WO2021095838A1 - HPV mRNAを封入した核酸脂質粒子ワクチン - Google Patents
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Definitions
- the present invention relates to a nucleic acid lipid particle vaccine encapsulating HPV mRNA.
- Non-Patent Document 1 Human papillomavirus is a virus that has circular double-stranded DNA as a genome without an envelope membrane, and there are about 200 genotypes (Non-Patent Document 1). Among them, there are genotypes that make infected cells cancerous, and in particular, genotypes 16 and 18 that have been proven to correlate with the development of cancer represented by cervical cancer are classified as high-risk types ( Non-Patent Document 2).
- L1 and L2 are structural proteins that serve as outer shells (capsids) of virus particles (Non-Patent Document 3).
- HPV infection is initiated by the adsorption of the L1 protein constituting the capsid on the heparan sulfate proteoglycan present on the surface of the host cell membrane (Non-Patent Document 4). Since neutralizing antibodies that protect against HPV infection target the L1 protein, the preventive vaccines currently on the market contain the VLP (virus-like particle) antigen of the L1 protein as a medicinal ingredient.
- all three existing prophylactic vaccines contain L1 VLP antigens of genotypes 16 and 18.
- Non-Patent Document 5 Non-Patent Document 5
- HPV-infected cells cause cell cycle abnormalities due to E6 and E7 carcinogenic proteins. This is because the functions of p53 and pRb, which are responsible for cell cycle and induction of apoptotic cell death, are inhibited by E6 and E7 (Non-Patent Documents 6 and 7). Areas important for the oncogenic activity of E6 and E7 have been clarified, and when using E6 and E7 as vaccine antigens, it is possible to insert inactivating mutations to enhance safety (non-). Patent Document 8-10).
- Non-Patent Document 11 Host defense immunity against HPV infection is the induction of neutralizing antibodies and the induction of cytotoxic T cells (CTL) and helper T cells.
- CTL cytotoxic T cells
- E6 and E7 are CTL-induced target antigens, and are attracting attention as therapeutic vaccine antigens for cervical cancer caused by HPV infection and cervical dysplasia (Non-Patent Document 11).
- Patent Document 1 describes the E6 and E7 fusion antigen gene sequences of HPV genotypes 6, 11, 16, 18, 31, 33, 39, 45, 52 and 58.
- an IgE leader sequence is added to the N-terminal of E6, and a furin peptidase cleavage site is inserted between the translation region sequences of E6 and E7.
- mutations are inserted in the p53-binding region of E6 and the pRb-binding region of E7 to inactivate the carcinogenic activity of E6 and E7.
- This gene sequence has been introduced into a mammalian expression plasmid, and the efficacy of the drug is being evaluated in a mouse model as a DNA gene vaccine against HPV. Immunity is intramuscular administration to the thigh of mice using electroporation.
- An object of the present invention is to provide a vaccine for preventing and / or treating infection by human papillomavirus.
- the present inventors have found that when lipid particles encapsulating mRNA encoding the E6-E7 antigen of human papillomavirus were administered to mice transplanted with cancer cells, a cancer degenerate effect was observed, and the present invention was developed. It came to be completed.
- the gist of the present invention is as follows.
- the particles containing a salt.
- R 1 and R 2 independently represents a C 1 -C 3 alkyl group
- L 1 represents a C 17- C 19 alkenyl group which may have one or more C 2- C 4 alkanoyloxy groups
- L 2 may have one or more C 2- C 4 alkanoyloxy groups, a C 10- C 19 alkyl group, or one or more C 2- C 4 alkanoyloxy groups.
- L 2 in the general formula (Ia) may have one or more acetoxy groups C 10- C 12 alkyl groups or C which may have one or more acetoxy groups. 17- C 19
- L 1 in the general formula (Ia) is (R) -11-acetyloxy-cis-8-heptadecenyl group, cis-8-heptadecenyl group, or (8Z, 11Z) -heptadecadienyl group.
- L 2 in the general formula (Ia) is a decyl group, a cis-7-decenyl group, a dodecyl group, or (R) -11-acetyloxy-cis-8-heptadecenyl group, (1) to The particle according to any one of (7).
- the structural formula of the cationic lipid is as follows: The particle according to (1) represented by. (10) The structural formula of the cationic lipid is as follows: The particle according to (1) represented by. (11) The cationic lipid has the following structural formula: The particle according to (1) represented by.
- the amphipathic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoil phosphatidylcholine and dioleoil phosphatidylethanolamine.
- the PEG lipid is 1,2-dimiristoyl-sn-glycerol methoxypolyethylene glycol and / or N- [methoxypoly (ethylene glycol) 2000] carbamoyl] -1,2-dimyristyloxypropyl-3-amine.
- the particle according to any one of (12) to (14).
- the lipid composition of amphoteric lipids, sterols, cationic lipids, and PEG lipids is 15% or less for amphoteric lipids, 20-55% for sterols, and 40 for cationic lipids in terms of molar amount.
- the lipid composition of amphoteric lipids, sterols, cationic lipids, and PEG lipids is 5 to 15% for amphoteric lipids, 35 to 50% for sterols, and cationic lipids in molar amounts.
- the lipid composition of amphoteric lipids, sterols, cationic lipids, and PEG lipids is 10 to 15% for amphoteric lipids, 35 to 45% for sterols, and cationic lipids in molar amounts.
- the lipid composition of amphoteric lipids, sterols, cationic lipids, and PEG lipids is 10 to 15% for amphoteric lipids, 35 to 45% for sterols, and cationic lipids in molar amounts.
- the human papillomavirus is HPV16 type, and the nucleic acid capable of expressing HPV16 type E6 antigen and E7 antigen is a cap structure (Cap), 5'untranslated region (5'-UTR), leader sequence (leader). Sequence), E6 translation region, protease cleavage sequence (Furin Cleavage site), E7 translation region, 3'untranslated region (3'-UTR) and polyA tail (polyA). 22) The particle according to any one of.
- the sequence of nucleic acids capable of expressing HPV16 type E6 antigen and E7 antigen consists of a nucleotide sequence having at least 90% identity with any of SEQ ID NOs: 2, 4 or 6 (23). Described particles.
- the modified nucleotide comprises at least one of a pyrimidine nucleotide substituted at the 5-position and / or a pseudouridine which may be substituted at the 1-position.
- (30) Use of the particles according to (29), wherein the infection is an infection with HPV16 type human papillomavirus.
- composition according to (32) The composition according to (31) for expressing the E6 and E7 antigens of human papillomavirus in vivo or in vitro.
- the composition according to (31) or (32) used as a medicine.
- the composition according to (33) for inducing an immune response against human papillomavirus.
- the composition according to (33) or (34) for preventing and / or treating human papillomavirus infection.
- a method for expressing the E6 and E7 antigens of human papillomavirus in vivo which comprises administering the composition according to any one of (31) to (35) to a mammal.
- a method for inducing an immune response against human papillomavirus which comprises administering the composition according to (33) or (34) to a mammal.
- a method for preventing and / or treating human papillomavirus infection which comprises administering the composition according to any one of (33) to (35) to a mammal.
- Lipid particles encapsulating nucleic acids capable of expressing the E6 and E7 antigens of human papillomavirus, wherein the lipid is a cationic lipid represented by the general formula (Ia), or pharmaceutically acceptable thereof.
- R 1 and R 2 independently represents a C 1 -C 3 alkyl group
- L 1 represents a C 17- C 19 alkenyl group which may have one or more C 2- C 4 alkanoyloxy groups
- L 2 may have one or more C 2- C 4 alkanoyloxy groups, a C 10- C 19 alkyl group, or one or more C 2- C 4 alkanoyloxy groups.
- L 2 in the general formula (Ia) is a decyl group, a cis-7-decenyl group, a dodecyl group, or (R) -11-acetyloxy-cis-8-heptadecenyl group, (1).
- -1) The particle according to any one of (1-7).
- Cationic lipid has the following structural formula: The particle according to (1-1) represented by. (1-10) Cationic lipid has the following structural formula: The particle according to (1-1) represented by. (1-11) Cationic lipid has the following structural formula: The particle according to (1-1) represented by.
- the particles according to (1-13), wherein the amphipathic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoil phosphatidylcholine and dioleoil phosphatidylethanolamine.
- PEG lipids are 1,2-dimiristoyl-sn-glycerol methoxypolyethylene glycol and / or N- [methoxypoly (ethylene glycol) 2000] carbamoyl] -1,2-dimyristyloxypropyl-3-
- PEG lipids are 1,2-dimiristoyl-sn-glycerol methoxypolyethylene glycol and / or N- [methoxypoly (ethylene glycol) 2000] carbamoyl] -1,2-dimyristyloxypropyl-3-
- the lipid composition of amphoteric lipids, sterols, cationic lipids, and PEG lipids is 22.5% or less for amphoteric lipids, 15-55% for sterols, and cations in terms of molar amount.
- the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is 5 to 15% for amphoteric lipids, 35 to 50% for sterols, and cationic in terms of molar amount. Lipids are 40-55%, PEG lipids are 1-3%, and the ratio of total lipid weight to nucleic acid weight is 15-30 (1-12), (1-14), (1-16), The particle according to any one of (1-18). (1-26) The particles according to (1-25), wherein the amphipathic lipid is 10 to 15%, the sterols are 35 to 45%, the cationic lipid is 40 to 50%, and the PEG lipid is 1 to 2%.
- the amphipathic lipid is 10 to 15%, sterols are 35 to 45%, cationic lipid is 45 to 50%, and PEG lipid is 1.5 to 2% (1-26). particle.
- the lipid composition of the amphoteric lipids, sterols, cationic lipids, and PEG lipids was 15 to 22.5% for the amphoteric lipids and 15 to 40% for the sterols in terms of molar amount.
- Cationic lipids are 40-60%, PEG lipids are 1-3%, and the ratio of total lipid weight to nucleic acid weight is 15-30 (1-13), (1-15), (1-17). ), (1-19).
- (1-34) The particle according to any one of (1-1) to (1-33), wherein the human papillomavirus is HPV16 type.
- (1-35) The particle according to (1-34), wherein the human papillomavirus is HPV16 type, and the HPV16 type E6 antigen has an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 8.
- (1-36) The human papillomavirus is HPV16 type, and the HPV16 type E7 antigen consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 9 (1-34) or (1-35). Described particles.
- the human papillomavirus is HPV16 type, and the nucleic acid capable of expressing the E6 and E7 antigens of the human papillomavirus comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 17.
- the particle according to any one of (1-34) to (1-36) encoding a fusion protein of HPV16 type E6 antigen and E7 antigen.
- the human papillomavirus is HPV16 type, and the nucleic acid capable of expressing HPV16 type E6 antigen and E7 antigen is a cap structure (Cap), 5'untranslated region (5'-UTR), leader sequence.
- the sequence of the nucleic acid capable of expressing the HPV16 type E6 antigen and the E7 antigen consists of a nucleotide sequence having at least 90% identity with any of the sequences of SEQ ID NOs: 2, 4 or 6 ((1-39). 1-38) The particles according to the above.
- the human papillomavirus is HPV18 type, and the HPV18 type E7 antigen consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 15 (1-40) or (1-41). Described particles.
- the human papillomavirus is HPV18 type, and the nucleic acid capable of expressing the E6 and E7 antigens of the human papillomavirus comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 18.
- the particle according to any one of (1-40) to (1-42) encoding a fusion protein of HPV18 type E6 antigen and E7 antigen.
- the human papillomavirus is HPV18 type, and the nucleic acid capable of expressing HPV18 type E6 antigen and E7 antigen is a cap structure (Cap), 5'untranslated region (5'-UTR), leader sequence.
- the sequence of the nucleic acid capable of expressing the HPV18 type E6 antigen and E7 antigen consists of a nucleotide sequence having at least 90% identity with the sequence of either SEQ ID NO: 11 or 13 (1-45).
- 44) The particles according to the above. (1-46) The particle according to any one of (1-1) to (1-45), wherein the nucleic acid contains at least one modified nucleotide.
- the particle according to (1-46), wherein the modified nucleotide comprises at least one of a pyrimidine nucleotide substituted at the 5-position and / or a pseudouridine optionally substituted at the 1-position.
- the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkyl pseudouridine (1-46).
- Particles. (1-49) The particle according to (1-46), wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine.
- (1-50) The particle according to any one of (1-1) to (1-49), which has an average particle diameter of 30 nm to 300 nm.
- (1-51) Use of the particles according to any one of (1-1) to (1-50) for producing a composition for preventing and / or treating infection by human papillomavirus.
- (1-52) Use of the particles according to (1-51), wherein the infection is an infection with HPV16 or HPV18 human papillomavirus.
- (1-53) A composition containing the particles according to any one of (1-1) to (1-50).
- (1-54) The composition according to (1-53) for expressing the E6 and E7 antigens of human papillomavirus in vivo or in vitro.
- (1-55) The composition according to (1-53) or (1-54) used as a medicine.
- (1-56) The composition according to (1-55) for inducing an immune response against human papillomavirus.
- (1-57) The composition according to (1-55) or (1-56) for preventing and / or treating human papillomavirus infection.
- (1-58) A method for expressing the E6 and E7 antigens of human papillomavirus in vitro, which comprises introducing the composition according to (1-53) or (1-54) into cells.
- (1-59) A method for expressing the E6 and E7 antigens of human papillomavirus in vivo, which comprises administering the composition according to any one of (1-53) to (1-57) to a mammal.
- (1-60) A method for inducing an immune response against human papillomavirus, which comprises administering the composition according to (1-55) or (1-56) to a mammal.
- (1-61) A method for preventing and / or treating human papillomavirus infection, which comprises administering the composition according to any one of (1-55) to (1-57) to a mammal.
- the present invention infection by human papillomavirus can be prevented and / or treated.
- the present invention makes it possible to prevent and / or treat diseases caused by infection with human papillomavirus (cervical cancer, cervical dysplasia, etc.).
- the particles of the present invention have excellent properties in terms of metabolic stability, in vitro activity, in vivo activity, rapid onset of drug effect, sustainability of drug effect, physical stability, drug interaction, safety and the like. It has and is useful as a medicine for treating or preventing the above-mentioned diseases.
- This specification includes the contents described in the Japanese patent application, Japanese Patent Application No. 2019-207001 and / or drawings which are the basis of the priority of the present application.
- the tumor size is expressed by the volume of the length (mm) ⁇ width (mm) ⁇ height (mm) of the tumor. OVA-specific antibody-inducing levels of mRNA-encapsulated nucleic acid lipid particles in C57BL / 6 mice. Examples 21-27; mRNA-encapsulated nucleic acid lipid particle administration group, NC; untreated negative control group.
- No peptides; negative control group without peptide treatment, HPV18E6 overlapping peptides; HPV18E6 pool peptide treatment group CTL induction levels of mRNA-encapsulated nucleic acid lipid particles in C57BL / 6 mice.
- the present invention is a lipid particle encapsulating a nucleic acid capable of expressing the E6 and E7 antigens of human papillomavirus, wherein the lipid is a cationic lipid represented by the general formula (Ia), or pharmaceutically acceptable thereof.
- the particles containing the salt are provided.
- R 1 and R 2 independently represents a C 1 -C 3 alkyl group
- L 1 represents a C 17- C 19 alkenyl group which may have one or more C 2- C 4 alkanoyloxy groups
- L 2 may have one or more C 2- C 4 alkanoyloxy groups, a C 10- C 19 alkyl group, or one or more C 2- C 4 alkanoyloxy groups. Shows good C 10- C 19 alkenyl groups;
- p is 3 or 4.
- R 1 and R 2 in the general formula (Ia) are independently exhibit C 1 -C 3 alkyl group, preferably both a methyl group.
- P in the general formula (Ia) is 3, or 4, but is preferably 3.
- L 1 in the general formula (Ia) represents a C 17- C 19 alkenyl group which may have one or more C 2- C 4 alkanoyloxy groups, preferably one or more acetoxy groups. It is a C 17- C 19 alkenyl group which may have one.
- Specific examples of L 1 include (R) -11-acetyloxy-cis-8-heptadecenyl group, cis-8-heptadecenyl group, (8Z, 11Z) -heptadecadienyl group and the like. it can.
- L 2 in the general formula (Ia) is, C 2 -C 4 alkanoyloxy group one or plurality which may have C 10 -C 19 alkyl group, or a C 2 -C 4 alkanoyloxy group one or It shows a C 10- C 19 alkenyl group which may have a plurality of C 10-C 19 alkenyl groups, preferably one or a plurality of C 10- C 12 alkyl groups which may have one or a plurality of acetoxy groups, or one or a plurality of acetoxy groups. It is a C 10- C 19 alkenyl group which may have one.
- L 2 in the general formula (Ia) may have one or more C 10- C 12 alkyl groups, which may have one or more acetoxy groups, or C which may have one or more acetoxy groups. It is also preferably a 17- C 19 alkenyl group. Specific examples of L 2 include a decyl group, a cis-7-decenyl group, a dodecyl group, and an (R) -11-acetyloxy-cis-8-heptadecenyl group.
- the cationic lipid which is a component constituting the particles of the present invention, the following structural formula: Diacetic acid (7R, 9Z, 26Z, 29R) -18-( ⁇ [3- (dimethylamino) propoxy] carbonyl ⁇ oxy) pentatriaconta-9,26-diene-7,29-diyl, respectively.
- 3-Dimethylaminopropyl carbonate (9Z, 12Z) -octacosa-19,22-diene-11-yl, acetic acid (7R, 9Z) -18-( ⁇ [3- (dimethylamino) propyloxy] carbonyl ⁇ oxy) octacosa -9-ene-7-yl can be exemplified.
- the cationic lipid represented by the general formula (Ia) may be one kind of compound or a combination of two or more kinds of compounds.
- the lipids of the present invention may further include amphipathic lipids, sterols and PEG lipids.
- the amphipathic lipid is a lipid that has an affinity for both polar and non-polar solvents. Specifically, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and combinations thereof are used. It can be exemplified.
- the amphipathic lipid used in the particles of the present invention is preferably distearoylphosphatidylcholine and / or dioleoil phosphatidylethanolamine, and more preferably distearoylphosphatidylcholine.
- the sterols are sterols having a hydroxy group, and specifically, cholesterol and the like can be exemplified.
- the PEG lipid is a PEG-modified lipid, specifically 1,2-dimiristoyl-sn-glycerol methoxypolyethylene glycol and / or N- [methoxypoly (ethylene glycol) 2000] carbamoyl] -1,2. -Dimyristyloxypropyl-3-amine, a combination thereof and the like can be exemplified, but 1,2-dimyristyl-sn-glycerol methoxypolyethylene glycol is preferable.
- the lipid composition of the amphoteric lipids, sterols, cationic lipids, and PEG lipids is not particularly limited, but the molar amount of the amphoteric lipids is 22.5% or less and the sterols are 15 to 15 to 55%, cationic lipids 40-65%, PEG lipids 1-5%, and the ratio of total lipid weight to nucleic acid weight is preferably 15-30, amphoteric lipids, sterols, cations.
- the lipid composition of sex lipids and PEG lipids is 5 to 22.5% for amphoteric lipids, 15 to 55% for sterols, 40 to 65% for cationic lipids, and 1 to 1 to PEG lipids in terms of molar amount. It is 5%, and the ratio of the total lipid weight to the nucleic acid weight is more preferably 15 to 30, and the lipid composition of the amphoteric lipid, sterols, cationic lipid, and PEG lipid is in molar amount.
- the ratio of total lipid weight to nucleic acid weight is 15: amphoteric lipids 10-22.5%, sterols 15-55%, cationic lipids 40-65%, PEG lipids 1-5%.
- the proportion of PEG lipid is more preferably 1 to 3%, further preferably 1 to 2%, and particularly preferably 1.5 to 2% in terms of molar amount. Further, in the above lipid composition, the ratio of the total lipid weight to the nucleic acid weight is more preferably 15 to 25, further preferably 15 to 22.5, and even more preferably 17.5 to 22.5. Especially preferable.
- cationic lipids 3-dimethylaminopropyl carbonate (9Z, 12Z) -octacosa-19,22-diene-11-yl, or diacetic acid (7R, 9Z, 26Z, 29R) -18-( ⁇ [3- (dimethyl) When amino) propoxy] carbonyl ⁇ oxy) pentatriaconta-9,26-diene-7,29-diyl is used, the lipid composition of amphoteric lipids, sterols, cationic lipids, and PEG lipids is particularly limited.
- the molar amount is 15% or less of the amphoteric lipid, 20 to 55% of the sterols, 40 to 65% of the cationic lipid, and 1 to 5% of the PEG lipid.
- the lipid composition of amphoteric lipids, sterols, cationic lipids, and PEG lipids is 5 to 15% for amphoteric lipids, 35 to 50% for sterols, and 40 to 55 for cationic lipids in terms of molar amount. %,
- the PEG lipid is more preferably 1 to 3%, and the lipid composition of the amphoteric lipid, sterols, cationic lipid, and PEG lipid is 10 to 15% in molar amount.
- the composition is 10 to 15% for amphoteric lipids, 35 to 45% for sterols, 45 to 50% for cationic lipids, and 1.5 to 2% for PEG lipids in terms of molar amount.
- the lipid composition of the amphoteric lipids, sterols, cationic lipids, and PEG lipids is 12.5% amphoteric lipids, 41% sterols, 45% cationic lipids in molar amounts.
- the PEG lipid content is 1.5%.
- the ratio of the total lipid weight to the nucleic acid weight is preferably 15 to 30, more preferably 15 to 25, still more preferably 15 to 22.5, and 17.5 to 22. It is particularly preferably .5.
- acetic acid (7R, 9Z) -18-( ⁇ [3- (dimethylamino) propyloxy] carbonyl ⁇ oxy) octacosa-9-en-7-yl is used as the cationic lipid, amphoteric lipids, sterols, etc.
- the lipid composition of the cationic lipid and the PEG lipid is not particularly limited, but in terms of molar amount, the amphoteric lipid is 12.5 to 22.5%, the sterols are 15 to 45%, and the cationic lipid is cationic. It is preferable that the lipid content is 40 to 65% and the PEG lipid content is 1 to 5%.
- the lipid composition of PEG lipids is 15 to 22.5% for amphoteric lipids, 15 to 40% for sterols, 45 to 60% for cationic lipids, and 1 to 2% for PEG lipids. More preferably, the lipid composition of the amphoteric lipids, sterols, cationic lipids, and PEG lipids is 17.5 to 22.5% for the amphoteric lipids and 15 to 15% for the sterols in molar amounts.
