LIPID NANOPARTICLE MRNA VACCINES
20210251898 · 2021-08-19
Assignee
Inventors
- Patrick Baumhof (Dusslingen, DE)
- Mariola Fotin-Mleczek (Sindelfingen, DE)
- Regina HEIDENREICH (Tübingen, DE)
- Michael J. Hope (Vancouver, CA)
- Edith JASNY (Stuttgart, DE)
- Sandra LAZZARO (Tübingen, DE)
- Paulo Jia Ching LIN (Vancouver, CA)
- Johannes LUTZ (Tübingen, DE)
- Barbara Mui (Vancouver, CA)
- Benjamin Petsch (Tübingen, DE)
- Susanne Rauch (Tübingen, DE)
- Kim Ellen SCHMIDT (Dettenhausen, DE)
- Ying Tam (Vancouver, CA)
Cpc classification
C12N2760/20134
CHEMISTRY; METALLURGY
A61K31/23
HUMAN NECESSITIES
A61K2039/55555
HUMAN NECESSITIES
A61K47/14
HUMAN NECESSITIES
A61K31/7105
HUMAN NECESSITIES
A61K31/40
HUMAN NECESSITIES
C12N2760/16134
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
C07C219/08
CHEMISTRY; METALLURGY
C07C229/16
CHEMISTRY; METALLURGY
A61K9/19
HUMAN NECESSITIES
C07C219/06
CHEMISTRY; METALLURGY
B82Y5/00
PERFORMING OPERATIONS; TRANSPORTING
A61K9/1272
HUMAN NECESSITIES
C07C233/36
CHEMISTRY; METALLURGY
A61K47/6929
HUMAN NECESSITIES
C07C255/24
CHEMISTRY; METALLURGY
C12N2760/16234
CHEMISTRY; METALLURGY
International classification
A61K9/127
HUMAN NECESSITIES
A61K31/23
HUMAN NECESSITIES
A61K31/40
HUMAN NECESSITIES
A61K31/7105
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
A61K47/14
HUMAN NECESSITIES
A61K47/69
HUMAN NECESSITIES
A61K9/19
HUMAN NECESSITIES
Abstract
The invention relates to mRNA comprising lipid nanoparticles and their medical uses. The lipid nanoparticles of the present invention comprise a cationic lipid according to formula (I), (II) or (III) and/or a PEG lipid according to formula (IV), as well as an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein. The invention further relates to the use of said lipid nanoparticles as vaccines or medicaments, in particular with respect to influenza or rabies vaccination.
Claims
1. A pharmaceutical composition comprising: (a) a mRNA comprising a coding sequence encoding a coronavirus spike (S) protein or an antigenic fragment thereof; and (b) a lipid nanoparticle carrier comprising: (i) a cationic lipid with the formula III: ##STR00179## or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L.sup.1 and L.sup.2 are each independently —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O).sub.x—, —S—S—, —C(═O)S—, —SC(═O)—, —NR.sup.aC(═O)—, —C(═O)NR.sup.a—, —NR.sup.aC(═O)NR.sup.a—, —OC(═O)NR.sup.a— or —NR.sup.aC(═O)O—; G.sup.1 and G.sup.2 are each independently unsubstituted C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene; G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.1-C.sub.24 alkenylene, C.sub.3-C.sub.8 cycloalkylene, or C.sub.3-C.sub.8 cycloalkenylene; R.sup.a is, at each occurrence, independently H or C.sub.1-C.sub.12, alkyl; R.sup.1 and R.sup.2 are each independently C.sub.6-C.sub.24 alkyl or C.sub.6-C.sub.24 alkenyl; R.sup.3 is OR.sup.5, CN, —C(═O)OR.sup.4, —OC(═O)R.sup.4 or —NR.sup.5C(═O)R.sup.4; R.sup.4 is C.sub.1-C.sub.12 alkyl; R.sup.5 is H or C.sub.1-C.sub.6 alkyl; and x is 0, 1 or 2; or (ii) a PEG lipid with the formula (IV) ##STR00180## wherein: R.sup.8 and R.sup.9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60; or (iii) a cationic lipid with the formula I: ##STR00181## or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L.sup.1 and L.sup.2 are each independently —O(C═O)—, —(C═O)O— or a carbon-carbon double bond; R.sup.1a and R.sup.1b are, at each occurrence, independently either (a) H or C.sub.1-C.sub.12, alkyl, or (b) R.sup.1a is H or C.sub.1-C.sub.12, alkyl, and R.sup.1b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.1b and the carbon atom to which it is bound to form a carbon-carbon double bond; R.sup.2a and R.sup.2b are, at each occurrence, independently either (a) H or C.sub.1-C.sub.12, alkyl, or (b) R.sup.2a is H or C.sub.1-C.sub.12, alkyl, and R.sup.2b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.2b and the carbon atom to which it is bound to form a carbon-carbon double bond; R.sup.1a and R.sup.3b are, at each occurrence, independently either (a) H or C.sub.1-C.sub.12, alkyl, or (b) R.sup.1a is H or C.sub.1-C.sub.12, alkyl, and R.sup.3b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.3b and the carbon atom to which it is bound to form a carbon-carbon double bond; R.sup.4a and R.sup.4b are, at each occurrence, independently either (a) H or C.sub.1-C.sub.12, alkyl, or (b) R.sup.4a is H or C.sub.1-C.sub.12, alkyl, and R.sup.4b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.4b and the carbon atom to which it is bound to form a carbon-carbon double bond; R.sup.5 and R.sup.6 are each independently methyl or cycloalkyl; R.sup.7 is, at each occurrence, independently H or C.sub.1-C.sub.12, alkyl; R.sup.8 and R.sup.9 are each independently C.sub.1-C.sub.12, alkyl; or R.sup.8 and R.sup.9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; and e is 1 or 2; or (iv) a cationic liquid with the formula H: ##STR00182## or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L.sup.1 and L.sup.2 are each independently —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S—S—, —C(═O)S—, —SC(═O)—, —NR.sup.aC(═O)—, —C(═O)NR.sup.a—, —NR.sup.aC(═O)NR.sup.a—, —OC(═O)NR.sup.a—, —NR.sup.aC(═O)O—, or a direct bond; G.sup.1 is C.sub.1-C.sub.2 alkylene, —(C═O)—, —O(C═O)—, —SC(═O)—, —NR.sup.aC(═O)— or a direct bond G.sup.2 is —C(═O)—, —(C═O)O—, —C(═O)S—, —C(═O)NR.sup.a— or a direct bond; G.sup.3 is C.sub.1-C.sub.6 alkylene; R.sup.a is, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl; R.sup.1a and R.sup.16 are, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.1a is H or C.sub.1-C.sub.12, alkyl, and R.sup.1b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.1b and the carbon atom to which it is bound to form a carbon-carbon double bond; R.sup.2a and R.sup.2b are, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12, alkyl; or (b) R.sup.2a is H or C.sub.1-C.sub.12, alkyl, and R.sup.2b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.2b and the carbon atom to which it is bound to form a carbon-carbon double bond; R.sup.1a and R.sup.3b are, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12, alkyl; or (b) R.sup.1a is H or C.sub.1-C.sub.12, alkyl, and R.sup.3b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.3b and the carbon atom to which it is bound to form a carbon-carbon double bond; R.sup.4a and R.sup.4b are, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12, alkyl; or (b) R.sup.4a is H or C.sub.1-C.sub.12, alkyl, and R.sup.4b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.4b and the carbon atom to which it is bound to form a carbon-carbon double bond; R.sup.5 and R.sup.6 are each independently H or methyl; R.sup.7 is C.sub.4-C.sub.20 alkyl; R.sup.8 and R.sup.9 are each independently C.sub.1-C.sub.12, alkyl; or R.sup.8 and R.sup.9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.
2. The pharmaceutical composition of claim 1, wherein the mRNA does not comprise a nucleoside having a base modification.
3. The pharmaceutical composition of claim 1, wherein the coding sequence of the mRNA consists of A, U, G and C nucleosides.
4. The pharmaceutical composition of claim 1, wherein the mRNA comprises at least one chemical modification that is a nucleoside modification
5. The pharmaceutical composition of claim 1, wherein the mRNA comprises a 1-methylpseudouridine substitution.
6. The pharmaceutical composition of claim 1, wherein the mRNA is encapsulated in or associated with said lipid nanoparticle.
7. The pharmaceutical composition of claim 1, wherein the mRNA coding sequence encodes a coronavirus S protein.
8. The pharmaceutical composition of claim 1, wherein the mRNA coding sequence encodes an antigenic fragment of a coronavirus S protein.
9. The pharmaceutical composition of claim 1, wherein the coronavirus S protein is from a SARS coronavirus.
10. The pharmaceutical composition of claim 1, wherein the lipid nanoparticle comprises the cationic lipid of any one of formulae (I), (II), and (III); and additionally comprises: (a) a PEG lipid with the formula (IV): ##STR00183## wherein: R.sup.8 and R.sup.9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60; or (b) a pegylated diacylglycerol (PEG-DAG).
11. The pharmaceutical composition of claim 10, comprising a PEG lipid with the formula (IV): ##STR00184## wherein: R.sup.8 and R.sup.9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
12. The pharmaceutical composition of claim 10, comprising a pegylated diacylglycerol (PEG-DAG).
13. The pharmaceutical composition of claim 12, wherein the PEG-DAG is a pegylated 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG).
14. The pharmaceutical composition of claim 1, wherein the lipid nanoparticle comprises a cationic lipid selected from the structures I-1 to I-41, II-1 to II-34 and III-1 to III-36: TABLE-US-00031 No. Structure I-1
15. The composition of claim 1, wherein in the PEG lipid R.sup.8 and R.sup.9 are saturated alkyl chains.
16. The lipid nanoparticle according to claim 14, wherein the PEG lipid is ##STR00298## wherein n is an integer selected such that the average molecular weight of the PEG lipid is about 2500 g/mol.
17. The pharmaceutical composition of claim 1, wherein the lipid nanoparticle comprises the cationic lipid of formula (I), (II) or (III), DSPC, cholesterol and a PEG-lipid.
18. The pharmaceutical composition of claim 1, wherein the mRNA sequence additionally comprises: a) a 5′-CAP structure; b) a poly(A) sequence; c) a poly (C) sequence; or d) two or more of a), b) and c).
19. The pharmaceutical composition of claim 1, wherein the mRNA sequence additionally comprises at least one histone stem loop.
20. The pharmaceutical composition of claim 19, wherein the mRNA sequence comprises, in 5′ to 3′-direction, the following elements: a) a 5′ m7GpppN CAP structure, b) a coding sequence encoding a coronavirus S protein or an antigenic fragment thereof, c) a poly(A) sequence of 10 to 200 adenosine nucleotides, d) optionally a poly(C) sequence, and e) optionally a histone stem-loop.
21. The pharmaceutical composition of claim 20, wherein the mRNA sequence comprises, in 5′ to 3′-direction, the following elements: a) a 5′ CAP1 structure, b) a coding sequence encoding a coronavirus S protein or an antigenic fragment thereof, c) a 3′-UTR element comprising a sequence from an alpha globin gene; d) a poly(A) sequence of 10 to 200 adenosine nucleotides, e) a poly(C) sequence of 10 to 200 cytosine nucleotides, and f) a histone stem-loop.
22. The pharmaceutical composition of claim 1, wherein the mRNA sequence comprises, in 5′ to 3′-direction, the following elements: a) a 5′ CAP1 structure, b) a 5′-UTR element which comprises a nucleic acid sequence from the 5′-UTR of a TOP gene; c) a coding sequence encoding a coronavirus S protein or an antigenic fragment thereof, d) a 3′-UTR sequence; e) optionally, a histone stem-loop; and f) a poly(A) sequence of 10 to 200 adenosine nucleotides.
23. The pharmaceutical composition of claim 22, wherein (b) the 5′-UTR sequence is a sequence from a HSD17B4 gene 5′-UTR.
