Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/5123—Organic compounds, e.g. fats, sugars
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
  • C12N2740/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
  • C12N2740/14034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present disclosure relates to a lipid complex in which a nucleic acid is encapsulated in a lipid, and a pharmaceutical composition containing the same.
• the present disclosure relates to a lipid complex that can be used to induce an immune response against HTLV-1, and a pharmaceutical composition containing the same.
• HTLV-1 human T-cell leukemia virus type-I
• ATL adult T-cell leukemia-lymphoma
• HAM HTLV-1 associated myelopathy
• HU HTLV-1 associated uveitis
• ATL is resistant to treatment and has a poor prognosis
• preventing HTLV-1 infection is important in preventing the onset of ATL.
• ATL patients who have undergone hematopoietic stem cell transplantation may experience a relapse of ATL after transplantation.
• inducing an immune response against HTLV-1 is also important in improving the prognosis of ATL treatment.
• effective treatments and methods of preventing the onset of these HTLV-1-related diseases have not yet been established.
• compositions and methods that can be used to induce an immune response against HTLV-1.
• the present disclosure is, for example, as follows:
• the nucleic acid is A nucleic acid comprising a polynucleotide encoding the HTLV-1 antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof;
• the nucleic acid comprises a nucleic acid comprising a polynucleotide encoding at least one selected from the group consisting of Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and Gag p24 or an immunogenic fragment thereof.
• the lipid complex according to any one of [1] to [3].
• nucleic acid (i) at least one amino acid sequence selected from SEQ ID NOs: 1, 4, 7, 8, 11-14; (ii) an amino acid sequence having at least 80% homology to at least one amino acid sequence selected from SEQ ID NOs: 1, 4, 7, 8, 11 to 14; (iii) an amino acid sequence in which 1 to 31 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 1, 4, 7, 8, 11 to 14; (iv) an amino acid sequence comprising at least one amino acid sequence selected from SEQ ID NO: 11 or 12, SEQ ID NO: 13, or SEQ ID NO: 14, and having at least 80% homology to SEQ ID NO: 1; (v) an amino acid sequence including at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1, the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1, or the amino acid sequence of positions 102 to 130 of SEQ ID NO: 1; (vi) an amino acid sequence comprising at least one of the amino acid sequence
• lipid complex according to any one of [1] to [6], wherein the lipid complex is a lipid nanoparticle (LNP).
• LNP lipid nanoparticle
• nucleic acid is mRNA.
• the nucleic acid is (i) a polynucleotide consisting of at least one base sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 9, 10, 15 to 17; (ii) a polynucleotide consisting of the base sequence of (i) above, in which 1 to 94 bases have been deleted, inserted, substituted, and/or added; (iii) a polynucleotide having a base sequence having 80% or more identity to the base sequence of (i); and (iv) a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a base sequence complementary to the base sequence of (i); (v) a polynucleotide consisting of at least one of the nucleotide sequences of bases 1 to 177 of SEQ ID NO: 3, bases 154 to 327 of SEQ ID NO: 3, or bases 304 to 390 of SEQ ID NO: 3; (vi) a polynucleot
• a pharmaceutical composition comprising the lipid complex according to any one of [1] to [11].
• [16] A method for preventing and/or treating an HTLV-1-associated disease, comprising administering to a subject the pharmaceutical composition according to [12] or [13].
• [17] Use of the lipid complex according to any one of [1] to [11] or the pharmaceutical composition according to [12] or [13] for the prevention and/or treatment of an HTLV-1-associated disease.
• lipid complexes and pharmaceutical compositions capable of inducing an immune response against HTLV-1 are provided.
• the pharmaceutical compositions of the present disclosure can be used for the prevention and/or treatment of HTLV-1-associated diseases because they can induce an immune response against HTLV-1.
• the pharmaceutical compositions of the present disclosure can be used as mRNA vaccines for the prevention of HTLV-1-associated diseases.
• the lipid complexes (particularly lipid nanoparticles) of the present disclosure have excellent stability.
• Fig. 1 is a graph showing the results of the ELISPOT assay in Example 2.
• Fig. 1 shows the results showing the number of spots in each peptide pool.
• Figure 2 is a graph showing the results of the ELISPOT assay in Example 2.
• Figure 2 shows the results showing the number of spots in each peptide pool.
• Figure 3 is a graph showing the results of the ELISPOT assay in Example 2.
• Figure 3 shows the results showing the number of spots in each peptide pool.
• FIG. 4 is a graph showing the results of the LIPS assay in Reference Example 1.
• FIG. 5 is a graph showing the results of RT-qPCR in Reference Example 1.
• FIG. 6 is a photograph showing the results of the proximity ligation assay in Reference Example 1.
• FIG. 7 is a photograph showing the results of the ELISPOT assay in Reference Example 1.
• FIG. 8 is a photograph showing the results of the ELISPOT assay in Reference Example 1.
• “Cationic lipid” refers to an amphipathic molecule having a lipophilic region containing one or more carbohydrate groups and a hydrophilic region containing polar groups that become protonated at a particular pH.
• Negtral lipid refers to a lipid that exists at physiological pH in either an uncharged or neutral zwitterionic form.
• Polyethylene glycol-modified lipid (PEG lipid) refers to a lipid bearing a polyethylene glycol (PEG) group.
• “Sterol” refers to an alcohol having a steroid skeleton.
• Alkyl means a straight-chain, cyclic or branched saturated aliphatic hydrocarbon group having the specified number of carbon atoms.
• Alkenyl means a straight or branched chain hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond, including, but not limited to, monoenes, dienes, trienes, and tetraenes.
• Alkylene means a linear, cyclic or branched divalent saturated aliphatic hydrocarbon group having the specified number of carbon atoms.
• a “lipid complex” refers to an aggregate containing multiple lipids that are physically bound to each other by intermolecular forces.
• a “lipid complex” is a “lipid particle.”
• a “lipid particle” is a particle consisting of an aggregate of lipid molecules.
• a “particle” typically has an average particle size of nano-size to micron-size (1 nm to 1 ⁇ m or um), and is called a nanosphere, microsphere, nanocapsule, microcapsule, etc.
• a particle can be, for example, a dispersed phase in an emulsion, or an internal phase in a suspension.
• a “lipid particle” is a microparticle consisting of a membrane-like molecular aggregate formed by the association of lipids.
• the term “lipid nanoparticles” (LNPs) refers to lipid particles having an average nano-sized particle size (eg, about 1 nm to 1000 nm).
• Protein refers to a peptide polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and/or artificial amino acids.
• the shape of the polymer can be, for example, linear, branched, or cyclic.
• the protein can also be called a peptide or a polypeptide.
• Retrovirus refers to a type of RNA virus that has reverse transcriptase. It is known that the retrovirus infects and grows in a host cell as follows. The retrovirus infects a host cell that is the target of infection, synthesizes double-stranded DNA by reverse transcription of viral RNA, and incorporates the viral DNA as a provirus into the genomic DNA of the host cell. The host cell then produces viral RNA and viral proteins, and the retroviral protease cleaves the immature viral proteins, and the mature viral proteins are assembled to become viral particles. The viral particles bud from the surface of the host cell.