- the ratio of the total lipid weight to the nucleic acid weight is preferably 15 to 30, more preferably 15 to 25, still more preferably 15 to 22.5, and 17.5 to 22. It is particularly preferably .5.
- Specific lipid combinations in the present invention include distearoyl phosphatidylcholine, dioleoylphosphatidylcholine, or dioleoylphosphatidylethanolamine as amphoteric lipids, cholesterol as sterols, and diacetic acid (7R, 9Z, as cationic lipids, 26Z, 29R) -18-( ⁇ [3- (dimethylamino) propoxy] carbonyl ⁇ oxy) pentatriaconta-9,26-diene-7,29-diyl, 3-dimethylaminopropyl carbonate (9Z, 12Z)- Octacosa-19,22-diene-11-yl, or acetic acid (7R, 9Z) -18- ( ⁇ [3- (dimethylamino) propyloxy] carbonyl ⁇ oxy) octacosa-9-ene-7-yl, PEG lipid 1,2-Dimyristyl-sn-glycerol me
- distearoylphosphatidylcholine or dioleoil phosphatidylethanolamine as a parental lipid
- cholesterol as a sterol
- the specific lipid combination in the present invention is distearoylphosphatidylcholine as a parental lipid, cholesterol as a sterol, and diacetic acid (7R, 9Z, 26Z, 29R) -18- ( ⁇ [3-]) as a cationic lipid.
- the nucleic acid encapsulated in the lipid particles can express the E6 antigen and the E7 antigen of human papillomavirus.
- the E6 antigen and E7 antigen of human papillomavirus expressing the nucleic acid enclosed in the lipid particles may be a fusion protein of both, and a protease cleavage sequence may be contained between the E6 antigen and the E7 antigen.
- the genotype of human papillomavirus is not particularly limited, but HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68. Can be exemplified. HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 are cancers represented by cervical cancer. Correlation with onset has been proven.
- HPV16 type E6 antigen The amino acid sequence of HPV16 type E6 antigen is shown in SEQ ID NO: 8.
- the nucleic acid encapsulated in the lipid particles encodes an HPV16 type E6 antigen consisting of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 8. It would be nice to have it.
- HPV16 type E7 antigen The amino acid sequence of HPV16 type E7 antigen is shown in SEQ ID NO: 9.
- the nucleic acid encapsulated in the lipid particles encodes an HPV16 type E7 antigen consisting of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 9. It would be nice to have it.
- HPV18 type E6 antigen The amino acid sequence of HPV18 type E6 antigen is shown in SEQ ID NO: 14.
- the nucleic acid encapsulated in the lipid particles encodes an HPV18 type E6 antigen consisting of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 14. It would be nice to have it.
- HPV18 type E7 antigen The amino acid sequence of HPV18 type E7 antigen is shown in SEQ ID NO: 15.
- the nucleic acid encapsulated in the lipid particles encodes an HPV18 type E7 antigen consisting of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 15. It would be nice to have it.
- the amino acid sequence of the protease cleavage sequence (Furin Cleavage site) is shown in SEQ ID NO: 16.
- the protease cleavage sequence may be any sequence that is cleaved by the Furin protein, and is represented by, for example, RX-K / R-R (R represents arginine, K represents lysine, and X represents any amino acid). (J. Biol. Chem. 1992, 267, 16396; J. Biol. Chem. 1991, 266, 12127).
- the amino acid sequence of the fusion protein of HPV16 type E6 antigen and E7 antigen is shown in SEQ ID NO: 17.
- the nucleic acid encapsulated in the lipid particle is a fusion of HPV16 type E6 antigen and E7 antigen consisting of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 17. It should encode a protein.
- the amino acid sequence of the fusion protein of HPV18 type E6 antigen and E7 antigen is shown in SEQ ID NO: 18.
- the nucleic acid encapsulated in the lipid particle is a fusion of HPV18 type E6 antigen and E7 antigen consisting of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 18. It should encode a protein.
- Amino acid sequence identity is a numerical value of the ratio of amino acids to the full-length sequence, with amino acids that completely match the corresponding amino acids as the same amino acid.
- the sequence identity in the present invention is calculated using the sequence analysis software GENETYX-SV / RC (manufactured by Genetics Co., Ltd.), and this algorithm is usually used in the art.
- the amino acids encoded by the nucleic acids encapsulated in the lipid particles of the present invention are mutated (substituted), deleted, inserted and / or amino acids as long as they maintain a certain level of identity with SEQ ID NOs: 8, 9, 14-18. Additions may have occurred.
- the amino acid encoded by the nucleic acid encapsulated in the lipid particle of the present invention retains the above-mentioned sequence identity and has several positions (preferably 5 or less, more preferably 5 or less) in the amino acid sequences of SEQ ID NOs: 8, 9, and 14-18. 3, 2 or 1 site), several amino acids (preferably 10 or less, more preferably 7 or less, still more preferably 5, 4, 3, 2 or 1) are substituted or deleted per site. , Insert and / or may be added.
- the nucleic acids capable of expressing the E6 and E7 antigens of human papillomavirus are cap structure (Cap), 5'untranslated region (5'-UTR), leader sequence. , E6 translation region, protease cleavage sequence (Furin Cleavage site), E7 translation region, 3'untranslated region (3'-UTR) and polyA tail (polyA).
- the cap structure (Cap) is a site that is present at the 5'end of many eukaryotic mRNAs and has a 7-methylguanosine structure. Examples of the cap structure include cap0, cap1, cap2, and ARCA (Anti-Reverse Cap Analog), and the cap structure is as shown by the following structural formula.
- the cap structure of the mRNA of the present invention is preferably cap0 or cap1, and more preferably cap1.
- the sequence of the 5'untranslated region (5'-UTR) is, for example, the sequence of base numbers 2 to 70 in the sequence of SEQ ID NO: 2, the sequence of base numbers 2 to 70 in the sequence of SEQ ID NO: 4, and the sequence number of SEQ ID NO: 4. It is the sequence of base numbers 2 to 70 in the sequence of 6, the sequence of base numbers 2 to 70 in the sequence of SEQ ID NO: 11, and the sequence of base numbers 2 to 70 in the sequence of SEQ ID NO: 13.
- the sequence of the leader sequence is, for example, the sequences of base numbers 71 to 124 in the sequence of SEQ ID NO: 2, the sequences of base numbers 71 to 124 in the sequence of SEQ ID NO: 4, and the sequence of SEQ ID NO: 6. It is the sequence of base numbers 71 to 124, the sequence of base numbers 71 to 124 in the sequence of SEQ ID NO: 11, and the sequence of base numbers 71 to 124 in the sequence of SEQ ID NO: 13.
- the sequence of the translation region of E6 is a sequence capable of expressing all or part of the amino acid sequence of the E6 antigen, and may include a start codon and / or a terminal codon, for example, the base in the sequence of SEQ ID NO: 2.
- the sequence of the protease cleavage sequence is, for example, the sequence of base numbers 575 to 595 in the sequence of SEQ ID NO: 2, the sequence of base numbers 575 to 595 in the sequence of SEQ ID NO: 4, and the sequence of SEQ ID NO: 6.
- sequences of base numbers 575 to 595 are the sequences of base numbers 590 to 610 in the sequence of SEQ ID NO: 11, and the sequences of base numbers 590 to 610 in the sequence of SEQ ID NO: 13.
- the sequence of the translation region of E7 is a sequence capable of expressing all or part of the amino acid sequence of the E7 antigen, and may include a start codon and / or a terminal codon, for example, the base in the sequence of SEQ ID NO: 2.
- sequences of base numbers 596 to 889 the sequences of base numbers 596 to 889 in the sequence of SEQ ID NO: 4, the sequences of base numbers 596 to 889 in the sequence of SEQ ID NO: 6, and the sequences of base numbers 611 to 925 in the sequence of SEQ ID NO: 11.
- the sequence of the 3'untranslated region (3'-UTR) is, for example, the sequence of base numbers 890 to 1021 in the sequence of SEQ ID NO: 2, the sequence of base numbers 890 to 1021 in the sequence of SEQ ID NO: 4, and the sequence number of SEQ ID NO: 4.
- polyA tail (polyA) sequence is, for example, the sequences of base numbers 1022 to 1123 in the sequence of SEQ ID NO: 2, the sequences of base numbers 1022 to 1123 in the sequence of SEQ ID NO: 4, and the sequence of SEQ ID NO: 6. It is the sequence of base numbers 1022 to 1123, the sequence of base numbers 1058 to 1159 in the sequence of SEQ ID NO: 11, and the sequence of base numbers 1058 to 1159 in the sequence of SEQ ID NO: 13.
- Cap structure Cap
- 5'untranslated region 5'-UTR
- leader sequence E6 translation region
- protease cleavage sequence Furin Cleavage site
- E7 translation region 3'untranslated region
- 3'-UTR and polyA tail (polyA) sequences may be modified, and the sequences of nucleic acids capable of expressing HPV16 type E6 and E7 antigens are SEQ ID NOs: 2, 4 or It may consist of a nucleotide sequence having at least 90%, preferably 95%, more preferably 97% identity with any of the sequences of 6.
- sequence of the nucleic acid capable of expressing the HPV18 type E6 antigen and E7 antigen has at least 90%, preferably 95%, more preferably 97% identity with the sequence of either SEQ ID NO: 11 or 13. It is preferable that it consists of a nucleotide sequence having.
- the nucleic acid encapsulated in the lipid particles may be in any form as long as it is a nucleic acid capable of expressing the E6 antigen and E7 antigen of human papillomavirus.
- single-stranded DNA single-stranded RNA (eg, mRNA), single-stranded polynucleotide in which DNA and RNA are mixed, double-stranded DNA, double-stranded RNA, hybrid polynucleotide of DNA-RNA, DNA and RNA.
- RNA single-stranded DNA
- RNA single-stranded RNA
- Examples thereof include a double-stranded polynucleotide composed of two types of polynucleotides in which the above is mixed, and is preferably mRNA.
- the nucleotides constituting the nucleic acid encapsulated in the lipid particles may be natural or modified nucleotides, but may contain at least one modified nucleotide.
- the modified nucleotide may be one in which any part of the base, sugar and phosphodiester bond is modified.
- the modification site may be one site or two or more sites.
- Examples of base modifications are cytosine 5-methylation, 5-fluorolation, N4-methylation, uracil 5-methylation (thymine), 5-fluorolation, adenine N6-methylation, guanine N2. -Methylation etc. can be mentioned.
- sugar modification is 2'-O-methylation of D-ribofuranose.
- An example of modification of a phosphate diester bond is a phosphorothioate bond.
- the modified nucleotide is preferably one in which the base portion is modified, for example, a pyrimidine nucleotide in which the 5-position is substituted, and a pseudouridine in which the 1-position may be substituted.
- Examples thereof include 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkyl pseudouridine.
- the 1-alkyl pseudouridine may be 1- (C1-C6 alkyl) pseudopseudouridine, preferably 1-methylpseudouridine or 1-ethylseudouridine.
- More preferred modified nucleotides include 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine.
- Particularly preferred as the modified nucleotide are a combination of 5-methylcytidine and 5-methyluridine, or a combination of 5-methylcytidine and 1-methylpseudouridine.
- Nucleic acids capable of expressing the E6 and E7 antigens of the human papillomavirus of the present invention can be produced by in vitro transcription reaction from DNA having a desired base sequence.
- Enzymes required for in-vitro transcription, buffer, and nucleoside-5'-triphosphate mixture (adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), cytidine-5'-triphosphate Acids (CTP) and uridine-5'-triphosphate (UTP)) are commercially available (AmpliScribeT7 High Yield Transcription Kit (Epicentre), mMESSAGE mMACHINE T7 Ultra Kit (Life thechnologies), etc.).
- plasmid DNA or DNA fragment As the DNA used for producing the single-stranded RNA, cloned DNA, for example, plasmid DNA or DNA fragment is used.
- the plasmid DNA or DNA fragment may be commercially available or can be produced by a method generally known in the art (eg, Sambrook, J. et al., Molecular Cloning a Laboratory). Manual second edition (1989), Rashtchian, A., Current Opinion in Biotechnology, 1995, 6 (1), 30-36, Gibson D. G. et al., Science, 2008, 319 (5867), 1215-1220 Method of description, etc.).
- a cap structure (Cap0 structure described above) can be introduced into the 5'end of the mRNA by a method using a capping enzyme after the in vitro transcription reaction. Further, Cap0 can be converted to Cap1 by a method of allowing 2'-O-methyltransferase to act on mRNA having Cap0.
- Commercially available products can be used for the capping enzyme and 2'-O-methyltransferase (for example, Vaccinia Capping System, M2080; mRNA Cap 2'-O-Methyltransferase, M0366, both manufactured by New England Biolab).
- mRNA having a cap structure can be produced according to the protocol attached to the product.
- the cap structure at the 5'end of mRNA can also be introduced by a method other than that using an enzyme. For example, by adding ARCA or CleanCap to the in vitro transcription reaction, the structure of the cap analog possessed by ARCA or the Cap1 structure derived from CleanCap can be introduced into mRNA.
- ARCA and CleanCap can be used for ARCA and CleanCap (ARCA, N-7003; CleanCap Reagent AG, N-7113, both manufactured by TriLink BioTechnologies).
- ARCA and CleanCap ARCA, N-7003; CleanCap Reagent AG, N-7113, both manufactured by TriLink BioTechnologies.
- mRNA having a cap structure can be produced according to the protocol attached to the product.
- the nucleic acid-encapsulated lipid particles of the present invention are produced by a thin film method, a reverse phase evaporation method, an ethanol injection method, an ether injection method, a dehydration-rehydration method, a surfactant dialysis method, a hydration method, a freeze-thaw method, or the like. can do.
- nucleic acid-encapsulated lipid particles can be produced by the method described in International Publication No. 2015/005253.
- the nucleic acid-encapsulated lipid particles of the present invention can also be produced by mixing a nucleic acid solution and a lipid solution in a microchannel.
- NanoAssemblr® from Precision Nanosystems can be used and manufactured according to the method described in the attached protocol.
- the particles of the present invention preferably have an average particle size of 30 nm to 300 nm, preferably 30 to 200 nm, and more preferably 30 to 100 nm.
- the average particle size can be obtained by measuring the volume average particle size based on the principle of dynamic light scattering using equipment such as Zeta Potential / Particle Sizer NICOMP (registered trademark) 380ZLS (PARTICLE SIZING SYSTEMS). it can.
- the particles of the present invention produce compositions for preventing and / or treating diseases caused by human papillomavirus infection (cervical cancer, cervical dysplasia, anal cancer, oropharyngeal cancer, condyloma acuminata).
- Can be used for Infection is caused by HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 6, and 11 human papillomaviruses. It is often an infection, preferably HPV16 and / or HPV18, and more preferably HPV16.
- the E6 and E7 antigens of human papillomavirus can be expressed in vivo or in vitro. Therefore, the present invention provides a method for expressing the E6 and E7 antigens of human papillomavirus in vitro, which comprises introducing a composition containing the above particles into cells. The present invention also provides a method for expressing the E6 and E7 antigens of human papillomavirus in vivo, which comprises administering a composition containing the above particles to a mammal. By expressing the E6 and E7 antigens of human papillomavirus in vivo, an immune response against human papillomavirus can be induced.
- the present invention provides a method for inducing an immune response against human papillomavirus, which comprises administering a composition containing the above particles to a mammal.
- the present invention also provides a method for preventing and / or treating human papillomavirus infection, which comprises administering a composition containing the above particles to a mammal.
- the particles of the present invention can be used as pharmaceuticals and as experimental reagents.
- the particles of the present invention are usually added to a carrier such as water, buffer, saline, etc., and the formulation (composition) can be introduced into cells (in vitro) or administered to mammals (in vitro). vivo).
- the carrier may be a pharmaceutically acceptable carrier (eg, saline).
- the particles of the present invention are creams and pastes based on fat, fatty oil, lanolin, petrolatum, paraffin, wax, resin, plastic, glycols, higher alcohol, glycerin, water, emulsifier, suspending agent and the like. , Ointment, gel, lotion and the like.
- the particles of the present invention are orally or intramuscularly or intravenously administered to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, pigs, monkeys, cats, dogs, horses, goats, sheep and cows.
- Parenteral administration can be performed by methods such as administration, rectal administration, transdermal administration, transmucosal administration, subcutaneous administration, and intradermal administration.
- the particles of the present invention are administered to humans, for example, at a dose of about 0.001 to 1 mg, preferably 0.01 to 0.2 mg per adult, once or several times, intramuscular injection, subcutaneous injection, intradermal injection. Injection, intravenous drip injection, or intravenous injection may be performed, but the dose and frequency of administration may be appropriately changed depending on the type of disease, symptoms, age, administration method, and the like.
- the particles of the present invention can be used in cells that want to express the E6 and E7 antigens of human papillomavirus (for example, HEK293 cells and their derivatives (HEK293T cells, FreeStyle293 cells and Expi293 cells), CHO cells, C2C12. It can be introduced into mouse myoblasts and immortalized mouse dendritic cells (MutuDC1940)) to express the E6 and E7 antigens of human papillomavirus in vitro.
- HEK293 cells and their derivatives HEK293T cells, FreeStyle293 cells and Expi293 cells
- CHO cells C2C12. It can be introduced into mouse myoblasts and immortalized mouse dendritic cells (MutuDC1940)) to express the E6 and E7 antigens of human papillomavirus in vitro.
- E6 and E7 antigens of human papillomavirus For the expression of E6 and E7 antigens of human papillomavirus, the E6 and E7 antigen proteins of human papillomavirus in the sample are detected by Western blotting, and the peptide fragments specific to E6 and E7 antigens of human papillomavirus are mass-analyzed. It can be analyzed by detecting it by a method.
- treatment refers to recovery of clinical symptoms of these diseases in a patient who has developed an infection caused by a virus or a bacterium, or a disease caused by the infection (for example, precancerous lesion or cancer). Means delay in remission, alleviation and / or exacerbation.
- prevention means reducing the incidence of diseases caused by infectious diseases such as viruses or bacteria. Prevention includes reducing the risk of disease progression due to infections such as viruses or bacteria, or reducing the severity of those diseases.
- the particles of the present invention are effective in the prevention and / or treatment of the above-mentioned diseases because they induce a protective immune response.
- Example 1 Preparation of HPV16 E6-E7 fusion2 mRNA -001 (1) Preparation of template DNA for in vitro translation (IVT) of HPV16 E6-E7 fusion2 A plasmid was constructed to prepare a template DNA for use in in vitro translation (IVT).
- GCTAGC (NheI site), T7 promoter sequence, human ⁇ -globin 5'-UTR sequence, KOZAK sequence, IgE leader sequence-HPV16 type E6-Furin Cleavage site-HPV16 type E7 translation region, human ⁇ -globin 3' A plasmid (pMA-HPV16_fusion2) was prepared into which a DNA fragment (SEQ ID NO: 1) containing a sequence in which a UTR sequence, a poly A tail, and an ACTAGT (SpeI site) were linked in order was introduced.
- SEQ ID NO: 1 containing a sequence in which a UTR sequence, a poly A tail, and an ACTAGT (SpeI site) were linked in order was introduced.
- RQ1 RNase-Free DNase 100 ⁇ L, Promega catalog # M6101
- 8 M LiCl solution 2000 ⁇ L, Sigma-Aldrich catalog # L7026
- the supernatant was discarded, 70% ethanol was added, and after centrifugation (4 ° C, 4000 ⁇ g, 10 minutes), the supernatant was discarded and air-dried.
- the obtained residue was dissolved in Nuclease-free water and then purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the attached manual.
- Example 2 Preparation of HPV16 E6-E7 fusion10 mRNA -001 (1) Preparation of template DNA for IVT of HPV16 E6-E7 fusion 10 A plasmid was constructed to prepare a template DNA to be used for the IVT template.
- GCTAGC (NheI site), T7 promoter sequence, ⁇ -globin 5'-UTR sequence, KOZAK sequence, IgE leader sequence-HPV16 type E6-Furin Cleavage site-HPV16 type E7 translation region, ⁇ -globin 3'-UTR
- pMA-HPV16_fusion10 into which a DNA fragment (SEQ ID NO: 3) containing a sequence in which a sequence, a poly A tail, and an ACTAGT (SpeI site) were linked in order was introduced was prepared.
- Example 1 -MRNA was obtained in the same manner as in (2) and (3).
- the obtained mRNA has the sequence of SEQ ID NO: 4. It was analyzed by Experion RNA Std Sens and confirmed to be the desired length.
- Example 3 Preparation of HPV16 E6-E7 fusion10 mRNA -002 Using the plasmid obtained in Example 2- (1) instead of the plasmid obtained in Example 1- (1), Example 1-( The plasmid DNA was obtained in the same manner as in 2). Then, the template DNA obtained in place of the template DNA of Example 1- (3) was used in Example using 100 mM Pseudo-UTP (Hongene catalog # R5-022) instead of 100 mM 5-Me-UTP. MRNA was obtained in the same manner as in 1- (3).
- the obtained mRNA has the sequence of SEQ ID NO: 4. It was analyzed by Experion RNA Std Sens and confirmed to be the desired length.
- Example 4 Preparation of HPV16 E6-E7 fusion10 mRNA -003 Using the plasmid obtained in Example 2- (1) instead of the plasmid obtained in Example 1- (1), Example 1-( The plasmid DNA was obtained in the same manner as in 2). Then, instead of 100 mM 5-Me-CTP, the template DNA obtained in place of the template DNA of Example 1- (3) was used instead of 100 mM CTP (Hongene catalog # R3331) and 100 mM 5-Me-UTP. Using 100 mM N1-methylpseudouridine-5'-triphosphate (TriLink catalog # N-1081), mRNA was obtained in the same manner as in Example 1- (3).
- TriLink catalog # N-1081 100 mM N1-methylpseudouridine-5'-triphosphate
- the obtained mRNA has the sequence of SEQ ID NO: 4. It was analyzed by Experion RNA Std Sens and confirmed to be the desired length.