24. The pharmaceutical composition of claim 1, wherein the lipid nanoparticle comprises the cationic lipid of formula (I).
25. The pharmaceutical composition of claim 1, wherein the lipid nanoparticle comprises the cationic lipid of formula (II).
26. The pharmaceutical composition of claim 1, wherein the lipid nanoparticle comprises the cationic lipid of formula (III).
27. A method of treating or preventing a disease or disorder in a subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition of claim 1.
28. A method for raising an immune response in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition of claim 1.
Description
FIGURES
[0967] The figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.
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[0990] (see section “Preferred sequences of the present invention”; Legend: First column: Protein or Nucleic Acid Accession No. (GenBank); second column (A): Protein Sequence wild type SEQ ID NO: third column (B): Nucleotide Sequence wild type SEQ ID NO: fourth column (C): Optimized Nucleotide Sequence SEQ ID NO:)
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[0992] (see section “Preferred sequences of the present invention”; Legend: First column: Protein or Nucleic Acid Accession No. (GenBank); second column (A): Protein Sequence wild type SEQ ID NO: third column (B): Nucleotide Sequence wild type SEQ ID NO: fourth column (C): Optimized Nucleotide Sequence SEQ ID NO:)
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[0994] (see section “Preferred sequences of the present invention”; Legend: First column: Protein or Nucleic Acid Accession No. (GenBank); second column (A): Protein Sequence wild type SEQ ID NO: third column (B): Nucleotide Sequence wild type SEQ ID NO: fourth column (C): Optimized Nucleotide Sequence SEQ ID NO:)
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[0996] (see section “Preferred sequences of the present invention”; Legend: First column: Protein or Nucleic Acid Accession No. (GenBank); second column (A): Protein Sequence wild type SEQ ID NO: third column (B): Nucleotide Sequence wild type SEQ ID NO: fourth column (C): Optimized Nucleotide Sequence SEQ ID NO:)
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[0998] (see section “Preferred sequences of the present invention”; Legend: First column: Protein or Nucleic Acid Accession No. (GenBank); second column (A): Protein Sequence wild type SEQ ID NO: third column (B): Nucleotide Sequence wild type SEQ ID NO: fourth column (C): Optimized Nucleotide Sequence SEQ ID NO:)
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[1000] (A/California/7/2009) of mice vaccinated with a combination of four mRNAs coding for different Influenza antigens (C-F) compared to controls injected with RiLa (A) or Influsplit (B). For each setting (A-F) three different time points are shown (d21, d35, and d49). Vaccination scheme, see Table A. A detailed description of the experiment is provided in Example 12.
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[1003] (A/HongKong/4801/2014) of mice vaccinated with a combination of four mRNAs coding for different Influenza antigens (C-F) compared to controls injected with RiLa (A) or Influsplit (B). For each setting (A-F) three different time points are shown (d21, d35, and d49). Vaccination scheme, see Table A. A detailed description of the experiment is provided in Example 12.
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[1016] (A/Hong Kong/4801/2014), influenza B (B/Brisbane/60/2008), H5N1 (A/Vietnam/1203/2004) (1-4 respectively). As a control, cells were stimulated with buffer (5). As further controls, mice were injected Rila (A) or Influsplit (B). Vaccination scheme, see Table B. A detailed description of the experiment is provided in Example 13.
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[1018] (A/Vietnam/1203/2004) (1 and 2). As a control, cells were stimulated with buffer (3). As further controls, mice were injected Rila (A) or Influsplit (B). Vaccination scheme, see Table B. A detailed description of the experiment is provided in Example 13.
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EXAMPLES
[1060] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
[1061] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
Example 1: Preparation of mRNA Constructs
[1062] For the present examples, DNA sequences encoding different proteins were prepared and used for subsequent RNA in vitro transcription reactions. The DNA sequences encoding the proteins were prepared by modifying the wild type encoding DNA sequence by introducing a GC-optimized sequence for stabilization. Sequences were introduced into a derived pUC19 vector. For further stabilization and/or increased translation UTR elements were introduced 5′- and/or 3′ of the coding region.
[1063] The following mRNA constructs were used in the examples:
[1064] Photinus pyralis luciferase: [1065] 5′-TOP-UTR derived from 32L4 ribosomal protein—GC-enriched coding sequence encoding PpLuc-3′-UTR derived from albumin gene—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224286).
[1066] Influenza Hemagglutinin (HA): [1067] 5′-TOP-UTR derived from 32L4 ribosomal protein—GC-enriched coding sequence encoding HA of Influenza A/California/07/2009 (H1N1)—3′-UTR derived from albumin gene—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224118) [1068] GC-enriched coding sequence encoding HA of Influenza A/California/07/2009 (H1N1)—3′-UTR derived from human alpha globin (muag)—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224117) [1069] GC-enriched coding sequence encoding HA of Influenza A/Hong Kong/4801/2014 (H3N2)—3′-UTR derived from human alpha globin (muag)—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224181) [1070] GC-enriched coding sequence encoding HA of Influenza B/Brisbane/60/2008-3′-UTR derived from human alpha globin (muag)—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224236). [1071] GC-enriched coding sequence encoding HA of Influenza A/Vietnam/1203/2004 (H5N1)-3′-UTR derived from human alpha globin (muag)—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224200). [1072] GC-enriched coding sequence encoding HA of Influenza A/Netherlands/602/2009 (H1N1)—3′-UTR derived from human alpha globin (muag)—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224166). [1073] GC-enriched coding sequence encoding HA of Influenza B/Brisbane/60/2008-3′-UTR derived from human alpha globin (muag)—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224236). [1074] GC-enriched coding sequence encoding HA of Influenza B/Phuket/3073/2013-3′-UTR derived from human alpha globin (muag)—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224246).
[1075] Influenza Neuraminidase (NA): [1076] GC-enriched coding sequence encoding NA of Influenza A/California/07/2009 (H1N1)—3′-UTR derived from human alpha globin (muag)—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224318). [1077] GC-enriched coding sequence encoding NA of Influenza A/Hong Kong/4801/2014 (H3N2)—3′-UTR derived from human alpha globin (muag)—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224336). [1078] GC-enriched coding sequence encoding NA of Influenza B/Brisbane/60/2008-3′-UTR derived from human alpha globin (muag)—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224348).
[1079] Rabies: [1080] RABV-G A: GC-enriched coding sequence encoding glycoprotein (RABV-G) of the Pasteur strain (GenBank accession number: AAA47218.1)—3′-UTR derived from human alpha globin (muag)—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224276) [1081] RABV-G B: 5′-TOP-UTR derived from 32L.sup.4 ribosomal protein—GC-enriched coding sequence encoding glycoprotein (RABV-G) of the Pasteur strain (GenBank accession number: AAA47218.1)—3′-UTR derived from albumin gene—a stretch of 64 adenosines—a stretch of 30 cytosines—a histone stem-loop sequence (SEQ ID NO: 224280).
[1082] Ebola: [1083] GP of Ebola virus: GC-enriched coding sequence encoding glycoprotein of ZEBOV Sierra Leone 2014; 5′-TOP-UTR derived from 32L.sup.4 ribosomal protein-3′-UTR derived from albumin gene (SEQ ID NO: 224362).
[1084] The obtained plasmid DNA constructs were transformed and propagated in bacteria (Escherichia coli) using common protocols known in the art. Subsequently, the DNA plasmids are enzymatically linearized using EcoRI and transcribed in vitro using DNA dependent T7 RNA polymerase in vitro run-off transcription in the presence of a nucleotide mixture and CAP analog (m7GpppG) under suitable buffer conditions. The obtained mRNAs were purified using PureMessenger® (CureVac, Tubingen, Germany; WO2008/077592 A1) and were used for further experimentation. NHP were administered with RABV-G A as protamine formulation and RABV-G B as LNP formulation. Compositions comprising more than one mRNA encoding different Influenza proteins/antigens may also be produced according to procedures as disclosed in the PCT application PCT/EP2016/082487.
[1085] Example LNP Formulation
[1086] Lipid nanoparticles, cationic lipids and polymer conjugated lipids (PEG-lipid) were prepared and tested according to the general procedures described in PCT Pub. Nos. WO 2015/199952, WO 2017/004143 and WO 2017/075531, the full disclosures of which are incorporated herein by reference. Lipid nanoparticle (LNP)-formulated mRNA was prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. LNPs were prepared as follows. Cationic lipid, DSPC, cholesterol and PEG-lipid were solubilized in ethanol at a molar ratio of approximately 50:10:38.5:1.5 or 47.5:10:40.8:1.7. LNPs for the Examples included, for example, cationic lipid compound III-3 and the foregoing components. Lipid nanoparticles (LNP) comprising compound III-3 were prepared at a ratio of mRNA to Total Lipid of 0.03-0.04 w/w. Briefly, the mRNA was diluted to 0.05 to 0.2 mg/mL in 10 to 50 mM citrate buffer, pH 4. Syringe pumps were used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol/vol) with total flow rates above 15 ml/min. The ethanol was then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter. Lipid nanoparticle particle diameter size was 60-90 nm as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK). For other cationic lipid compounds mentioned in the present specification, the formulation process is similar.
Example 2: Ppluc Expression after i.m. Application of LNP-Formulated mRNA
[1087] Expression of luciferase (Ppluc) in BALB/c mice was determined 24h and 48h after intramuscular injection (i.m.) into the M. tibialis.
[1088] Therefore, 0.1 μg, 1 μg and 10 μg mRNA coding for Ppluc were LNP-formulated to yield the respective LNP-formulation according to Table I. As a control served unformulated Ppluc mRNA (10 μg and 1 μg). At time point 0 h, four mice per group were transfected with Ppluc mRNA in accordance with the scheme shown in table I.
TABLE-US-00007 TABLE I (Example 2): Transfection scheme mRNA Group Treatment dose [μg] Route (Volume) Mice # A LNP-II-9- 10 i.m. (25 μl) 4 formulated Ppluc mRNA B LNP-II-9- 1 i.m. (25 μl) 4 formulated Ppluc mRNA C LNP-II-9- 0.1 i.m. (25 μl) 4 formulated Ppluc mRNA D LNP-II-10- 10 i.m. (25 μl) 4 formulated Ppluc mRNA E LNP-II-10- 1 i.m. (25 μl) 4 formulated Ppluc mRNA F LNP-II-10- 0.1 i.m. (25 μl) 4 formulated Ppluc mRNA G LNP-III-3- 10 i.m. (25 μl) 4 formulated Ppluc mRNA H LNP-III-3- 1 i.m. (25 μl) 4 formulated Ppluc mRNA I LNP-III-3- 0.1 i.m. (25 μl) 4 formulated Ppluc mRNA J unformulated 10 i.m. (25 μl) 4 Ppluc mRNA K unformulated 1 i.m. (25 μl) 5 Ppluc mRNA
[1089] After 24h and 48h, in vivo bioluminescence imaging of mice was performed with an IVIS Lumina II Imaging System 10 minutes after intraperitoneal Luciferin injection, using an exposure time of 60s. Bioluminescence values were quantified by measuring photon flux (photons/second) in the region of interest. After 48 hours, mice were sacrificed and muscle, lung, liver, spleen, brain, kidney and heart were collected, shock frozen in dry ice and stored at −80° C. for further analysis. Organ samples were lysed for 3 Minutes at full speed in a tissue lyser (Qiagen, Hilden, Germany). Afterwards 800 μl of lysis buffer were added and the resulting solutions were subjected another 6 minutes at full speed in the tissue lyser. After 10 minutes centrifugation at 13500 rpm at 4° C. the supernatants were mixed with luciferin buffer (25 mM glycylglycin, 15 mM MgSO4, 5 mM ATP, 62.5 μM luciferin) and luminescence was detected using a Chameleon plate reader (Hidex).