• HTLV-1 refers to a type of retrovirus, human T-cell leukemia virus type 1.
• the HTLV-1 is known as a virus that causes diseases such as adult T-cell leukemia/lymphoma (ATL (or ATLL)), HTLV-1 associated myelopathy (HAM, TSP), and HTLV-1 uveitis (HU).
• the HTLV-1 is also known as a virus that causes diseases such as adult T-cell leukemia/lymphoma (ATL, or ATLL), HTLV-1 associated myelopathy (HAM, TSP), and HTLV-1 uveitis (HU).
• HTLV-1 is a virus belonging to the Oncoviridae subfamily of the Roviridae family.
• HTLV-1 has viral structural genes such as Gag, Pol, and Env. HTLV-1 also has regulatory genes such as Tax and Rex. HTLV-1 also has accessory genes such as p12, p13, p30, and HBZ.
• An example of the HTLV-1 provirus is a protein consisting of an amino acid sequence registered in Genbank under the accession number AB513134.1.
• HTLV-1-associated disease is a general term for diseases caused by HTLV-1 infection.
• Examples of “HTLV-1-associated disease” include 1) ATL, 2) HAM, and 3) HU.
• ATL or “ATLL” means adult T-cell leukemia/lymphoma. It is known that in HTLV-1-infected individuals (carriers), ATL develops when HTLV-1 infects CD4 positive (+) T cells and the like, causing the infected cells to become cancerous.
• HAM stands for HTLV-1 associated myelopathy (TSP: tropical spastic paraparesis).
• HU means HTLV-1 uveitis or HTLV-1 associated uveitis.
• Gag protein refers to a retroviral capsid precursor protein. Gag is known to play a role in the assembly and budding of virus particles. Gag is processed by retroviral protease.
• HTLV-1 Gag is processed by protease into p15 (nucleocapsid protein), p19 (matrix protein), p24 (capsid protein), and the like.
• An example of the HTLV-1 Gag is a protein (SEQ ID NO: 1) consisting of an amino acid sequence registered in Genbank under the accession number AAA85841.1.
• Tax protein refers to a transcriptional activation factor that activates both the retroviral LTR (Long terminal repeat) and the intracellular promoter. Tax is known to play an important role in the infectivity of the virus and the oncogenic transformation of infected cells.
• An example of the TAX of HTLV-1 is a protein (SEQ ID NO: 4) consisting of the amino acid sequence registered in Genbank under the accession number P03409.2.
• HTLV-1 TAX (SEQ ID NO:4) MAHFPGGQSLLFGYPVYVFGDCVQGDWCPISGGLCSARLHRHALLATCPEHQITWDPIDGRVIGSALQFLIPRLPSFPTQRTSKTLKVLTPPITHTTPNIPPSFLQAMRKYSPFRNGYMEPTLGQHLPTLSFPDPGLRPQNLYTLWGGSVVCMYLYQLSPPITWPLLPHVIFCHP GQLGAFLTNVPYKRIEELLYKISLTTGALIILPEDCLPTTLFQPARAPVTLTAWQNGLLPFHSTLTTPGLIWTFTDGTPMISGPCPKDGQPSLVLQSSSFIFHKFQTKAYHPSFLLSHGLIQYSSFHSLHLLFEEYTNIPISLLFNEKEADDNDHEPQISPGGLEPPSEKHFRETEV
• HBZ protein (hereinafter, also referred to as HBZ)" refers to a retroviral bZIP factor. HBZ is known to contribute to the transcriptional control of viral RNA.
• the HTLV-1 HBZ is, for example, a protein consisting of the amino acid sequence registered in Genbank under accession number BAE06226.1 (SEQ ID NO: 7).
• the HTLV-1 HBZ may also be, for example, a protein consisting of the amino acid sequence represented by SEQ ID NO: 8.
• HTLV-1 HBZ Amino acid sequence of HTLV-1 HBZ (SEQ ID NO:7) MAASGLFRCLPVSCPEDLLVEELVDGLLSLEEELKDKEEEEAVLDGLLSLEEESRGRLRRGPPGEKAPPRGETHRDRQRRAEEKRKRKKEREKEEEKQTAEYL KRKEEEKARRRRRAEKKAADVARRKQEEQERRERKWRQGAEKAKQHSARKEKMQELGIDGYTRQLEGEVESLEAERRKLLQEKEDLMGEVNYWQGRLEAMWLQ
• HTLV-1 HBZ Amino acid sequence of HTLV-1 HBZ (SEQ ID NO:8) MAASGLFRCLPVSCPEDLLVEELVDGLLSLEEELKDKEEEEAVLDGLLSLEEESRGRLRRGPPGEKAPPRGETHRDRQRRAEEKRKRKKEREKEEEKQIAEYL KRKEEEKARRRRRAEKKAADVARRKQEEQERRERKWRQGAEKAKQHSARKEKMQELGIDGYTRQLEGEVESLEAERRKLLQEKEDLMGEVNYWQGRLEAMWLQ
• T cells refers to a type of white blood cell, classified as a lymphocyte, that expresses a T cell receptor (TCR).
• TCR T cell receptor
• Known examples of T cells include CD4+ helper T cells and CD8+ cytotoxic T cells.
• Antigenicity refers to the property of an antigen to induce immune responses such as antibody production and cellular immunity.
• Immunogenic refers to the property of an antigen to induce an immune response, such as the production of antibodies or cellular immunity.
• Immunogenic fragment also called an immunogenic portion, refers to a fragment or truncation of a protein or polypeptide that induces an immune response.
• Vaccine refers to a composition that generates an immune response for the prevention and/or treatment of a disease or condition. A vaccine is thus a pharmaceutical product that contains an immunogenic agent and is intended for use in generating specific defense and protection by vaccination in humans or animals.
• “Pharmaceutically acceptable carrier” means a solvent and/or additive that can be commonly used in the formulation technology of pharmaceutical compositions. It is preferable that the pharmaceutically acceptable carrier is one that is almost or completely non-toxic to living organisms.
• Nucleic acid refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and/or modified nucleotides.
• DNA deoxyribonucleotides
• RNA ribonucleotides
• nucleic acid refers to a polymer of nucleotides that encodes the amino acid sequence of the protein. Examples of the nucleic acid include genomic DNA, cDNA, and mRNA. The nucleic acid may be, for example, single-stranded or double-stranded.
• the nucleic acid can be interchangeably read as a "polynucleotide” or an "oligonucleotide.”
• the base sequence of the DNA can be, for example, the explanation of the example of the base sequence of an RNA polynucleotide shown in each sequence number described below can be used by replacing uracil (u) with thymine (t).
• Treating means ameliorating, alleviating, delaying the onset of, or inhibiting the progression of the diseases described herein.
• Prevention means reducing the possibility of onset of a disease or pathology, suppressing, delaying, or stopping onset of a disease or pathology, suppressing, alleviating, delaying, or stopping progression of a pathology, suppressing, reducing, delaying, or stopping aggravation, or suppressing, delaying, or stopping recurrence of a disease or pathology.
