VACCINATION IN ELDERLY PATIENTS

Abstract

The present invention relates to vaccines comprising at least one mRNA encoding at least one antigen for use in the treatment of a disease in an elderly patient preferably exhibiting an age of at least 50 years, more preferably of at least 55 years, 60 years, 65 years, 70 years, or older, wherein the treatment comprises vaccination of the patient and eliciting an immune response in said patient. The present invention is furthermore directed to kits and kits of parts comprising such a vaccine and/or its components and to methods applying such a vaccine or kit.

Claims

1. Vaccine comprising at least one mRNA encoding at least one antigen for use in the prophylaxis and/or treatment of a disease in an elderly patient exhibiting an age of at least 50 years, wherein the treatment comprises vaccination of the patient and eliciting an immune response in said patient.

2. Vaccine for use according to claim 1, wherein eliciting an immune response in a patient comprises eliciting a Th 1 immune response.

3. Vaccine for use according to any of claim 1 or 2, wherein the elderly patient is male or female and/or exhibits an age of at least 55 years, 60 years, 65 years, 70 years, or older.

4. Vaccine for use according to any of claims 1 to 3, wherein the disease is selected from infectious diseases, viral, bacterial or protozoological infectious diseases, autoimmune diseases, allergies or allergic diseases or cancer or tumour diseases.

5. Vaccine for use according to any of claims 1 to 4, wherein the antigen is selected from protein and peptide antigens, tumour antigens, self-antigens or auto-antigens, auto-immune self-antigens, pathogenic antigens, viral antigens, bacterial antigens, fungal antigens, protozoological antigens, animal antigens, allergy antigens.

6. Vaccine for use according to any of claims 1 to 5, wherein the vaccine is to be administered parenterally, orally, nasally, pulmonary, by inhalation, topically, rectally, buccally, vaginally, or via an implanted reservoir.

7. Vaccine for use according to any of claims 1 to 6, wherein the at least one mRNA encoding at least one antigen is to be administered in its naked form or is associated with or complexed with a cationic or polycationic compound.

8. Vaccine for use according to any of claims 1 to 6, wherein the at least one mRNA encoding at least one antigen is complexed with a polymeric carrier formed by disulfide-crosslinked cationic components selected from an oligopeptide having following sum formula (I):
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}(formula (Ia) 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 (Arginine), Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least 10% of all amino acids of the oligopeptide of formula (V); 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 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, provided, that the overall content of Xaa does not exceed 90% of all amino acids of the oligopeptide of formula (I), or from a disulfide-crosslinked cationic component comprising as a repeat unit an oligopeptide having following subformula (Ia):
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x(Cys).sub.y}formula (Ia) wherein (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o; and x are as defined above for formula (I), 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. or from a disulfide-crosslinked cationic component comprising as a repeat unit an oligopeptide having following subformula (Ib):
Cys.sup.1{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}Cys.sup.2;(formula (Ib)) wherein component {(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x} (formula (I)) within formula (Ib) is as defined herein and forms a core of subformula (Ib), and wherein Cys.sup.1 and Cys.sup.2 are Cysteines proximal to, or terminal to (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x.

9. Vaccine for use according to any of claims 1 to 6, wherein the at least one mRNA encoding at least one antigen is complexed with a polymeric carrier according to generic formula (VI):
L-P.sup.1S[SP.sup.2S].sub.nSP.sup.3-L wherein, P.sup.1 and P.sup.3 are different or identical to each other and represent a linear or branched hydrophilic polymer chain, each P.sup.1 and P.sup.3 exhibiting at least one SH-moiety, capable to form a disulfide linkage upon condensation with component P.sup.2, the linear or branched hydrophilic polymer chain selected independent from each other from polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine), hydroxyethylstarch or poly(hydroxyalkyl L-glutamine), wherein the hydrophilic polymer chain exhibits a molecular weight of about 1 kDa to about 100 kDa, P.sup.2 is a cationic or polycationic peptide or protein, having a length of about 3 to about 100 amino acids, or is a cationic or polycationic polymer, having a molecular weight of about 0.5 kDa to about 30 kDa, each P.sup.2 exhibiting at least two SH-moieties, capable to form a disulfide linkage upon condensation with further components P.sup.2 or component(s) P.sup.1 and/or P.sup.3; SS is a (reversible) disulfide bond; L is an optional ligand, which may be present or not, and may be selected independent from the other from RGD, Transferrin, Folate, a signal peptide or signal sequence, a localization signal or sequence, a nuclear localization signal or sequence (NLS), an antibody, a cell penetrating peptide (CPP), TAT, KALA, a ligand of a receptor, cytokines, hormones, growth factors, small molecules, carbohydrates, mannose, galactose, synthetic ligands, small molecule agonists, inhibitors or antagonists of receptors, or RGD peptidomimetic analogues; and n is an integer, selected from a range of about 1 to 50, preferably in a range of about 1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2, 3, or 4 to 9.