[1090] Results:
[1091] As can be seen in
[1092] As shown in
Example 3: Immunogenicity after Intramuscular (i.m.) Application of LNP-Formulated mRNA
[1093] LNP formulated HA-mRNA was used for testing the immunogenicity after intramuscular (i.m.) application. Specifically, a GC-enriched H1N1 (Netherlands 2009)-HA mRNA sequence as LNP formulation was applied as described above.
[1094] For vaccination, 8 BALB/c mice were intramuscularly injected into the M. tibialis of both legs (25 μl per leg) according to the vaccination scheme shown in Table II. As apparent, 10 μg mRNA encoding Influenza HA was LNP-formulated (as described above) to yield the respective LNP-formulation for vaccination; unformulated mRNA (10 μg) served as a control.
TABLE-US-00008 TABLE II (Example 3): Vaccination scheme Mice Group Treatment RNA dose Route (Volume) # A LNP-II-9- 10 μg i.m. 25 μl 8 formulated per leg HA mRNA B LNP-II-10- 10 μg i.m. 25 μl 8 formulated per leg HA mRNA C LNP-III-3- 10 μg i.m. 25 μl 8 formulated per leg HA mRNA D unformulated 10 μg i.m. 25 μl 8 HA mRNA per leg E RiLa buffer — i.m. 25 μl 8 per leg
[1095] On day 0, a prime vaccination was administered. Animals vaccinated with buffer served as negative control. On day 21, a blood sample was collected from the retrobulbar sinus and a boost vaccination was administered. After 35 days, the mice were sacrificed and blood and organ samples (spleen) were collected for further analysis. Splenocytes were isolated at day 35 and stimulated with an HA peptide library for T cell analysis. For immunogenicity assays, Hemagglutination inhibition (HI) titers were analyzed in the sera 3 weeks after prime and 2 weeks after boost. Frequencies of activated, HA-specific, multifunctional CD4+ and CD8+ T cells (IFN-γ+/TNF+) were measured by intracellular staining and flow cytometry. ELISA was applied for determining antibody titers.
[1096] Hemagglutination Inhibition Assay:
[1097] Hemagglutination inhibition (HI) assays were used for analyzing functional anti-HA antibody titers. Mouse sera were heat inactivated (56° C., 30 min), incubated with kaolin (Carl Roth, Germany) and pre-adsorbed to chicken red blood cells (CRBC; Lohmann Tierzucht, Germany). 50 μl of 2-fold dilutions of pre-treated sera were incubated for 45 min with 4 hemagglutination units (HAU) of inactivated Influenza A/California/7/2009 (H1N1) virus (NIBSC, UK) and 50 μl 0.5% CRBC was added. HI titers were determined by the reciprocal of the highest dilution of the serum able to inhibit hemagglutination.
[1098] ELISA:
[1099] Detection of an antigen-specific immune response (B-cell immune response) was carried out by detecting influenza specific IgG1 and IgG2a antibodies. Therefore, blood samples were taken from the vaccinated mice 21 days post prime and 14 days post boost and sera were prepared. MaxiSorb plates (Nalgene Nunc International) were coated with the inactivated virus. After blocking with 1×PBS containing 0.05% Tween-20 and 1% BSA the plates were incubated with diluted mouse serum (as indicated). Subsequently a biotin-coupled secondary antibody (anti-mouse-IgG1 and IgG2a, Pharmingen) was added. After washing, the plate was incubated with horseradish peroxidase-streptavidin and subsequently the conversion of the ABTS substrate (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was measured.
[1100] Intracellular Cytokine Staining:
[1101] Induction of antigen-specific T cells was determined 14 days after boost using intracellular cytokine staining (ICS). Splenocytes from vaccinated and control mice were isolated and stimulated with an HA peptide library (PepMix™ Influenza A (HA/California (H1N1)), JPT) and anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in the presence of the GolgiPlug containing protein transport inhibitor Brefeldin A (BD Biosciences). After stimulation, cells were washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: CD8-APC H7 (1:100), CD4-BD Horizon V450 (1:200) (BD Biosciences), Thy1.2-FITC (1:300), TNFα-PE (1:100), IFN-γ-APC (1:100) (eBioscience), and incubated with FcγR-block diluted 1:100. Aqua Dye was used to distinguish live/dead cells (Invitrogen). Cells were acquired using a Canto II flow cytometer (BD Biosciences) and flow cytometry data were analyzed using FlowJo software (Tree Star).
[1102] Results:
[1103] Results Hemagglutination Inhibition (HI) Assays:
[1104] LNP-formulated HA-mRNA led to very high HI titers already after prime vaccination as apparent from
[1105] Results ELISA Assays:
[1106] LNP-formulated HA-mRNA induced significantly higher functional antibody titers, both IgG1 and IgG2a subtypes, when compared to unformulated mRNA already after a single i.m. injection as apparent from
[1107] Results T Cell Assays:
[1108] As apparent from
Example 4: Immunogenicity after Intradermal (i.d.) Application of LNP-Formulated mRNA
[1109] LNP formulated HA-mRNA was used for testing the immunogenicity after intradermal (i.d.) application. Specifically, a LNP-formulated GC-enriched H1N1 (Netherlands 2009)-HA mRNA sequence was applied as described above.
[1110] Therefore, 8 mice per group were each vaccinated intradermally according to the vaccination scheme shown in Table III. As apparent, 10 μg HA-mRNA was LNP-formulated (as described above) to yield the respective LNP-formulation for the vaccination; unformulated HA mRNA (10 μg) served as a control.
TABLE-US-00009 TABLE III (Example 4): Vaccination scheme Mice Group Treatment RNA dose Route (Volume) # A LNP-II-9- 10 μg i.d. (25 μl) 8 formulated HA mRNA B LNP-II-10- 10 μg i.d. (25 μl) 8 formulated HA-mRNA C LNP-III-3- 10 μg i.d. (25 μl) 8 formulated HA-mRNA D unformulated 10 μg i.d. (25 μl) 8 HA-mRNA E RiLa buffer — i.d. (25 μl) 8
[1111] At time point 0 days, a prime vaccination was administered to 8 mice per group. On day 21, a blood sample was collected and a boost vaccination was administered. After 35 days, the mice were sacrificed and blood and organ samples (spleen) were collected for further analysis.
[1112] For immunogenicity assays, the HI titer was measured. ELISA was applied for determining antibody titers. Further, CD4 T cell immune response (IFNγ/TNFα producing CD4 T cells) and CD8 T cell immune response (IFNγ/TNFα producing CD8 T cells and CD107+IFNy producing CD8 T cells) was assessed 14 days after boost vaccination. Induction of antigen-specific T cells was determined using intracellular cytokine staining (ICS). Assays were performed as described above.
[1113] Results:
[1114] Results Hemagglutination Inhibition (HI) Assays:
[1115] LNP-formulated HA-mRNA led to very high HI titers already after prime vaccination compared to unformulated mRNA as apparent from
[1116] Results ELISA Assays:
[1117] LNP-formulated HA-mRNA induced significantly higher functional antibody titers, both IgG1 and IgG2a subtypes, when compared to unformulated mRNA already after a single i.d. injection as apparent from
[1118] Results T Cell Assays:
[1119] As apparent from
Example 5: Immunogenicity after Intramuscular (i.m.) Application of 1 μg LNP-Formulated mRNA
[1120] In the following example, the amount of 1 μg HA-mRNA (H1N1 (Netherlands 2009)-HA mRNA sequence LNP formulation, was used for testing the immunogenicity after intramuscular (i.m.) application as described above. Therefore, 8 BALB/c mice per group were each vaccinated intramuscularly in one leg according to the vaccination scheme shown in Table IV. As apparent, 1 μg HA-mRNA was LNP-formulated (as described above) to yield the respective LNP-formulation for the vaccination; unformulated HA mRNA (10 μg) served as a control.
TABLE-US-00010 TABLE IV (Example 5): Vaccination scheme Mice Group Treatment RNA dose Route (Volume) # A LNP-III-3- 1 μg i.m. (25 μl) 8 formulated HA-mRNA B unformulated 10 μg i.m. (25 μl) 8 HA-mRNA C Rila buffer — i.m. (25 μl) 8
[1121] At time point 0 days, a prime vaccination was administered to 8 mice per group. On day 21, a blood sample was collected and a boost vaccination was administered. After 35 days, the mice were sacrificed and blood and organ samples (spleen) were collected for further analysis.
[1122] For immunogenicity assays, the HI titer was measured. ELISA was applied for determining antibody titers. Further, frozen splenocytes were stimulated with a HA overlapping peptide library and T cell immune responses were assessed by measuring IFNγ production using Elispot.
[1123] Results:
[1124] Results Immunogenicity Assays:
[1125] 1 μg HA-mRNA, LNP-formulated, induced HI titers above the threshold of 1:40 already after prime vaccination as apparent from
[1126] Results ELISA Assays:
[1127] 1 μg HA-mRNA, LNP-formulated, induced significantly higher functional antibody titers, both IgG1 and IgG2a subtypes, when compared to non-formulated mRNA already after a single i.m. injection (see
[1128] Results T Cell Assays:
[1129] 1 μg HA-mRNA, LNP-formulated, led to increased IFNy production compared to non-formulated mRNA. The medium control as expected never generated a response (see
Example 6: HA-mRNA and RABV-G mRNA Vaccination of Monkeys
[1130] The rabies mRNA vaccine encoded the glycoprotein (RABV-G) of the Pasteur strain (GenBank accession number: AAA47218.1). Optimized mRNA constructs (RABV-G A and B) were used, which contain identical ORFs but different UTRs. The protamine-formulated RABV-G mRNA vaccines contained RABV-G mRNA A. The unformulated or LNP-formulated RABV-G mRNA vaccines contained RABV-G mRNA B (as described above) if not otherwise indicated.
[1131] Protamine RNA Formulation For the preparation of protamine complexed mRNA (“RNActive® formulation”), the obtained antigen mRNA constructs were complexed with protamine prior to use in in vivo vaccination experiments. The mRNA complexation consists 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 addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes were stably generated, free mRNA was added, and the final concentration of the vaccine was adjusted with Ringer's lactate solution.
[1132] LNP formulated HA-mRNA and RABV-G mRNA vaccines as prepared in the previous example were used for vaccination. Four nulliparous and nonpregnant cynomolgus monkeys (Macaca fascicularis) were vaccinated by intramuscular injection at days 1 and 29 with LNP-III-3-formulated HA-mRNA or RABV-G mRNA (1 μg or 10 μg, mRNA as described above) or protamine-formulated HA-mRNA or RABV-G mRNA (240 μg) into the biceps femoris muscle (500 μl). As negative control buffer was injected. Serum samples were collected from the femoral vein on days 0, 29 and 50.
[1133] Detection of an antigen-specific immune response was carried out on days 0, 29 and 50.
[1134] Hemagglutination Inhibition Assay:
[1135] Hemagglutination inhibition (HI) assays were used for analyzing functional anti-HA antibody titers. Non-human primate (NHP) sera were incubated with receptor destroying enzyme (RDEII, Denka Seiken, Japan) at 37° C. overnight, inactivated (56° C., 60 min) and incubated with kaolin. 50 μl of 2-fold dilutions of pre-treated sera were incubated for 45 min with 4 hemagglutination units (HAU) of inactivated Influenza A/California/7/2009 (H1N1) virus (NIBSC, UK) and 50 μl 0.5% CRBC was added. HI titers were determined by the reciprocal of the highest dilution of the serum able to inhibit hemagglutination.
[1136] Antibody Analysis:
[1137] HA-specific IgG titers in NHP sera were measured by ELISA using inactivated A/California/7/2009 (H1N1) virus for coating (1 μg/ml) and anti-human total IgG-HRP (ImmunoResearch) as detection antibody. Anti-rabies virus neutralizing titers (VNTs) in serum were analyzed by the Eurovir® Hygiene-Labor GmbH, Germany, using the FAVN test (Fluorescent Antibody Virus Neutralization test) and the Standard Challenge Virus CVS-11 according to WHO protocol.