- Example 5 Preparation of HPV16 E6-E7 fusion10 mRNA -004 Using the plasmid obtained in Example 2- (1) instead of the plasmid obtained in Example 1- (1), Example 1-( The plasmid DNA was obtained in the same manner as in 2). Then, the template DNA obtained in place of the template DNA of Example 1- (3) was used in place of 100 mM 5-Me-CTP using 100 mM CTP (Hongene catalog # R3331), and Example 1- (3) was used. ) was obtained in the same manner as in).
- the obtained mRNA has the sequence of SEQ ID NO: 4. It was analyzed by Experion RNA Std Sens and confirmed to be the desired length.
- GCTAGC (NheI site), T7 promoter sequence, ⁇ -globin 5'-UTR sequence, KOZAK sequence, IgE leader sequence-HPV16 type E6-Furin Cleavage site-HPV16 type E7 translation region, ⁇ -globin 3'-UTR
- pMA-HPV16_fusion10_opt2 a DNA fragment into which a DNA fragment (SEQ ID NO: 5) containing a sequence in which a sequence, a poly A tail, and an ACTAGT (SpeI site) were linked in order was introduced was prepared.
- RQ1 RNase-Free DNase 50 ⁇ L, Promega catalog # M6101
- 8 M LiCl solution 1000 ⁇ L, Sigma-Aldrich catalog # L7026
- the supernatant was discarded, 70% ethanol was added, and after centrifugation (4 ° C, 4000 ⁇ g, 10 minutes), the supernatant was discarded and air-dried.
- the obtained residue was dissolved in Nuclease-free water.
- Nuclease-free water (2818 ⁇ L) was added to this solution (962 ⁇ L, UV equivalent 5400 ⁇ g), heated at 70 ° C. for 20 minutes, and then cooled under ice cooling for 10 minutes.
- Nuclease 10 ⁇ capping buffer 500 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl 2 , 50 mM DTT) at 540 ⁇ L, 20 mM GTP (270 ⁇ L, 100 mM GTP, Hongene catalog # R2331) Dilute with free water), 20 mM SAM (270 ⁇ L, 32 mM SAM, New England Biolabs catalog # B9003S diluted with Nuclease-free water), Vaccinia Capping Enzyme (540 ⁇ L, Hongene catalog # ON-) 028) was added, and after incubation at 37 ° C.
- Nuclease 10 ⁇ capping buffer 500 mM Tris-HCl
- the obtained mRNA has the sequence of SEQ ID NO: 6. It was analyzed by Experion RNA Std Sens and confirmed to be the desired length.
- RQ1 RNase-Free DNase 100 ⁇ L, Promega catalog # M6101
- 8 M LiCl solution 2000 ⁇ L, Sigma-Aldrich catalog # L7026
- the supernatant was discarded, 70% ethanol was added, and after centrifugation (4 ° C, 4000 ⁇ g, 10 minutes), the supernatant was discarded and air-dried.
- the obtained residue was dissolved in Nuclease-free water.
- Nuclease-free water (2610 ⁇ L) was added to this solution (1590 ⁇ L, UV equivalent 6000 ⁇ g), heated at 70 ° C. for 10 minutes, and then cooled under ice cooling for 10 minutes.
- Nuclease 10 ⁇ capping buffer 500 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl 2 , 50 mM DTT) 600 ⁇ L, 20 mM GTP (300 ⁇ L, 100 mM GTP, Hongene catalog # R2331) Dilute with free water), 20 mM SAM (300 ⁇ L, S-adenosyl-L-methionine disulfate tosylate, OX-CHEM catalog # AX8250818 dissolved in 0.005 M sulfuric acid, 10% ethanol solution), Vaccinia Capping After adding Enzyme (600 ⁇ L, Hongene catalog # ON-028) and incubating at 37 ° C for 4 hours, 10
- the obtained mRNA has the sequence of SEQ ID NO: 6. Analysis was performed by LabChip GX Touch HT mRNA StdSens (PerkinElmer catalog # CLS960010), and it was confirmed that the length was the desired length.
- Example 7 Preparation of HPV16 E6-E7 fusion10 opt2 mRNA -002 Instead of 100 mM 5-Me-CTP in Example 6- (3), instead of 100 mM 5-Me-CTP, 100 mM N1-methylpseudouridine-5'-triphosphate. Using 100 mM 5-methyluridine triphosphate, mRNA was obtained in the same manner as in Example 6- (3). The obtained mRNA has the sequence of SEQ ID NO: 6. It was analyzed by Experion RNA StdSens and confirmed to be the desired length.
- Example 8 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles according to Example 1.
- LP 1,2-distearoyl-sn-glycerol methoxypolyethylene glycol
- HPV16 fusion2 mRNA-001 obtained in Example 1 was prepared to 51.8 ⁇ g / mL with citric acid buffer (20 mM Citrate Buffer, pH 4.0).
- lipid solution and mRNA solution were mixed in a microchannel using NanoAssemblr BenchTop (Precision Nanosystems Inc.) so that the volume ratio was 1: 3, and a crude dispersion of nucleic acid lipid particles was obtained.
- Dialysis (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) of the dispersion of nucleic acid lipid particles with about 25 to 50 times the amount of phosphate buffer (pH 7.4) for 12 to 18 hours. , Ethanol was removed to obtain a dispersion of purified mRNA-encapsulated nucleic acid lipid particles.
- LP was synthesized according to the method described in Example 23 of WO2015005253.
- the amount of each lipid in the dispersion liquid of the nucleic acid lipid particles was measured by reverse phase chromatography (System: DIONEX UltraMate 3000, MeOH: XSelect CSH (50 mm ⁇ 3 mm, 5 ⁇ m) (Thermo Fisher Scientific), Buffer A: 0.2. % Formic Acid, Buffer, B: 0.2% Formic Acid, Methanol, (B%): 75-95% (0-15 min), 95% (15-17 min), Flow Rate: 0.4 mL / min, Temperature: 50 ° C., Measurement: Colona CAD (Charged Aerosol Detector). The ratio of the total amount of lipid to mRNA was calculated by the following formula.
- the particle size of an average particle diameter of the nucleic acid-lipid particles was measured by Zeta Potential / Particle Sizer NICOMPTM 380ZLS ( PARTICLE SIZING SYSTEMS).
- the average particle size in the table represents the volume average particle size, and ⁇ or less represents the deviation.
- Example 9 to 13 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles
- Preparation and characteristic evaluation of mRNA-encapsulated nucleic acid lipid particles according to Examples 2, 3, 4, 5, and 6 in the same manner as in Example 8. was carried out.
- the results are shown in Table 1.
- Example 14 to 17 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles
- the mRNA-encapsulated nucleic acid lipid particles according to Examples 2, 4, 6 and 7 were prepared and their characteristics were evaluated in the same manner as in Example 8. did.
- Example 18 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles described in Example 2
- the mRNA-encapsulated nucleic acid lipid particles described in Example 2 were prepared and their characteristics were evaluated in the same manner as in Example 8.
- Table 1 The results are shown in Table 1.
- Example 19 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles described in Example 2
- the mRNA-encapsulated nucleic acid lipid particles described in Example 2 were prepared and their characteristics were evaluated in the same manner as in Example 8.
- the results are shown in Table 1.
- Example 20 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles described in Example 2 The mRNA-encapsulated nucleic acid lipid particles described in Example 2 were prepared and their characteristics were evaluated in the same manner as in Example 8. The results are shown in Table 1.
- Example 21 Preparation of OVA mRNA-encapsulated nucleic acid lipid particles
- Nucleic acid lipid particles encapsulating mRNA having the translation region of OVA (Ovalbumin) shown in SEQ ID NO: 7 were prepared in the same manner as in Example 8- (1).
- OVA ovalbuproin
- N- [methoxypoly (ethylene glycol) 2000] carbamoyl] -1,2-dimyristyloxypropyl-3-amine (N- [methoxy poly (ethylene)) having a polyethylene glycol molecular weight of about 2000 is used instead of PEG-DMG.
- N- [methoxypoly (ethylene glycol) 2000] carbamoyl] -1,2-dimyristyloxypropyl-3-amine (N- [methoxy poly (ethylene)) having a polyethylene glycol molecular weight of about 2000 is used instead of PEG-DMG.
- Ratio of mRNA to lipid The amount of mRNA in the dispersion liquid of nucleic acid lipid particles was defined as the "amount of mRNA in the presence of a surfactant" in (2-1). The amount of phospholipid in the dispersion of nucleic acid lipid particles was measured using phospholipid C-Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.) according to the package insert.
- the amount of phospholipid in the sample was measured in the presence of 2% Triton X-100 surfactant.
- the amount of cholesterol and LP in the dispersion liquid of nucleic acid lipid particles was measured by reverse phase chromatography (System: DIONEX UltiMate 3000, Volume: Chromolith Performance RP-18 endcapped 100-3 monolithic HPLC-colum. 1.52001.0001), Buffer A: 0.01% trifluoroacetic acid, Buffer, B: 0.01% trifluoroacetic acid, nucleic acid, (B%): 82-97% (0-17 min), Flow Rate: 2 mL / min, System: 50 ° C., Detection: Cholesterol CAD (Charged Aerosol Detector)).
- the total amount of lipid was calculated from the measured values of phospholipid, cholesterol and LP and the composition ratio of the lipid components constituting the nucleic acid lipid particles.
- the ratio of the total amount of lipid to mRNA was calculated by the following formula. [Total lipid concentration] / [MRNA concentration] (wt / wt) (2-3) Average particle size
- the particle size of the nucleic acid lipid particles was measured by Zeta Potential / Solid Sizer NICOMPTM 380ZLS (PARTICLE SIZING SYSTEMS).
- the average particle size in the table represents the volume average particle size, and ⁇ or less represents the deviation. The results are shown in Table 2.
- Example 22 Preparation of OVA mRNA-encapsulated nucleic acid lipid particles
- OVA Ovalbumin
- Example 23 Preparation of OVA mRNA-encapsulated nucleic acid lipid particles
- OVA Ovalbumin
- Example 24 Preparation of OVA mRNA-encapsulated nucleic acid lipid particles
- OVA Ovalbumin
- Example 25 Preparation of OVA mRNA-encapsulated nucleic acid lipid particles
- OVA ovalbuproin
- the results are shown in Table 2.
- Example 26 Preparation of OVA mRNA-encapsulated nucleic acid lipid particles
- OVA Ovalbumin
- Example 27 Preparation of OVA mRNA-encapsulated nucleic acid lipid particles
- OVA Ovalbumin
- Example 28 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles described in Example 4
- the mRNA-encapsulated nucleic acid lipid particles described in Example 4 were prepared and their characteristics were evaluated in the same manner as in Example 8.
- dipalmitoylphosphatidylcholine (1,2-Dioleoyl-sn-glycero-3-phosphocholine: hereinafter referred to as DOPC, NOF CORPORATION
- DOPC dipalmitoylphosphatidylcholine
- DOPC dipalmitoylphosphatidylcholine
- LP LP
- the molar ratio was 10: 43.5: 45: 1.5.
- Table 3 The results are shown in Table 3.
- Example 29 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles described in Example 4
- the mRNA-encapsulated nucleic acid lipid particles described in Example 4 were prepared and their characteristics were evaluated in the same manner as in Example 8.
- the results are shown in Table 3.
- Example 30 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles described in Example 4
- the mRNA-encapsulated nucleic acid lipid particles described in Example 4 were prepared and their characteristics were evaluated in the same manner as in Example 8.
- dioleoil phosphatidylethanolamine (1,2-Dioleoyl-sn-glycero-3-phosphatidylamine: hereinafter referred to as DOPE, NOF CORPORATION
- DOPE dioleoil phosphatidylethanolamine
- DOPE NOF CORPORATION
- Table 3 The results are shown in Table 3.
- Example 31 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles described in Example 4
- the mRNA-encapsulated nucleic acid lipid particles described in Example 4 were prepared and their characteristics were evaluated in the same manner as in Example 8.
- the results are shown in Table 3.
- Example 32 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles described in Example 4
- the mRNA-encapsulated nucleic acid lipid particles described in Example 4 were prepared and their characteristics were evaluated in the same manner as in Example 8.
- the results are shown in Table 3.
- Example 33 Preparation of HPV18 E6-E7 fusion1 opt1 mRNA-001 (1) Preparation of template DNA for in vitro translation (IVT) of HPV18 E6-E7 fusion1 opt1 A plasmid was constructed to prepare a template DNA for use in in vitro translation (IVT).
- GCTAGC (NheI site), T7 promoter sequence, human ⁇ -globin 5'-UTR sequence, KOZAK sequence, IgE leader sequence-HPV18 type E6-Furin Cleavage site-HPV18 type E7 ORF, human ⁇ -glob A plasmid (pMA-HPV18_fusion1_opt1) into which a DNA fragment (SEQ ID NO: 10) containing a sequence in which a UTR sequence, a poly A tail, and an ACTAGT (SpeI site) were linked in order was introduced was prepared.
- T-7113 100 mM ATP (150 ⁇ L, Hongene catalog # R1331), 100 mM GTP (150 ⁇ L, Hongene catalog # R2331), 100 mM CTP (150 ⁇ L, Hongene catalog # R3331), 100 mM UTP (150 ⁇ L, Hongene catalog # R5-027), Nuclease-free water (1200 ⁇ L, Thermo Fisher catalog # AM9937), T7 Transcription 5 ⁇ buffer (600 ⁇ L) 300 ⁇ L, Promega catalog # P137X) was mixed and incubated at 37 ° C. for 4 hours.
- RQ1 RNase-Free DNase (75 ⁇ L, Promega catalog # M6101) was mixed and incubated at 37 ° C. for 15 minutes.
- 8 M LiCl solution (1500 ⁇ L, Sigma-Aldrich catalog # L7026) was mixed and allowed to stand overnight at ⁇ 20 ° C. After centrifugation (4 ° C., 4000 xg, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (4 ° C., 4000 xg, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in Nuclease-free water and then purified using RNeasy Maxi kit (Qiagen catalog # 75162) according to the attached manual.
- Example 34 Preparation of HPV18 E6-E7 fusion1 opt1 mRNA-002 100 mM 5-Me-CTP (Hongene catalog # R3-029) instead of 100 mM CTP in Example 33- (3).
- MRNA was obtained in the same manner as in Example 33- (3) using 100 mM 5-methyllidene triphosphate instead of 100 mM N1-Me-Pseudo UTP.
- the resulting mRNA has the sequence of SEQ ID NO: 11. It was analyzed by LabChip GX Touch Stand RNA Reagent Kit (PerkinElmer catalog # CLS960010), and it was confirmed that the length was the desired length.
- Example 35 Preparation of HPV18 E6-E7 fusion1 opt2 mRNA-001 (1) Preparation of template DNA for IVT of HPV18 E6-E7 fusion1 opt2 A plasmid was constructed to prepare a template DNA to be used for the IVT template.
- GCTAGC (NheI site), T7 promoter sequence, ⁇ -globin 5'-UTR sequence, KOZAK sequence, IgE leader sequence-HPV18 type E6-Furin Cleavage site-HPV18 type E7 ORF, ⁇ -globin 3'-UTR ,
- a DNA fragment (SEQ ID NO: 12) containing a sequence in which the poly A tail and the ACTAGT (SpeI site) were linked in order was introduced into a plasmid (pMA-HPV18_fusion1_opt2).
- RQ1 RNase-Free DNase (75 ⁇ L, Promega catalog # M6101) was mixed and incubated at 37 ° C. for 15 minutes.
- 8 M LiCl solution (1500 ⁇ L, Sigma-Aldrich catalog # L7026) was mixed and allowed to stand overnight at ⁇ 20 ° C. After centrifugation (4 ° C., 4000 xg, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (4 ° C., 4000 xg, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in Nuclease-free water.
- Nuclease-free water (2780 ⁇ L) was added to a part of this solution (2470 ⁇ L, 7500 ⁇ g in terms of UV), heated at 70 ° C. for 10 minutes, and then cooled under ice cooling for 5 minutes.
- 10 ⁇ capping buffer 500 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl 2 , 50 mM DTT) 750 ⁇ L, 20 mM GTP (375 ⁇ L, 100 mM GTP, Hongene catalog #) Used by diluting with Nuclease-free water), 20 mM SAM (375 ⁇ L, 32 mM SAM, New England Biolabs catalog # B9003S diluted with Nuclease-free water # B9003S), Vac ON-028) was added, and after incubation at 37 ° C.
- 10 ⁇ capping buffer 500 mM Tris-HCl (pH 8.0), 50 mM KC
- Example 36 Preparation of HPV18 E6-E7 fusion1 opt2 mRNA-002 100 mM 5-Me-CTP (Hongene catalog # R3-029) instead of 100 mM CTP in Example 35- (3).
- MRNA was obtained in the same manner as in Example 35- (3) using 100 mM 5-methyllidene triphosphate instead of 100 mM N1-Me-Pseudo UTP.
- the obtained mRNA has the sequence of SEQ ID NO: 13. It was analyzed by LabChip GX Touch Stand RNA Reagent Kit (PerkinElmer catalog # CLS960010), and it was confirmed that the length was the desired length.
- Examples 37-40 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles (1) Preparation of mRNA-encapsulated nucleic acid lipid particles The mRNAs of Examples 33 to 36 were used instead of the mRNA of Example 1, and the mRNA was prepared in the same manner as in Example 8 according to the lipid molar ratio shown in Table 4. Encapsulation nucleic acid lipid particles were prepared and their characteristics were evaluated. The results are shown in Table 4.
- Example 41-52 Preparation of HPV mRNA-encapsulated nucleic acid lipid particles (1) Preparation of mRNA-encapsulated nucleic acid-lipid particles The mRNA-encapsulated nucleic acid-lipid particles described in Example 4 were prepared in the same manner as in Example 8.
- HEK293T cells which are 2 ⁇ 10 4 human fetal kidney cell lines, were seeded per well on a 96-well plate and cultured overnight at 37 ° C. and a CO 2 concentration of 5%. Then, the mRNA-encapsulated nucleic acid lipid particles of Examples 14 to 17 were added to HEK293T cells so that the final mRNA concentration was 0.3-10 ⁇ g / mL, and the cells were cultured at 37 ° C. and a CO 2 concentration of 5% for 48 hours.
- the 96-well plate was allowed to stand at 4 ° C. for 1 hour, and 300 ⁇ L of PBS (-) (PBST) containing 0.05% Tween 20 was added per well and washed 3 times.
- the HPV genotype 16 E7-expressing protein (16E7) immobilized on the well of the plate was then reacted at room temperature for 2 hours with the addition of an anti-16E7 antibody labeled horse radish peroxidase (HRP) to PBST. After washing the well 3 times with, HRP substrate was added and detected.
- the 16E7 protein expression level was calculated by subtracting the background absorbance at 540 nm from the absorbance at 450 nm for each well.
- the thigh muscles of 7-week-old C57BL / 6 mice were inoculated twice at 10-day intervals with 4-10 ⁇ g of mRNA-encapsulated nucleic acid lipid particles per animal.
- 40 ⁇ g of DNA was administered per animal by the electroporation method.
- the administration conditions for the electroporation method were 30 V, 50 ms ON / 100 ms OFF, and 3 cycles.
- the electroporation method was also inoculated twice at 10-day intervals.
- PBMCs Peripheral blood mononuclear cells
- spleen cells were prepared by collecting peripheral blood in the presence of heparin 1 week after the final immunization, or 1 week or 2 weeks after the final immunization, and evaluating samples. And said.
- Induction levels of cytotoxic T cells (CTL) specific for HPV genotype 16E7 vaccine antigens in PBMCs and spleen cells are measured by antibodies against T cell surface markers and immunohistochemistry by the tetramer complex of MHC class I molecules and 16E7 epitopes. After staining, it was measured using the FACS method.
- CTL cytotoxic T cells
- 16E7 recombinant protein was solid-phased on a 96-well plate at a concentration of 0.5 ⁇ g / mL at 4 ° C. overnight.
- a dilution series of mouse IgG proteins of known concentration was also prepared and immobilized on the same plate for a standard curve.
- blocking treatment was performed with PBST (1% BSA / PBST) containing 1% BSA for 1 hour. Plasma samples were serially diluted with 1% BSA / PBST, added to a 16E7 solid phase well, and reacted at room temperature for 2 hours.
- mice IgG standard curve were reacted in the same manner with the addition of 1% BSA / PBST. After washing 3 times with PBST, HRP-labeled anti-mouse IgG antibody was added to each well and reacted at room temperature for 1 hour. After washing with PBST three times, an HRP substrate was added to develop color, and a 1N sulfuric acid solution was added as a color stop solution. The mouse IgG concentration bound to 16E7 was calculated using a standard curve after subtracting the background absorbance at 540 nm from the absorbance at 450 nm in each well.
- TC-1 cancer cell transplantation experiment using C57BL / 6 mice and cancer regression effect of mRNA-encapsulated nucleic acid lipid particles (Fig. 4-6)
- a TC-1 cancer cell line expressing HPV genotype 16 E6 and E7 in C57BL / 6 mouse lung epithelial cells was administered subcutaneously to the abdomen of the mouse, and the tumor size was measured over time.
- the abdomen of the mouse which is a cancer transplantation site, was shaved before TC-1 cancer cell transplantation, and 1 ⁇ 10 5 TC-1 cells were transplanted per animal.
- mRNA-encapsulated nucleic acid lipid particles of Example 20 10 ⁇ g of mRNA per animal was intramuscularly administered 8 days after the TC-1 cell transplantation. Antibody administration for removing CD4-positive cells and CD8-positive cells was started 2 days before administration of mRNA-encapsulated nucleic acid lipid particles.
- a dilution series of mouse IgG proteins of known concentration was also prepared and immobilized on the same plate for a standard curve. Then, after washing with PBST three times, blocking treatment was performed with PBST (1% BSA / PBST) containing 1% BSA for 1 hour. Serum samples were serially diluted with 1% BSA / PBST, added to the OVA solid phase well, and reacted at room temperature for 2 hours. Wells for the mouse IgG standard curve were reacted in the same manner with the addition of 1% BSA / PBST. After washing 3 times with PBST, HRP-labeled anti-mouse IgG antibody was added to each well and reacted at room temperature for 1 hour.
- mice IgG concentration bound to 16E7 was calculated using a standard curve after subtracting the background absorbance at 540 nm from the absorbance at 450 nm in each well.
- the OVA antigen-specific cytokine production level was evaluated using the cytokine ELISA method using the culture supernatant as a sample.
- IFN- ⁇ which is a typical cytokine produced from CTL and Th1, was measured.