• the "prevention” may be, for example, treatment of a subject (patient) who develops a target disease, or treatment of a model animal of the target disease.
• Aggravation refers to an increase in the level (severity) of a disease or condition.
• Isolated means identified and separated and/or recovered from a component in its natural state.
• the “isolation” can be achieved, for example, by obtaining at least one purification step.
• the upper limit and lower limit of the numerical ranges described in this specification can be combined in any way.
• the ranges “A to D” and “C to B” are also included in the present disclosure as numerical ranges.
• the numerical range “lower limit to upper limit” described in this specification means a range equal to or greater than the lower limit and equal to or less than the upper limit.
• the expression “A and/or B” includes “A only,””Bonly,” and “both A and B.”
• numerical values used to indicate component contents, numerical ranges, and the like should be understood to be modified with the term “about” unless otherwise specified.
• Lipid Complex One aspect of the present disclosure relates to a lipid complex (hereinafter also referred to as the "lipid complex of the present disclosure” or “lipid complex") in which at least one type of nucleic acid selected from a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of a human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein, a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of a HTLV-1 antigenic Tax protein, and a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of a HTLV-1 antigenic HBZ protein is encapsulated in a lipid.
• HTLV-1 human T-cell leukemia virus 1
• the lipid complex of the present disclosure may include, for example, any one, more than one, or all of the following: a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of Gag, a nucleic acid comprising a polynucleotide encoding Tax or an immunogenic fragment thereof, or a nucleic acid comprising a polynucleotide encoding HBZ or an immunogenic fragment thereof.
• the lipid complex of the present disclosure is characterized by comprising a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of an HTLV-1 antigenic Gag protein, a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic Tax protein or an immunogenic fragment thereof, and a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic HBZ protein or an immunogenic fragment thereof. Since the lipid complex of the present disclosure comprises these nucleic acids, when administered to a subject, it can induce an immune response against HTLV-1, in particular an immune response against Gag, Tax and/or HBZ of HTLV-1. Therefore, the lipid complex of the present disclosure can be used to induce an immune response against HTLV-1. The lipid complex of the present disclosure can also be used to prevent and/or treat HTLV-1-related diseases.
• the nucleic acid is at least one selected from the following: - a nucleic acid comprising a polynucleotide encoding the HTLV-1 antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, - a nucleic acid comprising a polynucleotide encoding the HTLV-1 antigenic Gag protein p19 (Gag p19) or an immunogenic fragment thereof, - a nucleic acid comprising a polynucleotide encoding the HTLV-1 antigenic Gag protein p24 (Gag p24) or an immunogenic fragment thereof, - a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic Tax protein or an immunogenic fragment thereof, - A nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic HBZ protein or an immunogenic fragment thereof.
• the nucleic acid comprises a nucleic acid comprising a polynucleotide encoding at least one selected from Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and Gag p24 or an immunogenic fragment thereof.
• the nucleic acid may be composed of deoxynucleotide residues, ribonucleotide residues, or both.
• the nucleic acid may be composed of natural nucleic acid residues, non-natural nucleic acid residues, or both.
• Specific examples of the nucleic acid include DNA, RNA, and/or DNA/RNA composed of natural and/or non-natural nucleic acid residues.
• Examples of the non-natural nucleic acid residues include modified nucleotide residues or modified ribonucleotide residues in which the base, sugar residue, or sugar phosphate backbone of the nucleotide residue is modified.
• examples of the non-natural nucleic acid residue include cEt (constrained ethyl bicyclic nucleic acid acid, Ionis Pharmaceuticals), LNA (trademark, Locked Nucleic Acid), ENA (registered trademark, 2'-O,4'-C-Ethylene bridged Nucleic Acid), and the like.
• the nucleic acid may have a 5' cap at the 5' end, for example.
• the nucleic acid may be a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule.
• the nucleic acid containing the polynucleotide encoding each of the proteins can be designed, for example, by substituting the corresponding codons based on the amino acid sequence of each protein.
• the base sequence of the nucleic acid may be, for example, codon-optimized.
• nucleic acids and proteins can be synthesized, for example, by genetic engineering techniques or organic synthesis techniques, and can also be called synthetic DNA such as cDNA, or synthetic RNA.
• the nucleic acid may be RNA or DNA.
• the nucleic acid is mRNA.
• the nucleic acid when the nucleic acid is an mRNA, the nucleic acid may include a 5' cap structure, a polyA sequence, or the like at the end. The following describes nucleic acids that may be contained in the lipid complexes of the present disclosure.
• nucleic acid comprising a polynucleotide encoding an immunogenic fragment of HTLV-1 antigenic Gag protein
• the nucleic acid may be a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of HTLV-1 antigenic Gag protein (Gag).
• the polynucleotide encoding the immunogenic fragment of Gag is a polynucleotide essentially comprising a region encoding the immunogenic fragment of Gag.
• the immunogenic fragment of Gag may be any fragment as long as it is a protein having immunogenicity against HTLV-1.
• the nucleic acid may comprise a polynucleotide encoding an immunogenic fragment of Gag.
• the nucleic acid may also be composed of a polynucleotide encoding an immunogenic fragment of Gag.
• the nucleic acid when the nucleic acid is an mRNA, the nucleic acid may comprise a 5' cap structure, a 5'-UTR, a 3'-UTR, a polyA sequence, and the like, in addition to the polynucleotide encoding an immunogenic fragment of Gag.
• the immunogenic fragment of Gag may, for example, be at least 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 amino acids in length.
• the polynucleotide encoding an immunogenic fragment of Gag comprises a polynucleotide encoding at least one selected from HTLV-1 antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and Gag protein p24 (Gag p24) or an immunogenic fragment thereof.
• the nucleic acid is a nucleic acid comprising a polynucleotide encoding at least one selected from Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and Gag p24 or an immunogenic fragment thereof.
• the nucleic acid may include a polynucleotide encoding Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and/or Gag p24 or an immunogenic fragment thereof.
• the nucleic acid may also be a polynucleotide encoding Gag p19 or an immunogenic fragment thereof, and/or Gag p24 or an immunogenic fragment thereof.
• the nucleic acid when the nucleic acid is an mRNA, the nucleic acid may include a 5' cap structure, a 5'-UTR, a 3'-UTR, a polyA sequence, etc., in addition to the polynucleotide encoding Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and/or Gag p24 or an immunogenic fragment thereof.
• the Gag p15, Gag p19, and Gag p24 may also be further processed, for example to a length of 50 amino acids or 9 amino acids.
• the lipid membrane complex may encapsulate a nucleic acid containing a polynucleotide encoding the 50 amino acids or 9 amino acids obtained by further processing the Gag p15, Gag p19, or Gag p24.
• Gag p15 may be, for example, the following protein (a1), (a2), or (a3): (a1) a protein consisting of the amino acid sequence of SEQ ID NO: 11 or 12; (a2) a protein consisting of the amino acid sequence of SEQ ID NO: 11 or 12 in which one or more amino acids have been deleted, inserted, substituted, or added; (a3) a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 11 or 12.
• the amino acid sequence shown in SEQ ID NO: 11 or 12 is an amino acid sequence derived from HTLV-1.
