Polymeric carrier cargo complex for use as an immunostimulating agent or as an adjuvant

Abstract

The present invention is directed to a polymeric carrier cargo complex, comprising as a cargo at least one nucleic acid molecule and as a preferably non-toxic and non-immunogenic polymeric carrier disulfide-crosslinked cationic components for use as an immunostimulating agent or as an adjuvant, wherein the polymeric carrier cargo complex is administered in combination with at least one second nucleic acid molecule, which encodes a protein or peptide. The inventive polymeric carrier cargo complex administered in combination with the second nucleic acid molecule allows for both efficient transfection of nucleic acids into cells in vivo and in vitro and/or for induction of an innate and/or adaptive immune response, preferably dependent on the nucleic acid to be transported as a cargo and on the second nucleic acid molecule. The present invention also provides pharmaceutical compositions, particularly vaccines, comprising the inventive polymeric carrier cargo complex and the second nucleic acid molecule, as well as the use of the inventive polymeric carrier cargo complex and the second nucleic acid molecule for transfecting a cell, a tissue or an organism, as a medicament, for therapeutic purposes as disclosed herein, and/or as an immunostimulating agent or adjuvant, e.g. for eliciting an immune response for the treatment or prophylaxis of diseases as mentioned herein. Finally, the invention relates to kits containing the inventive polymeric carrier cargo complex and the second nucleic acid molecule, the inventive pharmaceutical composition and/or the inventive vaccine or any of its components in one or more parts of the kit.

1 . A polymeric carrier cargo complex, comprising:
 a) as a carrier a polymeric carrier formed by disulfide-crosslinked cationic components, and   b) as a cargo at least one first nucleic acid molecule,   for use as an immunostimulating agent or as an adjuvant,   wherein the polymeric carrier cargo complex is administered in combination with at least one second nucleic acid molecule encoding a protein or a peptide, and   wherein the polymeric carrier cargo complex and the second nucleic acid molecule are administered intramuscularly.   
     
     
         2 . A polymeric carrier cargo complex, comprising:
 a) as a carrier a polymeric carrier formed by disulfide-crosslinked cationic components, and   b) as a cargo at least one first nucleic acid molecule,   for use as an immunostimulating agent or as an adjuvant,   wherein the polymeric carrier cargo complex is administered in combination with at least one second nucleic acid molecule encoding a protein or a peptide, wherein the second nucleic acid molecule is an RNA molecule.   
     
     
         3 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to  claim 1 , wherein the second nucleic acid molecule is an RNA molecule. 
     
     
         4 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  3 , wherein the second nucleic acid molecule is either naked or complexed with a cationic component. 
     
     
         5 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  4 , wherein the second nucleic acid molecule is not packaged in a particle, such as a virus particle, an inactivated virus particle or a virus-like particle. 
     
     
         6 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  4 , wherein the second nucleic acid molecule is not comprised in the polymeric carrier cargo complex. 
     
     
         7 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  6 , wherein the polymeric carrier cargo complex and the second nucleic acid molecule are not administered together with a protein or peptide antigen selected from the group consisting of an antigen from a pathogen associated with infectious disease, an antigen associated with allergy or allergic disease, an antigen associated with autoimmune disease, an antigen associated with a cancer or tumour disease, or a fragment, variant and/or derivative of said protein or peptide antigen. 
     
     
         8 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  6 , wherein the polymeric carrier cargo complex and the second nucleic acid molecule are not administered together with a protein or peptide antigen. 
     
     
         9 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  8 , wherein the second nucleic acid molecule is an mRNA molecule. 
     
     
         10 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to  claim 9 , wherein the mRNA molecule comprises at least one selected from the group consisting of a 5′-UTR, a 3′-UTR, a poly(A) sequence, a poly(C) sequence and a histone stem-loop sequence. 
     
     
         11 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to  claim 10 , wherein the 3′-UTR comprises a nucleic acid sequence derived from the 3′-UTR of an albumin gene. 
     
     
         12 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to  claim 10  or  11 , wherein the 3′-UTR comprises the nucleic acid sequence corresponding to SEQ ID NO. 388. 
     
     
         13 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 10  to  12 , wherein the histone stem-loop sequence comprises a nucleic acid sequence corresponding to SEQ ID NO. 389. 
     
     
         14 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 10  to  13 , wherein the 5′-UTR comprises a nucleic acid sequence derived from a 5′-UTR of a TOP gene. 
     
     
         15 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 10  to  14 , wherein the 5′-UTR comprises a nucleic acid sequence derived from a ribosomal protein gene. 
     
     
         16 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 10  to  15 , wherein the 5′-UTR comprises a nucleic acid sequence derived from ribosomal protein 32L gene. 
     
     
         17 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 9  to  16 , wherein the mRNA molecule comprises a nucleic acid sequence derived from a 5′-TOP-UTR, a GC-optimized coding sequence, a nucleic acid sequence derived from the 3′-UTR of an albumin gene, a poly(A)-sequence, a poly(C)-sequence, and a histone stem loop. 
     
     
         18 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  17 , wherein the at least one first nucleic acid molecule is an RNA molecule. 
     