- HPV genotype 18E6E7 vaccine antigen-specific T cell cytokine-producing ability (Fig. 10) The C57BL / 6J mouse was purchased from Claire Japan. All treatments of the animals were performed under inhalation anesthesia with isoflurane. In the gastrocnemius muscle of 6-week-old C57BL / 6 mice, 5 ⁇ g of mRNA-encapsulated nucleic acid lipid particles per mRNA were administered twice at 2-week intervals. One week after the final administration, the spleen was collected and spleen cells were prepared.
- Spleen cells were treated with HPV18E6 pool peptide (JPT, catalog # PM-HPV18-E6), and the amount of IFN- ⁇ in the culture supernatant after culturing for 48 hours was measured by the cytokine ELISA method.
- HPV18E6 pool peptide JPT, catalog # PM-HPV18-E6
- cytotoxic T cells CTL
- HPV16E7 HPV genotype 16 E7
- MHC class I molecule-binding HPV16E7 epitopes and MHC class I molecules The levels of induction of cytotoxic T cells (CTL) specific for HPV genotype 16 E7 (HPV16E7) in spleen cells are antibody to T cell surface markers and MHC class I molecule-binding HPV16E7 epitopes and MHC class I molecules. Immunostaining was performed using the complex of the above, and the measurement was performed using the flow cytometry method.
- CTL cytotoxic T cells
- the mRNA-encapsulated nucleic acid lipid particles of Examples 9, 11 and 13 showed CTL induction levels equal to or higher than pDNA ( Right figure).
- mRNA-encapsulated nucleic acid lipid particles CTL-inducing ability in mice of Examples 14 to 17 (Fig. 3)
- 16E7-specific CTL induction levels of four mRNA-encapsulated nucleic acid lipid particles in C57BL / 6 mice The results are shown in FIG. 16E7-specific CTL induction levels were observed in all four mRNA-encapsulated nucleic acid lipid particles, although there were variations among individuals.
- the mRNA-encapsulated nucleic acid lipid particle administration group from which CD8-positive cells were removed also showed the same antibody induction level as the Example 20 (No-depletion) group.
- the mRNA-encapsulated nucleic acid lipid particle administration group from which CD4 positive cells were removed Comparably, compared with the Example 20 (No-depletion) group and the Example 20 (CD8-depletion) group, It was revealed that the 16E7 antibody induction level was decreased.
- the mRNA-encapsulated nucleic acid lipid particle administration group from which CD4 positive cells were removed showed a lower CTL induction level as compared with the Example 20 (No-depletion) group.
- the 16E7-specific CTL induction level was dramatically higher than that in the Example 20 (No-depletion) group. It became clear that it was decreasing.
- the mRNA-encapsulated nucleic acid lipid particle administration group (Example 20 (CD4-depletion)) from which CD4 positive cells were removed showed a TC-1 cancer growth inhibitory effect, similarly to the Example 20 (No-depletion) group. It was.
- TC-1 cancer growth was not suppressed as compared with the Example 20 (No-depletion) group.
- the tumor size was similar to that of the control group (No-depletion). From the above results, it was suggested that CD8-positive cells may be essential for the cancer growth inhibitory effect of administration of mRNA-encapsulated nucleic acid lipid particles.
- MRNA-encapsulated nucleic acid lipid particles having different lipid compositions OVA-specific antibody induction levels of Examples 21 to 27 (Fig. 7)
- the mRNA-encapsulated nucleic acid lipid particles of Examples 21 to 27 were intramuscularly administered to C57BL / 6 mice, and the blood OVA-specific antibody induction level one week after the final immunization was examined. The results are shown in Fig. 7.
- the OVA-specific antibody induction level was low in the mRNA-encapsulated nucleic acid-lipid particles of Example 22, and was comparable in the other mRNA-encapsulated nucleic acid-lipid particles-administered groups.
- MRNA-encapsulated nucleic acid lipid particles having different lipid compositions OVA-specific IFN- ⁇ induction levels of Examples 21 to 27 (Fig. 8) The mRNA-encapsulated nucleic acid lipid particles of Examples 21 to 27 were intramuscularly administered to C57BL / 6 mice, and the OVA-specific T cell cytokine induction level from spleen cells was examined one week after the final immunization. The results are shown in Fig. 8.
- Example 26 In the mRNA-encapsulated nucleic acid lipid particle immunity group of Example 26, the level of IFN- ⁇ induction to the stimulation of MHC class I-binding epitope peptide of OVA and OVA protein was the lowest, and Example 21, Example 22, Example 25, The OVA antigen-specific induction levels of Example 27 were comparable.
- RNA-encapsulated nucleic acid lipid particles with different mRNA modifications HPV18E6-specific cytokine production induction levels of Examples 37-40 (Fig. 10)
- the mRNA-encapsulated nucleic acid lipid particles of Examples 37 to 40 were intramuscularly administered to C57BL / 6 mice, and the amount of HPV18E6-specific T cell cytokines from spleen cells was examined one week after the final immunization. The results are shown in FIG. In each group of Examples 37 to 40, IFN- ⁇ production induction for HPV18E6 pool peptide treatment was observed as compared with the NC group (Fig. 10).
- MRNA-encapsulated nucleic acid lipid particles having different lipid composition ratios CTL-inducing ability of Examples 41 to 52 (Fig. 11)
- the mRNA-encapsulated nucleic acid lipid particles of Examples 41 to 52 were intramuscularly administered to C57BL / 6 mice, and 16E7-specific CTL induction levels in spleen cells were evaluated one week after the final immunization. The results are shown in FIG. 16E7-specific CTL induction levels were higher in all mRNA-encapsulated nucleic acid lipid particles evaluated compared to the NC group.
- MRNA-encapsulated nucleic acid lipid particles having different lipid composition ratios HPV16E7-specific IFN- ⁇ induction levels of Examples 41 to 52 (Fig. 12)
- the mRNA-encapsulated nucleic acid lipid particles of Examples 41 to 52 were intramuscularly administered to C57BL / 6 mice, and the HPV16E7-specific T cell cytokine induction level from spleen cells was examined one week after the final immunization. The results are shown in FIG. IFN- ⁇ production was enhanced by MHC class I-binding epitope peptide treatment of HPV16E7 in all mRNA-encapsulated nucleic acid lipid particle-administered groups compared to the NC group. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
- the present invention can be used for the prevention and / or treatment of infection caused by human papillomavirus.
- GCTAGC (NheI site): Base numbers 1 to 6 T7 promoter sequence: base numbers 8-27 5'-UTR sequence of human ⁇ -globin: base numbers 39-88 KOZAK sequence: base numbers 89-94 Translation region of IgE leader sequence: Base numbers 95-148 Translation region of HPV16 type E6: Base numbers 149-598 Furin cleavage site: Base numbers 599-619 Translation region of HPV16 type E7: Base numbers 620-913 3'-UTR sequence of human ⁇ -globin: base numbers 914 to 1045 poylA: Base numbers 1046 to 1147 ACTAGT (SpeI site): Base numbers 1152-1157 ⁇ SEQ ID NO: 2> HPV16 E6-E7 fusion2 mRNA -001.
- GCTAGC NheI site: Base numbers 1 to 6 T7 promoter sequence: base numbers 8-27 5'-UTR sequence of human ⁇ -globin: base numbers 39-88 KOZAK sequence: base numbers 89-94 Translation region of IgE leader sequence: Base numbers 95-148 Translation region of HPV16 type E6: Base numbers 149-598 Furin cleavage site: Base numbers 599-619 Translation region of HPV16 type E7: Base numbers 620-913 3'-UTR sequence of human ⁇ -globin: base numbers 914 to 1045 poylA: Base numbers 1046 to 1147 ACTAGT (SpeI site): Base numbers 1152-1157 ⁇ SEQ ID NO: 4> HPV16 E6-E7 fusion10 mRNA -001 to 006.
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Abstract
Description
(1)ヒトパピローマウイルスのE6抗原及びE7抗原を発現させることができる核酸を封入した脂質粒子であって、脂質が一般式(Ia)で表されるカチオン性脂質、又はその薬学的に許容される塩を含む前記粒子。
R1及びR2は、独立して、C1-C3アルキル基を示し;
L1は、C2-C4アルカノイルオキシ基を1若しくは複数個有していてもよいC17-C19アルケニル基を示し;
L2は、C2-C4アルカノイルオキシ基を1若しくは複数個有していてもよいC10-C19アルキル基、又はC2-C4アルカノイルオキシ基を1若しくは複数個有していてもよいC10-C19アルケニル基を示し;
pは、3、又は4である。
(2)一般式(Ia)中のR1及びR2が、共にメチル基である、(1)に記載の粒子。
(3)一般式(Ia)中のpが、3である(1)又は(2)に記載の粒子。
(4)一般式(Ia)中のL1が、アセトキシ基を1若しくは複数個有していてもよいC17-C19アルケニル基である、(1)~(3)のいずれかに記載の粒子。
(5)一般式(Ia)中のL2が、アセトキシ基を1若しくは複数個有していてもよいC10-C12アルキル基、又はアセトキシ基を1若しくは複数個有していてもよいC10-C19アルケニル基である、(1)~(4)のいずれかに記載の粒子。
(6)一般式(Ia)中のL2が、アセトキシ基を1若しくは複数個有していてもよいC10-C12アルキル基、又はアセトキシ基を1若しくは複数個有していてもよいC17-C19アルケニル基である、(1)~(4)のいずれかに記載の粒子。
(7)一般式(Ia)中のL1が、(R)-11-アセチルオキシ-cis-8-ヘプタデセニル基、cis-8-ヘプタデセニル基、又は(8Z,11Z)-ヘプタデカジエニル基である(1)~(6)のいずれかに記載の粒子。
(8)一般式(Ia)中のL2が、デシル基、cis-7-デセニル基、ドデシル基、又は(R)-11-アセチルオキシ-cis-8-ヘプタデセニル基である、(1)~(7)のいずれかに記載の粒子。
(9)カチオン性脂質が下記の構造式:
(10)カチオン性脂質が下記の構造式:
で表される(1)に記載の粒子。
(11)カチオン性脂質が下記の構造式:
(12)脂質が、さらに、両親媒性脂質、ステロール類及びPEG脂質を含む(1)~(11)のいずれかに記載の粒子。
(13)両親媒性脂質が、ジステアロイルホスファチジルコリン、ジオレオイルホスファチジルコリン及びジオレオイルホスファチジルエタノールアミンからなる群より選択される少なくとも1つである(12)記載の粒子。
(14)ステロール類がコレステロールである(12)又は(13)に記載の粒子。
(15)PEG脂質が、1、2-ジミリストイル-sn-グリセロール メトキシポリエチレン グリコール及び/又はN-[メトキシ ポリ(エチレングリコール)2000]カルバモイル]-1,2-ジミリスチルオキシプロピル-3-アミンである(12)~(14)のいずれかに記載の粒子。
(16)両親媒性脂質、ステロール類、カチオン性脂質、及びPEG脂質の脂質組成が、モル量にて、両親媒性脂質が15%以下、ステロール類が20~55%、カチオン性脂質が40~65%、PEG脂質が1~5%であり、核酸重量に対する総脂質重量の比率が、15~30である(12)~(15)のいずれかに記載の粒子。
(17)両親媒性脂質、ステロール類、カチオン性脂質、及びPEG脂質の脂質組成が、モル量にて、両親媒性脂質が5~15%、ステロール類が35~50%、カチオン性脂質が40~55%、PEG脂質が1~3%であり、核酸重量に対する総脂質重量の比率が、15~25である(16)記載の粒子。
(18)両親媒性脂質、ステロール類、カチオン性脂質、及びPEG脂質の脂質組成が、モル量にて、両親媒性脂質が10~15%、ステロール類が35~45%、カチオン性脂質が40~50%、PEG脂質が1~2%であり、核酸重量に対する総脂質重量の比率が、17.5~22.5である(17)記載の粒子。
(19)両親媒性脂質、ステロール類、カチオン性脂質、及びPEG脂質の脂質組成が、モル量にて、両親媒性脂質が10~15%、ステロール類が35~45%、カチオン性脂質が45~50%、PEG脂質が1.5~2%であり、核酸重量に対する総脂質重量の比率が、17.5~22.5である(18)記載の粒子。
(20)ヒトパピローマウイルスがHPV16型である(1)~(19)のいずれかに記載の粒子。
(21)ヒトパピローマウイルスがHPV16型であり、HPV16型のE6抗原が配列番号8のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる(20)記載の粒子。
(22)ヒトパピローマウイルスが、HPV16型であり、HPV16型のE7抗原が配列番号9のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる(20)又は(21)記載の粒子。
(23)ヒトパピローマウイルスがHPV16型であり、HPV16型のE6抗原及びE7抗原を発現させることができる核酸が、キャップ構造(Cap)、5’非翻訳領域(5’-UTR)、リーダー配列(leader sequence)、E6の翻訳領域、プロテアーゼ切断配列(Furin Cleavage site)、E7の翻訳領域、3’非翻訳領域(3’-UTR)及びポリA尾部(polyA)を含むmRNAである(20)~(22)のいずれかに記載の粒子。
(24)HPV16型のE6抗原及びE7抗原を発現させることができる核酸の配列が、配列番号2、4又は6のいずれかの配列と少なくとも90%の同一性を有するヌクレオチド配列からなる(23)記載の粒子。
(25)核酸が少なくとも1個の修飾ヌクレオチドを含む(1)~(24)のいずれかに記載の粒子。
(26)修飾ヌクレオチドが、5位が置換したピリミジンヌクレオチド及び/又は1位が置換していてもよいシュードウリジンの少なくとも1個を含む(25)記載の粒子。
(27)修飾ヌクレオチドが、5-メチルシチジン、5-メトキシウリジン、5-メチルウリジン、シュードウリジン、及び1-アルキルシュードウリジンからなる群から選ばれる少なくとも1個を含む(25)記載の粒子。
(28)平均粒子径が30nm~300nmである(1)~(27)のいずれかに記載の粒子。
(29)ヒトパピローマウイルスによる感染を予防及び/又は治療するための組成物を製造するための(1)~(28)のいずれかに記載の粒子の使用。
(30)感染がHPV16型のヒトパピローマウイルスによる感染である(29)に記載の粒子の使用。
(31)(1)~(28)のいずれかに記載の粒子を含有する、組成物。
(32)ヒトパピローマウイルスのE6抗原及びE7抗原をin vivo又はin vitroで発現させるための(31)記載の組成物。
(33)医薬として用いられる(31)又は(32)記載の組成物。
(34)ヒトパピローマウイルスに対する免疫反応を誘導するための(33)記載の組成物。
(35)ヒトパピローマウイルス感染を予防及び/又は治療するための(33)又は(34)記載の組成物。
(36)(31)又は(32)に記載の組成物を細胞に導入することを含む、ヒトパピローマウイルスのE6抗原及びE7抗原をin vitroで発現させる方法。
(37)(31)~(35)のいずれかに記載の組成物を哺乳動物に投与することを含む、ヒトパピローマウイルスのE6抗原及びE7抗原をin vivoで発現させる方法。
(38)(33)又は(34)に記載の組成物を哺乳動物に投与することを含む、ヒトパピローマウイルスに対する免疫反応を誘導する方法。
(39)(33)~(35)のいずれかに記載の組成物を哺乳動物に投与することを含む、ヒトパピローマウイルス感染を予防及び/又は治療する方法。
また、本発明の別の態様における要旨は以下である。
(1-1)ヒトパピローマウイルスのE6抗原及びE7抗原を発現させることができる核酸を封入した脂質粒子であって、脂質が一般式(Ia)で表されるカチオン性脂質、又はその薬学的に許容される塩を含む前記粒子。
R1及びR2は、独立して、C1-C3アルキル基を示し;
L1は、C2-C4アルカノイルオキシ基を1若しくは複数個有していてもよいC17-C19アルケニル基を示し;
L2は、C2-C4アルカノイルオキシ基を1若しくは複数個有していてもよいC10-C19アルキル基、又はC2-C4アルカノイルオキシ基を1若しくは複数個有していてもよいC10-C19アルケニル基を示し;
pは、3、又は4である。
(1-2)一般式(Ia)中のR1及びR2が、共にメチル基である、(1-1)に記載の粒子。
(1-3)一般式(Ia)中のpが、3である(1-1)又は(1-2)に記載の粒子。
(1-4)一般式(Ia)中のL1が、アセトキシ基を1若しくは複数個有していてもよいC17-C19アルケニル基である、(1-1)~(1-3)のいずれかに記載の粒子。
(1-5)一般式(Ia)中のL2が、アセトキシ基を1若しくは複数個有していてもよいC10-C12アルキル基、又はアセトキシ基を1若しくは複数個有していてもよいC10-C19アルケニル基である、(1-1)~(1-4)のいずれかに記載の粒子。
(1-6)一般式(Ia)中のL2が、アセトキシ基を1若しくは複数個有していてもよいC10-C12アルキル基、又はアセトキシ基を1若しくは複数個有していてもよいC17-C19アルケニル基である、(1-1)~(1-4)のいずれかに記載の粒子。
(1-7)一般式(Ia)中のL1が、(R)-11-アセチルオキシ-cis-8-ヘプタデセニル基、cis-8-ヘプタデセニル基、又は(8Z,11Z)-ヘプタデカジエニル基である(1-1)~(1-6)のいずれかに記載の粒子。
(1-8)一般式(Ia)中のL2が、デシル基、cis-7-デセニル基、ドデシル基、又は(R)-11-アセチルオキシ-cis-8-ヘプタデセニル基である、(1-1)~(1-7)のいずれかに記載の粒子。
(1-9)カチオン性脂質が下記の構造式:
(1-10)カチオン性脂質が下記の構造式:
で表される(1-1)に記載の粒子。
(1-11)カチオン性脂質が下記の構造式:
(1-12)脂質が、さらに、両親媒性脂質、ステロール類及びPEG脂質を含む(1-9)又は(1-10)に記載の粒子。
(1-13)脂質が、さらに、両親媒性脂質、ステロール類及びPEG脂質を含む(1-11)に記載の粒子。