10. Vaccine for use according to claim 9, wherein the at least one mRNA encoding at least one antigen is complexed with a polymeric carrier molecule according to formula (VIa)
L-P.sup.1S{[SP.sup.2S].sub.a[S(AA).sub.xS].sub.b}SP.sup.3-L, wherein S, L, P.sup.1, P.sup.2 and P.sup.3 are preferably as defined above for formula (VI), a and b are integers, wherein a+b=n and n is an integer as defined above for formula (VI), x is an integer selected from a range of about 1 to 100, and (AA) is selected from an aromatic, a hydrophilic, a lipophilic, or a weak basic amino acid or peptide, or is a signal peptide or signal sequence, a localization signal or sequence, a nuclear localization signal or sequence (NLS), an antibody, a cell penetrating peptide (CPP), or is selected from therapeutically active proteins or peptides, from antigens, tumour antigens, pathogenic antigens, animal antigens, viral antigens, protozoan antigens, bacterial antigens, allergic antigens, autoimmune antigens, from allergens, from antibodies, from immunostimulatory proteins or peptides, or from antigen-specific T-cell receptors.

11. Vaccine for use according to any of claims 1 to 10, wherein the vaccine is formulated to comprise a) an (adjuvant) component, comprising or consisting of at least one (m)RNA, complexed with a cationic or polycationic compound and/or with a polymeric carrier as defined according to any of claim 7 or 8, and b) at least one free mRNA encoding an antigen, as defined according to any of claims 1 and 5.

12. Vaccine for use according to claim 11, wherein the (m)RNA is an mRNA as defined according to any of claims 1 and 5, an immunostimulatory nucleic acid, a CpG nucleic acid, a CpGRNA, a CpG-DNA, or an immunostimulatory RNA (isRNA).

13. Vaccine for use according to any of claims 1 to 12, wherein the vaccine furthermore comprises a pharmaceutically acceptable carrier and/or vehicle.

14. Vaccine for use according to any of claims 1 to 13, wherein the vaccine furthermore comprises at least one adjuvant, an auxiliary substance selected from lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, 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-14, 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, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, a ligand of human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, a ligand of murine Toll-like receptor TLR1, TLR2, TLRs, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand of a NOD-like receptor, a ligand of a RIG-I like receptor, an immunostimulatory nucleic acid, an immunostimulatory RNA (isRNA), a CpG-DNA, an antibacterial agent, or an anti-viral agent.

15. Kit, comprising a vaccine as defined according to any of claims 1 to 15, wherein each mRNA encoding at least one antigen is provided in a different part of the kit.

Description

FIGURES

[0263] The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0264] FIGS. 1A, B: show the result of the vaccination of 18 months or 8 weeks old mice. The mice were vaccinated twice intradermally with 80 g mRNA coding for PR8 H1 HA (Hemagglutinin of influenza virus A/Puerto Rico/8/1934) or with mRNA coding for Gallus gallus ovalbumine as a control (control mRNA). Injections were done with an interval of 7 days. 5 weeks after the last vaccination the mice were challenged with a 10fold lethal dose of PR8 virus (10 LD50). The weight of the mice was controlled over 2 weeks and the mice were killed when they have lost more than 25% of their original weight. FIG. 1A shows the overall survival of the mice. FIG. 1B shows the weight of the mice.

[0265] FIGS. 1C, D: show the coding sequence of the mRNAs used for vaccination of 18 months or 8 weeks old mice (see FIGS. 1A, B) coding for PR8 H1 HA (Hemagglutinin of influenza virus A/Puerto Rico/8/1934) (SEQ ID NO: 384) (FIG. 2C) or for Gallus gallus ovalbumine as a control (control mRNA) (SEQ ID NO: 385) (FIG. 2D)

[0266] FIGS. 2A, B: show the results of the vaccination of 32 patients with an age between 52 and 74 with histologically confirmed diagnosis of adenocarcinoma of the prostate. These patients were vaccinated intradermally 5 times with a total of 1280 g mRNA per treatment coding for the tumour antigens PSA, PSCA, PSMA, and STEAP-1. Injections were done in study weeks 1, 3, 7, 15, and 23.2 weeks after the 3.sup.rd, 4.sup.th, and 5.sup.th vaccination blood samples of the patients were collected and analysed for the presence of an antigen specific immune response against the tumour antigens PSA, PSCA, PSMA and STEAP-1. As can be seen, patients older than 70 shows at least the same efficiency in generation of an antigen specific immune response as patients younger than 70. In FIG. 2B antigens against which a specific immune response was detected by ELISPOT, Tetramer staining, Intracellular Cytokine Staining (ICS) or ELISA are indicated for each patient included in the study.

[0267] FIGS. 2C-F: show the coding sequence of the mRNAs used for vaccination of 32 patients with an age between 52 and 74 with histologically confirmed diagnosis (see FIGS. 2A, B). The mRNA sequences code for the tumour antigens PSA, PSMA, PSCA, STEAP-1 (SEQ ID NOS: 386, 387, 388 and 389).