     
         19 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  18 , wherein the at least one first nucleic acid molecule is an immunostimulatory nucleic acid, preferably a non-coding immunostimulatory nucleic acid. 
     
     
         20 . A polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  19 , wherein the nitrogen/phosphate (N/P) ratio of the cationic components to the at least one first nucleic acid molecule is in the range of 0.1-20, or in the range of 0.1-5, or in the range of 0.1-1. 
     
     
         21 . A polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  20 , wherein the polymeric carrier comprises functional peptides or proteins additionally to the cationic components. 
     
     
         22 . A polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to  claim 21 , wherein the functional peptides or proteins are peptide or protein antigens or antigen epitopes. 
     
     
         23 . A polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  22 , wherein the polymeric carrier additionally comprises a ligand. 
     
     
         24 . A polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to  claim 23 , wherein the ligand is mannose. 
     
     
         25 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  24 , wherein the cationic components are cationic peptides, preferably selected from oligocationic and polycationic peptides. 
     
     
         26 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to  claim 25 , wherein the cationic peptides are selected from peptides according to formula (I)
   (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x ,   wherein   l+m+n+o+x=3-100, and   l, m, n or o=independently of each other is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90 and 91-100, provided that the overall content of Arg, Lys, His and Orn represents at least 10% of all amino acids of the cationic peptide; and Xaa is any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and
 x=any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided, that the overall content of Xaa does not exceed 90% of all amino acids of the cationic peptide, 
   or are selected from peptides according to subformula (Ia)
   {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa′) x (Cys) y }
 
   or from peptides according to subformula (Ib)
   Cys 1 {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x }Cys 2    
   wherein (Arg) l ; (Lys) m ; (His) n ; (Orn) o ; and x are as defined above; Xaa′ is any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His, Orn; or Cys and y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-90, provided that the overall content of Arg (Arginine), Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least 10% of all amino acids of the oligopeptide and wherein Cys 1  and Cys 2  are Cysteines proximal to, or terminal to (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x .   
     
     
         27 . The polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to  claim 25  or  26 , wherein the disulfide-bonds are formed by cysteine residues contained in the cationic peptides. 
     
     
         28 . A polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to  claim 27 , wherein the cysteine residue is located proximal to, preferably at the terminal ends of the cationic peptides. 
     
     
         29 . A polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  28 , wherein the cationic component comprises the peptide CysArg 12 Cys. 
     
     
         30 . A polymeric carrier cargo complex for use as an immunostimulating agent or an adjuvant according to any one of  claims 1  to  29  comprising:
 a. as a carrier a polymeric carrier formed by disulfide-crosslinked cationic polymers, and 
 b. as a cargo at least one nucleic acid (molecule), 
 in the treatment or prophylaxis of a disease selected from a tumour or a cancer disease, an infectious disease, an autoimmune disease or an allergy. 
 
     
     
         31 . A pharmaceutical composition comprising:
 (A) a polymeric carrier cargo complex, comprising:
 a) as a carrier a polymeric carrier formed by disulfide-crosslinked cationic components, and 
 b) as a cargo at least one first nucleic acid molecule, 
   and   (B) at least one second nucleic acid molecule, wherein the at least one second nucleic acid molecule is an RNA molecule encoding a protein or a peptide.   
     
     
         32 . The pharmaceutical composition according to  claim 31 , wherein the at least one second nucleic acid molecule encodes a protein or peptide antigen that is selected from the group consisting of an antigen from a pathogen associated with infectious disease; an antigen associated with allergy; an antigen associated with autoimmune disease; and an antigen associated with cancer or tumour disease;
 or a fragment, variant and/or derivative of said antigen.   
     
     
         33 . The pharmaceutical composition according to  claim 31  or  32 , wherein component (B) is not covalently linked to (A). 
     
     
         34 . The pharmaceutical composition according to any one of  claims 31  to  33 , which does not comprise a protein or peptide antigen selected from the group consisting of an antigen from a pathogen associated with infectious disease, an antigen associated with allergy or allergic disease, an antigen associated with autoimmune disease, an antigen associated with a cancer or tumour disease, or a fragment, variant and/or derivative of said protein or peptide antigen. 
     
     
         35 . The pharmaceutical composition according to any one of  claims 31  to  33 , which does not comprise a protein or peptide antigen. 
     
     
         36 . The pharmaceutical composition according to any one of  claims 31  to  35 , wherein the second nucleic acid molecule is either naked or complexed with a cationic component. 
     
     
         37 . The pharmaceutical composition according to any one of  claims 31  to  36 , wherein the second nucleic acid molecule is not packaged in a particle, such as a virus particle, an inactivated virus particle or a virus-like particle. 
     
     
         38 . The pharmaceutical composition according to any one of  claims 31  to  37 , wherein the antigen is derived from a pathogen, preferably a viral, bacterial, fungal or protozoan pathogen, preferably selected from the list consisting of: Rabies virus, Hepatitis B virus, human Papilloma virus (hPV),  Bacillus anthracis , Respiratory syncytial virus (RSV), Herpes simplex virus (HSV), Dengue virus, Rotavirus, Influenza virus and  Mycobacterium tuberculosis.    
     