• the amino acid sequence shown in SEQ ID NO: 11 is, for example, the sequence of endogenous mature Gag p15 that can be expressed in infected cells, which is generated when ribosomes undergo frameshift.
• the amino acid sequence shown in SEQ ID NO: 12 is, for example, the sequence of Gag p15 that is generated when ribosomes do not undergo frameshift.
• the amino acid sequence of (a1) for example, the amino acid sequence of SEQ ID NO: 12, which is predicted to be expressed in large amounts in vivo when made into a vaccine composition, is preferable.
• “one or several” may be within a range in which (a2) is a protein that has immunogenicity against HTLV-1.
• “one or several" in (a2) may be, for example, 1 to 31, 1 to 26, 1 to 25, 1 to 21, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.
• the numerical range of the number discloses, for example, all positive integers that fall within that range.
• the description "1 to 5" means the disclosure of all of "1, 2, 3, 4, and 5" (hereinafter the same).
• the substitution is preferably a conservative substitution.
• the conservative substitution means a substitution of an amino acid residue with an amino acid residue having a similar side chain.
• Examples of the conservative substitution include substitution between amino acid residues having a basic side chain such as lysine, arginine, histidine, etc.; substitution between amino acid residues having an acidic side chain such as aspartic acid, glutamic acid, etc.; substitution between amino acid residues having a non-charged polar side chain such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, etc.; substitution between amino acid residues having a non-polar side chain such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, etc.; substitution between amino acid residues having a ⁇ -branched side chain such as threonine, valine, isoleucine, etc.; substitution between amino acid residue
• the "identity” may be within a range in which the (a3) is a peptide having immunogenicity against HTLV-1.
• the “identity” of the (a3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the amino acid sequence of the (a1).
• the “identity” can be calculated, for example, by using the default parameters of the homology algorithm BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) of the National Center for Biotechnology Information (NCBI) (hereinafter the same).
• Gag p19 or an Immunogenic Fragment thereof
• the Gag p19 may be, for example, the following protein (b1), (b2), or (b3):
• (b1) a protein consisting of the amino acid sequence of SEQ ID NO: 13;
• (b2) a protein consisting of the amino acid sequence of SEQ ID NO: 13 in which one or more amino acids have been deleted, inserted, substituted, or added;
• (b3) a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 13.
• amino acid sequence of the amino acid sequence (b1) “one or several” may be, for example, 1 to 38, 1 to 32, 1 to 25, 1 to 19, 1 to 12, 1 to 6, 1 to 5, 1 to 3, 1 or 2, or 1.
• the "identity” may be within a range in which the (b3) is a peptide that has immunogenicity against HTLV-1.
• the "identity" of the (b3) to the amino acid sequence of the (b1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
• Gag p24 or an Immunogenic Fragment thereof
• the Gag p24 may be, for example, the following protein (c1), (c2), or (c3):
• (c1) a protein consisting of the amino acid sequence of SEQ ID NO: 14;
• (c2) a protein consisting of the amino acid sequence of SEQ ID NO: 14 in which one or more amino acids have been deleted, inserted, substituted, or added;
• (c3) a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 14.
• amino acid sequence of the amino acid sequence (c1) “one or several” may be, for example, 1 to 64, 1 to 53, 1 to 42, 1 to 32, 1 to 21, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 or 2, or 1.
• the "identity” may be within a range in which the (c3) is a peptide that has immunogenicity against HTLV-1.
• the "identity" of the (c3) to the amino acid sequence of the (c1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
• the immunogenic fragment of p15, p19, or p24 may be, for example, any protein fragment of the protein that is immunogenic to HTLV-1.
• the immunogenic fragment of p15 or p19 may be, for example, any protein fragment of the protein that is 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.
• the lipid complex of the present disclosure may, for example, contain a nucleic acid containing a polynucleotide encoding any one of the Gag p15 or an immunogenic fragment thereof, the Gag p19 or an immunogenic fragment thereof, and the Gag p24 or an immunogenic fragment thereof, or may contain a nucleic acid containing a polynucleotide encoding two of them, or may contain a nucleic acid containing a polynucleotide encoding all of them.
• the lipid complex of the present disclosure preferably contains a nucleic acid containing a polynucleotide encoding the Gag p15 or an immunogenic fragment thereof, the Gag p19 or an immunogenic fragment thereof, and the Gag p24 or an immunogenic fragment thereof.
• the lipid complex can, for example, efficiently induce an immune response against HTLV-1.
• Gag or Immunogenic Fragment Thereof The Gag p15, Gag p19, and Gag p24 are processed Gag proteins. Therefore, the Gag protein or its immunogenic fragment can be referred to as, for example, a precursor or progenitor protein of the Gag p15 or its immunogenic fragment, the Gag p19 or its immunogenic fragment, and/or the Gag p24 or its immunogenic fragment.
• the lipid membrane complex may encapsulate a nucleic acid, such as a polynucleotide encoding a Gag protein or an immunogenic fragment thereof.
• the Gag protein before processing
• the Gag may be, for example, the following protein (d1), (d2), or (d3).
• (d1) A protein consisting of the amino acid sequence of SEQ ID NO: 1.
• (d2) A protein consisting of an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted, or added in the amino acid sequence of SEQ ID NO: 1.
• (d3) A protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 1.
• amino acid sequence (d2) in the amino acid sequence (d1) may be, for example, 1 to 128, 1 to 107, 1 to 85, 1 to 64, 1 to 42, 1 to 31, 1 to 21, 1 to 14, 1 to 12, 1 to 8, 1 to 4, 1 or 2, or 1.
• the “identity” of the protein (d3) may be within a range in which the protein (d3) is a peptide that has immunogenicity against HTLV-1.
• the “identity” of the protein (d3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the amino acid sequence of the protein (d1).
• the immunogenic fragment of Gag may be, for example, any protein fragment of the protein that is immunogenic to HTLV-1.
• the immunogenic fragment of Gag may be, for example, any protein fragment of the protein that is 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.
• the polynucleotide encoding Gag p15 is, for example, a polynucleotide selected from the group consisting of the following (A1) to (A6) and (A7).
• (A1) a polynucleotide consisting of the base sequence of SEQ ID NO: 15; (A2) a polynucleotide consisting of the base sequence of (A1) above, in which one or several bases have been deleted, inserted, substituted, and/or added; (A3) a polynucleotide consisting of a base sequence having 80% or more identity to any of the base sequences of (A1); (A4) a polynucleotide having a nucleotide sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence of any one of (A1) to (A3); (A5) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 11 or 12; (A6) a polynucleotide encoding a protein consisting of the amino acid sequence of (A5) above in which one or several amino acids have been deleted,
• the base sequence of SEQ ID NO: 15 is as follows.
• the base sequence of SEQ ID NO: 15 is the coding sequence (mRNA) of Gag p15 consisting of the amino acid sequence of SEQ ID NO: 12.
• the polynucleotide (A1) of SEQ ID NO: 15 can be obtained, for example, from HTLV-1.
• the polynucleotide encoding Gag p15 is, for example, SEQ ID NO: 15, in which u is replaced with t.