(1-14)両親媒性脂質が、ジステアロイルホスファチジルコリン、ジオレオイルホスファチジルコリン及びジオレオイルホスファチジルエタノールアミンからなる群より選択される少なくとも1つである(1-12)記載の粒子。
(1-15)両親媒性脂質が、ジステアロイルホスファチジルコリン、ジオレオイルホスファチジルコリン及びジオレオイルホスファチジルエタノールアミンからなる群より選択される少なくとも1つである(1-13)記載の粒子。
(1-16)ステロール類がコレステロールである(1-12)又は(1-14)に記載の粒子。
(1-17)ステロール類がコレステロールである(1-13)又は(1-15)に記載の粒子。
(1-18)PEG脂質が、1、2-ジミリストイル-sn-グリセロール メトキシポリエチレン グリコール及び/又はN-[メトキシ ポリ(エチレングリコール)2000]カルバモイル]-1,2-ジミリスチルオキシプロピル-3-アミンである(1-12)、(1-14)、(1-16)のいずれかに記載の粒子。
(1-19)PEG脂質が、1、2-ジミリストイル-sn-グリセロール メトキシポリエチレン グリコール及び/又はN-[メトキシ ポリ(エチレングリコール)2000]カルバモイル]-1,2-ジミリスチルオキシプロピル-3-アミンである(1-13)、(1-15)、(1-17)のいずれかに記載の粒子。
(1-20)両親媒性脂質、ステロール類、カチオン性脂質、及びPEG脂質の脂質組成が、モル量にて、両親媒性脂質が22.5%以下、ステロール類が15~55%、カチオン性脂質が40~65%、PEG脂質が1~5%であり、核酸重量に対する総脂質重量の比率が、15~30である(1-12)~(1-19)のいずれかに記載の粒子。
(1-21)両親媒性脂質が5~22.5%である(1-20)に記載の粒子。
(1-22)両親媒性脂質が10~22.5%である(1-21)に記載の粒子。
(1-23)PEG脂質が1~3%である(1-20)~(1-22)のいずれかに記載の粒子。
(1-24)PEG脂質が1~2%である(1-23)に記載の粒子。
(1-25)両親媒性脂質、ステロール類、カチオン性脂質、及びPEG脂質の脂質組成が、モル量にて、両親媒性脂質が5~15%、ステロール類が35~50%、カチオン性脂質が40~55%、PEG脂質が1~3%であり、核酸重量に対する総脂質重量の比率が、15~30である(1-12)、(1-14)、(1-16)、(1-18)のいずれかに記載の粒子。
(1-26)両親媒性脂質が10~15%、ステロール類が35~45%、カチオン性脂質が40~50%、PEG脂質が1~2%である(1-25)記載の粒子。
(1-27)両親媒性脂質が10~15%、ステロール類が35~45%、カチオン性脂質が45~50%、PEG脂質が1.5~2%である(1-26)記載の粒子。
(1-28)両親媒性脂質、ステロール類、カチオン性脂質、及びPEG脂質の脂質組成が、モル量にて、両親媒性脂質が15~22.5%、ステロール類が15~40%、カチオン性脂質が40~60%、PEG脂質が1~3%であり、核酸重量に対する総脂質重量の比率が、15~30である(1-13)、(1-15)、(1-17)、(1-19)のいずれかに記載の粒子。
(1-29)カチオン性脂質が45~60%、PEG脂質が1~2%である(1-28)記載の粒子。
(1-30)両親媒性脂質が17.5~22.5%である(1-29)記載の粒子。
(1-31)核酸重量に対する総脂質重量の比率が、15~25である(1-20)~(1-30)のいずれかに記載の粒子。
(1-32)核酸重量に対する総脂質重量の比率が、15~22.5である(1-31)記載の粒子。
(1-33)核酸重量に対する総脂質重量の比率が、17.5~22.5である(1-32)記載の粒子。
(1-34)ヒトパピローマウイルスがHPV16型である(1-1)~(1-33)のいずれかに記載の粒子。
(1-35)ヒトパピローマウイルスがHPV16型であり、HPV16型のE6抗原が配列番号8のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる(1-34)記載の粒子。
(1-36)ヒトパピローマウイルスが、HPV16型であり、HPV16型のE7抗原が配列番号9のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる(1-34)又は(1-35)記載の粒子。
(1-37)ヒトパピローマウイルスがHPV16型であり、ヒトパピローマウイルスのE6抗原及びE7抗原を発現させることができる核酸が、配列番号17のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる、HPV16型のE6抗原及びE7抗原の融合タンパクをコードする(1-34)~(1-36)のいずれかに記載の粒子。
(1-38)ヒトパピローマウイルスがHPV16型であり、HPV16型のE6抗原及びE7抗原を発現させることができる核酸が、キャップ構造(Cap)、5’非翻訳領域(5’-UTR)、リーダー配列(leader sequence)、E6の翻訳領域、プロテアーゼ切断配列(Furin Cleavage site)、E7の翻訳領域、3’非翻訳領域(3’-UTR)及びポリA尾部(polyA)を含むmRNAである(1-34)~(1-37)のいずれかに記載の粒子。
(1-39)HPV16型のE6抗原及びE7抗原を発現させることができる核酸の配列が、配列番号2、4又は6のいずれかの配列と少なくとも90%の同一性を有するヌクレオチド配列からなる(1-38)記載の粒子。
(1-40)ヒトパピローマウイルスがHPV18型である(1-1)~(1-33)のいずれかに記載の粒子。
(1-41)ヒトパピローマウイルスがHPV18型であり、HPV18型のE6抗原が配列番号14のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる(1-40)記載の粒子。
(1-42)ヒトパピローマウイルスが、HPV18型であり、HPV18型のE7抗原が配列番号15のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からな(1-40)又は(1-41)記載の粒子。
(1-43)ヒトパピローマウイルスがHPV18型であり、ヒトパピローマウイルスのE6抗原及びE7抗原を発現させることができる核酸が、配列番号18のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる、HPV18型のE6抗原及びE7抗原の融合タンパクをコードする(1-40)~(1-42)のいずれかに記載の粒子。
(1-44)ヒトパピローマウイルスがHPV18型であり、HPV18型のE6抗原及びE7抗原を発現させることができる核酸が、キャップ構造(Cap)、5’非翻訳領域(5’-UTR)、リーダー配列(leader sequence)、E6の翻訳領域、プロテアーゼ切断配列(Furin Cleavage site)、E7の翻訳領域、3’非翻訳領域(3’-UTR)及びポリA尾部(polyA)を含むmRNAである(1-40)~(1-43)のいずれかに記載の粒子。
(1-45)HPV18型のE6抗原及びE7抗原を発現させることができる核酸の配列が、配列番号11又は13のいずれかの配列と少なくとも90%の同一性を有するヌクレオチド配列からなる(1-44)記載の粒子。
(1-46)核酸が少なくとも1個の修飾ヌクレオチドを含む(1-1)~(1-45)のいずれかに記載の粒子。
(1-47)修飾ヌクレオチドが、5位が置換したピリミジンヌクレオチド及び/又は1位が置換していてもよいシュードウリジンの少なくとも1個を含む(1-46)記載の粒子。
(1-48)修飾ヌクレオチドが、5-メチルシチジン、5-メトキシウリジン、5-メチルウリジン、シュードウリジン、及び1-アルキルシュードウリジンからなる群から選ばれる少なくとも1個を含む(1-46)記載の粒子。
(1-49)修飾ヌクレオチドが、5-メチルシチジン、5-メチルウリジン、及び1-メチルシュードウリジンからなる群から選ばれる少なくとも1個を含む(1-46)記載の粒子。
(1-50)平均粒子径が30nm~300nmである(1-1)~(1-49)のいずれかに記載の粒子。
(1-51)ヒトパピローマウイルスによる感染を予防及び/又は治療するための組成物を製造するための(1-1)~(1-50)のいずれかに記載の粒子の使用。
(1-52)感染がHPV16型又はHPV18型のヒトパピローマウイルスによる感染である(1-51)に記載の粒子の使用。
(1-53)(1-1)~(1-50)のいずれかに記載の粒子を含有する、組成物。
(1-54)ヒトパピローマウイルスのE6抗原及びE7抗原をin vivo又はin vitroで発現させるための(1-53)記載の組成物。
(1-55)医薬として用いられる(1-53)又は(1-54)記載の組成物。
(1-56)ヒトパピローマウイルスに対する免疫反応を誘導するための(1-55)記載の組成物。
(1-57)ヒトパピローマウイルス感染を予防及び/又は治療するための(1-55)又は(1-56)記載の組成物。
(1-58)(1-53)又は(1-54)に記載の組成物を細胞に導入することを含む、ヒトパピローマウイルスのE6抗原及びE7抗原をin vitroで発現させる方法。
(1-59)(1-53)~(1-57)のいずれかに記載の組成物を哺乳動物に投与することを含む、ヒトパピローマウイルスのE6抗原及びE7抗原をin vivoで発現させる方法。
(1-60)(1-55)又は(1-56)に記載の組成物を哺乳動物に投与することを含む、ヒトパピローマウイルスに対する免疫反応を誘導する方法。
(1-61)(1-55)~(1-57)のいずれかに記載の組成物を哺乳動物に投与することを含む、ヒトパピローマウイルス感染を予防及び/又は治療する方法。
本明細書は、本願の優先権の基礎である日本国特許出願、特願2019‐207001の明細書および/または図面に記載される内容を包含する。
R1及びR2は、独立して、C1-C3アルキル基を示し;
L1は、C2-C4アルカノイルオキシ基を1若しくは複数個有していてもよいC17-C19アルケニル基を示し;
L2は、C2-C4アルカノイルオキシ基を1若しくは複数個有していてもよいC10-C19アルキル基、又はC2-C4アルカノイルオキシ基を1若しくは複数個有していてもよいC10-C19アルケニル基を示し;
pは、3、又は4である。
本発明のmRNAのキャップ構造としては、好ましくはcap0、又はcap1であり、より好ましくはcap1である。5’非翻訳領域(5’-UTR)の配列は、例えば、配列番号2の配列中の塩基番号2~70の配列、配列番号4の配列中の塩基番号2~70の配列、及び配列番号6の配列中の塩基番号2~70の配列、配列番号11の配列中の塩基番号2~70の配列、配列番号13の配列中の塩基番号2~70の配列である。リーダー配列(leader sequence)の配列は、例えば、配列番号2の配列中の塩基番号71~124の配列、配列番号4の配列中の塩基番号71~124の配列、及び配列番号6の配列中の塩基番号71~124の配列、配列番号11の配列中の塩基番号71~124の配列、配列番号13の配列中の塩基番号71~124の配列である。E6の翻訳領域の配列は、E6抗原のアミノ酸配列の全て又は一部を発現できる配列であって、開始コドン及び/又は終止コドンを含んでいても良く、例えば、配列番号2の配列中の塩基番号125~574の配列、配列番号4の配列中の塩基番号125~574の配列、及び配列番号6の配列中の塩基番号125~574の配列、配列番号11の配列中の塩基番号125~589の配列、配列番号13の配列中の塩基番号125~589の配列である。プロテアーゼ切断配列(Furin Cleavage site)の配列は、例えば、配列番号2の配列中の塩基番号575~595の配列、配列番号4の配列中の塩基番号575~595の配列、及び配列番号6の配列中の塩基番号575~595の配列、配列番号11の配列中の塩基番号590~610の配列、配列番号13の配列中の塩基番号590~610の配列である。E7の翻訳領域の配列は、E7抗原のアミノ酸配列の全て又は一部を発現できる配列であって、開始コドン及び/又は終止コドンを含んでいても良く、例えば、配列番号2の配列中の塩基番号596~889の配列、配列番号4の配列中の塩基番号596~889の配列、及び配列番号6の配列中の塩基番号596~889の配列、配列番号11の配列中の塩基番号611~925の配列、配列番号13の配列中の塩基番号611~925の配列である。3’非翻訳領域(3’-UTR)の配列は、例えば、配列番号2の配列中の塩基番号890~1021の配列、配列番号4の配列中の塩基番号890~1021の配列、及び配列番号6の配列中の塩基番号890~1021の配列、配列番号11の配列中の塩基番号926~1057の配列、配列番号13の配列中の塩基番号926~1057の配列である。ポリA尾部(polyA)の配列は、例えば、配列番号2の配列中の塩基番号1022~1123の配列、配列番号4の配列中の塩基番号1022~1123の配列、及び配列番号6の配列中の塩基番号1022~1123の配列、配列番号11の配列中の塩基番号1058~1159の配列、配列番号13の配列中の塩基番号1058~1159の配列である。キャップ構造(Cap)、5’非翻訳領域(5’-UTR)、リーダー配列(leader sequence)、E6の翻訳領域、プロテアーゼ切断配列(Furin Cleavage site)、E7の翻訳領域、3’非翻訳領域(3’-UTR)及びポリA尾部(polyA)の配列には、改変がなされていてもよく、HPV16型のE6抗原及びE7抗原を発現させることができる核酸の配列は、配列番号2、4又は6のいずれかの配列と少なくとも90%、好ましくは95%、より好ましくは97%の同一性を有するヌクレオチド配列からなるとよい。また、HPV18型のE6抗原及びE7抗原を発現させることができる核酸の配列は、配列番号11又は13のいずれかの配列と少なくとも90%、好ましくは95%、より好ましくは97%の同一性を有するヌクレオチド配列からなるとよい。
[実施例1] HPV16 E6-E7 fusion2 mRNA -001の調製
(1)HPV16 E6-E7 fusion2のin vitro transcription(IVT)用の鋳型DNAの作製
in vitro transcription(IVT)に用いる鋳型DNAを作製するためにプラスミドを構築した。GCTAGC(NheIサイト)、T7プロモーター配列、human β-globinの5'-UTR配列、KOZAK配列、IgE leader sequence―HPV16型E6―Furin Cleavage site―HPV16型E7の翻訳領域、human β-globinの3’-UTR配列、ポリA尾部及びACTAGT(SpeIサイト)が順に連結した配列を含むDNA断片(配列番号1)を導入したプラスミド(pMA-HPV16_fusion2)を作製した。
実施例1-(1)で得られたプラスミド(250μg)を溶解したNuclease-free water (2200μL, Thermo Fisher catalog # AM9937)に10× CutSmart Buffer (250 μL、New England Biolabs catalog # B7204S)、SpeI-HF (30 μL、New England Biolabs catalog # R3133L)を加え、37℃で2時間インキュベーション後、65℃で20分インキュベーションした。7.5M酢酸アンモニウム(1250 μL)とエタノール(7500 μL)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をTE-Bufferで500μg/mLの溶液に調製した。
実施例1-(2)で得られた500 μg/mL 鋳型DNA (200μL)、100 mM CleanCap AG (200 μL, TriLink catalog # T-7113)、100 mM ATP (200 μL, Hongene catalog # R1331)、100 mM GTP (200 μL, Hongene catalog # R2331)、100 mM 5-Me-CTP (200 μL, Hongene catalog # R3-029)、100 mM 5-methyluridine triphosphate (200μL)、Nuclease-free water (1600 μL, Thermo Fisher catalog # AM9937)、T7 Transcription 5× buffer (800 μL, Promega catalog # P140X)、Enzyme mix, T7 RNA Polymerase (400 μL, Promega catalog # P137X)を混合し、37℃で4時間インキュベーションした。RQ1 RNase-Free DNase (100 μL, Promega catalog # M6101)を混合し、37℃で15分インキュベーションした。8 M LiCl溶液(2000 μL, Sigma-Aldrich catalog # L7026)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をNuclease-free waterに溶解後、RNeasy Maxi kit (Qiagen catalog # 75162)を用いて付属のマニュアル通りに精製した。得られた溶出液(10.3 mL、UV換算で17013 μg)とNuclease-free water (247 μL)、rApid Alkaline Phosphatase(Roche catalog # 04 898 141 001)の緩衝液(1550 μL)と酵素(3403 μL)を混合し、37℃で1時間インキュベーション後、75℃で15分インキュベーションした。8M LiCl溶液(7750 μL)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をNuclease-free waterに溶解後、逆相高速液体クロマトグラフィー(Chromolith Semi-Prep (Merck catalog # 1.52016.0001)を2本連結、5%アセトニトリル, 400 mM酢酸トリエチルアミン(pH7.0)/ 25%アセトニトリル, 400 mM酢酸トリエチルアミン(pH 7.0)、80℃)で精製することで目的とするmRNAを得た。
得られたmRNAは、配列番号2の配列を有する。Experion RNA StdSens (BIO-RAD catalog # 7007103JA)により分析し、目的の長さであることを確認した。
(1)HPV16 E6-E7 fusion10のIVT用の鋳型DNAの作製
IVT鋳型に用いる鋳型DNAを作成するためにプラスミドを構築した。GCTAGC(NheIサイト)、T7プロモーター配列、β-globinの5'-UTR配列、KOZAK配列、IgE leader sequence―HPV16型E6―Furin Cleavage site―HPV16型E7の翻訳領域,β-globinの3’-UTR配列、ポリA尾部及びACTAGT(SpeIサイト)が順に連結した配列を含むDNA断片(配列番号3)を導入したプラスミド(pMA-HPV16_fusion10)を作製した。
実施例1-(1)で得られたプラスミドの代わりに実施例2-(1)で得られたプラスミドを用い、実施例1-(2)及び(3)と同様の方法でmRNAを得た。
実施例1-(1)で得られたプラスミドの代わりに実施例2-(1)で得られたプラスミドを用い、実施例1-(2)と同様の方法で鋳型DNAを得た。その後、実施例1-(3)の鋳型DNAの代わりに得られた鋳型DNAを、100 mM 5-Me-UTPのかわりに100 mM Pseudo-UTP (Hongene catalog # R5-022)を用い、実施例1-(3)と同様の方法でmRNAを得た。
実施例1-(1)で得られたプラスミドの代わりに実施例2-(1)で得られたプラスミドを用い、実施例1-(2)と同様の方法で鋳型DNAを得た。その後、実施例1-(3)の鋳型DNAの代わりに得られた鋳型DNAを、100 mM 5-Me-CTPのかわりに100 mM CTP (Hongene catalog # R3331)、100mM 5-Me-UTPのかわりに100 mM N1-methylpseudouridine-5’-triphosphate (TriLink catalog # N-1081)を用い、実施例1-(3)と同様の方法でmRNAを得た。
実施例1-(1)で得られたプラスミドの代わりに実施例2-(1)で得られたプラスミドを用い、実施例1-(2)と同様の方法で鋳型DNAを得た。その後、実施例1-(3)の鋳型DNAの代わりに得られた鋳型DNAを、100 mM 5-Me-CTPのかわりに100 mM CTP (Hongene catalog # R3331)を用い、実施例1-(3)と同様の方法でmRNAを得た。
(1)HPV16 E6-E7 fusion10 opt2のIVT用の鋳型DNAの作製
IVT鋳型に用いる鋳型DNAを作成するためにプラスミドを構築した。GCTAGC(NheIサイト)、T7プロモーター配列、β-globinの5'-UTR配列、KOZAK配列、IgE leader sequence―HPV16型E6―Furin Cleavage site―HPV16型E7の翻訳領域,β-globinの3’-UTR配列、ポリA尾部及びACTAGT(SpeIサイト)が順に連結した配列を含むDNA断片(配列番号5)を導入したプラスミド(pMA-HPV16_fusion10_opt2)を作製した。
実施例6-(1)で得られたプラスミド(250μg)を溶解したNuclease-free water (2200μL, Thermo Fisher catalog # AM9937)に10× CutSmart Buffer (250 μL、New England Biolabs catalog # B7204S)、SpeI-HF (30 μL、New England Biolabs catalog # R3133L)を加え、37℃で2時間インキュベーション後、65℃で20分インキュベーションした。7.5M酢酸アンモニウム(1250 μL)とエタノール(7500 μL)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をTE-Bufferで500μg/mLの溶液に調製した。
実施例6-(2)で得られた500 μg/mL 鋳型DNA (100 μL)、100 mM ATP (150 μL, Hongene catalog # R1331)、100 mM GTP (150 μL, Hongene catalog # R2331)、100 mM CTP (150μL, Hongene catalog # R3331)、100 mM N1-methylpseudouridine-5’-triphosphate (150 μL, Hongene catalog # R5-027)、Nuclease-free water (700 μL, Thermo Fisher catalog # AM9937)、T7 Transcription 5× buffer (400 μL, Promega catalog # P140X)、Enzyme mix, T7 RNA Polymerase (200 μL, Promega catalog # P137X)を混合し、37℃で4時間インキュベーションした。RQ1 RNase-Free DNase (50 μL, Promega catalog # M6101)を混合し、37℃で15分インキュベーションした。8 M LiCl溶液(1000μL, Sigma-Aldrich catalog # L7026)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をNuclease-free waterに溶解した。この溶液(962 μL、UV換算で5400 μg)に、Nuclease-free water (2818 μL)を加え、70℃で20分加温後、氷冷下で10分冷却した。10× capping buffer(500 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl2, 50 mM DTT)を540 μL、20 mM GTP(270 μL, 100 mM GTP, Hongene catalog # R2331をNuclease-free waterで希釈して使用)、20 mM SAM(270 μL, 32 mM SAM, New England Biolabs catalog # B9003SをNuclease-free waterで希釈して使用)、Vaccinia Capping Enzyme(540 μL, Hongene catalog # ON-028)を加え、37℃で4時間インキュベーション後、10× capping buffer(90 μL)、20 mM SAM (270 μL)、2’-O-Methyltransferase(540 μL, Hongene catalog # ON-014)を加え、37℃で4時間インキュベーションした。8 M LiCl溶液(6300 μL)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をNuclease-free waterに溶解後、逆相高速液体クロマトグラフィー(Chromolith Semi-Prep (Merck catalog # 1.52016.0001)を2本連結、5%アセトニトリル, 400mM酢酸トリエチルアミン(pH 7.0)/ 25%アセトニトリル, 400mM酢酸トリエチルアミン(pH7.0)、80℃)で精製することで目的とするmRNAを得た。
実施例6-(2)で得られた500 μg/mL 鋳型DNA (200 μL)、100 mM ATP (300 μL, Hongene catalog # R1331)、100 mM GTP (300 μL, Hongene catalog # R2331)、100 mM CTP (300μL, Hongene catalog # R3331)、100 mM N1-methylpseudouridine-5’-triphosphate (300 μL, Hongene catalog # R5-027)、Nuclease-free water (1400 μL, APPLIED-BIO catalog # AM9937)、T7 Transcription 5× buffer, (400 mM HEPES-KOH (pH 7.5), 80 mM MgCl2, 10 mM spermidine, 200mM DTT)を800 μL、Enzyme mix, T7 RNA Polymerase (400 μL, Promega catalog # P137X)を混合し、37℃で8時間インキュベーションした。RQ1 RNase-Free DNase (100 μL, Promega catalog # M6101)を混合し、37℃で15分インキュベーションした。8 M LiCl溶液(2000μL, Sigma-Aldrich catalog # L7026)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をNuclease-free waterに溶解した。この溶液(1590 μL、UV換算で6000 μg)に、Nuclease-free water (2610 μL)を加え、70℃で10分加温後、氷冷下で10分冷却した。10× capping buffer(500 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl2, 50 mM DTT)を600 μL、20 mM GTP(300 μL, 100 mM GTP, Hongene catalog # R2331をNuclease-free waterで希釈して使用)、20 mM SAM (300 μL, S-adenosyl-L-methionine disulfate tosylate, OX-CHEM catalog # AX8250818を0.005M硫酸, 10%エタノール溶液に溶解して使用)、Vaccinia Capping Enzyme(600 μL, Hongene catalog # ON-028)を加え、37℃で4時間インキュベーション後、10× capping buffer(100 μL)、20 mM SAM (300 μL)、2’-O-Methyltransferase(600 μL, Hongene catalog # ON-014)を加え、37℃で4時間インキュベーションした。8 M LiCl溶液(7000 μL)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をNuclease-free waterに溶解後、逆相高速液体クロマトグラフィー(Chromolith Performance (Merck catalog # 1.02129.0001)、5%アセトニトリル, 400mM酢酸トリエチルアミン(pH 7.0)/ 25%アセトニトリル, 400mM酢酸トリエチルアミン(pH7.0)、45℃)で精製することで目的とするmRNAを得た。