[1138] Cytokine Analysis:
[1139] Blood samples of NHPs were collected on days 1 and 29 for HA immunization and day 1 for RABV-G immunization from all NHPs prior to administration and 6 and 24 hours after dosing to determine inflammation biomarkers (G-CSF, IFNγ, IL-1β, IL-2, IL 4, IL-5, IL-6, IL-8 and TNF). Plasma was analyzed using the Luminex-based PRCYTOMAG-40K kit (MD MILLIPORE).
[1140] Body Temperature:
[1141] Body temperature of NHPs was determined by rectal measurement right before and at 0.5, 2, 6 and 24 hours after each dose.
[1142] Results:
[1143] As a general result, a single intramuscular administration of a 1 μg mRNA dose, LNP-III-3-formulated, surprisingly induced protective antibody titers in all vaccinated non-human primates in the two indications rabies and influenza.
[1144] Results Antibody Titer Analysis:
[1145] The results from the neutralizing antibody titer analysis are shown in
[1146] Results Rabies Virus Neutralizing Titers (VNTs):
[1147] As shown in
[1148] Results HI-Titer:
[1149] LNP-III-3-formulated HA-mRNA led to very high HI titers already after prime vaccination as apparent from
[1150] Animals which received 1 μg of LNP-III-3-formulated HA-mRNA required a second vaccination to reach the protective titer in 3 of 4 animals, while none of the animals receiving 240 μg protamine-formulated HA-mRNA exhibited detectable HI titers.
[1151] Results Cytokine Analysis:
[1152] Systemic cytokine concentrations after vaccination with LNP-III-3-formulated mRNA stayed below detection level for IL-1β, IL 4, IL-5, IL-6, IFN-γ and TNF or did not increase for IL-2, IL-8 and G-CSF (
[1153] Importantly, lower concentrations of the pro-inflammatory cytokines TNF, IFN-γ and IL-6 were induced by LNP-formulations compared to protamine-formulations, which are generally well tolerated in humans.
[1154] Results body temperature-results and other results:
[1155] Intramuscular application of LNP-III-3-formulated mRNA in non-human primates showed no impact on body temperature or the body weight [data not shown]. Injection sites showed only slight erythema and/or edema in 1 of 4 animals in each group receiving the LNP-III-3-formulated HA-mRNA, which resolved 24 to 96 hours after injection and no injection site reactions after vaccination with the RABV-G-mRNA formulations [data not shown]. In summary, the LNP-III-3-formulated mRNA was well tolerated by NHPs.
Example 7: Immunogenicity after Intramuscular (i.m.) Application of Low Doses LNP-Formulated RABV-G MRNA
[1156] The amount of 0.5 μg LNP-formulated RABV-G-mRNA was used for testing the immunogenicity after intramuscular (i.m.) application. The sequence which was used is shown in SEQ ID NO: 224276. Therefore, BALB/c mice were each vaccinated according to the vaccination scheme shown in Table Va intramuscularly in one leg. Unformulated RABV-G mRNA (40 μg) served as a control.
TABLE-US-00011 TABLE Va (Example 7): Vaccination scheme Route, Immunisation Retrobular Strain sex Mice # Treatment RNA/mouse Volume schedule bleeding BALB/c 10 5 μg LNP-III-3 formulated i.m. d 0, d 21 d 1, d 21, d 35 Female RABV-G mRNA 1 × 25 μl BALB/c 10 1 μg LNP-III-3 formulated i.m. d 0, d 21 d 1, d 21, d 35 Female RABV-G mRNA 1 × 25 μl BALB/c 10 0.5 μg LNP-III-3 formulated i.m. d 0, d 21 d 1, d 21, d 35 Female RABV-G mRNA 1 × 25 μl BALB/c 10 0.1 μg LNP-III-3 formulated i.m. d 0, d 21 d 1, d 21, d 35 Female RABV-G mRNA 1 × 25 μl BALB/c 10 0.05 μg LNP-III-3 formulated i.m. d 0, d 21 d 1, d 21, d 35 Female RABV-G mRNA 1 × 25 μl BALB/c 10 0.01 μg LNP-III-3 formulated i.m. d 0, d 21 d 1, d 21, d 35 Female RABV-G mRNA 1 × 25 μl BALB/c 10 40 μg unformulated RABV-G i.m. d 0, d 21 d 1, d 21, d 35 Female mRNA 1 × 25 μl BALB/c 6 PBS i.m. d 0, d 21 d 1, d 21, d 35 Female 2 × 25 μl BALB/c 6 32 μg RNActive i.d. d 0, d 21 d 1, d 21, d 35 Female (protamine formulation) 2 × 50 μl
[1157] At time point 0 days, a prime vaccination was administered to 8 mice per group and a blood samples was taken one day later. On day 21, a blood sample was collected and a boost vaccination was administered. Prime and boost vaccinations were performed in different legs, i.e. left and right leg, respectively. After 35 days, the mice were sacrificed and blood and organ samples (spleen and liver) were collected for further analysis.
[1158] For immunogenicity assays, the VNT was measured as described before, i.e. anti-rabies virus neutralizing titers (VNTs) in serum were analyzed by the Eurovir® Hygiene-Labor GmbH, Germany, using the FAVN test (Fluorescent Antibody Virus Neutralization test) and the Standard Challenge Virus CVS-11 according to WHO protocol.
[1159] CD4 T cell immune response (IFNγ/TNFα producing CD4 T cells) and CD8 T cell immune response (IFNγ/TNFα producing CD8 T cells and CD107+IFNy producing CD8 T cells) were assessed. Assays were performed as described before. Histology of liver specimens from animals vaccinated with 5 μg LNP or buffer controls was examined. Serum levels of ALT/AST were examined in serum samples taken d1 and d21. AST/ALT and liver were analyzed by mfd Diagnostics GmbH, Germany.
[1160] Results:
[1161] Results rabies virus neutralizing titers (VNTs):
[1162] As shown in
[1163] As VNT-levels of
TABLE-US-00012 TABLE Vb (Example 7): Vaccination scheme Route, Immunisation Retrobular Strain sex Mice # Treatment RNA/mouse Volume schedule bleeding BALB/c 14 0.1 μg LNP-III-3 i.m. d 0, d 21 d 21, d 35 Female formulated RABV-G mRNA 1 × 25 μl BALB/c 14 0.3 μg LNP-III-3 i.m. d 0, d 21 d 21, d 35 Female formulated RABV-G mRNA 1 × 25 μl BALB/c 14 0.9 μg LNP-III-3 i.m. d 0, d 21 d 21, d 35 Female formulated RABV-G mRNA 1 × 25 μl BALB/c 6 PBS i.m. d 0, d 21 d 21, d 35 Female 2 × 25 μl
[1164] As proven in
[1165] Results:
[1166] Results T cell assays:
[1167] T cell responses were observed in mice immunized twice with 5 μg, 1 μg and 0.5 μg as apparent from
[1168] Results Liver damage analysis:
[1169] Histology of RABV-G LNP 5 μg mRNA and buffer administered control animals was performed. No pathological findings observed in any of the tested animals. AST levels were increased on day 1 post vaccination with the highest dose of LNPs (5 μg) but decreased over time and returned to background levels on day 21 (see
Example 8: Vaccination Experiment with a Combination of mRNAs Encoding HA of Four Different Influenza Viruses
[1170] For vaccination 9 mice per group were intramuscularly injected twice with a composition comprising LNP formulated mRNA encoding HA of 4 different influenza virus strains: A/California/7/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2), B/Brisbane/60/2008 (B) and A/Vietnam/1203/2004 (H5N1). Therefore, the four mRNAs were mixed in a ratio of 1:1:1:1 and then formulated as described above. As control, Fluarix Quadrivalent 2015-2016 was injected, a split virus vaccine comprising 4 different inactivated influenza virus strains (A/California/7/2009, A/Switzerland/9715293/2013, B/Phuket/3073/2013, and B/Brisbane/60/2008) indicated for active immunization for the prevention of disease caused by influenza A subtype viruses and type B viruses. As negative control Ringer lactate buffer was injected.
[1171] Detection of an HA-specific immune response (B-cell immune response) was carried out by detecting IgG2a antibodies directed against the particular influenza virus. Therefore, blood samples were taken from the vaccinated mice three weeks after vaccination and sera were prepared. MaxiSorb plates (Nalgene Nunc International) were coated with the particular recombinant HA protein. After blocking with 1×PBS containing 0.05% Tween-20 and 1% BSA the plates were incubated with diluted mice serum (as indicated). Subsequently a biotin-coupled secondary antibody (anti-mouse-IgG2a, Pharmingen) was added. After washing, the plate was incubated with horseradish peroxidase-streptavidin, followed by addition of the Amplex UltraRed Reagent (Invitrogen) and subsequent quantification of the fluorescent product.
[1172] Results:
[1173] The results shown in
[1174] These data proof that mRNA encoded antigens e.g. of different influenza viruses can be combined in one composition/vaccine.
[1175] In contrast to Fluarix, the LNP-III-3 formulated mRNA multivalent vaccine induced IgG2a antibody responses in naïve animals against all subtypes and the low dose of mRNA was immunogenic in mice surprisingly after single intramuscular injection.
Example 9: Vaccination Experiment with a Combination of mRNAs Encoding HA and NA of Different Influenza Viruses
[1176] For vaccination 8 mice per group are intramuscularly injected with a composition comprising LNP formulated mRNA encoding HA of 4 different influenza virus strains: A/California/7/2009 (H1N1) and/or A/Netherlands/602/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2), B/Brisbane/60/2008 and A/Vietnam/1203/2004 (H5N1) and NA of 3 different influenza virus strains: A/California/7/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2), and B/Brisbane/60/2008.
[1177] In another vaccination experiment, 8 mice per group are intramuscularly injected with a composition comprising mRNA sequences encoding HA of InfluenzaA/California/7/2009 (H1N1) and/or A/Netherlands/602/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2), B/Brisbane/60/2008 and B/Phuket/3073/2013 and NA of 3 different influenza virus strains: A/California/7/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2), and B/Brisbane/60/2008.
[1178] As control, Influvac® Tetravalent 2016-2017 is injected, a split virus vaccine comprising 4 different inactivated influenza virus strains (A/California/7/2009, A/Hong Kong/4801/2014, B/Phuket/3073/2013, and B/Brisbane/60/2008) indicated for active immunization for the prevention of disease caused by influenza A subtype viruses and type B viruses. As negative control Ringer lactate buffer is injected.
[1179] Detection of an HA-specific immune response (B-cell immune response) is carried out by detecting IgG2a antibodies directed against the particular influenza virus as described above.
[1180] NA-specific immune responses (B-cell immune response) directed against the particular influenza virus are determined using NA inhibition assay (NAI).
Example 10: Synthesis of Compound I-3
[1181] A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid (0.33 g) and 4-aminobutan-1-ol (0.23 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for two hours. The solution was washed with aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed down a silica gel column using a methanol/methylene chloride (0-8/100-92%) gradient, yielding compound 3 as a colorless oil (0.4 g).
Example 11: Synthesis of a Representative PEG Lipid
[1182] Pegylated lipid IVa (“PEG-DMA”) was prepared wherein n approximates the center of the range of ethylene oxide repeating units in the pegylated lipid.
[1183] Synthesis of IVa-1 and IVa-2:
[1184] To a solution of myristic acid (6 g, 26 mmol) in toluene (50 mL) was added oxalyl chloride (39 mmol, 1.5 equivalents, 5 g) at RT. Accordingly, 39 mmol of oxalyl chloride is 1.5 molar equivalents relative to the starting material myristic acid (26 mmoles×1.5=39 mmoles). After the resulting mixture was heated at 70° C. for 2h, the mixture was concentrated. The residue was taken up in toluene and concentrated again. The residual oil was added via a syringe to a concentrated ammonia solution (20 mL) at 10° C. The reaction mixture was filtered and washed with water. The white solid was dried in vacuo. The desired product was obtained as a white solid (3.47 g, 15 mmol, 58.7%).