EXAMPLES

[0268] The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

Example 1Preparation of MRNA Constructs

[0269] For the present examples DNA sequences, encoding PR8 H1 HA (Haemagglutinin of A/Puerto Rico/8/1934) (SEQ ID NO: 384), and Gallus gallus ovalbumine, respectively, as a control (control mRNA) (SEQ ID NO: 385), were prepared and used for subsequent in vitro transcription reactions.

[0270] According to a first preparation, the DNA sequence termed PR8 H1 HA (Haemagglutinin of A/Puerto Rico/8/1934) (SEQ ID NO: 384) (see FIG. 1C) was prepared by modifying the wildtype Haemagglutinin encoding DNA sequence by introducing a GC-optimized sequence for a better codon usage and stabilization. In SEQ ID NO: 384 (see FIG. 1C) the sequence of the corresponding mRNA is shown. The sequence was furthermore introduced into a pCV19 vector and modified to comprise stabilizing sequences derived from alpha-globin-3-UTR (muag (mutated alpha-globin-3-UTR)), a stretch of 70 adenosine at the 3-terminal end (poly-A-tail) and a stretch of 30 cytosine at the 3-terminal end (poly-C-tail). The sequence of the final DNA construct was termed PR8 H1 HA.

[0271] According to a second preparation, the DNA sequence termed Gallus gallus ovalbumine, respectively, as a control (control mRNA) (SEQ ID NO: 385) (see FIG. 1D) was prepared by modifying the wildtype Gallus gallus ovalbumine encoding DNA sequence by introducing a GC-optimized sequence for a better codon usage and stabilization. In SEQ ID NO: 385 (see FIG. 1D) the sequence of the corresponding mRNA is shown. The sequence was furthermore introduced into a pCV19 vector and modified to comprise stabilizing sequences derived from alpha-globin-3-UTR (muag (mutated alpha-globin-3-UTR)), a stretch of 70 adenosine at the 3-terminal end (poly-A-tail) and a stretch of 30 cytosine at the 3-terminal end (poly-C-tail). The sequence of the final DNA construct was termed Gallus gallus ovalbumine.

[0272] Likewise, DNA plasmids coding for the tumour antigens PSA, PSMA, PSCA, STEAP-1 were prepared. In SEQ ID NOs: 386, 387, 388 and 389, the sequence of the corresponding mRNAs are shown (see also FIGS. 2C-F).

[0273] In a further step, the respective DNA plasmids prepared above were transcribed into mRNA in vitro using T7-Polymerase. Subsequently the obtained mRNA was purified using PureMessenger (CureVac, Tbingen, Germany).

[0274] All obtained mRNAs used herein were furthermore complexed with protamine prior to use. The RNA complexation consisted of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA was complexed with protamine by slow addition of protamine-Ringer's lactate solution to mRNA. As soon as the complexes were stably generated, free mRNA was added, stirred shortly and the final concentration of the vaccine was adjusted with Ringer's lactate solution.

Example 2Vaccination of 18 Months or 8 Weeks Old Mice

[0275] In this experiment 18 months or 8 weeks old mice were vaccinated twice intradermally with 80 g mRNA coding for PR8 H1 HA (Hemagglutinin of A/Puerto Rico/8/1934; FIG. 1C) or with mRNA coding for Gallus gallus ovalbumine as a control (control mRNA; FIG. 1D). Injections were done with an interval of 7 days. 5 weeks after the last vaccination the mice were challenged with a 10fold lethal dose of PR8 virus (10 LD50). The weight of the mice was controlled over 2 weeks and the mice were killed when they have lost more than 25% of their original weight. The results are shown in FIGS. 1A and B. FIG. 1A shows the overall survival of the mice. FIG. 1B shows the weight of the mice. As can be seen in FIG. 1A, mice vaccinated with mRNA coding for PR8 H1 Hemagglutinin exhibited a significantly better survival (all mice survived) against influenza challenge infection with control mRNA only (all mice died about 7 days subsequent to vaccination with control mRNA encoding chicken ovalbumin, when vaccinated with 8 weeks and died about 9 days subsequent to vaccination with control mRNA, when vaccinated with 18 months).

Example 3Vaccination of Human Prostate Carcinoma Patients

[0276] In this experiment 32 patients with an age between 52 and 74 with histologically confirmed diagnosis of adenocarcinoma of the prostate were vaccinated intradermally 5 times with a total of 1280 g mRNA per treatment coding for the tumour antigens PSA, PSCA, PSMA, STEAP-1. Injections were done in study weeks 1, 3, 7, 15, and 23. 22 weeks after the 3.sup.rd, 4.sup.th, and 5.sup.th vaccination blood samples of the patients were collected and analysed for the presence of an antigen specific immune response against the tumour antigens PSA, PSCA, PSMA and STEAP-1. The results are shown in FIGS. 2A and 2B. As can be seen in FIG. 2A, patients older than 70 show at least the same efficiency in generation of an antigen specific immune response as patients younger than 70. In FIG. 2B antigens against which a specific immune response was detected by ELISPOT, Tetramer staining, Intracellular Cytokine Staining (ICS) or ELISA are indicated for each patient included in the study.