• “one or several” may be within a range in which the protein encoded by the polynucleotide of (A2) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 94, 1 to 78, 1 to 76, 1 to 63, 1 to 51, 1 to 47, 1 to 31, 1 to 25, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 7, 1 to 6, 1 to 5, 1 or 2, or 1 in the base sequence of (A1).
• the "identity” may be within a range in which the protein encoded by the polynucleotide of (A3) has immunogenicity against HTLV-1.
• the “identity” in (A3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (A1).
• the "hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (A1).
• the hybridization can be detected, for example, by various hybridization assays.
• the hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed.” edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used.
• the "stringent conditions” may be, for example, low stringent conditions, medium stringent conditions, or high stringent conditions.
• the "low stringent conditions” are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, and 32° C.
• the "medium stringent conditions” are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, and 42° C.
• the "high stringent conditions” are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, and 50° C.
• stringent conditions may be, for example, those described in "Molecular Cloning: A Laboratory Manual 2nd Ed.” edited by Sambrook et al. (Cold Spring Harbor Laboratory Press (1989)).
• the polynucleotide (A5) can be designed, for example, by substituting the corresponding codons based on the amino acid sequence of SEQ ID NO: 11 or 12.
• “one or several” may be within a range in which the protein encoded by the polynucleotide of (A6) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 31, 1 to 26, 1 to 25, 1 to 21, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (A5).
• the "identity” may be within a range in which the protein encoded by the polynucleotide of (A7) has immunogenicity against HTLV-1.
• the “identity” in (A7) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the amino acid sequence of (A5).
• the polynucleotide encoding Gag p19 is, for example, a polynucleotide selected from the group consisting of the following (B1) to (B6) and (B7).
• (B1) a polynucleotide consisting of the base sequence of SEQ ID NO: 16; (B2) a polynucleotide consisting of the base sequence of (B1) above, in which one or several bases have been deleted, inserted, substituted, and/or added; (B3) a polynucleotide consisting of a base sequence having 80% or more identity to any of the base sequences of (B1); (B4) a polynucleotide having a nucleotide sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence of any one of (B1) to (B3); (B5) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 13; (B6) a polynucleotide encoding a protein consisting of the amino acid sequence of (B5) above in which one or several amino acids have been deleted, inserted
• the base sequence of SEQ ID NO: 16 is as follows.
• the base sequence of SEQ ID NO: 16 is the coding sequence (mRNA) of Gag p19 consisting of the amino acid sequence of SEQ ID NO: 13.
• the polynucleotide (B1) of SEQ ID NO: 16 can be obtained, for example, from HTLV-1.
• the polynucleotide encoding Gag p19 is, for example, SEQ ID NO: 16, in which u is replaced with t.
• “one or several” may be within a range in which the protein encoded by the polynucleotide of (B2) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 116, 1 to 96, 1 to 94, 1 to 77, 1 to 58, 1 to 38, 1 to 19, 1 to 15, 1 to 11, 1 to 7, 1 to 3, 1 or 2, or 1 in the base sequence of (B1).
• the "identity” may be within a range in which the protein encoded by the polynucleotide of (B3) has immunogenicity against HTLV-1.
• the “identity” in (B3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (B1).
• the "hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (B1).
• the hybridization can be detected, for example, by various hybridization assays.
• the hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed.” edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be adopted.
• the stringent conditions can be the same as those described in (A4).
• the polynucleotide (B5) can be designed, for example, by substituting the corresponding codons based on the amino acid sequence of SEQ ID NO: 13.
• “one or several” may be within a range in which the protein encoded by the polynucleotide of (B6) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 38, 1 to 32, 1 to 31, 1 to 25, 1 to 19, 1 to 12, 1 to 6, 1 to 5, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (B5).
• the “identity” may be, for example, within a range in which the protein encoded by the polynucleotide of (B7) has immunogenicity against HTLV-1.
• the “identity” in (B7) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the amino acid sequence of (B5).
• the polynucleotide encoding Gag p24 is, for example, a polynucleotide selected from the group consisting of the following (C1) to (C6) and (C7).
• (C1) a polynucleotide consisting of the base sequence of SEQ ID NO: 17; (C2) a polynucleotide consisting of the base sequence of (C1) above, in which one or several bases have been deleted, inserted, substituted, and/or added; (C3) a polynucleotide consisting of a nucleotide sequence having 80% or more identity to any of the nucleotide sequences of (C1); (C4) a polynucleotide having a nucleotide sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence of any one of (C1) to (C3); (C5) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 14; (C6) a polynucleotide encoding a protein consisting of the amino acid sequence of (C5) above in which one or several
• the base sequence of SEQ ID NO:17 is as follows.
• the base sequence of SEQ ID NO:17 is a coding sequence (mRNA) of Gag p24 consisting of the amino acid sequence of SEQ ID NO:14.
• the polynucleotide (C1) of SEQ ID NO:17 can be obtained, for example, from HTLV-1.
• the polynucleotide encoding Gag p24 is, for example, SEQ ID NO: 17, in which u is replaced with t.
• “one or several” may be within a range in which the protein encoded by the polynucleotide of (C2) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 192, 1 to 160, 1 to 128, 1 to 96, 1 to 94, 1 to 64, 1 to 32, 1 to 25, 1 to 19, 1 to 12, 1 to 6, or 1 or 2 in the base sequence of (C1).
• the "identity” may be within a range in which the protein encoded by the polynucleotide of (C3) has immunogenicity against HTLV-1.
• the “identity” in (C3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (C1).
• the "hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (C1).
• the hybridization can be detected, for example, by various hybridization assays.
• the hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed.” edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be adopted.
• the stringent conditions can be the same as those described in (A4).
• the polynucleotide (C5) can be designed, for example, by substituting the corresponding codons based on the amino acid sequence of SEQ ID NO:14.
• “one or several” may be within a range in which the protein encoded by the polynucleotide of (C6) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 64, 1 to 53, 1 to 42, 1 to 32, 1 to 31, 1 to 21, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 or 2, or 1 in the amino acid sequence of (C5).
• the “identity” may be, for example, within a range in which the protein encoded by the polynucleotide of (C7) has immunogenicity against HTLV-1.
• the “identity” in (C7) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the amino acid sequence of (C5).
• Polynucleotides Encoding Immunogenic Fragments of Gag p15, p19, or p24 The polynucleotide encoding the immunogenic fragment of Gag p15, p19, or p24 may be any polynucleotide so long as the protein encoded by the polynucleotide has immunogenicity against HTLV-1.
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure preferably encodes at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO:1 (p19-1 in the Examples below), the amino acid sequence of positions 52 to 109 of SEQ ID NO:1 (p19-2 in the Examples below), or the amino acid sequence of positions 102 to 130 of SEQ ID NO:1 (p19-3 in the Examples below), and more preferably encodes at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO:1 (p19-1 in the Examples below) or the amino acid sequence of positions 52 to 109 of SEQ ID NO:1 (p19-2 in the Examples below).