実施例6-(3)の100 mM CTPのかわりに100 mM 5-Me-CTP、100mM N1-methylpseudouridine-5’-triphosphateのかわりに100 mM 5-methyluridine triphosphateを用い、実施例6-(3)と同様の方法でmRNAを得た。得られたmRNAは、配列番号6の配列を有する。Experion RNA StdSensにより分析し、目的の長さであることを確認した。
(1)mRNA封入核酸脂質粒子の調製
ジステアロイルホスファチジルコリン(1,2-Distearoyl-sn-glycero-3-phosphocholine:以下DSPCと表記,NOF CORPORATION)、コレステロール(Cholesterol:以下Cholと表記,Sigma-Aldrich,Inc.)、二酢酸(7R,9Z,26Z,29R)-18-({[3-(ジメチルアミノ)プロポキシ]カルボニル}オキシ)ペンタトリアコンタ-9,26-ジエン-7,29-ジイル(WO2015/005253の実施例23に記載の化合物)(以下LPと表記)、及びポリエチレングリコール分子量が約2000の1、2-ジミリストイル-sn-グリセロール メトキシポリエチレン グリコール(1,2-Dimyristoyl-sn-Glycero-3-Methoxypolyethylene Glycol、以下PEG-DMGと表記,NOF CORPORATION)を、DSPC:Chol:LP:PEG-DMG=10:43.5:45:1.5のモル比にて、総脂質濃度5mMになるようにエタノールに溶解した。
(1)で調製した核酸脂質粒子を含む分散液の特性評価を行った。それぞれの特性評価の方法について説明する。
mRNAの封入率は、Quant-iT RiboGreen RNA Assay kit (Invitrogen)を用い、添付文書に準じて測定した。
すなわち、0.015% Triton X-100界面活性剤存在下及び非存在下において、核酸脂質粒子の分散液中のmRNAを定量し、次式により封入率を算出した。
{[界面活性剤存在下におけるmRNA量]-[界面活性剤非存在下におけるmRNA量]}/[界面活性剤存在下におけるmRNA量]}x 100(%)
核酸脂質粒子の分散液中のmRNA量を逆相クロマトグラフィーにて測定した(System:Agilent 1100series,Column:Bioshell A400 Protein C4(10cm×4.6mm,3.4μm)(SUPELCO),Buffer A:0.1M 酢酸トリエチルアミン(pH7.0),Buffer B:アセトニトリル,(B%):5-50%(0-15min),Flow Rate:1mL/min,Temperature:70℃,Detection:260nm)。
mRNAに対する総脂質量の比率を次式により算出した。
核酸脂質粒子の粒子径は、Zeta Potential/Particle Sizer NICOMPTM 380ZLS (PARTICLE SIZING SYSTEMS)にて測定した。表中の平均粒子径は体積平均粒子径を表し、±以下は、偏差を表す。
実施例8と同様の方法にて、実施例2、3、4、5、乃至6に記載のmRNA封入核酸脂質粒子の調製、特性評価を実施した。ただし、構成脂質組成をDSPC:Chol:LP:PEG-DMG=12.5:41:45:1.5のモル比とした。結果を表1に示した。
実施例8と同様の方法にて、実施例2、4、6、乃至7に記載のmRNA封入核酸脂質粒子の調製、特性評価を実施した。ただし、構成脂質組成をDSPC:Chol:LP:PEG-DMG=12.5:41:45:1.5のモル比とした。結果を表1に示した。
実施例8と同様の方法にて、実施例2に記載のmRNA封入核酸脂質粒子の調製、特性評価を実施した。ただし、構成脂質組成をDSPC:Chol:LP:PEG-DMG=12.5:41:45:1.5のモル比とし、mRNA重量に対する全脂質重量の比率を25とした。結果を表1に示した。
実施例8と同様の方法にて、実施例2に記載のmRNA封入核酸脂質粒子の調製、特性評価を実施した。ただし、構成脂質組成をDSPC:Chol:LP:PEG-DMG=12.5:41:45:1.5のモル比とし、mRNA重量に対する全脂質重量の比率を30とした。結果を表1に示した。
実施例8と同様の方法にて、実施例2に記載のmRNA封入核酸脂質粒子の調製、特性評価を実施した。結果を表1に示した。
mRNA封入核酸脂質粒子の調製
実施例8-(1)と同様の方法にて、配列番号7に記載のOVA(Ovalbumin)の翻訳領域をもつmRNAを封入した核酸脂質粒子の調製を実施した。ただし、PEG-DMGの代わりに、ポリエチレングリコール分子量が約2000のN-[メトキシ ポリ(エチレングリコール)2000]カルバモイル]-1,2-ジミリスチルオキシプロピル-3-アミン(N-[methoxy poly(ethylene glycol)2000 carbamyl]-1,2-dimyristyloxlpropyl-3-amine):以下PEG-C-DMAと表記, Journal of Controlled Release 112 (2006) 280-290に記載の化合物12)を用い、構成脂質組成をDSPC:Chol:LP:PEG-C-DMA=10:38.5:50:1.5のモル比とした。
(2)mRNA封入核酸脂質粒子の特性評価
(1)で調製した核酸脂質粒子を含む分散液の特性評価を行った。それぞれの特性評価の方法について説明する。
(2-1)mRNAの封入率
mRNAの封入率は、Quant-iT RiboGreen RNA Assay kit (Invitrogen)を用い、添付文書に準じて測定した。
すなわち、0.015% Triton X-100界面活性剤存在下及び非存在下において、核酸脂質粒子の分散液中のmRNAを定量し、次式により封入率を算出した。
{[界面活性剤存在下におけるmRNA量]-[界面活性剤非存在下におけるmRNA量]}/[界面活性剤存在下におけるmRNA量]}x 100(%)
(2-2)mRNAと脂質の比率
核酸脂質粒子の分散液中のmRNA量を(2-1)の「界面活性剤存在下におけるmRNA量」とした。
核酸脂質粒子の分散液中のリン脂質量をリン脂質C-テストワコー(富士フイルム和光純薬株式会社)を用い、添付文書に準じて測定した。すなわち、2% Triton X-100界面活性剤存在下において、試料中のリン脂質量を測定した。
核酸脂質粒子の分散液中のコレステロール及びLP量を逆相クロマトグラフィーにて測定した(System:DIONEX UltiMate 3000,Column:Chromolith Performance RP-18 endcapped 100-3 monolithic HPLC-column(Merck, Cat.#: 1.52001.0001),Buffer A:0.01%トリフルオロ酢酸,Buffer,B:0.01%トリフルオロ酢酸,メタノール,(B%):82-97%(0-17min),Flow Rate:2mL/min,Temperature:50℃,Detection:Corona CAD(Charged Aerosol Detector))。
リン脂質、コレステロール及びLPの測定値と核酸脂質粒子を構成する脂質成分の組成比から、総脂質量を算出した。
mRNAに対する総脂質量の比率を次式により算出した。
[総脂質濃度]/[mRNA濃度] (wt/wt)
(2-3)平均粒子径
核酸脂質粒子の粒子径は、Zeta Potential/Particle Sizer NICOMPTM 380ZLS (PARTICLE SIZING SYSTEMS)にて測定した。表中の平均粒子径は体積平均粒子径を表し、±以下は、偏差を表す。
結果を表2に示した。
実施例21と同様の方法にて、配列番号7に記載のOVA(Ovalbumin)の翻訳領域をもつmRNAを封入した核酸脂質粒子の調製、特性評価を実施した。ただし、構成脂質組成をDSPC:Chol:LP:PEG-C-DMAG=10:35:50:5のモル比とした。結果を表2に示した。
実施例21と同様の方法にて、配列番号7に記載のOVA(Ovalbumin)の翻訳領域をもつmRNAを封入した核酸脂質粒子の調製、特性評価を実施した。ただし、構成脂質組成をDSPC:Chol:LP:PEG-C-DMA=10:23.5:65:1.5のモル比とした。結果を表2に示した。
実施例21と同様の方法にて、配列番号7に記載のOVA(Ovalbumin)の翻訳領域をもつmRNAを封入した核酸脂質粒子の調製、特性評価を実施した。ただし、構成脂質組成をDSPC:Chol:LP:PEG-C-DMA=10:48.5:40:1.5のモル比とした。結果を表2に示した。
実施例21と同様の方法にて、配列番号7に記載のOVA(Ovalbumin)の翻訳領域をもつmRNAを封入した核酸脂質粒子の調製、特性評価を実施した。ただし、構成脂質組成をDSPC:Chol:LP:PEG-C-DMA=5:43.5:50:1.5のモル比とした。結果を表2に示した。
実施例21と同様の方法にて、配列番号7に記載のOVA(Ovalbumin)の翻訳領域をもつmRNAを封入した核酸脂質粒子の調製、特性評価を実施した。ただし、構成脂質組成をDSPC:Chol:LP:PEG-C-DMA=15:33.5:50:1.5のモル比とした。結果を表2に示した。
実施例21と同様の方法にて、配列番号7に記載のOVA(Ovalbumin)の翻訳領域をもつmRNAを封入した核酸脂質粒子の調製、特性評価を実施した。ただし、構成脂質組成をChol:LP:PEG-C-DMA=53.5:45:1.5のモル比とした。結果を表2に示した。
実施例8と同様の方法にて、実施例4に記載のmRNA封入核酸脂質粒子の調製、特性評価を実施した。ただし、DSPCの代わりに、ジオレオイルホスファチジルコリン(1,2-Dioleoyl-sn-glycero-3-phosphocholine:以下DOPCと表記,NOF CORPORATION)を用い、構成脂質組成をDOPC:Chol:LP:PEG-DMG=10:43.5:45:1.5のモル比とした。結果を表3に示した。
実施例8と同様の方法にて、実施例4に記載のmRNA封入核酸脂質粒子の調製、特性評価を実施した。ただし、DSPCの代わりに、DOPCを用い、構成脂質組成をDOPC:Chol:LP:PEG-DMG=15:38.5:45:1.5のモル比とした。結果を表3に示した。
実施例8と同様の方法にて、実施例4に記載のmRNA封入核酸脂質粒子の調製、特性評価を実施した。ただし、DSPCの代わりに、ジオレオイルホスファチジルエタノールアミン(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine:以下DOPEと表記,NOF CORPORATION)を用い、構成脂質組成をDOPE:Chol:LP:PEG-DMG=10:43.5:45:1.5のモル比とした。結果を表3に示した。
実施例8と同様の方法にて、実施例4に記載のmRNA封入核酸脂質粒子の調製、特性評価を実施した。ただし、DSPCの代わりに、DOPEを用い、構成脂質組成をDOPE:Chol:LP:PEG-DMG=15:38.5:45:1.5のモル比とした。結果を表3に示した。
実施例8と同様の方法にて、実施例4に記載のmRNA封入核酸脂質粒子の調製、特性評価を実施した。ただし、構成脂質組成をDSPC:Chol:LP:PEG-DMG=12.5:41:45:1.5のモル比とした。結果を表3に示した。
(1)HPV18 E6-E7 fusion1 opt1のin vitro transcription (IVT) 用の鋳型DNAの作製
in vitro transcription(IVT)に用いる鋳型DNAを作製するためにプラスミドを構築した。GCTAGC(NheIサイト)、T7プロモーター配列、human β-globinの5’-UTR配列、KOZAK配列、IgE leader sequence―HPV18型E6―Furin Cleavage site―HPV18型E7のORF、human β-globinの3’-UTR配列、ポリA尾部及びACTAGT(SpeIサイト)が順に連結した配列を含むDNA断片(配列番号10)を導入したプラスミド(pMA-HPV18_fusion1_opt1)を作製した。
(2)鋳型DNAのリニアライズ化
実施例33-(1)で得られたプラスミド(350μg)を溶解したNuclease-free water (3080μL, Thermo Fisher catalog # AM9937)に10× CutSmart Buffer (350 μL、New England Biolabs catalog # B7204S)、SpeI-HF (70 μL、New England Biolabs catalog # R3133L)を加え、37℃で2時間インキュベーション後、65℃で20分インキュベーションした。7.5M酢酸アンモニウム(1750 μL)とエタノール(10500 μL)を混合し、-80℃で終夜静置した。遠心分離(-10℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(-10℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をTE-Bufferで500μg/mLの溶液に調製した。
(3)in vitro transcriptionによるHPV18 E6-E7 fusion1 opt1 mRNA -001の調製
実施例33-(2)で得られた500 μg/mL 鋳型DNA (150μL)、100 mM CleanCap AG (150 μL, TriLink catalog # T-7113)、100 mM ATP (150 μL, Hongene catalog # R1331)、100 mM GTP (150 μL, Hongene catalog # R2331)、100 mM CTP (150μL, Hongene catalog # R3331)、100 mM N1-Me-Pseudo UTP (150 μL, Hongene catalog # R5-027)、Nuclease-free water (1200 μL, Thermo Fisher catalog # AM9937)、T7 Transcription 5× buffer (600 μL, Promega catalog # P140X)、Enzyme mix, T7 RNA Polymerase (300 μL, Promega catalog # P137X)を混合し、37℃で4時間インキュベーションした。RQ1 RNase-Free DNase (75 μL, Promega catalog # M6101)を混合し、37℃で15分インキュベーションした。8 M LiCl溶液(1500 μL, Sigma-Aldrich catalog # L7026)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をNuclease-free waterに溶解後、RNeasy Maxi kit (Qiagen catalog # 75162)を用いて付属のマニュアル通りに精製した。得られた溶出液の一部(11.0 mL、UV換算で9813 μg)とNuclease-free water (537 μL)、rApid Alkaline Phosphatase(Roche catalog # 04 898 141 001)の緩衝液(1500 μL)と酵素(1963 μL)を混合し、37℃で30分インキュベーション後、75℃で3分インキュベーションした。8M LiCl溶液(15000 μL)を混合し、-20℃で3時間静置した。遠心分離(-8℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(-8℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をNuclease-free waterに溶解後、逆相高速液体クロマトグラフィー(YMC Triart-C8 5μm 10×150mm (YMC # TO12S05-1510WT)、5%アセトニトリル, 400 mM酢酸トリエチルアミン(pH7.0)/ 25%アセトニトリル, 400 mM酢酸トリエチルアミン(pH 7.0)、75℃)で精製することで目的とするmRNAを得た。
得られたmRNAは、配列番号11の配列を有する。LabChip GX Touch Standard RNA Reagent Kit(PerkinElmer catalog #CLS960010)により分析し、目的の長さであることを確認した。
実施例33-(3)の100 mM CTPのかわりに100 mM 5-Me-CTP (Hongene catalog # R3-029)を用い、100 mM N1-Me-Pseudo UTPのかわりに100 mM 5-methyluridine triphosphateを用い、実施例33-(3)と同様の方法でmRNAを得た。
得られたmRNAは、配列番号11の配列を有する。LabChip GX Touch Standard RNA Reagent Kit(PerkinElmer catalog #CLS960010)により分析し、目的の長さであることを確認した。
(1)HPV18 E6-E7 fusion1 opt2のIVT用の鋳型DNAの作製
IVT鋳型に用いる鋳型DNAを作製するためにプラスミドを構築した。GCTAGC(NheIサイト)、T7プロモーター配列、β-globinの5’-UTR配列、KOZAK配列、IgE leader sequence―HPV18型E6―Furin Cleavage site―HPV18型E7のORF,β-globinの3’-UTR配列、ポリA尾部及びACTAGT(SpeIサイト)が順に連結した配列を含むDNA断片(配列番号12)を導入したプラスミド(pMA-HPV18_fusion1_opt2)を作製した。
(2)鋳型DNAのリニアライズ化
実施例35-(1)で得られたプラスミド(400μg)を溶解したNuclease-free water (3520μL, Thermo Fisher catalog # AM9937)に10× CutSmart Buffer (400 μL、New England Biolabs catalog # B7204S)、SpeI-HF (80 μL、New England Biolabs catalog # R3133L)を加え、37℃で2時間インキュベーション後、65℃で20分インキュベーションした。7.5M酢酸アンモニウム(2000 μL)とエタノール(12000 μL)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をTE-Bufferで500μg/mLの溶液に調製した。
(3) in vitro transcriptionによるHPV18 E6-E7 fusion1 opt2 mRNA -001 の調製
実施例35-(2)で得られた500 μg/mL 鋳型DNA (150 μL)、100 mM ATP (225 μL, Hongene catalog # R1331)、100 mM GTP (225 μL, Hongene catalog # R2331)、100 mM CTP (225 μL, Hongene catalog # R3331)、100 mM N1-Me-Pseudo UTP (225 μL, Hongene catalog # R5-027)、Nuclease-free water (1050 μL, Thermo Fisher catalog # AM9937)、T7 Transcription 5× buffer (600 μL, Promega catalog # P140X)、Enzyme mix, T7 RNA Polymerase (300 μL, Promega catalog # P137X)を混合し、37℃で4時間インキュベーションした。RQ1 RNase-Free DNase (75 μL, Promega catalog # M6101)を混合し、37℃で15分インキュベーションした。8 M LiCl溶液(1500μL, Sigma-Aldrich catalog # L7026)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をNuclease-free waterに溶解した。この溶液の一部(2470 μL、UV換算で7500 μg)に、Nuclease-free water (2780 μL)を加え、70℃で10分加温後、氷冷下で5分冷却した。10× capping buffer(500 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl2, 50 mM DTT)を750 μL、20 mM GTP(375 μL, 100 mM GTP, Hongene catalog # R2331をNuclease-free waterで希釈して使用)、20 mM SAM(375 μL, 32 mM SAM, New England Biolabs catalog # B9003SをNuclease-free waterで希釈して使用)、Vaccinia Capping Enzyme(750 μL, Hongene catalog # ON-028)を加え、37℃で4時間インキュベーション後、10× capping buffer(125 μL)、20 mM SAM (375 μL)、2’-O-Methyltransferase(750 μL, Hongene catalog # ON-014)を加え、37℃で4時間インキュベーションした。8 M LiCl溶液(8750 μL)を混合し、-20℃で終夜静置した。遠心分離(4℃、4000×g、30分)後、上澄みを廃棄、70%エタノールを加え、遠心分離(4℃、4000×g、10分)後、上澄みを廃棄し風乾した。得られた残渣をNuclease-free waterに溶解後、逆相高速液体クロマトグラフィー(YMC Triart-C8 5μm 10×150mm (YMC # TO12S05-1510WT)、5%アセトニトリル, 400 mM酢酸トリエチルアミン(pH7.0)/ 25%アセトニトリル, 400 mM酢酸トリエチルアミン(pH 7.0)、75℃)で精製することで目的とするmRNAを得た。
得られたmRNAは、配列番号13の配列を有する。LabChip GX Touch Standard RNA Reagent Kit(PerkinElmer catalog #CLS960010)により分析し、目的の長さであることを確認した。
実施例35-(3)の100 mM CTPのかわりに100 mM 5-Me-CTP (Hongene catalog # R3-029)を用い、100 mM N1-Me-Pseudo UTPのかわりに100 mM 5-methyluridine triphosphateを用い、実施例35-(3)と同様の方法でmRNAを得た。
得られたmRNAは、配列番号13の配列を有する。LabChip GX Touch Standard RNA Reagent Kit(PerkinElmer catalog #CLS960010)により分析し、目的の長さであることを確認した。
(1)mRNA封入核酸脂質粒子の調製
実施例1のmRNAの代わりに実施例33から実施例36のmRNAをそれぞれ用い、表4に記載の脂質モル比に従って実施例8と同様の方法にてmRNA封入核酸脂質粒子の調製、特性評価を実施した。結果を表4に示した。
(1)mRNA封入核酸脂質粒子の調製
実施例8と同様の方法にて、実施例4に記載のmRNA封入核酸脂質粒子の調製を実施した。ただし、二酢酸(7R,9Z,26Z,29R)-18-({[3-(ジメチルアミノ)プロポキシ]カルボニル}オキシ)ペンタトリアコンタ-9,26-ジエン-7,29-ジイルの代わりに、酢酸(7R,9Z)-18-({[3-(ジメチルアミノ)プロピルオキシ]カルボニル}オキシ)オクタコサ-9-エン-7-イル(WO2015/005253の実施例28に記載の化合物)(以下LP2と表記)を用い、構成脂質組成を表5に記載のモル比とした。
尚、LP2はWO2015005253の実施例28に記載の方法に従い合成した。
(2)mRNA封入核酸脂質粒子の特性評価
実施例8と同様の方法にて、mRNA封入核酸脂質粒子の特性評価を実施した。ただし、mRNA量は以下の通りに分析した。
核酸脂質粒子分散液を90%メタノールに希釈溶解し、核酸脂質粒子中のmRNA量を紫外可視分光光度計(パーキンエルマー社製、LAMBDATM 465)にて測定した。mRNA濃度を次式により算出した。
{[260nmにおける吸光度]-[350nmにおける吸光度]}x 40 x 希釈倍率(μg/mL)
結果を表5に示した。
J. Yan et al. / Vaccine 27 (2009) 431-440を参考に、HPV16型E6,E7の融合タンパクを発現するプラスミドを構築した。翻訳領域をGenBank Accession Number:Fj229356に記載の配列とした。
mRNA封入核酸脂質粒子を導入した培養細胞からのHPV遺伝子型16E7ワクチン抗原の発現レベル(図1)
96 well plateの1wellあたり、2×104個のヒト胎児腎臓細胞株であるHEK293T細胞を播種し、37℃、CO2濃度5%の条件で一晩培養した。その後、mRNA終濃度が0.3-10 μg/mLとなるように、実施例14~17のmRNA封入核酸脂質粒子をHEK293T細胞へ加え、37℃、CO2濃度5%の条件で48時間培養した。培養後、96 well plateを4℃で1時間静置し、0.05% Tween 20を含むPBS (-)(PBST)を1wellあたり300 μL加えて3回洗浄を行った。その後、プレートのwell上に固相化されたHPV遺伝子型16のE7発現タンパク(16E7)は、horse radish peroxidase(HRP)がラベルされた抗16E7抗体を加えて、室温で2時間反応させ、PBSTでwellを3回洗浄した後、HRP基質を加えて検出した。16E7タンパク発現レベルは、各wellの450 nmの吸光度からバックグラウンドとなる540 nmの吸光度を差し引いて算出した。
HPV遺伝子型16E7ワクチン抗原に特異的な細胞傷害性T細胞(CTL)の誘導レベル(図2、3、5、9)
C57BL/6Jマウスは、日本クレアから購入した。全ての動物実験は、医薬基盤・健康・栄養研究所の機関ガイドラインに従って実施した。動物のあらゆる処置は麻酔下で行われ、イソフルランの吸入麻酔下、あるいはケタラール/セラクタールの皮下投与麻酔下で行った。