     
         39 . The pharmaceutical composition according to any one of  claims 31  to  37 , wherein the antigen is associated with allergy or allergic disease and is derived from a source selected from the list consisting of: grass pollen, tree pollen, flower pollen, herb pollen, dust mite, mold, animals, food, and insect venom. 
     
     
         40 . The pharmaceutical composition according to any one of  claims 31  to  37 , wherein the antigen is associated with a cancer or tumour disease and is selected from the list consisting of: p53, CA125, EGFR, Her2/neu, hTERT, PAP, MAGE-A1, MAGE-A3, MAGE-C1, MAGE-C2, Mesothelin, MUC-1, NY-ESO-1, GP100, MART-1, Tyrosinase, PSA, PSCA, PSMA, VEGF, VEGFR1, VEGFR2, Ras, CEA, Survivin, 5T4, STEAP and WT1. 
     
     
         41 . The pharmaceutical composition according to any one of  claims 31  to  40 , wherein the polymeric carrier cargo complex is for use as an immunostimulating agent or as an adjuvant. 
     
     
         42 . The pharmaceutical composition according to any one of  claims 31  to  41 , wherein the at least one first nucleic acid molecule is an RNA. 
     
     
         43 . The pharmaceutical composition according to any one of  claims 31  to  42 , wherein the at least one first nucleic acid molecule is an immunostimulatory nucleic acid. 
     
     
         44 . The pharmaceutical composition according to any one of  claims 31  to  43 , wherein the nitrogen/phosphate (N/P) ratio of the cationic components to the at least one first nucleic acid molecule is in the range of 0.1-20, or in the range of 0.1-5, or in the range of 0.1-1. 
     
     
         45 . The pharmaceutical composition according to any one of  claims 31  to  44 , wherein the polymeric carrier additionally comprises a ligand. 
     
     
         46 . The pharmaceutical composition according to  claim 45 , wherein the ligand is mannose. 
     
     
         47 . The pharmaceutical composition according to any one of  claims 31  to  46 , wherein the cationic components are cationic peptides. 
     
     
         48 . The pharmaceutical composition according to any one of  claims 31  to  47 , wherein the cationic peptides are selected from peptides according to formula (I)
   (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x , 
 wherein 
 l+m+n+o+x=3-100, and 
 l, m, n or o=independently of each other is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90 and 91-100, provided that the overall content of Arg, Lys, His and Orn represents at least 10% of all amino acids of the cationic peptide; and Xaa is any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and
 x=any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided, that the overall content of Xaa does not exceed 90% of all amino acids of the cationic peptide, 
 
 or are selected from peptides according to subformula (Ia)
   {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa′) x (Cys) y }
 
 
 or from peptides according to subformula (Ib)
   Cys 1 {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x }Cys 2    
 
 wherein (Arg) l ; (Lys) m ; (His) n ; (Orn) o ; and x are as defined above; Xaa′ is any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His, Orn; or Cys and y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-90, provided that the overall content of Arg (Arginine), Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least 10% of all amino acids of the oligopeptide and wherein Cys 1  and Cys 2  are Cysteines proximal to, or terminal to (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x . 
 
     
     
         49 . The pharmaceutical composition according to  claim 47  or  48 , wherein the disulfide-bonds are formed by cysteine residues contained in the cationic peptides. 
     
     
         50 . The pharmaceutical composition according to  claim 49 , wherein the cysteine residue is located proximal to, preferably at the terminal ends of the cationic peptides. 
     
     
         51 . The pharmaceutical composition according to  claim 49  or  50 , wherein the cationic component comprises the peptide CysArg 12 Cys. 
     
     
         52 . A vaccine, comprising a pharmaceutical composition according to any one of  claims 31  to  51 . 
     
     
         53 . The vaccine according to  claim 52 , wherein the pharmaceutical composition according to any of  claims 31  to  51  elicits an adaptive immune response. 
     
     
         54 . The vaccine according to  claim 52  or  53 , wherein the polymeric carrier cargo complex is used as an immunostimulating agent or adjuvant. 
     
     
         55 . The vaccine according to any one of  claims 52  to  54 , wherein the vaccine further comprises a pharmaceutically acceptable carrier. 
     
     
         56 . A kit, preferably a kit of parts, comprising the pharmaceutical composition according to any one of  claims 31  to  51 , and/or the vaccine according to any one of  claims 52  to  55 , and optionally a liquid vehicle for solubilising and optionally technical instructions with information on the administration and dosage of the active composition and/or the vaccine. 
     
     
         57 . The kit according to  claim 56 , wherein the at least one second nucleic acid molecule, which encodes a protein or peptide, is provided in lyophilized form as a separate part. 
     
     
         58 . The kit according to  claim 56  or  57 , wherein the kit contains as a part Ringer-Lactate solution. 
     
     
         59 . The pharmaceutical composition according to any one of  claims 31  to  51  or the kit according to any one of  claims 56  to  58 , for use in the treatment or prophylaxis of an infectious disease; an allergy; an autoimmune disease; or a cancer or tumour disease.