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure particularly encodes at least one of the amino acid sequence of amino acids 1 to 59 of SEQ ID NO:1 (p19-1 in the Example described below), the amino acid sequence of amino acids 52 to 109 of SEQ ID NO:1 (p19-2 in the Example described below), or the amino acid sequence of amino acids 102 to 130 of SEQ ID NO:1 (p19-3 in the Example described below), and it is preferable that the region other than the above polynucleotide consists of an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to an amino acid sequence other than the above polynucleotide in SEQ ID NO:1.
• the region other than the polynucleotide is preferably composed of an amino acid sequence other than the polynucleotide in SEQ ID NO:1, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the polynucleotide in SEQ ID NO:1.
• the nucleic acid encapsulated in the lipid complex of the present disclosure is preferably a polynucleotide encoding at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO:1 (p19-1 in the Examples below), the amino acid sequence of positions 52 to 109 of SEQ ID NO:1 (p19-2 in the Examples below), or the amino acid sequence of positions 102 to 130 of SEQ ID NO:1 (p19-3 in the Examples below), and more preferably a polynucleotide encoding at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO:1 (p19-1 in the Examples below) or the amino acid sequence of positions 52 to 109 of SEQ ID NO:1 (p19-2 in the Examples below).
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure preferably contains at least one of the base sequences of bases 1 to 177 of SEQ ID NO: 3 (polynucleotide encoding p19-1 in the Examples described below), bases 154 to 327 of SEQ ID NO: 3 (polynucleotide encoding p19-2 in the Examples described below), or bases 304 to 390 of SEQ ID NO: 3 (polynucleotide encoding p19-3 in the Examples described below).
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure contains at least one of the base sequences from base 1 to base 177 of SEQ ID NO: 3 (the polynucleotide encoding p19-1 in the Examples described below) or the base sequence from base 154 to base 327 of SEQ ID NO: 3 (the polynucleotide encoding p19-2 in the Examples described below).
• the polynucleotide contained in the nucleic acid to be encapsulated in the lipid complex of the present disclosure is, in particular, a polynucleotide consisting of at least one of the nucleotide sequences of 1 to 177 of SEQ ID NO: 3 (polynucleotide encoding p19-1 in the Examples described below), 154 to 327 of SEQ ID NO: 3 (polynucleotide encoding p19-2 in the Examples described below), or 304 to 390 of SEQ ID NO: 3 (polynucleotide encoding p19-3 in the Examples described below), and the region other than the polynucleotide in the nucleic acid is preferably a nucleotide sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleotide sequence other than the polynucleotide in SEQ
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure is at least one of the base sequences of bases 1 to 177 of SEQ ID NO: 3 (polynucleotide encoding p19-1 in the Examples described below) or base sequences of bases 154 to 327 of SEQ ID NO: 3 (polynucleotide encoding p19-2 in the Examples described below), and the region other than the polynucleotide in the nucleic acid is preferably a base sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence other than the polynucleotide in SEQ ID NO: 3.
• the polynucleotide contained in the nucleic acid to be encapsulated in the lipid complex of the present disclosure encodes at least one of the amino acid sequence of 1 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below), the amino acid sequence of 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below), or the amino acid sequence of 102 to 130 of SEQ ID NO: 1 (p19-3 in the Examples described below), and the region other than the polynucleotide in the nucleic acid preferably consists of a base sequence encoding an amino acid sequence other than the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the amino acid sequence of SEQ ID NO: 1.
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure encodes at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below) or the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below), and the region of the nucleic acid other than the polynucleotide is preferably composed of a base sequence encoding an amino acid sequence other than the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the amino acid sequence of SEQ ID NO: 1.
• the lipid complexes of the present disclosure may comprise a nucleic acid comprising a polynucleotide encoding any one, two, or all of, for example, the Gag p15 or an immunogenic fragment thereof, the Gag p19 or an immunogenic fragment thereof, and the Gag p24 or an immunogenic fragment thereof.
• the lipid complex of the present disclosure preferably comprises, for example, a nucleic acid comprising a polynucleotide encoding the Gag p15 or an immunogenic fragment thereof, the Gag p19 or an immunogenic fragment thereof, and a nucleic acid comprising a polynucleotide encoding the Gag p24 or an immunogenic fragment thereof.
• nucleic acid encoding Gag examples include a nucleic acid comprising a polynucleotide selected from the group consisting of (D1) to (D6) and (D7) below.
• (D1) a polynucleotide consisting of the base sequence of SEQ ID NO: 2 or 3;
• D2 a polynucleotide consisting of the base sequence of (D1) above, in which one or several bases have been deleted, inserted, substituted, and/or added;
• D3 a polynucleotide consisting of a base sequence having 80% or more identity to any of the base sequences of (D1);
• (D4) a polynucleotide having a nucleotide sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence of any one of (D1) to (D3);
• D5 a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO:1;
• D6 a polynucleotide encoding a protein consisting of the amino acid sequence of (D5) above in which one or several
• the base sequences of SEQ ID NO:2 and SEQ ID NO:3 are as follows.
• the base sequences of SEQ ID NO:2 and SEQ ID NO:3 are coding sequences (mRNA) of Gag before processing, consisting of the amino acid sequence of SEQ ID NO:1.
• the polynucleotides (D1) of SEQ ID NO:2 and SEQ ID NO:3 can be obtained, for example, from HTLV-1.
• “one or several” may be within a range in which the protein encoded by the polynucleotide of (D2) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 387, 1 to 322, 1 to 258, 1 to 192, 1 to 129, 1 to 94, 1 to 64, 1 to 51, 1 to 42, 1 to 38, 1 to 25, 1 to 12, 1 to 6, 1 to 3, 1 or 2, or 1 in the base sequence of (D1).
• the "identity” may be within a range in which the protein encoded by the polynucleotide of (D3) has immunogenicity against HTLV-1.
• the “identity” in (D3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (D1).
• the "hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (D1).
• the hybridization can be detected, for example, by various hybridization assays.
• the hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed.” edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be adopted.
• the stringent conditions can be the same as those described in (A4).
• the polynucleotide (D5) can be designed, for example, by substituting the corresponding codons based on the amino acid sequence of SEQ ID NO:1.
• “one or several” may be within a range in which the protein encoded by the polynucleotide of (D6) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 128, 1 to 107, 1 to 85, 1 to 64, 1 to 42, 1 to 21, 1 to 14, 1 to 12, 1 to 8, 1 to 4, 1 or 2, or 1 in the amino acid sequence of (D5).
• the "identity” may be, for example, within a range in which the protein encoded by the polynucleotide of (D7) has immunogenicity against HTLV-1.
• the "identity" of (D7) to the amino acid sequence of (D5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
• polynucleotide of the immunogenic fragment of Gag may be, for example, within the scope of the protein encoded by the polynucleotide having immunogenicity against HTLV-1.
• nucleic acid comprising a polynucleotide encoding an immunogenic fragment of HTLV-1 antigenic Tax
• the nucleic acid may be a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of HTLV-1 antigenic Tax protein (Tax).
• the polynucleotide encoding the immunogenic fragment of Tax is a polynucleotide essentially comprising a region encoding the immunogenic fragment of Tax.