[1185] Synthesis of IVa-3:
[1186] To suspension of 20-2 (3.47 g, 15 mmol) in THF (70 mL) was added in portions of lithium aluminium hydride (1.14 g, 30 mmol) at RT during 30 min period of time. Then the mixture was heated to reflux gently (oil bath at 65° C.) overnight. The mixture was cooled to 5° C. and sodium sulphate 9 hydrate was added. The mixture was stirred for 2h, filtered through a layer of celite, washed with 15% of MeOH in DCM (200 mL). The filtrate and washings were combined and concentrated. The residual solid was dried in vacuo. The desired product was obtained as a white solid (2.86g 13.4 mmol, 89.5%).
[1187] Synthesis of IVa-4: To a solution of myristic acid (3.86 g, 16.9 mmol) in benzene (40 mL) and DMF (1 drop) was added oxalyl chloride (25.35 mmol, 1.5 equivalents, 3.22 g) at RT. Accordingly, 25.35 mmol of oxalyl chloride is 1.5 molar equivalents relative to the starting material myristic acid (16.9 mmol×1.5=25.35 mmol). The mixture was stirred at RT for 1.5h. Heated at 60° C. for 30 min. The mixture was concentrated. The residue was taken up in toluene and concentrated again. The residual oil (light yellow) was taken in 20 mL of benzene and added via syringe to a solution of 20-3 (2.86g 13.4 mmol) and triethylamine (3.53 mL, 1.5 equivalents) in benzene (40 mL) at 10° C. Accordingly, 3.53 mL of triethylamine is 1.5 molar equivalents with respect to the 13.4 mmol of 20-3 i.e. a 50% molar excess of this reagent compared to the starting material for this step. After addition, the resulting mixture was stirred at RT overnight. The reaction mixture was diluted with water and was adjusted to pH 6-7 with 20% H2504. The mixture was filtered and washed with water. A pale solid was obtained. The crude product was recrystallized from methanol. This gave the desired product as an off-white solid (5.65 g, 13 mmol, 100%).
[1188] Synthesis of IVa-5:
[1189] To suspension of 20-4 (5.65 g, 13 mmol) in THF (60 mL) was added in portions lithium aluminium hydride (0.99 g, 26 mmol) at RT during 30 min period of time. Then the mixture was heated to reflux gently overnight. The mixture was cooled to 0° C. and sodium sulphate 9 hydrate. The mixture was stirred for 2h, then filtered through a pad of celite and silica gel and washed with ether first. The filtrate turned cloudy and precipitation formed. Filtration gave a white solid. The solid was recrystallized from MeOH and a colorless crystalline solid (2.43 g).
[1190] The pad of celite and silica gel was then washed 5% of MeOH in DCM (400 mL) and then 10% of MeOH in DCM with 1% of triethylamine (300 mL). The fractions containing the desired product were combined and concentrated. A white solid was obtained. The solid was recrystalized from MeOH and a colorless crystalline solid (0.79 g). The above two solids (2.43g and 0.79 g) were combined and dried in vacuo (3.20 g, 60%).
[1191] 1HNMR (CDCl3 at 7.27 ppm) δ: 2.58 (t-like, 7.2 Hz, 4H), 1.52-1.44 (m, 4H), 1.33-1.24 (m, 44H), 0.89 (t-like, 6.6 Hz, 6H), 2.1-1.3 (very broad, 1H).
[1192] Synthesis of Iva:
[1193] To a solution of 20-5 (7 mmol, 2.87 g) and triethylamine (30 mmol, 4.18 mL) in DCM (100 mL) was added a solution of mPEG-NHS (from NOF, 5.0 mmol, 9.97 g, PEG MW approx. 2,000, n=about 45) in DCM (120 mL,).
[1194] After 24h the reaction solution was washed with water (300 mL). The aqueous phase was extracted twice with DCM (100 mL×2). DCM extracts were combined, washed with brine (100 mL). The organic phase was dried over sodium sulfate, filtered, concentrated partially. The concentrated solution (ca 300 mL) was cooled at approximatelyl5 C. Filtration gave a white solid (1.030 g, the unreacted starting amine). To the filtration was added Et3N (1.6 mmol, 0.222 mL, 4 equivalents) and acetic anhydride (1.6 mmol, 164 mg). The mixture was stirred at RT for 3h and then concentrated to a solid. The residual solid was purified by column chromatography on silica gel (0-8% methanol in DCM). This gave the desired product as a white solid (9.211 g). 1HNMR (CDCl3 at 7.27 ppm) δ: 4.19 (s, 2H), 3.83-3.45 (m, 180200H), 3.38 (s, 3H), 3.28 (t-like, 7.6 Hz, 2H, CH2N), 3.18 (t-like, 7.8 Hz, 2H, CH2N), 1.89 (s, 6.6 H, water), 1.58-1.48 (m, 4H), 1.36-1.21 (m, 48-50H), 0.88 (t-like, 6.6 Hz, 6H).
Example 12: Vaccination Experiment with a Combination of LNP-III-3 Formulated mRNAs Encoding HA of Different Influenza Viruses
[1195] Female BALB/c mice were immunized intramuscularly (i.m.) with LNP-III-3 formulated mRNA vaccine compositions with doses, application routes and vaccination schedules as indicated in Table A (mRNA sequences according to Example 1). As a negative control, one group of mice was injected with buffer (ringer lactate, Rila). As a positive control, one group of mice was vaccinated with an approved Influenza vaccine (Influsplit® tetra 2016/2017; A/California/07/2009, A/Hong Kong/4801/2014, B/Brisbane/60/2008, B/Phuket/3073/2013). All animals were injected with the respective composition on day 0 and day 21. Blood samples were collected on day 21, 35, and 49 for the determination of binding antibody titers (using ELISA) and blocking antibody titers (using a HI assay). T cell responses were analyzed by intracellular cytokine staining (ICS) using splenocytes isolated on day 49. Detailed descriptions of the performed experiments are provided below.
TABLE-US-00013 TABLE A Immunization regimen (Example 12) No. of Treatment groups (control Dose/ Group mice and mRNA compositions) formulation Treatment A 6 Rila buffer — i.m., 2 × 25 μl B 6 Influsplit ® 1/10 human dose i.m., 2 × 25 μl C 9 H1N1 A/Netherlands/602/2009 1 μg i.m., H3N2 A/Hong Kong/4801/2014 (0.25 μg each) 1 × 25 μl HA B/Brisbane/60/2008 LNP-III-3 HA B/Phuket/3073/2013 formulated D 9 H1N1 A/Netherlands/602/2009 4 μg i.m., H3N2 A/Hong Kong/4801/2014 (1 μg each) 1 × 25 μl HA B/Brisbane/60/2008 LNP-III-3 HA B/Phuket/3073/2013 formulated E 9 H1N1 A/California/07/2009 1 μg i.m., H3N2 A/Hong Kong/4801/2014 (0.25 μg each) 1 × 25 μl HA B/Brisbane/60/2008 LNP-III-3 HA B/Phuket/3073/2013 formulated F 9 H1N1 A/California/07/2009 4 μg i.m., H3N2 A/Hong Kong/4801/2014 (1 μg each) 1 × 25 μl HA B/Brisbane/60/2008 LNP-III-3 HA B/Phuket/3073/2013 formulated
[1196] 12.1. Determination of anti HA protein specific IgG1 and IgG2a antibodies by ELISA:
[1197] ELISA assay was performed essentially as commonly known in the art, or as described above. ELISA was performed for each antigen comprised in the mRNA vaccine composition (as indicated in Table A). Results are shown in
[1198] 12.2. Hemagglutination inhibition assay (HI):
[1199] In a 96-well plate, the obtained sera were mixed with HA H1N1 antigen (A/California/07/2009 (H1N1); NIBSC) or HA H3N2 antigen and red blood cells (4% erythrocytes; Lohmann Tierzucht). In the presence of HA neutralizing antibodies, an inhibition of hemagglutination of erythrocytes can be observed. The lowest level of titered serum that resulted in a visible inhibition of hemagglutination was the assay result. The results are shown in
[1200] 12.3. Detection of T-cell responses:
[1201] Splenocytes from vaccinated mice were isolated according to a standard protocol known in the art. Briefly, isolated spleens were grinded through a cell strainer and washed in PBS/1% FBS followed by red blood cell lysis. After an extensive washing step with PBS/1% FBS splenocytes were seeded into 96-well plates (2×10.sup.6 cells per well). The cells were stimulated with a pool of overlapping 15mer peptides of H1N1 (A/California/07/2009) for determining CD8+ T-cell responses or they were stimulated with recombinant HA protein for determining CD4+ T-cell responses. After stimulation, cells were washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fcγ-block diluted 1:100. Aqua Dye was used to distinguish live/dead cells (Invitrogen). Cells were acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data was analyzed using FlowJo software package (Tree Star, Inc.). Results for CD4+ T-cells are shown in
[1202] Results:
[1203] The data shows that IgG1 and IgG2a antibodies could be detected after vaccination with the LNP formulated mRNA combination vaccines. Notably, for all mRNA encoded antigens comprised in the respective combination, specific IgG1 and IgG2a antibodies could be detected demonstrating that all mRNAs comprised in the respective compositions are translated into protein and trigger a humoral immune response in mice as shown in
[1204] Functional neutralizing antibodies were demonstrated for H1N1 (A/California/7/2009) and H3N2 (A/HongKong/4801/2014) (see
[1205]
[1206]
[1207] Overall, the data demonstrates that LNP formulated mRNA based combination vaccines for HA antigens derived from different influenza viruses (A types and B types) induce strong and durable humoral immune responses and T-cell mediated cellular immune responses.
Example 13: Vaccination Experiment with a Combination of LNP-III-3 Formulated mRNAs Encoding HA of Different Influenza Viruses
[1208] Female BALB/c mice were immunized intramuscularly (i.m.) with LNP-III-3 formulated mRNA vaccine compositions with doses, application routes and vaccination schedules as indicated in Table B (mRNA Sequences according to Example 1). As a negative control, one group of mice was injected with buffer (ringer lactate, Rila).). As a positive control, one group of mice was vaccinated with an approved Influenza vaccine (Fluarix®; A/California/07/2009, A/Switzerland/9715293/2013, B/Brisbane/60/2008, B/Phuket/3073/2013). All animals were vaccinated on day 0 and day 21. Blood samples were collected on day 21, 35, and 49 for the determination of binding antibody titers (using ELISA), blocking antibody titers (using a HI assay) and the determination of virus neutralizing titers (VNTs). T cell responses were analyzed by intracellular cytokine staining (ICS) using splenocytes isolated on day 49. Detailed descriptions of the performed experiments are provided below.