HPV遺伝子型16E7ワクチン抗原に対する抗体価測定(図4)
7週齢のC57BL/6マウスの大腿部筋肉内に、一匹あたりmRNA量で 10 μgのmRNA封入核酸脂質粒子を10日間隔で2回接種した。最終免疫から1週後にヘパリン存在下での末梢血の回収と血漿の調製を行い、評価試料とした。血漿中のHPV遺伝子型16E7ワクチン抗原に結合するtotal IgGは、ELISA法を用いて測定した。ELISA法の固相化処理は、96 wellプレートに16E7組換えタンパクを0.5 μg/mLの濃度で4℃、一晩固相化した。同時に、濃度既知のマウスIgGタンパクの希釈系列も作製し、標準曲線用に同一プレート上に固相化した。その後、PBSTで3回洗浄後、1% BSAを含むPBST(1% BSA/PBST)で1時間、ブロッキング処理を行った。血漿試料は、1% BSA/PBSTで段階希釈系列を作製し、16E7固相化wellに添加し、室温で2時間反応させた。マウスIgG標準曲線用のwellsは、1% BSA/PBSTを添加し、同様に反応させた。PBSTで3回洗浄後、HRPラベルされた抗マウスIgG抗体を各wellに添加し、室温で1時間反応させた。PBSTで3回洗浄後、HRP基質を加えて発色し、発色停止液として1Nの硫酸溶液を加えた。16E7に結合したマウスIgG濃度は、各wellの450 nmの吸光度からバックグラウンドとなる540 nmの吸光度を差し引いた後、標準曲線を用いて算出した。
C57BL/6マウスを用いたTC-1がん細胞の移植実験とmRNA封入核酸脂質粒子のがん退縮効果(図4-6)
C57BL/6マウスの肺上皮細胞にHPV遺伝子型16のE6およびE7が発現するTC-1がん細胞株を、マウス脇腹の皮下に投与し、腫瘍サイズを経時的に測定した。がん移植サイトであるマウス脇腹はTC-1がん細胞移植前に剃毛し、1匹あたり1×105個のTC-1細胞を移植した。実施例20のmRNA封入核酸脂質粒子は、TC-1細胞移植から8日後に、1匹あたり10 μgのmRNAを筋肉内に投与した。CD4陽性細胞及びCD8陽性細胞を除去するための抗体投与は、mRNA封入核酸脂質粒子投与の2日前から開始した。
抗CD4抗体、抗CD8抗体の投与によるCD4陽性及びCD8陽性細胞の除去(図4-6)
7週齢のC57BL/6マウスの腹腔内に、1日1匹あたり100 μgの抗CD4抗体(GK1.5)、及び抗CD8抗体(53-6.72)を、mRNA封入核酸脂質粒子投与の2日前から3日間連続で投与した。抗体投与3日目に、実施例20のmRNA封入核酸脂質粒子を1匹あたりmRNA量で10 μg、大腿部筋肉内に投与した。
OVAワクチン抗原に対する抗体価測定(図7)
7週齢のC57BL/6マウスの尾根部皮内に、一匹あたりmRNA量で 15 μgのmRNA封入核酸脂質粒子を2週間隔で2回接種した。最終免疫から1週後に末梢血の回収と血清の調製を行い、評価試料とした。血漿中のOVAワクチン抗原に結合するtotal IgGは、ELISA法を用いて測定した。ELISA法の固相化処理において、96 wellプレートにOVA組換えタンパクを1 μg/mLの濃度で4℃、一晩固相化した。同時に、濃度既知のマウスIgGタンパクの希釈系列も作製し、標準曲線用に同一プレート上に固相化した。その後、PBSTで3回洗浄後、1% BSAを含むPBST(1% BSA/PBST)で1時間、ブロッキング処理を行った。血清試料は、1% BSA/PBSTで段階希釈系列を作製し、OVA固相化wellに添加し、室温で2時間反応させた。マウスIgG標準曲線用のwellsは、1% BSA/PBSTを添加し、同様に反応させた。PBSTで3回洗浄後、HRPラベルされた抗マウスIgG抗体を各wellに添加し、室温で1時間反応させた。PBSTで3回洗浄後、HRP基質を加えて発色し、発色停止液として1Nの硫酸溶液を加えた。16E7に結合したマウスIgG濃度は、各wellの450 nmの吸光度からバックグラウンドとなる540 nmの吸光度を差し引いた後、標準曲線を用いて算出した。
OVAワクチン抗原特異的T細胞サイトカイン産生能(図8)
7週齢のC57BL/6マウスの尾根部皮内に、一匹あたりmRNA量で 15 μgのmRNA封入核酸脂質粒子を2週間隔で2回接種した。最終免疫から1週後に脾臓を回収し、脾臓細胞を調製した。96 well培養プレートに播種した脾臓細胞を、OVA抗原のMHC class I拘束性エピトープペプチド、OVAワクチン抗原タンパク質それぞれで刺激し、24時間培養した。OVA抗原特異的サイトカイン産生レベルは、培養上清を試料とし、サイトカインELISA法を用いて評価した。本検討では、CTLやTh1から産生される代表的なサイトカインであるIFN-γを測定した。
HPV遺伝子型18E6E7ワクチン抗原特異的T細胞サイトカイン産生能(図10)
C57BL/6Jマウスは、日本クレアから購入した。動物のあらゆる処置はイソフルランの吸入麻酔下で行った。
6週齢のC57BL/6マウスの腓腹筋内に、一匹あたりmRNA換算で5 μgのmRNA封入核酸脂質粒子を2週間隔で2回投与した。最終投与から1週間後に脾臓を採取し、脾臓細胞を調製した。HPV18E6プールペプチド(JPT社製、catalog # PM-HPV18-E6)で脾臓細胞を処理し、48時間培養後の培養上清中のIFN-γ量を、サイトカインELISA法によって測定した。
HPV遺伝子型16E6E7ワクチン抗原に特異的な細胞傷害性T細胞(CTL)の誘導レベル(図11)
C57BL/6Jマウスは、日本クレアから購入した。動物のあらゆる処置はイソフルランの吸入麻酔下で行った。
6週齢のC57BL/6マウスの腓腹筋内に、一匹あたりmRNA換算で5 μgのmRNA封入核酸脂質粒子を2週間隔で2回投与した。最終投与から1週間後に脾臓を採取し、脾臓細胞を調製した。脾臓細胞中のHPV遺伝子型16のE7(HPV16E7)に特異的な細胞傷害性T細胞(CTL)の誘導レベルは、T細胞表面マーカーに対する抗体及びMHC class I分子拘束性HPV16E7エピトープとMHC class I分子の複合体によって免疫染色を行い、フローサイトメトリー法を用いて測定した。
HPV遺伝子型16E6E7ワクチン抗原特異的T細胞サイトカイン産生能(図12)
6週齢のC57BL/6マウスの腓腹筋内に、一匹あたりmRNA換算で5 μgのmRNA封入核酸脂質粒子を2週間隔で2回投与した。最終投与から1週間後に脾臓を採取し、脾臓細胞を調製した。96 well培養プレートに脾臓細胞を播種し、HPV16E7のMHC class I拘束性エピトープペプチド処理下で24時間培養した後、培養上清中のIFN-γ量をサイトカインELISA法によって測定した。
実施例14~17のmRNA封入核酸脂質粒子によるHPV遺伝子型16のE7発現レベル(図1)
HEK293T細胞を用いて、培養細胞におけるmRNA封入核酸脂質粒子からの抗原発現レベルを検討した。結果を図1に示す。実施例15、16においては、実施例14、17に比べてHPV血清型16のE7(16E7)タンパク発現レベルが高い傾向が認められた。一方で、mRNA封入核酸脂質粒子未処理のwellと比較すると、いずれも16E7タンパク発現を誘導していることから、培養細胞におけるmRNA封入核酸脂質粒子のタンパク発現誘導能が明らかとなった。
参考例1のDNAワクチンモデルを構築し、マウスモデルにおける実施例9、11、13のmRNA封入核酸脂質粒子のCTL誘導能と比較検討を行った。結果を図2に示す。最終免疫から1週後に末梢血中の16E7特異的CTL誘導レベルを評価した結果、3種のmRNA封入核酸脂質粒子は、いずれもDNAワクチンモデル(pDNA)に比べて高いCTL誘導能を示した(左図)。また、最終免疫から2週後の脾臓細胞中の16E7特異的CTL誘導レベルを評価した結果、実施例9、11、13のmRNA封入核酸脂質粒子はpDNAと同等以上のCTL誘導レベルを示した(右図)。
C57BL/6マウスにおける4種類のmRNA封入核酸脂質粒子の16E7特異的CTL誘導レベルを検討した。結果を図3に示す。16E7特異的CTL誘導レベルは、個体間のばらつきはあるものの、4種のmRNA封入核酸脂質粒子全てにおいて認められた。
TC-1がん細胞を移植後、CD4陽性細胞及びCD8陽性細胞を除去したマウスにおけるmRNA封入核酸脂質粒子の抗体誘導レベルを検討した。結果を図4に示す。細胞除去抗体を投与していない対照群(対照群(No-depletion))と実施例20のmRNA封入核酸脂質粒子を投与した群(実施例20(No-depletion))を比較すると、実施例20(No-depletion)群で16E7特異的抗体レベルが認められた。また、CD8陽性細胞を除去したmRNA封入核酸脂質粒子投与群(実施例20(CD8-depletion))も、実施例20(No-depletion)群と同等の抗体誘導レベルを示した。しかしながら、CD4陽性細胞を除去したmRNA封入核酸脂質粒子投与群(実施例20(CD4-depletion))では、実施例20(No-depletion)群と実施例20(CD8-depletion)群に比べて、16E7抗体誘導レベルが減少していることが明らかとなった。以上の結果から、mRNA封入核酸脂質粒子投与による16E7抗原特異的抗体誘導には、CD4陽性細胞が重要である可能性が示唆された。
TC-1がん細胞を移植後、CD4陽性細胞及びCD8陽性細胞を除去したマウスにおけるmRNA封入核酸脂質粒子の16E7特異的CTL誘導レベルを検討した。結果を図5に示す。細胞除去抗体を投与していない対照群(対照群(No-depletion))と実施例20のmRNA封入核酸脂質粒子を投与した群(実施例20(No-depletion))を比較すると、実施例20(No-depletion)群で16E7特異的CTLレベルが認められた。一方、CD4陽性細胞を除去したmRNA封入核酸脂質粒子投与群(実施例20(CD4-depletion))は、実施例20(No-depletion)群と比較すると低いCTL誘導レベルを示した。更に、CD8陽性細胞を除去したmRNA封入核酸脂質粒子投与群(実施例20(CD8-depletion))では、実施例20(No-depletion)群と比べて、16E7特異的CTL誘導レベルが劇的に減少していることが明らかとなった。以上の結果から、mRNA封入核酸脂質粒子投与による16E7抗原特異的CTL誘導には、CD8陽性細胞が必須である可能性が示唆された。
TC-1がん細胞を移植後、CD4陽性細胞及びCD8陽性細胞を除去したマウスにおけるmRNA封入核酸脂質粒子のがん退縮効果を検討した。結果を図6に示す。細胞除去抗体を投与していない対照群(対照群(No-depletion))と実施例20のmRNA封入核酸脂質粒子を投与した群(実施例20(No-depletion))を比較すると、実施例20(No-depletion)群でTC-1がん腫瘍の増殖抑制効果が認められた。また、CD4陽性細胞を除去したmRNA封入核酸脂質粒子投与群(実施例20(CD4-depletion))は、実施例20(No-depletion)群と同様に、TC-1がん増殖抑制効果を示した。しかしながら、CD8陽性細胞を除去したmRNA封入核酸脂質粒子投与群(実施例20(CD8-depletion))では、実施例20(No-depletion)群と比べて、TC-1がん増殖が抑制されず、腫瘍サイズは対照群(No-depletion)と同等であった。以上の結果から、mRNA封入核酸脂質粒子投与によるがん増殖抑制効果には、CD8陽性細胞が必須である可能性が示唆された。
C57BL/6マウスに実施例21から実施例27のmRNA封入核酸脂質粒子を筋肉内投与し、最終免疫から1週後の血中OVA特異的抗体誘導レベルを検討した。結果を図7に示す。OVA特異的抗体誘導レベルは、実施例22のmRNA封入核酸脂質粒子で低く、その他のmRNA封入核酸脂質粒子投与群では同等であった。
C57BL/6マウスに実施例21から実施例27のmRNA封入核酸脂質粒子を筋肉内投与し、最終免疫から1週後に、脾臓細胞からのOVA特異的T細胞サイトカイン誘導レベルを検討した。結果を図8に示す。実施例26のmRNA封入核酸脂質粒子免疫群では、OVAのMHC class I拘束性エピトープペプチド、及びOVAタンパクの刺激に対するIFN-γ誘導レベルが最も低く、実施例21、実施例22、実施例25、実施例27のOVA抗原特異的誘導レベルは同等であった。
C57BL/6マウスにおける4種類のmRNA封入核酸脂質粒子の16E7特異的CTL誘導レベルを検討した。結果を図9に示す。16E7特異的CTL誘導レベルは、リン脂質種がDOPCである実施例28、実施例29、DOPEである実施例30、実施例31、そしてDSPCである実施例32と同等であった。
C57BL/6マウスに実施例37から実施例40のmRNA封入核酸脂質粒子を筋肉内投与し、最終免疫から1週後に、脾臓細胞からのHPV18E6特異的T細胞サイトカイン量を検討した。結果を図10に示す。実施例37から実施例40の各群では、NC群と比較して、HPV18E6プールペプチド処理に対するIFN-γ産生誘導が見られた(図10)。
C57BL/6マウスに実施例41から実施例52のmRNA封入核酸脂質粒子を筋肉内投与し、最終免疫から1週後に、脾臓細胞中の16E7特異的CTL誘導レベルを評価した。結果を図11に示す。16E7特異的CTL誘導レベルは、NC群に比べて、評価した全てのmRNA封入核酸脂質粒子において高かった。
C57BL/6マウスに実施例41から実施例52のmRNA封入核酸脂質粒子を筋肉内投与し、最終免疫から1週後に、脾臓細胞からのHPV16E7特異的T細胞サイトカイン誘導レベルを検討した。結果を図12に示す。NC群に比べて、いずれのmRNA封入核酸脂質粒子投与群においても、HPV16E7のMHC class I拘束性エピトープペプチド処理によってIFN-γ産生が増強された。
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。
GCTAGC(NheIサイト):塩基番号1~6
T7プロモーター配列:塩基番号8~27
human β-globinの5'-UTR配列:塩基番号39~88
KOZAK配列:塩基番号89~94
IgE leader sequenceの翻訳領域:塩基番号95~148
HPV16型E6の翻訳領域:塩基番号149~598
Furin cleavage site:塩基番号599~619
HPV16型E7の翻訳領域:塩基番号620~913
human β-globinの3’-UTR配列:塩基番号914~1045
poylA:塩基番号1046~1147
ACTAGT(SpeIサイト):塩基番号1152~1157
gctagcgtaatacgactcactataaggagacccaagctacatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccgccaccatggactggacctggatcctgtttctggtggccgctgccacacgggtgcacagctttcaggaccctcaagagaggcccagaaagctgcctcagctgtgtaccgagctgcagaccaccatccacgacatcatcctggaatgcgtgtactgcaagcagcagctcctgcggagagaggtgtacgatttcgccttccgggacctgtgcatcgtgtacagagatggcaacccctacgccgtgtgcgacaagggcctgaagttctacagcaagatcagcgagtaccggcactactgctacagcctgtacggcaccacactggaacagcagtacaacaagcccctgtgcgacctgctgatccggtgcatcaactgccagaaacctctgtgccccgaggaaaagcagcggcacctggacaagaagcagcggttccacaacatcagaggccggtggacaggcagaggcatgagctgttgtcggagcagccggaccagaagagaaacccagctgagaggccggaagagaagaagccacggcgatacccctacactgcacgagtacatgctggacctgcagcctgagacaaccgatctgtacggctacggccagctgaacgacagctctgaggaagaggacgagatcgacggacctgctggacaggccgaacctgatagagcccactacaatatcgtgaccttctgctgcaagtgcgacagcaccctgagactgtgtgtgcagagcacccacgtggacatcagaaccctggaagatctgctgatgggcaccctgggaatcgtgggccctatctgtagccagaagccttgagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagagcactagt
<配列番号2>HPV16 E6-E7 fusion2 mRNA -001。
aggagacccaagcuacauuugcuucugacacaacuguguucacuagcaaccucaaacagacaccgccaccauggacuggaccuggauccuguuucugguggccgcugccacacgggugcacagcuuucaggacccucaagagaggcccagaaagcugccucagcuguguaccgagcugcagaccaccauccacgacaucauccuggaaugcguguacugcaagcagcagcuccugcggagagagguguacgauuucgccuuccgggaccugugcaucguguacagagauggcaaccccuacgccgugugcgacaagggccugaaguucuacagcaagaucagcgaguaccggcacuacugcuacagccuguacggcaccacacuggaacagcaguacaacaagccccugugcgaccugcugauccggugcaucaacugccagaaaccucugugccccgaggaaaagcagcggcaccuggacaagaagcagcgguuccacaacaucagaggccgguggacaggcagaggcaugagcuguugucggagcagccggaccagaagagaaacccagcugagaggccggaagagaagaagccacggcgauaccccuacacugcacgaguacaugcuggaccugcagccugagacaaccgaucuguacggcuacggccagcugaacgacagcucugaggaagaggacgagaucgacggaccugcuggacaggccgaaccugauagagcccacuacaauaucgugaccuucugcugcaagugcgacagcacccugagacugugugugcagagcacccacguggacaucagaacccuggaagaucugcugaugggcacccugggaaucgugggcccuaucuguagccagaagccuugagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagagcacuag
<配列番号3>HPV16 E6-E7 fusion10のIVT用の鋳型DNA。
GCTAGC(NheIサイト):塩基番号1~6
T7プロモーター配列:塩基番号8~27
human β-globinの5'-UTR配列:塩基番号39~88
KOZAK配列:塩基番号89~94
IgE leader sequenceの翻訳領域:塩基番号95~148
HPV16型E6の翻訳領域:塩基番号149~598
Furin cleavage site:塩基番号599~619
HPV16型E7の翻訳領域:塩基番号620~913
human β-globinの3’-UTR配列:塩基番号914~1045
poylA:塩基番号1046~1147
ACTAGT(SpeIサイト):塩基番号1152~1157
gctagcgtaatacgactcactataaggagacccaagctacatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccgccaccatggactggacctggatcctgttcctggtggccgccgccacacgggtgcacagcttccaggacccccaagagaggcccagaaagctgccccagctgtgcaccgagctgcagaccaccatccacgacatcatcctggaatgcgtgtactgcaagcagcagctcctgcggagagaggtgtacgacttcgccttccgggacctgtgcatcgtgtacagagacggcaacccctacgccgtgtgcgacaagggcctgaagttctacagcaagatcagcgagtaccggcactactgctacagcctgtacggcaccacactggaacagcagtacaacaagcccctgtgcgacctgctgatccggtgcatcaactgccagaaacccctgtgccccgaggaaaagcagcggcacctggacaagaagcagcggttccacaacatcagaggccggtggacaggcagaggcatgagctgctgccggagcagccggaccagaagagaaacccagctgagaggccggaagagaagaagccacggcgacacccccacactgcacgagtacatgctggacctgcagcccgagacaaccgacctgtacggctacggccagctgaacgacagcagcgaggaagaggacgagatcgacggacccgccggacaggccgaacccgacagagcccactacaacatcgtgaccttctgctgcaagtgcgacagcaccctgagactgtgcgtgcagagcacccacgtggacatcagaaccctggaagacctgctgatgggcaccctgggaatcgtgggccccatctgcagccagaagccctgagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagagcactagt
<配列番号4>HPV16 E6-E7 fusion10 mRNA -001~006。
aggagacccaagcuacauuugcuucugacacaacuguguucacuagcaaccucaaacagacaccgccaccauggacuggaccuggauccuguuccugguggccgccgccacacgggugcacagcuuccaggacccccaagagaggcccagaaagcugccccagcugugcaccgagcugcagaccaccauccacgacaucauccuggaaugcguguacugcaagcagcagcuccugcggagagagguguacgacuucgccuuccgggaccugugcaucguguacagagacggcaaccccuacgccgugugcgacaagggccugaaguucuacagcaagaucagcgaguaccggcacuacugcuacagccuguacggcaccacacuggaacagcaguacaacaagccccugugcgaccugcugauccggugcaucaacugccagaaaccccugugccccgaggaaaagcagcggcaccuggacaagaagcagcgguuccacaacaucagaggccgguggacaggcagaggcaugagcugcugccggagcagccggaccagaagagaaacccagcugagaggccggaagagaagaagccacggcgacacccccacacugcacgaguacaugcuggaccugcagcccgagacaaccgaccuguacggcuacggccagcugaacgacagcagcgaggaagaggacgagaucgacggacccgccggacaggccgaacccgacagagcccacuacaacaucgugaccuucugcugcaagugcgacagcacccugagacugugcgugcagagcacccacguggacaucagaacccuggaagaccugcugaugggcacccugggaaucgugggccccaucugcagccagaagcccugagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagagcacuag
<配列番号5>HPV16 E6-E7 fusion10 opt2 鋳型DNA。
GCTAGC(NheIサイト):塩基番号1~6
T7プロモーター配列:塩基番号8~27
human β-globinの5'-UTR配列:塩基番号39~88
KOZAK配列:塩基番号89~94
IgE leader sequenceの翻訳領域:塩基番号95~148
HPV16型E6の翻訳領域:塩基番号149~598
Furin cleavage site:塩基番号599~619
HPV16型E7の翻訳領域:塩基番号620~913
human β-globinの3’-UTR配列:塩基番号914~1045
poylA:塩基番号1046~1147
ACTAGT(SpeIサイト):塩基番号1152~1157
gctagcgtaatacgactcactatagggagacccaagctacatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccgccaccatggactggacctggatcctgttcctggtggccgccgccacacgggtgcacagcttccaggacccccaagagaggcccagaaagctgccccagctgtgcaccgagctgcagaccaccatccacgacatcatcctggaatgcgtgtactgcaagcagcagctcctgcggagagaggtgtacgacttcgccttccgggacctgtgcatcgtgtacagagacggcaacccctacgccgtgtgcgacaagggcctgaagttctacagcaagatcagcgagtaccggcactactgctacagcctgtacggcaccacactggaacagcagtacaacaagcccctgtgcgacctgctgatccggtgcatcaactgccagaaacccctgtgccccgaggaaaagcagcggcacctggacaagaagcagcggttccacaacatcagaggccggtggacaggcagaggcatgagctgctgccggagcagccggaccagaagagaaacccagctgagaggccggaagagaagaagccacggcgacacccccacactgcacgagtacatgctggacctgcagcccgagacaaccgacctgtacggctacggccagctgaacgacagcagcgaggaagaggacgagatcgacggacccgccggacaggccgaacccgacagagcccactacaacatcgtgaccttctgctgcaagtgcgacagcaccctgagactgtgcgtgcagagcacccacgtggacatcagaaccctggaagacctgctgatgggcaccctgggaatcgtgggccccatctgcagccagaagccctgagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagagcactagt
<配列番号6>HPV16 E6-E7 fusion10 opt2 mRNA -001。