• the immunogenic fragment of Tax may be any within the scope of a protein having immunogenicity against HTLV-1.
• the nucleic acid may comprise a polynucleotide encoding Tax or an immunogenic fragment thereof.
• the nucleic acid may also be a polynucleotide encoding Tax or an immunogenic fragment thereof.
• the nucleic acid when the nucleic acid is an mRNA, the nucleic acid may comprise a 5' cap structure, a 5'-UTR, a 3'-UTR, a polyA sequence, and the like, in addition to the polynucleotide encoding the immunogenic fragment of Tax.
• the immunogenic fragment of Tax may be, for example, at least 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 amino acids in length.
• the polynucleotide encoding an immunogenic fragment of Tax comprises a polynucleotide encoding Tax or an immunogenic fragment thereof. That is, in some embodiments, the nucleic acid is a nucleic acid comprising a polynucleotide encoding Tax or an immunogenic fragment thereof.
• Tax or an immunological fragment thereof
• the Tax may be, for example, the following protein (e1), (e2), or (e3): (e1) a protein consisting of the amino acid sequence of SEQ ID NO: 4; (e2) a protein consisting of the amino acid sequence of SEQ ID NO: 4 in which one or more amino acids have been deleted, inserted, substituted, or added; (e3) a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 4.
• amino acid sequence of (e1) “one or several” in (e2) may be, for example, 1 to 105, 1 to 88, 1 to 70, 1 to 52, 1 to 35, 1 to 17, 1 to 14, 1 to 10, 1 to 7, 1 to 3, 1 or 2, or 1.
• the "identity” may be within the range that the (e3) is a peptide having immunogenicity against HTLV-1.
• the "identity” of the (e3) to the amino acid sequence of the (e1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
• the immunogenic fragment of Tax may be, for example, any protein fragment that is immunogenic to HTLV-1.
• the immunogenic fragment of Tax may be, for example, any protein fragment of the protein that is 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.
• the polynucleotide encoding Tax is, for example, a polynucleotide selected from the group consisting of the following (E1) to (E6) and (E7).
• (E1) a polynucleotide consisting of the base sequence of SEQ ID NO: 5 or 6;
• E2 a polynucleotide consisting of the base sequence of (E1) above, in which one or several bases have been deleted, inserted, substituted, and/or added;
• E3 a polynucleotide consisting of a nucleotide sequence having 80% or more identity to any of the nucleotide sequences of (E1);
• (E4) a polynucleotide having a nucleotide sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence of any one of (E1) to (E3);
• (E5) a polynucleot
• the base sequences of SEQ ID NO:5 and SEQ ID NO:6 are as follows.
• the base sequences of SEQ ID NO:5 and SEQ ID NO:6 are the coding sequence (mRNA) of Tax consisting of the amino acid sequence of SEQ ID NO:4.
• the polynucleotides (E1) of SEQ ID NO:5 and SEQ ID NO:6 can be obtained, for example, from HTLV-1.
• “one or several” may be within a range in which the protein encoded by the polynucleotide of (E2) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 318, 1 to 265, 1 to 212, 1 to 159, 1 to 106, 1 to 53, 1 to 42, 1 to 31, 1 to 21, 1 to 10, 1 to 6, 1 or 2, or 1 in the base sequence of (E1).
• the "identity” may be within a range in which the protein encoded by the polynucleotide of (E3) has immunogenicity against HTLV-1.
• the “identity” of (E3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (E1).
• the "hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (E1).
• the hybridization can be detected, for example, by various hybridization assays.
• the hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed.” edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be adopted.
• the stringent conditions can be the same as those described in (A4).
• the polynucleotide (E5) can be designed, for example, by substituting the corresponding codons based on the amino acid sequence of SEQ ID NO:4.
• one or several may be within a range in which the protein encoded by the polynucleotide of (E6) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 105, 1 to 88, 1 to 70, 1 to 52, 1 to 35, 1 to 31, 1 to 17, 1 to 14, 1 to 10, 1 to 7, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (E5).
• the "identity” may be, for example, within a range in which the protein encoded by the polynucleotide of (E7) has immunogenicity against HTLV-1.
• the "identity" of (E7) to the amino acid sequence of (E5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
• polynucleotide encoding the immunogenic fragment of Tax may be any polynucleotide so long as the protein encoded by the polynucleotide has immunogenicity against HTLV-1.
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure preferably encodes at least one of the amino acid sequence from 151 to 208 of SEQ ID NO:4 (Tax-4 in the Examples below) or the amino acid sequence from 251 to 308 of SEQ ID NO:4 (Tax-6 in the Examples below).
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure particularly encodes at least one of the amino acid sequence of 151-208th bases of SEQ ID NO:4 (Tax-4 in the Examples described below) or the amino acid sequence of 251-308th bases (Tax-6 in the Examples described below), and the region other than the above polynucleotide preferably consists of an amino acid sequence other than the above polynucleotide in SEQ ID NO:4, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the above polynucleotide in SEQ ID NO:4.
• the nucleic acid encapsulated in the lipid complex of the present disclosure preferably encodes at least one of the amino acid sequence from 151 to 208 of SEQ ID NO:4 (Tax-4 in the Examples below) or the amino acid sequence from 251 to 308 of SEQ ID NO:4 (Tax-6 in the Examples below).
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure preferably contains at least one of the base sequences of bases 451 to 624 of SEQ ID NO: 6 (polynucleotide encoding Tax-4 in the Examples described below) or bases 751 to 924 of SEQ ID NO: 6 (polynucleotide encoding Tax-6 in the Examples described below).
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure is particularly a polynucleotide consisting of at least one of the base sequences of bases 451 to 624 of SEQ ID NO: 6 (polynucleotide encoding Tax-4 in the Examples described below) or base sequences of bases 751 to 924 of SEQ ID NO: 6 (polynucleotide encoding Tax-6 in the Examples described below), and it is preferable that the region other than the above polynucleotide in the nucleic acid is a base sequence other than the above polynucleotide in SEQ ID NO: 6, or a base sequence that has at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the above polynucleotide in SEQ ID NO: 6.
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure particularly codes for at least one of the amino acid sequence from 151 to 208 of SEQ ID NO: 4 (Tax-4 in the Examples described below) or the amino acid sequence from 251 to 308 of SEQ ID NO: 4 (Tax-6 in the Examples described below), and the region of the nucleic acid other than the polynucleotide preferably consists of a base sequence that codes for an amino acid sequence other than the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the amino acid sequence of SEQ ID NO: 4.
• nucleic acid comprising a polynucleotide encoding an immunogenic fragment of an HTLV-1 antigenic HBZ protein
• the nucleic acid may be a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of an HTLV-1 antigenic HBZ protein (HBZ).
• the polynucleotide encoding the immunogenic fragment of HBZ is a polynucleotide essentially comprising a region encoding the immunogenic fragment of HBZ.
• the immunogenic fragment of HBZ may be any within the scope of a protein having immunogenicity against HTLV-1.
• the nucleic acid may comprise a polynucleotide encoding HBZ or an immunogenic fragment thereof.
