.g.
  • telomeres selected from pathogenic proteins as defined above or from animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens), autoimmune antigens, or further antigens, from allergens, from antibodies, from immunostimulatory proteins or peptides, from antigen-specific T-cell receptors, or from any other protein or peptide suitable for a specific (therapeutic) application, wherein the coding DNA or RNA may be transported into a cell, a tissue or an organism and the protein may be expressed subsequently in this cell, tissue or organism.
  • Therapeutically active proteins as defined above or from animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens), autoimmune antigens, or further antigens, from allergens, from antibodies, from immunostimulatory proteins or peptides, from antigen-specific T-cell receptors, or from any other protein or peptide suitable for a specific (therapeutic) application, wherein the coding
  • therapeutically active proteins may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention. These may be selected from any naturally occurring recombinant or isolated protein known to a skilled person from the prior art. Without being restricted thereto therapeutically active proteins may comprise proteins, capable of stimulating or inhibiting the signal transduction in the cell, e.g. cytokines, antibodies, etc.
  • Therapeutically active proteins may thus comprise cytokines of class I of the family of cytokines, having 4 positionally conserved cysteine residues (CCCC) and comprising a conserved sequence motif Trp-Ser- X-Trp-Ser (WSXWS), wherein X is a non-conserved amino acid.
  • Cytokines of class I of the family of cytokines comprise the GM-CSF subfamily, e.g. IL-3, IL-5, GM-CSF, the IL- 6-subfamily, e.g. IL-6, IL-1 1 , IL-12, or the IL-2-subfamily, e.g.
  • Therapeutically active proteins may also comprise cytokines of class II of the family of cytokines, which also comprise 4 positionally conserved cystein residues (CCCC), but no conserved sequence motif Trp- Ser-X-Trp-Ser (WSXWS). Cytokines of class II of the family of cytokines comprise e.g. IFN-alpha, IFN-beta, IFN-gamma, etc. Therapeutically active proteins may additionally comprise cytokines of the family of tumor necrose factors, e.g.
  • TNF-alpha TNF-beta, etc.
  • cytokines of the family of chemokines which comprise 7 transmembrane helices and interact with G-protein, e.g. IL-8, MIP-1 , RANTES, CCR5, CXR4, etc., or cytokine specific receptors, such as TNF-RI, TNF-RII, CD40, OX40 (CD134), Fas, etc.
  • G-protein e.g. IL-8, MIP-1 , RANTES, CCR5, CXR4, etc.
  • cytokine specific receptors such as TNF-RI, TNF-RII, CD40, OX40 (CD134), Fas, etc.
  • Therapeutically active proteins which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be selected from any of the proteins given in the following: 0ATL3, 0FC3, 0PA3, 0PD2, 4-1 BBL, 5T4, 6Ckine, 707-AP, 9D7, A2M, AA, AAAS, AACT, AASS, ABAT, ABCA1, ABCA4, ABCB1 , ABCB11, ABCB2, ABCB4, ABCB7, ABCC2, ABCC6, ABCC8, ABCD1, ABCD3, ABCG5, ABCG8, ABU, ABO, ABR ACAAl, ACACA, ACADL, ACADM, ACADS, ACADVL, ACAT1, ACCPN, ACE, ACHE, ACHM3, ACHM1, ACLS, ACPI, ACTA1, ACTC, ACTN4, ACVRL1, AD2, ADA, ADAMTS13, ADAMTS2, ADFN, ADH
  • Therapeutically active proteins which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may further be selected from apoptotic factors or apoptosis related proteins including AIF, Apaf e.g. Apaf-1 , Apaf-2, Apaf-3, oder APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl- x L , Bcl-x s , bik, CAD, Calpain, Caspase e.g.
  • AIF Apaf e.g. Apaf-1 , Apaf-2, Apaf-3, oder APO-2 (L), APO-3 (L)
  • Apopain Bad, Bak, Bax, Bcl-2, Bcl- x L , Bcl-x s , bik, CAD, Calpain, Caspase e.g.
  • a therapeutically active protein which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, can also be an adjuvant protein.
  • an adjuvant protein is preferably to be understood as any protein, which is capable to elicit an innate immune response as defined herein.
  • an innate immune response comprises activation of a pattern recognition receptor, such as e.g. a receptor selected from the Toll-like receptor (TLR) familiy, including e.g. a Toll like receptor selected from human TLR1 to TLR10 or from murine Toll like receptors TLR1 to TLR13.
  • TLR Toll-like receptor
  • an innate immune response is elicited in a mammal as defined above.
  • the adjuvant protein is selected from human adjuvant proteins or from pathogenic adjuvant proteins, in particular from bacterial adjuvant proteins.
  • mRNA encoding huma proteins involved in adjuvant effects may be used as well.
  • Human adjuvant proteins which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, typically comprise any huma protein, which is capable of eliciting an innate immune response (in a mammal), e.g. as a reaction of the binding of an exogenous TLR ligand to a TLR.
  • human adjuvant proteins encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be selected from the group consisting of, without being limited thereto, cytokines which induce or enhance an innate immune response, including IL-2, IL-12, IL-15, IL-18, IL-21 CCL21, GM-CSF and TNF-alpha; cytokines which are released from macrophages, including IL-1 , IL-6, IL-8, IL- 12 and TNF-alpha; from components of the complement system including C1 q, MBL, C1 r, C1 s, C2b, Bb, D, MASP-1 , MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1 INH, C4bp, MCP, DAF, H
  • NF- ⁇ , C-FOS, c-Jun, c-Myc e.g. IL-1 alpha, 1L-1 beta, Beta-Defensin, IL-6, IFN gamma, IFN alpha and IFN beta; from costimulatory molecules, including CD28 or CD40-ligand or PDl ; protein domains, including LAMP; cell surface proteins; or human adjuvant proteins including CD80, CD81 , CD86, trif, flt-3 ligand, thymopentin, Gp96 or fibronectin, etc., or any species homolog of any of the above human adjuvant proteins.
  • costimulatory molecules including CD28 or CD40-ligand or PDl
  • protein domains including LAMP
  • cell surface proteins or human adjuvant proteins including CD80, CD81 , CD86, trif, flt-3 ligand, thymopentin, Gp96 or fibronectin, etc., or any species homolog of any of the
  • Pathogenic adjuvant proteins which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, typically comprise any pathogenic (adjuvant) protein, which is capable of eliciting an innate immune response (in a mammal), more preferably selected from pathogenic (adjuvant) proteins derived from bacteria, protozoa, viruses, or fungi, animals, etc., and even more preferably from pathogenic adjuvant proteins selected from the group consisting of, without being limited thereto, bacterial proteins, protozoa proteins (e.g. profilin - like protein of Toxoplasma gondii), viral proteins, or fungal proteins, animal proteins, etc.
  • pathogenic (adjuvant) protein which is capable of eliciting an innate immune response (in a mammal)
  • pathogenic (adjuvant) proteins derived from bacteria, protozoa, viruses, or fungi, animals, etc. and even more preferably from pathogenic adj
  • bacterial (adjuvant) proteins which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may comprise any bacterial protein, which is capable of eliciting an innate immune response (preferably in a mammal) or shows an adjuvant character.
  • such bacterial (adjuvant) proteins are selected from the group consisting of bacterial heat shock proteins or chaperons, including Hsp60, Hsp70, Hsp90, Hsp100; OmpA (Outer membrane protein) from gram-negative bacteria; bacterial porins, including OmpF; bacterial toxins, including pertussis toxin (PT) from Bordetella pertussis, pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis, PT-9K 129G mutant from pertussis toxin, pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis, tetanus toxin, cholera toxin (CT), cholera toxin B-subunit, CTK63 mutant from cholera toxin, CTE1 12K mutant from CT, Escherichia coli heat-labile entero
  • CT
  • Bacterial (adjuvant) proteins which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may also be selected from bacterial adjuvant proteins, even more preferably selected from the group consisting of, without being limited thereto, bacterial flagellins, including flagellins from organisms including Agrobacterium, Aquifex, Azospirillum, Bacillus, Bartonella, Bordetella, Borrelia, Burkholderia, Campylobacter, Caulobacte, Clostridium, Escherichia, Helicobacter, Lachnospiraceae, Legionella, Listeria, Proteus, Pseudomonas, Rhizobium, Rhodobacter, Roseburia, Salmonella, Serpulina, Serratia, Shigella, Treponema, Vibrio, Wolinella, Yersinia, more preferably flagellins from the species, without being limited
  • Bacterial flagellins which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, even more preferably comprise a sequence selected from the group comprising any of the following sequences as referred to their accession numbers: organism species gene name accession No Gl No
  • Rhizobium Rhizobium meliloti flaA M24526 Gl:152220 flaB
  • Protozoa proteins which may also be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may be selected from any protozoa protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii, Protozoan heat shock proteins, LeIF from Leishmania spp., profi linlike protein from Toxoplasma gondii, etc.
  • Viral proteins which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may be selected from any viral protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Respiratory Syncytial Virus fusion glycoprotein (F-protein), envelope protein from MMT virus, mouse leukemia virus protein, Hemagglutinin protein of wild type measles virus, etc.
  • F-protein Respiratory Syncytial Virus fusion glycoprotein
  • Fungal proteins which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may be selected from any fungal protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, fungal immunomodulatory protein (FIP; LZ-8), etc.
  • pathogenic adjuvant proteins which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may finally be selected from any further pathogenic protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Keyhole limpet hemocyanin (KLH), OspA, etc.
  • KLH Keyhole limpet hemocyanin
  • OspA OspA
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may alternatively encode an antigen.
  • the term "antigen" refers to a substance which is recognized by the immune system and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies as part of an adaptive immune response.
  • the first step of an adaptive immune response is the activation of naive antigen-specific T cells by antigen- presenting cells. This occurs in the lymphoid tissues and organs through which naive T cells are constantly passing.
  • the three cell types that can serve as antigen-presenting cells are dendritic cells, macrophages, and B cells.
  • Tissue dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by infection to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents to express MHC class II molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may be important to induce T cells. By presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells.
  • effector T cells The most important function of effector T cells is the killing of infected cells by CD8 + cytotoxic T cells and the activation of macrophages by TH1 cells which together make up cell-mediated immunity, and the activation of B cells by both TH2 and TH1 cells to produce different classes of antibody, thus driving the humoral immune response.
  • T cells recognize an antigen by their T cell receptors which does not recognize and bind antigen directly, but instead recognize short peptide fragments e.g. of pathogens' protein antigens, which are bound to MHC molecules on the surfaces of other cells.
  • T cells fall into two major classes that have different effector functions. The two classes are distinguished by the expression of the cell-surface proteins CD4 and CD8.
  • T cells differ in the class of MHC molecule that they recognize.
  • MHC class I and MHC class II- differ in their structure and expression pattern on tissues of the body.
  • CD4 + T cells bind to the MHC class II molecule and CD8 + T cells to the MHC class I molecule.
  • MHC class I and MHC class II have distinct distributions among cells that reflect the different effector functions of the T cells that recognize them.
  • MHC class I molecules present peptides from pathogens, commonly viruses to CD8 + T cells, which differentiate into cytotoxic T cells that are specialized to kill any cell that they specifically recognize. Almost all cells express MHC class I molecules, although the level of constitutive expression varies from one cell type to the next.
  • MHC class I molecules bind peptides from proteins degraded in the cytosol and transported in the endoplasmic reticulum. Thereby MHC class I molecules on the surface of cells infected with viruses or other cytosolic pathogens display peptides from these pathogen.
  • the CD8 + T cells that recognize MHC class hpeptide complexes are specialized to kill any cells displaying foreign peptides and so rid the body of cells infected with viruses and other cytosolic pathogens.
  • CD4 + T cells CD4 + helper T cells
  • MHC class II molecules are normally found on B lymphocytes, dendritic cells, and macrophages, cells that participate in immune responses, but not on other tissue cells. Macrophages, for example, are activated to kill the intravesicular pathogens they harbour, and B cells to secrete immunoglobulins against foreign molecules. MHC class II molecules are prevented from binding to peptides in the endoplasmic reticulum and thus MHC class II molecules bind peptides from proteins which are degraded in endosomes.
  • TH1 cells can capture peptides from pathogens that have entered the vesicular system of macrophages, or from antigens internalized by immature dendritic cells or the immunoglobulin receptors of B cells.
  • Pathogens that accumulate in large numbers inside macrophage and dendritic cell vesicles tend to stimulate the differentiation of TH1 cells, whereas extracellular antigens tend to stimulate the production of TH2 cells.
  • TH1 cells activate the microbicidal properties of macrophages and induce B cells to make IgG antibodies that are very effective of opsonising extracellular pathogens for ingestion by phagocytic cells
  • TH2 cells initiate the humoral response by activating naive B cells to secrete IgM, and induce the production of weakly opsonising antibodes such as IgGI and lgG3 (mouse) and lgG2 and lgG4 (human) as well as IgA and IgE (mouse and human).
  • antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention typically comprise any antigen, falling under the above definition, more preferably protein and peptide antigens, e.g. tumor antigens, allergy antigens, auto-immune self-antigens, pathogens, etc.
  • antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be antigens generated outside the cell, more typically antigens not derived from the host organism (e.g. a human) itself (i.e.
  • non-self antigens but rather derived from host cells outside the host organism, e.g. viral antigens, bacterial antigens, fungal antigens, protozoological antigens, animal antigens (preferably selected from animals or organisms as disclosed herein), allergy antigens, etc.
  • Allergy antigens are typically antigens, which cause an allergy in a human and may be derived from either a human or other sources.
  • Antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be furthermore antigens generated inside the cell, the tissue or the body, e.g. by secretion of proteins, their degradation, metabolism, etc.
  • antigens include antigens derived from the host organism (e.g. a human) itself, e.g. tumor antigens, self-antigens or auto-antigens, such as auto-immune self-antigens, etc., but also (non-self) antigens as defined above, which have been originally been derived from host cells outside the host organism, but which are fragmented or degraded inside the body, tissue or cell, e.g. by (protease) degradation, metabolism, etc.
  • host organism e.g. a human
  • tumor antigens e.g. tumor antigens, self-antigens or auto-antigens, such as auto-immune self-antigens, etc.
  • non-self antigens as defined above, which have been originally been derived from host cells outside the host organism, but which are fragmented or degraded inside the body, tissue or cell, e.g. by (protease) degradation, metabolism, etc.
  • Tumor antigens are preferably located on the surface of the (tumor) cell. Tumor antigens may also be selected from proteins, which are overexpressed in tumor cells compared to a normal cell. Furthermore, tumor antigens also includes antigens expressed in cells which are (were) not themselves (or originally not themselves) degenerate but are associated with the supposed tumor. Antigens which are connected with tumor-supplying vessels or (re)formation thereof, in particular those antigens which are associated with neovascularization, e.g.
  • Antigens connected with a tumor furthermore include antigens from cells or tissues, typically embedding the tumor. Further, some substances (usually proteins or peptides) are expressed in patients suffering (knowingly or not-knowingly) from a cancer disease and they occur in increased concentrations in the body fluids of said patients. These substances are also referred to as “tumor antigens", however they are not antigens in the stringent meaning of an immune response inducing substance.
  • the class of tumor antigens can be divided further into tumor-specific antigens (TSAs) and tumor-associated- antigens (TAAs). TSAs can only be presented by tumor cells and never by normal "healthy" cells.
  • TAAs which are more common, are usually presented by both tumor and healthy cells. These antigens are recognized and the antigen-presenting cell can be destroyed by cytotoxic T cells. Additionally, tumor antigens can also occur on the surface of the tumor in the form of, e.g., a mutated receptor. In this case, they can be recognized by antibodies.
  • tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention are shown in Tables 1 and 2 below. These tables illustrate specific (protein) antigens (i.e. "tumor antigens") with respect to the cancer disease, they are associated with. According to the invention, the terms “cancer diseases” and “tumor diseases” are used synonymously herein.
  • colorectal cancer gastric cancer
  • lung cancer head and neck cancer
  • leukemia esophageal cancer
  • gastric cancer cervical cancer
  • ovarian adenocarcinoma antigen cancer breast cancer
  • bladder cancer head and neck cancer, lung cancer, melanoma,
  • gastric cancer pancreatic cancer
  • liver cancer breast cancer
  • gallbladder cancer colon cancer
  • ovarian cancer colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, uterus cancer, cervix carcinoma, colon cancer,
  • gut carcinoma colorectal cancer, colon cancer, hepatocellular cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, liver cancer cervix cancer, bladder
  • bladder cancer lung cancer, T-cell cyp-B cyclophilin B leukemia, squamous cell carcinoma,
  • DAM-10/MAGE- differentiation antigen melanoma melanoma skin tumors, ovarian B1 10 cancer, lung cancer
  • DAM-6/MAGE- differentiation antigen melanoma melanoma skin tumors, ovarian B2 6 cancer, lung cancer
  • lung cancer ovarian cancer, head and neck cancer, colon cancer
  • EpCam epithelial cell adhesion molecule cancer EpCam epithelial cell adhesion molecule cancer, lung cancer
  • EZH2 (enhancer of Zeste homolog 2) prostate cancer, breast cancer
  • Fra-1 Fos-related antigen- 1 renal cell carcinoma thyroid cancer leukemia, renal cell carcinoma, head and neck cancer, colon cancer,
  • G250/CAIX glycoprotein 250 ovarian cancer, cervical cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • GAGE-6 G antigen 6 cancer bladder cancer, lung cancer,
  • bladder cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
  • breast cancer bladder cancer, human epidermal receptor- melanoma, ovarian cancer, pancreas
  • Her2/neu/ErbB2 2/neurological cancer gastric cancer
  • breast cancer melanoma
  • lung cancer ovarian cancer
  • sarcoma human telomerase reverse Non-Hodgkin-lymphoma
  • acute hTERT transcriptase leukemia acute hTERT transcriptase leukemia
  • tongue cancer hepatocellular carcinomas, melanoma, gastric cancer, esophageal, colon cancer, M-HN-1 pancreatic cancer
  • livin bladder cancer melanoma bladder cancer, head and neck cancer, melanoma, colon cancer,
  • bladder cancer bladder cancer, head and neck cancer, melanoma, colon cancer,
  • MAGE-A2 melanoma antigen-A2 lung cancer sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
  • MAGE-A4 melanoma antigen-A4 lung cancer sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
  • MAGE-A6 melanoma antigen-A6 lung cancer sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
  • melanoma lung cancer, leukemia, preferentially expressed antigen of head and neck cancer, renal cell
  • bladder cancer bladder cancer, melanoma, renal
  • bladder cancer bladder cancer, melanoma, sarcoma, brain tumor, esophagel cancer, renal
  • SART-1 1 cancer lung cancer, uterine cancer head and neck cancer, lung cancer, squamous antigen rejecting tumor renal cell carcinoma, melanoma,
  • gastric cancer colon cancer
  • lung cancer breast cancer
  • breast cancer ovarian
  • the tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention are selected from the group consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1 , alpha-5-beta-1 -integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha- methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1 , BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52
  • the tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention are selected from the group consisting of MAGE-A1 (e.g. MAGE-A1 according to accession number M77481 ), MAGE-A2, MAGE- A3, MAGE-A6 (e.g. MAGE-A6 according to accession number NM_005363), MAGE-C1 , MAGE-C2, melan-A (e.g. melan-A according to accession number NM_00551 1 ), GP100 (e.g. GP100 according to accession number M77348), tyrosinase (e.g.
  • tyrosinase according to accession number NM_000372
  • surviving e.g. survivin according to accession number AF077350
  • CEA e.g. CEA according to accession number NM_004363
  • Her-2/neu e.g. Her-2/neu according to accession number M11730
  • WT1 e.g. WT1 according to accession number NM_0003708
  • PRAME e.g. PRAME according to accession number NM_0061 15
  • EGFRI epidermal growth factor receptor 1
  • EGFRI epidermal growth factor receptor 1
  • MUC1 mucin-1
  • mucin-1 according to accession number NM_002456
  • SEC61 G e.g. SEC61 G according to accession number NM_014302
  • hTERT e.g. hTERT accession number NM_198253
  • 5T4 e.g. 5T4 according to accession number NM_006670
  • NY-Eso-1 e.g. NY-Eso1 according to accession number NM_001327)
  • TRP-2 e.g. TRP-2 according to accession number NM_001922
  • STEAP PCA
  • PSA PSMA
  • PSMA etc.
  • the tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may form a cocktail of antigens, e.g. in a vaccine, a pharmaceutical composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment of prostate cancer (PCa), preferably of neoadjuvant and/or hormone-refractory prostate cancers, and diseases or disorders related thereto.
  • a cocktail of antigens e.g. in a vaccine, a pharmaceutical composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment of prostate cancer (PCa), preferably of neoadjuvant and/or hormone-refractory prostate cancers, and diseases or disorders related thereto.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, which may encode at least two, three or four (preferably different) antigens of the following combinations of antigens: ⁇ PSA and PSMA, or
  • PSA PSMA and PSCA
  • PSA PSA
  • PSMA PSMA
  • STEAP STEAP
  • PSMA PSMA
  • PSCA PSCA
  • STEAP STEAP
  • PSA PSA
  • PSCA PSCA
  • STEAP STEAP
  • PSA PSMA
  • PSCA PSCA
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, which may encode at least two, three or four (preferably different) antigens:
  • At least one antigen is selected from:
  • PSA Prostate-Specific Antigen
  • PSMA Prostate-Specific Membrane Antigen
  • PSCA Prostate Stem Cell Antigen
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, encoding four (preferably different) antigens selected from PSA, PSMA, PSCA and STEAP.
  • the tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may form a cocktail of antigens, e.g. in a vaccine, a pharmaceutical composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment 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.
  • NSCLC non- small cell lung cancers
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention is preferably at least one RNA, more preferably at least one mRNA, which may encode at least one, preferably two, three, four, five, six, seven, eight, nine, ten eleven or twelve (preferably different) antigens of the following group of antigens:
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, which may encode at least two, three, five or six (preferably different) antigens of the following combinations of antigens:
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, which may encode at least one, preferably two, three, four, five, six, seven, eight, nine, ten eleven or twelve (preferably different) antigens of the following combinations of antigens:
  • VVT1 and MAGE-C2 are VVT1 and MAGE-C2, or
  • VVT1 VVT1 , 5T4 and MAGE-C2, or
  • VVT1 VVT1 , NY-ESO-1 and Survivin, or
  • hTERT, WT1 , 5T4 and NY-ESO-1 or hTERT, WT1 , 5T4 and Survivin, or hTERT, VVT1 , 5T4 and MAGE-C2, or hTERT, 5T4, NY-ESO-1 and Survivin, or hTERT, 5T4, NY-ESO-1 and MAGE-C2, or hTERT, NY-ESO-1 , Survivin and MAGE-C2, or WT1 , 5T4, NY-ESO-1 , and Survivin, or WT1 , 5T4, NY-ESO-1 , and MAGE-C2, or WT1 , 5T4, Survivin, and MAGE-C2, or 5T4, NY-ESO-1 , Survivin, and MAGE-C2, or 5T4, NY-ESO-1 , Survivin, and MAGE-C2, or 5T4, NY-ESO-1 , Survivin, and MAGE-C2, or 5T4, NY-ESO-1
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, which may encode at least two (preferably different) antigens exclusively selected from any of the antigens of the above mentioned group(s) or subgroup(s) comprising (at least) any one of the following combinations of antigens:
  • the tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may form a cocktail of antigens, e.g. in in a vaccine, a pharmaceutical composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment 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.
  • NSCLC non- small cell lung cancers
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention is preferably at least one RNA, more preferably at least one mRNA, which may encode at least two (preferably different) antigens,
  • At least one, preferably at least two, three, four, five or even six, of these at least two antigens is (are) selected from:
  • the further antigen(s) is (are) selected from at least one antigen as defined herein, preferably in any of the herein mentioned combinations, groups or subgroups of antigens, e.g. the further antigen(s) is (are) selected from, e.g.:
  • the at least one antigen(s) according to a) is (are) selected from:
  • the at least one antigen(s) according to a) is (are) selected from:
  • the at least one antigen(s) according to b) is (are) selected from an antigen (antigens) as defined in one of the following combinations:
  • WT1 and MAGE- A3 WT1 and MAGE- A3; or WT1 and MUC1 ; or
  • WT1 and Her-2/neu WT1 and NY-ESO-1 ; or WT1 and CEA; or
  • WT1 and MAGE-C1 WT1 and MAGE-C1 ; or WT1 and MAGE-C2; or MAGE-A2 and 5T4; or MAGE-A2 and MAGE-A3; or MAGE-A2 and MUC1 ; or MAGE-A2 and Her-2/neu; or MAGE-A2 and NY-ESO-1 ; or MAGE-A2 and CEA; or MAGE-A2 and Survivin; or MAGE-A2 and MAGE-C1 ; or MAGE-A2 and MAGE-C2; or 5T4 and MAGE-A3; or 5T4 and MUC1 ; or
  • 5T4 and Her-2/neu or 5T4 and NY-ESO-1 ; or 5T4 and CEA; or
  • MUC1 and Survivin or MUC1 and MAGE-C1 ; or MUC1 and MAGE-C2; or HER-2/NEU and NY-ESO-1 ; or HER-2/NEU and CEA; or HER-2/NEU and Survivin; or HER-2/NEU and MAGE-C1 ; or HER-2/NEU and MAGE-C2; or NY-ESO-1 and CEA; or NY-ESO-1 and Survivin; or NY-ESO-1 and MAGE-C1 ; or NY-ESO-1 and MAGE-C2; or CEA and Survivin; or
  • hTERT WT1 and MAGE-A2; or hTERT, WT1 and 5T4; or
  • the at least one antigen(s) according to b) is (are) selected from the following combination:
  • each of the at least two (preferably different) antigens as defined herein may be encoded by one (monocistronic) RNA, preferably one (monocistronic) mRNA.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may comprise at least two (monocistronic) RNAs, preferably mRNAs, wherein each of these at least two (monocistronic) RNAs, preferably mRNAs, may encode just one (preferably different) antigen, preferably selected from one of the above mentioned combinations.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may comprise (at least) one bi- or even multicistronic RNA, preferably mRNA, i.e. (at least) one RNA which carries two or even more of the coding sequences of at the least two (preferably different) antigens, preferably selected from one of the above mentioned combinations.
  • Such coding sequences of the at least two (preferably different) antigens of the (at least) one bi- or even multicistronic RNA may be separated by at least one IRES (internal ribosomal entry site) sequence, as defined below.
  • the term "encoding at least two (preferably different) antigens” may mean, without being limited thereto, that the (at least) one (bi- or even multicistronic) RNA, preferably a mRNA, may encode e.g. at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve (preferably different) antigens of the above mentioned group(s) of antigens or their fragments or variants. More preferably, without being limited thereto, the (at least) one (bi- or even multicistronic) RNA, preferably mRNA, may encode e.g.
  • IRES internal ribosomal entry site
  • IRES sequences can function as a sole ribosome binding site, but it can also serve to provide a bi- or even multicistronic RNA as defined above which codes for several proteins, which are to be translated by the ribosomes independently of one another.
  • IRES sequences which can be used according to the invention are those from picornaviruses (e.g.
  • FMDV pestiviruses
  • CFFV pestiviruses
  • PV polioviruses
  • ECMV encephalomyocarditis viruses
  • FMDV foot and mouth disease viruses
  • HCV hepatitis C viruses
  • CSFV classical swine fever viruses
  • MLV mouse leukoma virus
  • SIV simian immunodeficiency viruses
  • CrPV cricket paralysis viruses
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may comprise a mixture of at least one monocistronic RNA, preferably mRNA, as defined above, and at least one bi- or even multicistronic RNA, preferably mRNA, as defined above.
  • the at least one monocistronic RNA and/or the at least one bi- or even multicistronic RNA preferably encode different antigens or their fragments or variants, the antigens preferably being selected from one of the above mentioned groups or subgroups of antigens, more preferably in one of the above mentioned combinations.
  • the at least one monocistronic RNA and the at least one bi- or even multicistronic RNA may preferably also encode (in part) identical antigens selected from one of the above mentioned groups or subgroups of antigens, preferably in one of the above mentioned combinations, provided that the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention as a whole provides at least two (preferably different) antigens as defined above.
  • Such an aspect may be advantageous e.g. for a staggered, e.g. time dependent, administration of e.g. a vaccine, a pharmaceutical composition or a kit of the present invention to a patient in need thereof.
  • RNAs encoding the at least two (preferably different) antigens may be e.g. contained in (different parts of) a kit of parts composition or may be e.g. administered separately as components of different embodiments or aspects according to the present invention.
  • one further class of antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention comprises allergy antigens.
  • allergy antigens may be selected from antigens derived from different sources, e.g. from animals, plants, fungi, bacteria, etc. Allergens in this context include e.g. grasses, pollens, molds, drugs, or numerous environmental triggers, etc. Allergy antigens typically belong to different classes of compounds, such as nucleic acids and their fragments, proteins or peptides and their fragments, carbohydrates, polysaccharides, sugars, lipids, phospholipids, etc.
  • antigens which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, i.e. protein or peptide antigens and their fragments or epitopes, or nucleic acids and their fragments, particularly nucleic acids and their fragments, encoding such protein or peptide antigens and their fragments or epitopes.
  • antigens derived from animals which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may include antigens derived from, without being limited thereto, insects, such as mite (e.g. house dust mites), mosquito, bee (e.g. honey bee, bumble bee), cockroache, tick, moth (e.g.
  • Antigens derived from plants which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may include antigens derived from, without being limited thereto, fruits, such as kiwi, pineapple, jackfruit,papaya, lemon, orange, mandarin, melon, sharon fruit, strawberry, lychee, apple, cherry compassion apple, mango, passion fruit, plum, apricot, nectarine, pear, passion fruit, raspberry, grape, from vegetables, such as garlic, onion, leek, soya bean, celery, cauliflower, turnip, paprika, chickpea, fennel, zucchini, cucumber, carrot, yam, bean, pea, olive, tomato, potato, lentil, lettuce, avocado, parsley, horseradish, chirimoya, beet, pumkin, spinach, from spices, such as mustard, coriander, saffron, pepper, aniseed, from crop
  • Antigens derived from fungi which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may include antigens derived from, without being limited thereto, e.g.
  • Alternia sp. Aspergillus sp., Beauveria sp., Candida sp., Cladosporium sp., Endothia sp., Curcularia sp., Embellisia sp., Epicoccum sp., Fusarium sp., Malassezia sp., Penicillum sp., Pleospora sp., Saccharomyces sp., etc.
  • Antigens derived from bacteria which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may include antigens derived from, without being limited thereto, e.g. Bacillus tetani, Staphylococcus aureus, Streptomyces griseus, etc. Antibodies
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may encode an antibody.
  • an antibody may be selected from any antibody, e.g. any recombinantly produced or naturally occurring antibodies, known in the art, in particular antibodies suitable for therapeutic, diagnostic or scientific purposes, or antibodies which have been identified in relation to specific cancer diseases.
  • the term “antibody” is used in its broadest sense and specifically covers monoclonal and polyclonal antibodies (including agonist, antagonist, and blocking or neutralizing antibodies) and antibody species with polyepitopic specificity.
  • “antibody” typically comprises any antibody known in the art (e.g.
  • IgM, IgD, IgG, IgA and IgE antibodies such as naturally occurring antibodies, antibodies generated by immunization in a host organism, antibodies which were isolated and identified from naturally occurring antibodies or antibodies generated by immunization in a host organism and recombinantly produced by biomolecular methods known in the art, as well as chimeric antibodies, human antibodies, humanized antibodies, bispecific antibodies, intrabodies, i.e. antibodies expressed in cells and optionally localized in specific cell compartments, and fragments and variants of the aforementioned antibodies.
  • an antibody consists of a light chain and a heavy chain both having variable and constant domains.
  • the light chain consists of an N-terminal variable domain, V L , and a C-terminal constant domain, Q.
  • the heavy chain of the IgG antibody for example, is comprised of an N-terminal variable domain, V H , and three constant domains, C H 1 , C H 2 und C H 3.
  • Single chain antibodies may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention as well.
  • the nucleic acid (sequence), lyophilized or to be lyophilized,according to the present invention may encode a polyclonal antibody.
  • polyclonal antibody typically means mixtures of antibodies directed to specific antigens or immunogens or epitopes of a protein which were generated by immunization of a host organism, such as a mammal, e.g. including goat, cattle, swine, dog, cat, donkey, monkey, ape, a rodent such as a mouse, hamster and rabbit.
  • Polyclonal antibodies are generally not identical, and thus usually recognize different epitopes or regions from the same antigen.
  • each (lyophilized) nucleic acid (sequence) encoding a specific (monoclonal) antibody being directed to specific antigens or immunogens or epitopes of a protein.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may encode a monoclonal antibody.
  • monoclonal antibody typically refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed to a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen.
  • monoclonal antibodies as defined above may be made by the hybridoma method first described by Kohler and Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods, e.g. as described in U.S. Pat. No. 4,81 6,567.
  • “Monoclonal antibodies” may also be isolated from phage libraries generated using the techniques described in McCafferty et a/., Nature, 348:552-554 (1 990), for example.
  • an immunogen (antigen) of interest is injected into a host such as a mouse and B-cell lymphocytes produced in response to the immunogen are harvested after a period of time.
  • the B-cells are combined with myeloma cells obtained from mouse and introduced into a medium which permits the B-cells to fuse with the myeloma cells, producing hybridomas. These fused cells (hybridomas) are then placed into separate wells of microtiter plates and grown to produce monoclonal antibodies. The monoclonal antibodies are tested to determine which of them are suitable for detecting the antigen of interest. After being selected, the monoclonal antibodies can be grown in cell cultures or by injecting the hybridomas into mice.
  • the peptide sequences of these monoclonal antibodies have to be sequenced and the nucleic acid sequences encoding these antibodies can be present as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention.
  • non-human monoclonal or polyclonal antibodies such as murine antibodies may also be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention.
  • such antibodies are typically only of limited use, since they generally induce an immune response by production of human antibodies directed to the said non-human antibodies, in the human body. Therefore, a particular non-human antibody can only be administered once to the human.
  • chimeric, humanized non- human and human antibodies are also envisaged encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention.
  • "Chimeric" antibodies which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophi lized, according to the present invention, are preferably antibodies in which the constant domains of an antibody described above are replaced by sequences of antibodies from other organisms, preferably human sequences.
  • nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention are antibodies in which the constant and variable domains (except for the hypervariable domains) described above of an antibody are replaced by human sequences.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may encode human antibodies, i.e. antibodies having only human sequences.
  • human antibodies can be isolated from human tissues or from immunized non-human host organisms which are transgene for the human IgG gene locus, and nucleic acid sequences may be prepared according to procedures well known in the art. Additionally, human antibodies can be provided by the use of a phage display.
  • nucleic acid may encode bispecific antibodies.
  • Bispecific antibodies in context of the invention are preferably antibodies which act as an adaptor between an effector and a respective target by two different F ⁇ -domains, e.g. for the purposes of recruiting effector molecules such as toxins, drugs, cytokines etc., targeting effector cells such as CTL, NK cells, makrophages, granulocytes, etc. (see for review: Kontermann R.E., Acta Pharmacol. Sin, 2005, 26(1 ): 1 -9).
  • Bispecific antibodies as described herein are, in general, configured to recognize by two different F ⁇ -domains, e.g.
  • bispecificity means herewith that the antigen-binding regions of the antibodies are specific for two different epitopes.
  • different antigens, immunogens or epitopes, etc. can be brought close together, what, optionally, allows a direct interaction of the two components.
  • different cells such as effector cells and target cells can be connected via a bispecific antibody.
  • antibodies or fragments thereof which bind, on the one hand, a soluble antigen as described herein, and, on the other hand, an antigen or receptor on the surface of a tumor cell.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also encode intrabodies, wherein these intrabodies may be antibodies as defined above. Since these antibodies are intracellular expressed antibodies, i.e. antibodies which may be encoded by nucleic acids localized in specific areas of the cell and also expressed there, such antibodies may be termed intrabodies.
  • Antibodies as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may preferably comprise full-length antibodies, i.e. antibodies composed of the full heavy and full light chains, as described above. However, derivatives of antibodies such as antibody fragments, variants or adducts may also be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also encode antibody fragments selected from Fab, Fab', F(ab') 2 , Fc, Facb, pFc', Fd and Fv fragments of the aforementioned (full-length) antibodies.
  • antibody fragments are known in the art.
  • a Fab fragment, antigen binding
  • fragment is composed of one constant and one variable domain of each of the heavy and the light chain. The two variable domains bind the epitope on specific antigens.
  • the two chains are connected via a disulfide linkage.
  • a scFv (“single chain variable fragment”) fragment typically consists of the variable domains of the light and heavy chains.
  • the domains are linked by an artificial linkage, in general a polypeptide linkage such as a peptide composed of 15-25 glycine, proline and/or serine residues.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be in the form of dsRNA, preferably siRNA.
  • a dsRNA, or a siRNA is of interest particularly in connection with the phenomenon of RNA interference.
  • RNAi RNA interference
  • the in vitro technique of RNA interference (RNAi) is based on double- stranded RNA molecules (dsRNA), which trigger the sequence-specific suppression of gene expression (Zamore (2001 ) Nat. Struct. Biol. 9: 746-750; Sharp (2001 ) Genes Dev. 5:485- 490: Hannon (2002) Nature 41 : 244-251 ).
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may thus be a double-stranded RNA (dsRNA) having a length of from 17 to 29, preferably from 19 to 25, and preferably being at least 90%, more preferably 95% and especially 100% (of the nucleotides of a dsRNA) complementary to a section of the nucleic acid sequence of a (therapeutically relevant) protein or antigen described (as active ingredient) hereinbefore, either a coding or a non-coding section, preferably a coding section.
  • dsRNA double-stranded RNA
  • nucleic acid sequence
  • lyophilized or to be lyophilized may be a dsRNA having the general structure 5'-(N 17 .
  • each N is a (preferably different) nucleotide of a section of the mRNA of a therapeutically relevant protein or antigen described hereinbefore, preferably being selected from a continuous number of 17 to 29 nucleotides of the mRNA of a therapeutically relevant protein or antigen and being present in the general structure 5'-(N 1 7 .2 9 )-3' in their natural order.
  • dsRNAs used as nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention can also be directed against nucleotide sequences of a (therapeutically relevant) protein or antigen described (as active ingredient) hereinbefore that do not lie in the coding region, in particular in the 5' non-coding region of the mRNA, for example, therefore, against non-coding regions of the mRNA having a regulatory function.
  • the target sequence of the dsRNA used as nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention can therefore lie in the translated and untranslated region of the mRNA and/or in the region of the control elements of a protein or antigen described hereinbefore.
  • the target sequence of a dsRNA used as nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention can also lie in the overlapping region of untranslated and translated sequence; in particular, the target sequence can comprise at least one nucleotide upstream of the start triplet of the coding region of the mRNA.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be in the form of a CpG nucleic acid, in particular CpG-RNA or CpG-DNA.
  • a CpG-RNA or CpG-DNA used according to the invention can be a single-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA).
  • the CpG nucleic acid used according to the invention is preferably in the form of CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). Also preferably, such CpG nucleic acids have a length as described above. Preferably, the CpG motifs are unmethylated.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be in the form of an immunostimulatory RNA.
  • the immunostimulatory RNA of the complexed RNA of the present invention may be any (double-stranded or single-stranded) RNA, e.g. a coding RNA, as defined above.
  • the immunostimulatory RNA may be a single-stranded, a double-stranded or a partially double-stranded RNA, more preferably a single-stranded RNA, and or a circular or linear RNA, more preferably a linear RNA.
  • the immunostimulatory RNA may be a (linear) single-stranded RNA. Even more preferably, the immunostimulatory RNA may be a ((linear) single-stranded) messenger RNA (mRNA). An immunostimulatory RNA may also occur as a short RNA oligonucleotide as defined above. An immunostimulatory RNA as used herein may furthermore be selected from any class of RNA molecules, found in nature or being prepared synthetically, and which can induce an immune response. In this context, an immune response may occur in various ways. A substantial factor for a suitable immune response is the stimulation of different T-cell sub-populations.
  • T- lymphocytes are typically divided into two sub-populations, the T-helper 1 (Th1 ) cells and the T-helper 2 (Th2) cells, with which the immune system is capable of destroying intracellular (Th1 ) and extracellular (Th2) pathogens (e.g. antigens).
  • the two Th cell populations differ in the pattern of the effector proteins (cytokines) produced by them.
  • Th1 cells assist the cellular immune response by activation of macrophages and cytotoxic T- cells.
  • Th2 cells promote the humoral immune response by stimulation of the B-cells for conversion into plasma cells and by formation of antibodies (e.g. against antigens).
  • the Th1/Th2 ratio is therefore of great importance in the immune response.
  • the Th1/Th2 ratio of the immune response is preferably shifted in the direction towards the cellular response (Thl response) and a cellular immune response is thereby induced.
  • the immune system may be activated by ligands of Toll-like receptors (TLRs).
  • TLRs are a family of highly conserved pattern recognition receptor (PRR) polypeptides that recognize pathogen- associated molecular patterns (PAMPs) and play a critical role in innate immunity in mammals.
  • PRR pattern recognition receptor
  • TLR1 - TLR13 Toll-like receptors: TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13
  • TLR1 - TLR13 Toll-like receptors: TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13
  • CpG DNA unmethylated bacterial DNA and synthetic analogs thereof
  • ligands for certain TLRs include certain nucleic acid molecules and that certain types of RNA are immunostimulatory in a sequence-independent or sequence-dependent manner, wherein these various immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, or intracellular receptors such as RIG-I, MDA-5, etc.
  • these various immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, or intracellular receptors such as RIG-I, MDA-5, etc.
  • Lipford eta/ determined certain G,U- containing oligoribonucleotides as immunostimulatory by acting via TLR7 and TLR8 (see WO 03/086280).
  • the immunostimulatory G,U-containing oligoribonucleotides described by Lipford et a/ were believed to be derivable from RNA sources including ribosomal RNA, transfer RNA, messenger RNA, and viral RNA.
  • RNA molecule
  • immunostimulatory properties i.e. enhance the immune response.
  • RNA as defined above and being the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may thus be used to enhance (unspecific) immunostimulation, if suitable and desired for a specific treatment.
  • the at least one (immunostimulatory) RNA (molecule) used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may thus comprise any RNA sequence known to be immunostimulatory, including, without being limited thereto, RNA sequences representing and/or encoding ligands of TLRs, preferably selected from family members TLR1 - TLR13, more preferably from TLR7 and TLR8, ligands for intracellular receptors for RNA (such as RIG-I or MAD-5, etc.) (see e.g. Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA helicases: two parallel ways to trigger antiviral responses. Mol.
  • immunostimulatory RNA molecules used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may include any other RNA capable of eliciting an immune response.
  • immunostimulatory RNA may include ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA).
  • Such further (classes of) immunostimulatory RNA molecules which may be used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, without being limited thereto, may comprise e.g. an RNA molecule of formula (I):
  • G is guanosine, uracil or an analogue of guanosine or uracil;
  • X is guanosine, uracil, adenosine, thymidine, cytosine or an analogue of the above- mentioned nucleotides
  • I is an integer from 1 to 40
  • n is an integer and is at least 3;
  • n is an integer from 1 to 40
  • RNA molecules which may be used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may comprise, without being limited thereto, e.g. an RNA molecule of formula (II):
  • C is cytosine, uracil or an analogue of cytosine or uracil;
  • X is guanosine, uracil, adenosine, thymidine, cytosine or an analogue of the above- mentioned nucleotides
  • I is an integer from 1 to 40
  • n is an integer and is at least 3;
  • n is an integer from 1 to 40
  • n > 1 at least 50% of the nucleotides are cytosine or an analogue thereof.
  • the immunostimulatory RNA molecules used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention comprise a length as defined above in general for RNA molecules of the RNA of the present invention, more preferably a length of 5 to 5000, of 500 to 5000 or, more preferably, of 1000 to 5000 or, alternatively, of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or, more preferably, of 5 to 30 nucleotides.
  • the immunostimulatory RNA used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be furthermore modified, preferably "chemically modified” in order to enhance the immunostimulatory properties of said DNA.
  • chemical modification means that the immuostimulatory RNA is modified by replacement, insertion or removal of individual or several atoms or atomic groups compared with naturally occurring RNA species.
  • the chemical modification of the immunostimulatory RNA comprises at least one analogue of naturally occurring nucleotides.
  • examples which may be mentioned for nucleotide analogues and which may be used herein for modification are analogues of guanosine, uracil, adenosine, thymidine, cytosine.
  • the modifications may refer to modifications of the base, the ribose moiety and/or the phosphate backbone moiety.
  • analogues of guanosine, uracil, adenosine, and cytosine include, without implying any limitation, any naturally occurring or non-naturally occurring guanosine, uracil, adenosine, thymidine or cytosine that has been altered chemically, for example by acetylation, methylation, hydroxylation, etc., including 1- methyl-adenosine, 1 -methyl-guanosine, 1 -methyl-inosine, 2,2-dimethyl-guanosine, 2,6- diaminopurine, 2'-Amino-2'-deoxyadenosine, 2'-Amino-2'-deoxycytidine, 2'-Amino-2'- deoxyguanosine, 2'-Amino-2'-deoxyuridine, 2-Amino-6-chloropurineriboside, 2- Aminopurine-riboside, 2'-Araadenos
  • analogue as described above, particular preference is given according to the invention to those analogues that increase the immunogenicity of the immunostimulatory RNA sequence used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention and/or do not interfere with a further modification that has been introduced into said immunostimulatory RNA.
  • nucleic acid sequence
  • lyophilized or to be lyophilized may also occur in the form of a modified nucleic acid, wherein any modification, as defined herein, may be introduced into the nucleic acid prior to lyophilization. Modifications as defined herein preferably lead to a further stabilized nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention as defined above may thus be provided as a "stabilized nucleic acid", preferably as a stabilized RNA, more preferably as an RNA that is essentially resistant to in vivo degradation (e.g. by an exo- or endo-nuclease).
  • a stabilized nucleic acid preferably as a stabilized RNA, more preferably as an RNA that is essentially resistant to in vivo degradation (e.g. by an exo- or endo-nuclease).
  • Such stabilization can be effected, for example, by a modified phosphate backbone of the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention.
  • a backbone modification in connection with the present invention is a modification in which phosphates of the backbone of the nucleotides contained in the nucleic acid (sequence), lyophilized or to be lyophilized, are chemically modified.
  • Nucleotides that may be preferably used in this connection contain e.g. a phosphorothioate-modified phosphate backbone, preferably at least one of the phosphate oxygens contained in the phosphate backbone being replaced by a sulfur atom.
  • Stabilized lyophilized nucleic acids may further include, for example: non-ionic phosphate analogues, such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiesters and alkylphosphotriesters, in which the charged oxygen residue is present in alkylated form.
  • non-ionic phosphate analogues such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group
  • phosphodiesters and alkylphosphotriesters in which the charged oxygen residue is present in alkylated form.
  • backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5'-0-(1 -thiophosphate)).
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may additionally or alternatively also contain sugar modifications.
  • a sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the nucleic acid (sequence), lyophilized or to be lyophilized, and typically includes, without implying any limitation, sugar modifications selected from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2 '-fluoro-2'-deoxycytidine-5'- triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate), 2'-deoxy-2'-deamine oligoribonucleotide (2'-amino-2 '-deoxycytidine-5'-triphosphate, 2 '-amino-2 '-deoxyuridine- 5 '-triphosphate), 2 '-0-alkyl oligoribonucleotide,
  • Significant in this case means an increase in the expression of the protein compared with the expression of the native nucleic acid sequence by at least 20%, preferably at least 30%, 40%, 50% or 60%, more preferably by at least 70%, 80%, 90% or even 100% and most preferably by at least 1 50%, 200% or even 300% or more.
  • a nucleotide having such a base modification is preferably selected from the group of the base-modified nucleotides consisting of 2-amino- 6-chloropurineriboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2-thiocytidi Res' -triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-tr ' iphosphate, 5- aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine- 5 '-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5- iodouridine-5 '-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'- triphosphate, 6-azacytidine-5 '-triphosphate, 6-azauridine-5'-triphosphate, 6-aza
  • nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5- methylcytidine-5 '-triphosphate, 7-deazaguanosine-5 '-triphosphate, 5-bromocytidine-5'- triphosphate, and pseudouridine-5'-triphosphate.
  • nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention can likewise be modified (and preferably stabilized) by introducing further modified nucleotides containing modifications of their ribose or base moieties.
  • nucleotide analogues are defined as non-natively occurring variants of naturally occurring nucleotides.
  • analogues are chemically derivatized nucleotides with non-natively occurring functional groups, which are preferably added to or deleted from the naturally occurring nucleotide or which substitute the naturally occurring functional groups of a nucleotide. Accordingly, each component of the naturally occurring nucleotide may be modified, namely the base component, the sugar (ribose) component and/or the phosphate component forming the backbone (see above) of the nucleic acid sequence.
  • Exemplary analogues of guanosine, uracil, adenosine, and cytosine include, without implying any limitation, any naturally occurring or non-naturally occurring guanosine, uracil, adenosine, thymidine or cytosine that has been altered chemically, for example by acetylation, methylation, hydroxylation, etc., including 1 - methyl-adenosine, 1 -methyl-guanosine, 1 -methyl-inosine, 2,2-dimethyl-guanosine, 2,6- diaminopurine, 2'-Amino-2'-deoxyadenosine, 2'-Amino-2'-deoxycytidine, 2'-Amino-2'- deoxyguanosine, 2 , -Amino-2'-deoxyuridine, 2-Amino-6-chloropurineriboside, 2-Aminopurine- riboside, 2'-Ara
  • analogue as described above, particular preference may be given according to the invention to those analogues that do not interfere with a further modification of the nucleic acid (sequence), lyophilized or to be lyophilized, that has been introduced.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention can contain a lipid modification.
  • a lipid-modified nucleic acid (sequence), lyophilized or to be lyophilized typically comprises a nucleic acid as defined herein.
  • Such a lipid-modified nucleic acid (sequence), lyophilized or to be lyophilized typically further comprises at least one linker covalently linked with that nucleic acid, and at least one lipid covalently linked with the respective linker.
  • the lipid-modified nucleic acid (sequence), lyophilized or to be lyophilized comprises an at least one nucleic acid as defined herein and at least one (Afunctional) lipid covalently linked (without a linker) with that nucleic acid.
  • the lipid-modified nucleic acid (sequence), lyophilized or to be lyophilized comprises a nucleic acid RNA as defined herein, at least one linker covalently linked with that nucleic acid, and at least one lipid covalently linked with the respective linker, and also at least one (bifunctional) lipid covalently linked (without a linker) with that nucleic acid.
  • the lipid contained in the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention (complexed or covalently bound thereto) is typically a lipid or a lipophilic residue that preferably is itself biologically active.
  • Such lipids preferably include natural substances or compounds such as, for example, vitamins, e.g. alpha-tocopherol (vitamin E), including RRR-alpha-tocopherol (formerly D-alpha-tocopherol), L-alpha-tocopherol, the racemate D,L-alpha-tocopherol, vitamin E succinate (VES), or vitamin A and its derivatives, e.g.
  • retinoic acid retinol
  • vitamin D and its derivatives e.g. vitamin D and also the ergosterol precursors thereof
  • vitamin E and its derivatives vitamin K and its derivatives, e.g. vitamin K and related quinone or phytol compounds, or steroids, such as bile acids, for example cholic acid, deoxycholic acid, dehydrocholic acid, cortisone, digoxygenin, testosterone, cholesterol or thiocholesterol.
  • bile acids for example cholic acid, deoxycholic acid, dehydrocholic acid, cortisone, digoxygenin, testosterone, cholesterol or thiocholesterol.
  • Further lipids or lipophilic residues within the scope of the present invention include, without implying any limitation, polyalkylene glycols (Oberhauser et al., Nucl.
  • Acids Res. 1 992, 20, 533), aliphatic groups such as, for example, Cl -C20-alkanes, C1 -C20-alkenes or C1 -C20-alkanol compounds, etc., such as, for example, dodecanediol, hexadecanol or undecyl residues (Saison-Behmoaras et a/., EMBO J, 1 991 , 10, 1 1 1 1 ; Kabanov et a/., FEBS Lett., 1 990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), phospholipids such as, for example, phosphatidylglycerol, diacylphosphatidylglycerol, phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylserine,
  • polyamines or polyalkylene glycols such as, for example, polyethylene glycol (PEG) (Manoharan et al, Nucleosides & Nucleotides, 1995, 14, 969), hexaethylene glycol (HEG), palmitin or palmityl residues (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), octadecylamines or hexylamino- carbonyl-oxycholesterol residues (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277, 923), and also waxes, terpenes, alicyclic hydrocarbons, saturated and mono- or poly-unsaturated fatty acid residues, etc.
  • PEG polyethylene glycol
  • HEG hexaethylene glycol
  • HOG hexaethylene glycol
  • palmitin or palmityl residues Mishra et al.,
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may likewise be stabilized in order to prevent degradation of the nucleic acid by various approaches, particularly, when RNA or mRNA is used as a lyophilized nucleic acid.
  • RNAases ribonucleases
  • the natural degradation of mRNA in the cytoplasm of cells is very finely regulated and RNase contaminations may be generally removed by special treatment prior to use of said compositions, in particular with diethyl pyrocarbonate (DEPC).
  • DEPC diethyl pyrocarbonate
  • a number of mechanisms of natural degradation are known in this connection in the prior art, which may be utilized as well.
  • the terminal structure is typically of critical importance for a mRNA.
  • cap structure a modified guanosine nucleotide
  • the so-called poly-A tail is typically a sequence of up to 200 adenosine nucleotides
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, particularly if provided as a mRNA, can therefore be stabilized against degradation by RNases by the addition of a so-called "5' cap” structure.
  • a so-called "5' cap” structure Particular preference is given in this connection to an m7G(5')ppp (5'(A,G(5')ppp(5')A or G(5')ppp(5')G as the 5' cap" structure.
  • a modification is introduced only if a modification, for example a lipid modification, has not already been introduced at the 5' end of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention if provided as a mRNA or if the modification does not interfere with the immunogenic properties of the (unmodified or chemically modified) nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention.
  • a modification for example a lipid modification
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may contain, especially if the nucleic acid is in the form of a mRNA, a poly-A tail on the 3' terminus of typically about 10 to 200 adenosine nucleotides, preferably about 10 to 100 adenosine nucleotides, more preferably about 20 to 100 adenosine nucleotides or even more preferably about 40 to 80 adenosine nucleotides.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may contain, especially if the nucleic acid is in the form of a mRNA, a poly-C tail on the 3' terminus of typically about 10 to 200 cytosine nucleotides, preferably about 10 to 100 cytosine nucleotides, more preferably about 20 to 70 cytosine nucleotides or even more preferably about 20 to 60 or even 10 to 40 cytosine nucleotides.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may be modified, and thus stabilized, especially if the nucleic acid is in the form of a mRNA, by modifying the G/C content of the nucleic acid, particularly an mRNA, preferably of the coding region thereof.
  • the G/C content of the coding region of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention especially if the nucleic acid is in the form of a mRNA, is modified, particularly increased, compared to the G/C content of the coding region of its particular wild type mRNA, i.e. the unmodified mRNA.
  • the encoded amino acid sequence of the at least one mRNA is preferably not modified compared to the coded amino acid sequence of the particular wild type mRNA.
  • nucleic acid sequence
  • lyophilized or to be lyophilized of the present invention, especially if the nucleic acid is in the form of a mRNA, is based on the fact that the sequence of any mRNA region to be translated is important for efficient translation of that mRNA.
  • composition and the sequence of various nucleotides is important.
  • sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content.
  • the codons of the mRNA are therefore varied compared to its wild type mRNA, while retaining the translated amino acid sequence, such that they include an increased amount of G/C nucleotides.
  • the most favorable codons for the stability can be determined (so-called alternative codon usage).
  • nucleic acid lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, there are various possibilities for modification of the at least one mRNA sequence, compared to its wild type sequence.
  • amino acids which are encoded by codons which contain exclusively G or C nucleotides no modification of the codon is necessary.
  • the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) require no modification, since no A or U is present.
  • codons which contain A and/or U nucleotides can be modified by substitution of other codons which code for the same amino acids but contain no A and/or U. Examples of these are: the codons for Pro can be modified from CCU or CCA to CCC or CCG;
  • the codons for Arg can be modified from CGU or CGA or AGA or AGG to CGC or CGG; the codons for Ala can be modified from GCU or GCA to GCC or GCG;
  • the codons for Gly can be modified from GGU or GGA to GGC or GGG.
  • a or U nucleotides cannot be eliminated from the codons, it is however possible to decrease the A and U content by using codons which contain a lower content of A and/or U nucleotides. Examples of these are: the codons for Phe can be modified from UUU to UUC;
  • the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUC or CUG;
  • the codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyr can be modified from UAU to UAC;
  • the codon for Cys can be modified from UGU to UGC; the codon for His can be modified from CAU to CAC;
  • the codon for Gin can be modified from CAA to CAG;
  • the codons for lie can be modified from AUU or AUA to AUC;
  • codons for Thr can be modified from ACU or ACA to ACC or ACG;
  • the codon for Asn can be modified from AAU to AAC;
  • the codon for Lys can be modified from AAA to AAG;
  • the codons for Val can be modified from GUU or GUA to GUC or GUG;
  • the codon for Asp can be modified from GAU to GAC;
  • the codon for Glu can be modified from GAA to GAG;
  • the stop codon UAA can be modified to UAG or UGA.
  • the G/C content of the coding region of nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is increased by at least 7%, more preferably by at least 15%, particularly preferably by at least 20%, compared to the G/C content of the coded region of the wild type mRNA.
  • nucleic acid sequence
  • lyophilized or to be lyophilized of the present invention, especially if the nucleic acid is in the form of a mRNA, to the maximum (i.e. 100% of the substitutable codons), in particular in the region coding for a protein, compared to the wild type sequence.
  • nucleic acid sequence
  • lyophilized or to be lyophilized of the present invention, especially if the nucleic acid is in the form of a mRNA, is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells.
  • nucleic acid sequence
  • lyophilized or to be lyophilized of the present invention, especially if the nucleic acid is in the form of a mRNA, to an increased extent, the corresponding modified nucleic acid sequence is translated to a significantly poorer degree than in the case where codons coding for relatively "frequent" tRNAs are present.
  • the modified nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention is in the form of a mRNA
  • the coding region of the modified nucleic acid (sequence), lyophilized or to be lyophilized is preferably modified compared to the corresponding region of the wild type mRNA such that at least one codon of the wild type sequence which codes for a tRNA which is relatively rare in the cell is exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and carries the same amino acid as the relatively rare tRNA.
  • sequences of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is modified such that codons for which frequently occurring tRNAs are available are inserted.
  • all codons of the wild type sequence which code for a tRNA which is relatively rare in the cell can in each case be exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and which, in each case, carries the same amino acid as the relatively rare tRNA.
  • the sequential G/C content which is increased, in particular maximized, in the modified nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention especially if the nucleic acid is in the form of a mRNA, with the "frequent" codons without modifying the amino acid sequence of the protein encoded by the coding region of the nucleic acid.
  • This preferred aspect allows provision of a particularly efficiently translated and stabilized (modified) nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA.
  • nucleotide sequence of any desired nucleic acid or mRNA can be modified with the aid of the genetic code or the degenerative nature thereof 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, and the amino acid sequence coded by the modified nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention preferably not being modified compared to the non-modified sequence.
  • the source code in Visual Basic 6.0 development environment used: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3
  • Microsoft Visual Studio Enterprise 6.0 with Servicepack 3 is also described in WO 02/098443.
  • the A/U content in the environment of the ribosome binding site of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is increased compared to the A U content in the environment of the ribosome binding site of its particular wild type mRNA.
  • This modification an increased A/U content around the ribosome binding site increases the efficiency of ribosome binding to the nucleic acid.
  • nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention especially if the nucleic acid is in the form of a mRNA, may be modified with respect to potentially destabilizing sequence elements.
  • the coding region and/or the 5' and/or 3' untranslated region of this nucleic acid (sequence), lyophilized or to be lyophilized may be modified compared to the particular wild type nucleic acid such that is contains no destabilizing sequence elements, the coded amino acid sequence of the modified nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, preferably not being modified compared to its particular wild type nucleic acid.
  • DSE destabilizing sequence elements
  • nucleic acid for further stabilization of the modified nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, optionally in the region which encodes for a protein or a peptide as defined herein, one or more such modifications compared to the corresponding region of the wild type nucleic acid can therefore be carried out, so that no or substantially no destabilizing sequence elements are contained there.
  • DSE present in the untranslated regions (3 '- and/or 5'-UTR) can also be eliminated from the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, by such modifications.
  • Such destabilizing sequences are e.g. AU-rich sequences (AURES), which occur in 3 '-UTR sections of numerous unstable RNAs (Caput et al, Proc. Natl. Acad. Sci. USA 1 986, 83: 1 670 to 1 674).
  • AURES AU-rich sequences
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is therefore preferably modified compared to the wild type nucleic acid such that the modified nucleic acid contains no such destabilizing sequences.
  • sequence motifs which are recognized by possible endonucleases, e.g.
  • sequence GAACAAG which is contained in the 3'-UTR segment of the gene which codes for the transferrin receptor (Binder et al., EMBO J. 1994, 1 3: 1 969 to 1 980).
  • sequence motifs are also preferably removed in the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention especially if the nucleic acid is in the form of a mRNA, has, in a modified form, at least one IRES as defined above and/or at least one 5 ' and/or 3' stabilizing sequence, in a modified form, e.g. to enhance ribosome binding or to allow expression of different encoded proteins located on the at least one (bi- or even multicistronic) RNA of the nucleic acid (sequence), lyophi lized or to be lyophilized, of the present invention.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, as described herein, especially if the nucleic acid is in the form of a mRNA, furthermore preferably has at least one 5' and/or 3' stabilizing sequence.
  • These stabilizing sequences in the 5' and/or 3' untranslated regions have the effect of increasing the half-life of the nucleic acid in the cytosol.
  • These stabilizing sequences can have 100% sequence identity to naturally occurring sequences which occur in viruses, bacteria and eukaryotes, but can also be partly or completely synthetic.
  • the untranslated sequences (UTR) of the (alpha-)globin gene e.g.
  • stabilizing sequences which can be used in the present invention for a stabilized lyophilized nucleic acid.
  • Another example of a stabilizing sequence has the general formula (C/U)CCAN x CCC(U/A)Py x UC(C/U)CC (SEQ ID NO: 4), which is contained in the 3'UTR of the very stable RNA which codes for (alpha-)globin, type(l)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf. Holcik et a/., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414).
  • nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention is therefore preferably present as (alpha-)globin UTR (untranslated regions)-stabilized RNA, in particular as (alpha- )globin UTR-stabilized RNA.
  • nucleic acid sequence
  • lyophilized or to be lyophilized of the present invention
  • nucleic acid is in the form of a mRNA
  • sequence sequence
  • lyophilized or to be lyophilized of the present invention by techniques of the well known site directed mutagenesis or with an oligonucleotide ligation strategy (see e.g. Maniatis et a/., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., Cold Spring Harbor, NY, 2001).
  • a corresponding DNA molecule may be transcribed in vitro.
  • This DNA matrix preferably comprises a suitable promoter, e.g. a T7 or SP6 promoter, for in vitro transcription, which is followed by the desired nucleotide sequence for the nucleic acid, e.g. mRNA, to be prepared and a termination signal for in vitro transcription.
  • the DNA molecule, which forms the matrix of at least one RNA of interest may be prepared by fermentative proliferation and subsequent isolation as part of a plasmid which can be replicated in bacteria.
  • Plasmids which may be mentioned as suitable for the present invention are e.g. the plasmids pT7Ts (GenBank accession number U26404; Lai et a/., Development 1 995, 121 : 2349 to 2360), pGEM ® series, e.g. pGEM ® -1 (GenBank accession number X65300; from Promega) and pSP64 (GenBank accession number X65327); cf. also Mezei and Storts, Purification of PCR Products, in: Griffin and Griffin (ed.), PCR Technology: Current Innovation, CRC Press, Boca Raton, FL, 2001 .
  • Nucleic acid molecules used according to the invention as defined above may be prepared using any method known in the art, including synthetic methods such as e.g. solid phase synthesis, as well as in vitro methods, such as in vitro transcription reactions.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, may additionally or alternatively encode a secretory signal peptide.
  • signal peptides are sequences, which typically exhibit a length of about 15 to 30 amino acids and are preferably located at the N-terminus of the encoded peptide, without being limited thereto.
  • Signal peptides as defined herein preferably allow the transport of the protein or peptide as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, into a defined cellular association, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.
  • secretory signal peptide sequences as defined herein include, without being limited thereto, signal sequences of classical or non- classical MHC-molecules (e.g. signal sequences of MHC I and II molecules, e.g.
  • signal sequences of cytokines or immunoglobulines as defined herein signal sequences of the invariant chain of immunoglobulines or antibodies as defined herein, signal sequences of Lampl , Tapasin, Erp57, Calretikulin, Calnexin, and further membrane associated proteins or of proteins associated with the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.
  • signal sequences of MHC class I molecule HLA-A*0201 may be used according to the present invention.
  • nucleic acid sequence
  • lyophilized or to be lyophilized of the present invention, especially if the nucleic acid is in the form of a mRNA, and further to any nucleic acid (sequence), lyophilized or to be lyophilized, as used in the context of the present invention and may be, if suitable or necessary, be combined with each other in any combination, provided, these combinations of modifications do not interfere with each other in the respective lyophilized nucleic acid.
  • a person skilled in the art will be able to take his choice accordingly.
  • nucleic acid sequence
  • proteins or peptides as encoded by the nucleic acid sequence
  • lyophilized or to be lyophilized of the present invention as defined above, may comprise fragments or variants of those sequences.
  • Such fragments or variants may typically comprise a sequence having a sequence identity with one of the above mentioned nucleic acids, or with one of the proteins or peptides or sequences, if encoded by the nucleic acid sequences of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably at least 70%, more preferably at least 80%, equally more preferably at least 85%, even more preferably at least 90% and most preferably at least 95% or even 97%, to the entire wild type sequence, either on nucleic acid level or on amino acid level.
  • “Fragments” of proteins or peptides in the context of the present invention may comprise a sequence of a protein or peptide as defined above, which is, with regard to its amino acid sequence (or its encoded nucleic acid sequence), N-terminally, C-terminally and/or intrasequentially truncated compared to the amino acid sequence of the original (native) protein (or its encoded nucleic acid sequence). Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level.
  • a sequence identity with respect to such a fragment as defined above may therefore preferably refer to the entire protein or peptide as defined above or to the entire (coding) nucleic acid sequence of such a protein or peptide.
  • fragments of nucleic acids in the context of the present invention may comprise a sequence of a nucleic acid as defined above, which is, with regard to its nucleic acid sequence 5'-, 3'- and/or intrasequentially truncated compared to the nucleic acid sequence of the original (native) nucleic acid sequence.
  • a sequence identity with respect to such a fragment as defined above may therefore preferably refer to the entire nucleic acid as defined above.
  • Fragments of proteins or peptides in the context of the present invention may furthermore comprise a sequence of a protein or peptide as defined above, which has a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 1 1 , or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g.
  • fragments may be selected from any part of the amino acid sequence.
  • These fragments are typically recognized by T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form.
  • Fragments of proteins or peptides as defined herein may also comprise epitopes of those proteins or peptides.
  • Epitopes also called "antigen determinants" in the context of the present invention are typically fragments located on the outer surface of (native) proteins or peptides as defined herein, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies or B-cell receptors, i.e. in their native form.
  • Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides.
  • antigenic determinants can be conformational or discontinous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.
  • “Variants” of proteins or peptides as defined above may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, wherein nucleic acids of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, encoding the protein or peptide as defined above, are exchanged.
  • a protein or peptide may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s).
  • these fragments and/or variants have the same biological function or specific activity compared to the full-length native potein, e.g.
  • nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may also encode a protein or peptide as defined above, wherein the encoded amino acid sequence comprises conservative amino acid substitution(s) compared to its physiological sequence.
  • Those encoded amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined above.
  • Substitutions in which amino acids which originate from the same class are exchanged for one another are called conservative substitutions.
  • these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function.
  • an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)).
  • Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three- dimensional structure or do not affect the binding region. Modifications to a three- dimensional structure by insertion(s) or deletion(s) can easily be determined e.g.
  • CD spectra circular dichroism spectra
  • variants of proteins or peptides as defined above which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophi lized, of the present invention, may also comprise those sequences, wherein nucleic acids of the nucleic acid (sequence), lyophilized or to be lyophilized, are exchanged according to the degeneration of the genetic code, without leading to an alteration of respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least part thereof may not differ from the original sequence in one or more mutation(s) within the above meaning.
  • nucleic acid sequences e.g.
  • nucleic acid sequences as defined herein, or amino acid sequences, preferably their encoded amino acid sequences, e.g. the amino acid sequences of the proteins or peptides as defined above) are identical, the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. gaps can be inserted into the sequence of the first sequence and the component at the corresponding position of the second sequence can be compared. If a position in the first sequence is occupied by the same component as is the case at a position in the second sequence, the two sequences are identical at this position.
  • the percentage to which two sequences are identical is a function of the number of identical positions divided by the total number of positions. The percentage to which two sequences are identical can be determined using a mathematical algorithm.
  • a preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et a/. (1993), PNAS USA, 90:5873-5877 or Altschul et a/. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.
  • the nucleic acid as defined above is typically present in a lactate containing solution prior to lyophilization.
  • the lactate is typically already contained in or added to the nucleic acid as defined above to form such a solution, or vice versa.
  • the nucleic acid and lactate containing solution prior to lyophilization may additionally contain water, preferably water for injection (WFI).
  • WFI water for injection
  • WFI water for injection
  • WFI Water for injection
  • WFI Water for Injection
  • EU endotoxin units
  • Endotoxins are a class of pyrogens that are components of the cell wall of Gram-negative bacteria (the most common type of bacteria in water), preferably in an action limit of 10 cfu/100 ml.
  • the microbial quality may be tested by membrane filtration of a 100 ml sample and plate count agar at an incubation temperature of 30 to 35 degrees Celsius for a 48-hour period.
  • the chemical purity requirements of WFI are typically the same as of PW (purified water).
  • the (residual) water content of the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein is typically reduced to a content of about 0.5 % (w/w) to about 10 % (w/w), more preferably to a content of about 1 % (w/w) to about 5 % (w/w), even more preferably to a content of about 2 % (w/w) to about 4% (w/w), most preferably to a content of about 3 % (w/w), e.g. 3 % (w/w) ⁇ 2 % (w/w), or 3 % (w/w) ⁇ 1 % (w/w).
  • nucleic acid sequence
  • nucleic acid and lactate containing solution prior to lyophilization may contain, additional to the nucleic acid and the lactate, further components or additives, e.g. a cryoprotectant, a lyoprotectant or any further suitable additive, preferably as defined in the following.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may additionally contain at least one suspending agent, preferably mannit, preferably in a concentration of about 1 to 15% (w/w), more preferably in a concentration of about 3 to 10% (w/w), and even more preferably in a concentration of about 4 to 6% (w/w).
  • at least one suspending agent preferably mannit, preferably in a concentration of about 1 to 15% (w/w), more preferably in a concentration of about 3 to 10% (w/w), and even more preferably in a concentration of about 4 to 6% (w/w).
  • the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may additionally contain at least one component or additive selected, e.g., from proteins, amino acids, alcohols, carbohydrates, mannose, mannit, metals or metal ions, surfactants, polymers or complexing agents, buffers, etc., or a combination thereof.
  • one preferred component or additive may also be selected from the group of amino acids.
  • Such group may comprise, without being limited thereto, any naturally occurring amino acid, including alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine, more preferably glycine, arginine, and alanine.
  • Cryoprotectants and/or lyoprotectants selected from the group of amino acids may additionally comprise any modification of a naturally occurring amino acid as defined above.
  • a further component or additive may be selected from the group of alcohols.
  • group may comprise, without being limited thereto, any alcohol suitable for the preparation of a pharmaceutical composition, preferably, without being limited thereto, mannitol, polyethyleneglycol, polypropyleneglycol, sorbitol, etc.
  • a further component or additive may be selected from the group of (free) carbohydrates.
  • group may comprise, without being limited thereto, any (free) carbohydrate, suitable for the preparation of a pharmaceutical composition, preferably, without being limited thereto, (free) monosaccharides, such as e.g.
  • (free) glucose, (free) fructose, (free) mannose preferably means unbound or unconjugated, e.g. the mannose is not covalently bound to the nucleic acid(sequence) to be lyophilized, or in other words, the mannose is unconjugated, preferably with respect to the nucleic acid (sequence) to be lyophilized), etc., disaccharides, such as e.g. lactose, maltose, sucrose, trehalose, etc., and polysaccharides, such as e.g. dextran, HP-beta CD, etc.
  • disaccharides such as e.g. lactose, maltose, sucrose, trehalose, etc.
  • polysaccharides such as e.g. dextran, HP-beta CD, etc.
  • mannose as a further component or additive is a D-Mannose.
  • D-Mannose may be depicted according to at least one of the D-Mannose isomers a-D-Mannofuranose, ⁇ -D-Mannofuranose, a-D-Mannopyranose and ⁇ -D- Mannopyranose.
  • the occurrence of the different mannose isomers in nature significantly differs.
  • D-Mannose forms anomers, wherein a-D-Mannofuranose ocurrs in a concentration/frequency of less than 1 %, ⁇ -D-Mannofuranose in a concentration/frequency of less than 1 %, a-D-Mannopyranose in a concentration/frequency of about 67 % and ⁇ -D- Mannopyranose in a concentration/frequency of about 33 %.
  • D-Mannose may be selected more preferably from at least one, two, three or four of the anomers a-D- Mannofuranose, ⁇ -D-Mannofuranose, ⁇ -D-Mannopyranose and/or ⁇ -D-Mannopyranose.
  • mannose upon solubilization in an aqueous solution mannose typically forms the above anomers in an equilibrity reaction, typically in the above concentrations.
  • mannose as a further component or additive is selected from an anomeric mixture of D-Mannose, preferably an anomeric mixture comprising a-D- Mannofuranose, ⁇ -D-Mannofuranose, ⁇ -D-Mannopyranose and ⁇ -D-Mannopyranose, more preferably in the above concentrations/frequencies.
  • mannose as a further component or additive may be selected from L-mannose or a racemic mixture of D-Mannose and/or L-Mannose, wherein D-mannose is preferably as described above.
  • Such mixtures may be obtained e.g. by a non-selective synthesis of mannose, e.g. by non-selective oxidation of mannitol.
  • An anomeric mixture may furthermore be obtained by solubilization of mannose in an aqueous solution, e.g. in water, WFI, or any buffer or solution as defined herein.
  • mannose as a further component or additive is typically present in the inventive solution for lyophilization, transfection and/or injection in a concentration of about 0.01 to about 10% (w/w), preferably in a concentration of about 0.01 to about 10% (w/w), more preferably in a concentration of about 0.1 to about 7.5% (w/w), even more preferably in a concentration of about 0.5 to about 5% (w/w), and most preferably in a concentration of about 1 to about 4% (w/w), e.g. a concentration of about 2 to about 4% (w/w), such as about 2.5 % (w/w).
  • a concentration of about 1 % (w/w) mannose corresponds to a concentration of about 55,506 mM mannose.
  • a further suitable component or additive may be selected from the group of proteins.
  • Such group may comprise, without being limited thereto, proteins such as albumin, gelatine, therapeutically active proteins as defined above, antibodies as defined above, antigens as defined above, or any further protein encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention as defined above.
  • a component or additive which may be contained in the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and accordingly in the nucleic acid and lactate containing solution prior to lyophilization may be selected from the group of metals or metal ions, typically comprising, without being limited thereto, metals or metal ions or salts selected from
  • alkali metals including members of group 1 of the periodic table: lithium (Li), sodium (Na), potassium (K), rubidium ( b), caesium (Cs), and francium (Fr), and their (monovalent) metal alkali metal ions and salts; preferably lithium (Li), sodium (Na), potassium (K), and their (monovalent) metal alkali metal ions and salts;
  • alkaline earth metals including members of group 2 of the periodic table: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba) and radium (Ra), and their (divalent) alkaline earth metal ions and salts; preferably magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba) and their (divalent) alkaline earth metal ions and salts;
  • transition metals including members of groups 3 to 13 of the periodic table and their metal ions and salts;.
  • the transition metals typically comprise the 40 chemical elements 21 to 30, 39 to 48, 71 to 80, and 103 to 1 12.
  • the name transition originates from their position in the periodic table of elements. In each of the four periods in which they occur, these elements represent the successive addition of electrons to the d atomic orbitals of the atoms. In this way, the transition metals represent the transition between subgroup 2 elements and subgroup 12 (or 13) elements.
  • Transition metals in the context of the present invention particularly comprise members of subgroup 3 of the periodic table: including Scandium (Sc), Yttrium (Y), and Lutetium (Lu), members of subgroup 4 of the periodic table: including Titan (Ti), Zirconium (Zr), and Hafnium (HO, members of subgroup 5 of the periodic table: including Vanadium (V), Niobium (Nb), and Tantalum (Ta), members of subgroup 6 of the periodic table: including Chrome (Cr), Molybdenum (Mo), and Tungsten (W), members of subgroup 7 of the periodic table: including Manganese (Mn), Technetium (Tc), and Rhenium (Re), members of subgroup 8 of the periodic table: including Iron (Fe), Ruthenium (Ru), and Osmium (Os), members of subgroup 9 of the periodic table: including Cobalt (Co), Rhodium (Rh), and Iridium (Ir), members of subgroup 10 of the periodic table: including Nickel (Ni),
  • earth metals or members of the boron group including members of group 3 of the periodic table: including Boron (B), Aluminium (Al), Gallium (Ga), Indium (In) and Thallium (TI) and their metal ions and salts; preferably Boron (B) and Aluminium (Al) and their metal ions and salts;
  • metalloids or semi metals including Boron (B), Silicon (Si), Germanium (Ge), Arsenic (As), Antimony (Sb), Tellurium (Te).and Polonium (Po), and their semi metal ions and salts; preferably Boron (B) and Silicon (Si) and their semi metal ions and salts;
  • a further component or additive may be selected from the group of surfactant may comprise, without being limited thereto, any surfactant, suitable for the preparation of a pharmaceutical composition, preferably, without being limited thereto, Tween, e.g. Tween 80 (0.2%), Pluronics, e.g. Pluronic L121 (1 .25%), Triton-X, SDS, PEG, LTAB, Saponin, Cholate, etc.
  • nucleic acid lyophilized or to be lyophilized, as defined herein and accordingly in the nucleic acid and lactate containing solution prior to lyophilization
  • polymers or complexing agents typically comprising, without being limited thereto, any polymer suitable for the preparation of a pharmaceutical composition, such as minor/major groove binders, nucleic acid binding proteins, lipoplexes, nanoplexes, non-cationic or non- polycationic compounds, such as PLGA, Polyacetate, Polyacrylate, PVA, Dextran, hydroxymethylcellulose, starch, MMP, PVP, heparin, pectin, hyaluronic acid, and derivatives thereof, or cationic or polycationic compound, particularly cationic or polycationic polymers or cationic or polycationic lipids, preferably a cationic or polycationic polymers.
  • such a cationic or polycationic compound is typically selected from any cationic or polycationic compound, suitable for complexing and thereby stabilizing a nucleic acid as defined herein, e.g. by associating the nucleic acid as defined herein with the cationic or polycationic compound.
  • cationic or polycationic compounds are selected from cationic or polycationic peptides or proteins, including protamine, nucleoline, spermin or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1 , L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophil), PLL
  • oligoarginines in this context are e.g. Arg 7 , Argg, Arg 9 , Arg 7 , H 3 R 9 , R 9 H 3 , H 3 R 9 H 3 , YSSR 9 SSY, (RKH) 4 , Y(RKH) 2 R, etc.
  • Further preferred cationic or polycationic compounds, which can be used for complexing the nucleic acid as defined herein may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
  • PEI polyethyleneimine
  • DOTMA [1 -(2,3-sioleyloxy)propyl)]-N,N,N- trimethylammonium chloride
  • DMRIE di-C14-amidine
  • DOTIM DOTIM
  • SAINT DC-Choi
  • BGTC CTAP
  • DOPC DODAP
  • DOPE Dioleyl phosphatidylethanol-amine
  • DOSPA DODAB
  • DOIC DOIC
  • DMEPC DOGS: Dioctadecylamidoglicylspermin
  • DIMRI Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide
  • DOTAP dioleoyloxy-3- (trimethylammonio)propane
  • DC-6-14 O,O-ditetradecanoyl-N-(a- trimethylammonioacetyl)diethanolamine chloride
  • CLIP1 rac-[(2,3- dioctadecyloxypropyl)(2-hydroxyethyl)
  • modified polyaminoacids such as ⁇ -aminoacid- polymers or reversed polyamides, etc.
  • modified polyethylenes such as PVP (poly(N-ethyl- 4-vinylpyridinium bromide)), etc.
  • modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.
  • modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modified polybetaaminoester (PBAE), such as diamine end modified 1 ,4 butanediol diacrylate-co-5-amino-1 -pentanol polymers, etc.
  • dendrimers such as polypropylamine dendrimers or pAMAM based dendrimers, etc.
  • polyimine(s) such as PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, sugar backbone based poly
  • association or complexing the nucleic acid as defined herein with cationic or polycationic compounds preferably provides adjuvant properties to the RNA and confers a stabilizing effect to the nucleic acid as defined herein by complexation.
  • the procedure for stabilizing the nucleic acid as defined herein is in general described in EP-A-1 083232, the disclosure of which is incorporated by reference into the present invention in its entirety.
  • cationic or polycationic compounds are compounds selected from the group consisting of protamine, nucleoline, spermin, spermidine, oligoarginines as defined above, such as Arg 7 , Arge, Arg 9 , Arg 7 , H 3 R 9 , R 9 H 3 , H 3 R 9 H 3 , YSSR 9 SSY, (RKH) 4/ Y(RKH) 2 R, etc.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may additionally contain water, water for injection (WFI), or a buffer, preferably selected from a buffer as defined above, e.g.
  • a buffer containing 2- hydroxypropanoic acid preferably including at least one of its optical isomers L-(+)-lactic acid, (5)-lactic acid, D-(-)-lactic acid or (A)-lactic acid, more preferably its biologically active optical isomer L-(+)-lactic acid, or a salt or an anion thereof, preferably selected from sodium-lactate, potassium-lactate, or Al 3 + -lactate, NH 4 + -lactate, Fe-lactate, Li-lactate, Mg- lactate, Ca-lactate, Mn-lactate or Ag-lactate, or a buffer selected from Ringer's lactate (RiLa), lactated Ringer's solution (main content sodium lactate, also termed "Hartmann's Solution” in the UK), acetated Ringer's solution, or ortho-lactate-containing solutions (e.g.
  • a buffer as defined herein may also be a mannose containing buffer, an isotonic buffer or solution, preferably selected from isotonic saline, a lactate or ortho-lactate-containing isotonic solution, a isotonic buffer or solution selected from phosphate-buffered saline (PBS), TRIS-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS), Grey's balanced salt solution (GBSS), or normal saline (NaCI), hypotonic (saline) solutions with addition of glucose or dextrose, or any solution as defined herein, etc.
  • PBS phosphate-buffered saline
  • TRIS-buffered saline TRIS-buffered saline
  • HBSS Hank's balanced salt solution
  • EBSS Earle's balanced salt
  • isotonic buffers or solutions are preferably prepared by a skilled person preferably as defined herein or according to definitions preparation protocols well known in the art for these specific isotonic buffers or solutions. More preferably, the lactate containing solution prior to lyophilization as defined above may contain these isotonic buffers or solutions or (all) its contents in isotonic concentrations, preferably as defined herein or in the art for these specific isotonic solutions.
  • the lactate containing solution prior to lyophilization as defined above and accordingly its components, additives, isotonic buffers or solutions, may be present in an osmolality or osmolarity comparable to that of blood plasma, preferably in an osmolarity as generally defined herein for the nucleic acid and lactate containing solution prior to lyophilization (as well as after reconstituting the lyophilized nucleic acid).
  • a buffer may be used, more preferably an aqueous (isotonic solution or aqueous) buffer, containing a sodium salt, preferably at least 50 mM of a sodium salt, a calcium salt, preferably at least 0.01 mM of a calcium salt, and optionally a potassium salt, preferably at least 3 mM of a potassium salt.
  • the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc.
  • examples of sodium salts include e.g.
  • examples of the optional potassium salts include e.g. KCI, Kl, KBr, K 2 C0 3 , KHC0 3 , K 2 S0 4
  • examples of calcium salts include e.g. CaCI 2 , Cal 2 , CaBr 2 , CaC0 3 , CaS0 4 , Ca(OH) 2 .
  • the salts are present in such an (isotonic solution or) buffer in a concentration of at least 50 mM sodium chloride (NaCI), at least 3 mM potassium chloride (KCI) and at least 0.01 mM calcium chloride (CaCl 2 ).
  • organic anions of the aforementioned cations may be contained in the buffer.
  • the buffer may contain salts selected from sodium chloride (NaCI), calcium chloride (CaCI 2 ) and optionally potassium chloride (KCI), wherein further anions may be present additional to the chlorides.
  • CaCl 2 can also be replaced by another salt like KCI.
  • the buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the aforementioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects.
  • Reference media are e.g.
  • liquids occurring in "in vivd' methods such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in "in vitrd' methods, such as common buffers or liquids.
  • common buffers or liquids are known to a skilled person.
  • the inventive nucleic acid (sequence), lyophilized or to be lyophilized, when lyophilized may again be reconstituted after lyophilization in a buffer as defined herein, preferably as defined above, e.g. as a further step of a method for lyophilization as defined herein.
  • the inventive lyophilized nucleic acid when lyophilized, may alternatively be reconstituted in water, a buffer as defined above, or a buffer containing mannose, to obtain the desired salt concentration or alternatively the desired buffer conditions.
  • the reconstitution of the lyophilized nucleic acid is carried out in WFI (water for injection), if the nucleic acid has been lyophilized in Ringer Lactate solution which represents an isotonic solution for injection.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may additionally contain one or more compatible solid or liquid fillers or diluents or encapsulating compounds, which are suitable for administration to a patient to be treated.
  • compatible means that these constituents are capable of being mixed with the nucleic acid (sequence), lyophilized or to be lyophilized, as defined according to the present invention in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the inventive nucleic acid (sequence), lyophilized or to be lyophilized, under typical use conditions.
  • Pharmaceutically acceptable carriers, fillers and diluents must, of course, have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a person to be treated.
  • Some examples of compounds which can be used as pharmaceutically acceptable carriers, fillers or constituents thereof are sugars, such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oi l, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol,
  • the nucleic acid and lactate containing solution prior to lyophilization may occur as a liquid, a semi-liquid or even a semi-solid or a solid sample, preferably as a liquid, a semi- liquid or a even a semi-solid sample, more preferably as a liquid or a semi-liquid sample.
  • the pH of the nucleic acid and lactate containing solution prior to lyophilization may be in the range of about 4 to 8, preferably in the range of about 6 to about 8, more preferably from about 7 to about 8.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein typically comprises a final (residual) water content preferably in the range of about 0.5 % (w/w) to about 10 % (w/w), more preferably of about 1 % (w/w) to about 5 % (w/w), even more preferably of about 2 % (w/w) to about 4% (w/w), and most preferably of about 3 % (w/w), e.g. 3 % (w/w) ⁇ 2 % (w/w), or 3 % (w/w) ⁇ 1 % (w/w).
  • the nucleic acid and lactate containing solution prior to lyophilization may contain the herein defined contents, optional components, additives, etc. in such a concentration so as to lead to an osmolarity comparable to that of blood plasma.
  • osmolarity is typically to be understood as a measure of all contents, optional components, additives, etc. of the nucleic acid and lactate containing solution prior to lyophilization (as well as after reconstituting the lyophilized nucleic acid) as defined herein.
  • osmolarity is typically the measure of solute concentration, defined as the number of osmoles (Osm) of all solubilized contents, optional components, additives, etc. per liter (I) of solution (osmol/l or osm/l).
  • the nucleic acid and lactate containing solution prior to lyophilization may comprise an osmolarity preferably in the range of about 200 mosmol/l to about 400 mosmol/l, more preferably in the range of about 250 mosmol/l to about 350 mosmol/l, even more preferably in the range of about 270 mosmol/l to about 330 mosmol/l or in the range of about 280 mosmol/l to about 320 mosmol/l, or in the range of about e.g. about 290 mosmol/l to about 310 mosmol/l, e.g.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein typically comprises an excellent enhanced storage-stability, when compared to a nucleic acid (sequence), lyophilized or to be lyophilized, of the art, which has been lyophilized without the presence of lactate, e.g. in the presence of water for injection (WFI) as described herein.
  • the nucleic acid (sequence), lyophilized or to be lyophilized, as defined and as prepared herein advantageously can be stored in a temperature range of about -80°C to +60°C significantly longer, when compared to a nucleic acid (sequence), lyophilized or to be lyophilized, of the art.
  • nucleic acid sequence
  • lyophilized or to be lyophilized as defined herein even allows presence of additional ingredients, such as salts, metalloids, metal anions or further ingredients as defined above, which have been formerly regarded as problematic or even instabilizing the lyophilized nucleic acid.
  • the storage-stability of the nucleic acid (sequence), lyophilized or to be lyophilized is calculated on the basis of the relative integrity of the nucleic acid.
  • the relative integrity of the nucleic acid in the nucleic acid is typically defined as the relative content of the nucleic acid exhibiting a correct length when compared to the total content of nucleic acid in the sample.
  • the relative integrity of the mRNA in the lyophilized mRNA is typically defined as the relative content of the mRNA exhibiting a correct length when compared to the total content of mRNA in the sample.
  • the storage-stability of a nucleic acid is typically determined on the basis of the relative integrity (over a defined or not defined period of time), wherein the nucleic acid typically exhibits an unchangend biological activity.
  • the storage stability is preferably regarded as complied with, if the relative integrity of the (lyophilized) nucleic acid(s) is at least about 60 to 80%, preferably at least about 70 %.
  • a relative integrity of more than 70 % meets the quality criteria of CureVac GmbH for mRNA exhibiting a GC-content of more than 60 % and a base length of ⁇ 2000 nt in RNA containing formulations. This criterium may be applied to the above definition.
  • the present invention also provides a method of lyophilization of a nucleic acid, preferably for preparation of lyophilized nucleic acid (sequence) according to the present invention as defined above.
  • the inventive lyophilized nucleic acid (sequence) as defined according to the present invention is preferably prepared according to the herein described inventive method for lyophilization of a nucleic acid.
  • the inventive method of lyophilization of a nucleic acid preferably leads to an enhanced storage stability of the nucleic acid. The method typically comprises the following steps:
  • nucleic acid containing sample which has been supplemented with a lactate as defined herein, and optionally supplemented with further ingredients as defined above;
  • step b) freezing the nucleic acid containing sample, obtained according to step a);
  • step b) drying the frozen nucleic acid containing sample, obtained according to step b), via sublimation;
  • step c) optionally floating the (lyophilized) nucleic acid (sequence) (obtained according to step c)) with an inert gas, such as nitrogen, etc., or a noble gas, such as helium, neon, argon, xenon, krypton;
  • an inert gas such as nitrogen, etc., or a noble gas, such as helium, neon, argon, xenon, krypton;
  • the inventive method is directed to a method of lyophilization of a nucleic acid.
  • Lyophilization also termed cryodesiccation
  • a frozen sample containing at least one nucleic acid and a lactate containing solution, preferably as defined above
  • sublimation is typically understood as a process, which allows removing water from a frozen sample (containing at least one nucleic acid and a lactate containing solution, preferably as defined above) in one or more steps via sublimation.
  • lyophilization is typically carried out by freeze-drying a sample first freezing a nucleic acid containing sample, which has been supplemented with a lactate as defined herein, and then drying the nucleic acid containing sample via sublimation, optionally by reducing the surrounding pressure and/or adding enough heat to allow the frozen water in the sample to sublime directly from the solid phase to gas.
  • nucleic acid containing sample which has been supplemented with a lactate as defined herein, and optionally supplemented with further ingredients as defined above, is provided.
  • the nucleic acid and the lactate are preferably as defined above for the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention.
  • the nucleic acid containing sample may be prepared either by adding a lactate as defined above, preferably in the above defined concentrations, to a sample containing a nucleic acid as defined above, or by adding a sample containing a nucleic acid as defined above to a lactate containing sample, preferably a lactate as defined above in the above defined concentrations.
  • the nucleic acid containing sample which has been supplemented with a lactate as defined herein, may additionally be supplemented with further ingredients, preferably as defined above for the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention.
  • the sample, containing the at least one nucleic acid and a lactate as defined herein, is frozen.
  • the freezing process may be carried out by any method, which allows to (entirely) freeze the sample. In a lab, this may be done by placing the material in a freeze-drying flask and rotating the flask in a bath, called a shell freezer, which is cooled by mechanical refrigeration, dry ice and methanol, or liquid nitrogen. On a larger- scale, freezing is usually carried out using a freeze-drying machine. In this step, it is important to cool the material below its triple point, the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting will occur in the following steps.
  • the freezing temperatures are in the range between -0 °C and -80 °C, or between -20 °C and -80 °C, preferably in the range between -30 °C and -60 °C, even more preferably in the range between -40 °C and -50 °C, most preferably about -47 °C.
  • the frozen sample is dried, typically using two drying steps, primary drying step c1) and secondary drying step c2).
  • primary drying step cl free, i.e. unbound, water surrounding the nucleic acid and optionally further components, escapes from the solution.
  • Subsequent thereto water being bound on a molecular basis by the nucleic acids may be removed in a secondary drying step c2) by adding thermal energy. In both cases the hydration sphere around the nucleic acids is lost.

Abstract

The present invention is directed to the lyophilization of nucleic acids in a lactate-containing solution or formulation. The present invention is particularly suitable for enhancing and improving storage and shipping capabilities of nucleic acids for multiple purposes. The present invention is furthermore directed to methods of lyophilization suitable to prepare such inventive lyophilized nucleic acids, to the use of a lactate-containing solution or formulation for the preparation of such lyophilized nucleic acids or the use of a solution containing at least one nucleic acid (sequence) and (free) lactate for lyophilization of at least one nucleic acid (sequence), to the use of such lyophilized nucleic acids in the preparation of pharmaceutical compositions, to first and second medical indications using such lyophilized nucleic acids and to kits, particularly to kit of parts, comprising such lyophilized nucleic acids.

Description

Lyophilization of nucleic acids in lactate-containing solutions
The present invention is directed to the lyophilization of nucleic acids in a lactate- containing solution or formulation. The present invention is particularly suitable for enhancing and improving storage and shipping capabilities of nucleic acids for multiple purposes. The present invention is furthermore directed to methods of lyophilization suitable to prepare such inventive lyophilized nucleic acids, to the use of a lactate- containing solution or formulation for the preparation of such lyophilized nucleic acids or the use of a solution containing at least one nucleic acid (sequence) and (free) lactate for lyophilization of at least one nucleic acid (sequence), to the use of such lyophilized nucleic acids in the preparation of pharmaceutical compositions, to first and second medical indications using such lyophilized nucleic acids and to kits, particularly to kit of parts, comprising such lyophilized nucleic acids.
In gene therapy and many other therapeutically relevant biochemical and biotechnological applications the use of nucleic acids for therapeutic and diagnostic purposes is of major importance. As an example, rapid progress has occurred in recent years in the field of gene therapy and promising results have been achieved. In this context, the final dosage form providing these nucleic acids but also production, transport and storage thereof are of particular interest. It is understood that nucleic acids, e.g., naked DNA, introduced into a patient' circulatory system are typically not stable and therefore have little chance of affecting most disease processes (see e.g. Poxon et a/., Pharmaceutical development and Technology, 5(1 ), 1 15- 122 (2000)). This obstacle has led to the development of a number of gene delivery systems, e.g. liposomal delivery, gene delivery vectors, etc., which allow increasing metabolic activities. However, these systems and formulations are typically not suitable for long-term storage or storage above temperatures of -20°C or even -80°C and thus require methods such as lyophilization to increase shelf life. Lyophilization is a worldwide known and recognized method in the art to enhance storage stability of temperature sensitive biomolecules, such as nucleic acids. During lyophilization, typically water is removed from a frozen sample containing nucleic acids via sublimation. The process of lyophilization is usually characterized by a primary and a secondary drying step. During the primary drying step, free, i.e. unbound, water surrounding the nucleic acid and optionally further components, escapes from the solution. Subsequent thereto water being bound on a molecular basis by the nucleic acids may be removed in a secondary drying step by adding thermal energy. In both cases the hydration sphere around the nucleic acids is lost.