TABLE-US-00014 TABLE B Immunization regimen (Example 13) No. of Treatment groups Dose/ Group mice (control/mRNA compositions) formulation Treatment A 6 Rila buffer — i.m., 2 × 25 μl B 6 Fluarix ® 1/10 human dose i.m., 2 × 25 μl C 9 H1N1 A/Netherlands/602/2009 0.25 μg i.m., H3N2 A/Hong Kong/4801/2014 (0.06 μg each) 1 × 25 μl HA B/Brisbane/60/2008 LNP-III-3 H5N1 A/Vietnam/1203/2004 formulated D 9 H1N1 A/Netherlands/602/2009 1 μg i.m., H3N2 A/Hong Kong/4801/2014 (0.25 μg each) 1 × 25 μl HA B/Brisbane/60/2008 LNP-III-3 H5N1 A/Vietnam/1203/2004 formulated E 9 H1N1 A/Netherlands/602/2009 4 μg i.m., H3N2 A/Hong Kong/4801/2014 (1 μg each) 1 × 25 μl HA B/Brisbane/60/2008 LNP-III-3 H5N1 A/Vietnam/1203/2004 formulated
[1209] 13.1. Determination of anti HA protein specific IgG1 and IgG2a antibodies by ELISA:
[1210] ELISA assay was performed essentially as commonly known in the art, or as described above. ELISA was performed for each antigen comprised in the mRNA vaccine composition (as indicated in Table B). Results are shown in
[1211] 13.2. Hemagglutination inhibition assay (HI) and virus neutralizing assay:
[1212] HI-assay was performed as described above. The results are shown in
[1213] 13.3. Detection of T-cell responses:
[1214] Splenocytes from vaccinated mice were isolated according to a standard protocol known in the art. ICS experiment was performed essentially as described in Example 12. Results for CD4+ T-cells are shown in
[1215] Results:
[1216] The data shows that IgG1 and IgG2a antibodies could be detected after vaccination with the LNP formulated mRNA combination vaccines. Notably, for all mRNA encoded antigens comprised in the respective combination, specific IgG1 and IgG2a antibodies could be detected demonstrating that all mRNAs comprised in the respective compositions are translated into protein and trigger a humoral immune response in mice as shown in
[1217] Functional neutralizing antibodies were demonstrated for H1N1 (A/California/7/2009) and H3N2 (A/HongKong/4801/2014) (see
[1218]
[1219]
[1220] Overall, the data demonstrates that LNP formulated mRNA based combination vaccines for HA antigens derived from different influenza viruses (A types and B types) induce strong and durable humoral immune responses and T-cell mediated cellular immune responses.
Example 14: Vaccination Experiment with LNP-III-3 Formulated mRNA Encoding Neuraminidase NA1 of Influenza Virus
[1221] Female BALB/c mice were immunized intramuscularly (i.m.) with LNP-III-3 formulated mRNA vaccine compositions with doses, application routes and vaccination schedules as indicated in Table C (mRNA Sequences according to Example 1). As a negative control, one group of mice was injected with buffer (ringer lactate, Rila). As positive control, one group of mice was injected i.m. with Influsplit Tetra® 2016/2017 (A/California/7/2009 (H1N1); A/Hong Kong/4801/2014 (H3N2); B/Brisbane/60/2008; B/Phuket/3073/2013). All animals were vaccinated on day 0 and day 21. Blood samples were collected on day 21, 35, and 49 for determination of immune responses. T cell responses were analyzed by intracellular cytokine staining (ICS) using splenocytes isolated on day 49.
TABLE-US-00015 TABLE C Immunization regimen (Example 14) No. of Treatment groups Dose/ Route, Setup mice (control/mRNA compositions) formulation Volume A 6 Rila — i.m., 1 × 25 μl B 6 Influsplit Tetra ® 2016/2017 1/10 of human dose i.m., 2 × 25 μl C 6 NA1 A/California/07/2009 1 μg; i.m., LNP-III-3 1 × 25 μl formulated D 6 NA1 A/California/07/2009 2.5 μg; i.m., LNP-III-3 1 × 25 μl formulated
[1222] 14.1. Determination of immune responses for N1 NA (A/California/7/2009):
[1223] Functional NA-specific antibodies were analyzed using an enzyme-linked lectin assay (ELLA), essentially performed as previously described in the art. ELLA was performed in 96 well plates coated with a large glycoprotein substrate fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure functional NA inhibiting (NI) antibody titers, serial dilutions of sera were incubated on fetuin-coated plates with A/California/7/2009(H1N1) virus (pre-treated with Triton-X-100). The reciprocal of the highest serum dilution that results in ≥50% inhibition of NA activity is designated as the NI antibody titer. The result is shown in
[1224] To determine T-cell responses, an ICS experiment was performed, essentially as outlined above. Cells were stimulated with NA specific peptide mixture and CD8+ T-cell responses and CD4+ T-cell responses were determined. The results are shown in
[1225] Results:
[1226] As shown in
[1227] As shown in
Example 15: Vaccination Experiment with LNP-III-3 Formulated mRNAs Encoding Rabies Virus Antigen in Mice and Evaluation of Pro-Inflammatory Environment and Injection of LNP-III-3 Formulated Fluorescently Labeled mRNA (F*-mRNA) in Mice and Analysis of Composition and Activation Status of Immune Cells in Draining Lymph-Nodes (dLNs)
[1228] F*-mRNA corresponds to a fluorescently labeled PpLuc mRNA (60%-UTP-40%-5-Aminoallyl-UTP) which cannot be translated into a protein due to the labeling (SEQ ID NO: 224286).
[1229] The activation of the innate immune system is required to mount efficacious adaptive immune responses after vaccination. Therefore, the inventive LNP-III-3-formulated mRNA according to the invention was tested for their potency to (transiently) induce pro-inflammatory cytokines and chemokines after i.m. administration of LNP-formulated RABV-G mRNA in mice.
[1230] BALB/c mice (n=6/group) were vaccinated i.m. with 10 μg non-formulated (mRNA) or LNP-formulated RABV-G-mRNA (mRNA/LNP), or with buffer control. Muscle tissues, dLNs and serum samples were isolated 4h, 14h, 24h and 96h after i.m. application and cytokine or chemokine content was measured in protein lysates and sera by Cytometric Bead Array (CBA). The results are shown in
[1231] To elucidate whether the pro-inflammatory environment translates into activation and changes in the composition of immune cells, the number and activation status of leukocytes in the dLNs was analyzed. To ensure that any observed effect was independent of the mRNA-encoded protein, a fluorescently labeled mRNA that cannot be translated (F*-mRNA) was used. BALB/c mice (n=3/group) were injected i.m. in both legs with 10 μg non-formulated (F*mRNA) or LNP-formulated F*mRNA (F*mRNA/LNP), or with buffer. Right and left dLNs were isolated 4h, 24h and 48h after i.m. application and analyzed separately by flow cytometry. Numbers of each cell population (50A) and frequency of activated immune cells (50B) in the dLNs are shown in
[1232] Results:
[1233] As shown in
[1234] As shown in
[1235] As shown in
[1236] Taken together, these results suggest that i.m. injection of LNP-formulated mRNA vaccines induces a broad but transient local immunostimulatory milieu, which is relevant for the induction of strong adaptive immune responses.
Example 16: Vaccination Experiment with LNP-III-3 Formulated mRNAs Encoding Rabies Virus Antigen in Non-Human Primates
[1237] LNP-III-3 formulated RABV-G mRNA vaccines as prepared in the previous example were used for vaccination.
[1238] Studies with cynomolgus monkeys (Macaca fascicularis) were conducted at Envigo CRS, S.A.U., Santa Perpetua de Mogoda, Spain. Animals were of Vietnamese origin, bred in captivity, nulliparous and not pregnant. Animals had at treatment start an age of 2.5 to 3.5 years and a body weight of 2.2-3.3 kg. NHPs were vaccinated i.m. at days 0 and 28 into the biceps femoris muscle with a single dose of 500 μl. Vaccination with the licensed human rabies vaccine Rabipur® (Novartis) was performed i.m. in NHPs with the full human dose according to the pre-exposure prophylaxis schedule on days 0, 7, and 28 or on a reduced schedule on days 0 and 28. VNTs of vaccinated monkeys were analyzed as described above. T-cell responses (CD 4+ and CD8+) were analyzed as described above. The results are shown in
[1239] Results:
[1240]
[1241]
[1242]
[1243]
[1244]
Example 17: Vaccination Experiment with LNP-III-3 Formulated mRNAs Encoding Influenza H1N1 or Influenza H3N2 HA Antigens in Non-Human Primates
[1245] LNP-III-3 formulated HA-mRNA Influenza A/California/7/2009 (H1N1) or HA-mRNA Influenza A/Hong Kong/4801/2014 (H3N2) vaccine as described in the previous example were used for vaccination.
[1246] Studies with cynomolgus monkeys (Macaca fascicularis) were conducted at Envigo CRS, S.A.U., Santa Perpetua de Mogoda, Spain. Animals were of Vietnamese origin, bred in captivity, nulliparous and not pregnant. Animals had at treatment start an age of 2.5 to 3.5 years and a body weight of 2.2-3.3 kg. NHPs were vaccinated with 10 μg of either the H1N1-HA or the H3N2-HA vaccine and measured functional antibodies against the respective viruses by hemagglutination inhibition (HI) assays. As control, a group of animals was vaccinated with a full human dose of Fluad®.
[1247] Naïve NHPs were vaccinated i.m. with 10 μg of either the H1N1-HA or the H3N2-HA vaccine at days 0 and 28 into the biceps femoris muscle with a single dose of 500 μl. Functional antibodies against the respective viruses were measured by hemagglutination inhibition (HI) assays. The results are shown in
[1248] Results:
[1249]
[1250]
[1251]
[1252]
Example 18: Vaccination Experiment with a Combination of mRNAs Encoding Different Influenza Antigens in Non-Human Primates
[1253] Non-human primates (NHPs) are immunized (6 animals per group) with LNP-III-3 formulated mRNA vaccines with doses, application routes and vaccination schedules as indicated in Table D (mRNA Sequences preferably according to Example 1). As vaccines, an mRNA composition comprising four HA antigens is used (“tetravalent HA”) or an mRNA composition comprising seven HA+NA antigens (four HA, three NA; heptavalent or “septavalent HA+NA”) is used. All animals are vaccinated on day 0 and day 28. Blood samples are collected on day 0, 14, 28, 56, 77, and 84 for determination of antibody responses. T cell responses are analyzed by intracellular cytokine staining (ICS) using isolated splenocytes. Analysis of immune responses performed essentially as described above (ELLA, HI assay, ELISA, ICS, VNTs).
TABLE-US-00016 TABLE D Immunization regimen Dose/ Treatment formulation Route, Volume HA A/California/7/2009 H1N1 40 μg* i.m., 500 μl HA A/Hong Kong/4801/2014 H3N2 LNP-III-3 HA B/Brisbane/60/2008 HA B/Phuket/3073/2013 “Tetravalent HA” HA A/California/7/2009 H1N1 200 μg* i.m., 500 μl HA A/Hong Kong/4801/2014 H3N2 LNP-III-3 HA B/Brisbane/60/2008 HA B/Phuket/3073/2013 “Tetravalent HA” NA1 A/California/07/2009 70 μg* i.m., 500 μl NA2 A/Hong Kong/4801/2014 LNP-III-3 NA B/Brisbane/60/2008) HA A/California/7/2009 H1N1 HA A/Hong Kong/4801/2014 H3N2 HA B/Brisbane/60/2008 HA B/Phuket/3073/2013 “Septavalent HA + NA” NA1 A/California/07/2009 350 μg* i.m., 500 μl NA2 A/Hong Kong/4801/2014 LNP-III-3 NA B/Brisbane/60/2008) HA A/California/7/2009 H1N1 HA A/Hong Kong/4801/2014 H3N2 HA B/Brisbane/60/2008 HA B/Phuket/3073/2013 “Septavalent HA + NA” Licensed vaccines 1 human dose i.m., 500 μl *Each mRNA represented equally in the composition, i.e. 4 × 10 μg, 4 × 50 μg, 7 × 10 μg, or 7 × 50 μg
Example 19: Clinical Development of an LNP-III-3 Formulated Influenza mRNA Vaccine
[1254] To demonstrate safety and efficiency of the Influenza mRNA vaccine composition, a randomized, double blind, placebo-controlled clinical trial (phase I) is initiated.
[1255] For clinical development, GMP-grade RNA is produced using an established GMP process, implementing various quality controls on DNA level and RNA level as described in detail in WO 2016/180430 A1.
[1256] In the clinical trial, human volunteers (adult subjects, 18-45 years of age) are intramuscularly (i.m.) injected for at least two times with an mRNA composition comprising one mRNA coding for one influenza antigen as specified herein (“monovalent”, H3N2 A/Hong Kong/4801/2014), or with an mRNA composition comprising four HA influenza antigens as specified herein (“tetravalent HA”), or with an mRNA composition comprising four HA and three NA influenza antigens as specified herein(“septavalent HA+NA”) or with an mRNA composition comprising multiple HA and multiple NA influenza antigens as specified herein (“multivalent HA+NA”). In addition, a group of elderly volunteers is treated (elderly adults >65 years of age). The design of the studies is indicated in Tables E-H below.