gggagacccaagcuacauuugcuucugacacaacuguguucacuagcaaccucaaacagacaccgccaccauggacuggaccuggauccuguuccugguggccgccgccacacgggugcacagcuuccaggacccccaagagaggcccagaaagcugccccagcugugcaccgagcugcagaccaccauccacgacaucauccuggaaugcguguacugcaagcagcagcuccugcggagagagguguacgacuucgccuuccgggaccugugcaucguguacagagacggcaaccccuacgccgugugcgacaagggccugaaguucuacagcaagaucagcgaguaccggcacuacugcuacagccuguacggcaccacacuggaacagcaguacaacaagccccugugcgaccugcugauccggugcaucaacugccagaaaccccugugccccgaggaaaagcagcggcaccuggacaagaagcagcgguuccacaacaucagaggccgguggacaggcagaggcaugagcugcugccggagcagccggaccagaagagaaacccagcugagaggccggaagagaagaagccacggcgacacccccacacugcacgaguacaugcuggaccugcagcccgagacaaccgaccuguacggcuacggccagcugaacgacagcagcgaggaagaggacgagaucgacggacccgccggacaggccgaacccgacagagcccacuacaacaucgugaccuucugcugcaagugcgacagcacccugagacugugcgugcagagcacccacguggacaucagaacccuggaagaccugcugaugggcacccugggaaucgugggccccaucugcagccagaagcccugagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagagcacuag
<配列番号7>配列番号7 OVA(Ovalbumin) の翻訳領域
atgggctccatcggcgcagcaagcatggaattttgttttgatgtattcaaggagctcaaagtccaccatgccaatgagaacatcttctactgccccattgccatcatgtcagctctagccatggtatacctgggtgcaaaagacagcaccaggacacagataaataaggttgttcgctttgataaacttccaggattcggagacagtattgaagctcagtgtggcacatctgtaaacgttcactcttcacttagagacatcctcaaccaaatcaccaaaccaaatgatgtttattcgttcagccttgccagtagactttatgctgaagagagatacccaatcctgccagaatacttgcagtgtgtgaaggaactgtatagaggaggcttggaacctatcaactttcaaacagctgcagatcaagccagagagctcatcaattcctgggtagaaagtcagacaaatggaattatcagaaatgtccttcagccaagctccgtggattctcaaactgcaatggttctggttaatgccattgtcttcaaaggactgtgggagaaaacatttaaggatgaagacacacaagcaatgcctttcagagtgactgagcaagaaagcaaacctgtgcagatgatgtaccagattggtttatttagagtggcatcaatggcttctgagaaaatgaagatcctggagcttccatttgccagtgggacaatgagcatgttggtgctgttgcctgatgaagtctcaggccttgagcagcttgagagtataatcaactttgaaaaactgactgaatggaccagttctaatgttatggaagagaggaagatcaaagtgtacttacctcgcatgaagatggaggaaaaatacaacctcacatctgtcttaatggctatgggcattactgacgtgtttagctcttcagccaatctgtctggcatctcctcagcagagagcctgaagatatctcaagctgtccatgcagcacatgcagaaatcaatgaagcaggcagagaggtggtagggtcagcagaggctggagtggatgctgcaagcgtctctgaagaatttagggctgaccatccattcctcttctgtatcaagcacatcgcaaccaacgccgttctcttctttggcagatgtgtttccccttaa
<配列番号8>HPV16型のE6抗原のアミノ酸配列。
FQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKGLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRGMSCCRSSRTRRETQL
<配列番号9>HPV16型のE7抗原のアミノ酸配列。
HGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKP
<配列番号10> pMA-HPV18_fusion1_opt1
GCTAGCGTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCCGCCACAAGAGTGCACAGCTTCGAGGACCCCACCAGACGGCCCTACAAGCTGCCCGACCTGTGCACCGAGCTGAACACCAGCCTGCAGGACATCGAGATCACCTGCGTGTACTGCAAGACCGTGCTGGAACTGACCGAGGTGTTCGAGTTCGCCTTCAAGGACCTGTTCGTGGTGTACCGGGACAGCATCCCCCACGCCGCCTGCCACAAGGGCATCGACTTCTACAGCCGGATCAGAGAGCTGCGGCACTACAGCGACAGCGTGTACGGCGACACCCTGGAAAAGCTGACCAACACCGGCCTGTACAACCTGCTGATCCGGTGCCTGAGATGCCAGAAGCCCCTGAACCCCGCCGAGAAGCTGAGACACCTGAACGAGAAGCGGCGGTTCCACAACATCGCCGGCCACTACAGAGGCCAGGGCCACAGCTGCTGCAACCGGGCCAGACAAGAGAGACTGCAGCGGCGGAGAGAAACCCAAGTGCGGGGCAGAAAGAGAAGAAGCCACGGCCCCAAGGCCACACTGCAGGACATCGTGCTGCACCTGGAACCCCAGAACGAGATCCCCGTGGACCTGCTCGGACACGGCCAGCTGAGCGACAGCGAGGAAGAGAACGACGAGATCGACGGCGTGAACCACCAGCACCTGCCCGCCAGAAGGGCCGAACCACAGAGACACACCATGCTGTGCATGTGCTGCAAGTGCGAGGCCCGGATCAAGCTGGTGGTGGAAAGCAGCGCCGACGACCTGAGAGCCTTCCAGCAGCTGTTCCTGAACACCCTGAGCTTCGTGGGACCCTGGTGCGCCAGCCAGCAGTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAGCACTAGT
NheI配列:塩基番号1~6
T7プロモーター:塩基番号7~24
A:転写開始点:塩基番号25
5‘-UTR:塩基番号39~88
Kozak配列:塩基番号89~94
IgE leader配列:塩基番号95~148
HPV18 E6配列:塩基番号149~613
Furin認識配列:塩基番号614~634
HPV18 E7配列:塩基番号635~949
3‘-UTR:塩基番号950~1081
polyA配列:塩基番頭1082~1183
SpeI配列:塩基番号1188~1193
<配列番号11> HPV18 E6-E7 fusion1 opt1 mRNA -001及び002
AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGGACUGGACCUGGAUCCUGUUCCUGGUGGCCGCCGCCACAAGAGUGCACAGCUUCGAGGACCCCACCAGACGGCCCUACAAGCUGCCCGACCUGUGCACCGAGCUGAACACCAGCCUGCAGGACAUCGAGAUCACCUGCGUGUACUGCAAGACCGUGCUGGAACUGACCGAGGUGUUCGAGUUCGCCUUCAAGGACCUGUUCGUGGUGUACCGGGACAGCAUCCCCCACGCCGCCUGCCACAAGGGCAUCGACUUCUACAGCCGGAUCAGAGAGCUGCGGCACUACAGCGACAGCGUGUACGGCGACACCCUGGAAAAGCUGACCAACACCGGCCUGUACAACCUGCUGAUCCGGUGCCUGAGAUGCCAGAAGCCCCUGAACCCCGCCGAGAAGCUGAGACACCUGAACGAGAAGCGGCGGUUCCACAACAUCGCCGGCCACUACAGAGGCCAGGGCCACAGCUGCUGCAACCGGGCCAGACAAGAGAGACUGCAGCGGCGGAGAGAAACCCAAGUGCGGGGCAGAAAGAGAAGAAGCCACGGCCCCAAGGCCACACUGCAGGACAUCGUGCUGCACCUGGAACCCCAGAACGAGAUCCCCGUGGACCUGCUCGGACACGGCCAGCUGAGCGACAGCGAGGAAGAGAACGACGAGAUCGACGGCGUGAACCACCAGCACCUGCCCGCCAGAAGGGCCGAACCACAGAGACACACCAUGCUGUGCAUGUGCUGCAAGUGCGAGGCCCGGAUCAAGCUGGUGGUGGAAAGCAGCGCCGACGACCUGAGAGCCUUCCAGCAGCUGUUCCUGAACACCCUGAGCUUCGUGGGACCCUGGUGCGCCAGCCAGCAGUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAGCACUAG
<配列番号12> pMA-HPV18_fusion1_opt2
GCTAGCGTAATACGACTCACTATAGGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCCGCCACAAGAGTGCACAGCTTCGAGGACCCCACCAGACGGCCCTACAAGCTGCCCGACCTGTGCACCGAGCTGAACACCAGCCTGCAGGACATCGAGATCACCTGCGTGTACTGCAAGACCGTGCTGGAACTGACCGAGGTGTTCGAGTTCGCCTTCAAGGACCTGTTCGTGGTGTACCGGGACAGCATCCCCCACGCCGCCTGCCACAAGGGCATCGACTTCTACAGCCGGATCAGAGAGCTGCGGCACTACAGCGACAGCGTGTACGGCGACACCCTGGAAAAGCTGACCAACACCGGCCTGTACAACCTGCTGATCCGGTGCCTGAGATGCCAGAAGCCCCTGAACCCCGCCGAGAAGCTGAGACACCTGAACGAGAAGCGGCGGTTCCACAACATCGCCGGCCACTACAGAGGCCAGGGCCACAGCTGCTGCAACCGGGCCAGACAAGAGAGACTGCAGCGGCGGAGAGAAACCCAAGTGCGGGGCAGAAAGAGAAGAAGCCACGGCCCCAAGGCCACACTGCAGGACATCGTGCTGCACCTGGAACCCCAGAACGAGATCCCCGTGGACCTGCTCGGACACGGCCAGCTGAGCGACAGCGAGGAAGAGAACGACGAGATCGACGGCGTGAACCACCAGCACCTGCCCGCCAGAAGGGCCGAACCACAGAGACACACCATGCTGTGCATGTGCTGCAAGTGCGAGGCCCGGATCAAGCTGGTGGTGGAAAGCAGCGCCGACGACCTGAGAGCCTTCCAGCAGCTGTTCCTGAACACCCTGAGCTTCGTGGGACCCTGGTGCGCCAGCCAGCAGTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAGCACTAGT
NheI配列:塩基番号1~6
T7プロモーター:塩基番号7~24
G:転写開始点:塩基番号25
5‘-UTR:塩基番号39~88
Kozak配列:塩基番号89~94
IgE leader配列:塩基番号95~148
HPV18 E6配列:塩基番号149~613
Furin認識配列:塩基番号614~634
HPV18 E7配列:塩基番号635~949
3‘-UTR:塩基番号950~1081
polyA配列:塩基番頭1082~1183
SpeI配列:塩基番号1188~1193
<配列番号13> HPV18 E6-E7 fusion1 opt2 mRNA -001及び002
GGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGGACUGGACCUGGAUCCUGUUCCUGGUGGCCGCCGCCACAAGAGUGCACAGCUUCGAGGACCCCACCAGACGGCCCUACAAGCUGCCCGACCUGUGCACCGAGCUGAACACCAGCCUGCAGGACAUCGAGAUCACCUGCGUGUACUGCAAGACCGUGCUGGAACUGACCGAGGUGUUCGAGUUCGCCUUCAAGGACCUGUUCGUGGUGUACCGGGACAGCAUCCCCCACGCCGCCUGCCACAAGGGCAUCGACUUCUACAGCCGGAUCAGAGAGCUGCGGCACUACAGCGACAGCGUGUACGGCGACACCCUGGAAAAGCUGACCAACACCGGCCUGUACAACCUGCUGAUCCGGUGCCUGAGAUGCCAGAAGCCCCUGAACCCCGCCGAGAAGCUGAGACACCUGAACGAGAAGCGGCGGUUCCACAACAUCGCCGGCCACUACAGAGGCCAGGGCCACAGCUGCUGCAACCGGGCCAGACAAGAGAGACUGCAGCGGCGGAGAGAAACCCAAGUGCGGGGCAGAAAGAGAAGAAGCCACGGCCCCAAGGCCACACUGCAGGACAUCGUGCUGCACCUGGAACCCCAGAACGAGAUCCCCGUGGACCUGCUCGGACACGGCCAGCUGAGCGACAGCGAGGAAGAGAACGACGAGAUCGACGGCGUGAACCACCAGCACCUGCCCGCCAGAAGGGCCGAACCACAGAGACACACCAUGCUGUGCAUGUGCUGCAAGUGCGAGGCCCGGAUCAAGCUGGUGGUGGAAAGCAGCGCCGACGACCUGAGAGCCUUCCAGCAGCUGUUCCUGAACACCCUGAGCUUCGUGGGACCCUGGUGCGCCAGCCAGCAGUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAGCACUAG
<配列番号14> HPV18型のE6抗原のアミノ酸配列。
FEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKGIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQGHSCCNRARQERLQRRRETQV
<配列番号15> HPV18型のE7抗原のアミノ酸配列。
HGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVGPWCASQQ
<配列番号16> プロテアーゼ切断配列のアミノ酸配列
RGRKRRS
<配列番号17> HPV16型のE6抗原及びE7抗原融合タンパクのアミノ酸配列
MDWTWILFLVAAATRVHSFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKGLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRGMSCCRSSRTRRETQLRGRKRRSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKP
<配列番号18> HPV18型のE6抗原及びE7抗原融合タンパクのアミノ酸配列
MDWTWILFLVAAATRVHSFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKGIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQGHSCCNRARQERLQRRRETQVRGRKRRSHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVGPWCASQQ
Claims (56)
- 一般式(Ia)中のR1及びR2が、共にメチル基である、請求項1に記載の粒子。
- 一般式(Ia)中のpが、3である請求項1又は2に記載の粒子。
- 一般式(Ia)中のL1が、アセトキシ基を1若しくは複数個有していてもよいC17-C19アルケニル基である、請求項1~3のいずれかに記載の粒子。
- 一般式(Ia)中のL2が、アセトキシ基を1若しくは複数個有していてもよいC10-C12アルキル基、又はアセトキシ基を1若しくは複数個有していてもよいC10-C19アルケニル基である、請求項1~4のいずれかに記載の粒子。
- 一般式(Ia)中のL2が、アセトキシ基を1若しくは複数個有していてもよいC10-C12アルキル基、又はアセトキシ基を1若しくは複数個有していてもよいC17-C19アルケニル基である、請求項1~4のいずれかに記載の粒子。
- 一般式(Ia)中のL1が、(R)-11-アセチルオキシ-cis-8-ヘプタデセニル基、cis-8-ヘプタデセニル基、又は(8Z,11Z)-ヘプタデカジエニル基である請求項1~6のいずれかに記載の粒子。
- 一般式(Ia)中のL2が、デシル基、cis-7-デセニル基、ドデシル基、又は(R)-11-アセチルオキシ-cis-8-ヘプタデセニル基である、請求項1~7のいずれかに記載の粒子。
- 脂質が、さらに、両親媒性脂質、ステロール類及びPEG脂質を含む請求項9又は10に記載の粒子。
- 脂質が、さらに、両親媒性脂質、ステロール類及びPEG脂質を含む請求項11に記載の粒子。
- 両親媒性脂質が、ジステアロイルホスファチジルコリン、ジオレオイルホスファチジルコリン及びジオレオイルホスファチジルエタノールアミンからなる群より選択される少なくとも1つである請求項12記載の粒子。
- 両親媒性脂質が、ジステアロイルホスファチジルコリン、ジオレオイルホスファチジルコリン及びジオレオイルホスファチジルエタノールアミンからなる群より選択される少なくとも1つである請求項13記載の粒子。
- ステロール類がコレステロールである請求項12又は14に記載の粒子。
- ステロール類がコレステロールである請求項13又は15に記載の粒子。
- PEG脂質が、1、2-ジミリストイル-sn-グリセロール メトキシポリエチレン グリコール及び/又はN-[メトキシ ポリ(エチレングリコール)2000]カルバモイル]-1,2-ジミリスチルオキシプロピル-3-アミンである請求項12、14、16のいずれかに記載の粒子。
- PEG脂質が、1、2-ジミリストイル-sn-グリセロール メトキシポリエチレン グリコール及び/又はN-[メトキシ ポリ(エチレングリコール)2000]カルバモイル]-1,2-ジミリスチルオキシプロピル-3-アミンである請求項13、15、17のいずれかに記載の粒子。
- 両親媒性脂質、ステロール類、カチオン性脂質、及びPEG脂質の脂質組成が、モル量にて、両親媒性脂質が22.5%以下、ステロール類が15~55%、カチオン性脂質が40~65%、PEG脂質が1~5%であり、核酸重量に対する総脂質重量の比率が、15~30である請求項12~19のいずれかに記載の粒子。
- 両親媒性脂質が5~22.5%である請求項20に記載の粒子。
- 両親媒性脂質が10~22.5%である請求項21に記載の粒子。
- 両親媒性脂質、ステロール類、カチオン性脂質、及びPEG脂質の脂質組成が、モル量にて、両親媒性脂質が5~15%、ステロール類が35~50%、カチオン性脂質が40~55%、PEG脂質が1~3%であり、核酸重量に対する総脂質重量の比率が、15~30である請求項12、14、16、18のいずれかに記載の粒子。
- 両親媒性脂質が10~15%、ステロール類が35~45%、カチオン性脂質が40~50%、PEG脂質が1~2%である請求項23記載の粒子。
- 両親媒性脂質、ステロール類、カチオン性脂質、及びPEG脂質の脂質組成が、モル量にて、両親媒性脂質が15~22.5%、ステロール類が15~40%、カチオン性脂質が40~60%、PEG脂質が1~3%であり、核酸重量に対する総脂質重量の比率が、15~30である請求項13、15、17、19のいずれかに記載の粒子。
- カチオン性脂質が45~60%、PEG脂質が1~2%である請求項25記載の粒子。
- 核酸重量に対する総脂質重量の比率が15~25である請求項20~26のいずれかに記載の粒子。
- 核酸重量に対する総脂質重量の比率が15~22.5である請求項27記載の粒子。
- ヒトパピローマウイルスがHPV16型である請求項1~28のいずれかに記載の粒子。
- ヒトパピローマウイルスがHPV16型であり、HPV16型のE6抗原が配列番号8のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる請求項29記載の粒子。
- ヒトパピローマウイルスが、HPV16型であり、HPV16型のE7抗原が配列番号9のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる請求項29又は30記載の粒子。
- ヒトパピローマウイルスがHPV16型であり、ヒトパピローマウイルスのE6抗原及びE7抗原を発現させることができる核酸が、配列番号17のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる、HPV16型のE6抗原及びE7抗原の融合タンパクをコードする請求項29~31のいずれかに記載の粒子。
- ヒトパピローマウイルスがHPV16型であり、HPV16型のE6抗原及びE7抗原を発現させることができる核酸が、キャップ構造(Cap)、5’非翻訳領域(5’-UTR)、リーダー配列(leader sequence)、E6の翻訳領域、プロテアーゼ切断配列(Furin Cleavage site)、E7の翻訳領域、3’非翻訳領域(3’-UTR)及びポリA尾部(polyA)を含むmRNAである請求項29~32のいずれかに記載の粒子。
- HPV16型のE6抗原及びE7抗原を発現させることができる核酸の配列が、配列番号2、4又は6のいずれかの配列と少なくとも90%の同一性を有するヌクレオチド配列からなる請求項33記載の粒子。
- ヒトパピローマウイルスがHPV18型である請求項1~28のいずれかに記載の粒子。
- ヒトパピローマウイルスがHPV18型であり、HPV18型のE6抗原が配列番号14のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる請求項35記載の粒子。
- ヒトパピローマウイルスが、HPV18型であり、HPV18型のE7抗原が配列番号15のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる請求項35又は36記載の粒子。
- ヒトパピローマウイルスがHPV18型であり、ヒトパピローマウイルスのE6抗原及びE7抗原を発現させることができる核酸が、配列番号18のアミノ酸配列と少なくとも95%の同一性を有するアミノ酸配列からなる、HPV18型のE6抗原及びE7抗原の融合タンパクをコードする請求項35~37のいずれかに記載の粒子。
- ヒトパピローマウイルスがHPV18型であり、HPV18型のE6抗原及びE7抗原を発現させることができる核酸が、キャップ構造(Cap)、5’非翻訳領域(5’-UTR)、リーダー配列(leader sequence)、E6の翻訳領域、プロテアーゼ切断配列(Furin Cleavage site)、E7の翻訳領域、3’非翻訳領域(3’-UTR)及びポリA尾部(polyA)を含むmRNAである請求項35~38のいずれかに記載の粒子。
- HPV18型のE6抗原及びE7抗原を発現させることができる核酸の配列が、配列番号11又は13のいずれかの配列と少なくとも90%の同一性を有するヌクレオチド配列からなる請求項39記載の粒子。
- 核酸が少なくとも1個の修飾ヌクレオチドを含む請求項1~40のいずれかに記載の粒子。
- 修飾ヌクレオチドが、5位が置換したピリミジンヌクレオチド及び/又は1位が置換していてもよいシュードウリジンの少なくとも1個を含む請求項41記載の粒子。
- 修飾ヌクレオチドが、5-メチルシチジン、5-メトキシウリジン、5-メチルウリジン、シュードウリジン、及び1-アルキルシュードウリジンからなる群から選ばれる少なくとも1個を含む請求項41記載の粒子。
- 修飾ヌクレオチドが、5-メチルシチジン、5-メチルウリジン、及び1-メチルシュードウリジンからなる群から選ばれる少なくとも1個を含む請求項41記載の粒子。
- 平均粒子径が30nm~300nmである請求項1~44のいずれかに記載の粒子。
- ヒトパピローマウイルスによる感染を予防及び/又は治療するための組成物を製造するための請求項1~45のいずれかに記載の粒子の使用。
- 感染がHPV16型又はHPV18型のヒトパピローマウイルスによる感染である請求項46に記載の粒子の使用。
- 請求項1~45のいずれかに記載の粒子を含有する、組成物。
- ヒトパピローマウイルスのE6抗原及びE7抗原をin vivo又はin vitroで発現させるための請求項48記載の組成物。
- 医薬として用いられる請求項48又は49記載の組成物。
- ヒトパピローマウイルスに対する免疫反応を誘導するための請求項50記載の組成物。
- ヒトパピローマウイルス感染を予防及び/又は治療するための請求項50又は51記載の組成物。
- 請求項48又は49記載の組成物を細胞に導入することを含む、ヒトパピローマウイルスのE6抗原及びE7抗原をin vitroで発現させる方法。
- 請求項48~52のいずれかに記載の組成物を哺乳動物に投与することを含む、ヒトパピローマウイルスのE6抗原及びE7抗原をin vivoで発現させる方法。
- 請求項50又は51に記載の組成物を哺乳動物に投与することを含む、ヒトパピローマウイルスに対する免疫反応を誘導する方法。
- 請求項50~52のいずれかに記載の組成物を哺乳動物に投与することを含む、ヒトパピローマウイルス感染を予防及び/又は治療する方法。
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US17/776,743 US20220409540A1 (en) | 2019-11-15 | 2020-11-13 | Nucleic acid lipid particle vaccine encapsulating hpv mrna |
EP20887577.3A EP4059515A4 (en) | 2019-11-15 | 2020-11-13 | LIPID AND NUCLEIC ACID PARTICLE VACCINE ENCAPSULATING HPV MRNA |
CN202080077953.1A CN114650841A (zh) | 2019-11-15 | 2020-11-13 | 封装有HPV mRNA的核酸脂质颗粒疫苗 |
KR1020227016803A KR20220102617A (ko) | 2019-11-15 | 2020-11-13 | HPV mRNA 를 봉입한 핵산 지질 입자 백신 |
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