During lyophilization the sample containing nucleic acids is initially cooled below the freezing point of the solution and accordingly of the water contained therein. As a result, the water freezes. Dependent on temperature, rate of cooling down (freezing rate), and the time for freezing, the crystal structure of water is changed. This exhibits physical stress on the nucleic acid and other components of the solution, which may lead to a damage of the nucleic acid, e.g. breakage of strands, loss of supercoiling, etc. Furthermore, due to the decrease of volume and loss of the hydration sphere, autocatalytic degradation processes are favored e.g. by traces of transition metals. Additionally, significant changes of pH are possible by concentration of traces of acids and bases.
As discussed above, lyophilization involves two stresses, freezing and drying. Both are known to damage nucleic acids, such as non-viral vectors or plasmid DNA but also DNA. In the literature, a number of cryoprotectants and lyoprotectants are discussed for lyophilization purposes to prevent these damages. In this context, cryoprotectants are understood as excipients, which allow influencing the structure of the ice and/or the eutectical temperature of the mixture. Lyoprotectants are typically excipients, which partially or totally replace the hydration sphere around a molecule and thus prevent catalytic and hydrolytic processes. In this context, carbohydrates such as sugars play a central role as lyoprotectants. However, when using cryoprotectants and lyoprotectants, no general rule may be applied with respect to their impact on different groups of compounds. Therefore, an optimal formulation has to be found using empirical methods.
In the specific context of DNA, lyophilization causes the removal of the hydration sphere around the DNA, wherein it appears that there are approximately 20 water molecules per nucleotide pair bound most tightly to DNA. These water molecules do not form an ice-like structure upon low-temperature cooling. Upon DNA dehydration over hygroscopic salts at 0% relative humidity, only five or six water molecules remain (see e.g. Tao et a/., Biopolymers, 28, 101 9-1030 (1 989)). Lyophilization may increase the stability of DNA under long-term storage, but may also cause some damage upon the initial lyophilization process, potentially through changes in the DNA secondary structure, breaks of the nucleic acid chain(s) or the concentration of reactive elements such as contaminating metals. Lyophilization can also cause damage upon the initial lyophilization process in other nucleic acid molecules, e.g. RNA. Agents that can substitute for non-freezable water, such as trehalose, can demonstrate cryoprotective properties for DNA and other molecules during lyophilization of intact bacteria (see e.g. Israeli et a/, Cryobiology, 30, 51 9-523 (1 993); or Rudolph et a/, Arch. Biochem. Biophys., 245, 1 34-143 (1986)). Other cryoprotective agents, such as polyols, amino acids, sugars, and lyotropic salts, are preferentially excluded from contact with protein surfaces but are also capable of stabilizing enzymes during lyophilization by undefined mechanisms (see e.g. Carpenter et a/., Cryobiology 27, 21 9-231 (1 990)). It is possible that these agents that act as cryoprotectants to proteins may also act to stabilize nucleic acids upon lyophilization.
The complex of DNA and cationic liposomes requires lyoprotectants to maintain transfection efficiency after lyophilization (Anchordoquy et a/., Pharm. Res., 1 4(1 1 ), S641 - S642 (1 997)). Moreover, other gene delivery systems containing polyethylenimine, polylysine, and adenovirus particles require cryoprotection to maintain transfection efficiency (Talsma et a/., Int. J. Pharm., 157(2), 233-238 (1997)). However, it is unclear if this effect is due only to stabilization of the liposomal component of the mixture or the plasmid DNA component may itself be damaged by lyophilization in the absence of carbohydrate lyoprotectants, because lyoprotectants may serve a variety of protective roles.
In this context, Poxon et a/. (2000, supra) investigated the effect of lyophilization on plasmid DNA activity. Poxon et a/. (2000, supra) hypothetized, that a change in the DNA conformation from supercoi led to open circular and linear form would be indicative of damage of the plasmid DNA. However, the percentage of supercoiled DNA did not change after lyophi lization and subsequent DMED treatment, suggesting that other effects drew responsible for the loss of transfection efficiency. Poxon et a/. (2000, supra) found that a decrease in plasmid DNA activity as measured by an in vitro transfection assay can be ameliorated by the use of carbohydrates during lyophilization of the plasmid DNA. As a particular example, a statistically significant loss of transfection efficiency (p<0.05) by lyophilization of pRL-CMV plasmid DNA could completely be restored by using mono- and disaccharides during lyophilization. As lyoprotectants, glucose (monosaccaride), sucrose and lactose (disaccharides) were used. Poxon eta/. (2000, supra), however, only carried out investigations with lyophilized plasmid DNA using carbohydrate lyoprotectants. No other nucleic acids, such as RNA or PNA, were discussed. Poxon et a/. (2000, supra), did also not investigate if the addition of sugars to the lyophilization affects the stability of the lyophilized plasmid DNA.
A similar approach was carried out by Molina et al. (see e.g. Molina et al., J. Pharm. Sci. 2004 Sep, 93(9), 2259-73). Molina et al. (2004, supra) lyophilized lipid/DNA complexes in the presence of the carbohydrates glucose, sucrose and trehalose, i.e. mono- or disaccharide sugars. Molina et al. (2004, supra) furthermore used a specific liposome preparation, preferably liposomes containing DOTAP in a 1 :1 (w/w) ratio with the zwitterionic lipid DOPE, wherein a 3:1 lipid:DNA weight ratio was applied. The results in Molina et al. (2004, supra) showed that even though lyophilized samples prepared using these carbohydrates maintained their particle size at low temperatures (i.e. -20°C, 4°C) the transfection activity of these lyophilized lipoplexes gradually declined with time regardless of their storage temperature. In other words, no long-term storage is possible using these carbohydrates, particularly glucose, sucrose and trehalose, i.e. mono- or disaccharide sugars. Surprisingly, the loss of activity was observed in all formulations and sometimes even under conditions that maintained particle size. Molina et al. (2004, supra) concluded that due to the glass like structure of the lyophilized DNA and considering that reactions requiring positional specificity would be sharply inhibited by the reduced molecular mobility in the glass, a highly reactive species, e.g. free radicals, may have a severe impact on transfection activity. They suspected that mobility of such highly reactive species is not dramatically restricted in a glassy formulation, wherein the high free volume of a glass might facilitate diffusion of small free radical species (e.g. OH*, O2 *), and thereby permitting significant levels of strand breakage to occur during storage. They also hypothesized that trace amounts of transition metals (e.g. Cu2+, Fe3+) in excipients are responsible for this phenomenon and may catalyze the generation of reactive oxygen species (ROS) via the Fenton reaction (see Molina et ai, 2004, supra). Maitani et al. (see Maitani et al., Int. J. Pharm. 356 (2008),' 69-75) disclose cationic lipid- based gene delivery systems and investigate, similar to Molina etal. (2004, supra), the effect of sugars on the storage stability of lyophilized liposome/DNA complexes with high transfection efficiency. Particularly, Maitani et al. (2008, supra) carried out lyophilization experiments and examined stability of lyophilized liposome/DNA complexes with sucrose, isomaltose and isomaltotriose at different temperatures over 50 days, and determined which sugars could inhibit aggregation and maintain the transfection activity of plasmid DNA during preservation at temperatures above their glass transition temperatures (Tg). As a result, Maitani et al. (2008, supra) found that liposome/DNA complexes with sucrose, prepared according to the dehydration rehydration vesicle (DRV) method, could be stored even at 50°C without a large loss of transfection activity. Isomaltose and isomaltotriose were selected as excipients because their Tg values were higher than that of sucrose and therefore, there were expected to exhibit a greater stabilizing effect.
In the publication of Quaak et al. (see Quaak et al, Eur. J. Pharm. Biopharm. 70 (2008) 4329-438) the authors investigate the GMP production of a plasmid DNA construct pDERMATT (plasmid DNA encoding recombinant MART-1 and Tetanus toxin fragment-c) for vaccination against melanoma in a phase I clinic trial. One purpose of the study was the provision of a reproducible process to manufacture pharmaceutical grade plasmid DNA but also a stable dosage form for the use in clinical trials. As requirements, the plasmid product must be of high purity, essentially in its supercoiled form and free of host-cell proteins, chromosomal RNA, RNA, preferably without the use of RNase A, and endotoxins. Quaak et al. (2008, supra) used as excipients sucrose, trehalose, mannitol and polyvinylpyrrolidone (PVP), wherein lyophilization of formulations containing sucrose as a bulking agent in a concentration of (2%) turned out to result in a stable product. The resulting product showed immunogenicity and a strong MART-1 specific CTL response in a murine model.
Even though many available prior art documents discuss the stabilization of nucleic acids during lyophilization and long-term storage in the context of plasmid DNA, only few publications focus on stabilization of other nucleic acids, such as RNAs. In this context, provision of stabilization of RNAs during lyophilization and long-term storage is particularly important. As generally known, the physico chemical stability of RNAs in solution is extremely low. RNA is typically completely degraded even in the absence of RNases when stored a few days at room temperature. To avoid such degradation and a loss of function, particularly when regarding coding RNAs such as mRNAs the RNA is to be stored at -20°C or even -80°C. Furthermore, when using nucleic acids, particularly RNAs, as an active agent in a pharmaceutical composition or a vaccine, Yadava et a/, (see Yadava et a/., AAPS Pharm. Sci. Tech., Vol. 9, No. 2, June 2008, pp. 335- 341 ) discuss the effect of lyophilization and freeze-thawing on the stability of siRNA- liposome complexes. The lipoplexes in Yadava et a/. (2008, supra), when lyophilized in the presence of sugars, such as glucose or sucrose, could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions, they lost 65-75% of their biological functionality. The lyophilization process thus did not appear to alter siRNA's intrinsic biological activity. However, the underlying principle of RNA interference (RNAi) is the post-transcriptional gene silencing due to the cleavage of mRNA, triggered by small double stranded RNA molecules (small interfering RNAs, siRNAs), homologous in sequence to the target mRNA, which are typically less sensitive to loss of biological activity due to breakage and (partial) degradation than mRNAs or RNAs in general, as these siRNAs may even exhibit a biological activity, when their (short) sequence is partially degraded. Yadava et a/. (2008, supra), did not investigate if the addition of sugars or salts affects the stability of the lyophilized siRNA. Despite little success shown for some DNA derived nucleic acids, all the above approaches utilize carbohydrates, particularly sugars such as mono- or disaccharides, as a lyoprotectants to stabilize these nucleic acids during lyophilization. However, when using sugars, particularly when using mono- or disaccharides, a theoretical sugar/nucleic acid mass ratio of about 1000 to about 4000 is required to optimally conserve the biological activity of the nucleic acids (see Molina et a/. (2004, supra); and Poxon et a/. (2000, supra)). Unfortunately, this mass ratio, even if preferred, is not possible for nucleic acids above a concentration of 2 g l due to the high concentration and due to the restricted solubility of sugar in water. When using more suitable concentrations of sugar or sugar/nucleic acid mass ratios of about 4, a decrease of biological activity could be observed. Data furthermore show that during (long-term) storage of lyophilized DNA vectors the content of supercoiled DNA is accompanied by a loss of biological activity.
An additional problem originates from the use of salts in such solutions. The use of salts generally exhibits a destabilizing effect on peptides and complexes (see e.g. Yadava et at, (2008 supra)), but also on many nucleic acid solutions, and is thus avoided. However, when neutralizing the electrostatic interactions of biomolecules or their complexes, aggregation may occur quite easily and also may be accompanied by a loss of activity and degradation. Additionally, even though storage at -20°C or even -80°C is technically possible, such storage does not allow long-term storage without a significant loss of biological activity. It also requires extraordinary effort and costs. Such costs, however, cannot be afforded by all commercial and sometimes private parties. This particularly applies to tropical regions or third-world countries, where expenses have to be kept low for economical reasons and due to limited energy available in these regions. Furthermore, there may be the need to transport such nucleic acids in cases, where not much space (or energy) is available for storage, e.g. in cases of the situation "production to bedside" or even "bench to bedside". This also may be the case for short but also for long-term expeditions, for long-term storage of nucleic acids in databases, registers or deposit institutions, e.g. databases for biological researchers, governmental or national databases of criminal offenders, etc. Long-term storage capablities of RNA furthermore opens a various field of applications and treatments involving RNAs as active substances, e.g. (tumor)vaccination using RNAs, gene therapy using RNAs, etc. Storage at temperatures above 4°C, for both DNA and RNA and any further nucleic acid, such as PNAs, therefore may entail a valuable economical and logistical advantage in all of these different aspects.
Summarizing the above, there is a long-lasting and urgent need to provide means, which allow (a skilled person) to lyophilize nucleic acids more efficiently without (a significant) loss of function of these nucleic acids and which allow for a (long-term) storage of these nucleic acids, particularly of RNAs, at temperatures above -80°C or -20°C, preferably above 4°C or even at room temperature or higher temperatures.
The object of the present invention is solved by the attached claims. According to a particular embodiment, the problem underlying the present invention is solved by a lyophilized nucleic acid, which has been lyophilized from a lactate containing solution. Preferably, the inventive nucleic acid (sequence), lyophilized or to be lyophilized, is prepared using a method as described herein, particularly a method of lyophilization of a nucleic acid according to the present invention. In other words, the present invention also provides the use of a lactate containing solution, preferably as defined herein for lyophilizing a nucleic acid (sequence) preferably as defined herein, or the use of a solution containing at least one nucleic acid (sequence) as defined herein and (free) lactate as defined herein for lyophilization of at least one nucleic acid (sequence). In this context, "free" preferably means unbound or unconjugated, e.g. the lactate is not covalently bound to the nucleic acid(sequence) to be lyophilized, or in other words, the lactate is unconjugated, preferably with respect to the nucleic acid (sequence) to be lyophilized).
This solution is particularly surprising and was not suggested by any of the above prior art documents. A skilled person, bearing in mind that salts typically destabilize a nucleic acid during lyophilization, always would have expected that lactate, representing a salt, would rather destabilize than stabilize a nucleic acid during lyophilization. Even more surprisingly, it turned out to the inventors, that the use of lactate retrieved better results than the use of glucose, which typically has been used during lyophilization. In the context of the present invention lyophilization (also termed cryodesiccation) is typically understood as a freeze-drying process, which allows removing water from a frozen sample (containing at least one nucleic acid and a lactate containing solution) via sublimation as described below in further detail. For the inventive purpose, a lactate as defined herein may be any lactate available in the art. Preferably, a lactate within the context of the present invention is defined as a chemical compound, particularly a salt, derived from (free) lactic acid (lUPAC systematic name: 2- hydroxypropanoic acid), also known as milk acid, including its optical isomers L-(+)-lactic acid, (5)-lactic acid, D-(-)-lactic acid or (A)-lactic acid, more preferably its biologically active optical isomer L-(+)-lactic acid, wherein the salt or an anion thereof, preferably may be selected from sodium-lactate, potassium- lactate, or Al3 +-lactate, NH4 +-lactate, Fe-lactate, Li-lactate, Mg-lactate, Ca-lactate, Mn-lactate or Ag-lactate, or selected from Ringer's lactate (RiLa), lactated Ringer's solution (main content sodium lactate, also termed "Hartmann's Solution" in the UK), acetated Ringer's solution, or selected from lactate containing water, or ortho-lactate-containing (isotonic) solutions (e.g. for injection purposes), etc. The chemical structure of lactic acid is as follows:
Figure imgf000010_0001
Lactic acid is a chemical compound that plays a role in several biochemical processes. It was first isolated in 1 780 by a Swedish chemist, Carl Wilhelm Scheele, and is a carboxylic acid with a chemical formula of C3H603. It has a hydroxyl group adjacent to the carboxyl group, making it an alpha hydroxy acid (AHA). In solution, it can lose a proton from the acidic group, producing the lactate ion CH3CH(OH)COO". Lactic acid is chiral and has two optical isomers. One is known as L-(+)-lactic acid or (5)-lactic acid and the other, its mirror image, is D-(-)-lactic acid or (^-lactic acid, wherein L-(+)-lactic acid is the biologically important isomer. L-lactate is constantly produced in animals from pyruvate via the enzyme lactate dehydrogenase (LDH) in a process of fermentation during normal metabolism and exercise. Industrially, lactic acid is typically produced via fermentation using among others bacteria such as Lactobacillus bacteria, etc. The present invention preferably uses lactate as a monomer and therefore preferably excludes polymeric forms of lactic acid, in particular poly lactic acid (PLA), or salts therefrom from the scope of the present invention.
The lactate containing solution as defined herein, which is used for lyophilizing a nucleic acid (sequence) as defined herein, typically comprises a lactate concentration prior to lyophilization in the range of about 3 mM to about 300 mM, preferably in the range of about 5 mM to about 200 mM, more preferably in the range of about 10 mM to about 150 mM, even more preferably about 15 mM to about 35 mM, and most preferably 20 mM to about 31 mM.
Alternatively, the lactate containing solution as defined herein, which is used for preparation of the inventive lyophilizing a nucleic acid (sequence) as defined herein, typically comprises a Ringer's lactate concentration (or a concentration of any of the afore mentioned lactate containing solutions) prior to lyophilization e.g. in the range of about 10% (w/w) to about 100% (w/w), e.g. in the range of about 20% (w/w) to about 100% (w/w), in the range of about 30% (w/w) to about 100% (w/w), in the range of about 40% (w/w) to about 100% (w/w), in the range of about 50% (w/w) to about 90% (w/w), preferably in the range of about 60% (w/w) to about 90% (w/w), more preferably in the range of about 70% (w/w) to about 90% (w/w), e.g. about 80% (w/w), of Ringer's lactate (or the afore mentioned lactate containing solution). In this context, Ringer's lactate (100 % (w/w)) is typically defined as a solution comprising 131 mM Na+, 5,36 mM K+, 1 ,84 mM Ca2+, and 28,3 mM Lactate).
The present invention is directed to a nucleic acid, which has been lyophilized or may be lyophilized from a lactate containing solution. In the context of the present invention, such a nucleic acid (sequence), lyophilized or to be lyophilized, may be any suitable nucleic acid, selected e.g. from any (double-stranded or single-stranded) DNA, preferably, without being limited thereto, e.g. genomic DNA, single-stranded DNA molecules, double-stranded DNA molecules, coding DNA, DNA primers, DNA probes, immunostimulatory DNA, a (short) DNA oligonucleotide ((short) oligodesoxyribonucleotides), or may be selected e.g. from any PNA (peptide nucleic acid) or may be selected e.g. from any (double-stranded or single-stranded) RNA, preferably, without being limited thereto, a (short) RNA oligonucleotide ((short) oligoribonucleotide), a coding RNA, a messenger RNA (mRNA), an immunostimulatory RNA, a siRNA, an antisense RNA, a micro RNA, or riboswitches, ribozymes or aptamers; etc. The nucleic acid (sequence), lyophilized or to be lyophilized, may also be a ribosomal RNA (rRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), or a viral RNA (vRNA). Preferably, the nucleic acid (sequence), lyophilized or to be lyophilized, is an RNA. More preferably, the nucleic acid (sequence), lyophilized or to be lyophilized, may be a (linear) single-stranded RNA, even more preferably an mRNA. In the context of the present invention, an mRNA is typically an RNA, which is composed of several structural elements, e.g. an optional 5'-UTR region, an upstream positioned ribosomal binding site followed by a coding region, an optional 3'-UTR region, which may be followed by a poly-A tail (and/or a poly-C-tail). An mRNA may occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA which carries the coding sequences of one, two or more proteins or peptides. Such coding sequences in di-, or even multicistronic mRNA may be separated by at least one IRES sequence, e.g. as defined herein.
Furthermore, the nucleic acid (sequence), lyophilized or to be lyophilized, may be a single- or a double-stranded nucleic acid (molecule) (which may also be regarded as a nucleic acid (molecule) due to non-covalent association of two single-stranded nucleic acid(s) (molecules)) or a partially double-stranded or partially single stranded nucleic acid, which are at least partially self complementary (both of these partially double-stranded or partially single stranded nucleic acid molecules are typically formed by a longer and a shorter single- stranded nucleic acid molecule or by two single stranded nucleic acid molecules, which are about equal in length, wherein one single-stranded nucleic acid molecule is in part complementary to the other single-stranded nucleic acid molecules molecule and both thus form a double-stranded nucleic acid molecules molecule in this region, i.e. a partially double-stranded or partially single stranded nucleic acid molecules). Preferably, the nucleic acid (sequence), lyophilized or to be lyophilized, may be a single-stranded nucleic acid molecule. Furthermore, the nucleic acid (sequence), lyophilized or to be lyophilized, may be a circular or linear nucleic acid molecule, preferably a linear nucleic acid molecule.
According to one alternative, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be a coding nucleic acid, e.g. a DNA or RNA. Such a coding DNA or RNA may be any DNA or RNA as defined above. Preferably, such a coding DNA or RNA may be a single- or a double-stranded DNA or RNA, more preferably a single-stranded DNA or RNA, and/or a circular or linear DNA or RNA, more preferably a linear DNA or RNA. Even more preferably, the coding DNA or RNA may be a (linear) single-stranded DNA or RNA. Most preferably, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be a ((linear) single-stranded) messenger RNA (mRNA). Such an mRNA may occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA which carries the coding sequences of one, two or more proteins or peptides. Such coding sequences in di-, or even multicistronic mRNA may be separated by at least one IRES sequence, e.g. as defined herein. Most preferably, the lyophilized nucleic acid, which has been lyophilized or which is to be lyophilized from a lactate containing solution, is an mRNA (sequence), preferably as defined herein. Accordingly, an inventive lyophilized mRNA (sequence) may be prepared, preferably using a method as described herein, e.g. by the use of a lactate containing solution as defined herein and an mRNA (sequence) as defined herein or a solution containing at least one mRNA (sequence) as defined herein and (free) lactate as defined herein, for lyophilization of at least one mRNA (sequence).
The nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may encode a protein or a peptide, which may be selected, without being restricted thereto, e.g. from therapeutically active proteins or peptides, from antigens, e.g. tumor antigens, pathogenic antigens (e.g. selected from pathogenic proteins as defined above or from animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens), autoimmune antigens, or further antigens, from allergens, from antibodies, from immunostimulatory proteins or peptides, from antigen-specific T-cell receptors, or from any other protein or peptide suitable for a specific (therapeutic) application, wherein the coding DNA or RNA may be transported into a cell, a tissue or an organism and the protein may be expressed subsequently in this cell, tissue or organism. a) Therapeutically active proteins
In this context, therapeutically active proteins may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention. These may be selected from any naturally occurring recombinant or isolated protein known to a skilled person from the prior art. Without being restricted thereto therapeutically active proteins may comprise proteins, capable of stimulating or inhibiting the signal transduction in the cell, e.g. cytokines, antibodies, etc. Therapeutically active proteins may thus comprise cytokines of class I of the family of cytokines, having 4 positionally conserved cysteine residues (CCCC) and comprising a conserved sequence motif Trp-Ser- X-Trp-Ser (WSXWS), wherein X is a non-conserved amino acid. Cytokines of class I of the family of cytokines comprise the GM-CSF subfamily, e.g. IL-3, IL-5, GM-CSF, the IL- 6-subfamily, e.g. IL-6, IL-1 1 , IL-12, or the IL-2-subfamily, e.g. IL-2, IL-4, IL-7, IL-9, IL-15, etc., or the cytokines IL-1 alpha, IL-1 beta, IL-10 etc. Therapeutically active proteins may also comprise cytokines of class II of the family of cytokines, which also comprise 4 positionally conserved cystein residues (CCCC), but no conserved sequence motif Trp- Ser-X-Trp-Ser (WSXWS). Cytokines of class II of the family of cytokines comprise e.g. IFN-alpha, IFN-beta, IFN-gamma, etc. Therapeutically active proteins may additionally comprise cytokines of the family of tumor necrose factors, e.g. TNF-alpha, TNF-beta, etc., or cytokines of the family of chemokines, which comprise 7 transmembrane helices and interact with G-protein, e.g. IL-8, MIP-1 , RANTES, CCR5, CXR4, etc., or cytokine specific receptors, such as TNF-RI, TNF-RII, CD40, OX40 (CD134), Fas, etc.
Therapeutically active proteins, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be selected from any of the proteins given in the following: 0ATL3, 0FC3, 0PA3, 0PD2, 4-1 BBL, 5T4, 6Ckine, 707-AP, 9D7, A2M, AA, AAAS, AACT, AASS, ABAT, ABCA1, ABCA4, ABCB1 , ABCB11, ABCB2, ABCB4, ABCB7, ABCC2, ABCC6, ABCC8, ABCD1, ABCD3, ABCG5, ABCG8, ABU, ABO, ABR ACAAl, ACACA, ACADL, ACADM, ACADS, ACADVL, ACAT1, ACCPN, ACE, ACHE, ACHM3, ACHM1, ACLS, ACPI, ACTA1, ACTC, ACTN4, ACVRL1, AD2, ADA, ADAMTS13, ADAMTS2, ADFN, ADH1B, ADH1C, ADLDH3A2, ADRB2, ADRB3, ADSL, AEZ, AFA, AFD1, AFP, AGA, AGL, AGMX2, AGPS, AGS1, AGT, AGTR1, AG XT, AH02, AHCY, AHDS, AHHR, AHSG, AIC, AIED, AIH2, AIH3, AIM-2, AIPL1, AIRE, AK1, ALAD, ALAS2, ALB, HPG1, ALDH2, ALDH3A2, ALDH4A1, ALDH5A1, ALDH1A1, ALDOA, ALDOB, ALMS1, ALPL, ALPP, ALS2, ALX4, AMACR, AM BP, AMCD, AMCD1, AMCN, AMELX, AMELY, AMGL, AMH, AMHR2, AMPD3, AMPD1, AMT, ANC, ANCR, ANK1, ANOP1, AOM, AP0A4, AP0C2, AP0C3, AP3B1, APC, aPKC, APOA2, APOA1, APOB, APOC3, APOC2, APOE, APOH, APP, APRT, APS1, AQP2, AR, ARAF1, ARG1, ARHGEF12, ARMET, ARSA, ARSB, ARSC2, ARSE, ART-4, ARTC1/m, ARTS, ARVD1, ARX, AS, ASAH, ASAT, ASD1, ASL, ASMD, ASMT, ASNS, ASPA, ASS, ASSP2, ASSP5, ASSP6, AT3, ATD, ATHS, ATM, ATP2A1, ATP2A2, ATP2C1, ATP6B1, ATP7A, ATP7B, ATP8B1, ATPSK2, ATRX, ATXN1, ATXN2, ATXN3, AUTS1, AVMD, AVP, AVPR2, AVSD1, AXIN1, AXIN2, AZF2, B2M, B4GALT7, B7H4, BAGE, BAGE-1, BAX, BBS2, BBS3, BBS4, BCA225, BCAA, BCH, BCHE, BCKDHA, BCKDHB, BCL10, BCL2, BCL3, BCL5, BCL6, BCPM, BCR, BCR/ABL, BDC, BDE, BDMF, BDMR, BEST1, beta-Catenin/m, BF, BFHD, BFIC, BFLS, BFSP2, BGLAP,BGN, BHD, BHR1, BING-4, BIRC5, BJS, BLM, BLMH, BLNK, BMPR2, BPGM, BRAF, BRCA1, BRCA1/m, BRCA2, BRCA2/m, BRCD2, BRCD1, BRDT, BSCL, BSCL2, BTAA, BTD, BTK, BUB1, BWS, BZX, C0L2A1, C0L6A1, C1NH, C1QA, C1QB, C1QG, C1S, C2, C3, C4A, C4B, C5, C6, C7, C7orf2, C8A, C8B, C9, CAT 25, CA15-3/CA 27-29, CA195, CA19-9, CA72-4, CA2, CA242, CA50, CABYR, CACD, CACNA2D1, CACNA1A, CACNA1F, CACNA1S, CACNB2, CACNB4, CAGE, CA1, CALB3, CALCA, CALCR, CALM, CALR, CAM43, CAMEL, CAP-1, CAPN3, CARD15, CASP-5/m, CASP-8, CASP-8/m, CASR, CAT, CATM, CAV3, CB1, CBBM, CBS, CCA1, CCAL2, CCAL1, CCAT, CCL-1, CCL-11, CCL-12, CCL- 13, CCL-14, CCL-15, CCL-16, CCL-17, CCL-18, CCL-19, CCL-2, CCL-20, CCL-21, CCL- 22, CCL-23, CCL-24, CCL-25, CCL-27, CCL-3, CCL-4, CCL-5, CCL-7, CCL-8, CCM1, CCNB1, CCND1, CCO, CCR2, CCR5, CCT, CCV, CCZS, CD1, CD19, CD20, CD22, CD25, CD27, CD27L, cD3, CD30, CD30, CD30L, CD33, CD36, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD44v, CD44v6, CD52, CD55, CD56, CD59, CD80, CD86, CDAN1, CDAN2, CDAN3, CDC27, CDC27/m, CDC2L1, CDH1, CDK4, CDK4/m, CDKN1C, CDKN2A, CDKN2Am, CDKN1 A, CDKN1C, CDL1, CDPD1, CDR1, CEA, CEACAMl , CEACAM5, CECR, CECR9, CEPA, CETP, CFNS, CFTR, CGF1, CHAC, CHED2, CHEDI, CHEK2, CHM, CHML, CHR39C, CHRNA4, CHRNA1, CHRNB1, CHRNE, CHS, CHS1, CHST6, CHX10, CIAS1, CIDX, CKN1, CLA2, CLA3, CLA1, CLCA2, CLCN1, CLCN5, CLCNKB, CLDN16, CLP, CLN2, CLN3, CLN4, CLN5, CLN6, CLN8, C1QA, C1QB, C1QG, C1R, CLS, CMCWTD, CMDJ, CMD1A, CMD1B, CMH2, MH3, CMH6, CMKBR2, CMKBR5, CML28, CML66, CMM, CMT2B, CMT2D, CMT4A, CMT1A, CMTX2, CMTX3, C-MYC, CNA1, CND, CNGA3, CNGA1, CNGB3, CNSN, CNTF, COA- 1/m, COCH, COD2, COD1, COH1, COL10A, COL2A2, COL11A2, COL17A1, COL1 Al, COL1A2, COL2A1, COL3A1, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL7A1, COL8A2, COL9A2, COL9A3, COL11A1, COL1A2, COL23A1, COL1A1, COLQ, COMP, COMT, CORD5, CORD1, COX10, COX-2, CP, CPB2, CPO, CPP, CPS1, CPT2, CPT1A, CPX, CRAT, CRB1, CRBM, CREBBP, CRH, CRHBP, CRS, CRV, CRX, CRYAB, CRYBA1, CRYBB2, CRYGA, CRYGC, CRYGD, CSA, CSE, CSF1 R, CSF2RA, CSF2RB, CSF3R, CSF1 R, CST3, CSTB, CT, CT7, CT- 9/BRD6, CTAA1, CTACK, CTEN, CTH, CTHM, CTLA4, CTM, CTNNB1, CTNS, CTPA, CTSB, CTSC, CTSK, CTSL, CTS1, CUBN, CVD1, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CYB5, CYBA, CYBB, CYBB5, CYFRA 21-1, CYLD, CYLD1, CYMD, CYP11B1, CYP11B2, CYP17, CYP17A1 , CYP19, CYP19A1, CYP1A2, CYP1B1, CYP21A2, CYP27A1, CYP27B1, CYP2A6, CYP2C, CYP2C19, CYP2C9, CYP2D, CYP2D6, CYP2D7P1, CYP3A4, CYP7B1, CYPBl, CYP11B1, CYP1A1, CYP1B1, CYRAA, D40,DADI, DAM, DAM-10/MAGE-B1 , DAM-6/MAGE-B2, DAX1, DAZ, DBA, DBH, DBI, DBT, DCC, DC- CK1, DCK, DCR, DCX, DDB 1, DDB2, DDIT3, DDU, DECR1, DEK-CAN, DEM, DES, DF,DFN2, DFN4, DFN6, DFNA4, DFNA5, DFNB5, DGCR, DHCR7, DHFR, DHOF, DHS, DIA1, DIAPH2, DIAPH1, DIH1, DIO1, DISCI, DKC1, DLAT, DLD, DLL3, DLX3, DMBT1, DMD, DM1, DMPK, DMWD, DNAI1, DNASE1, DNMT3B, DPEP1, DPYD, DPYS, DRD2, DRD4, DRPLA, DSCR1, DSG1, DSP, DSPP, DSS, DTDP2, DTR, DURS1, DWS, DYS, DYSF, DYT2, DYT3, DYT4, DYT2, DYT1, DYX1, EBAF, EBM, EBNA, EBP, EBR3, EBS1, ECA1, ECB2, ECE1, ECGF1, ECT, ED2, ED4, EDA, EDAR, ECA1, EDN3, EDNRB, EEC1, EEF1A1L14, EEGV1, EFEMP1, EFTUD2/m, EGFR, EGFR/Her1, EGI, EGR2, EIF2AK3, elF4G, EKV, El IS, ELA2, ELF2, ELF2M, ELK1, ELN, ELONG, EMD, EML1, EMMPRIN, EMX2, ENA-78, ENAM, END3, ENG, ENO1, ENPP1, ENUR2, ENUR1, EOS, EP300, EPB41, EPB42, EPCAM, EPD, EphAI, EphA2, EphA3, EphrinA2, EphrinA3, EPHX1, EPM2A, EPO,EPOR, EPX, ERBB2, ERCC2 ERCC3,ERCC4, ERCC5, ERCC6, ERVR, ESR1, ETFA, ETFB, ETFDH, ETM1, ETV6-AML1 , ETV1, EVC, EVR2, EVR1, EWSR1, EXT2, EXT3, EXT1, EYA1, EYCL2, EYCL3, EYCL1, EZH2, F10, F11, F12, F13A1, F13B, F2, F5, F5F8D, F7, F8, F8C, F9, FABP2, FACL6, FAH, FANCA, FANCB, FANCC, FANCD2, FANCF, FasL,FBN2, FBN1, FBP1, FCG3RA,FCGR2A, FCGR2B, FCGR3A, FCHL, FCMD, FCP1, FDPSL5, FECH, FEO, FEOM1, FES, FGA, FGB, FGD1, FGF2, FGF23, FGF5, FGFR2, FGFR3, FGFR1, FGG, FGS1, FH, FIC1, FIH, F2, FKBP6, FLNA, FLT4, FM03,FM04, FMR2, FMR1, FN, FN1/m, FOXC1, FOXE1, FOXL2, FOX01A, FPDMM, FPF, Fra-1, FRAXF, FRDA, FSHB, FSHMD1A, FSHR, FTH1, FTHU7, FTL, FTZF1, FUCA1, FUT2, FUT6, FUT1, FY, G250, G250/CAIX, G6PC, G6PD, G6PT1, G6PT2, GAA, GABRA3, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GALC, GALE, GALK1, GALNS, GALT, GAMT, GAN, GAST, GASTRIN 17, GATA3, GATA, GBA, GBE, GC, GCDH, GCGR, GCH1, GCK, GCP-2, GCS1, G-CSF, GCSH, GCSL, GCY, GDEP,GDF5, GDI1, GDNF, GDXY, GFAP, GFND, GGCX, GGT1, GH2, GH1, GHR, GHRHR, GHS, GIF, GINGF, GIP, GJA3, GJA8, GJB2, GJB3, GJB6, GJB1, GK, GLA, GLB, GLB1, GLC3B, GLC1B, GLC1C, GLDC, GLB, GLP1, GLRA1, GLUD1, GM1 (fuc-GMI), GM2A, GM-CSF, GMPR, GNAI2, GNAS, GNAT1, GNB3, GNE, GNPTA, GNRH, GNRH1, GNRHR, GNS, GnT-V, gplOO, GP1BA, GP1 BB, GP9, GPC3, GPD2, GPDS1, GPI, GP1 BA, GPN1 LW, GPNMB/m, GPSC, GPX1 , GRHPR, GRK1 , GROa, GROp, GROy, GRPR, GSE, GSM1, GSN, GSR, GSS, GTD, GTS, GUCA1A, GUCY2D, GULOP, GUSB, GUSM, GUST, GYPA, GYPC, GYS1, GYS2, H0KPP2, H0MG2, HADHA, HADHB, HAGE, HAGH, HAL, HAST-2, HB 1, HBA2, HBA1, HBB, HBBP1, HBD, HBE1, HBG2, HBG1, HBHR, HBP1, HBQ1, HBZ, HBZP, HCA, HCC-1, HCC-4, HCF2, HCG, HCL2, HCL1, HCR, HCVS, HD, HPN, HER2, HER2/NEU, HER3, HERV-K-MEL, HESX1, HEX A, HEXB, HF1, HFE, HF1, HGD, HHC2, HHC3, HHG, HK1 HLA-A, HLA-A*0201 -R170I, HLA-A11/m, HLA-A2/m, HLA-DPB1 HLA-DRA, HLCS, HLXB9, HMBS, HMGA2, HMGCL, HMI, HMN2, HMOX1, HMS1 HMW-MAA, HND, HNE, HNF4 , HOAC, HOMEOBOX NKX 3.1, HOM-TES-14/SCP-1, HOM-TES-85, HOXA1 HOXD13, HP, HPC1, HPD, HPE2, HPE1, HPFH, HPFH2, HPRT1, HPS1, HPT, HPV-E6, HPV-E7, HR, HRAS, HRD, HRG, HRPT2, HRPT1, HRX, HSD11B2, HSD17B3, HSD17B4, HSD3B2, HSD3B3, HSN1, HSP70-2M, HSPG2, HST-2, HTC2, HTC1, hTERT, HTN3, HTR2C, HVBS6, HVBS1, HVEC, HV1S, HYAL1, HYR, I-309, IAB, IBGC1, IBM2, ICAM1, ICAM3, iCE, ICHQ, ICR5, ICR1, ICS 1, IDDM2, IDDM1, IDS, IDUA, IF, IFNa/b, IFNGR1, IGAD1, IGER, IGF-1R, IGF2R, 1GF1, IGH, IGHC, 1GHG2, IGHG1, IGHM, IGHR, IG C, IHG1, IHH, IKBKG, IL1, IL-1 RA, IL10, IL-11, IL12, IL12RB1, IL13, IL-13Ra2, IL-15, IL-16, IL-17, IL18, IL-1a, IL-1a, IL-1 b, IL-1 p, IL1RAPL1, IL2, IL24, IL-2R, IL2RA, IL2RG, IL3, IL3RA,IL4, IL4R,IL4R, IL-5, IL6, IL-7, IL7R, IL-8, IL-9, Immature laminin receptor, IMMP2L, INDX, INFGR1, INFGR2, IN Fa, IFNp, INFy, INS, INSR, INVS, IP-10, IP2, IPF1, IP1, IRF6, IRS1, ISCW, ITGA2, ITGA2B, ITGA6, ITGA7, ITGB2, ITGB3, ITGB4, ITIH1, ITM2B, IV, IVD, JAG1, JAK3, JBS, JBTS1, JMS, JPD, AL1, KAL2, KALI, KLK2, KLK4, KCNA1, KCNE2, KCNE1, KCNH2, KCNJ1, KCNJ2, KCNJ1, KCNQ2, KCNQ3, KCNQ4, KCNQ1, KCS, KERA, KFM, KFS, KFSD, KHK, ki-67, KIAA0020, KIAA0205, KIAA0205/m, KIF1B, KIT, KK-LC-1, KLK3, KLKB1, KM-HN-1, KMS, KNG, KNO, K-RAS/m, KRAS2, KREV1, KRT1, KRT10, KRT12, KRT13, KRT14, KRT14L1, KRT14L2, KRT14L3,KRT16, KRT16L1, KRT16L2, KRT17, KRT18, KRT2A, KRT3, KRT4, KRT5, KRT6 A, KRT6B, KRT9, KRTHB1, KRTHB6, KRT1, KSA, KSS, KWE, KYNU, L0H19CR1, L1CAM, LAGE, LAGE-1, LALL, LAMA2, LAMA3, LAMB3, LAMB1, LAMC2, LAMP2, LAP, LCA5, LCAT, LCCS, LCCS 1, LCFS2, LCS1, LCT, LDHA, LDHB, LDHC, LDLR, LDLR/FUT, LEP, LEWISY, LGCR, LGGF-PBP, LGI1, LGMD2H, LGMD1A, LGMD1B, LHB, LHCGR, LHON, LHRH, LHX3, LIF, LIG1, LIMM, LIMP2, LIPA, LIPA, LIPB, LIPC, LIVIN, L1 CAM, LMAN1, LMNA, LMX1B, LOLR, LOR, LOX, LPA, LPL, LPP, LQT4, LRP5, LRS 1, LSFC, LT- β, LTBP2, LTC4S, LYL1, XCL1, LYZ, M344, MA50, MAA, MADH4, MAFD2, MAFD1, MAGE, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGEB1, MAGE-B10, MAGE-B16, MAGE-B17, 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-E2, MAGE-F1,MAGE-H1, MAGEL2, MGB1, MGB2, MAN2A1, MAN2B1, MANBA, MANBB, MAOA, MAOB, MAPK8IP1, MAPT, MART-1, MART-2, MART2/m, MAT1A, MBL2, MBP, MBS1, MC1 R, MC2R, MC4R, MCC, MCCC2, MCCC1, MCDR1, MCF2, MCKD, MCL1, MC1R, MCOLN1, MCOP, MCOR, MCP-1, MCP-2, MCP-3, MCP-4, MCPH2, MCPH1, MCS, M-CSF, MDB, MDCR, MDM2, MDRV, MDS 1, ME1, ME1/m, ME2, ME20, ME3, MEAX, MEB, MEC CCL-28, MECP2, MEFV, MELANA, MELAS, MEN1 MSLN, MET, MF4, MG50, MG50/PXDN, MGAT2, MGAT5, MGC1 MGCR, MGCT, MG1, MGP, MHC2TA, MHS2, MHS4, MIC2, M1C5, MIDI, MIF, MIP, MIP-5/HCC-2, MITF, MJD, MKI67, MKKS, MKS1, MLH1, MLL, MLLT2, MLLT3, MLLT7, MLLT1, MLS, MLYCD, MMAIa, MMP 11, MMVP1, MN/CA IX-Antigen, MNG1, MN1, MOC31, MOCS2, MOCS1, MOG, MORC, MOS, MOV18, MPD1, MPE, MPFD, MPl, MPIF-1, MPL, MPO, MPS3C, MPZ, MRE11A, MROS, MRP1, MRP2, MRP3, MRSD, MRX14, MRX2, MRX20, MRX3, MRX40, MRXA, MRX1, MS, MS4A2, MSD, MSH2, MSH3, MSH6, MSS, MSSE, MSX2, MSX1, MTATP6, MTC03, MTCO1, MTCYB, MTHFR, MTM1, MTMR2, MTND2, MTND4, MTND5, MTND6, MTND1, MTP, MTR, MTRNR2, MTRNR1, MTRR,MTTE, MTTG, MTTI, MTTK, MTTL2, MTTL1, MTTN, MTTP, MTTS1, MUC1,MUC2, MUC4, MUC5AC, MUM-1, MUM-1/m, MUM-2, MUM-2/m, MUM-3, MUM-3/m, MUT, mutant p21 ras, MUTYH, MVK, MX2, MXI1, MY05A, MYB, MYBPC3, MYC, MYCL2, MYH6, MYH7, MYL2, MYL3, MYMY, MYO15A, MYO1G, MYO5A, MYO7A, MYOC, Myosin/m, MYP2, MYP1, NA88-A, N-acetylglucosaminyltransferase-V, NAGA, NAGLU, NAMSD, NAPB, NAT2, NAT, NBIA1, NBS1, NCAM, NCF2, NCF1, NDN, NDP, NDUFS4, NDUFS7, NDUFS8, NDUFVl, NDUFV2, NEB, NEFH, NEM1, Neo-PAP, neo-PAP/m, NEU1, NEUROD1, NF2, NF1, NFYC/m, NGEP, NHS, NKS1, NKX2E, NM, NME1, NMP22, NMTC, NODAL, NOG, NOS3, NOTCH3, NOTCH 1, NP, NPC2, NPC1, NPHL2, NPHP1, NPHS2, NPHS1, NPM/ALK, NPPA, NQO1, NR2E3, NR3C1, NR3C2, NRAS, NRAS/m, NRL, NROB1, NRTN, NSE, NSX, NTRK1, NUMA1, NXF2, NY-COl, NY-ESO1, NY-ESO-B, NY-LU-12, ALDOA, NYS2, NYS4, NY-SAR-35, NYS1, NYX, OA3, OA1, OAP, OASD, OAT, OCA1, OCA2, OCD1, OCRL, OCRL1, OCT, ODDD, ODT1, OFC1, OFD1, OGDH, OGT, OGT/m, OPA2, OPA1, OPD1, OPEM, OPG, OPN, OPN1LW, OPN1MW, OPN1SW, OPPG, OPTB1, TTD, ORM1, ORP1, OS- 9, OS-9/m, OSM LIF, OTC, OTOF, OTSC1, OXCT1, OYTES1 , P15, PI 90 MINOR BCR- ABL, P2RY12, P3, P16, P40, P4HB, P-501, P53, P53/m, P97, PABPN1, PAFAH1B1, PAFAH1P1, PAGE-4, PAGE-5, PAH, PAI-1, PAI-2, PAK3, PAP, PAPPA, PARK2, PART-1, PATE, PAX2, PAX3, PAX6, PAX7, PAX8, PAX9, PBCA, PBCRA1, PBT, PBX1, PBXP1, PC, PCBD, PCCA, PCCB, PCK2, PCK1, PCLD, PCOS1 , PCSK1 , PDB1, PDCN, PDE6A, PDE6B, PDEF, PDGFB, PDGFR, PDGFRL, PDHA1, PDR, PDX1, PECAM1, PEE1, PEO1, PEPD, ΡΕΧΊ0, PEX12, PEX13, PEX3, PEX5, PEX6, PEX7, PEX1, PF4, PFBI, PFC, PFKFB1, PFKM, PGAM2, PGD, PGK1, PGK1P1, PGL2, PGR, PGS, PHA2A, PHB, PHEX, PHGDH, PHKA2, PHKA1, PHKB, PHKG2, PHP, PHYH, PI, PI3, PIGA, PIM1 -KINASE, PIN1, PIP5K1B, PITX2, PITX3, PKD2, PKD3, PKD1, PKDTS, PKHD1, PKLR, PKP1, PKU1, PLA2G2A, PLA2G7, PLAT, PLEC1, PLG, PLI, PLOD, PLP1, PMEL17, PML, PML/RARa, PMM2, PMP22, PMS2, PMS1, PNKD, PNLIP, POF1, POLA, POLH, POMC, PON2, PON1, PORC, POTE, POU1F1, POU3F4, POU4F3, POU1F1, PPAC, PPARG, PPCD, PPGB, PPH1, PPKB, PPMX, PPOX, PPP1R3A, PPP2R2B, PPT1, PRAME, PRB, PRB3, PRCA1 , PRCC, PRD, PRDX5/m, PRF1, PRG4, PRKAR1 A, PR CA, PRKDC, PRKWNK4, PRNP, PROC, PRODH, PROM1, PROP1, PROS1, PRST, PRP8, PRPF31, PRPF8, PRPH2, PRPS2, PRPS1, PRS, PRSS7, PRSS1, PRTN3, PRX, PSA, PSAP, PSCA, PSEN2, PSEN1, PSG1, PSGR, PSM, PSMA, PSORS1, PTC, PTCH, PTCH1, PTCH2, PTEN, PTGS1, PTH, PTHR1, PTLAH, PTOS1, PTPN12, PTPNI I, PTPRK, PTPRKm, PTS, PUJO, PVR, PVRL1, PWCR, PXE, PXMP3, PXR1, PYGL, PYGM, QDPR, RAB27A, RAD54B, RAD54L, RAG2, RAGE, RAGE-1, RAG1, RAP1, RARA, RASA1 , RBAF600/m, RB1, RBP4, RBP4, RBS, RCA1, RCAS1, RCCP2, RCD1, RCV1, RDH5, RDPA, RDS, RECQL2, RECQL3, RECQL4, REG1A, REHOBE, REN, RENBP, RENS1, RET, RFX5, RFXANK, RFXAP, RGR, RHAG, RHAMM/CD168, RHD, RHO, Rip-1, RLBP1, RLN2, RLN1, RLS, RMD1, RMRP, ROM1, ROR2, RP, RP1, RP14, RP17, RP2, RP6, RP9, RPD1, RPE65, RPGR, RPGRIP1, RP1, RP10, RPS19, RPS2, RPS4X, RPS4Y, RPS6KA3, RRAS2, RS1, RSN, RSS, RU1, RU2, RUNX2,RUNXI, RWS, RYR1, S-100, SAA1, SACS, SAG, SAGE, SALL1, SARDH, SART1, SART2, SART3, SAS, SAX1, SCA2, SCA4, SCA5, SCA7, SCA8, SCA1, SCC, SCCD, SCF, SCLC1, SCN1A, SCN1B, SCN4A, SCN5A, SCNN1A, SCNN1B, SCNN1G, SC02, SCP1, SCZD2, SCZD3, SCZD4, SCZD6, SCZD1, SDF-Ια/β, SDHA, SDHD, SDYS, SEDL, SERPENA7, SERPINA3, SERPINA6, SERPINA1, SERPINC1, SERPIND1, SERPINE1, SERPINF2, SERPING1, SERPINI1, SFTPA1, SFTPB, SFTPC, SFTPD, SGCA, SGCB, SGCD, SGCE, SGM1, SGSH, SGY-1, SH2D1A, SHBG, SHFM2, SHFM3, SHFM1, SHH, SHOX, SI, SIAL, SIALYL LEWISX, SIASD, S11, SIM1, SIRT2/m, SIX3, SJS1, SKP2, SLC10A2, SLC12A1, SLC12A3, SLC17A5, SLC19A2, SLC22A1L, SLC22A5, SLC25A13, SLC25A15, SLC25A20, SLC25A4, SLC25A5, SLC25A6, SLC26A2, SLC26A3, SLC26A4, SLC2A1, SLC2A2, SLC2A4, SLC3A1, SLC4A1, SLC4A4, SLC5A1, SLC5A5, SLC6A2, SLC6A3, SLC6A4, SLC7A7, SLC7A9, SLC11A1, SLOS, SMA, SMAD1, SMAL, SMARCB1, SMAX2, SMCR, SMCY, SM1, SMN2, SMN1, SMPD1, SNCA, SNRPN, SOD2, SOD3, SOD1, SOS1, SOST, SOX9, SOX10, Sp17, SPANXC, SPG23, SPG3A, SPG4, SPG5A, SPG5B, SPG6, SPG7, SPINK1, SPINK5, SPPK, SPPM, SPSMA, SPTA1, SPTB, SPTLC1, SRC, SRD5A2, SRPX, SRS, SRY, BhCG, SSTR2, SSX1, SSX2 (HOM-MEL-40/SSX2), SSX4, ST8, STAMP-1, STAR, STARP1, STATH, STEAP, STK2, STK11, STn/ KLH, STO, STOM, STS, SUOX, SURF1, SURVIVIN-2B, SYCP1, SYM1, SYNl, SYNS1, SYP, SYT/SSX, SYT-SSX-1, SYT-SSX-2, TA-90, TAAL6, TACSTD1, TACSTD2, TAG 72, TAF7L, TAF1, TAGE, TAG-72, TALI, TAM, TAP2, TAP1, TAPVR1, TARC, TARP, TAT, TAZ, TBP, TBX22, TBX3, TBX5, TBXA2R, TBXAS1 , TCAP, TCF2, TCF1 , TCIRG1 , TCL2, TCL4, TCL1 A, TCN2, TCOF1 , TCR, TCRA, TDD, TDFA, TDRD1 , TECK, TECTA, TEK, TEIJAML1 , TELAB1 , TEX15, TF, TFAP2B, TFE3, TFR2, TG, TGFA, TGF-β, TGFBI, TGFB1 , TGFBR2, TGFBRE, TGFp, TGFPRII, TGIF, TGM-4, TGM1 , TH, THAS, THBD, THC, THC2, THM, THPO, THRA, THRB, TIMM8A, TIMP2, TIMP3, ΤΙΜΡΊ , TITF1 , TKCR, TKT, TLP, TLR1 , TLR10, TLR2, TLR3, TLR4, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLX1 , TM4SF1 , TM4SF2, TMC1 , TMD, TMIP, TNDM, TNF, TNFRSF1 1 A, TNFRSF1 A, TNFRSF6, TNFSF5, TNFSF6, TNFa, TNF , TNNI3, TNNT2, TOC, TOP2A, TOP1 , TP53, TP63, TPA, TPBG, TPI, TPI/m, TPI1 , TPM3, TPM1 , TPMT, TPO, TPS, TPTA, TRA, TRAG3, TRAPPC2, TRC8, TREH, TRG, TRH, TRIM32, TRIM37, TRP1 , TRP2, TRP-2/6b, TRP-2/INT2, Trp-p8, TRPS1 , TS, TSC2, TSC3, TSC1 , TSG101 , TSHB, TSHR, TSP-180, TST, TTGA2B, TTN, TTPA, TTR, TU M2-PK, TULP1 , TWIST, TYH, TYR, TYROBP, TYROBP, TYRP1 , TYS, UBE2A, UBE3A, UBE1 , UCHL1 , UFS, UGT1 A, ULR, UMPK, UMPS, UOX, UPA, UQCRC1 , UR05, UROD, UPK1 B, UROS, USH2A, USH3A, USH1 A, USH1 C, USP9Y, UV24, VBCH, VCF, VDI, VDR, VEGF, VEGFR-2, VEGFR-1 , VEGFR-2/FLK-1 , VHL, VIM, VMD2, VMD1 , VMGLOM, VNEZ, VNF, VP, VRNI, VWF, VWS, WAS, WBS2, WFS2, WFS1 , WHCR, WHN, WISP3, WMS, WRN, WS2A, WS2B, WSN, WSS, WT2, WT3, WT1 , WTS, WWS, XAGE, XDH, XIC, XIST, XK, XM, XPA, XPC, XRCC9, XS, ZAP70, ZFHX1 B, ZFX, ZFY, ZIC2, ZIC3, ZNF145, ZNF261 , ZNF35, ZNF41 , ZNF6, ZNF198, ZWS1 .