TABLE-US-00017 TABLE E Clinical design of a tetravalent HA influenza study Clinical No. Total dose human mRNA per Formulation/ volume of adult Group Treatment dose (μg) Route (ml) subjects 1 Control 0 — 0.5 30 (saline) 2 tetravalent 20* LNP-III-3 0.5 30 HA (i.m.) 3 tetravalent 40* LNP-III-3 0.5 30 HA (i.m.) 4 tetravalent 80* LNP-III-3 0.5 30 HA (i.m.) 5 Licensed — i.m. 0.5 30 vaccine control 6 mRNA 40 or LNP-III-3 0.5 30 elderly vaccine 80* (i.m.) *each mRNA represented equally in the composition
TABLE-US-00018 TABLE F Clinical design of a monovalent influenza study (H3N2) Clinical No. Total dose human mRNA per Formulation/ volume of adult Group Treatment dose (μg) Route (mL) subjects 1 Control 0* i.m. 0.5 30 (saline) 2 monovalent 20* LNP-III-3 0.5 30 HA (i.m.) 3 monovalent 40* LNP-III-3 0.5 30 HA (i.m.) 4 monovalent 80* LNP-III-3 0.5 30 HA (i.m.) 5 Licensed — i.m. 0.5 30 vaccine control 6 monovalent 40 or LNP-III-3 0.5 30 elderly HA 80* (i.m.) *each mRNA represented equally in the composition
TABLE-US-00019 TABLE G Clinical design of a heptavalent/septavalent HA + NA influenza study Clinical No. Total dose human mRNA per Formulation/ volume of adult Group Treatment dose (μg) Route (ml) subjects 1 Control 0 i.m. 0.5 30 (saline) 2 septavalent 20* LNP-III-3 0.5 30 HA + NA (i.m.) 3 septavalent 40* LNP-III-3 0.5 30 HA + NA (i.m.) 4 septavalent 80* LNP-III-3 0.5 30 HA + NA (i.m.) 5 Licensed — i.m. 0.5 30 vaccine control 6 septavalent 40 or LNP-III-3 0.5 30 elderly HA + NA 80* (i.m.) *each mRNA represented equally in the composition
TABLE-US-00020 TABLE H Clinical design of a multivalent HA + NA influenza study Clinical No. Total dose human mRNA per Formulation/ volume of adult Group Treatment dose (μg) Route (ml) subjects 1 Control 0 i.m. 0.5 30 (saline) 2 Multivalent 20* LNP-III-3 0.5 30 HA + NA (i.m.) 3 Multivalent 40* LNP-III-3 0.5 30 HA + NA (i.m.) 4 Multivalent 80* LNP-III-3 0.5 30 HA + NA (i.m.) 5 Licensed — i.m. 0.5 30 vaccine control 6 Multivalent 40 or LNP-III-3 0.5 30 elderly HA + NA 80* (i.m.) *each mRNA represented equally in the composition
[1257] In order to assess the safety profile of the Influenza vaccine compositions according to the invention, subjects are monitored after administration (vital signs, vaccination site tolerability assessments, hematologic analysis).
[1258] The efficacy of the immunization is analysed by determination of HI-titers and ELLA assay. Blood samples are collected on day 0 as baseline and after completed vaccination. Sera are analyzed for functional antibodies (HI assay, ELLA, VNTs (FAVN test)). In addition, a RFFIT assay is performed to analyze the presence of early VNTs using the rapid fluorescent foci inhibition test using the cell culture-adapted challenge virus strain CVS 11 as recommended by the World Organization for Animal Health. In brief, heat-inactivated sera are tested in serial two-fold dilutions for their potential to neutralize a 100 tissue culture infectious dose 50% of CVS. Sera dilutions are incubated with virus for about 70 min at 37° C. (in a water-jacket incubator with 5% CO2). 30,000 BHK-21 cells are added per well. Infected cell cultures are incubated for 22 hours at 37° C. and 5% CO2. Cells are fixed using 80% acetone/20% PBS at −20° C. for 10 min and stained using FITC-conjugated anti-rabies globulin. Plates are washed twice using PBS and excess of PBS is removed. Cell cultures are scored positive or negative for the presence of rabies virus detected by FITC-positive signals. Negative scored cells in sera treated wells represent neutralization of rabies virus. Each RFFIT test includes WHO or OIE standard serum (positive reference serum), which serves as reference for standardisation of the assay. Neutralization activity of test sera is calculated with reference to the standard serum provided by the WHO and displayed as International Units/ml (IU/ml).
[1259] Furthermore, a subset of healthy subjects is challenged with live Influenza virus or placebo by oral administration. Subjects are followed post-challenge for symptoms of Influenza associated illness, infection and immune responses.
Example 20: Stability of LNPs (LNP-III-3) Stored at 5° C. for 3 Months
[1260] To compare immunogenicity and reactogenicity of LNP-III-3 formulated RABV-G mRNA stored at 5° C. for 3 months, immunogenicity was assessed by determining humoral responses, including functional antibodies and cellular immune responses two weeks post boost vaccination (see Table J).
[1261] RABV-G-specific antibody titers were determined by Virus Neutralization Assay as described above.
TABLE-US-00021 TABLE J Immunization protocol (of Example 20): Route, Immunisation Retrobular Strain sex Mice # Treatment RNA/mouse Volume schedule bleeding BALB/c 8 0.9 μg LNP-III-3 formulated i.m. d 0, d 21 d 0, d 21, d 35 Female RABV-G mRNA (LNP-batch 1 × 25 μl freshly prepared) BALB/c 8 0.9 μg LNP-III-3 formulated i.m. d 0, d 21 d 0, d 21, d 35 Female RABV-G mRNA (LNP-batch 1 × 25 μl stored at 5° C. for three month) BALB/c 8 0.3 μg LNP-III-3 formulated i.m. d 0, d 21 d 0, d 21, d 35 Female RABV-G mRNA (LNP-batch 1 × 25 μl freshly prepared) BALB/c 8 0.9 μg LNP-III-3 formulated i.m. d 0, d 21 d 0, d 21, d 35 Female RABV-G mRNA (LNP-batch 1 × 25 μl stored at 5° C. for three month) BALB/c 6 PBS i.m. d 0, d 21 d 0, d 21, d 35 Female 2 × 25 μl
[1262] As apparent from
Example 21: Toxicity Analysis of LNPs (LNP-III-3)
[1263] The aim of this example was to evaluate the toxicity of the inventive LNPs (LNP-III-3). To this end, several in vivo toxicity studies were carried out in different animal models (e.g. mice, rats, or rabbits) with different mRNA doses (f.e. 1 μg, l0 μg, 40 μg, 100 μg, or 200 μg). The results showed that the inventive LNPs showed no significant toxicity in vivo, evidenced by analysis of local reactions, pain, food consumption, body weight, organ weight, clinical chemistry (i.e. no adverse test substance related changes observed) and hematology (i.e. no adverse test substance related changes observed). Only minor local reactions like erythemas and edemas, i.e. normal reactions to vaccines which usually occur within 1-3 days, were found in a minority of the animals vaccinated with e.g. 10 μg and 100 μg.
Example 22: Vaccination Experiment with a Combination of LNP-III-3 Formulated mRNAs Encoding HA of Different Influenza Viruses in Ferrets
[1264] Ferrets (Mustela putorius furσ, 6-12 months old) were immunized intramuscularly (i.m.) with LNP-III-3 formulated mRNA vaccine compositions comprising mRNA constructs encoding H1N1 A/California/07/2009, H3N2 A/Hong Kong/4801/2014, HA B/Brisbane/60/2008, and HA B/Phuket/3073/2013, herein referred to as “tetravalent mRNA vaccine” (see Table K). Respective mRNA sequences according to Example 1. As a positive control, one group of ferrets was vaccinated with an approved Influenza vaccine (Fluad® tetra 2016/2017). All animals were injected with the respective composition on day 0 and day 21. Blood samples were collected on day 0, 21, 35, and 49 for the determination of blocking antibody titers for each encoded antigen (using a HI assay as described above and MN assay). The results are shown in
TABLE-US-00022 TABLE K Immunization regimen (Example 22) No. of Group ferrets Treatment groups Dose Treatment A 3 tetravalent 160 μg i.m., mRNA vaccine (40 μg each mRNA) 1 × 250 μl B 3 tetravalent 40 μg i.m., mRNA vaccine (10 μg each mRNA) 1 × 250 μl C 3 tetravalent 10 μg i.m., mRNA vaccine (2.5 μg each mRNA) 1 × 250 μl D 6 Fluarix ® full human dose i.m., 2 × 250 μl
[1265] Results:
[1266] As shown in
[1267] Overall, the results demonstrate that the herein used tetravalent mRNA vaccine induces functional antibody responses for all four antigens. Moreover, the antibody responses were comparable to those observed for the approved vaccine Fluad, showing the enormous potential of the inventive LNP-formulated vaccine.
Example 23: Challenge Vaccination Experiment with a Combination of LNP-III-3 Formulated mRNAs Encoding HA of Different Influenza Viruses in Ferrets
[1268] Ferrets (Mustela putorius furσ, 6-12 months old) are immunized intramuscularly (i.m.) with LNP-III-3 formulated tetravalent mRNA vaccine of Example 22. As a positive control, groups are vaccinated withFluad® tetra 2016/2017. As negative control, groups are injected with placebo. For each group, 6 animals are treated (“immunization phase” in Table L).
[1269] To simulate a past season infection with influenza virus, some groups are also infected prior to the vaccination experiment with influenza virus. Group 5-8 are infected with H3N2 A/Fukui/20/2004 virus and group 13-16 were infected with B/Massachusetts/2/2012 Yamagata lineage (see “prime phase” in Table L)
[1270] After immunization, ferrets are challenged intratracheal with influenza virus. Group 5-8 were challenged with HA A/Netherlands/602/2009 virus and group 13-16 were challenged with B/Brisbane/60/2008 Victoria lineage (see “challenge phase” in Table L). Day 1-3 post virus challenge ferrets are analysed for virus load (nose, throat, swabs) and health parameters (fever, body weight). 4 days after virus challenge, animals are euthanized and analysed for immune responses (HI titers, IgG, Mn etc).