Therapeutically active proteins, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may further be selected from apoptotic factors or apoptosis related proteins including AIF, Apaf e.g. Apaf-1 , Apaf-2, Apaf-3, oder APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl- xL, Bcl-xs, bik, CAD, Calpain, Caspase e.g. Caspase-1 , Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-1 1 , ced-3, ced-9, c-Jun, c-Myc, crm A, cytochrom C, CdR1 , DcR1 , DD, DED, DISC, DNA-PKcS, DR3, DR4, DR5, FADD/MORT-1 , FAK, Fas (Fas-ligand CD95/fas (receptor)), FLICE/MACH, FLIP, fodrin, fos, G-Actin, Gas-2, gelsolin, granzyme A/B, ICAD, ICE, JNK, lamin A/B, MAP, MCL-1 , Mdm-2, MEKK-1 , MORT-1 , NEDD, NF-kappaB, NuMa, p53, PAK- 2, PARP, perforin, PITSLRE, PKCdelta, pRb, presenilin, prICE, RAIDD, Ras, RIP, sphingomyelinase, thymidinkinase from herpes simplex, TRADD, TRAF2, TRAIL-R1 , TRAIL-R2, TRAIL-R3, transglutaminase, etc. A therapeutically active protein, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, can also be an adjuvant protein. In this context, an adjuvant protein is preferably to be understood as any protein, which is capable to elicit an innate immune response as defined herein. Preferably, such an innate immune response comprises activation of a pattern recognition receptor, such as e.g. a receptor selected from the Toll-like receptor (TLR) familiy, including e.g. a Toll like receptor selected from human TLR1 to TLR10 or from murine Toll like receptors TLR1 to TLR13. Preferably, an innate immune response is elicited in a mammal as defined above. More preferably, the adjuvant protein is selected from human adjuvant proteins or from pathogenic adjuvant proteins, in particular from bacterial adjuvant proteins. In addition, mRNA encoding huma proteins involved in adjuvant effects may be used as well.
Human adjuvant proteins, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, typically comprise any huma protein, which is capable of eliciting an innate immune response (in a mammal), e.g. as a reaction of the binding of an exogenous TLR ligand to a TLR. More preferably, human adjuvant proteins encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be selected from the group consisting of, without being limited thereto, cytokines which induce or enhance an innate immune response, including IL-2, IL-12, IL-15, IL-18, IL-21 CCL21, GM-CSF and TNF-alpha; cytokines which are released from macrophages, including IL-1 , IL-6, IL-8, IL- 12 and TNF-alpha; from components of the complement system including C1 q, MBL, C1 r, C1 s, C2b, Bb, D, MASP-1 , MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1 INH, C4bp, MCP, DAF, H, I, P and CD59; from proteins which are components of the signalling networks of the pattern recognition receptors including TLR and IL-1 R1 , whereas the components are ligands of the pattern recognition receptors including IL-1 alpha, IL-1 beta, Beta-defensin, heat shock proteins, such as HSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, Typlll repeat extra domain A of fibronectin; the receptors, including IL-1 RI, TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; the signal transducers including components of the Small-GTPases signalling (RhoA, Ras, Rac1 , Cdc42 etc.), components of the PIP signalling (PI3K, Src-Kinases, etc.), components of the MyD88-dependent signalling (MyD88, IRAKI, IRAK2, etc.), components of the MyD88-independent signalling (TICAM1 , TICAM2 etc.); activated transcription factors including e.g. NF-κΒ, C-FOS, c-Jun, c-Myc; and induced target genes including e.g. IL-1 alpha, 1L-1 beta, Beta-Defensin, IL-6, IFN gamma, IFN alpha and IFN beta; from costimulatory molecules, including CD28 or CD40-ligand or PDl ; protein domains, including LAMP; cell surface proteins; or human adjuvant proteins including CD80, CD81 , CD86, trif, flt-3 ligand, thymopentin, Gp96 or fibronectin, etc., or any species homolog of any of the above human adjuvant proteins.
Pathogenic adjuvant proteins, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, typically comprise any pathogenic (adjuvant) protein, which is capable of eliciting an innate immune response (in a mammal), more preferably selected from pathogenic (adjuvant) proteins derived from bacteria, protozoa, viruses, or fungi, animals, etc., and even more preferably from pathogenic adjuvant proteins selected from the group consisting of, without being limited thereto, bacterial proteins, protozoa proteins (e.g. profilin - like protein of Toxoplasma gondii), viral proteins, or fungal proteins, animal proteins, etc.
In this context, bacterial (adjuvant) proteins, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may comprise any bacterial protein, which is capable of eliciting an innate immune response (preferably in a mammal) or shows an adjuvant character. More preferably, such bacterial (adjuvant) proteins are selected from the group consisting of bacterial heat shock proteins or chaperons, including Hsp60, Hsp70, Hsp90, Hsp100; OmpA (Outer membrane protein) from gram-negative bacteria; bacterial porins, including OmpF; bacterial toxins, including pertussis toxin (PT) from Bordetella pertussis, pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis, PT-9K 129G mutant from pertussis toxin, pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis, tetanus toxin, cholera toxin (CT), cholera toxin B-subunit, CTK63 mutant from cholera toxin, CTE1 12K mutant from CT, Escherichia coli heat-labile enterotoxin (LT), B subunit from heat-labile enterotoxin (LTB) Escherichia coli heat-labile enterotoxin mutants with reduced toxicity, including LTK63, LTR72; phenol-soluble modulin; neutrophil-activating protein (HP-NAP) from Helicobacter pylori; Surfactant protein D; Outer surface protein A lipoprotein from Borrelia burgdorferi, Ag38 (38 kDa antigen) from Mycobacterium tuberculosis; proteins from bacterial fimbriae; Enterotoxin CT of Vibrio cholerae, Pi lin from pili from gram negative bacteria, and Surfactant protein A; etc., or any species homolog of any of the above bacterial (adjuvant) proteins.
Bacterial (adjuvant) proteins, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may also be selected from bacterial adjuvant proteins, even more preferably selected from the group consisting of, without being limited thereto, bacterial flagellins, including flagellins from organisms including Agrobacterium, Aquifex, Azospirillum, Bacillus, Bartonella, Bordetella, Borrelia, Burkholderia, Campylobacter, Caulobacte, Clostridium, Escherichia, Helicobacter, Lachnospiraceae, Legionella, Listeria, Proteus, Pseudomonas, Rhizobium, Rhodobacter, Roseburia, Salmonella, Serpulina, Serratia, Shigella, Treponema, Vibrio, Wolinella, Yersinia, more preferably flagellins from the species, without being limited thereto, Agrobacterium tumefaciens, Aquifex pyrophilus, Azospirillum brasilense, Bacillus subtilis, Bacillus thuringiensis, Bartonella bacilliformis, Bordetella bronchiseptica, Borrelia burgdorferi, Burkholderia cepacia, Campylobacter jejuni, Caulobacter crescentus, Clostridium botulinum strain Bennett clone I, Escherichia coli, Helicobacter pylori, Lachnospiraceae bacterium, Legionella pneumophila, Listeria monocytogenes, Proteus mirabilis, Pseudomonas aeroguinosa, Pseudomonas syringae, Rhizobium meliloti, Rhodobacter sphaeroides, Roseburia cecicola, Roseburis hominis, Salmonella typhimurium, Salmonella bongori, Salmonella typhi, Salmonella enteritidis, Serpulina hyodysenteriae, Serratia marcescens, Shigella boydii, Treponema phagedenis, Vibrio alginolyticus, Vibrio cholerae, Vibrio parahaemolyticus, Wolinella succinogenes and Yersinia enterocolitica. Bacterial flagellins, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, even more preferably comprise a sequence selected from the group comprising any of the following sequences as referred to their accession numbers: organism species gene name accession No Gl No
Agrobacterium Agrobacterium FlaD (flaD) U95165 Gl:14278870
tumefaciens FlhB (flhB)
FliG (fliG)
FliN (fliN)
FliM (fliM)
MotA (motA)
FlgF (flgF) Flil (flil)
FIgB (flgB)
FIgC (flgC)
FliE (fliE)
FIgG (flgG)
FIgA (flgA)
FIgl (flgl)
FlgH (flgH)
FliL (fliL)
FliP (fliP)
FlaA (flaA)
FlaB (flaB)
FlaC (flaC)
Aquifex Aquifex pyrophilus U1 7575 Gl:596244
Azospirillum Azospirillum Laf1 U26679 Gl:1 1 73509 brasilense
Bacillus Bacillus subtilis hag AB033501 Gl:14278870
Bacillus Bacillus flab X67138 Gl:46019718 thuringiensis
Bartonella Bartonella L20677 Gl:304184 bacilliformis
Bordetella Bordetella flaA L13034 Gl:289453 bronchiseptica
Borrelia Borrelia burgdorferi X16833 Gl:39356
Burkholderia Burkholderia fliC AF01 1370 Gl:2935154 cepacia
Campylobacter Campylobacter flaA J05635 Gl:144197 jejuni flaB
Caulobacter Caulobacter 101556 Gl:144239 crescentus
Clostridium Clostridium FlaA DQ845000 Gl:1 14054886 botulinum strain
Bennett clone 1
Escherichia Escherichia coli hag M14358 Gl:14631 1
AJ 884569
(E BL-SVA)
Helicobacter Helicobacter pylori flaA X60746 Gl:43631
Lachnospiraceae Lachnospiraceae DQ789131 Gl:1 1391 1615 bacterium
Legionella Legionella flaA X83232 GL602877 pneumophila
Listeria Listeria flaA X65624 GL44097 monocytogenes
Proteus Proteus mirabilis FlaD (flaD) AF221596 GI.6959881
FlaA (flaA)
FlaB (flaB)
FliA (fliA) FliZ
(fliZ)
Pseudomonas Pseudomonas flaA M57501 Gl:151225 aeroguinosa
Pseudomonas Pseudomonas fliC EF544882 Gl:146335619 syringae
Rhizobium Rhizobium meliloti flaA M24526 Gl:152220 flaB
Rhodobacter Rhodobacter fliC AF274346 Gl:10716972 sphaeroides
Roseburia Roseburia cecicola M20983 Gl:152535
Roseburia Roseburis hominis Fla2 DQ789141 Gl:1 1391 1632
Salmonella Salmonella D13689 (NCBI Gl:217062
typhimurium ID)
Salmonella Salmonella bongori fliC AY603412 Gl:51342390
Salmonella Salmonella typhi flag L21912 Gl:397810
Salmonella Salmonella fliC M84980 Gl:154015
enteritidis
Serpulina Serpulina flaB2 X63513 Gl:450669
hyodysenteriae
Serratia Serratia hag M27219 Gl:152826
marcescens
Shigella Shigella boydii fliC-SB D26165 Gl:442485
Treponema Treponema flaB2 M94015 Gl:155060
phagedenis
Vibrio Vibrio alginolyticus flaA EF125175 Gl:119434395
Vibrio s Vibrio AF069392 Gl:7327274 parahaemolyticus
Wolinella Wolinella flag M82917 Gl:155337
succinogenes
Yersinia Yersinia L33467 Gl:496295
enterocolitica
Protozoa proteins, which may also be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may be selected from any protozoa protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii, Protozoan heat shock proteins, LeIF from Leishmania spp., profi linlike protein from Toxoplasma gondii, etc.
Viral proteins, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may be selected from any viral protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Respiratory Syncytial Virus fusion glycoprotein (F-protein), envelope protein from MMT virus, mouse leukemia virus protein, Hemagglutinin protein of wild type measles virus, etc.
Fungal proteins, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may be selected from any fungal protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, fungal immunomodulatory protein (FIP; LZ-8), etc. Finally, pathogenic adjuvant proteins, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may finally be selected from any further pathogenic protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Keyhole limpet hemocyanin (KLH), OspA, etc. b) Antigens
The nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may alternatively encode an antigen. According to the present invention, the term "antigen" refers to a substance which is recognized by the immune system and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies as part of an adaptive immune response. In this context, the first step of an adaptive immune response is the activation of naive antigen-specific T cells by antigen- presenting cells. This occurs in the lymphoid tissues and organs through which naive T cells are constantly passing. The three cell types that can serve as antigen-presenting cells are dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses. Tissue dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by infection to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents to express MHC class II molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may be important to induce T cells. By presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells. The most important function of effector T cells is the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by TH1 cells which together make up cell-mediated immunity, and the activation of B cells by both TH2 and TH1 cells to produce different classes of antibody, thus driving the humoral immune response. T cells recognize an antigen by their T cell receptors which does not recognize and bind antigen directly, but instead recognize short peptide fragments e.g. of pathogens' protein antigens, which are bound to MHC molecules on the surfaces of other cells. T cells fall into two major classes that have different effector functions. The two classes are distinguished by the expression of the cell-surface proteins CD4 and CD8. These two types of T cells differ in the class of MHC molecule that they recognize. There are two classes of MHC molecule- MHC class I and MHC class II- which differ in their structure and expression pattern on tissues of the body. CD4+ T cells bind to the MHC class II molecule and CD8+ T cells to the MHC class I molecule. MHC class I and MHC class II have distinct distributions among cells that reflect the different effector functions of the T cells that recognize them. MHC class I molecules present peptides from pathogens, commonly viruses to CD8+ T cells, which differentiate into cytotoxic T cells that are specialized to kill any cell that they specifically recognize. Almost all cells express MHC class I molecules, although the level of constitutive expression varies from one cell type to the next. But not only pathogenic peptides from viruses are presented by MHC class I molecules, also self-antigens like tumour antigens are presented by them. MHC class I molecules bind peptides from proteins degraded in the cytosol and transported in the endoplasmic reticulum. Thereby MHC class I molecules on the surface of cells infected with viruses or other cytosolic pathogens display peptides from these pathogen. The CD8+ T cells that recognize MHC class hpeptide complexes are specialized to kill any cells displaying foreign peptides and so rid the body of cells infected with viruses and other cytosolic pathogens. The main function of CD4+ T cells (CD4+ helper T cells) that recognize MHC class II molecules is to activate other effector cells of the immune system. Thus MHC class II molecules are normally found on B lymphocytes, dendritic cells, and macrophages, cells that participate in immune responses, but not on other tissue cells. Macrophages, for example, are activated to kill the intravesicular pathogens they harbour, and B cells to secrete immunoglobulins against foreign molecules. MHC class II molecules are prevented from binding to peptides in the endoplasmic reticulum and thus MHC class II molecules bind peptides from proteins which are degraded in endosomes. They can capture peptides from pathogens that have entered the vesicular system of macrophages, or from antigens internalized by immature dendritic cells or the immunoglobulin receptors of B cells. Pathogens that accumulate in large numbers inside macrophage and dendritic cell vesicles tend to stimulate the differentiation of TH1 cells, whereas extracellular antigens tend to stimulate the production of TH2 cells. TH1 cells activate the microbicidal properties of macrophages and induce B cells to make IgG antibodies that are very effective of opsonising extracellular pathogens for ingestion by phagocytic cells, whereas TH2 cells initiate the humoral response by activating naive B cells to secrete IgM, and induce the production of weakly opsonising antibodes such as IgGI and lgG3 (mouse) and lgG2 and lgG4 (human) as well as IgA and IgE (mouse and human).
In the context of the present invention, antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention typically comprise any antigen, falling under the above definition, more preferably protein and peptide antigens, e.g. tumor antigens, allergy antigens, auto-immune self-antigens, pathogens, etc. In accordance with the invention, antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be antigens generated outside the cell, more typically antigens not derived from the host organism (e.g. a human) itself (i.e. non-self antigens) but rather derived from host cells outside the host organism, e.g. viral antigens, bacterial antigens, fungal antigens, protozoological antigens, animal antigens (preferably selected from animals or organisms as disclosed herein), allergy antigens, etc. Allergy antigens are typically antigens, which cause an allergy in a human and may be derived from either a human or other sources. Antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be furthermore antigens generated inside the cell, the tissue or the body, e.g. by secretion of proteins, their degradation, metabolism, etc. Such antigens include antigens derived from the host organism (e.g. a human) itself, e.g. tumor antigens, self-antigens or auto-antigens, such as auto-immune self-antigens, etc., but also (non-self) antigens as defined above, which have been originally been derived from host cells outside the host organism, but which are fragmented or degraded inside the body, tissue or cell, e.g. by (protease) degradation, metabolism, etc.
One class of antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention comprises tumor antigens. "Tumor antigens" are preferably located on the surface of the (tumor) cell. Tumor antigens may also be selected from proteins, which are overexpressed in tumor cells compared to a normal cell. Furthermore, tumor antigens also includes antigens expressed in cells which are (were) not themselves (or originally not themselves) degenerate but are associated with the supposed tumor. Antigens which are connected with tumor-supplying vessels or (re)formation thereof, in particular those antigens which are associated with neovascularization, e.g. growth factors, such as VEGF, bFGF etc., are also included herein. Antigens connected with a tumor furthermore include antigens from cells or tissues, typically embedding the tumor. Further, some substances (usually proteins or peptides) are expressed in patients suffering (knowingly or not-knowingly) from a cancer disease and they occur in increased concentrations in the body fluids of said patients. These substances are also referred to as "tumor antigens", however they are not antigens in the stringent meaning of an immune response inducing substance. The class of tumor antigens can be divided further into tumor-specific antigens (TSAs) and tumor-associated- antigens (TAAs). TSAs can only be presented by tumor cells and never by normal "healthy" cells. They typically result from a tumor specific mutation. TAAs, which are more common, are usually presented by both tumor and healthy cells. These antigens are recognized and the antigen-presenting cell can be destroyed by cytotoxic T cells. Additionally, tumor antigens can also occur on the surface of the tumor in the form of, e.g., a mutated receptor. In this case, they can be recognized by antibodies.
Examples of tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention are shown in Tables 1 and 2 below. These tables illustrate specific (protein) antigens (i.e. "tumor antigens") with respect to the cancer disease, they are associated with. According to the invention, the terms "cancer diseases" and "tumor diseases" are used synonymously herein.
Table 1 : Antigens expressed in cancer diseases
Tumor antigen Name of tumor antigen Cancers or cancer diseases related thereto
colorectal cancer, gastric cancer,
5T4 ovarian cancer
707-AP 707 alanine proline Melanoma
9D7 renal cell carcinoma
hepatocellular carcinoma, gallbladder cancer, testicular cancer,
AFP alpha-fetoprotein ovarian cancer, bladder cancer
AlbZIP HPG1 prostate cancer
alpha5beta1 - Integrin
alpha5beta6- Integrin colon cancer
alpha- methylacyl- coenzyme A prostate cancer racemase
lung cancer, head and neck cancer, leukemia, esophageal cancer, gastric cancer, cervical cancer, ovarian adenocarcinoma antigen cancer, breast cancer, squamous cell
ART-4 recognized by T cells 4 carcinoma
B7H4 ovarian cancer
bladder cancer, head and neck cancer, lung cancer, melanoma,
BAGE-1 B antigen squamous cell carcinoma
BCL-2 leukemia
BING-4 melanoma
CA 15-3/CA 27- breast cancer, ovary cancer, lung 29 cancer, prostate cancer
gastric cancer, pancreatic cancer, liver cancer, breast cancer, gallbladder cancer, colon cancer,
CA 19-9 ovary cancer, lung cancer
CA 72-4 ovarian cancer
ovarian cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, uterus cancer, cervix carcinoma, colon cancer,
CA125 breast cancer, lung cancer calreticulin bladder cancer
CTL-recognized antigen on
CAMEL melanoma melanoma
CASP-8 caspase-8 head and neck cancer
cathepsin B breast cancer
cathepsin L breast cancer
CD19 B-cell malignancies
CD20
CD22
CD25
CD30
CD33
CD4
CD52
CD55
CD56
CD80
gut carcinoma, colorectal cancer, colon cancer, hepatocellular cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, liver cancer cervix cancer, bladder
CEA carcinoembryonic antigen cancer, melanoma
calcium-activated chloride
CLCA2 channel-2 lung cancer
CML28 leukemia
Coactosin-like
protein pancreatic cancer
Collagen XXIII prostate cancer ovarian cancer, breast cancer,
COX-2 colorectal cancer
bromodomain testis-specific
CT-9/BRD6 protein
Cten C-terminal tensin-like protein prostate cancer
cyclin B1
cyclin D1 ovarian cancer
bladder cancer, lung cancer, T-cell cyp-B cyclophilin B leukemia, squamous cell carcinoma,
CYPB1 cytochrom P450 1 B1 leukemia
DAM-10/MAGE- differentiation antigen melanoma melanoma, skin tumors, ovarian B1 10 cancer, lung cancer
DAM-6/MAGE- differentiation antigen melanoma melanoma, skin tumors, ovarian B2 6 cancer, lung cancer
lung cancer, ovarian cancer, head and neck cancer, colon cancer,
EGFR Her1 pancreatic cancer, breast cancer lung cancer, breast cancer, bladder tumor cell-associated extracellular cancer, ovarian cancer, brain
EM PRIN matrix metalloproteinase inducer/ cancer, lymphoma
ovarian cancer, breast cancer, colon
EpCam epithelial cell adhesion molecule cancer, lung cancer
EphA2 ephrin type-A receptor 2 glioma
EphA3 ephrin type-A receptor 2 melanoma, sarcoma, lung cancer
ErbB3 breast cancer
endometrium cancer, melanoma,
EZH2 (enhancer of Zeste homolog 2) prostate cancer, breast cancer
renal cell carcinoma, breast cancer,
FGF-5 fibroblast growth factor-5 prostate cancer
FN fibronectin melanoma
breast cancer, esophageal cancer,
Fra-1 Fos-related antigen- 1 renal cell carcinoma, thyroid cancer leukemia, renal cell carcinoma, head and neck cancer, colon cancer,
G250/CAIX glycoprotein 250 ovarian cancer, cervical cancer bladder cancer, lung cancer, sarcoma, melanoma, head and neck
GAGE-1 G antigen 1 cancer
bladder cancer, lung cancer, sarcoma, melanoma, head and neck
GAGE-2 G antigen 2 cancer
bladder cancer, lung cancer, sarcoma, melanoma, head and neck
GAGE-3 G antigen 3 cancer
bladder cancer, lung cancer, sarcoma, melanoma, head and neck
GAGE-4 G antigen 4 cancer
bladder cancer, lung cancer, sarcoma, melanoma, head and neck
GAGE-5 G antigen 5 cancer
bladder cancer, lung cancer, sarcoma, melanoma, head and neck
GAGE-6 G antigen 6 cancer bladder cancer, lung cancer,
sarcoma, melanoma, head and neck
GAGE-7b G antigen 7b cancer
bladder cancer, lung cancer, sarcoma, melanoma, head and neck
CAGE-8 G antigen 8 cancer
gene differentially expressed in
GDEP prostate prostate cancer
GnT-V N-acetylglucosaminyltransferase V glioma, melanoma
SP100 glycoprotein 100 kDa melanoma
hepatocellular carcinoma,
GPC3 glypican 3 melanoma
HAGE helicase antigen bladder cancer
HAST-2 human signet ring tumor-2
hepsin prostate
breast cancer, bladder cancer, human epidermal receptor- melanoma, ovarian cancer, pancreas
Her2/neu/ErbB2 2/neurological cancer, gastric cancer
HERV-K-MEL melanoma
HNE human neutrophil elastase leukemia
homeobox NKX
3.1 prostate cancer
HOM-TES-
1 /SCP-1 ovarian cancer
HOM-TES-85
HPV-E6 cervical cancer
HPV-E7 cervical cancer
HST-2 gastric cancer
breast cancer, melanoma, lung cancer, ovarian cancer, sarcoma, human telomerase reverse Non-Hodgkin-lymphoma, acute hTERT transcriptase leukemia
iCE intestinal carboxyl esterase renal cell carcinoma
IGF-1 R colorectal cancer
interleukin 13 receptor alpha 2
IL-13Ra2 chain glioblastoma
IL-2R colorectal cancer
IL-5
immature laminin
receptor renal cell carcinoma
kallikrein 2 prostate cancer
kallikrein 4 prostate cancer
prostate cancer, breast cancer, Non-
Ki67 Hodgkin-lymphoma, melanoma IAA0205 bladder cancer
K -LC-1 Kita-kyushu lung cancer antigen 1 lung cancer
tongue cancer, hepatocellular carcinomas, melanoma, gastric cancer, esophageal, colon cancer, M-HN-1 pancreatic cancer
bladder cancer, head and neck
LAGE-1 L antigen cancer, melanoma
livin bladder cancer, melanoma bladder cancer, head and neck cancer, melanoma, colon cancer,
MAGE-A1 melanoma antigen-A1 lung cancer, sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
MAGE-A10 melanoma antigen-A10 lung cancer, sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer, lung cancer, sarcoma, leukemia, prostate cancer, myeloma, brain
MAGE-A12 melanoma antigen-A12 tumors
bladder cancer, head and neck cancer, melanoma, colon cancer,
MAGE-A2 melanoma antigen-A2 lung cancer, sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
MAGE- A3 melanoma antigen-A3 lung cancer, sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
MAGE-A4 melanoma antigen-A4 lung cancer, sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
MAGE-A6 melanoma antigen-A6 lung cancer, sarcoma, leukemia bladder cancer, head and neck cancer, melanoma, colon cancer,
MAGE-A9 melanoma-antigen-A9 lung cancer, sarcoma, leukemia
MAGE-B1 melanoma-antigen-B1 melanoma
MAGE-B10 melanoma-antigen-B10 melanoma
MAGE-B16 melanoma-antigen-B16 melanoma
MAGE-B17 melanoma-antigen-B17 melanoma
MAGE-B2 melanoma-antigen-B2 melanoma
MAGE-B3 melanoma-antigen-B3 melanoma
MAGE-B4 melanoma-antigen-B4 melanoma
MAGE-B5 melanoma-antigen-B5 melanoma
MAGE-B6 melanoma-antigen-B6 melanoma
MAGE-C1 melanoma-antigen-C1 bladder cancer, melanoma
MAGE-C2 melanoma-antigen-C2 melanoma
MAGE-C3 melanoma-antigen-C3 melanoma
MAGE-D1 melanoma-antigen-D1 melanoma
MAGE-D2 melanoma-antigen-D2 melanoma
MAGE-D4 melanoma-antigen-D4 melanoma
MAGE-E1 melanoma-antigen-E1 bladder cancer, melanoma
MAGE-E2 melanoma-antigen-E2 melanoma
MAGE-F1 melanoma-antigen-F1 melanoma
MAGE-H1 melanoma-antigen-H 1 melanoma
MAGEL2 MAGE- 1 ike 2 melanoma
mammaglobin A breast cancer
melanoma antigen recognized by
MART-1/Melan-A T cells-1/melanoma antigen A melanoma
melanoma antigen recognized by
MART-2 T cells-2 melanoma
matrix protein 22 bladder cancer
MC1 R melanocortin 1 receptor melanoma
Figure imgf000034_0001
PDEF prostate cancer
Pim-1 -Kinase
Pinl Propyl isomerase prostate cancer
POTE prostate cancer
melanoma, lung cancer, leukemia, preferentially expressed antigen of head and neck cancer, renal cell
P AME melanoma carcinoma, sarcoma
prostein prostate cancer
proteinase-3
PSA prostate-specific antigen prostate cancer
PSCA prostate cancer
PSGR prostate cancer
PSM
prostate-specific membrane
PSMA antigen prostate cancer
bladder cancer, renal cancer,
RAGE-1 renal antigen sarcoma, colon cancer
receptor for hyaluronic acid
RHAMM/CD1 68 mediated motility leukemia
bladder cancer, melanoma, renal
RU1 renal ubiquitous 1 cancer
bladder cancer, melanoma, sarcoma, brain tumor, esophagel cancer, renal
RU2 renal ubiquitous 1 cancer, colon cancer, breast cancer
S-100 melanoma
SAGE sarcoma antigen
squamous antigen rejecting tumor esophageal cancer, head and neck
SART-1 1 cancer, lung cancer, uterine cancer head and neck cancer, lung cancer, squamous antigen rejecting tumor renal cell carcinoma, melanoma,
SART-2 1 brain tumor
head and neck cancer, lung cancer, squamous antigen rejecting tumor leukemia, melanoma, esophageal
SART-3 1 cancer
see squamous cell carcinoma antigen lung cancer
Sp1 7 sperm protein 1 7 multiple myeloma
hepatocellular cell carcinom, breast
SSX-1 synovial sarcoma X breakpoint 1 cancer
SSX-2/HOM-
MEL-40 synovial sarcoma X breakpoint 2 breast cancer
bladder cancer, hepatocellular cell
SSX-4 synovial sarcoma X breakpoint 4 carcinoma, breast cancer
STAMP-1 prostate cancer
six transmembrane epithelial
STEAP antigen prostate prostate cancer
survivin bladder cancer
survivin-2B intron 2-retaining survivin bladder cancer
TA-90 melanoma
TAG-72 prostate carcinoma
TARP prostate cancer
TGFb TGFbeta
TCFbRII TGFbeta receptor II
TGM-4 prostate-specific transglutaminase prostate cancer breast cancer, leukemia, and
TRAG-3 taxol resistant associated protein 3 melanoma
TRC testin-related gene
TRP-1 tyrosine related protein 1 melanoma
TRP-2/6b TRP-2/novel exon 6b melanoma, glioblastoma
TRP-2/INT2 TRP-2/intron 2 melanoma, glioblastoma
Trp-p8 prostate cancer
Tyrosinase melanoma
urokinase-type plasminogen
UPA activator breast cancer
VEGF vascular endothelial growth factor
vascular endothelial growth factor
VEGFR-2/FLK-1 receptor-2
gastric cancer, colon cancer, lung cancer, breast cancer, ovarian
WT1 Wilm' tumor gene cancer, leukemia
Table 2: Mutant antigens expressed in cancer diseases
Figure imgf000036_0001
Figure imgf000037_0001
In a preferred aspect of the present invention, the tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention are selected from the group consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1 , alpha-5-beta-1 -integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha- methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1 , BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1 , cyclin D1 , cyp-B, CYPB1 , DAM-10, DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6- AML1 , EZH2, FGF-5, FN, Frau-1 , G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201 -R17I, HLA-A1 1/m, HLA-A2/m, HNE, homeobox NKX3.1 , HOM-TES-14/SCP-1 , HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1 R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein- 2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m, KK-LC-1 , K-Ras/m, LAGE-A1 , LDLR-FUT, MAGE-A1 , MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE- A12, MAGE-B1 , MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B1 6, MAGE-B1 7, MAGE-C1 , MAGE-C2, MAGE-C3, MAGE-D1 , MAGE-D2, MAGE-D4, MAGE-E1 , MAGE-E2, MAGE-F1 , MAGE-H1 , MAGEL2, mammaglobin A, MART-1 /melan-A, MART-2, MART-2/m, matrix protein 22, MC1 R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP1 1 , MN/CA IX-antigen, MRP-3, MUC-1 , MUC-2, MUM- 1/m, MUM-2/m, MUM-3/m, myosin class l/m, NA88-A, N-acetylglucosaminyltransferase- V, Neo-PAP, Neo-PAP/m, NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO- 1 , NY-ESO-B, OA1 , OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, pi 5, p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI-1 , PAI-2, PART-1 , PATE, PDEF, Pim- 1 -Kinase, Pin-1 , Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1 , RBAF600/m, RHAMM/CD168, RU1 , RU2, S-100, SAGE, SART-1 , SART-2, SART-3, SCC, SIRT2/m, Sp1 7, SSX-1 , SSX-2/H OM-M E L- 40, SSX-4, STAMP-1 , STEAP, survivin, survivin-2B, SYT-SSX-1 , SYT-SSX-2, TA-90, TAG- 72, TARP, TEL-AML1 , TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1 , TRP- 2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGF, VEGFR-2/FLK-1 , and WT1 .
In a particularly preferred aspect, the tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention are selected from the group consisting of MAGE-A1 (e.g. MAGE-A1 according to accession number M77481 ), MAGE-A2, MAGE- A3, MAGE-A6 (e.g. MAGE-A6 according to accession number NM_005363), MAGE-C1 , MAGE-C2, melan-A (e.g. melan-A according to accession number NM_00551 1 ), GP100 (e.g. GP100 according to accession number M77348), tyrosinase (e.g. tyrosinase according to accession number NM_000372), surviving (e.g. survivin according to accession number AF077350), CEA (e.g. CEA according to accession number NM_004363), Her-2/neu (e.g. Her-2/neu according to accession number M11730), WT1 (e.g. WT1 according to accession number NM_000378), PRAME (e.g. PRAME according to accession number NM_0061 15), EGFRI (epidermal growth factor receptor 1 ) (e.g. EGFRI (epidermal growth factor receptor 1 ) according to accession number AF288738), MUC1 , mucin-1 (e.g. mucin-1 according to accession number NM_002456), SEC61 G (e.g. SEC61 G according to accession number NM_014302), hTERT (e.g. hTERT accession number NM_198253), 5T4 (e.g. 5T4 according to accession number NM_006670), NY-Eso-1 (e.g. NY-Eso1 according to accession number NM_001327), TRP-2 (e.g. TRP-2 according to accession number NM_001922), STEAP, PCA, PSA, PSMA, etc.
According to a further particularly preferred aspect, the tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may form a cocktail of antigens, e.g. in a vaccine, a pharmaceutical composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment of prostate cancer (PCa), preferably of neoadjuvant and/or hormone-refractory prostate cancers, and diseases or disorders related thereto. For this purpose, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention is preferably at least one RNA, more preferably at least one mRNA, which may encode at least one, preferably two, three or even four (preferably different) antigens of the following group of antigens: · PSA (Prostate-Specific Antigen) = KLK3 (Kallikrein-3),
• PSMA (Prostate-Specific Membrane Antigen),
• PSCA (Prostate Stem Cell Antigen),
• STEAP (Six Transmembrane Epithelial Antigen of the Prostate). More preferably, in the latter aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, which may encode at least two, three or four (preferably different) antigens of the following combinations of antigens: · PSA and PSMA, or
• PSA and PSCA, or
• PSA and STEAP, or
• PSMA and PSCA, or
• PSMA and STEAP, or
· PSCA and STEAP,
or PSA, PSMA and PSCA, or
PSA, PSMA and STEAP, or
PSMA, PSCA and STEAP, or
PSA, PSCA and STEAP, or
or
PSA, PSMA, PSCA and STEAP
Even more preferably, in the latter aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, which may encode at least two, three or four (preferably different) antigens:
a) wherein at least one antigen is selected from:
• STEAP (Six Transmembrane Epithelial Antigen of the Prostate); and b) wherein the further antigen(s) is (are) selected from at least one antigen of any of the following specific antigens or combinations thereof:
• PSA (Prostate-Specific Antigen), or
• PSMA (Prostate-Specific Membrane Antigen), or
• PSCA (Prostate Stem Cell Antigen);
or
• PSA and PSMA, or
• PSA and PSCA, or
• PSMA and PSCA;
or
• PSA, PSMA and PSCA.
Most preferably, in the latter aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, encoding four (preferably different) antigens selected from PSA, PSMA, PSCA and STEAP.
According to another particularly preferred aspect, the tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may form a cocktail of antigens, e.g. in a vaccine, a pharmaceutical composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment 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. For this purpose, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention is preferably at least one RNA, more preferably at least one mRNA, which may encode at least one, preferably two, three, four, five, six, seven, eight, nine, ten eleven or twelve (preferably different) antigens of the following group of antigens:
• hTERT,
• WT1 ,
• MAGE-A2,
· 5T4,
• MAGE- A3,
• MUC1 ,
• Her-2/neu,
• NY-ESO-1 ,
· CEA,
• Survivin,
• MAGE-C1 , and/or
• MAGE-C2,
wherein any combination of these antigens is possible.
More preferably, in the latter aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, which may encode at least two, three, five or six (preferably different) antigens of the following combinations of antigens:
· hTERT,
• WT1 ,
• 5T4,
• NY-ESO-1 ,
• Survivin, and/or
· MAGE-C2,
wherein any combination of these antigens is possible.
Even more preferably, in the latter aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, which may encode at least one, preferably two, three, four, five, six, seven, eight, nine, ten eleven or twelve (preferably different) antigens of the following combinations of antigens:
• hTERT and WT1 , or
• hTERT and 5T4, or hTERT and NY-ESO-1 , or hTERT and Survivin, or
hTERT and MAGE-C2, or
WT1 and 5T4, or
WT1 and NY-ESO-1 , or
WTl and Survivin, or
VVT1 and MAGE-C2, or
5T4 and NY-ESO-1 , or
5T4 and Survivin, or
5T4 and MAGE-C2, or
NY-ESO-1 and Survivin, or
NY-ESO-1 and MAGE-C2, or
Survivin and MAGE-C2,
or
hTERT, WT1 and 5T4, or
hTERT, WT1 and NY-ESO-1 , or
hTERT, WT1 and Survivin, or
hTERT, WT1 and MAGE-C2, or
hTERT, 5T4, and NY-ESO-1 , or
hTERT, 5T4, and Survivin, or
hTERT, 5T4, and MAGE-C2, or
hTERT, NY-ESO-1 and Survivin, or hTERT, NY-ESO-1 and MAGE-C2, or hTERT, Survivin and MAGE-C2, or
WT1 , 5T4 and NY-ESO-1 , or
WT1 , 5T4 and Survivin, or
VVT1 , 5T4 and MAGE-C2, or
VVT1 , NY-ESO-1 and Survivin, or
WT1 , NY-ESO-1 and MAGE-C2, or
WT1 , Survivin and MAGE-C2, or
5T4, NY-ESO-1 and Survivin, or
5T4, NY-ESO-1 and MAGE-C2, or
5T4, Survivin and MAGE-C2, or
NY-ESO-1 , Survivin, and MAGE-C2, or
hTERT, WT1 , 5T4 and NY-ESO-1 , or hTERT, WT1 , 5T4 and Survivin, or hTERT, VVT1 , 5T4 and MAGE-C2, or hTERT, 5T4, NY-ESO-1 and Survivin, or hTERT, 5T4, NY-ESO-1 and MAGE-C2, or hTERT, NY-ESO-1 , Survivin and MAGE-C2, or WT1 , 5T4, NY-ESO-1 , and Survivin, or WT1 , 5T4, NY-ESO-1 , and MAGE-C2, or WT1 , 5T4, Survivin, and MAGE-C2, or 5T4, NY-ESO-1 , Survivin, and MAGE-C2, or • hTERT, WT1 , 5T4, NY-ESO-1 and Survivin, or
• hTERT, WT1 , 5T4, NY-ESO-1 and MAGE-C2, or
• WT1 , 5T4, NY-ESO-1 , Survivin and MAGE-C2,
or
· hTERT, WT1 , 5T4, NY-ESO-1, Survivin, and MAGE-C2.
Preferably, in the latter aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also be at least one RNA, more preferably at least one mRNA, which may encode at least two (preferably different) antigens exclusively selected from any of the antigens of the above mentioned group(s) or subgroup(s) comprising (at least) any one of the following combinations of antigens:
• hTERT and WT1 , or
• hTERT and 5T4, or
• hTERT and NY-ESO-1, or
· hTERT and Survivin, or
• hTERT and MAGE-C2, or
• WT1 and 5T4, or
• WT1 and NY-ESO-1 , or
• WT1 and Survivin, or
· WT1 and MAGE-C2, or
• 5T4 and NY-ESO-1 , or
• 5T4 and Survivin, or
• 5T4 and MAGE-C2, or
• NY-ESO-1 and Survivin, or
· NY-ESO-1 and MAGE-C2, or
• Survivin and MAGE-C2,
or
• hTERT, WT1 and 5T4, or
• hTERT, WT1 and NY-ESO-1, or
· hTERT, WT1 and Survivin, or
• hTERT, WT1 and MAGE-C2, or
• hTERT, 5T4, and NY-ESO-1 , or
• hTERT, 5T4, and Survivin, or
• hTERT, 5T4, and MAGE-C2, or
· hTERT, NY-ESO-1 and Survivin, or
• hTERT, NY-ESO-1 and MAGE-C2, or
• hTERT, Survivin and MAGE-C2, or
• WT1 , 5T4 and NY-ESO-1 , or
• WT1 , 5T4 and Survivin, or
· WT1 , 5T4 and MAGE-C2, or
• WT1 , NY-ESO-1 and Survivin, or
• WT1 , NY-ESO-1 and MAGE-C2, or
• WT1 , Survivin and MAGE-C2, or
• 5T4, NY-ESO-1 and Survivin, or • 5T4, NY-ESO-1 and MAGE-C2, or
• 5T4, Survivin and MAGE-C2, or
• NY-ESO-1 , Survivin, and MAGE-C2,
or
· hTERT, VVT1 , 5T4 and NY-ESO-1 , or
• hTERT, WT1 , 5T4 and Survivin, or
• hTERT, VVT1 , 5T4 and MAGE-C2, or
• hTERT, 5T4, NY-ESO-1 and Survivin, or
• hTERT, 5T4, NY-ESO-1 and MAGE-C2, or
· hTERT, NY-ESO-1 , Survivin and MAGE-C2, or
• WT1 , 5T4, NY-ESO-1 , and Survivin, or
• WT1 , 5T4, NY-ESO-1 , and MAGE-C2, or
• WT1, 5T4, Survivin, and MAGE-C2, or
• 5T4, NY-ESO-1 , Survivin, and MAGE-C2,
or
• hTERT, WT1 , 5T4, NY-ESO-1 and Survivin, or
• hTERT, WT1, 5T4, NY-ESO-1 and MAGE-C2, or
• WT1 , 5T4, NY-ESO-1 , Survivin and MAGE-C2,
or
· hTERT, WT1 , 5T4, NY-ESO-1 , Survivin, and MAGE-C2.
According to a further particularly preferred aspect, the tumor antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may form a cocktail of antigens, e.g. in in a vaccine, a pharmaceutical composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment 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. For this purpose, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention is preferably at least one RNA, more preferably at least one mRNA, which may encode at least two (preferably different) antigens,
a) wherein at least one, preferably at least two, three, four, five or even six, of these at least two antigens is (are) selected from:
· 5T4
• NY-ESO-1 ,
• MAGE-A2,
• MAGE-A3,
• MAGE-C1 , and/or
· MAGE-C2, and b) wherein the further antigen(s) is (are) selected from at least one antigen as defined herein, preferably in any of the herein mentioned combinations, groups or subgroups of antigens, e.g. the further antigen(s) is (are) selected from, e.g.:
• hTERT,
· WT1 ,
• MAGE-A2,
• 5T4,
• MAGE-A3,
• MUC1 ,
· Her-2/neu,
• NY-ESO-1,
• CEA,
• Survivin,
• MAGE-C1 , and/or
· MAGE-C2.
Preferably, in the latter aspect, the at least one antigen(s) according to a) is (are) selected from:
• NY-ESO-1,
· MAGE-C1 , and/or
• MAGE-C2.
More preferably, in the latter aspect, the at least one antigen(s) according to a) is (are) selected from:
· MAGE-C1 , and/or
• MAGE-C2.
Preferably, in the latter aspect, the at least one antigen(s) according to b) is (are) selected from an antigen (antigens) as defined in one of the following combinations:
· hTERT and WT1 ; or
• hTERT and MAGE-A2; or
• hTERT and 5T4; or
• hTERT and MAGE- A3; or
• hTERT and MUC1 ; or
· hTERT and Her-2/neu; or
• hTERT and NY-ESO-1 ; or
. hTERT and CEA; or
• hTERT and Survivin; or
• hTERT and MAGE-C1 ; or
· hTERT and MAGE-C2; or
• WT1 and MAGE-A2; or WT1 and 5T4; or
WT1 and MAGE- A3; or WT1 and MUC1 ; or
WT1 and Her-2/neu; or WT1 and NY-ESO-1 ; or WT1 and CEA; or
WT1 and Survivin; or
WT1 and MAGE-C1 ; or WT1 and MAGE-C2; or MAGE-A2 and 5T4; or MAGE-A2 and MAGE-A3; or MAGE-A2 and MUC1 ; or MAGE-A2 and Her-2/neu; or MAGE-A2 and NY-ESO-1 ; or MAGE-A2 and CEA; or MAGE-A2 and Survivin; or MAGE-A2 and MAGE-C1 ; or MAGE-A2 and MAGE-C2; or 5T4 and MAGE-A3; or 5T4 and MUC1 ; or
5T4 and Her-2/neu; or 5T4 and NY-ESO-1 ; or 5T4 and CEA; or
5T4 and Survivin; or
5T4 and MAGE-C1 ; or 5T4 and MAGE-C2; or MAGE- A3 and MUC1 ; or MAGE-A3 and Her-2/neu; or MAGE- A3 and NY-ESO-1 ; or MAGE- A3 and CEA; or - MAGE-A3 and Survivin; or MAGE- A3 and MAGE-C1 MAGE- A3 and MAGE-C2 MUC1 and Her-2/neu; or MUC1 and NY-ESO-1 ; or MUC1 and CEA; or
MUC1 and Survivin; or MUC1 and MAGE-C1 ; or MUC1 and MAGE-C2; or HER-2/NEU and NY-ESO-1 ; or HER-2/NEU and CEA; or HER-2/NEU and Survivin; or HER-2/NEU and MAGE-C1 ; or HER-2/NEU and MAGE-C2; or NY-ESO-1 and CEA; or NY-ESO-1 and Survivin; or NY-ESO-1 and MAGE-C1 ; or NY-ESO-1 and MAGE-C2; or CEA and Survivin; or
CEA and MAGE-C1 ; or
CEA and MAGE-C2; or
Survivin and MAGE-C1 ; or
Survivin and MAGE-C2; or
MAGE-C1 and MAGE-C2; or
hTERT, WT1 and MAGE-A2; or hTERT, WT1 and 5T4; or
hTERT, WT1 and MAGE- A3; or hTERT, WTl and MUCl ; or hTERT, WT1 and Her-2/neu; or hTERT, WT1 and NY-ESO-1 ; or hTERT, WT1 and CEA; or
hTERT, WT1 and Survivin; or hTERT, WT1 and MAGE-C1 ; or hTERT, WT1 and MAGE-C2; or WT1 , MAGE-A2 and 5T4; or WT1 , MAGE-A2 and MAGE- A3; or WT1 , MAGE-A2 and MUC1 ; or WT1 , MAGE-A2 and Her-2/neu; or WT1 , MAGE-A2 and NY-ESO-1 ; or WT1 , MAGE-A2 and CEA; or WT1 , MAGE-A2 and Survivin; or WT1 , MAGE-A2 and MAGE-C1 ; or WTl , MAGE-A2 and MAGE-C2; or MAGE-A2, 5T4 and MAGE-A3; or MAGE-A2, 5T4 and MUC1 ; or MAGE-A2, 5T4 and Her-2/neu; or MAGE-A2, 5T4 and NY-ESO-1 ; or MAGE-A2, 5T4 and CEA; or
MAGE-A2, 5T4 and Survivin; or MAGE-A2, 5T4 and MAGE-C1 ; or MAGE-A2, 5T4 and MAGE-C2; or 5T4, MAGE- A3 and MUCl ; or 5T4, MAGE- A3 and Her-2/neu; or 5T4, MAGE- A3 and NY-ESO-1 ; or 5T4, MAGE- A3 and CEA; or 5T4, MAGE-A3 and Survivin; or 5T4, MAGE- A3 and MAGE-C1 ; or 5T4, MAGE-A3 and MAGE-C2; or MAGE- A3, MUC1 and Her-2/neu; or • MAGE- A3, MUC1 and NY-ESO-1 ; or
• MAGE-A3, MUC1 and CEA; or
• MAGE-A3, MUC1 and Survivin; or
• MAGE-A3, MUC1 and MAGE-C1 ; or
• MAGE-A3, MUC1 and MAGE-C2; or
• MUC1 , Her-2/neu and NY-ESO-1 ; or
• MUC1 , Her-2/neu and CEA; or
• MUC1 , Her-2/neu and Survivin; or
• MUC1 , Her-2/neu and MAGE-C1 ; or
• MUC1 , Her-2/neu and MAGE-C2; or
• HER-2/NEU, NY-ESO-1 and CEA; or
• HER-2/NEU, NY-ESO-1 and Survivin; or
• HER-2/NEU, NY-ESO-1 and MAGE-C1 ; or
• HER-2/NEU, NY-ESO-1 and MAGE-C2; or
• NY-ESO-1 , CEA and Survivin; or
• NY-ESO-1 , CEA and MAGE-C1 ; or
• NY-ESO-1 , CEA and MAGE-C2; or
• CEA, Survivin and MAGE-C1 ; or
• CEA, Survivin and MAGE-C2; or
• Survivin, MAGE-C1 and MAGE-C2; or
• hTERT, WT1 , MAGE-A2 and 5T4; or
• hTERT, WT1 , MAGE-A2 and MAGE-A3; or
• hTERT, WT1 , MAGE-A2 and MUC1 ; or
• hTERT, WT1 , MAGE-A2 and Her-2/neu; or
• hTERT, WT1 , MAGE-A2 and NY-ESO-1 ; or
• hTERT, WT1 , MAGE-A2 and CEA; or
• hTERT, WT1 , MAGE-A2 and Survivin; or
• hTERT, WT1 , MAGE-A2 and MAGE-C1 ; or
• hTERT, WT1 , MAGE-A2 and MAGE-C2; or
• WT1 , MAGE-A2, 5T4 and MAGE- A3; or
• WT1 , MAGE-A2, 5T4 and MUC1 ; or
• WT1 , MAGE-A2, 5T4 and Her-2/neu; or
• WT1 , MAGE-A2, 5T4 and NY-ESO-1 ; or
• WT1 , MAGE-A2, 5T4 and CEA; or
• WT1 , MAGE-A2, 5T4 and Survivin; or
• WT1 , MAGE-A2, 5T4 and MAGE-C1 ; or
• WT1 , MAGE-A2, 5T4 and MAGE-C2; or
• MAGE-A2, 5T4, MAGE- A3 and MUC1 ; or
• MAGE-A2, 5T4, MAGE- A3 and Her-2/neu; or
• MAGE-A2, 5T4, MAGE- A3 and NY-ESO-1 ; or
• MAGE-A2, 5T4, MAGE-A3 and CEA; or
• MAGE-A2, 5T4, MAGE-A3 and Survivin; or
• MAGE-A2, 5T4, MAGE-A3 and MAGE-C1 ; or • MAGE-A2, 5T4, MAGE- A3 and MAGE-C2; or
• 5T4, MAGE- 3, MUC1 , and Her-2/neu; or
• 5T4, MAGE-A3, MUC1 and NY-ESO-1 ; or
• 5T4, MAGE-A3, MUCl and CEA; or
• 5T4, MAGE-A3, MUCl and Survivin; or
• 5T4, MAGE- A3, MUC1 and MAGE-C1 ; or
• 5T4, MAGE- A3, MUC1 and MAGE-C2; or
• MAGE- A3, MUC1 , Her-2/neu and NY-ESO-1 ; or
• MAGE- A3, MUC1 , Her-2/neu and CEA; or
• MAGE-A3, MUC1 , Her-2/neu and Survivin; or
• MAGE-A3, MUC1 , Her-2/neu and MAGE-C1 ; or
• MAGE- A3, MUC1 , Her-2/neu and MAGE-C2; or
• MUC1 , Her-2/neu, NY-ESO-1 and CEA; or
• MUC1 , Her-2/neu, NY-ESO-1 and Survivin; or
• MUC1 , Her-2/neu, NY-ESO-1 and MAGE-C1 ; or
• MUC1 , Her-2/neu, NY-ESO-1 and MAGE-C2; or
• HER-2/NEU, NY-ESO-1 , CEA and Survivin; or
• HER-2/NEU, NY-ESO-1 , CEA and MAGE-C1 ; or
• HER-2/NEU, NY-ESO-1 , CEA and MAGE-C2; or
• NY-ESO-1 , CEA, Survivin and MAGE-C1 ; or
• NY-ESO-1 , CEA, Survivin and MAGE-C2; or
• CEA, Survivin, MAGE-C1 and MAGE-C2;
or
• hTERT, WT1 , MAGE-A2, 5T4 and MAGE- A3; or
• hTERT, WT1 , MAGE-A2, 5T4 and MUC1 ; or
• hTERT, WT1 , MAGE-A2, 5T4 and Her-2/neu; or
• hTERT, WT1 , MAGE-A2, 5T4 and NY-ESO-1 ; or
• hTERT, WT1 , MAGE-A2, 5T4 and CEA; or
• hTERT, WT1 , MAGE-A2, 5T4 and Survivin; or
• hTERT, WT1 , MAGE-A2, 5T4 and MAGE-C1 ; or
• hTERT, WT1 , MAGE-A2, 5T4 and MAGE-C2; or
• WT1 , MAGE-A2, 5T4, MAGE-A3 and MUC1 ; or
• WT1 , MAGE-A2, 5T4, MAGE-A3 and Her-2/neu; or
• WT1 , MAGE-A2, 5T4, MAGE-A3 and NY-ESO-1 ; or
• WT1 , MAGE-A2, 5T4, MAGE-A3 and CEA; or
• WT1 , MAGE-A2, 5T4, MAGE-A3 and Survivin; or
• WT1 , MAGE-A2, 5T4, MAGE-A3 and MAGE-C1 ; or
• WT1 , MAGE-A2, 5T4, MAGE- A3 and MAGE-C2; or
• MAGE-A2, 5T4, MAGE- A3, MUC1 and Her-2/neu; or
• MAGE-A2, 5T4, MAGE- A3, MUC1 and NY-ESO-1 ; or
• MAGE-A2, 5T4, MAGE-A3, MUC1 and CEA; or
• MAGE-A2, 5T4, MAGE- A3, MUC1 and Survivin; or
• MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C1 ; or
• MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C2; or • 5T4, MAGE- A3, MUCl , Her-2/neu and NY-ESO-1 ; or
• 5T4, MAGE- A3, MUC1 , Her-2/neu and CEA; or
• 5T4, MAGE- A3, MUC1 , Her-2/neu and Survivin; or
• 5T4, MAGE- A3, MUC1 , Her-2/neu and MAGE-C1 ; or
· 5T4, MAGE- A3, MUC1 , Her-2/neu and MAGE-C2; or
• MAGE-A3, MUCl , Her-2/neu, NY-ESO-1 and CEA; or
• MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 and Survivin; or
• MAGE-A3, MUCl , Her-2/neu, NY-ESO-1 and MAGE-Cl ; or
• MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 and MAGE-C2; or · MUC1 , Her-2/neu, NY-ESO-1 , CEA and Survivin; or
• MUC1 , Her-2/neu, NY-ESO-1 , CEA and MAGE-C1 ; or
• MUC1 , Her-2/neu, NY-ESO-1 , CEA and MAGE-C2; or
• HER-2/NEU, NY-ESO-1 , CEA, Survivin and MAGE-C1 ; or
• HER-2/NEU, NY-ESO-1 , CEA, Survivin and MAGE-C2; or
· NY-ESO-1 , CEA, Survivin, MAGE-C1 and MAGE-C2;
or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE-A3 and MUC1 ; or
· hTERT, WT1 , MAGE-A2, 5T4, MAGE- A3 and Her-2/neu; or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE- A3 and NY-ESO-1 ; or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE- A3 and CEA; or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE- A3 and Survivin; or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE- A3 and MAGE-C1 ; or · hTERT, WT1 , MAGE-A2, 5T4, MAGE- A3 and MAGE-C2; or
• WT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 and Her-2/neu; or
• WT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 and NY-ESO-1; or
• WT1 , MAGE-A2, 5T4, MAGE- A3, MUC1 and CEA; or
• WT1 , MAGE-A2, 5T4, MAGE- A3, MUC1 and Survivin; or · WT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-Cl ; or
• WT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C2; or
• MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu and NY-ESO-1 ; or
• MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu and CEA; or
• MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu and Survivin; or · MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu and MAGE-C1 ; or
• MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu and MAGE-C2; or
• 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 and CEA; or
• 5T4, MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 and Survivin; or
• 5T4, MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 and MAGE-C1 ; or · 5T4, MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 and MAGE-C2; or
• MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and Survivin; or
• MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and MAGE-C1 ; or
• MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and MAGE-C2; or
• MUC1 , Her-2/neu, NY-ESO-1 , CEA, Survivin and MAGE-C1 ; or · MUCl , Her-2/neu, NY-ESO-1 , CEA, Survivin and MAGE-C2; or
• HER-2/NEU, NY-ESO-1 , CEA, Survivin, MAGE-C1 and MAGE-C2; • hTERT, WTl , MAGE-A2, 5T4, MAGE-A3, MUC1 and Her-2/neu; or
· hTERT, WTl , MAGE-A2, 5T4, MAGE-A3, MUC1 and NY-ESO-1 ; or
• hTERT, WTl , MAGE-A2, 5T4, MAGE-A3, MUC1 and CEA; or
• hTERT, WTl , MAGE-A2, 5T4, MAGE- A3, MUC1 and Survivin; or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE- A3, MUC1 and MAGE-C1 ; or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE- A3, MUC1 and MAGE-C2; or
· WT1 , MAGE-A2, 5T4, MAGE- A3, MUCl , Her-2/neu and NY-ESO-1 ; or
• WTl , MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu and CEA; or
• WT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu and Survivin; or
• WT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu and MAGE-C1 ; or
• WT1 , MAGE-A2, 5T4, MAGE-A3, MUCl , Her-2/neu and MAGE-C2; or
· MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 and CEA; or
• MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 and Survivin; or
• MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 and MAGE-C1 ; or
• MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 and MAGE-C2; or
• 5T4, MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and Survivin; or
· 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and MAGE-C1 ; or
• 5T4, MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and MAGE-C2; or
• MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA, Survivin and MAGE-C1 ; or
• MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA, Survivin and MAGE-C2; or
• MUC1 , Her-2/neu, NY-ESO-1 , CEA, Survivin, MAGE-C1 and MAGE-C2;
or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE-A3, MUCl , Her-2/neu and NY-ESO-1 ; or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu and CEA; or
· hTERT, WTl , MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu and Survivin; or
• hTERT, WTl , MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu and MAGE-C1 ; or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu and MAGE-C2; or
• WT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 and CEA; or
• WTl , MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 and Survivin; or · WT1 , MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 and MAGE-C1 ; or
• WT1 , MAGE-A2, 5T4, MAGE-A3, MUCl , Her-2/neu, NY-ESO-1 and MAGE-C2; or
• MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and Survivin; or · MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and MAGE-C1 ; or
• MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and MAGE-C2; or
• 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA, Survivin and MAGE-C1 ; or · 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA, Survivin and MAGE-C2; or
• MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA, Survivin, MAGE-C1 and MAGE- C2; • hTERT, WTl, MAGE-A2, 5T4, MAGE- A3, MUCl, Her-2/neu, NY-ESO-1 and CEA; or
• hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and Survivin; or
• hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1; or
· hTERT, WTl, MAGE-A2, 5T4, MAGE- A3, MUC1, Her-2/neu, NY-ESO-1 and
MAGE-C2; or
• WTl, MAGE-A2, 5T4, MAGE- A3, MUC1, Her-2/neu, NY-ESO-1, CEA and Survivin; or
• WTl, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1;or
• WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2; or
• MAGE-A2, 5T4, MAGE- A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C1;or
· MAGE-A2, 5T4, MAGE- A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and
MAGE-C2; or
• 5T4, MAGE- A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1 and MAGE-C2; or
• hTERT, WTl, MAGE-A2, 5T4, MAGE- A3, MUC1, Her-2/neu, NY-ESO-1, CEA and Survivin; or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE- A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and MAGE-C1;or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA and MAGE-C2; or
• WTl, MAGE-A2, 5T4, MAGE- A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C1; or
· WT1, MAGE-A2, 5T4, MAGE- A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C2; or
• MAGE-A2, 5T4, MAGE- A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE- C1 and MAGE-C2; or
• hTERT, WT1, MAGE-A2, 5T4, MAGE- A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C1 ; or
• hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C2; or
• WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1 and MAGE-C2; or
• hTERT, WT1 , MAGE-A2, 5T4, MAGE-A3, MUC1 , Her-2/neu, NY-ESO-1 , CEA, Survivin, MAGE-C1 and MAGE-C2.
More preferably, in the latter aspect, the at least one antigen(s) according to b) is (are) selected from the following combination:
• Survivin and 5T4 In the above aspects, each of the at least two (preferably different) antigens as defined herein may be encoded by one (monocistronic) RNA, preferably one (monocistronic) mRNA. In other words, the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may comprise at least two (monocistronic) RNAs, preferably mRNAs, wherein each of these at least two (monocistronic) RNAs, preferably mRNAs, may encode just one (preferably different) antigen, preferably selected from one of the above mentioned combinations.
According to another particularly preferred aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may comprise (at least) one bi- or even multicistronic RNA, preferably mRNA, i.e. (at least) one RNA which carries two or even more of the coding sequences of at the least two (preferably different) antigens, preferably selected from one of the above mentioned combinations. Such coding sequences of the at least two (preferably different) antigens of the (at least) one bi- or even multicistronic RNA may be separated by at least one IRES (internal ribosomal entry site) sequence, as defined below. Thus, the term "encoding at least two (preferably different) antigens" may mean, without being limited thereto, that the (at least) one (bi- or even multicistronic) RNA, preferably a mRNA, may encode e.g. at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve (preferably different) antigens of the above mentioned group(s) of antigens or their fragments or variants. More preferably, without being limited thereto, the (at least) one (bi- or even multicistronic) RNA, preferably mRNA, may encode e.g. at least two, three, four, five or six (preferably different) antigens of the above mentioned subgroup(s) of antigens or their fragments or variants within the above definitions. In this context, a so-called IRES (internal ribosomal entry site) sequence as defined above can function as a sole ribosome binding site, but it can also serve to provide a bi- or even multicistronic RNA as defined above which codes for several proteins, which are to be translated by the ribosomes independently of one another. Examples of IRES sequences which can be used according to the invention are those from picornaviruses (e.g. FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), mouse leukoma virus (MLV), simian immunodeficiency viruses (SIV) or cricket paralysis viruses (CrPV).
According to a further particularly preferred aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may comprise a mixture of at least one monocistronic RNA, preferably mRNA, as defined above, and at least one bi- or even multicistronic RNA, preferably mRNA, as defined above. The at least one monocistronic RNA and/or the at least one bi- or even multicistronic RNA preferably encode different antigens or their fragments or variants, the antigens preferably being selected from one of the above mentioned groups or subgroups of antigens, more preferably in one of the above mentioned combinations. However, the at least one monocistronic RNA and the at least one bi- or even multicistronic RNA may preferably also encode (in part) identical antigens selected from one of the above mentioned groups or subgroups of antigens, preferably in one of the above mentioned combinations, provided that the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention as a whole provides at least two (preferably different) antigens as defined above. Such an aspect may be advantageous e.g. for a staggered, e.g. time dependent, administration of e.g. a vaccine, a pharmaceutical composition or a kit of the present invention to a patient in need thereof. The components of such a vaccine, a pharmaceutical composition or a kit of the present invention, particularly the different RNAs encoding the at least two (preferably different) antigens, may be e.g. contained in (different parts of) a kit of parts composition or may be e.g. administered separately as components of different embodiments or aspects according to the present invention.
According to another aspect, one further class of antigens as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention comprises allergy antigens. Such allergy antigens may be selected from antigens derived from different sources, e.g. from animals, plants, fungi, bacteria, etc. Allergens in this context include e.g. grasses, pollens, molds, drugs, or numerous environmental triggers, etc. Allergy antigens typically belong to different classes of compounds, such as nucleic acids and their fragments, proteins or peptides and their fragments, carbohydrates, polysaccharides, sugars, lipids, phospholipids, etc. Of particular interest in the context of the present invention are antigens, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, i.e. protein or peptide antigens and their fragments or epitopes, or nucleic acids and their fragments, particularly nucleic acids and their fragments, encoding such protein or peptide antigens and their fragments or epitopes.
Particularly preferred, antigens derived from animals, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may include antigens derived from, without being limited thereto, insects, such as mite (e.g. house dust mites), mosquito, bee (e.g. honey bee, bumble bee), cockroache, tick, moth (e.g. silk moth), midge, bug, flea, wasp, caterpillar, fruit fly, migratory locust, grasshopper, ant aphide, from crustaceans, such as shrimps, crab, krill, lobster, prawn, crawfish, scampi, from birds, such as duck, goose, seagull, turkey, ostrich, chicken, from fishes, such as eel, herring, carp, seabream, codfish, halibut, catfish, beluga, salmon, flounder, mackerel, cuttlefish, perch, form molluscs, such as scallop, octopus, abalone, snail, whelk, squid, clam, mussel, from spiders, from mammals, such as cow, rabbit, sheep, lion, jaguar, leopard, rat, pig, buffalo, dog, loris, hamster, guinea pig, fallow deer, horse, cat, mouse, ocelot, serval, from arthropod, such as spider, or silverfish, from worms, such as nematodes, from trichinella species, or roundworm, from amphibians, such as frogs, or from sea squirt, etc. Antigens derived from plants, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may include antigens derived from, without being limited thereto, fruits, such as kiwi, pineapple, jackfruit,papaya, lemon, orange, mandarin, melon, sharon fruit, strawberry, lychee, apple, cherry paradise apple, mango, passion fruit, plum, apricot, nectarine, pear, passion fruit, raspberry, grape, from vegetables, such as garlic, onion, leek, soya bean, celery, cauliflower, turnip, paprika, chickpea, fennel, zucchini, cucumber, carrot, yam, bean, pea, olive, tomato, potato, lentil, lettuce, avocado, parsley, horseradish, chirimoya, beet, pumkin, spinach, from spices, such as mustard, coriander, saffron, pepper, aniseed, from crop, such as oat, buckwheat, barley, rice, wheat, maize, rapeseed, sesame, from nuts, such as cashew, walnut, butternut, pistachio, almond, hazelnut, peanut, brazil nut, pecan, chestnut, from trees, such as alder, hornbeam, cedar, birch, hazel, beech, ash, privet, oak, plane tree, cypress, palm, from flowers, such as ragweed, carnation, forsythia, sunflower, lupine, chamomile, lilac, passion flower, from grasses, such as quack grass, common bent, brome grass, Bermuda grass, sweet vernal grass, rye grass, or from other plants, such as opium poppy, pellitory, ribwort, tobacco, asparagus, mugwort, cress, etc.
Antigens derived from fungi, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may include antigens derived from, without being limited thereto, e.g. Alternia sp., Aspergillus sp., Beauveria sp., Candida sp., Cladosporium sp., Endothia sp., Curcularia sp., Embellisia sp., Epicoccum sp., Fusarium sp., Malassezia sp., Penicillum sp., Pleospora sp., Saccharomyces sp., etc.
Antigens derived from bacteria, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may include antigens derived from, without being limited thereto, e.g. Bacillus tetani, Staphylococcus aureus, Streptomyces griseus, etc. Antibodies
According to a further alternative, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may encode an antibody. According to the present invention, such an antibody may be selected from any antibody, e.g. any recombinantly produced or naturally occurring antibodies, known in the art, in particular antibodies suitable for therapeutic, diagnostic or scientific purposes, or antibodies which have been identified in relation to specific cancer diseases. Herein, the term "antibody" is used in its broadest sense and specifically covers monoclonal and polyclonal antibodies (including agonist, antagonist, and blocking or neutralizing antibodies) and antibody species with polyepitopic specificity. According to the invention, "antibody" typically comprises any antibody known in the art (e.g. IgM, IgD, IgG, IgA and IgE antibodies), such as naturally occurring antibodies, antibodies generated by immunization in a host organism, antibodies which were isolated and identified from naturally occurring antibodies or antibodies generated by immunization in a host organism and recombinantly produced by biomolecular methods known in the art, as well as chimeric antibodies, human antibodies, humanized antibodies, bispecific antibodies, intrabodies, i.e. antibodies expressed in cells and optionally localized in specific cell compartments, and fragments and variants of the aforementioned antibodies. In general, an antibody consists of a light chain and a heavy chain both having variable and constant domains. The light chain consists of an N-terminal variable domain, VL, and a C-terminal constant domain, Q. In contrast, the heavy chain of the IgG antibody, for example, is comprised of an N-terminal variable domain, VH, and three constant domains, CH1 , CH2 und CH3. Single chain antibodies may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention as well.
According to a first alternative, the nucleic acid (sequence), lyophilized or to be lyophilized,according to the present invention may encode a polyclonal antibody. In this context, the term, "polyclonal antibody" typically means mixtures of antibodies directed to specific antigens or immunogens or epitopes of a protein which were generated by immunization of a host organism, such as a mammal, e.g. including goat, cattle, swine, dog, cat, donkey, monkey, ape, a rodent such as a mouse, hamster and rabbit. Polyclonal antibodies are generally not identical, and thus usually recognize different epitopes or regions from the same antigen. Thus, in such a case, typically a mixture (a composition) of different (lyophilized) nucleic acids according to the present invention will be used, each (lyophilized) nucleic acid (sequence) encoding a specific (monoclonal) antibody being directed to specific antigens or immunogens or epitopes of a protein. According to a further alternative, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may encode a monoclonal antibody. The term "monoclonal antibody" herein typically refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed to a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. For example, monoclonal antibodies as defined above may be made by the hybridoma method first described by Kohler and Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods, e.g. as described in U.S. Pat. No. 4,81 6,567. "Monoclonal antibodies" may also be isolated from phage libraries generated using the techniques described in McCafferty et a/., Nature, 348:552-554 (1 990), for example. According to Kohler and Milstein, an immunogen (antigen) of interest is injected into a host such as a mouse and B-cell lymphocytes produced in response to the immunogen are harvested after a period of time. The B-cells are combined with myeloma cells obtained from mouse and introduced into a medium which permits the B-cells to fuse with the myeloma cells, producing hybridomas. These fused cells (hybridomas) are then placed into separate wells of microtiter plates and grown to produce monoclonal antibodies. The monoclonal antibodies are tested to determine which of them are suitable for detecting the antigen of interest. After being selected, the monoclonal antibodies can be grown in cell cultures or by injecting the hybridomas into mice. However, for the purposes of the present invention, the peptide sequences of these monoclonal antibodies have to be sequenced and the nucleic acid sequences encoding these antibodies can be present as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention. For therapeutical purposes in humans, non-human monoclonal or polyclonal antibodies, such as murine antibodies may also be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention. However, such antibodies are typically only of limited use, since they generally induce an immune response by production of human antibodies directed to the said non-human antibodies, in the human body. Therefore, a particular non-human antibody can only be administered once to the human. To solve this problem, chimeric, humanized non- human and human antibodies are also envisaged encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention. "Chimeric" antibodies, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophi lized, according to the present invention, are preferably antibodies in which the constant domains of an antibody described above are replaced by sequences of antibodies from other organisms, preferably human sequences. „Humanized" (non-human) antibodies, which may be also encoded by nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, are antibodies in which the constant and variable domains (except for the hypervariable domains) described above of an antibody are replaced by human sequences. According to another alternative, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may encode human antibodies, i.e. antibodies having only human sequences. Such human antibodies can be isolated from human tissues or from immunized non-human host organisms which are transgene for the human IgG gene locus, and nucleic acid sequences may be prepared according to procedures well known in the art. Additionally, human antibodies can be provided by the use of a phage display.
In addition, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may encode bispecific antibodies. "Bispecific" antibodies in context of the invention are preferably antibodies which act as an adaptor between an effector and a respective target by two different F^-domains, e.g. for the purposes of recruiting effector molecules such as toxins, drugs, cytokines etc., targeting effector cells such as CTL, NK cells, makrophages, granulocytes, etc. (see for review: Kontermann R.E., Acta Pharmacol. Sin, 2005, 26(1 ): 1 -9). Bispecific antibodies as described herein are, in general, configured to recognize by two different F^-domains, e.g. two different antigens, immunogens, epitopes, drugs, cells (or receptors on cells), or other molecules (or structures) as described above. Bispecificity means herewith that the antigen-binding regions of the antibodies are specific for two different epitopes. Thus, different antigens, immunogens or epitopes, etc. can be brought close together, what, optionally, allows a direct interaction of the two components. For example, different cells such as effector cells and target cells can be connected via a bispecific antibody. Encompassed, but not limited, by the present invention are antibodies or fragments thereof which bind, on the one hand, a soluble antigen as described herein, and, on the other hand, an antigen or receptor on the surface of a tumor cell.
According to the invention, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also encode intrabodies, wherein these intrabodies may be antibodies as defined above. Since these antibodies are intracellular expressed antibodies, i.e. antibodies which may be encoded by nucleic acids localized in specific areas of the cell and also expressed there, such antibodies may be termed intrabodies.
Antibodies as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may preferably comprise full-length antibodies, i.e. antibodies composed of the full heavy and full light chains, as described above. However, derivatives of antibodies such as antibody fragments, variants or adducts may also be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention.
The nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may also encode antibody fragments selected from Fab, Fab', F(ab')2, Fc, Facb, pFc', Fd and Fv fragments of the aforementioned (full-length) antibodies. In general, antibody fragments are known in the art. For example, a Fab ("fragment, antigen binding") fragment is composed of one constant and one variable domain of each of the heavy and the light chain. The two variable domains bind the epitope on specific antigens. The two chains are connected via a disulfide linkage. A scFv ("single chain variable fragment") fragment, for example, typically consists of the variable domains of the light and heavy chains. The domains are linked by an artificial linkage, in general a polypeptide linkage such as a peptide composed of 15-25 glycine, proline and/or serine residues.
According to a further alternative, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be in the form of dsRNA, preferably siRNA. A dsRNA, or a siRNA, is of interest particularly in connection with the phenomenon of RNA interference. The in vitro technique of RNA interference (RNAi) is based on double- stranded RNA molecules (dsRNA), which trigger the sequence-specific suppression of gene expression (Zamore (2001 ) Nat. Struct. Biol. 9: 746-750; Sharp (2001 ) Genes Dev. 5:485- 490: Hannon (2002) Nature 41 : 244-251 ). In the transfection of mammalian cells with long dsRNA, the activation of protein kinase R and RnaseL brings about unspecific effects, such as, for example, an interferon response (Stark et a/. (1998) Annu. Rev. Biochem. 67: 227- 264; He and Katze (2002) Viral Immunol. 15: 95-1 19). These unspecific effects are avoided when shorter, for example 21 - to 23-mer, so-called siRNA (small interfering RNA), is used, because unspecific effects are not triggered by siRNA that is shorter than 30 bp (Elbashir et al. (2001 ) Nature 41 1 : 494-498).
The nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may thus be a double-stranded RNA (dsRNA) having a length of from 17 to 29, preferably from 19 to 25, and preferably being at least 90%, more preferably 95% and especially 100% (of the nucleotides of a dsRNA) complementary to a section of the nucleic acid sequence of a (therapeutically relevant) protein or antigen described (as active ingredient) hereinbefore, either a coding or a non-coding section, preferably a coding section. 90% complementary means that with a length of a dsRNA described herein of, for example, 20 nucleotides, this contains not more than 2 nucleotides without corresponding complementarity with the corresponding section of the mRNA. The sequence of the double- stranded RNA used according to the invention is, however, preferably wholly complementary in its general structure with a section of the nucleic acid of a therapeutically relevant protein or antigen described hereinbefore. In this context the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be a dsRNA having the general structure 5'-(N17.29)-3', preferably having the general structure 5'- (Ν,9_25)-3', more preferably having the general structure 5I-(N,9.24)-3I, or yet more preferably having the general structure 5'-(N21.23)-3', wherein for each general structure each N is a (preferably different) nucleotide of a section of the mRNA of a therapeutically relevant protein or antigen described hereinbefore, preferably being selected from a continuous number of 17 to 29 nucleotides of the mRNA of a therapeutically relevant protein or antigen and being present in the general structure 5'-(N1 7.29)-3' in their natural order. In principle, all the sections having a length of from 17 to 29, preferably from 19 to 25, base pairs that occur in the mRNA can serve as target sequence for a dsRNA herein. Equally, dsRNAs used as nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention can also be directed against nucleotide sequences of a (therapeutically relevant) protein or antigen described (as active ingredient) hereinbefore that do not lie in the coding region, in particular in the 5' non-coding region of the mRNA, for example, therefore, against non-coding regions of the mRNA having a regulatory function. The target sequence of the dsRNA used as nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention can therefore lie in the translated and untranslated region of the mRNA and/or in the region of the control elements of a protein or antigen described hereinbefore. The target sequence of a dsRNA used as nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention can also lie in the overlapping region of untranslated and translated sequence; in particular, the target sequence can comprise at least one nucleotide upstream of the start triplet of the coding region of the mRNA.
According to another alternative, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be in the form of a CpG nucleic acid, in particular CpG-RNA or CpG-DNA. A CpG-RNA or CpG-DNA used according to the invention can be a single-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic acid used according to the invention is preferably in the form of CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). Also preferably, such CpG nucleic acids have a length as described above. Preferably, the CpG motifs are unmethylated.
Likewise, according to a further alternative, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be in the form of an immunostimulatory RNA. The immunostimulatory RNA of the complexed RNA of the present invention may be any (double-stranded or single-stranded) RNA, e.g. a coding RNA, as defined above. Preferably, the immunostimulatory RNA may be a single-stranded, a double-stranded or a partially double-stranded RNA, more preferably a single-stranded RNA, and or a circular or linear RNA, more preferably a linear RNA. More preferably, the immunostimulatory RNA may be a (linear) single-stranded RNA. Even more preferably, the immunostimulatory RNA may be a ((linear) single-stranded) messenger RNA (mRNA). An immunostimulatory RNA may also occur as a short RNA oligonucleotide as defined above. An immunostimulatory RNA as used herein may furthermore be selected from any class of RNA molecules, found in nature or being prepared synthetically, and which can induce an immune response. In this context, an immune response may occur in various ways. A substantial factor for a suitable immune response is the stimulation of different T-cell sub-populations. T- lymphocytes are typically divided into two sub-populations, the T-helper 1 (Th1 ) cells and the T-helper 2 (Th2) cells, with which the immune system is capable of destroying intracellular (Th1 ) and extracellular (Th2) pathogens (e.g. antigens). The two Th cell populations differ in the pattern of the effector proteins (cytokines) produced by them. Thus, Th1 cells assist the cellular immune response by activation of macrophages and cytotoxic T- cells. Th2 cells, on the other hand, promote the humoral immune response by stimulation of the B-cells for conversion into plasma cells and by formation of antibodies (e.g. against antigens). The Th1/Th2 ratio is therefore of great importance in the immune response. In connection with the present invention, the Th1/Th2 ratio of the immune response is preferably shifted in the direction towards the cellular response (Thl response) and a cellular immune response is thereby induced. According to one example, the immune system may be activated by ligands of Toll-like receptors (TLRs). TLRs are a family of highly conserved pattern recognition receptor (PRR) polypeptides that recognize pathogen- associated molecular patterns (PAMPs) and play a critical role in innate immunity in mammals. Currently at least thirteen family members, designated TLR1 - TLR13 (Toll-like receptors: TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13), have been identified. Furthermore, a number of specific TLR ligands have been identified. It was e.g. found that unmethylated bacterial DNA and synthetic analogs thereof (CpG DNA) are ligands for TLR9 (Hemmi H et a/. (2000) Nature 408:740-5; Bauer S et a/. (2001 ) Proc NatlAcadSci USA 98, 9237-42). Furthermore, it has been reported that ligands for certain TLRs include certain nucleic acid molecules and that certain types of RNA are immunostimulatory in a sequence-independent or sequence-dependent manner, wherein these various immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, or intracellular receptors such as RIG-I, MDA-5, etc. E.g. Lipford eta/, determined certain G,U- containing oligoribonucleotides as immunostimulatory by acting via TLR7 and TLR8 (see WO 03/086280). The immunostimulatory G,U-containing oligoribonucleotides described by Lipford et a/, were believed to be derivable from RNA sources including ribosomal RNA, transfer RNA, messenger RNA, and viral RNA.
According to the present invention, it was found that any RNA (molecule) as e.g. defined above (irrespective of its specific length, strandedness, modification and/or nucleotide sequence) may have immunostimulatory properties, i.e. enhance the immune response. RNA as defined above and being the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may thus be used to enhance (unspecific) immunostimulation, if suitable and desired for a specific treatment. The at least one (immunostimulatory) RNA (molecule) used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may thus comprise any RNA sequence known to be immunostimulatory, including, without being limited thereto, RNA sequences representing and/or encoding ligands of TLRs, preferably selected from family members TLR1 - TLR13, more preferably from TLR7 and TLR8, ligands for intracellular receptors for RNA (such as RIG-I or MAD-5, etc.) (see e.g. Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA helicases: two parallel ways to trigger antiviral responses. Mol. Cell 22, 561 -569), or any other immunostimulatory RNA sequence. Furthermore, (classes of) immunostimulatory RNA molecules, used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, may include any other RNA capable of eliciting an immune response. Without being limited thereto, such immunostimulatory RNA may include ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA). Such further (classes of) immunostimulatory RNA molecules, which may be used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention, without being limited thereto, may comprise e.g. an RNA molecule of formula (I):
G|XmGn wherein:
G is guanosine, uracil or an analogue of guanosine or uracil;
X is guanosine, uracil, adenosine, thymidine, cytosine or an analogue of the above- mentioned nucleotides;
I is an integer from 1 to 40,
wherein when I = 1 G is guanosine or an analogue thereof,
when I > 1 at least 50% of the nucleotides are guanosine or an analogue thereof;
m is an integer and is at least 3;
wherein when m = 3 X is uracil or an analogue thereof,
when m > 3 at least 3 successive uracils or analogues of uracil occur;
n is an integer from 1 to 40,
wherein when n = 1 G is guanosine or an analogue thereof, when n > 1 at least 50% of the nucleotides are guanosine or an analogue thereof.
In addition, such further (classes of) immunostimulatory RNA molecules, which may be used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may comprise, without being limited thereto, e.g. an RNA molecule of formula (II):
QXmC„ wherein:
C is cytosine, uracil or an analogue of cytosine or uracil;
X is guanosine, uracil, adenosine, thymidine, cytosine or an analogue of the above- mentioned nucleotides;
I is an integer from 1 to 40,
wherein when I = 1 C is cytosine or an analogue thereof,
when I > 1 at least 50% of the nucleotides are cytosine or an analogue thereof;
m is an integer and is at least 3;
wherein when m = 3 X is uracil or an analogue thereof,
when m > 3 at least 3 successive uracils or analogues of uracil occur;
n is an integer from 1 to 40,
wherein when n = 1 C is cytosine or an analogue thereof,
when n > 1 at least 50% of the nucleotides are cytosine or an analogue thereof.
Preferably, the immunostimulatory RNA molecules used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention comprise a length as defined above in general for RNA molecules of the RNA of the present invention, more preferably a length of 5 to 5000, of 500 to 5000 or, more preferably, of 1000 to 5000 or, alternatively, of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or, more preferably, of 5 to 30 nucleotides. The immunostimulatory RNA used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention may be furthermore modified, preferably "chemically modified" in order to enhance the immunostimulatory properties of said DNA. The term "chemical modification" means that the immuostimulatory RNA is modified by replacement, insertion or removal of individual or several atoms or atomic groups compared with naturally occurring RNA species.
Preferably, the chemical modification of the immunostimulatory RNA comprises at least one analogue of naturally occurring nucleotides. In a list which is in no way conclusive, examples which may be mentioned for nucleotide analogues and which may be used herein for modification are analogues of guanosine, uracil, adenosine, thymidine, cytosine. The modifications may refer to modifications of the base, the ribose moiety and/or the phosphate backbone moiety. In this context, analogues of guanosine, uracil, adenosine, and cytosine include, without implying any limitation, any naturally occurring or non-naturally occurring guanosine, uracil, adenosine, thymidine or cytosine that has been altered chemically, for example by acetylation, methylation, hydroxylation, etc., including 1- methyl-adenosine, 1 -methyl-guanosine, 1 -methyl-inosine, 2,2-dimethyl-guanosine, 2,6- diaminopurine, 2'-Amino-2'-deoxyadenosine, 2'-Amino-2'-deoxycytidine, 2'-Amino-2'- deoxyguanosine, 2'-Amino-2'-deoxyuridine, 2-Amino-6-chloropurineriboside, 2- Aminopurine-riboside, 2'-Araadenosine, 2'-Aracytidine, 2'-Arauridine, 2'-Azido-2'- deoxyadenosine, 2'-Azido-2'-deoxycytidine, 2'-Azido-2'-deoxyguanosine, 2'-Azido-2'- deoxyuridine, 2-Chloroadenosine, 2'-Fluoro-2'-deoxyadenosine, 2'-Fluoro-2'- deoxycytidine, 2'-Fluoro-2'-deoxyguanosine, 2'-Fluoro-2'-deoxyuridine, 2'- Fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isopenenyl- adenosine, 2'-0-Methyl-2-aminoadenosine, 2'-0-Methyl-2'-deoxyadenosine, 2'-0-Methyl- 2'-deoxycytidine, 2'-0-Methyl-2'-deoxyguanosine, 2'-0-Methyl-2'-deoxyuridine, 2'-0- Methyl-5-methyluridine, 2'-0-Methylinosine, 2'-0-Methylpseudouridine, 2-Thiocytidine, 2- thio-cytosine, 3-methyl-cytosine, 4-acety I -cytosine, 4-Thiouridine, 5- (carboxyhydroxymethyl)-uracil, 5,6-Dihydrouridine, 5-Aminoallylcytidine, 5-Aminoallyl- deoxy-uridine, 5-Bromouridine, 5-carboxymehtylaminomethyl-2-thio-uracil, 5- carboxymethylamonomethyl-uracil, 5-Chloro-Ara-cytosine, 5-Fluoro-uridine, 5-lodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6- Azacytidine, 6-Azauridine, 6-Chloro-7-deaza-guanosine, 6-Chloropurineriboside, 6- Mercapto-guanosine, 6-Methyl-mercaptopurine-riboside, 7-Deaza-2 '-deoxy-guanosine, 7- Deazaadenosine, 7-methyl-guanosine, 8-Azaadenosine, 8-Bromo-adenosine, 8-Bromo- guanosine, 8-Mercapto-guanosine, 8-Oxoguanosine, Benzimidazole-riboside, Beta-D- mannosyl-queosine, Dihydro-uracil, Inosine, N1 -Methyladenosine, N6-([6- Aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl- adenosine, N7-Methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester, Puromycin, Queosine, Uracil-5-oxyacetic acid, Uracil-5-oxyacetic acid methyl ester, Wybutoxosine, Xanthosine, and Xylo-adenosine. The preparation of such analogues is known to a person skilled in the art, for example from US Patents 4,373,071 , US 4,401 ,796, US 4,41 5,732, US 4,458,066, US 4,500,707, US 4,668,777, US 4,973,679, US 5,047,524, US 5, 132,418, US 5,1 53,31 9, US 5,262,530 and 5,700,642. In the case of an analogue as described above, particular preference is given according to the invention to those analogues that increase the immunogenicity of the immunostimulatory RNA sequence used as the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention and/or do not interfere with a further modification that has been introduced into said immunostimulatory RNA.
In general, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention as defined above may also occur in the form of a modified nucleic acid, wherein any modification, as defined herein, may be introduced into the nucleic acid prior to lyophilization. Modifications as defined herein preferably lead to a further stabilized nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention.
According to a first aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention as defined above may thus be provided as a "stabilized nucleic acid", preferably as a stabilized RNA, more preferably as an RNA that is essentially resistant to in vivo degradation (e.g. by an exo- or endo-nuclease). Such stabilization can be effected, for example, by a modified phosphate backbone of the nucleic acid (sequence), lyophilized or to be lyophilized, according to the present invention. A backbone modification in connection with the present invention is a modification in which phosphates of the backbone of the nucleotides contained in the nucleic acid (sequence), lyophilized or to be lyophilized, are chemically modified. Nucleotides that may be preferably used in this connection contain e.g. a phosphorothioate-modified phosphate backbone, preferably at least one of the phosphate oxygens contained in the phosphate backbone being replaced by a sulfur atom. Stabilized lyophilized nucleic acids may further include, for example: non-ionic phosphate analogues, such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiesters and alkylphosphotriesters, in which the charged oxygen residue is present in alkylated form. Such backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5'-0-(1 -thiophosphate)).
The nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may additionally or alternatively also contain sugar modifications. A sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the nucleic acid (sequence), lyophilized or to be lyophilized, and typically includes, without implying any limitation, sugar modifications selected from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2 '-fluoro-2'-deoxycytidine-5'- triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate), 2'-deoxy-2'-deamine oligoribonucleotide (2'-amino-2 '-deoxycytidine-5'-triphosphate, 2 '-amino-2 '-deoxyuridine- 5 '-triphosphate), 2 '-0-alkyl oligoribonucleotide, 2 '-deoxy-2 '-C-alkyl oligoribonucleotide (2'-0-methylcytidine-5'-triphosphate, 2 '-methyluridine-5'-triphosphate), 2 '-C-alkyl oligoribonucleotide, and isomers thereof (2 '-aracytidine-5'-triphosphate, 2'-arauridine-5'- triphosphate), or azidotriphosphate (2 '-azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2 '- deoxyuridine-5'-triphosphate).
The nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may additionally or alternatively also contain at least one base modification, which is preferably suitable for increasing the expression of the protein coded for by the nucleic acid (sequence), lyophilized or to be lyophilized, as compared with the unaltered, i.e. natural (= native), nucleic acid sequence. Significant in this case means an increase in the expression of the protein compared with the expression of the native nucleic acid sequence by at least 20%, preferably at least 30%, 40%, 50% or 60%, more preferably by at least 70%, 80%, 90% or even 100% and most preferably by at least 1 50%, 200% or even 300% or more. In connection with the present invention, a nucleotide having such a base modification is preferably selected from the group of the base-modified nucleotides consisting of 2-amino- 6-chloropurineriboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2-thiocytidi Res' -triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-tr'iphosphate, 5- aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine- 5 '-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5- iodouridine-5 '-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'- triphosphate, 6-azacytidine-5 '-triphosphate, 6-azauridine-5'-triphosphate, 6- chloropurineriboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7- deazaguanosi ne-5 '-triphosphate, 8-azaadenosi ne-5 '-tri phosphate, 8-azidoadenosi ne-5 ' - triphosphate, benzimidazole-riboside-5'-triphosphate, Nl -methyladenosine-5'-triphosphate, N1 -methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 06- methylguanosi ne-5 '-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'- triphosphate, xanthosine-5 '-triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5- methylcytidine-5 '-triphosphate, 7-deazaguanosine-5 '-triphosphate, 5-bromocytidine-5'- triphosphate, and pseudouridine-5'-triphosphate.