TABLE-US-00023 TABLE L Experimental procedure (Example 23) prime phase Vaccination shedule Challenge phase 1 No treatment placebo day 49 H1N1 Nose, 2 Fluad day 49 influenza A Throat, 3 tetravalent day 49 Swabs, 4 mRNA vaccine day 28 Body day 49 weight 5 H3N2 placebo day 49 6 day 21 Fluad day 49 7 tetravalent day 49 8 H3N2 mRNA vaccine day 28 day 0 day 49 9 No treatment placebo day 49 B Brisbane Nose, 10 Fluad day 49 Throat, 11 tetravalent day 49 Swabs, 12 mRNA vaccine day 28 Body day 49 weight 13 B placebo day 49 14 Massachusetts Fluad day 49 15 day 21 tetravalent day 49 16 B mRNA vaccine day 28 Massachusetts day 49 day 0
Example 24: Vaccination Experiment with LNP-III-3 Formulated mRNAs Encoding Three Different NA Antigens (Trivalent NA mRNA Vaccine)
[1271] As exemplarily shown in Example 14, LNP-III-3 formulated mRNA encoding neuraminidase induces strong and effective immune responses. In the present example, a trivalent mRNA composition comprising mRNA encoding NA of Influenza A/California/07/2009 (H1N1), mRNA encoding NA Influenza A/Hong Kong/4801/2014 (H3N2) and mRNA encoding NA of Influenza B/Brisbane/60/2008 was vaccinated (herein referred to as “Trivalent NA mRNA vaccine”). Respective mRNA sequences according to Example 1
[1272] Female BALB/c mice were injected at day 0 and day 21 with a trivalent NA mRNA vaccine, Influsplit Tetra (2016/2017) as positive control or a buffer control (RiLa) according to a regimen as provided in Table M below. Serum samples were taken for the determination of specific antibody titers (ELLA assay, performed according to Example 14). Results of the ELLA assay are shown in
TABLE-US-00024 TABLE M Immunization regimen (Example 24): No. of Group mice Treatment groups Dose Treatment 1 13 trivalent NA 7.5 μg i.m., mRNA vaccine (2.5 μg each mRNA) 1 × 25 μl LNP-III-3 formulated 2 6 Influsplit Full human dose i.m., Tetra 2016/2017 1 × 25 μl 3 3 RiLa buffer i.m., 1 × 25 μl
[1273] Results:
[1274] As shown in
Example 25: Vaccination Experiment with LNP-III-3 Formulated mRNAs Encoding Three Different NA Antigens and Four Different HA Antigens (Septavalent mRNA Vaccine)
[1275] As exemplarily shown in the Examples above, LNP-III-3 formulated tetravalent HA mRNA vaccines and trivalent NA mRNA vaccines induce strong and effective immune responses for each encoded antigen. In the present example, an LNP-III-3 formulated mRNA composition encoding three different NA antigens (mRNA encoding NA of Influenza A/California/07/2009 (H1N1), mRNA encoding NA of Influenza A/Hong Kong/4801/2014 (H3N2) and mRNA encoding NA of Influenza B/Brisbane/60/2008 was vaccinated) and four different HA antigens (mRNA encoding HA of Influenza A/California/07/2009 (H1N1), mRNA encoding HA of Influenza A/Hong Kong/4801/2014 (H3N2) and mRNA encoding HA of Influenza B/Brisbane/60/2008 and mRNA encoding HA of Influenza B) was vaccinated (herein referred to as “septavalent HA/NA mRNA vaccine”). Respective mRNA sequences according to Example 1.
[1276] Female BALB/c mice were injected i.m. at day 0 and day 21 with LNP-III-3 formulated tetravalent HA mRNA vaccine, LNP-III-3 formulated trivalent NA mRNA vaccine, or LNP-III-3 formulated septavalent HA/NA mRNA vaccine. As positive control, one group of mice was injected with Influsplit Tetra® 2016/2017. As negative control, one group of mice was injected with RiLa buffer. Serum samples for the analysis of immune responses (HI titer, ELISA) were collected at day 21, 35, 49 (assays performed as described above). Splenocytes collected at day 49 (ICS). Experimental details provided in Table N. ELISA and HI-titer results are shown in
TABLE-US-00025 TABLE N Immunization regimen (Example 25) No. of Group mice Treatment groups Dose Treatment 1 8 Tetravalent HA 4 μg i.m., mRNA vaccine (1 μg each) 1 × 25 μl 2 8 Trivalent NA 3 μg i.m., mRNA vaccine (1 μg each) 1 × 25 μl 3 8 septavalent HA/ 7 μg i.m., NA mRNA vaccine (1 μg each) 1 × 25 μl 4 8 septavalent HA/ 7 μg NA mRNA vaccine (0.5 μg each) 5 6 Influsplit Tetra 1/10 human i.m., dose 2 × 25 μl 6 6 RiLa — i.m., 1 × 25 μl
[1277] Results:
[1278] The results show that the LNP-III-3 formulated septavalent HA/NA mRNA vaccine induces strong and effective immune responses in vaccinated mice.
[1279]
[1280]
[1281]
Example 26: Vaccination Experiment with LNP-III-3 Formulated mRNAs Encoding Ebola GP
[1282] In the present example, the inventive mRNA LNP-III-3 formulation was compared with an established mRNA vaccine format (Protamine formulation; see Example 6) using mRNA encoding glycoprotein GP of Ebola virus (ZEBOV GP Sierra Leone 2014).
[1283] Protamine formulation of mRNA encoding GP of Ebola virus (SEQ ID NO: 224362) as described in Example 6. LNP formulation of mRNA encoding GP of Ebola virus (SEQ ID NO: 224362) as described above.
[1284] Immunogenicity of Ebola GP mRNA vaccine in mice:
[1285] Female BALB/c mice were injected at day 0 and day 21 with a LNP-III-3 formulated mRNA encoding GP of Ebola virus (RNA ID “R3875”; intramuscular (i.m.)) or protamine formulated mRNA encoding GP of Ebola virus (RNA ID “R3875”; intradermal (i.d.)). As negative control, one group of mice was injected with RiLa buffer. Serum samples for the analysis of IgG endpoint titers (ELISA) were collected at day 35. ELISA was performed using recombinant ZEBOV Mayinga GP protein (lacking the transmamebrane domain) for coating. ELISA results are shown in
TABLE-US-00026 TABLE O Immunization regimen (Example 26) No. of Group mice Treatment groups Dose Treatment 1 8 GP Ebola, Protamine 80 μg i.d., formulated 1 × 50 μl 2 8 GP Ebola; LNP 5 μg i.m., formulated 1 × 25 μl 3 8 GP Ebola; LNP 7 μg i.m., formulated (1 μg each) 1 × 25 μl 4 8 RiLa 7 μg i.d., (0.5 μg each) 1 × 50 μl
[1286] Results:
[1287]
Example 27: Vaccination Experiment with LNP-III-3 Formulated mRNAs Encoding H3N2 Administered Subcutaneously
[1288] As described in the previous examples, LNP-III-3 formulated mRNA vaccines induce strong and effective immune responses when administered intramuscularily or intradermally (e.g. see Example 4). To evaluate the effectivity of the inventive vaccine format for other suitable administration routes, sub-cutaneous injection was tested.
[1289] Non-human primates (Macaca fascicularis) were injected with LNP-III-3 formulated mRNA encoding Influenza A/Hong Kong/4801/2014 H3N2 (mRNA sequences according to Example 1). Three groups were vaccinated subcutaneously (sc) with different vaccine doses (10 μg, 50 μg, 100 μg) and one control group was vaccinated intramuscularily. HI titers at day 0, day 28, day 49 and day 70 were determined as described herein. Results of the experiment are shown in
TABLE-US-00027 TABLE P Immunization regimen (Example 27) No. of Group NHP Treatment groups Dose and route Treatment 1 8 LNP-III-3 10 μg, Day 0, formulated H3N2 subcutaneous day 28 2 8 LNP-III-3 50 μg, Day 0, formulated H3N2 subcutaneous day 28 3 8 LNP-III-3 100 μg, Day 0, formulated H3N2 subcutaneous day 28 4 8 LNP-III-3 100 μg, i.m. Day 0, formulated H3N2 day 28
[1290] Results: As shown in
Example 28: Vaccination Experiment with LNP-III-3 Formulated OVA mRNA Vaccine
[1291] For vaccination 9 mice (C57 BL/6) per group were intradermally injected 3 times within 3 weeks with 1 μg LNP formulated Ova mRNA (Component A) and 32 μg protamine formulated Ova mRNA (Component B), as negative control RiLa was injected (see Table Q).
[1292] Levels of circulating antigen-specific CD8 positive T cells were measured with OVA-specific dextramers (bind to antigen specific T cell receptors of CD8 positive cells) at day 7 and 21. 1 μg LNP formulated Ova mRNA (Component A) vaccine induces high and boostable levels of circulating antigen-specific CD8 positive T cells after intradermal application (see
TABLE-US-00028 TABLE Q Components, treatment and RNA dilution (Example 28) Mice Component Treatment RNA dose Route (Volume) # A OVA mRNA 1 μg i.d (25 μl)/1 9 formulated in LNP site of injection B Ova mRNA 32 μg i.d (100 μl)/4 9 formulated with sites of injection protamine (state of the art) C Rila buffer — i.d (25 μl)/1 3 site of injection
[1293] Levels of multifunctional antigen-specific CD8 positive T cells (IFNγ/TNFα) were measured by intracellular cytokine staining (ICS). Therefore splenocytes were isolated from the vaccinated mice one week after the last vaccination and CD8 positive T cells were stimulated with OVA specific peptides (SIINFEKL and TEWTSSNVMEERKIKV) (see
[1294] Detection of B-cell immune responses was carried out by detecting OVA-specific IgG2c titers. Therefore, serum samples were taken from the vaccinated mice one week after the last vaccination and analyzed by ELISA. 1 μg of LNP-formulated OVA-mRNA vaccine leads to increased OVA-specific IgG2c titers after intradermal application compare to protamine formulated Ova mRNA (see
[1295] The results demonstrate that the inventive LNP-III-3 formulated mRNA vaccine format is suitable to induce anti-tumor responses in vivo and therefore useable for vaccination against tumor.
Example 29: Tumor Challenge Experiment with LNP-III-3 Formulated OVA mRNA Vaccine
[1296] C57BL/6 mice were injected subcutaneously (s.c.) with 3×105 E.G7-OVA cells per mouse (in a volume of 100 μl PBS) on the right flank on day 0 of the experiment. Intradermal (i.d.) therapy started at day 4 and continued twice a week for three weeks. Mice were injected with 1 μg OVA mRNA and an irrelevant PpLuc mRNA formulated in LNPs. To control for anti-tumor effects due to injection procedure, mice were injected with buffer (RiLa).
[1297] The results of the experiment are shown in
[1298] Tumor growth was monitored by measuring the tumor size in three dimensions using a calliper. Tumor volume was calculated according to the following formula:
[1299] The results in
TABLE-US-00029 TABLE R Components, treatment and RNA dilution (Example 29) RNA Mice Component Treatment dose Route (Volume) Schedule # A OVA mRNA 1 μg i.d (25 μl)/1 2 × week 10 formulated in LNP site of injection B PpLuc mRNA 1 μg i.d (25 μl)/1 2 × week 10 formulated in LNP site of injection C RiLa — i.d (25 μl)/1 2 × week 6 site of injection
[1300] The results demonstrate that the inventive LNP-III-3 formulated mRNA vaccine format is suitable for tumor vaccination.
Example 30: Vaccination Experiment with LNP-III-3 Formulated Endogene Tumor Associated Antigens mRNA Vaccine in Combination with Checkpoint Inhibitor
[1301] C57BL/6 mice were injected subcutaneously (s.c.) with 1×10.sup.5 B16.F10 cells (murine melanoma cell line) per mouse on the right flank on day 0 of the experiment. At day 6 after tumor challenge, mice were vaccinated intradermal with LNP formulated Trp2 mRNA (Component A) and irrelevant LNP formulated PpLuc mRNA (Component B). In addition immune checkpoint inhibitors anti-PD1 (Clone: RMP1-14) and anti-CTLA4 (clone: 9H10) (both BioXCell) were administered intraperitoneal (i.p.), median tumor size and survival rates were analyzed. To exclude an anti-tumor effect due to the checkpoint inhibitors, mice were injected with component B and a control antibody (rat IgG2a, BioXCell).
[1302] Tumor-bearing mice treated with vaccine against mTrp2 and checkpoint inhibitors anti-PD1 and anti-CTLA4 show delayed tumor growth and increased survival compared to other treatments with irrelevant mRNA (Component B) in combination with checkpoint inhibitors anti-PD1 and anti-CTLA4 or a control antibody (see
TABLE-US-00030 TABLE S Components, treatment and RNA/Antibody dilution RNA dose (i.d)/ Mice Treatment volume Antibody (i.p) Schedule # A mTrp2 mRNA 1 μg/25 μl Anti-PD1 + 2 × week 10 formulated in LNP anti-CTLA4 B PpLuc mRNA 1 μg/25 μl Isotype 2 × week 10 formulated in LNP C PpLuc mRNA 1 μg/25 μl Anti-PD1 + 2 × week 10 formulated in LNP anti-CTLA4
[1303] The results demonstrate that the inventive LNP-III-3 formulated mRNA vaccine format is suitable for the combination with checkpoint inhibitors for anti-tumor therapy.