According to another aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention can likewise be modified (and preferably stabilized) by introducing further modified nucleotides containing modifications of their ribose or base moieties. Generally, the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may contain any native (= naturally occurring) nucleotide, e.g. guanosine, uracil, adenosine, and/or cytosine or an analogue thereof. In this connection, nucleotide analogues are defined as non-natively occurring variants of naturally occurring nucleotides. Accordingly, analogues are chemically derivatized nucleotides with non-natively occurring functional groups, which are preferably added to or deleted from the naturally occurring nucleotide or which substitute the naturally occurring functional groups of a nucleotide. Accordingly, each component of the naturally occurring nucleotide may be modified, namely the base component, the sugar (ribose) component and/or the phosphate component forming the backbone (see above) of the nucleic acid sequence. Exemplary analogues of guanosine, uracil, adenosine, and cytosine include, without implying any limitation, any naturally occurring or non-naturally occurring guanosine, uracil, adenosine, thymidine or cytosine that has been altered chemically, for example by acetylation, methylation, hydroxylation, etc., including 1 - methyl-adenosine, 1 -methyl-guanosine, 1 -methyl-inosine, 2,2-dimethyl-guanosine, 2,6- diaminopurine, 2'-Amino-2'-deoxyadenosine, 2'-Amino-2'-deoxycytidine, 2'-Amino-2'- deoxyguanosine, 2,-Amino-2'-deoxyuridine, 2-Amino-6-chloropurineriboside, 2-Aminopurine- riboside, 2'-Araadenosine, 2'-Aracytidine, 2'-Arauridine, 2'-Azido-2'-deoxyadenosine, 2'- Azido-2'-deoxycytidine, 2'-Azido-2'-deoxyguanosine, 2'-Azido-2'-deoxyuridine, 2- Chloroadenosine, 2'-Fluoro-2'-deoxyadenosine, 2'-Fluoro-2'-deoxycytidine, 2'-Fluoro-2'- deoxyguanosine, 2'-Fluoro-2'-deoxyuridine, 2'-Fluorothymidine, 2-methyl-adenosine, 2- methyl-guanosine, 2-methyl-thio-N6-isopenenyl-adenosine, 2'-0-Methyl-2-aminoadenosine, 2'-0-Methyl-2'-deoxyadenosine, 2'-0-Methyl-2'-deoxycytidine, 2'-0-Methyl-2'- deoxyguanosine, 2'-0-Methyl-2'-deoxyuridine, 2'-0-Methyl-5-methyluridine, 2'-0- Methyl inosine, 2'-0-Methylpseudouridine, 2-Thiocytidine, 2-thio-cytosine, 3-methyl-cytosine,
4- acetyl-cytosine, 4-Thiouridine, 5-(carboxyhydroxymethyl)-uracil, 5,6-Dihydrouridine, 5- Aminoallylcytidine, 5-Aminoallyl-deoxy-uridine, 5-Bromouridine, 5- carboxymehtylaminomethyl-2-thio-uracil, 5-carboxymethylamonomethyl-uracil, 5-Chloro-Ara- cytosine, 5-Fluoro-uridine, 5-lodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy- uridine, 5-methyl-2-thio-uridine, 6-Azacytidine, 6-Azauridine, 6-Chloro-7-deaza-guanosine, 6- Chloropurineriboside, 6-Mercapto-guanosine, 6-Methyl-mercaptopurine-riboside, 7-Deaza-2'- deoxy-guanosine, 7-Deazaadenosine, 7-methyl-guanosine, 8-Azaadenosine, 8-Bromo- adenosine, 8-Bromo-guanosine, 8-Mercapto-guanosine, 8-Oxoguanosine, Benzimidazole- riboside, Beta-D-mannosyl-queosine, Dihydro-uracil, Inosine, N1-Methyladenosine, N6-Q6- Aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-Methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester, Puromycin, Queosine, Uracil-
5- oxyacetic acid, Uracil-5-oxyacetic acid methyl ester, Wybutoxosine, Xanthosine, and Xylo- adenosine. The preparation of such analogues is known to a person skilled in the art, for example from US Patents 4,373,071 , US 4,401 ,796, US 4,415,732, US 4,458,066, US 4,500,707, US 4,668,777, US 4,973,679, US 5,047,524, US 5,132,418, US 5,153,319, US 5,262,530 and 5,700,642. In the case of an analogue as described above, particular preference may be given according to the invention to those analogues that do not interfere with a further modification of the nucleic acid (sequence), lyophilized or to be lyophilized, that has been introduced.
According to a particular aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention can contain a lipid modification. Such a lipid-modified nucleic acid (sequence), lyophilized or to be lyophilized, typically comprises a nucleic acid as defined herein. Such a lipid-modified nucleic acid (sequence), lyophilized or to be lyophilized, typically further comprises at least one linker covalently linked with that nucleic acid, and at least one lipid covalently linked with the respective linker. Alternatively, the lipid-modified nucleic acid (sequence), lyophilized or to be lyophilized, comprises an at least one nucleic acid as defined herein and at least one (Afunctional) lipid covalently linked (without a linker) with that nucleic acid. According to a third alternative, the lipid-modified nucleic acid (sequence), lyophilized or to be lyophilized, comprises a nucleic acid RNA as defined herein, at least one linker covalently linked with that nucleic acid, and at least one lipid covalently linked with the respective linker, and also at least one (bifunctional) lipid covalently linked (without a linker) with that nucleic acid.
The lipid contained in the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention (complexed or covalently bound thereto) is typically a lipid or a lipophilic residue that preferably is itself biologically active. Such lipids preferably include natural substances or compounds such as, for example, vitamins, e.g. alpha-tocopherol (vitamin E), including RRR-alpha-tocopherol (formerly D-alpha-tocopherol), L-alpha-tocopherol, the racemate D,L-alpha-tocopherol, vitamin E succinate (VES), or vitamin A and its derivatives, e.g. retinoic acid, retinol, vitamin D and its derivatives, e.g. vitamin D and also the ergosterol precursors thereof, vitamin E and its derivatives, vitamin K and its derivatives, e.g. vitamin K and related quinone or phytol compounds, or steroids, such as bile acids, for example cholic acid, deoxycholic acid, dehydrocholic acid, cortisone, digoxygenin, testosterone, cholesterol or thiocholesterol. Further lipids or lipophilic residues within the scope of the present invention include, without implying any limitation, polyalkylene glycols (Oberhauser et al., Nucl. Acids Res., 1 992, 20, 533), aliphatic groups such as, for example, Cl -C20-alkanes, C1 -C20-alkenes or C1 -C20-alkanol compounds, etc., such as, for example, dodecanediol, hexadecanol or undecyl residues (Saison-Behmoaras et a/., EMBO J, 1 991 , 10, 1 1 1 ; Kabanov et a/., FEBS Lett., 1 990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), phospholipids such as, for example, phosphatidylglycerol, diacylphosphatidylglycerol, phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, di- hexadecy!-rac-glycerol, sphingolipids, cerebrosides, gangliosides, or triethylammonium 1 ,2- di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651 ; Shea et al., Nucl. Acids Res., 1990, 1 8, 3777), polyamines or polyalkylene glycols, such as, for example, polyethylene glycol (PEG) (Manoharan et al, Nucleosides & Nucleotides, 1995, 14, 969), hexaethylene glycol (HEG), palmitin or palmityl residues (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), octadecylamines or hexylamino- carbonyl-oxycholesterol residues (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277, 923), and also waxes, terpenes, alicyclic hydrocarbons, saturated and mono- or poly-unsaturated fatty acid residues, etc.
The nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may likewise be stabilized in order to prevent degradation of the nucleic acid by various approaches, particularly, when RNA or mRNA is used as a lyophilized nucleic acid. It is known in the art that instability and (fast) degradation of mRNA or of RNA in general may represent a serious problem in the application of RNA based compositions. This instability of RNA is typically due to RNA-degrading enzymes, "RNAases" (ribonucleases), wherein contamination with such ribonucleases may sometimes completely degrade RNA in solution. Accordingly, the natural degradation of mRNA in the cytoplasm of cells is very finely regulated and RNase contaminations may be generally removed by special treatment prior to use of said compositions, in particular with diethyl pyrocarbonate (DEPC). A number of mechanisms of natural degradation are known in this connection in the prior art, which may be utilized as well. E.g., the terminal structure is typically of critical importance for a mRNA. As an example, at the 5' end of naturally occurring mRNAs there is usually a so-called "cap structure" (a modified guanosine nucleotide), and at the 3' end is typically a sequence of up to 200 adenosine nucleotides (the so-called poly-A tail).
The nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, particularly if provided as a mRNA, can therefore be stabilized against degradation by RNases by the addition of a so-called "5' cap" structure. Particular preference is given in this connection to an m7G(5')ppp (5'(A,G(5')ppp(5')A or G(5')ppp(5')G as the 5' cap" structure. However, such a modification is introduced only if a modification, for example a lipid modification, has not already been introduced at the 5' end of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention if provided as a mRNA or if the modification does not interfere with the immunogenic properties of the (unmodified or chemically modified) nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention. According to a further preferred aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may contain, especially if the nucleic acid is in the form of a mRNA, a poly-A tail on the 3' terminus of typically about 10 to 200 adenosine nucleotides, preferably about 10 to 100 adenosine nucleotides, more preferably about 20 to 100 adenosine nucleotides or even more preferably about 40 to 80 adenosine nucleotides.
According to a further preferred aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may contain, especially if the nucleic acid is in the form of a mRNA, a poly-C tail on the 3' terminus of typically about 10 to 200 cytosine nucleotides, preferably about 10 to 100 cytosine nucleotides, more preferably about 20 to 70 cytosine nucleotides or even more preferably about 20 to 60 or even 10 to 40 cytosine nucleotides. According to another aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may be modified, and thus stabilized, especially if the nucleic acid is in the form of a mRNA, by modifying the G/C content of the nucleic acid, particularly an mRNA, preferably of the coding region thereof. In a particularly preferred aspect of the present invention, the G/C content of the coding region of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is modified, particularly increased, compared to the G/C content of the coding region of its particular wild type mRNA, i.e. the unmodified mRNA. The encoded amino acid sequence of the at least one mRNA is preferably not modified compared to the coded amino acid sequence of the particular wild type mRNA.
This modification of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is based on the fact that the sequence of any mRNA region to be translated is important for efficient translation of that mRNA. Thus, the composition and the sequence of various nucleotides is important. In particular, sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content. According to the invention, the codons of the mRNA are therefore varied compared to its wild type mRNA, while retaining the translated amino acid sequence, such that they include an increased amount of G/C nucleotides. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage).
Depending on the amino acid to be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, there are various possibilities for modification of the at least one mRNA sequence, compared to its wild type sequence. In the case of amino acids which are encoded by codons which contain exclusively G or C nucleotides, no modification of the codon is necessary. Thus, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) require no modification, since no A or U is present.
In contrast, codons which contain A and/or U nucleotides can be modified by substitution of other codons which code for the same amino acids but contain no A and/or U. Examples of these are: the codons for Pro can be modified from CCU or CCA to CCC or CCG;
the codons for Arg can be modified from CGU or CGA or AGA or AGG to CGC or CGG; the codons for Ala can be modified from GCU or GCA to GCC or GCG;
the codons for Gly can be modified from GGU or GGA to GGC or GGG. In other cases, although A or U nucleotides cannot be eliminated from the codons, it is however possible to decrease the A and U content by using codons which contain a lower content of A and/or U nucleotides. Examples of these are: the codons for Phe can be modified from UUU to UUC;
the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUC or CUG;
the codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyr can be modified from UAU to UAC;
the codon for Cys can be modified from UGU to UGC; the codon for His can be modified from CAU to CAC;
the codon for Gin can be modified from CAA to CAG;
the codons for lie can be modified from AUU or AUA to AUC;
the codons for Thr can be modified from ACU or ACA to ACC or ACG;
the codon for Asn can be modified from AAU to AAC;
the codon for Lys can be modified from AAA to AAG;
the codons for Val can be modified from GUU or GUA to GUC or GUG;
the codon for Asp can be modified from GAU to GAC;
the codon for Glu can be modified from GAA to GAG;
the stop codon UAA can be modified to UAG or UGA.
In the case of the codons for Met (AUG) and Trp (UGG), on the other hand, there is no possibility of sequence modification. The substitutions listed above can be used either individually or in all possible combinations to increase the G/C content of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, compared to its particular wild type mRNA (i.e. the original sequence). Thus, for example, all codons for Thr occurring in the wild type sequence can be modified to ACC (or ACG). Preferably, however, for example, combinations of the above substitution possibilities are used: substitution of all codons coding for Thr in the original sequence (wild type mRNA) to ACC (or ACG) and
substitution of a codons originally coding for Ser to UCC (or UCG or AGC);
substitution of a codons coding for lie in the original sequence to AUC and
substitution of a codons originally coding for Lys to AAG and
substitution of a codons originally coding for Tyr to UAC;
substitution of a codons coding for Val in the original sequence to GUC (or GUG) and substitution of a codons originally coding for Glu to GAG and
substitution of all codons originally coding for Ala to GCC (or GCG) and
substitution of all codons originally coding for Arg to CGC (or CGG);
substitution of a codons coding for Val in the original sequence to GUC (or GUG) and substitution of all codons originally coding for Glu to GAG and
substitution of all codons originally coding for Ala to GCC (or GCG) and
substitution of all codons originally coding for Gly to GGC (or GGG) and
substitution of all codons originally coding for Asn to AAC;
substitution of all codons coding for Val in the original sequence to GUC (or GUG) and substitution of all codons originally coding for Phe to UUC and
substitution of all codons originally coding for Cys to UGC and
substitution of all codons originally coding for Leu to CUG (or CUC) and
substitution of all codons originally coding for Gin to CAG and
substitution of all codons originally coding for Pro to CCC (or CCG); etc.
Preferably, the G/C content of the coding region of nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is increased by at least 7%, more preferably by at least 15%, particularly preferably by at least 20%, compared to the G/C content of the coded region of the wild type mRNA. According to a specific aspect at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70 %, even more preferably at least 80% and most preferably at least 90%, 95% or even 100% of the substitutable codons in the region coding for a protein or peptide as defined herein or its fragment or variant thereof or the whole sequence of the wild type mRNA sequence are substituted, thereby increasing the GC content of said sequence.
In this context, it is particularly preferable to increase the G/C content of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, to the maximum (i.e. 100% of the substitutable codons), in particular in the region coding for a protein, compared to the wild type sequence.
According to the invention, a further preferred modification of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, if so-called "rare codons" are present in the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, to an increased extent, the corresponding modified nucleic acid sequence is translated to a significantly poorer degree than in the case where codons coding for relatively "frequent" tRNAs are present.
Especially if the modified nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention is in the form of a mRNA, the coding region of the modified nucleic acid (sequence), lyophilized or to be lyophilized, is preferably modified compared to the corresponding region of the wild type mRNA such that at least one codon of the wild type sequence which codes for a tRNA which is relatively rare in the cell is exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and carries the same amino acid as the relatively rare tRNA. By this modification, the sequences of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is modified such that codons for which frequently occurring tRNAs are available are inserted. In other words, according to the invention, by this modification all codons of the wild type sequence which code for a tRNA which is relatively rare in the cell can in each case be exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and which, in each case, carries the same amino acid as the relatively rare tRNA.
Which tRNAs occur relatively frequently in the cell and which, in contrast, occur relatively rarely is known to a person skilled in the art; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001 , 1 1 (6): 660-666. The codons which use for the particular amino acid the tRNA which occurs the most frequently, e.g. the Gly codon, which uses the tRNA which occurs the most frequently in the (human) cell, are particularly preferred.
According to the invention, it is particularly preferable to link the sequential G/C content which is increased, in particular maximized, in the modified nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, with the "frequent" codons without modifying the amino acid sequence of the protein encoded by the coding region of the nucleic acid. This preferred aspect allows provision of a particularly efficiently translated and stabilized (modified) nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA.
The determination of a modified nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention as described above (increased G/C content; exchange of tRNAs) can be carried out using the computer program explained in WO 02/098443 - the disclosure content of which is included in its full scope in the present invention. Using this computer program, the nucleotide sequence of any desired nucleic acid or mRNA can be modified with the aid of the genetic code or the degenerative nature thereof 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, and the amino acid sequence coded by the modified nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention preferably not being modified compared to the non-modified sequence. Alternatively, it is also possible to modify only the G/C content or only the codon usage compared to the original sequence. The source code in Visual Basic 6.0 (development environment used: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3) is also described in WO 02/098443.
In a further preferred aspect of the present invention, the A/U content in the environment of the ribosome binding site of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is increased compared to the A U content in the environment of the ribosome binding site of its particular wild type mRNA. This modification (an increased A/U content around the ribosome binding site) increases the efficiency of ribosome binding to the nucleic acid. An effective binding of the ribosomes to the ribosome binding site (Kozak sequence: GCCGCCACCAUGG (SEQ ID NO: 3), the AUG forms the start codon) in turn has the effect of an efficient translation of the nucleic acid.
According to a further aspect the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, may be modified with respect to potentially destabilizing sequence elements. Particularly, the coding region and/or the 5' and/or 3' untranslated region of this nucleic acid (sequence), lyophilized or to be lyophilized, may be modified compared to the particular wild type nucleic acid such that is contains no destabilizing sequence elements, the coded amino acid sequence of the modified nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, preferably not being modified compared to its particular wild type nucleic acid. It is known that, for example, in sequences of eukaryotic RNAs destabilizing sequence elements (DSE) occur, to which signal proteins bind and regulate enzymatic degradation of RNA. For further stabilization of the modified nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, optionally in the region which encodes for a protein or a peptide as defined herein, one or more such modifications compared to the corresponding region of the wild type nucleic acid can therefore be carried out, so that no or substantially no destabilizing sequence elements are contained there. According to the invention, DSE present in the untranslated regions (3 '- and/or 5'-UTR) can also be eliminated from the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, by such modifications.
Such destabilizing sequences are e.g. AU-rich sequences (AURES), which occur in 3 '-UTR sections of numerous unstable RNAs (Caput et al, Proc. Natl. Acad. Sci. USA 1 986, 83: 1 670 to 1 674). The nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is therefore preferably modified compared to the wild type nucleic acid such that the modified nucleic acid contains no such destabilizing sequences. This also applies to those sequence motifs which are recognized by possible endonucleases, e.g. the sequence GAACAAG, which is contained in the 3'-UTR segment of the gene which codes for the transferrin receptor (Binder et al., EMBO J. 1994, 1 3: 1 969 to 1 980). These sequence motifs are also preferably removed in the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA.
Also preferably, the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, has, in a modified form, at least one IRES as defined above and/or at least one 5 ' and/or 3' stabilizing sequence, in a modified form, e.g. to enhance ribosome binding or to allow expression of different encoded proteins located on the at least one (bi- or even multicistronic) RNA of the nucleic acid (sequence), lyophi lized or to be lyophilized, of the present invention. According to the invention, the nucleic acid (sequence), lyophilized or to be lyophilized, as described herein, especially if the nucleic acid is in the form of a mRNA, furthermore preferably has at least one 5' and/or 3' stabilizing sequence. These stabilizing sequences in the 5' and/or 3' untranslated regions have the effect of increasing the half-life of the nucleic acid in the cytosol. These stabilizing sequences can have 100% sequence identity to naturally occurring sequences which occur in viruses, bacteria and eukaryotes, but can also be partly or completely synthetic. The untranslated sequences (UTR) of the (alpha-)globin gene, e.g. from Homo sapiens or Xenopus laevis may be mentioned as an example of stabilizing sequences which can be used in the present invention for a stabilized lyophilized nucleic acid. Another example of a stabilizing sequence has the general formula (C/U)CCANxCCC(U/A)PyxUC(C/U)CC (SEQ ID NO: 4), which is contained in the 3'UTR of the very stable RNA which codes for (alpha-)globin, type(l)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf. Holcik et a/., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Such stabilizing sequences can of course be used individually or in combination with one another and also in combination with other stabilizing sequences known to a person skilled in the art. The nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, is therefore preferably present as (alpha-)globin UTR (untranslated regions)-stabilized RNA, in particular as (alpha- )globin UTR-stabilized RNA.
Nevertheless, substitutions, additions or eliminations of bases are preferably carried out with the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, using a DNA matrix for preparation of the nucleic acid nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention by techniques of the well known site directed mutagenesis or with an oligonucleotide ligation strategy (see e.g. Maniatis et a/., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., Cold Spring Harbor, NY, 2001). In such a process, for preparation of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, a corresponding DNA molecule may be transcribed in vitro. This DNA matrix preferably comprises a suitable promoter, e.g. a T7 or SP6 promoter, for in vitro transcription, which is followed by the desired nucleotide sequence for the nucleic acid, e.g. mRNA, to be prepared and a termination signal for in vitro transcription. The DNA molecule, which forms the matrix of at least one RNA of interest, may be prepared by fermentative proliferation and subsequent isolation as part of a plasmid which can be replicated in bacteria. Plasmids which may be mentioned as suitable for the present invention are e.g. the plasmids pT7Ts (GenBank accession number U26404; Lai et a/., Development 1 995, 121 : 2349 to 2360), pGEM® series, e.g. pGEM®-1 (GenBank accession number X65300; from Promega) and pSP64 (GenBank accession number X65327); cf. also Mezei and Storts, Purification of PCR Products, in: Griffin and Griffin (ed.), PCR Technology: Current Innovation, CRC Press, Boca Raton, FL, 2001 .
Nucleic acid molecules used according to the invention as defined above may be prepared using any method known in the art, including synthetic methods such as e.g. solid phase synthesis, as well as in vitro methods, such as in vitro transcription reactions.
According to another particularly preferred aspect, the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, may additionally or alternatively encode a secretory signal peptide. Such signal peptides are sequences, which typically exhibit a length of about 15 to 30 amino acids and are preferably located at the N-terminus of the encoded peptide, without being limited thereto. Signal peptides as defined herein preferably allow the transport of the protein or peptide as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, into a defined cellular compartiment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartiment. Examples of secretory signal peptide sequences as defined herein include, without being limited thereto, signal sequences of classical or non- classical MHC-molecules (e.g. signal sequences of MHC I and II molecules, e.g. of the MHC class I molecule HLA-A*0201 ), signal sequences of cytokines or immunoglobulines as defined herein, signal sequences of the invariant chain of immunoglobulines or antibodies as defined herein, signal sequences of Lampl , Tapasin, Erp57, Calretikulin, Calnexin, and further membrane associated proteins or of proteins associated with the endoplasmic reticulum (ER) or the endosomal-lysosomal compartiment. Particularly preferably, signal sequences of MHC class I molecule HLA-A*0201 may be used according to the present invention.
Any of the above modifications may be applied to the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, especially if the nucleic acid is in the form of a mRNA, and further to any nucleic acid (sequence), lyophilized or to be lyophilized, as used in the context of the present invention and may be, if suitable or necessary, be combined with each other in any combination, provided, these combinations of modifications do not interfere with each other in the respective lyophilized nucleic acid. A person skilled in the art will be able to take his choice accordingly.
The nucleic acid (sequence), lyophilized or to be lyophilized, as well as proteins or peptides as encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention as defined above, may comprise fragments or variants of those sequences. Such fragments or variants may typically comprise a sequence having a sequence identity with one of the above mentioned nucleic acids, or with one of the proteins or peptides or sequences, if encoded by the nucleic acid sequences of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably at least 70%, more preferably at least 80%, equally more preferably at least 85%, even more preferably at least 90% and most preferably at least 95% or even 97%, to the entire wild type sequence, either on nucleic acid level or on amino acid level.
"Fragments" of proteins or peptides in the context of the present invention (encoded by a nucleic acid as defined herein) may comprise a sequence of a protein or peptide as defined above, which is, with regard to its amino acid sequence (or its encoded nucleic acid sequence), N-terminally, C-terminally and/or intrasequentially truncated compared to the amino acid sequence of the original (native) protein (or its encoded nucleic acid sequence). Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level. A sequence identity with respect to such a fragment as defined above may therefore preferably refer to the entire protein or peptide as defined above or to the entire (coding) nucleic acid sequence of such a protein or peptide. Likewise, "fragments" of nucleic acids in the context of the present invention may comprise a sequence of a nucleic acid as defined above, which is, with regard to its nucleic acid sequence 5'-, 3'- and/or intrasequentially truncated compared to the nucleic acid sequence of the original (native) nucleic acid sequence. A sequence identity with respect to such a fragment as defined above may therefore preferably refer to the entire nucleic acid as defined above.
Fragments of proteins or peptides in the context of the present invention (encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein) may furthermore comprise a sequence of a protein or peptide as defined above, which has a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 1 1 , or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 1 7, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form.
Fragments of proteins or peptides as defined herein (encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein) may also comprise epitopes of those proteins or peptides. Epitopes (also called "antigen determinants") in the context of the present invention are typically fragments located on the outer surface of (native) proteins or peptides as defined herein, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies or B-cell receptors, i.e. in their native form. Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides. In this context antigenic determinants can be conformational or discontinous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain. "Variants" of proteins or peptides as defined above may be encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, wherein nucleic acids of the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention, encoding the protein or peptide as defined above, are exchanged. Thereby, a protein or peptide may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s). Preferably, these fragments and/or variants have the same biological function or specific activity compared to the full-length native potein, e.g. its specific antigenic property. The nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention may also encode a protein or peptide as defined above, wherein the encoded amino acid sequence comprises conservative amino acid substitution(s) compared to its physiological sequence. Those encoded amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined above. Substitutions in which amino acids which originate from the same class are exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function. This means that e.g. an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three- dimensional structure or do not affect the binding region. Modifications to a three- dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra) (Urry, 1 85, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam).
Furthermore, variants of proteins or peptides as defined above, which may be encoded by the nucleic acid (sequence), lyophilized or to be lyophi lized, of the present invention, may also comprise those sequences, wherein nucleic acids of the nucleic acid (sequence), lyophilized or to be lyophilized, are exchanged according to the degeneration of the genetic code, without leading to an alteration of respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least part thereof may not differ from the original sequence in one or more mutation(s) within the above meaning. In order to determine the percentage to which two sequences (nucleic acid sequences, e.g. nucleic acid sequences as defined herein, or amino acid sequences, preferably their encoded amino acid sequences, e.g. the amino acid sequences of the proteins or peptides as defined above) are identical, the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. gaps can be inserted into the sequence of the first sequence and the component at the corresponding position of the second sequence can be compared. If a position in the first sequence is occupied by the same component as is the case at a position in the second sequence, the two sequences are identical at this position. The percentage to which two sequences are identical is a function of the number of identical positions divided by the total number of positions. The percentage to which two sequences are identical can be determined using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et a/. (1993), PNAS USA, 90:5873-5877 or Altschul et a/. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.
In the context of the present invention, the nucleic acid as defined above is typically present in a lactate containing solution prior to lyophilization. In this context, the lactate is typically already contained in or added to the nucleic acid as defined above to form such a solution, or vice versa.
The nucleic acid and lactate containing solution prior to lyophilization may additionally contain water, preferably water for injection (WFI). In this context, the term "water for injection" (WFI) is a term defined by standard USP 23. USP 23 monograph states that " Water for Injection (WFI) is water purified by distillation or reverse osmosis." WFI is typically produced by either distillation or 2-stage reverse osmosis. WFI typically does not contain more than 0.25 USP endotoxin units (EU) per ml. Endotoxins are a class of pyrogens that are components of the cell wall of Gram-negative bacteria (the most common type of bacteria in water), preferably in an action limit of 10 cfu/100 ml. The microbial quality may be tested by membrane filtration of a 100 ml sample and plate count agar at an incubation temperature of 30 to 35 degrees Celsius for a 48-hour period. The chemical purity requirements of WFI are typically the same as of PW (purified water). Upon lyophilization, the (residual) water content of the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein is typically reduced to a content of about 0.5 % (w/w) to about 10 % (w/w), more preferably to a content of about 1 % (w/w) to about 5 % (w/w), even more preferably to a content of about 2 % (w/w) to about 4% (w/w), most preferably to a content of about 3 % (w/w), e.g. 3 % (w/w) ± 2 % (w/w), or 3 % (w/w) ± 1 % (w/w). The nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may contain, additional to the nucleic acid and the lactate, further components or additives, e.g. a cryoprotectant, a lyoprotectant or any further suitable additive, preferably as defined in the following.
As a particularly preferred component or additive, the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may additionally contain at least one suspending agent, preferably mannit, preferably in a concentration of about 1 to 15% (w/w), more preferably in a concentration of about 3 to 10% (w/w), and even more preferably in a concentration of about 4 to 6% (w/w).
As a further component, the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may additionally contain at least one component or additive selected, e.g., from proteins, amino acids, alcohols, carbohydrates, mannose, mannit, metals or metal ions, surfactants, polymers or complexing agents, buffers, etc., or a combination thereof. In the context of the present invention, one preferred component or additive may also be selected from the group of amino acids. Such group may may comprise, without being limited thereto, any naturally occurring amino acid, including alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine, more preferably glycine, arginine, and alanine. Cryoprotectants and/or lyoprotectants selected from the group of amino acids may additionally comprise any modification of a naturally occurring amino acid as defined above. Furthermore, in the context of the present invention, a further component or additive may be selected from the group of alcohols. Such group may comprise, without being limited thereto, any alcohol suitable for the preparation of a pharmaceutical composition, preferably, without being limited thereto, mannitol, polyethyleneglycol, polypropyleneglycol, sorbitol, etc. Additionally, in the context of the present invention, a further component or additive may be selected from the group of (free) carbohydrates. Such group may comprise, without being limited thereto, any (free) carbohydrate, suitable for the preparation of a pharmaceutical composition, preferably, without being limited thereto, (free) monosaccharides, such as e.g. (free) glucose, (free) fructose, (free) mannose ("free" preferably means unbound or unconjugated, e.g. the mannose is not covalently bound to the nucleic acid(sequence) to be lyophilized, or in other words, the mannose is unconjugated, preferably with respect to the nucleic acid (sequence) to be lyophilized), etc., disaccharides, such as e.g. lactose, maltose, sucrose, trehalose, etc., and polysaccharides, such as e.g. dextran, HP-beta CD, etc.
According to a particularly preferred aspect, mannose as a further component or additive is a D-Mannose. D-Mannose may be depicted according to at least one of the D-Mannose isomers a-D-Mannofuranose, β-D-Mannofuranose, a-D-Mannopyranose and β-D- Mannopyranose. Typically, the occurrence of the different mannose isomers in nature significantly differs. D-Mannose forms anomers, wherein a-D-Mannofuranose ocurrs in a concentration/frequency of less than 1 %, β-D-Mannofuranose in a concentration/frequency of less than 1 %, a-D-Mannopyranose in a concentration/frequency of about 67 % and β-D- Mannopyranose in a concentration/frequency of about 33 %. Thus, D-Mannose may be selected more preferably from at least one, two, three or four of the anomers a-D- Mannofuranose, β-D-Mannofuranose, α-D-Mannopyranose and/or β-D-Mannopyranose. Importantly, upon solubilization in an aqueous solution mannose typically forms the above anomers in an equilibrity reaction, typically in the above concentrations. According to an even more preferred aspect, mannose as a further component or additive is selected from an anomeric mixture of D-Mannose, preferably an anomeric mixture comprising a-D- Mannofuranose, β-D-Mannofuranose, α-D-Mannopyranose and β-D-Mannopyranose, more preferably in the above concentrations/frequencies. Alternatively, but less preferred, mannose as a further component or additive may be selected from L-mannose or a racemic mixture of D-Mannose and/or L-Mannose, wherein D-mannose is preferably as described above. Such mixtures may be obtained e.g. by a non-selective synthesis of mannose, e.g. by non-selective oxidation of mannitol. An anomeric mixture may furthermore be obtained by solubilization of mannose in an aqueous solution, e.g. in water, WFI, or any buffer or solution as defined herein. According to a more particular aspect, mannose as a further component or additive is typically present in the inventive solution for lyophilization, transfection and/or injection in a concentration of about 0.01 to about 10% (w/w), preferably in a concentration of about 0.01 to about 10% (w/w), more preferably in a concentration of about 0.1 to about 7.5% (w/w), even more preferably in a concentration of about 0.5 to about 5% (w/w), and most preferably in a concentration of about 1 to about 4% (w/w), e.g. a concentration of about 2 to about 4% (w/w), such as about 2.5 % (w/w). Herein, a concentration of about 1 % (w/w) mannose corresponds to a concentration of about 55,506 mM mannose. Any of the above and herein mentioned values and concentrations for mannose in % (w/w) may thus be calculated in mM on the above basis. Also, in the context of the present invention, a further suitable component or additive may be selected from the group of proteins. Such group may comprise, without being limited thereto, proteins such as albumin, gelatine, therapeutically active proteins as defined above, antibodies as defined above, antigens as defined above, or any further protein encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention as defined above.
A component or additive, which may be contained in the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and accordingly in the nucleic acid and lactate containing solution prior to lyophilization may be selected from the group of metals or metal ions, typically comprising, without being limited thereto, metals or metal ions or salts selected from
alkali metals, including members of group 1 of the periodic table: lithium (Li), sodium (Na), potassium (K), rubidium ( b), caesium (Cs), and francium (Fr), and their (monovalent) metal alkali metal ions and salts; preferably lithium (Li), sodium (Na), potassium (K), and their (monovalent) metal alkali metal ions and salts;
alkaline earth metals, including members of group 2 of the periodic table: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba) and radium (Ra), and their (divalent) alkaline earth metal ions and salts; preferably magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba) and their (divalent) alkaline earth metal ions and salts;
transition metals, including members of groups 3 to 13 of the periodic table and their metal ions and salts;. The transition metals typically comprise the 40 chemical elements 21 to 30, 39 to 48, 71 to 80, and 103 to 1 12. The name transition originates from their position in the periodic table of elements. In each of the four periods in which they occur, these elements represent the successive addition of electrons to the d atomic orbitals of the atoms. In this way, the transition metals represent the transition between subgroup 2 elements and subgroup 12 (or 13) elements. Transition metals in the context of the present invention particularly comprise members of subgroup 3 of the periodic table: including Scandium (Sc), Yttrium (Y), and Lutetium (Lu), members of subgroup 4 of the periodic table: including Titan (Ti), Zirconium (Zr), and Hafnium (HO, members of subgroup 5 of the periodic table: including Vanadium (V), Niobium (Nb), and Tantalum (Ta), members of subgroup 6 of the periodic table: including Chrome (Cr), Molybdenum (Mo), and Tungsten (W), members of subgroup 7 of the periodic table: including Manganese (Mn), Technetium (Tc), and Rhenium (Re), members of subgroup 8 of the periodic table: including Iron (Fe), Ruthenium (Ru), and Osmium (Os), members of subgroup 9 of the periodic table: including Cobalt (Co), Rhodium (Rh), and Iridium (Ir), members of subgroup 10 of the periodic table: including Nickel (Ni), Palladium (Pd), and Platin (Pt), members of subgroup 1 1 of the periodic table: including Copper (Cu), Silver (Ag), and Gold (Au), members of subgroup 12 of the periodic table: including Zinc (Zn), Cadmium (Cd), and Mercury (Hg); preferably members of period 4 of any of subgroups 1 to 12 of the periodic table: including Scandium (Sc), Titanium (Ti), Vanadium (V), Chromium (Cr), Manganese (Mn), Iron (Fe), Cobalt (Co), Nickel (Ni), Copper (Cu) and Zinc (Zn) and their metal ions and salts;
earth metals or members of the boron group, including members of group 3 of the periodic table: including Boron (B), Aluminium (Al), Gallium (Ga), Indium (In) and Thallium (TI) and their metal ions and salts; preferably Boron (B) and Aluminium (Al) and their metal ions and salts;
metalloids or semi metals: including Boron (B), Silicon (Si), Germanium (Ge), Arsenic (As), Antimony (Sb), Tellurium (Te).and Polonium (Po), and their semi metal ions and salts; preferably Boron (B) and Silicon (Si) and their semi metal ions and salts;
In the context of the present invention, a further component or additive may be selected from the group of surfactant may comprise, without being limited thereto, any surfactant, suitable for the preparation of a pharmaceutical composition, preferably, without being limited thereto, Tween, e.g. Tween 80 (0.2%), Pluronics, e.g. Pluronic L121 (1 .25%), Triton-X, SDS, PEG, LTAB, Saponin, Cholate, etc.
Another component or additive, which may be contained in the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and accordingly in the nucleic acid and lactate containing solution prior to lyophilization may be selected from the group of polymers or complexing agents, typically comprising, without being limited thereto, any polymer suitable for the preparation of a pharmaceutical composition, such as minor/major groove binders, nucleic acid binding proteins, lipoplexes, nanoplexes, non-cationic or non- polycationic compounds, such as PLGA, Polyacetate, Polyacrylate, PVA, Dextran, hydroxymethylcellulose, starch, MMP, PVP, heparin, pectin, hyaluronic acid, and derivatives thereof, or cationic or polycationic compound, particularly cationic or polycationic polymers or cationic or polycationic lipids, preferably a cationic or polycationic polymers. In the context of the present invention, such a cationic or polycationic compound is typically selected from any cationic or polycationic compound, suitable for complexing and thereby stabilizing a nucleic acid as defined herein, e.g. by associating the nucleic acid as defined herein with the cationic or polycationic compound. Particularly preferred, cationic or polycationic compounds are selected from cationic or polycationic peptides or proteins, including protamine, nucleoline, spermin or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1 , L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1 ), pVEC, hCT-derived peptides, SAP, protamine, spermine, spermidine, or histones. Additionally, preferred cationic or polycationic proteins or peptides may be selected from following proteins or peptides having the following total formula: (Arg)i;(Lys)m;(His)n;(Orn)0;(Xaa)x, wherein I + m + n +o + x = 8-15, and I, m, n or o independently of each other may be any number selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 50% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native (= naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and x may be any number selected from 0, 1 , 2, 3 or 4, provided, that the overall content of Xaa does not exceed 50 % of all amino acids of the oligopeptide. Particularly preferred oligoarginines in this context are e.g. Arg7, Argg, Arg9, Arg7, H3R9, R9H3, H3R9H3, YSSR9SSY, (RKH)4, Y(RKH)2R, etc. Further preferred cationic or polycationic compounds, which can be used for complexing the nucleic acid as defined herein may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA: [1 -(2,3-sioleyloxy)propyl)]-N,N,N- trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Choi, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3- (trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(a- trimethylammonioacetyl)diethanolamine chloride, CLIP1 : rac-[(2,3- dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3- dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9: rac-[2(2,3- dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, e.g. modified polyaminoacids, such as β-aminoacid- polymers or reversed polyamides, etc., modified polyethylenes, such as PVP (poly(N-ethyl- 4-vinylpyridinium bromide)), etc., modified acrylates, such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc., modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modified polybetaaminoester (PBAE), such as diamine end modified 1 ,4 butanediol diacrylate-co-5-amino-1 -pentanol polymers, etc., dendrimers, such as polypropylamine dendrimers or pAMAM based dendrimers, etc., polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, sugar backbone based polymers, such as cyclodextrin based polymers, dextran based polymers, Chitosan, etc., si Ian backbone based polymers, such as PMOXA-PDMS copolymers, etc., Blockpolymers consisting of a combination of one or more cationic blocks (e.g. selected of a cationic polymer as mentioned above) and of one or more hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole); etc. Association or complexing the nucleic acid as defined herein with cationic or polycationic compounds preferably provides adjuvant properties to the RNA and confers a stabilizing effect to the nucleic acid as defined herein by complexation. The procedure for stabilizing the nucleic acid as defined herein is in general described in EP-A-1 083232, the disclosure of which is incorporated by reference into the present invention in its entirety. Particularly preferred as cationic or polycationic compounds are compounds selected from the group consisting of protamine, nucleoline, spermin, spermidine, oligoarginines as defined above, such as Arg7, Arge, Arg9, Arg7, H3R9, R9H3, H3R9H3, YSSR9SSY, (RKH)4/ Y(RKH)2R, etc. As a further component, the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may additionally contain water, water for injection (WFI), or a buffer, preferably selected from a buffer as defined above, e.g. a buffer containing 2- hydroxypropanoic acid, preferably including at least one of its optical isomers L-(+)-lactic acid, (5)-lactic acid, D-(-)-lactic acid or (A)-lactic acid, more preferably its biologically active optical isomer L-(+)-lactic acid, or a salt or an anion thereof, preferably selected from sodium-lactate, potassium-lactate, or Al3 +-lactate, NH4 +-lactate, Fe-lactate, Li-lactate, Mg- lactate, Ca-lactate, Mn-lactate or Ag-lactate, or a buffer selected from Ringer's lactate (RiLa), lactated Ringer's solution (main content sodium lactate, also termed "Hartmann's Solution" in the UK), acetated Ringer's solution, or ortho-lactate-containing solutions (e.g. for injection purposes), or lactate containing water. A buffer as defined herein may also be a mannose containing buffer, an isotonic buffer or solution, preferably selected from isotonic saline, a lactate or ortho-lactate-containing isotonic solution, a isotonic buffer or solution selected from phosphate-buffered saline (PBS), TRIS-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS), Grey's balanced salt solution (GBSS), or normal saline (NaCI), hypotonic (saline) solutions with addition of glucose or dextrose, or any solution as defined herein, etc. These isotonic buffers or solutions are preferably prepared by a skilled person preferably as defined herein or according to definitions preparation protocols well known in the art for these specific isotonic buffers or solutions. More preferably, the lactate containing solution prior to lyophilization as defined above may contain these isotonic buffers or solutions or (all) its contents in isotonic concentrations, preferably as defined herein or in the art for these specific isotonic solutions. Preferably, the lactate containing solution prior to lyophilization as defined above and accordingly its components, additives, isotonic buffers or solutions, may be present in an osmolality or osmolarity comparable to that of blood plasma, preferably in an osmolarity as generally defined herein for the nucleic acid and lactate containing solution prior to lyophilization (as well as after reconstituting the lyophilized nucleic acid). In the above context a buffer may be used, more preferably an aqueous (isotonic solution or aqueous) buffer, containing a sodium salt, preferably at least 50 mM of a sodium salt, a calcium salt, preferably at least 0.01 mM of a calcium salt, and optionally a potassium salt, preferably at least 3 mM of a potassium salt. According to a preferred aspect, the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Without being limited thereto, examples of sodium salts include e.g. NaCI, Nal, NaBr, Na2C03, NaHC03, Na2S04, examples of the optional potassium salts include e.g. KCI, Kl, KBr, K2C03, KHC03, K2S04, and examples of calcium salts include e.g. CaCI2, Cal2, CaBr2, CaC03, CaS04, Ca(OH)2. Typically, the salts are present in such an (isotonic solution or) buffer in a concentration of at least 50 mM sodium chloride (NaCI), at least 3 mM potassium chloride (KCI) and at least 0.01 mM calcium chloride (CaCl2). Furthermore, organic anions of the aforementioned cations may be contained in the buffer. According to a more preferred aspect, the buffer may contain salts selected from sodium chloride (NaCI), calcium chloride (CaCI2) and optionally potassium chloride (KCI), wherein further anions may be present additional to the chlorides. CaCl2 can also be replaced by another salt like KCI. The buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the aforementioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects. Reference media are e.g. liquids occurring in "in vivd' methods, such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in "in vitrd' methods, such as common buffers or liquids. Such common buffers or liquids are known to a skilled person. Furthermore, according to a particularly preferred aspect, the inventive nucleic acid (sequence), lyophilized or to be lyophilized, when lyophilized, may again be reconstituted after lyophilization in a buffer as defined herein, preferably as defined above, e.g. as a further step of a method for lyophilization as defined herein. The inventive lyophilized nucleic acid, when lyophilized, may alternatively be reconstituted in water, a buffer as defined above, or a buffer containing mannose, to obtain the desired salt concentration or alternatively the desired buffer conditions. Most preferred, the reconstitution of the lyophilized nucleic acid is carried out in WFI (water for injection), if the nucleic acid has been lyophilized in Ringer Lactate solution which represents an isotonic solution for injection.
As another component, the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein and preferably the nucleic acid and lactate containing solution prior to lyophilization may additionally contain one or more compatible solid or liquid fillers or diluents or encapsulating compounds, which are suitable for administration to a patient to be treated. The term "compatible" as used here means that these constituents are capable of being mixed with the nucleic acid (sequence), lyophilized or to be lyophilized, as defined according to the present invention in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the inventive nucleic acid (sequence), lyophilized or to be lyophilized, under typical use conditions. Pharmaceutically acceptable carriers, fillers and diluents must, of course, have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a person to be treated. Some examples of compounds which can be used as pharmaceutically acceptable carriers, fillers or constituents thereof are sugars, such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oi l, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.
The nucleic acid and lactate containing solution prior to lyophilization may occur as a liquid, a semi-liquid or even a semi-solid or a solid sample, preferably as a liquid, a semi- liquid or a even a semi-solid sample, more preferably as a liquid or a semi-liquid sample.
The pH of the nucleic acid and lactate containing solution prior to lyophilization (and optionally after lyophilization) may be in the range of about 4 to 8, preferably in the range of about 6 to about 8, more preferably from about 7 to about 8.
Additionally, the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein typically comprises a final (residual) water content preferably in the range of about 0.5 % (w/w) to about 10 % (w/w), more preferably of about 1 % (w/w) to about 5 % (w/w), even more preferably of about 2 % (w/w) to about 4% (w/w), and most preferably of about 3 % (w/w), e.g. 3 % (w/w) ± 2 % (w/w), or 3 % (w/w) ± 1 % (w/w).
Finally, the nucleic acid and lactate containing solution prior to lyophilization (as well as after reconstituting the lyophilized nucleic acid) may contain the herein defined contents, optional components, additives, etc. in such a concentration so as to lead to an osmolarity comparable to that of blood plasma. In this context, the term "osmolarity" is typically to be understood as a measure of all contents, optional components, additives, etc. of the nucleic acid and lactate containing solution prior to lyophilization (as well as after reconstituting the lyophilized nucleic acid) as defined herein. More precisely, osmolarity is typically the measure of solute concentration, defined as the number of osmoles (Osm) of all solubilized contents, optional components, additives, etc. per liter (I) of solution (osmol/l or osm/l). In the present context, the nucleic acid and lactate containing solution prior to lyophilization (as well as after reconstituting the lyophilized nucleic acid) as defined herein may comprise an osmolarity preferably in the range of about 200 mosmol/l to about 400 mosmol/l, more preferably in the range of about 250 mosmol/l to about 350 mosmol/l, even more preferably in the range of about 270 mosmol/l to about 330 mosmol/l or in the range of about 280 mosmol/l to about 320 mosmol/l, or in the range of about e.g. about 290 mosmol/l to about 310 mosmol/l, e.g. about 295 mosmol/l, about mosmol/l, about 296 mosmol/l, about 297 mosmol/l, about 298 mosmol/l, about 299 mosmol/l, about, 300 mosmol/l, about 301 mosmol/l, about 302 mosmol/l, about 303 mosmol/l, about 304 mosmol/l, about 305 mosmol/l, about 306 mosmol/l, about 307 mosmol/l, about 308 mosmol/l.
The nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein typically comprises an excellent enhanced storage-stability, when compared to a nucleic acid (sequence), lyophilized or to be lyophilized, of the art, which has been lyophilized without the presence of lactate, e.g. in the presence of water for injection (WFI) as described herein. The nucleic acid (sequence), lyophilized or to be lyophilized, as defined and as prepared herein advantageously can be stored in a temperature range of about -80°C to +60°C significantly longer, when compared to a nucleic acid (sequence), lyophilized or to be lyophilized, of the art. The nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein even allows presence of additional ingredients, such as salts, metalloids, metal anions or further ingredients as defined above, which have been formerly regarded as problematic or even instabilizing the lyophilized nucleic acid. According to the present invention, the storage-stability of the nucleic acid (sequence), lyophilized or to be lyophilized, is calculated on the basis of the relative integrity of the nucleic acid. The relative integrity of the nucleic acid in the nucleic acid (sequence), lyophilized or to be lyophilized, is typically defined as the relative content of the nucleic acid exhibiting a correct length when compared to the total content of nucleic acid in the sample. In the context of an mRNA, the relative integrity of the mRNA in the lyophilized mRNA is typically defined as the relative content of the mRNA exhibiting a correct length when compared to the total content of mRNA in the sample. The storage-stability of a nucleic acid is typically determined on the basis of the relative integrity (over a defined or not defined period of time), wherein the nucleic acid typically exhibits an unchangend biological activity. In the context of the present invention the storage stability is preferably regarded as complied with, if the relative integrity of the (lyophilized) nucleic acid(s) is at least about 60 to 80%, preferably at least about 70 %. A relative integrity of more than 70 % meets the quality criteria of CureVac GmbH for mRNA exhibiting a GC-content of more than 60 % and a base length of <2000 nt in RNA containing formulations. This criterium may be applied to the above definition.
According to a further embodiment, the present invention also provides a method of lyophilization of a nucleic acid, preferably for preparation of lyophilized nucleic acid (sequence) according to the present invention as defined above. I.e., the inventive lyophilized nucleic acid (sequence) as defined according to the present invention is preferably prepared according to the herein described inventive method for lyophilization of a nucleic acid. The inventive method of lyophilization of a nucleic acid preferably leads to an enhanced storage stability of the nucleic acid. The method typically comprises the following steps:
a) optionally providing a nucleic acid containing sample, which has been supplemented with a lactate as defined herein, and optionally supplemented with further ingredients as defined above;
b) freezing the nucleic acid containing sample, obtained according to step a);
c) drying the frozen nucleic acid containing sample, obtained according to step b), via sublimation;
d) optionally floating the (lyophilized) nucleic acid (sequence) (obtained according to step c)) with an inert gas, such as nitrogen, etc., or a noble gas, such as helium, neon, argon, xenon, krypton;
e) optionally sealing the lyophilized nucleic acid. The inventive method is directed to a method of lyophilization of a nucleic acid. Lyophilization (also termed cryodesiccation) is typically understood as a process, which allows removing water from a frozen sample (containing at least one nucleic acid and a lactate containing solution, preferably as defined above) in one or more steps via sublimation. In the context of the present invention, lyophilization is typically carried out by freeze-drying a sample first freezing a nucleic acid containing sample, which has been supplemented with a lactate as defined herein, and then drying the nucleic acid containing sample via sublimation, optionally by reducing the surrounding pressure and/or adding enough heat to allow the frozen water in the sample to sublime directly from the solid phase to gas.
According to an optional first step a) of the inventive method of lyophilization a nucleic acid containing sample, which has been supplemented with a lactate as defined herein, and optionally supplemented with further ingredients as defined above, is provided. The nucleic acid and the lactate are preferably as defined above for the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention. The nucleic acid containing sample may be prepared either by adding a lactate as defined above, preferably in the above defined concentrations, to a sample containing a nucleic acid as defined above, or by adding a sample containing a nucleic acid as defined above to a lactate containing sample, preferably a lactate as defined above in the above defined concentrations. The nucleic acid containing sample, which has been supplemented with a lactate as defined herein, may additionally be supplemented with further ingredients, preferably as defined above for the nucleic acid (sequence), lyophilized or to be lyophilized, of the present invention.
According to the second step b) the sample, containing the at least one nucleic acid and a lactate as defined herein, is frozen. The freezing process may be carried out by any method, which allows to (entirely) freeze the sample. In a lab, this may be done by placing the material in a freeze-drying flask and rotating the flask in a bath, called a shell freezer, which is cooled by mechanical refrigeration, dry ice and methanol, or liquid nitrogen. On a larger- scale, freezing is usually carried out using a freeze-drying machine. In this step, it is important to cool the material below its triple point, the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting will occur in the following steps. Larger crystals are easier to freeze-dry. Usually, the freezing temperatures are in the range between -0 °C and -80 °C, or between -20 °C and -80 °C, preferably in the range between -30 °C and -60 °C, even more preferably in the range between -40 °C and -50 °C, most preferably about -47 °C. According to a third step c), the frozen sample is dried, typically using two drying steps, primary drying step c1) and secondary drying step c2). During the primary drying step cl), free, i.e. unbound, water surrounding the nucleic acid and optionally further components, escapes from the solution. Subsequent thereto water being bound on a molecular basis by the nucleic acids may be removed in a secondary drying step c2) by adding thermal energy. In both cases the hydration sphere around the nucleic acids is lost.
The primary drying step cl) may be carried out at normal pressure, e.g. in the range of about 980 to about 1045 millibar (mbar), e.g. about 1013 mbar, but also may be carried out by lowering the pressure, usually to the range of a few millibar, e.g. in the range of about 0.001 mbar to about 0.2 mbar, preferably in the range of about 0.01 mbar to about 0.1 mbar, even more preferably in the range of about 0.025 mbar to about 0.075 mbar, e.g. about 0.05 mbar. In this primary drying step, pressure is typically controlled through the application of partial vacuum. The vacuum allows speeding up sublimation, making it useful as a deliberate drying process. Furthermore, a cold condenser chamber and/or condenser plates may be used to provide (a) surface(s) for the water vapor to re-solidify on. Condenser temperatures are typically below -50 °C (-60 °F). Alternatively, instead of lowering the pressure, heat may be supplied to the sample to allow for the water to sublimate. The amount of heat necessary can be calculated using the sublimating molecules' latent heat of sublimation. In this initial drying phase, about 95% (w/w) of the water in the material is sublimated. This phase may be carried out slow to avoid applying too much heat and possible alteration or damage of the structure of the nucleic acid to be lyophilized. The heat, if applied, may be in the range of about -40 °C to about +20 °C, e.g. in the range of about -30 °C to about +20 °C, in the range of about -20 °C to about +20 °C, in the range of about -10 °C to about +10 °C, in the range of about -40 °C to about +10 °C, in the range of about -30 °C to about +10 °C, in the range of about -20 °C to about +10 °C, in the range of about -20 °C to about +/-0 °C, or in the range of about -10 °C to about +/-0 °C. As a further alternative, heat and low pressure may be applied, preferably heat in the range as defined above and a low pressure in the range as defined above.
The secondary drying step c2) typically aims to remove unfrozen water molecules bound in the structure of the nucleic acids, since the ice (frozen water molecules) is usually removed in the primary drying step cl ) above. In this secondary drying step c2), the temperature is typically raised higher than in the primary drying step, and can even be above 0 °C, to break any physico-chemical interactions that have formed between the water molecules and the frozen material. Alternatively, the pressure may be lowered in this stage to encourage desorption. According to a further alternative, heat can be applied and pressure can be lowered, preferably in the above ranges. More preferably, the heat, if applied, may be in the range of about +10 °C to about +40 °C, preferably in the range of about +25 °C to about + 35°C, e.g. about 30 °C. The pressure, if lowered, is usually lowered to the range of a few millibars, e.g. as defined above, more preferably in the range of about 0.001 mbar to about 0.05 mbar, preferably in the range of about 0.001 mbar to about 0.025 mbar, even more preferably in the range of about 0.005 mbar to about 0.01 5 mbar, e.g. about 0.01 mbar. As a further alternative, heat and low pressure may be applied, preferably in the ranges as defined above.
After the freeze-drying process is complete, i.e. steps b) and c), particularly c1 ) and c2), are finished, the lyophiiized nucleic acid (sequence) obtained according to steps b) and c), particularly c1 ) and c2), is typically floated in an optional step d) with an inert gas, such as nitrogen, etc., or a noble gas, such as helium, neon, argon, xenon, krypton, and/or the nucleic acid (sequence), lyophiiized or to be lyophiiized, is typically sealed. For this purpose, the vacuum is usually broken, e.g. to atmospheric pressure (preferably about 101 3 mbar), if low pressure was applied, and the temperature is typically adjusted to room temperature, if heat was used.
Subsequently or alternatively to step d) of the inventive method of lyophilization, the lyophiiized nucleic acid (sequence) is optionally sealed in step e) of the inventive method of lyophilization with or without an inert gas. For this purpose, the nucleic acid (sequence), lyophiiized or to be lyophiiized, is advantageously contained in any of the above mentioned steps a), b), c), and d) (and more preferably already lyophiiized) in a sealabie container.
At the end of the lyophilization method as defined above, typically comprising optionally step a), step b) step c), particularly steps c1 ) and c2), and optionally step d) and/or step e), a lyophiiized nucleic acid (sequence) is preferably obtained, wherein the final residual water content in the inventive lyophiiized nucleic acid (sequence) is preferably in the range of about 0.5 % (w/w) to about 10 % (w/w), more preferably in the range of about 1 % (w/w) to about 5 % (w/w), even more preferably in the range of about 2 % (w/w) to about 4% (w/w), most preferably in the range of about 3 % (w/w), e.g. 3 % (w/w) ± 2 % (w/w), or 3 % (w/w) ± 1 % (w/w).
The inventive lyophilized nucleic acid (sequence) may additionally or alternatively to steps d) and/or e) be reconstituted in a solution to obtain a product which is ready to be used in any of the herein mentioned applications. Therefore, according to a particularly preferred aspect, the lyophilized nucleic acid (sequence) may again be reconstituted in a buffer or a solution as defined above for reconstitution. Preferably, such a solution for reconstitution is a solution as defined above for the solution prior to lyophilization of the nucleic acid as defined herein, wherein the solution for reconstitution may contain at least one of the components as defined above for said solution except of the nucleic acid. Most preferred is an isotonic solution as defined herein for reconstitution. The reconstitution may occur, e.g., after lyophilization, e.g. as a further step f) of the abovementioned method for lyophilization.
According to a further embodiment, the present invention furthermore provides a pharmaceutical composition, comprising the inventive nucleic acid (sequence), lyophilized or to be lyophilized, as defined above and optionally a pharmaceutically acceptable carrier and/or vehicle. The inventive pharmaceutical composition may optionally be supplemented with further components as defined above for the inventive lyophilized nucleic acid (sequence). The inventive pharmaceutical composition may be lyophilized as a whole as discussed for the lyophilized nucleic acid (sequence) as defined herein.
As a first ingredient, the inventive pharmaceutical composition comprises the inventive nucleic acid (sequence), lyophilized or to be lyophilized, as defined above.
As a second ingredient the inventive pharmaceutical composition may comprise another class of compounds, which may be added to the inventive pharmaceutical composition in this context, may be selected from at least one pharmaceutically active component. A pharmaceutically active component in this context is a compound that has a therapeutic effect against a particular medical indication, preferably cancer diseases, autoimmune disease, allergies, infectious diseases or a further disease as defined herein. Such compounds include, without implying any limitation, preferably compounds including, without implying any limitation, peptides or proteins (e.g. as defined herein), nucleic acids, (therapeutically active) low molecular weight organic or inorganic compounds (molecular weight less than 5000, preferably less than 1000), sugars, antigens or antibodies (e.g. as defined herein), therapeutic agents already known in the prior art, antigenic cells, antigenic cellular fragments, cellular fractions; modified, attenuated or de-activated (e.g. chemically or by irridation) pathogens (virus, bacteria etc.), etc.
Furthermore, the inventive pharmaceutical composition may comprise a pharmaceutically acceptable carrier and/or vehicle. In the context of the present invention, a pharmaceutically acceptable carrier typically includes the liquid or non-liquid basis of the inventive pharmaceutical composition. If the inventive pharmaceutical composition is provided in liquid form, the carrier will typically be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g phosphate, citrate etc. buffered solutions. Particularly for injection of the inventive inventive pharmaceutical composition, water or preferably a buffer, more preferably an aqueous buffer, may be used, containing a sodium salt, preferably at least 50 mM of a sodium salt, a calcium salt, preferably at least 0.01 mM of a calcium salt, and optionally a potassium salt, preferably at least 3 mM of a potassium salt. According to a preferred aspect, the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Without being limited thereto, examples of sodium salts include e.g. NaCI, Nal, NaBr, Na2C03, NaHC03, Na2S04, examples of the optional potassium salts include e.g. KCI, Kl, KBr, K2C03, KHC03, K2S04, and examples of calcium salts include e.g. CaCI2, Cal2, CaBr2, CaC03, CaS04, Ca(OH)2. Furthermore, organic anions of the aforementioned cations may be contained in the buffer. According to a more preferred aspect, the buffer suitable for injection purposes as defined above, may contain salts selected from sodium chloride (NaCI), calcium chloride (CaCI2) and optionally potassium chloride (KCI), wherein further anions may be present additional to the chlorides. CaCI2 can also be replaced by another salt like KCI. Typically, the salts in the injection buffer are present in a concentration of at least 50 mM sodium chloride (NaCI), at least 3 mM potassium chloride (KCI) and at least 0,01 mM calcium chloride (CaCI2). The injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects. Reference media are e.g. liquids occurring in "in vivd' methods, such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in "in vitrd' methods, such as common buffers or liquids. Such common buffers or liquids are known to a skilled person and may be as defined above. However, one or more compatible solid or liquid fillers or diluents or encapsulating compounds may be used as well for the inventive pharmaceutical composition, which are suitable for administration to a patient to be treated. The term "compatible" as used here means that these constituents of the inventive pharmaceutical composition are capable of being mixed with the (lyophilized) nucleic acid (sequence) as defined herein in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the inventive pharmaceutical composition under typical use conditions. Pharmaceutically acceptable carriers, fillers and diluents must, of course, have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a person to be treated. Some examples of compounds which can be used as pharmaceutically acceptable carriers, fillers or constituents thereof are sugars, such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.
The inventive pharmaceutical composition may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual injection or infusion techniques.
Preferably, the inventive pharmaceutical composition may be administered by parenteral injection, more preferably by subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual injection or via infusion techniques. Sterile injectable forms of the inventive pharmaceutical compositions may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di- glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutical ly-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation of the inventive pharmaceutical composition.
The inventive pharmaceutical composition as defined above may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient, i.e. the at least one nucleic acid as defined above, e.g. the nucleic acid (sequence), lyophilized or to be lyophilized, as defined above, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
The inventive pharmaceutical composition may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g. including diseases of the skin or of any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the inventive pharmaceutical composition may be formulated in a suitable ointment, containing the components as defined above suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, pol oxypropylene compound, emulsifying wax and water. Alternatively, the inventive pharmaceutical composition can be formulated in a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
The inventive pharmaceutical composition typically comprises a "safe and effective amount" of the components of the inventive pharmaceutical composition as defined above, particularly of the at least one (lyophilized) nucleic acid (sequence). As used herein, a "safe and effective amount" means an amount of the at least one (lyophilized) nucleic acid (sequence) that is sufficient to significantly induce a positive modification of a disease or disorder as defined herein. At the same time, however, a "safe and effective amount" is small enough to avoid serious side-effects, that is to say to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment. A "safe and effective amount" of the components of the inventive pharmaceutical composition, particularly of the at least one (lyophilized) nucleic acid (sequence) will furthermore vary in connection with the particular condition to be treated and also with the age and physical condition of the patient to be treated, the body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the activity of the specific (lyophilized) nucleic acid (sequence) employed, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutically acceptable carrier used, and similar factors, within the knowledge and experience of the accompanying doctor. The inventive pharmaceutical composition may be used for human and also for veterinary medical purposes, preferably for human medical purposes, as a pharmaceutical composition in general or as a vaccine.
According to a specific aspect, the inventive pharmaceutical composition may be provided as a vaccine. Such an inventive vaccine is typically composed like the inventive pharmaceutical composition, i.e. it contains a (lyophilized) nucleic acid (sequence) as defined above and optionally a pharmaceutically acceptable carrier and/or vehicle. Further components may be as defined above for the inventive pharmaceutical composition. The inventive vaccine preferably supports at least an innate immune response of the immune system of a patient to be treated. Additionally, the inventive vaccine furthermore may also elicit an adaptive immune response, preferably, if the at least one nucleic acid (sequence) of the inventive vaccine encodes any of the above mentioned antigens (or antibodies), which elicit an adaptive immune response or any antigen as defined above is added to the inventive vaccine which can effectively induce an adaptive immune response.
The inventive vaccine may also comprise a pharmaceutically acceptable carrier, adjuvant, and/or vehicle as defined above for the inventive pharmaceutical composition. In the specific context of the inventive vaccine, the choice of a pharmaceutically acceptable carrier is determined in principle by the manner in which the inventive vaccine is administered. The inventive vaccine can be administered, for example, systemically or locally. Routes for systemic administration in general include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injections and/or intranasal administration routes. Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardiac and sublingual injections. More preferably, vaccines herein may be administered by an intradermal, subcutaneous, or intramuscular route. Inventive vaccines are therefore preferably formulated in liquid (or sometimes in solid) form. The suitable amount of the inventive vaccine to be administered can be determined by routine experiments with animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models. Preferred unit dose forms for injection include sterile solutions of water, physiological saline or mixtures thereof. The pH of such solutions should be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices. Suitable pharmaceutically acceptable carriers for topical application include those which are suitable for use in lotions, creams, gels and the like. If the inventive vaccine is to be administered orally, tablets, capsules and the like are the preferred unit dose form. The pharmaceutically acceptable carriers for the preparation of unit dose forms which can be used for oral administration are well known in the prior art. The choice thereof will depend on secondary considerations such as taste, costs and storability, which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art. The inventive vaccine can additionally contain one or more auxiliary substances in order to further increase its immunogenicity. A synergistic action of the at least one (lyophilized) nucleic acid sequence of the inventive vaccine and of an auxiliary substance, which may be optionally contained in the inventive vaccine as described above, is preferably achieved thereby. Depending on the various types of auxiliary substances, various mechanisms can come into consideration in this respect. For example, compounds that permit the maturation of dendritic cells (DCs), for example lipopolysaccharides, TNF-alpha or CD40 ligand, form a first class of suitable auxiliary substances. In general, it is possible to use as auxiliary substance any agent that influences the immune system in the manner of a "danger signal" (LPS, GP96, etc.) or cytokines, such as GM-CFS, which allow an immune response produced by the immune-stimulating adjuvant according to the invention to be enhanced and/or influenced in a targeted manner. Particularly preferred auxiliary substances are cytokines, such as monokines, lymphokines, interleukins or chemokines, that further promote the innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-12, IL-13, IL-1 , IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL- 24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, INF-alpha, IFN-beta, INF- gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.
Further additives which may be included in the inventive vaccine are emulsifiers, such as, for example, Tween®; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; taste-imparting agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; preservatives.
The inventive vaccine can also additionally contain any further compound, which is known to be immune-stimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its binding affinity (as ligands) to murine Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13. Another class of compounds, which may be added to an inventive vaccine in this context, may be CpG nucleic acids, in particular CpG-RNA or CpG-DNA. A CpG-RNA or CpG- DNA can be a single-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic acid is preferably in the form of CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). The CpG nucleic acid preferably contains at least one or more (mitogenic) cytosine/guanine dinucleotide sequence(s) (CpG motif(s)). According to a first preferred alternative, at least one CpG motif contained in these sequences, that is to say the C (cytosine) and the G (guanine) of the CpG motif, is unmethylated. All further cytosines or guanines optionally contained in these sequences can be either methylated or unmethylated. According to a further preferred alternative, however, the C (cytosine) and the G (guanine) of the CpG motif can also be present in methylated form. The CpG nucleic acids may be provided either in solubilized or in lyophilized form, e.g. lyophilized using a method likewise as described herein for the inventive (lyophilized) nucleic acid (sequence).
The present invention furthermore provides several applications and uses of the inventive lyophilized nucleic acid, preferably prepared according to the inventive method for lyophilization of a nucleic acid.
According to one aspect, the present invention is directed to the first medical use of at least one inventive nucleic acid (sequence), lyophi lized or to be lyophilized, as defined herein, preferably to the use of the inventive nucleic acid (sequence), lyophilized or to be lyophilized, as a medicament. The medicament may be in the form of a pharmaceutical composition or in the form of a vaccine as a specific form of pharmaceutical compositions. A pharmaceutical composition in the context of the present invention typically comprises at least one nucleic acid (sequence), lyophilized or to be lyophilized, as defined above, optionally further ingredients, e.g. as defined above for the inventive lyophilized nucleic acid, and optionally a pharmaceutically acceptable carrier and/or vehicle, preferably as defined above.
According to one further aspect, the present invention is directed to the use of at least one inventive nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein for the prophylaxis, treatment and/or amelioration of diseases as defined herein, preferably selected from cancer or tumor diseases, infectious diseases, preferably (viral, bacterial or protozoological) infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general, diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws, cardiovascular diseases, neuronal diseases, or any further disease mentioned herein.
According to another aspect, the present invention is directed to the second medical use of at least one inventive nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein for the treatment of diseases as defined herein, preferably to the use of the inventive nucleic acid (sequence), lyophilized or to be lyophilized, for the preparation of a medicament for the prophylaxis, treatment and/or amelioration of various diseases as defined herein, preferably selected from cancer or tumor diseases, infectious diseases, preferably (viral, bacterial or protozoological) infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general, diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws, cardiovascular diseases, neuronal diseases, or any further disease mentioned herein. According to one specific aspect, diseases as defined herein comprise cancer or tumor diseases, preferably selected from melanomas, malignant melanomas, colon carcinomas, lymphomas, sarcomas, blastomas, renal carcinomas, gastrointestinal tumors, gliomas, prostate tumors, bladder cancer, rectal tumors, stomach cancer, oesophageal cancer, pancreatic cancer, liver cancer, mammary carcinomas (= breast cancer), uterine cancer, cervical cancer, acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), hepatomas, various virus- induced tumors such as, for example, papilloma virus-induced carcinomas (e.g. cervical carcinoma = cervical cancer), adenocarcinomas, herpes virus-induced tumors (e.g. Burkitt's lymphoma, EBV-induced B-cell lymphoma), heptatitis B-induced tumors (hepatocell carcinomas), HTLV-1- and HTLV-2-induced lymphomas, acoustic neuroma, lung carcinomas (= lung cancer = bronchial carcinoma), small-cell lung carcinomas, pharyngeal cancer, anal carcinoma, glioblastoma, rectal carcinoma, astrocytoma, brain tumors, retinoblastoma, basalioma, brain metastases, medulloblastomas, vaginal cancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome, meningiomas, Schneeberger disease, hypophysis tumor, Mycosis fungoides, carcinoids, neurinoma, spinalioma, Burkitt's lymphoma, laryngeal cancer, renal cancer, thymoma, corpus carcinoma, bone cancer, non- Hodgkin's lymphomas, urethral cancer, CUP syndrome, head/neck tumors, oligodendroglioma, vulval cancer, intestinal cancer, colon carcinoma, oesophageal carcinoma (= oesophageal cancer), wart involvement, tumors of the small intestine, craniopharyngeomas, ovarian carcinoma, genital tumors, ovarian cancer (= ovarian carcinoma), pancreatic carcinoma (= pancreatic cancer), endometrial carcinoma, liver metastases, penile cancer, tongue cancer, gall bladder cancer, leukaemia, plasmocytoma, lid tumor, prostate cancer (= prostate tumors), etc.
According to one further specific aspect, diseases as defined herein comprise infectious diseases, preferably (viral, bacterial or protozoological) infectious diseases. Such infectious diseases, preferably to (viral, bacterial or protozoological) infectious diseases, are typically selected from influenza, malaria, SARS, yellow fever, AIDS, Lyme borreliosis, Leishmaniasis, anthrax, meningitis, viral infectious diseases such as AIDS, Condyloma acuminata, hollow warts, Dengue fever, three-day fever, Ebola virus, cold, early summer meningoencephalitis (FSME), flu, shingles, hepatitis, herpes simplex type I, herpes simplex type II, Herpes zoster, influenza, Japanese encephalitis, Lassa fever, Marburg virus, measles, foot-and-mouth disease, mononucleosis, mumps, Norwalk virus infection, Pfeiffer's glandular fever, smallpox, polio (childhood lameness), pseudo-croup, fifth disease, rabies, warts, West Nile fever, chickenpox, cytomegalic virus (CMV), bacterial infectious diseases such as miscarriage (prostate inflammation), anthrax, appendicitis, borreliosis, botulism, Camphylobacter, Chlamydia trachomatis (inflammation of the urethra, conjunctivitis), cholera, diphtheria, donavanosis, epiglottitis, typhus fever, gas gangrene, gonorrhoea, rabbit fever, Heliobacter pylori, whooping cough, climatic bubo, osteomyelitis, Legionnaire's disease, leprosy, listeriosis, pneumonia, meningitis, bacterial meningitis, anthrax, otitis media, Mycoplasma hominis, neonatal sepsis (Chorioamnionitis), noma, paratyphus, plague, Reiter's syndrome, Rocky Mountain spotted fever, Salmonella paratyphus, Salmonella typhus, scarlet fever, syphilis, tetanus, tripper, tsutsugamushi disease, tuberculosis, typhus, vaginitis (colpitis), soft chancre, and infectious diseases caused by parasites, protozoa or fungi, such as amoebiasis, bilharziosis, Chagas disease, Echinococcus, fish tapeworm, fish poisoning (Ciguatera), fox tapeworm, athlete's foot, canine tapeworm, candidosis, yeast fungus spots, scabies, cutaneous Leishmaniosis, lambliasis (giardiasis), lice, malaria, microscopy, onchocercosis (river blindness), fungal diseases, bovine tapeworm, schistosomiasis, porcine tapeworm, toxoplasmosis, trichomoniasis, trypanosomiasis (sleeping sickness), visceral Leishmaniosis, nappy/diaper dermatitis or miniature tapeworm. According to another specific aspect, diseases as defined herein comprise autoimmune diseases as defined in the following. Autoimmune diseases can be broadly divided into systemic and organ-specific or localised autoimmune disorders, depending on the principal clinico-pathologic features of each disease. Autoimmune diseases may be divided into the categories of systemic syndromes, including systemic lupus erythematosus (SLE), Sjogren's syndrome, Scleroderma, Rheumatoid Arthritis and polymyositis or local syndromes which may be endocrinologic (type I diabetes (Diabetes mellitus Type 1 ), Hashimoto's thyroiditis, Addison's disease etc.), dermatologic (pemphigus vulgaris), haematologic (autoimmune haemolytic anaemia), neural (multiple sclerosis) or can involve virtually any circumscribed mass of body tissue. The autoimmune diseases to be treated may be selected from the group consisting of type I autoimmune diseases or type II autoimmune diseases or type III autoimmune diseases or type IV autoimmune diseases, such as, for example, multiple sclerosis (MS), rheumatoid arthritis, diabetes, type I diabetes (Diabetes mellitus Type Ί ), chronic polyarthritis, Basedow's disease, autoimmune forms of chronic hepatitis, colitis ulcerosa, type I allergy diseases, type II allergy diseases, type III allergy diseases, type IV allergy diseases, fibromyalgia, hair loss, Bechterew's disease, Crohn's disease, Myasthenia gravis, neurodermitis, Polymyalgia rheumatica, progressive systemic sclerosis (PSS), Reiter's syndrome, rheumatic arthritis, psoriasis, vasculitis, etc, or type II diabetes. While the exact mode as to why the immune system induces an immune reaction against autoantigens has not been elucidated so far, there are several findings with regard to the etiology. Accordingly, the autoreaction may be due to a T-Cell bypass. A normal immune system requires the activation of B-cells by T-cells before the former can produce antibodies in large quantities. This requirement of a T-cell can be by-passed in rare instances, such as infection by organisms producing super-antigens, which are capable of initiating polyclonal activation of B-cells, or even of T-cells, by directly binding to the β-subunit of T-cell receptors in a non-specific fashion. Another explanation deduces autoimmune diseases from a Molecular Mimicry. An exogenous antigen may share structural similarities with certain host antigens; thus, any antibody produced against this antigen (which mimics the self-antigens) can also, in theory, bind to the host antigens and amplify the immune response. The most striking form of molecular mimicry is observed in Group A beta- haemolytic streptococci, which shares antigens with human myocardium, and is responsible for the cardiac manifestations of rheumatic fever.
Additionally, according to one further specific aspect, diseases as defined herein comprise allergies or allergic diseases, i.e. diseases related to allergies. Allergy is a condition that typically involves an abnormal, acquired immunological hypersensitivity to certain foreign antigens or allergens, such as the allergy antigens as defined above. Such allergy antigens or allergens may be selected from allergy antigens as defined above antigens derived from different sources, e.g. from animals, plants, fungi, bacteria, etc. Allergens in this context include e.g. grasses, pollens, molds, drugs, or numerous environmental triggers, etc. Allergies normally result in a local or systemic inflammatory response to these antigens or allergens and lead to immunity in the body against these allergens. Without being bound to theory, several different disease mechanisms are supposed to be involved in the development of allergies. According to a classification scheme by P. Cell and R. Coombs the word "allergy" was restricted to type I hypersensitivities, which are caused by the classical IgE mechanism. Type I hypersensitivity is characterised by excessive activation of mast cells and basophils by IgE, resulting in a systemic inflammatory response that can result in symptoms as benign as a runny nose, to life-threatening anaphylactic shock and death. Well known types of allergies include, without being limited thereto, allergic asthma (leading to swelling of the nasal mucosa), allergic conjunctivitis (leading to redness and itching of the conjunctiva), allergic rhinitis ("hay fever"), anaphylaxis, angiodema, atopic dermatitis (eczema), urticaria (hives), eosinophilia, respiratory, allergies to insect stings, skin allergies (leading to and including various rashes, such as eczema, hives (urticaria) and (contact) dermatitis), food allergies, allergies to medicine, etc. Treatment of such allergic disorders or diseases may occur preferably by desensitizing the immune reaction which triggers a specific immune response. Such a desensitizing may be carried out by administering an effective amount of the allergen or allergic antigen encoded by the nucleic acid (sequence), lyophilized or to be lyophilized, as defined herein, preferably, when formulated as a pharmaceutical composition, to induce a slight immune reaction. The amount of the allergen or allergic antigen may then be raised step by step in subsequent administrations until the immune system of the patient to be treated tolerates a specific amount of allergen or allergic antigen. Additionally, diseases to be treated in the context of the present invention likewise include (hereditary) diseases, or genetic diseases in general monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general. Such (mono-)genetic diseases, (hereditary) diseases, or genetic diseases in general are typically caused by genetic defects, e.g. due to gene mutations resulting in loss of protein activity or regulatory mutations which do not allow transcription or translation of the protein. Frequently, these diseases lead to metabolic disorders or other symptoms, e.g. muscle dystrophy. The present invention allows treating the following (hereditary) diseases or genetic diseases: 3-beta-hydroxysteroid dehydrogenase deficiency (type II); 3-ketothiolase deficiency; 6-mercaptopurine sensitivity; Aarskog-Scott syndrome; Abetalipoproteinemia; Acatalasemia; Achondrogenesis; Achondrogenesis-hypochondrogenesis; Achondroplasia; Achromatopsia; Acromesomelic dysplasia (Hunter-Thompson type); ACTH deficiency; Acyl-CoA dehydrogenase deficiency (short-chain, medium chain, long chain); Adenomatous polyposis coli; Adenosin-deaminase deficiency; Adenylosuccinase deficiency; Adhalinopathy; Adrenal hyperplasia, congenital (due to 11 -beta-hydroxylase deficiency; due to 17-alpha-hydroxylase deficiency; due to 21 - hydroxylase deficiency); Adrenal hypoplasia, congenital, with hypogonadotropic hypogonadism; Adrenogenital syndrom; Adrenoleukodystrophy; Adrenomyeloneuropathy; Afibrinogenemia; Agammaglobulinemia; Alagille syndrome; Albinism (brown, ocular, oculocutaneous, rufous); Alcohol intolerance, acute; Aldolase A deficiency; Aldosteronism, glucocorticoid-remediable; Alexander disease; Alkaptonuria; Alopecia universalis; Alpha-1 - antichymotrypsin deficiency; Alpha-methylacyl-CoA racemase deficiency; Alpha- thalassemia/mental retardation syndrome; Alport syndrome; Alzheimer disease-1 (APP- related); Alzheimer disease-3; Alzheimer disease-4; Amelogenesis imperfecta; Amyloid neuropathy (familial, several allelic types); Amyloidosis (Dutch type; Finnish type; hereditary renal; renal; senile systemic); Amytrophic lateral sclerosis; Analbuminemia; Androgen insensitivity; Anemia (Diamond-Blackfan); Anemia (hemolytic, due to PK deficiency); Anemia (hemolytic, Rh-null, suppressor type); Anemia (neonatal hemolytic, fatal and nearfatal); Anemia (sideroblastic, with ataxia); Anemia (sideroblastic/hypochromic); Anemia due to G6PD deficiency; Aneurysm (familial arterial); Angelman syndrome; Angioedema; Aniridia; Anterior segment anomalies and cataract; Anterior segment mesenchymal dysgenesis; Anterior segment mesenchymal dysgenesis and cataract; Antithrombin III deficiency; Anxiety-related personality traits; Apert syndrome; Apnea (postanesthetic); ApoA-l and apoC-lll deficiency (combined); Apolipoprotein A-ll deficiency; Apolipoprotein B-100 (ligand-defective); Apparent mineralocorticoid excess (hypertension due to); Argininemia; Argininosuccinicaciduria; Arthropathy (progressive pseudorheumatoid, of childhood); Aspartylglucosaminuria; Ataxia (episodic); Ataxia with isolated vitamin E deficiency; Ataxia-telangiectasia; Atelosteogenesis II; ATP-dependent DNA ligase I deficiency; Atrial septal defect with atrioventricular conduction defects; Atrichia with papular lesions; Autism (succinylpurinemic); Autoimmune polyglandular disease, type I; Autonomic nervous system dysfunction; Axenfeld anomaly; Azoospermia; Bamforth-Lazarus syndrome; Bannayan-Zonana syndrome; Barthsyndrome; Bartter syndrome (type 2 or type 3); Basal cell carcinoma ; Basal cell nevus syndrome; BCG infection; Beare-Stevenson cutis gyrata syndrome; Becker muscular dystrophy; Beckwith- Wiedemann syndrome; Bernard-Sou I ier syndrome (type B; type C); Bethlem myopathy; Bile acid malabsorption, primary ; Biotinidase deficiency; Bladder cancer; Bleeding disorder due to defective thromboxane A2 receptor; Bloom syndrome; Brachydactyly (type B1 or type C); Branchiootic syndrome; Branchiootorenal syndrome; Breast cancer (invasive intraductal; lobular; male, with Reifenstein syndrome; sporadic); Breast cancer-1 (early onset); Breast cancer-2 (early onset); Brody myopathy; Brugada syndrome; Brunner syndrome; Burkitt lymphoma; Butterfly dystrophy (retinal); C1 q deficiency (type A; type B; type C ); C1 r/C1 s deficiency; C1 s deficiency, isolated; C2 deficiency; C3 deficiency; C3b inactivator deficiency; C4 deficiency; C8 deficiency, type II; C9 deficiency; Campomelic dysplasia with autosomal sex reversal; Camptodactyly-arthropathy-coxa varapericarditis syndrome; Canavan disease; Carbamoylphosphate synthetase I deficiency; Carbohydrate-deficient glycoprotein syndrome (type I; type lb; type II); Carcinoid tumor of lung; Cardioencephalomyopathy (fatal infantile, due to cytochrome c oxidase deficiency); Cardiomyopathy (dilated; X-linked dilated; familial hypertrophic; hypertrophic); Carnitine deficiency (systemic primary); Carnitine-acylcarnitine translocase deficiency; Carpal tunnel syndrome (familial); Cataract (cerulean; congenital; crystalline aculeiform; juvenile-onset; polymorphic and lamellar; punctate; zonular pulverulent); Cataract, Coppock-like; CD59 deficiency; Central core disease; Cerebellar ataxia; Cerebral amyloid angiopathy; Cerebral arteriopathy with subcortical infarcts and leukoencephalopathy; Cerebral cavernous malformations-1 ; Cerebrooculofacioskeletal syndrome; Cerebrotendinous xanthomatosis; Cerebrovascular disease; Ceroid lipofuscinosis (neuronal, variant juvenile type, with granular osmiophilic deposits); Ceroid lipofuscinosis (neuronal-1 , infantile); Ceroid- lipofuscinosis (neuronal-3, juvenile); Char syndrome; Charcot-Marie-Tooth disease; Charcot-Marie-Tooth neuropathy; Charlevoix-Saguenay type; Chediak-Higashi syndrome; Chloride diarrhea (Finnish type); Cholestasis (benign recurrent intrahepatic); Cholestasis (familial intrahepatic); Cholestasis (progressive familial intrahepatic); Cholesteryl ester storage disease; Chondrodysplasia punctata (brachytelephalangic; rhizomelic; X-linked dominant; X-linked recessive; Grebe type); Chondrosarcoma; Choroideremia; Chronic granulomatous disease (autosomal, due to deficiency of CYBA); Chronic granulomatous disease (X-linked); Chronic granulomatous disease due to deficiency of NCF-1; Chronic granulomatous disease due to deficiency of NCF-2; Chylomicronemia syndrome, familial; Citrullinemia; classical Cockayne syndrome-1; Cleft lip, cleft jaw, cleft palate; Cleft lip/palate ectodermal dysplasia syndrome; Cleidocranial dysplasia; CMO II deficiency; Coats disease; Cockayne syndrome-2, type B; Coffin-Lowry syndrome; Colchicine resistance; Colon adenocarcinoma; Colon cancer; Colorblindness (deutan; protan; tritan); Colorectal cancer; Combined factor V and VIII deficiency; Combined hyperlipemia (familial); Combined immunodeficiency (X-linked, moderate); Complex I deficiency; Complex neurologic disorder; Cone dystrophy-3; Cone-rod dystrophy 3; Cone-rod dystrophy 6; Cone-rod retinal dystrophy-2; Congenital bilateral absence of vas deferens; Conjunctivitis, ligneous; Contractural arachnodactyly; Coproporphyria; Cornea plana congenita; Corneal clouding; Corneal dystrophy (Avellino type; gelatinous drop-like; Groenouw type I; lattice type I; Reis-Bucklers type); Cortisol resistance; Coumarin resistance; Cowden disease; CPT deficiency, hepatic (type I; type II); Cramps (familial, potassium-aggravated); Craniofacial-deafness-hand syndrome; Craniosynostosis (type 2); Cretinism; Creutzfeldt-Jakob disease ; Crigler-Najjar syndrome; Crouzon syndrome; Currarino syndrome; Cutis laxa; Cyclic hematopoiesis; Cyclic ichthyosis; Cylindromatosis; Cystic fibrosis; Cystinosis (nephropathic); Cystinuria (type II; type III); Daltonism; Darier disease; D-bifunctional protein deficiency; Deafness, autosomal dominant 1 ; Deafness, autosomal dominant 1 1 ; Deafness, autosomal dominant 12; Deafness, autosomal dominant 15; Deafness, autosomal dominant 2; Deafness, autosomal dominant 3; Deafness, autosomal dominant 5; Deafness, autosomal dominant 8; Deafness, autosomal dominant 9; Deafness, autosomal recessive 1 ; Deafness, autosomal recessive 2; Deafness, autosomal recessive 21 ; Deafness, autosomal recessive 3; Deafness, autosomal recessive 4; Deafness, autosomal recessive 9; Deafness, nonsyndromic sensorineural 13; Deafness, X-linked 1 ; Deafness, X-linked 3; Debrisoquine sensitivity; Dejerine-Sottas disease; Dementia (familial Danish); Dementia (frontotemporal, with parkinsonism); Dent disease; Dental anomalies; Dentatorubro-pallidoluysian atrophy; Denys-Drash syndrome; Dermatofibrosarcoma protuberans; Desmoid disease; Diabetes insipidus (nephrogenic ); Diabetes insipidus (neurohypophyseal); Diabetes mellitus (insulin-resistant); Diabetes mellitus (rare form); Diabetes mellitus (type II); Diastrophic dysplasia; Dihydropyrimidinuria; Dosage-sensitive sex reversal; Doyne honeycomb degeneration of retina; Dubin-Johnson syndrome; Duchenne muscular dystrophy; Dyserythropoietic anemia with thrombocytopenia; Dysfibrinogenemia (alpha type; beta type; gamma type); Dyskeratosis congenita-1 ; Dysprothrombinemia; Dystonia (DOPAresponsive); Dystonia (myoclonic); Dystonia-1 (torsion); Ectodermal dysplasia; Ectopia lentis; Ectopia pupillae; Ectrodactyly (ectodermal dysplasia, and cleft lip/palate syndrome 3); Ehlers-Danlos syndrome (progeroid form); Ehlers-Danlos syndrome (type I; type II; type III; type IV; type VI; type VII); Elastin Supravalvar aortic stenosis; Elliptocytosis-1 ; Elliptocytosis-2; Elliptocytosis-3; Ellis-van Creveld syndrome; Emery-Dreifuss muscular dystrophy; Emphysema; Encephalopathy; Endocardial fibroelastosis-2; Endometrial carcinoma; Endplate acetylcholinesterase deficiency; Enhanced S-cone syndrome; Enlarged vestibular aqueduct; Epidermolysis bullosa; Epidermolysis bullosa dystrophica (dominant or recessive); Epidermolysis bullosa simplex; Epidermolytic hyperkeratosis; Epidermolytic palmoplantar keratoderma; Epilepsy (generalize; juvenile; myoclonic; nocturnal frontal lobe; progressive myoclonic); Epilepsy, benign, neonatal (typel or type2); Epiphyseal dysplasia (multiple); Episodic ataxia (type 2); Episodic ataxia/myokymia syndrome; Erythremias (alpha-; dysplasia); Erythrocytosis; Erythrokeratoderma; Estrogen resistance; Exertional myoglobinuria due to deficiency of LDH-A; Exostoses, multiple (type 1 ; type 2); Exudative vitreoretinopathy, X-linked; Fabry disease; Factor H deficiency; Factor VII deficiency; Factor X deficiency; Factor XI deficiency; Factor XII deficiency; Factor XIIIA deficiency; Factor XIIIB deficiency; Familial Mediterranean fever; Fanconi anemia; Fanconi-Bickel syndrome; Farber lipogranulomatosis; Fatty liver (acute); Favism; Fish-eye disease; Foveal hypoplasia; Fragile X syndrome; Frasier syndrome; Friedreich ataxia; fructose-bisphosphatase Fructose intolerance; Fucosidosis; Fumarase deficiency; Fundus albipunctatus; Fundus flavimaculatus; G6PD deficiency; GABA-transaminase deficiency; Galactokinase deficiency with cataracts; Galactose epimerase deficiency; Galactosemia; Galactosialidosis; GAMT deficiency; Gardner syndrome; Gastric cancer; Gaucher disease; Generalized epilepsy with febrile seizures plus; Germ cell tumors; Gerstmann-Straussler disease; Giant cell hepatitis (neonatal); Giant platelet disorder; Giant-cell fibroblastoma; Gitelman syndrome; Glanzmann thrombasthenia (type A; type B); Glaucoma 1 A; Glaucoma 3A; Glioblastoma multiforme; Glomerulosclerosis (focal segmental); Glucose transport defect (blood-brain barrier); Glucose/galactose malabsorption; Glucosidase I deficiency; Glutaricaciduria (type I; type MB; type IIC); Gluthation synthetase deficiency; Glycerol kinase deficiency; Glycine receptor (alpha-1 polypeptide); Glycogen storage disease I; Glycogen storage disease II; Glycogen storage disease III; Glycogen storage disease IV; Glycogen storage disease VI; Glycogen storage disease VII; Glycogenosis (hepatic, autosomal); Glycogenosis (X-linked hepatic); GM1 -gangliosidosis; GM2-gangliosidosis; Goiter (adolescent multinodular); Goiter (congenital); Goiter (nonendemic, simple); Gonadal dysgenesis (XY type); Granulomatosis, septic; Graves disease; Greig cephalopolysyndactyly syndrome; Griscelli syndrome; Growth hormone deficient dwarfism; Growth retardation with deafness and mental retardation; Gynecomastia (familial, due to increased aromatase activity); Gyrate atrophy of choroid and retina with ornithinemia (B6 responsive or unresponsive); Hailey-Hailey disease; Haim- Munk syndrome; Hand-foot-uterus syndrome; Harderoporphyrinuria; HDL deficiency (familial); Heart block (nonprogressive or progressive); Heinz body anemia; HELLP syndrome; Hematuria (familial benign); Heme oxygenase-1 deficiency; Hemiplegic migraine; Hemochromotosis; Hemoglobin H disease; Hemolytic anemia due to ADA excess; Hemolytic anemia due to adenylate kinase deficiency; Hemolytic anemia due to band 3 defect; Hemolytic anemia due to glucosephosphate isomerase deficiency; Hemolytic anemia due to glutathione synthetase deficiency; Hemolytic anemia due to hexokinase deficiency; Hemolytic anemia due to PGK deficiency; Hemolytic-uremic syndrome; Hemophagocytic lymphohistiocytosis; Hemophilia A; Hemophilia B; Hemorrhagic diathesis due to factor V deficiency; Hemosiderosis (systemic, due to aceruloplasminemia); Hepatic lipase deficiency; Hepatoblastoma; Hepatocellular carcinoma; Hereditary hemorrhagic telangiectasia-1 ; Hereditary hemorrhagic telangiectasia- 2; Hermansky-Pudlak syndrome; Heterotaxy (X-linked visceral); Heterotopia (periventricular); Hippel-Lindau syndrom; Hirschsprung disease; Histidine-rich glycoprotein Thrombophilia due to HRG deficiency; HMG-CoA lyase deficiency; Holoprosencephaly-2; Holoprosencephaly-3; Holoprosencephaly-4; Holoprosencephaly-5; Holt-Oram syndrome; Homocystinuria; Hoyeraal-Hreidarsson; HPFH (deletion type or nondeletion type); HPRT- related gout; Huntington disease; Hydrocephalus due to aqueductal stenosis; Hydrops fetalis; Hyperbetalipoproteinemia; Hypercholesterolemia, familial; Hyperferritinemia- cataract syndrome; Hyperglycerolemia; Hyperglycinemia; Hyperimmunoglobulinemia D and periodic fever syndrome; Hyperinsulinism; Hyperinsulinism-hyperammonemia syndrome; Hyperkalemic periodic paralysis; Hyperlipoproteinemia; Hyperlysinemia; Hypermethioninemia (persistent, autosomal, dominant, due to methionine, adenosyltransferase l/lll deficiency); Hyperornithinemia- hyperammonemiahomocitrullinemia syndrome; Hyperoxaluria; Hyperparathyroidism; Hyperphenylalaninemia due to pterin-4acarbinolamine dehydratase deficiency; Hyperproinsulinemia; Hyperprolinemia; Hypertension; Hyperthroidism (congenital); Hypertriglyceridemia; Hypoalphalipoproteinemia; Hypobetalipoproteinemia;
Hypocalcemia; Hypochondroplasia; Hypochromic microcytic anemia; Hypodontia; Hypofibrinogenemia; Hypoglobulinemia and absent B cells; Hypogonadism (hypergonadotropic); Hypogonadotropic (hypogonadism); Hypokalemic periodic paralysis; Hypomagnesemia; Hypomyelination (congenital); Hypoparathyroidism; Hypophosphatasia (adult; childhood; infantile; hereditary); Hypoprothrombinemia; Hypothyroidism (congenital; hereditary congenital; nongoitrous); lchthyosiform erythroderma ; Ichthyosis ; Ichthyosis bullosa of Siemens ; lgG2 deficiency; Immotile cilia syndrome-1 ; Immunodeficiency (T-cell receptor/CD3 complex); Immunodeficiency (X-linked, with hyper-lgM); Immunodeficiency due to defect in CD3-gamma; Immunodeficiency- centromeric i nstabi I ityfacial anomalies syndrome; Incontinentia pigmenti; Insensitivity to pain (congenital, with anhidrosis); Insomnia (fatal familial); lnterleukin-2 receptor deficiency (alpha chain); Intervertebral disc disease; Iridogoniodysgenesis; Isolated growth hormone deficiency (lllig type with absent GH and Kowarski type with bioinactive GH); Isovalericacidemia ; Jackson-Weiss sydnrome; Jensen syndrome; Jervell and Lange-Nielsen syndrome; Joubert syndrom; Juberg-Marsidi syndrome; Kallmann syndrome; Kanzaki disease; Keratitis; Keratoderma (palmoplantar); Keratosis palmoplantaris striata I; Keratosis palmoplantaris striata II; Ketoacidosis due to SCOT deficiency; Keutel syndrome; Klippel- Trenaurnay syndrom; Kniest dysplasia; Kostmann neutropenia; Krabbe disease; Kurzripp- Polydaktylie syndrom; Lacticacidemia due to PDX1 deficiency; Langer mesomelic dysplasia; Laron dwarfism; Laurence-Moon-Biedl-Bardet syndrom; LCHAD deficiency; Leber congenital amaurosis; Left-right axis malformation; Leigh syndrome; Leiomyomatosis (diffuse, with Alport syndrome); Leprechaunism; Leri- Weill dyschondrosteosis; Lesch- Nyhan syndrome; Leukemia (acute myeloid; acute promyelocytic; acute T-cell lymphoblastic; chronic myeloid; juvenile myelomonocytic; Leukemia-1 (T-cell acute lymphocytic); Leukocyte adhesion deficiency; Leydig cell adenoma; Lhermitte-Duclos syndrome; Liddle syndrome; Li-Fraumeni syndrome; Lipoamide dehydrogenase deficiency; Lipodystrophy; Lipoid adrenal hyperplasia; Lipoprotein lipase deficiency; Lissencephaly (X- linked); Lissencephaly-1; liver Glycogen storage disease (type 0); Long QT syndrome-1; Long QT syndrome-2; Long QT syndrome-3; Long QT syndrome-5; Long QT syndrome-6; Lowe syndrome; Lung cancer; Lung cancer (nonsmall cell); Lung cancer (small cell); Lymphedema; Lymphoma (B-cell non-Hodgkin); Lymphoma (diffuse large cell); Lymphoma (follicular); Lymphoma (MALT); Lymphoma (mantel cell); Lymphoproliferative syndrome (X- linked); Lysinuric protein intolerance; Machado-Joseph disease; Macrocytic anemia refractory (of 5q syndrome); Macular dystrophy; Malignant mesothelioma; Malonyl-CoA decarboxylase deficiency ; Mannosidosis, (alpha- or beta- ); Maple syrup urine disease (type la; type lb; type II); Marfan syndrome; Maroteaux-Lamy syndrome; Marshall syndrome; MASA syndrome; Mast cell leukemia; Mastocytosis with associated hematologic disorder; McArdle disease; McCune-Albright polyostotic fibrous dysplasia; McKusick-Kaufman syndrome; McLeod phenotype ; Medullary thyroid carcinoma; Medulloblastoma; Meesmann corneal dystrophy; Megaloblastic anemia-1; Melanoma; Membroproliferative glomerulonephritis ; Meniere disease; Meningioma (NF2-related; SIS-related); Menkes disease; Mental retardation (X-linked); Mephenytoin poor metabolizer; Mesothelioma; Metachromatic leukodystrophy; Metaphyseal chondrodysplasia (Murk Jansen type; Schmid type); Methemoglobinemia; Methionine adenosyltransferase deficiency (autosomal recessive); Methylcobalamin deficiency (cbl G type); Methylmalonicaciduria (mutase deficiency type); Mevalonicaciduria; MHC class II deficiency; Microphthalmia (cataracts, and iris abnormalities); Miyoshi myopathy; MODY; Mohr-Tranebjaerg syndrome; Molybdenum cofactor deficiency (type A or type B); Monilethrix; Morbus Fabry; Morbus Gaucher; Mucopolysaccharidosis; Mucoviscidosis; Muencke syndrome; Muir-Torre syndrome; Mulibrey nanism; Multiple carboxylase deficiency (biotinresponsive); Multiple endocrine neoplasia; Muscle glycogenosis; Muscular dystrophy (congenital merosindeficient); Muscular dystrophy (Fukuyama congenital); Muscular dystrophy (limb- girdle); Muscular dystrophy) Duchenne-like); Muscular dystrophy with epidermolysis bullosa simplex; Myasthenic syndrome (slow-channel congenital); Mycobacterial infection (atypical, familial disseminated); Myelodysplastic syndrome; Myelogenous leukemia; Myeloid malignancy; Myeloperoxidase deficiency; Myoadenylate deaminase deficiency; Myoglobinuria/hemolysis due to PGK deficiency; Myoneurogastrointestinal encephalomyopathy syndrome; Myopathy (actin; congenital; desmin-related; cardioskeletal; distal; nemaline); Myopathy due to CPT II deficiency; Myopathy due to phosphoglycerate mutase deficiency; Myotonia congenita; Myotonia levior; Myotonic dystrophy; Myxoid liposarcoma; NAGA deficiency; Nailpatella syndrome; Nemaline myopathy 1 (autosomal dominant); Nemaline myopathy 2 (autosomal recessive); Neonatal hyperparathyroidism; Nephrolithiasis; Nephronophthisis (juvenile); Nephropathy (chronic hypocomplementemic); Nephrosis-1 ; Nephrotic syndrome; Netherton syndrome; Neuroblastoma; Neurofibromatosis (type 1 or type 2); Neurolemmomatosis; neuronal-5 Ceroid-lipofuscinosis; Neuropathy; Neutropenia (alloimmune neonatal); Niemann-Pick disease (type A; type B; type C1 ; type D); Night blindness (congenital stationary); Nijmegen breakage syndrome; Noncompaction of left ventricular myocardium; Nonepidermolytic palmoplantar keratoderma; Norrie disease; Norum disease; Nucleoside phosphorylase deficiency; Obesity; Occipital hornsyndrome; Ocular albinism (Nettleship-Falls type); Oculopharyngeal muscular dystorphy; Oguchi disease; Oligodontia; Omenn syndrome; Opitz G syndrome; Optic nerve coloboma with renal disease; Ornithine transcarbamylase deficiency; Oroticaciduria; Orthostatic intolerance; OSMED syndrome; Ossification of posterior longitudinal ligament of spine; Osteoarthrosis; Osteogenesis imperfecta; Osteolysis; Osteopetrosis (recessive or idiopathic); Osteosarcoma; Ovarian carcinoma; Ovarian dysgenesis; Pachyonychia congenita (Jackson-Lawler type or Jadassohn-Lewandowsky type); Paget disease of bone; Pallister-Hall syndrome; Pancreatic agenesis; Pancreatic cancer; Pancreatitis; Papillon- Lefevre syndrome; Paragangliomas ; Paramyotonia congenita; Parietal foramina; Parkinson disease (familial or juvenile); Paroxysmal nocturnal hemoglobinuria; Pelizaeus-Merzbacher disease; Pendred syndrome; Perineal hypospadias; Periodic fever; Peroxisomal biogenesis disorder; Persistent hyperinsulinemic hypoglycemia of infancy; Persistent Mullerian duct syndrome (type II); Peters anomaly; Peutz-Jeghers syndrome ; Pfeiffer syndrome; Phenylketonuria; Phosphoribosyl pyrophosphate synthetaserelated gout; Phosphorylase kinase deficiency of liver and muscle; Piebaldism; Pilomatricoma; Pinealoma with bilateral retinoblastoma; Pituitary ACTH secreting adenoma; Pituitary hormone deficiency; Pituitary tumor; Placental steroid sulfatase deficiency; Plasmin inhibitor deficiency; Plasminogen deficiency (types I and II); Plasminogen Tochigi disease; Platelet disorder; Platelet glycoprotein IV deficiency; Platelet-activating factor acet l hydrolase deficiency; Polycystic kidney disease; Polycystic lipomembranous osteodysplasia with sclerosing leukenencephalophathy; Polydactyly, postaxial; Polyposis; Popliteal pterygium syndrome; Porphyria (acute hepatic or acute intermittent or congenital erythropoietic); Porphyria cutanea tarda; Porphyria hepatoerythropoietic ; Porphyria variegata; Prader-Willi syndrome; Precocious puberty; Premature ovarian failure; Progeria Typ 1; Progeria Typ II; Progressive external ophthalmoplegia; Progressive intrahepatic cholestasis-2; Prolactinoma (hyperparathyroidism, carcinoid syndrome); Prolidase deficiency; Propionicacidemia; Prostate cancer; Protein S deficiency; Proteinuria; Protoporphyria (erythropoietic); Pseudoachondroplasia; Pseudohermaphroditism; Pseudohypoaldosteronism;
Pseudohypoparathyroidism; Pseudovaginal perineoscrotal hypospadias; Pseudovitamin D deficiency rickets; Pseudoxanthoma elasticum (autosomal dominant; autosomal recessive); Pulmonary alveolar proteinosis; Pulmonary hypertension; Purpura fulminans; Pycnodysostosis; Pyropoikilocytosis; Pyruvate carboxylase deficiency; Pyruvate dehydrogenase deficiency; Rabson-Mendenhall syndrome; Refsum disease; Renal cell carcinoma; Renal tubular acidosis; Renal tubular acidosis with deafness; Renal tubular acidosis-osteopetrosis syndrome; Reticulosis (familial histiocytic); Retinal degeneration; Retinal dystrophy; Retinitis pigmentosa; Retinitis punctata albescens; Retinoblastoma; Retinol binding protein deficiency; Retinoschisis; Rett syndrome; Rh(mod) syndrome; Rhabdoid predisposition syndrome; Rhabdoid tumors ; Rhabdomyosarcoma; Rhabdomyosarcoma (alveolar); Rhizomelic chondrodysplasia punctata; Ribbing-Syndrom; Rickets (vitamin D-resistant); Rieger anomaly; Robinow syndrome; Rothmund-Thomson syndrome; Rubenstein-Taybi syndrome; Saccharopinuria; Saethre-Chotzen syndrome; Salla disease; Sandhoff disease (infantile, juvenile, and adult forms); Sanfilippo syndrome (type A or type B); Schindler disease; Schizencephaly; Schizophrenia (chronic); Schwannoma (sporadic); SCID (autosomal recessive, T-negative/Bpositive type); Secretory pathway w TMD; SED congenita; Segawa syndrome; Selective T-cell defect; SEMD (Pakistani type); SEMD (Strudwick type); Septooptic dysplasia; Severe combined immunodeficiency (B cellnegative); Severe combined immunodeficiency (T-cell negative, B-cel l/natural killer cell- positive type); Severe combined immunodeficiency (Xlinked); Severe combined immunodeficiency due to ADA deficiency; Sex reversal (XY, with adrenal failure); Sezary syndrome; Shah-Waardenburg syndrome; Short stature; Shprintzen-Goldberg syndrome; Sialic acid storage disorder; Sialidosis (type I or type II); Sialuria; Sickle cell anemia; Simpson-Golabi-Behmel syndrome; Situs ambiguus; Sjogren-Larsson syndrome; Smith- Fineman-Myers syndrome; Smith-Lemli-Opitz syndrome (type I or type II); Somatotroph! noma; Sorsby fundus dystrophy; Spastic paraplegia; Spherocytosis; Spherocytosis-1 ; Spherocytosis-2; Spinal and bulbar muscular atrophy of Kennedy; Spinal muscular atrophy; Spinocerebellar ataxia; Spondylocostal dysostosis; Spondyloepiphyseal dysplasia tarda; Spondylometaphyseal dysplasia (Japanese type); Stargardt disease-1 ; Steatocystoma multiplex; Stickler syndrome; Sturge-Weber syndrom; Subcortical laminal heteropia; Subcortical laminar heterotopia; Succinic semialdehyde dehydrogenase deficiency; Sucrose intolerance; Sutherland-Haan syndrome; Sweat chloride elevation without CF; Symphalangism; Synostoses syndrome; Synpolydactyly; Tangier disease; Tay- Sachs disease; T-cell acute lymphoblastic leukemia; T-cell immunodeficiency; T-cell prolymphocyte leukemia; Thalassemia (alpha- or delta-); Thalassemia due to Hb Lepore; Thanatophoric dysplasia (types I or II); Thiamine-responsive megaloblastic anemia syndrome; Thrombocythemia; Thrombophilia (dysplasminogenemic); Thrombophilia due to heparin cofactor II deficiency; Thrombophilia due to protein C deficiency; Thrombophilia due to thrombomodulin defect; Thyroid adenoma; Thyroid hormone resistance; Thyroid iodine peroxidase deficiency; Tietz syndrome; Tolbutamide poor metabolizer; Townes- Brocks syndrome; Transcobalamin II deficiency; Treacher Collins mandibulofacial dysostosis; Trichodontoosseous syndrome; Trichorhinophalangeal syndrome; Trichothiodystrophy; Trifunctional protein deficiency (type I or type II); Trypsinogen deficiency; Tuberous sclerosis-1 ; Tuberous sclerosis-2; Turcot syndrome; Tyrosine phosphatase; Tyrosinemia; Ulnar-mammary syndrome; Urolithiasis (2,8-dihydroxyadenine); Usher syndrome (type 1 B or type 2A); Venous malformations; Ventricular tachycardia; Virilization; Vitamin K-dependent coagulation defect; VLCAD deficiency; Vohwinkel syndrome; von Hippel-Lindau syndrome; von Willebrand disease; Waardenburg syndrome; Waardenburg syndrome/ocular albinism; Waardenburg-Shah neurologic variant; Waardenburg-Shah syndrome; Wagner syndrome; Warfarin sensitivity; Watson syndrome; Weissenbacher-Zweymuller syndrome; Werner syndrome; Weyers acrodental dysostosis; White sponge nevus; Williams-Beuren syndrome; Wilms tumor (typel ); Wilson disease; Wiskott-Aldrich syndrome; Wolcott-Rallison syndrome; Wolfram syndrome; Wolman disease; Xanthinuria (type I); Xeroderma pigmentosum; X-SCID; Yemenite deaf-blind hypopigmentation syndrome; ypocalciuric hypercalcemia (type I); Zellweger syndrome; Zlotogora-Ogur syndrome.
Diseases to be treated in the context of the present invention likewise also include diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws are preferably selected from the group consisting of autosomal-recessive inherited diseases, such as, for example, adenosine deaminase deficiency, familial hypercholesterolaemia, Canavan's syndrome, Gaucher's disease, Fanconi anaemia, neuronal ceroid lipofuscinoses, mucoviscidosis (cystic fibrosis), sickle cell anaemia, phenylketonuria, alcaptonuria, albinism, hypothyreosis, galactosaemia, alpha-1 -anti-trypsin deficiency, Xeroderma pigmentosum, Ribbing's syndrome, mucopolysaccharidoses, cleft lip, jaw, palate, Laurence Moon Biedl Bardet sydrome, short rib polydactylia syndrome, cretinism, Joubert's syndrome, type II progeria, brachydact lia, adrenogenital syndrome, and X-chromosome inherited diseases, such as, for example, colour blindness, e.g. red/green blindness, fragile X syndrome, muscular dystrophy (Duchenne and Becker-Kiener type), haemophilia A and B, G6PD deficiency, Fabry's disease, mucopolysaccharidosis, Nome's syndrome, Retinitis pigmentosa, septic granulomatosis, X-SCID, ornithine transcarbamylase deficiency, Lesch-Nyhan syndrome, or from autosomal-dominant inherited diseases, such as, for example, hereditary angiooedema, Marfan syndrome, neurofibromatosis, type I progeria, Osteogenesis imperfecta, Klippel-Trenaurnay syndrome, Sturge-Weber syndrome, Hippel-Lindau syndrome and tuberosis sclerosis.
The present invention also allows treatment of diseases, which have not been inherited, or which may not be summarized under the above categories. Such diseases may include e.g. the treatment of patients, which are in need of a specific protein factor, e.g. a specific therapeutically active protein as mentioned above. This may e.g. include dialysis patients, e.g. patients which undergo a (regular) a kidney or renal dialysis, and which may be in need of specific therapeutically active proteins as defined above, e.g. erythropoietin (EPO), etc.
Likewise, diseases in the context of the present invention may include cardiovascular diseases chosen from, without being limited thereto, coronary heart disease, arteriosclerosis, apoplexy and hypertension, etc.
Finally, diseases in the context of the present invention may be chosen from neuronal diseases including e.g. Alzheimer's disease, amyotrophic lateral sclerosis, dystonia, epilepsy, multiple sclerosis and Parkinson's disease etc.
According to a final embodiment, the present invention also provides kits, particularly as kit of parts. Such kit of parts may contain e.g. a pharmaceutical composition or a vaccine as defined above, preferably divided into different parts of the kit. As an example, the inventive pharmaceutical composition or the inventive vaccine may be prepared as a kit of parts, e.g. by incorporating into one or more parts of the kit (all or at least some components of) the inventive pharmaceutical composition or the inventive vaccine as described herein (whereby at least the lyophilized nucleic acid is included), or the lyophilized nucleic acid as defined above, as a dry formulation, i.e. devoid of any liquid component, and in at least one further separate part of the kit a liquid and/or a buffer as described herein for the preparation of the inventive nucleic acid (sequence), lyophilized or to be lyophilized,, the inventive pharmaceutical composition or the inventive vaccine or any further liquid and/or buffer as described herein for lyophilization, transfection and/or injection. Alternatively, the inventive pharmaceutical composition or the inventive vaccine may be prepared as a kit of parts, e.g. by incorporating into one or more parts of the kit only the nucleic acid (sequence), lyophilized or to be lyophilized, as described herein, and in at least one further separate part of the kit a liquid and/or a buffer as described herein for the preparation of the inventive nucleic acid (sequence), lyophilized or to be lyophilized, for the inventive pharmaceutical composition or the inventive vaccine or any further liquid and/or buffer as described herein for lyophilization, transfection and/or injection. Kit of parts may also comprise as components alone or in combination with further ingredients at least one inventive nucleic acid (sequence), lyophilized or to be lyophilized, preferably as defined according to the present invention, i.e. at least one inventive lyophilized nucleic acid, which has been supplemented with lactate. Without being limited thereto, further ingredients may include components as defined above, e.g. (solutions comprising) proteins, amino acids, alcohols, carbohydrates, metals or metal ions, surfactants, polymers or complexing agents, and/or buffers, preferably all as defined above. These further ingredients may be contained in different parts of the kit (kit of parts). Any of the kits or kit of parts as described above may contain optionally technical instructions with information on the administration and dosage of the inventive lyophilized nucleic acid. Such kits, preferably kit of parts, may be applied, e.g., for any of the above mentioned applications or uses. The kit may optionally contain technical instructions with information on the administration and dosage of the lyophilized nucleic acid. As an example, the kit of parts may comprise in one or more parts of the kit at least one lyophilized nucleic acid as defined herein, and optionally in one or more parts of the kit further additives as defined herein, and in one or more parts of the kit water or a buffer as defined herein, and optionally technical instructions with information on the administration and dosage of the lyophilized nucleic acid.
Figures:
The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.
Figure 1 : shows the dependency of the relative integrity of mRNA in different solutions stored at 37°C for 0 to 8 weeks, particularly when solved in water for injection (WFI), when solved in 80% (w/w) Ringer-Lactate solution and lyophilized or when solved in WFI and lyophilized. As can be seen in Figure 1 , mRNA obtained by lyophilization of mRNA solved in 80% (w/w) Ringer-
Lactate solution exhibits a significantly better median relative integrity of > 70% (w/w) within a period of 8 weeks. In comparison, mRNA solved in WFI exhibits a significant degradation and decrease of median relative integrity at a temperature of 37°C within 3 weeks and mRNA solved in WFI and lyophilized exhibits a significant degradation and decrease of median relative integrity at a temperature of 37°C already within 2 weeks.
Figure 2: displays the dependency of the relative integrity of mRNA coding for prostate specific antigen (PSA/KLK3 mRNA) in different solutions stored at 60°C for 0 or 5 weeks, particularly when solved and lyophilized in 80% (w/w) Ringer-
Lactate solution, when solved and lyophilized in water for injection (WFI), or when solved and lyophilized in a salt solution containing 5 mM K, 130 mM Na, 2 mM Ca, 2 mM Mg). As can be seen in Figure 2, mRNA obtained by lyophilization of mRNA solved in 80% (w/w) Ringer-Lactate solution exhibits a significantly better median relative integrity of > 70% within a period of at least 4 weeks. In comparison, mRNA obtained by lyophilization of mRNA solved in WFI exhibits a significant degradation and decrease of median relative integrity at a temperature of 60°C already within 3 weeks and mRNA obtained by lyophilization of mRNA solved in the above salt solution exhibits a significant degradation and decrease of median relative integrity at a temperature of 60°C already within 1 week. Use of a salt solution alone containing alkali and earth-alkali salts alone thus shows the expected degradation. Use of salts of lactic acid (Ringer's lactate), however, shows a surprisingly good increase in long-term storage capabilities. shows the relative integrity of mRNA lyophilized from sodium lactate containing solutions at 37°C within a period of 3 weeks, particularly when solved and lyophilized in water for injection (WFI), when solved and lyophilized in 80% (w/w) Ringer-Lactate solution (22 mM sodium lactate), when solved and lyophilized in 30 mM sodium lactate or when solved and lyophilized in 3 mM sodium lactate. As can be seen in Figure 3 the stabilizing effect clearly results from use of lactate for lyophilization compared to use of WFI for lyophilization. Free mRNA lyophilized from different sodium lactate containing solutions or buffers, such as Ringer- Lactate, showed nearly the same effect. illustrates the in vivo expression of freshly prepared luciferase encoded by mRNA versus in vivo expression of luciferase encoded by lyophilized mRNA after storage of 8 weeks, wherein the lyophilized mRNA was lyophilized in 80% (w/w) Ringer-Lactate solution and stored for 8 weeks at 37°C. As can be seen in Figure 4, the storage of mRNA lyophilized in 80% (w/w) Ringer- Lactate solution does not exhibit a negative effect on the biological activity of the mRNA. The expression levels remain constant and does not show any significant difference with respect to freshly thawn mRNA in 80% (w/w) Ringer-Lactate solution. shows the relative integrity of lyophilizates from different salt containing injection solutions stored at room temperature: The results show an enhanced stability of mRNA coding for prostate specific antigen PSA (KLK3 mRNA) lyophilized from lactate containing buffer (80% (w/w) Ringer's lactate (RiLa)) in contrast to lyophilisates from other buffers and injection solutions (200 mM sodium acetat at pH 5.5 (Acetate), 100 mM potassium phosphate at pH 7.4 (Phosphate), 80 mM HEPES (2-(4-(2-Hydroxyethyl)-1 - piperazinyl)-ethansulfonsaure) at pH7.4 (HEPES), PBS (phosphate buffered saline) at pH 7.4 (PBS), 100 mM Tris (Tris(hydroxymethyl)-aminomethan) at pH 7.4 (TRIS), 100mM Ammonium chloride at pH 8.5) (Ammonium).
Figure 6: shows the relative integrity of mRNA lyophilized from a glucose-containing solution at 60°C for 0 to 33 days (d). As can be seen, the relative integrity of mRNA coding for prostate specific antigen PSA (KLK3 mRNA) lyophilzed in a glucose containing solution stored at 60°C for 0 to 33 days (d) significantly decreases below 70% already at day 21 , i.e. 3 weeks.
Figure 7: shows the tumour growth after immunization with mRNA complexed with protamine in a solution containing ringer lactate (middle curve (from top to bottom)) or from the lyophilized version from the same solution (lowest curve (from top to bottom)) within 29 days. The highest curve (from top to bottom) represents the vaccination with Ringer lactate buffer without mRNA as control (the curve is also represented by triangles).
Figure 8: shows the mRNA sequence encoding Photinus pyralis luciferase (SEQ ID
NO: 1 ) in the mRNA construct pCV19-Pp luc(GC)-muag-A70-C30; which exhibits a length of 1857 nucleotides. The mRNA sequence contains following sequence elements:
• the coding sequence encoding Photinus pyralis luciferase;
• stabilizing sequences derived from alpha-globin-3'-UTR (muag (mutated alpha-globin-3'-UTR));
• 70 x adenosine at the 3'-terminal end (poly-A-tail);
• 30 x cytosine at the 3'- terminal end (poly-C-tail).
The ORF is indicated in italic letters, muag (mutated alpha-globin-3'-UTR is indicated with a dotted line, the poly-A-tail is underlined with a single line and the poly-C-tail is underlined with a double line.
Figure 9: shows the mRNA sequence encoding human KLK3 (PSA) (SEQ ID NO: 2) in the mRNA construct pCV19-HsKLK3(GC)-muag-A70C30; which exhibits a length of 990 nucleotides. The mRNA sequence contains following sequence elements:
• the coding sequence encoding KLK3 (PSA);
• stabilizing sequences derived from alpha-globin-3'-UTR (muag (mutated alpha-globin-3'-UTR));
• 70 x adenosine at the 3'-terminal end (poly-A-tail);
• 30 x cytosine at the 3'- terminal end (poly-C-tail).
The ORF is indicated in italic letters, muag (mutated alpha-globin-3'-UTR is indicated with a dotted line, the poly-A-tail is underlined with a single line and the poly-C-tail is underlined with a double line. Examples:
The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.
Example 1 - Preparation of plasmids
For the present examples DNA sequences, encoding Photinus pyralis luciferase, as well as DNA sequences, encoding human prostate antigen KLK3/PSA, were prepared and used for subsequent in vitro transcription reactions and stability studies.
According to a first preparation, the DNA sequence corresponding to pCV19-Ppluc(GQ- muag-A70-C30 was prepared, which encodes the Photinus pyralis luciferase. The DNA constructs were prepared by modifying the wild type Photinus pyralis luciferase encoding DNA sequence by introducing a GC-optimized sequence for a better codon usage and stabilization, stabilizing sequences derived from alpha-globin-3'-UTR (muag (mutated alpha-globin-3'-UTR)), a stretch of 70 x adenosine at the 3'-terminal end (poly-A-tail) and a stretch of 30 x cytosine at the 3'- terminal end (poly-C-tail) corresponding to SEQ ID NO: 1 (see Figure 8). The sequence of the final DNA construct had a length of 1857 nucleotides. The corresponding mRNA sequence was termed "pCV19-Ppluc(GC)-muag-A70-C30" (SEQ ID NO: 1) (see Figure 8).
According to a second preparation, the DNA sequence corresponding to pCV19- HsKLK3(GQ-muag-A70C30 DNA was prepared, encoding human prostate antigen KLK3/PSA. Therefore, a basic DNA construct was prepared termed pCV19-HsKLK3(GQ- muag-A70C30 by introducing into the underlying wild type sequence construct stabilizing sequences derived from alpha-globin-3'-UTR (muag (mutated alpha-globin-3'-UTR)), a stretch of 70 x adenosine at the 3'-terminal end (poly-A-tail) and a stretch of 30 x cytosine at the 3'- terminal end (poly-C-tail), leading to a sequence corresponding to SEQ ID NO: 2 (see Figure 9). The corresponding mRNA sequence was termed pCV19-HsKLK3(GC)-muag- A70C30 (SEQ ID NO: 2) (see Figure 9).
Both sequences contain following sequence elements:
• the coding sequence encoding Photinus pyralis luciferase (SEQ ID NO:
1 ) or human prostate antigen KLK3/PSA (SEQ ID NO: 2); • stabilizing sequences derived from alpha-globin-3'-UTR (muag (mutated alpha-globin-3'-UTR));
• 70 x adenosine at the 3'-terminal end (poly-A-tail);
• 30 x cytosine at the 3'- terminal end (poly-C-tail).
Example 2 - In vitro transcription:
The respective DNA plasmids prepared according to Example 1 were transcribed in vitro using T7-Polymerase (T7-Opti mRNA Kit, CureVac, Tubingen, Germany) following the manufactures instructions. Subsequently the mRNA was purified using PureMessenger® (CureVac, Tubingen, Germany).
Example 3 - Lyophylisation:
The PureMessenger® purified and precipitated mRNA obtained according to Example 1 coding for prostate specific antigen PSA (KLK3 mRNA) or Photinus pyralis luciferase (Luc mRNA) were prepared for transfection (see Example 2) and expression purposes and for stability tests either as free mRNA or as complexed mRNA.
Free mRNA:
The PureMessenger® purified and precipitated mRNA obtained according to Example 1 coding for prostate specific antigen PSA (KLK3 mRNA) or Photinus pyralis luciferase (Luc mRNA) was dissolved in water for injection (WFI) to 5 g/l. Subsequently the mRNA was diluted with WFI (water for injection), Ringer's lactate (80% (w/w) Ringer's lactate: 104,8 mM Na+; 4,29 mM K+; 1 ,47 mM Ca2+; 22,64 mM Lactate; wherein Ringer's lactate (100% (w/w)) comprises 131 mM Na+, 5,36 mM K+, 1 ,84 mM Ca2+, and 28,3 mM Lactate) sodium lactate solutions (3 mM and 30 mM sodium lactate), 200 mM sodium acetate pH 5.5, 100mM potassium phosphate pH 7.4, 80mM HEPES (2-(4-(2-Hydroxyethyl)-1 -piperazinyl)- ethansulfonic acid) pH 7.4, PBS (phosphate buffered saline) pH7.4, 1 00mM Tris (Tris(hydroxymethyl)-aminomethan) pH 7.4, 1 00mM Ammonium chloride pH 8.5, salt solution (5mM K+, 130mM Na+, 2mM Ca2+, 2mM Mg2+) or a Glucose-containing buffer (5% (w/w) Glucose in WFI) to a concentration of 0.1 g l RNA. Aliquots of these solutions were lyophilized (Controls were frozen in liquid nitrogen or kept in solution). The locked cups were stored for the indicated time at room temperature, at 37°C or at 60°C. The resuspension was conducted with WFI. Complexed mRNA: The PureMessenger® purified and precipitated mRNA coding for prostate specific antigen PSA (KLK3 mRNA) or Photinus pyralis luciferase (Luc mRNA) was dissolved in water (WF1) to 5 g/l. Subsequently the mRNA was diluted with WFI, Ringer's lactate (80% (w/w) Ringer's lactate) or salt solution (5mM K+, 130mM Na+, 2mM Ca2+, 2mM Mg2) to a concentration of 0.4 g/l and (optionally) mixed with protamine in a ratio of 4:1 RNA/Protamine (w/w) and stored for the indicated time at room temperature, at 37°C or at 60°C. The resuspension was conducted with WFI.
Gel electrophoresis:
1 pg RNA was loaded on an agarose gel and analyzed by the Biolmager and the software Launch VisionWorks LS (UVP LLC, Upland Canada).
Example 4 - Transfection and expression of luciferase:
HeLa cells were trypsinized and washed in opti-MEM. 1 x10s cells per sample were electroporated with 3 pg of Luc mRNA. The transcripts were either (A) 3 aliquotes of 8 week stored or (B) of freshly prepared mRNA. Electroporated cells were seeded in 24-well plates in 1 ml complete medium (RPMI 1640, 10% FCS, 100 units/ml each of Penicillin and Streptomycin, 10 mM L-Glutamin). The transfection solution was sucked off 24h after transfection and the cells were lysed in 200 μΙ lysis buffer (25 mM Tris-P04, 2 mM EDTA, 10% glycerol, 1 % Triton-X 100, 2 mM DTT). The supernatants were then mixed with luciferin buffer (25 mM Glycylglycin, 15 mM MgS04, 5 mM ATP, 62,5 μΜ luciferin) and luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany)). The results of these experiments are shown in Figures 1 to 6.
According to a first experiment the dependency of the relative integrity of mRNA in different solutions stored at 37°C for 0 to 8 weeks were investigated, particularly when solved in water for injection (WFI), when solved in 80% (w/w) Ringer-Lactate solution and lyophilized or when solved in WFI and lyophilized. As can be seen in Figure 1 , mRNA obtained by lyophilization of mRNA solved in 80% (w/w) Ringer-Lactate solution exhibits a significantly better median relative integrity of > 70% within a period of 8 weeks. In comparison, mRNA solved in WFI exhibits a significant degradation and decrease of median relative integrity at a temperature of 37°C within 3 weeks and mRNA solved in WFI and lyophilized exhibits a significant degradation and decrease of median relative integrity at a temperature of 37°C already within 2 weeks.
According to a second experiment the dependency of the relative integrity of mRNA coding for prostate specific antigen (PSA/KLK3 mRNA) in different solutions stored at 60°C for 0 or 5 weeks was investigated, particularly when solved and lyophilized in 80% (w/w) Ringer- Lactate solution, when solved and lyophilized in water for injection (WFI), or when solved and lyophilized in a salt solution containing 5 mM K, 130 mM Na, 2 mM Ca, 2 mM Mg). As can be seen in Figure 2, mRNA obtained by lyophilization of mRNA solved in 80% (w/w) Ringer-Lactate solution exhibits a significantly better median relative integrity of > 70% within a period of at least 4 weeks. In comparison, mRNA obtained by lyophilization of mRNA solved in WFI exhibits a significant degradation and decrease of median relative integrity at a temperature of 60°C already within 3 weeks and mRNA obtained by lyophilization of mRNA solved in the above salt solution exhibits a significant degradation and decrease of median relative integrity at a temperature of 60°C already within 1 week. Use of a salt solution alone containing alkali and earth-alkali salts alone thus shows the expected degradation. Use of salts of lactic acid (Ringer's lactate), however, shows a surprisingly good increase in long-term storage capabilities. According to a further experiment the relative integrity of mRNA lyophilized from sodium lactate containing solutions at 37°C within a period of 3 weeks was investigated, particularly when solved and lyophilized in water for injection (WFI), when solved and lyophilized in 80% (w/w) Ringer-Lactate solution (22 mM sodium lactate), when solved and lyophilized in 30 mM sodium lactate or when solved and lyophilized in 3 mM sodium lactate. As can be seen in Figure 3 the stabilizing effect clearly results from use of lactate for lyophilization compared to use of WFI for lyophilization. Free mRNA lyophilized from different sodium lactate containing solutions or buffers, such as Ringer-Lactate, showed nearly the same effect. According to a fourth experiment illustrates the in vitro expression (in HeLa cells) of freshly prepared luciferase encoded by mRNA versus in vitro expression of luciferase encoded by lyophilized mRNA after storage of 8 weeks was determined, wherein the lyophilized mRNA was lyophilized in 80% (w/w) Ringer-Lactate solution and stored for 8 weeks at 37°C. As can be seen in Figure 4, the storage of mRNA lyophilized in 80% (w/w) Ringer-Lactate solution does not exhibit a negative effect on the biological activity of the mRNA. The expression levels remain constant and does not show any significant difference with respect to freshly thawn mRNA in 80% (w/w) Ringer-Lactate solution. According to another experiment the relative integrity of lyophilizates from different salt containing injection solutions stored at room temperature were investigated. The results are shown in Figure 5. They show an enhanced stability of mRNA coding for prostate specific antigen PSA (KLK3 mRNA) lyophilized from lactate containing buffer (80% (w/w) Ringer's lactate (RiLa)) in contrast to lyophilisates from other buffers and injection solutions (200 mM sodium acetat at pH 5.5 (Acetate), 100 mM potassium phosphate at pH 7.4 (Phosphate), 80 mM HEPES (2-(4-(2-Hydroxyethyl)-1 -piperazinyl)-ethansulfonsaure) at pH7.4 (HEPES), PBS (phosphate buffered saline) at pH 7.4 (PBS), 100 mM Tris (Tris(hydroxymethyl)- aminomethan) at pH 7.4 (TRIS), 100mM Ammonium chloride at pH 8.5) (Ammonium). According to a last experiment the relative integrity of mRNA lyophilized from a glucose- containing solution at 60°C for 0 to 33 days (d) was determined. The results are shown in Figure 6. As can be seen, the relative integrity of mRNA coding for prostate specific antigen PSA (KLK3 mRNA) lyophilzed in a glucose containing solution stored at 60°C for 0 to 33 days (d) significantly decreases below 70% already at day 21 , i.e. 3 weeks.
Example 5: Tumor challenge using mRNA coding for ovalbumine
For the present experiment, mRNA coding for ovalbumine was complexed with protamine in the following protocol. RNA was mixed in a ratio of 4:1 RNA Protamine solution (w/w) with a diluted protamine solution containing protamine, WFI and Ringer lactate were added to a final RNA concentration of 0.8 g/l and 80% (w/w) Ringer lactate. The formulations were aliquoted into borosilicate glas typ I and half of the samples were frozen by liquid nitrogen for at least 5 min and lyophilized at 0.055 mbar for 22 h. Sample plates were kept at room temperature for 1 7 h and were than elevated to 35°C for another 5 h. The chamber was floatet with dry argon and the samples were closed under this atmosphere by a bromobutyl stopper. The lyophilized and non-lyophilized samples were stored in an exsiccator at 4-8°C and were dissolved in WFI prior to use (Prime 7 d / Boost 15 d after drying). Prior to use the samples were controlled for relative integrity by agarose gel chromatography and complex size by dynamic light scattering using a Zetasizer Nano (Malvern Instruments, Malvern, UK). For tumor challenge experiments, 7 week old C57BL/6 mice were vaccinated with 2 cycles (Prime day Ί / Boost day 9) of intradermal injections of 80 μΙ formulations or the lyophilized RNA and the and non-lyophilized RNA. As a negative control 80 μΙ 80% Ringer lactate without any RNA were injected. At day 15 1 x105 E.G7-OVA cells per mice were implanted subcutaneously. Tumour size was measured in 3 dimensions using a calliper.
The surprising results are shown in Figure 7. Figure 7 shows the tumour growth after immunization with mRNA complexed with protamine in a solution containing ringer lactate (middle curve (from top to bottom)) or from the lyophilized version from the same solution (lowest curve (from top to bottom)) within 29 days. The highest curve (from top to bottom) represents the vaccination with Ringer lactate buffer without mRNA as control (the curve is also represented by triangles). It is very surpring that lyophilized and subsequently resonstituted mRNA is more effective preventing tumor growth than the sample without the prior step of lyophilization. Since all RNAs were controlled regarding particle size and integrity it is guaranteed that all RNAs were completely intact. This is a surprising and not obvious advantage of the present invention over the prior art.

Claims

Claims
1 . Lyophilized nucleic acid, which has been lyophilized from a lactate containing solution.
2. Lyophilized nucleic acid (sequence) according to claim 1 , wherein the lactate concentration of the lactate containing solution prior to lyphilization is in the range of about 3 mM to about 300 mM, including a range of about 5 mM to about 200 mM, a range of about 10 mM to about 1 50 mM, or a range of about 15 mM to about 35 mM, including a range of about 20 mM to about 31 mM.
3. Lyophilized nucleic acid (sequence) according to any of claims 1 or 2, wherein the pH of the nucleic acid and lactate containing solution prior to lyophilization is in the range of about 4 to 8, including a range of about 6 to about 8, or a range of about 7 to about 8.
4. Lyophilized nucleic acid (sequence) according to any of claims 1 to 3, wherein the water content of the lyophilized nucleic acid (sequence) is reduced to a content of about 0.5 % (w/w) to about 10 % (w/w), including a range of about 1 % (w/w) to about 5 % (w/w), a range of about 2 % (w/w) to about 4% (w/w), a range of about 3 % (w/w) or a value of about 3 % (w/w) ± 2 % (w/w), or 3 % (w/w) ± 1 % (w/w).
5. Lyophilized nucleic acid (sequence) according to any of claims 1 to 4, wherein the relative integrity of the lyophilized nucleic acid (sequence) is at least about 70 %.
6. Lyophilized nucleic acid (sequence) according to any of claims 1 to 5, wherein the lactate of the lactate containing solution is derived from free lactic acid (lUPAC systematic name: 2-hydroxypropanoic acid), also known as milk acid, including its optical isomers L-(+)-lactic acid, (5)-lactic acid, D-(-)-lactic acid or (^-lactic acid, or its biologically active optical isomer L-(+)-lactic acid, a salt or an anion thereof, selected from sodium-lactate, potassium-lactate, or Al3 +-lactate, NH4 +-lactate, Fe- lactate, Li-lactate, Mg-lactate, Ca-lactate, Mn-lactate or Ag-lactate, or is selected from Ringer's lactate (RiLa), lactated Ringer's solution (main content sodium lactate, also termed "Hartmann's Solution" in the UK), acetated Ringer's solution, or is selected from lactate containing water, or an ortho-lactate-containing solution.
7. Lyophilized nucleic acid (sequence) according to any of claims 1 to 6, wherein the lactate concentration of the lactate containing solution prior to lyophilization comprises a Ringer's lactate concentration prior to lyophilization in the range of about 1 0% (w/w) to about 100% (w/w), including a range of about 20% (w/w) to about 100% (w/w), in the range of about 30% (w/w) to about 100% (w/w), in the range of about 40% (w/w) to about 100% (w/w), in the range of about 50% (w/w) to about 90% (w/w), preferably in the range of about 60% (w/w) to about 90% (w/w), more preferably in the range of about 70% (w/w) to about 90% (w/w), including about 80% (w/w), of Ringer's lactate.
8. Lyophilized nucleic acid (sequence) according to any of claims 1 to 7, wherein the nucleic acid is selected from DNA, including genomic DNA, single-stranded DNA molecules, double-stranded DNA molecules, coding DNA, DNA primers, DNA probes, immunostimulatory DNA, (short) DNA oligonucleotides ((short) oligodesoxyribonucleotides), or is selected from PNA (peptide nucleic acid) or is selected from RNA, including (short) RNA oligonucleotides ((short) oligoribonucleotides), a coding RNA, a messenger RNA (mRNA), an immunostimulatory RNA, a siRNA, a micro RNA an antisense RNA, or riboswitches, ribozymes aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), a viral RNA (vRNA), preferably mRNA.
9. Lyophilized nucleic acid (sequence) according to any of claims 1 to 8, wherein the lactate containing solution additionally comprises an additive selected from mannit, proteins, amino acids, alcohols, carbohydrates, metals or metal ions, surfactants, polymers or complexing agents, or a buffer,
the proteins comprising albumin, gelatine, therapeutically active proteins, antibodies, and antigens,
the amino acids comprising any naturally occurring amino acid, including alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine, more preferably glycine, arginine, and alanine. Cryoprotectants and/or lyoprotectants selected from the group of amino acids may additionally comprise any modification of a naturally occurring amino acid as defined above,
the alcohols comprising mannitol, polyethyleneglycol, polypropyleneglycol, sorbitol,
the carbohydrates comprising monosaccharides, including glucose, fructose and mannose, disaccharides, including lactose, maltose, sucrose, and trehalose, and polysaccharides, including dextran and HP-beta CD,
the metals or metal ions comprising metals or metal ions or salts selected from alkali metals, including lithium (Li), sodium (Na), potassium (K), and their (monovalent) metal alkali metal ions and salts;
alkaline earth metals, including magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba) and their (divalent) alkaline earth metal ions and salts;
transition metals, including members of period 4 of any of subgroups 1 to 12 of the periodic table including Scandium (Sc), Titanium (Ti), Vanadium (V), Chromium (Cr), Manganese (Mn), Iron (Fe), Cobalt (Co), Nickel (Ni), Copper (Cu) and Zinc (Zn) and their metal ions and salts;
earth metals or members of the boron group, including Boron (B) and Aluminium (Al) and their metal ions and salts;
metalloids or semi metals, including Boron (B) and Silicon (Si) and their semi metal ions and salts;
the surfactants comprising Tween, Tween 80 (0.2%), Pluronics, Pluronic L121 (1 .25%);
the polymers or complexing agents comprising lipoplexes, nanoplexes, cationic or polycationic compound, particularly cationic or polycationic polymers or cationic or polycationic lipids, including protamine, nucleoline, spermin or spermidine, or other cationic peptides or proteins, including poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1 , L-oligomers, Calcitonin peptide(s), Antennapedia- derived peptides (particularly from Drosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1 ), pVEC, hCT-derived peptides, SAP, protamine, spermine, spermidine, or histones, or proteins or peptides having the following total formula: (Arg^iLys^iHis^iOrn^Xaa)^ wherein I + m + n +o + x = 8-15, and I, m, n or o independently of each other may be any number selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 50% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native (= naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and x may be any number selected from 0, 1 , 2, 3 or 4, provided, that the overall content of Xaa does not exceed 50 % of all amino acids of the oligopeptide, or are selected from cationic polysaccharides, including chitosan, polybrene, cationic polymers, polyethyleneimine (PEI), cationic lipids, DOTMA: [1 -(2,3-sioleyloxy)propyl)]-N,N,N- trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Choi, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA,
DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(a- trimethylammonioacetyl)diethanolamine chloride, CLIP1 : rac-[(2,3- dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-
[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethyIammonium, CLIP9: rac- [2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, e.g. modified polyaminoacids, including β-aminoacid-polymers or reversed polyamides, modified polyethylenes, including PVP (poly(N-ethyl-4-vinylpyridinium bromide)), modified acrylates, including pDMAEMA (poly(dimethylaminoethyl methylacrylate)), modified Amidoamines including pAMAM (poly(amidoamine)), modified polybetaaminoester (PBAE), including diamine end modified 1 ,4 butanediol diacrylate-co-5-amino-1 - pentanol polymers, dendrimers, including polypropylamine dendrimers or pAMAM based dendrimers, polyimine(s), including PEI: poly(ethyleneimine), poly(propyleneimine), polyallylamine, sugar backbone based polymers, including cyclodextrin based polymers, dextran based polymers, Chitosan, silan backbone based polymers, including PMOXA-PDMS copolymers, Blockpolymers consisting of a combination of one or more cationic blocks (e.g. selected of a cationic polymer as mentioned above) and of one or more hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole);
the buffers selected from isotonic saline or buffered (aqueous) solutions, including phosphate or citrate buffered solutions, including an aqueous buffer, containing a sodium salt in a concentration of at least 50 mM of a sodium salt, a calcium salt in a concentration of at least 0.01 mM of a calcium salt, and optionally a potassium salt, in a concentration of at least 3 mM of a potassium salt, or sodium, calcium and, optionally, potassium salts in the form of their halogenides, including chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, or include NaCI, Nal, NaBr, Na2C03, NaHC03, Na2S04, KCl, Kl, KBr, K2C03, KHC03, K2S04, CaCI2, Cal2, CaBr2, CaC03, CaS04, or Ca(OH)2, or a buffer exhibiting salts selected from sodium chloride (NaCI), calcium chloride (CaCl2) and optionally potassium chloride (KCl), present in a concentration of at least 50 mM sodium chloride (NaCI), at least 3 mM potassium chloride (KCl) and at least 0.01 mM calcium chloride (CaCl2).
10. Method of preparation of a lyophilized nucleic acid (sequence) according to any of claims 1 to 9, comprising the following steps:
a) optionally providing a nucleic acid containing sample, which has been supplemented with a lactate as defined according to any of claims 2 and/or 6, and optionally supplemented with further ingredients as defined according to claim 9;
b) freezing the nucleic acid containing sample, obtained according to step a);
c) drying the frozen nucleic acid containing sample, obtained according to step b), via sublimation;
d) optionally floating the lyophilized nucleic acid (sequence) with an inert gas, including nitrogen, or a noble gas, including helium, neon, or argon; e) optionally sealing the lyophilized nucleic acid.
11. Method according to claim 10, wherein freezing in step b) occurs at a temperature in the range between -20 °C and -80 °C, including a range between -30 °C and -60 °C, a range between -40 °C and -50 °C, or a temperature of about -47 °C.
2. Method according to claim 10 or 1 1 , wherein drying in step c) occurs in a primary drying step c1 ) and a secondary drying step c2).
3. Method according to claim 1 2, wherein in the primary drying step c1 ) is carried out at a pressure including a pressure in a range of about 980 to about 1 045 mbar, or a pressure in a range of about 0.001 mbar to about 0.2 mbar, a range of about 0.01 mbar to about 0.1 mbar, or a range of about 0.025 mbar to about 0.075 mbar, or a pressure of about 0.05 mbar and/or is carried out at a temperature of about -40 °C to about +20 °C.
4. Method according to claim 12, wherein in the secondary drying step c2) is carried out at a temperatur range of about +10 °C to about +40 °C, inclusing a range of about +25 °C to about + 35°C, or a temperatur of about 30 °C, and/or a pressure of about 0.001 mbar to about 0.05 mbar, including a range of about 0.001 mbar to about 0.025 mbar, a range of about 0.005 mbar to about 0.01 5 mbar, or a pressure of about 0.01 mbar.
5. At least one lyophi lized nucleic acid (sequence) according to any of claims 1 to 9 for use as a medicament.
6. At least one lyophilized nucleic acid (sequence) as defined according to any of claims 1 to 9 for use in the prophylaxis, treatment and/or amelioration of diseases selected from cancer or tumor diseases, infectious diseases, including viral, bacterial or protozoological infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, including hereditary diseases, or genetic diseases, diseases which have a genetic inherited background and which are typically caused by a single gene defect, cardiovascular diseases or neuronal diseases.
7. Kit comprising at least one lyophi lized nucleic acid (sequence) according to any of claims 1 to 9, and optionally technical instructions with information on the administration and dosage of the lyophilized nucleic acid. Kit of parts, comprising in one or more parts of the kit at least one lyophilized nucleic acid as defined according to any of claims 1 to 9, and optionally in one or more parts of the kit further additives as defined according to claim 9, and in one or more parts of the kit water or a buffer, and optionally technical instructions with information on the administration and dosage of the lyophilized nucleic acid.
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