• the nucleic acid may also be a polynucleotide encoding HBZ or an immunogenic fragment thereof.
• the nucleic acid when the nucleic acid is an mRNA, the nucleic acid may comprise a 5' cap structure, a 5'-UTR, a 3'-UTR, a polyA sequence, and the like, in addition to the polynucleotide encoding the immunogenic fragment of HBZ.
• the HBZ immunogenic fragment may, for example, be at least 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 amino acids in length.
• the polynucleotide encoding an immunogenic fragment of HBZ comprises a polynucleotide encoding HBZ or an immunogenic fragment thereof. That is, in some embodiments, the nucleic acid is a nucleic acid comprising a polynucleotide encoding HBZ or an immunogenic fragment thereof.
• the HBZ may be, for example, the following protein (f1), (f2), or (f3).
• (f1) a protein consisting of the amino acid sequence of SEQ ID NO: 7 or 7;
• (f2) a protein consisting of the amino acid sequence of SEQ ID NO: 7 or 7 in which one or more amino acids have been deleted, inserted, substituted, or added;
• (f3) a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 7 or 7.
• amino acid sequence (f2) in the amino acid sequence (f1) may be, for example, 1 to 62, 1 to 52, 1 to 41, 1 to 31, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 or 2.
• the "identity” may be within a range in which the (f3) is a peptide that has immunogenicity against HTLV-1.
• the "identity" of the (f3) to the amino acid sequence of the (f1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
• the immunogenic fragment of HBZ may be, for example, any protein fragment of the protein that is immunogenic to HTLV-1.
• the immunogenic fragment of HBZ may be, for example, any protein fragment of the protein that is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.
• the polynucleotide encoding HBZ is, for example, a polynucleotide selected from the group consisting of (F1) to (F6) and (F7) below.
• (F1) a polynucleotide consisting of the base sequence of SEQ ID NO: 9 or 10
• (F2) a polynucleotide consisting of the base sequence of (F1) above, in which one or several bases have been deleted, inserted, substituted, and/or added
• (F3) a polynucleotide consisting of a nucleotide sequence having 80% or more identity to any one of the nucleotide sequences (F1) to (F3)
• (F4) a polynucleotide having a nucleotide sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide having any one of the nucleotide sequences of (F1); (F5)
• the base sequences of SEQ ID NO:9 and SEQ ID NO:10 are as follows.
• the base sequences of SEQ ID NO:9 and SEQ ID NO:10 are coding sequences (mRNA) of HBZ consisting of the amino acid sequence of SEQ ID NO:8.
• the polynucleotides (F1) of SEQ ID NO:9 and SEQ ID NO:10 can be obtained, for example, from HTLV-1.
• “one or several” may be within a range in which the protein encoded by the polynucleotide of (F2) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 186, 1 to 156, 1 to 123, 1 to 94, 1 to 93, 1 to 60, 1 to 30, 1 to 24, 1 to 18, 1 to 12, 1 to 6, 1 to 3, 1 or 2, or 1 in the base sequence of (F1).
• the "identity” may be within a range in which the protein encoded by the polynucleotide of (F3) has immunogenicity against HTLV-1.
• the “identity” of (F3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (F1).
• the "hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (F1).
• the hybridization can be detected, for example, by various hybridization assays.
• the hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed.” edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be adopted.
• the stringent conditions can be the same as those described in (A4).
• the polynucleotide (F5) can be designed, for example, by substituting the corresponding codons based on the amino acid sequence of SEQ ID NO: 7 or 8.
• “one or several” may be within a range in which the protein encoded by the polynucleotide of (F6) has immunogenicity against HTLV-1.
• “one or several” may be, for example, 1 to 62, 1 to 52, 1 to 41, 1 to 31, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 or 2 in the amino acid sequence of (F5).
• the "identity” may be, for example, within a range in which the protein encoded by the polynucleotide of (F7) has immunogenicity against HTLV-1.
• the "identity" of (F7) to the amino acid sequence of (F5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
• Polynucleotides encoding immunogenic fragments of HBZ may be any polynucleotide as long as the protein encoded by the polynucleotide has immunogenicity against HTLV-1.
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure is preferably one that encodes the amino acid sequence from 1 to 58 of SEQ ID NO:8 (HBZ-1 in the Examples described below).
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure particularly encodes the amino acid sequence from 1 to 58 of SEQ ID NO:8 (HBZ-1 in the Examples described below), and the region other than the polynucleotide preferably consists of an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to an amino acid sequence other than the polynucleotide in SEQ ID NO:1 or an amino acid sequence other than the polynucleotide in SEQ ID NO:8.
• the nucleic acid to be encapsulated in the lipid complex of the present disclosure is preferably one that encodes the amino acid sequence from 1 to 58 of SEQ ID NO:8 (HBZ-1 in the Examples below).
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure preferably contains the base sequence from base 1 to base 174 of SEQ ID NO: 10 (the polynucleotide encoding HBZ-1 in the Examples described below).
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure is particularly a polynucleotide consisting of the 1st to 174th base sequence of SEQ ID NO: 10 (the polynucleotide encoding HBZ-1 in the Examples described below), and the region of the nucleic acid other than the above polynucleotide is preferably a base sequence that has at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to a base sequence other than the above polynucleotide in SEQ ID NO: 10, or a base sequence other than the above polynucleotide in SEQ ID NO: 10.
• the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure encodes the amino acid sequence from 1 to 58 of SEQ ID NO: 1 (HBZ-1 in the Examples described below), and the region of the nucleic acid other than the polynucleotide preferably consists of a base sequence encoding an amino acid sequence other than the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the amino acid sequence of SEQ ID NO: 10.
• the nucleic acid encapsulated in the lipid complex of the present disclosure preferably contains a polynucleotide consisting of a polynucleotide encoding the 1st to 174th base sequence of SEQ ID NO: 10 (the polynucleotide encoding HBZ-1 in the Examples described below).
• Each protein for obtaining the nucleic acid can be synthesized by genetic engineering techniques, specifically, by using a transformant as described below.
• the protein may be produced, for example, by in vitro synthesis.
• the transformant is cultured, and Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and/or Gag p24 or an immunogenic fragment thereof of the present disclosure is collected from the culture.
• the culture may be a culture supernatant, or a transformant such as cultured cells or cultured bacterial cells, or a processed or disrupted product thereof.
• the production method of the present disclosure involves extracting Gag p15 or its immunogenic fragment, Gag p19 or its immunogenic fragment, and/or Gag p24 or its immunogenic fragment by disrupting the host.
• the production method of the present disclosure involves using the culture solution as is or removing the host by centrifugation or the like.
• the production method of the present disclosure can use, for example, general biochemical methods used for isolating and purifying proteins, specifically, concentration using an ultrafiltration membrane; salting out such as ammonium sulfate precipitation; chromatography using various columns such as gel filtration, ion exchange chromatography, and affinity chromatography; alone or in appropriate combination, to isolate or purify proteins such as Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and/or Gag p24 or an immunogenic fragment thereof.
• general biochemical methods used for isolating and purifying proteins specifically, concentration using an ultrafiltration membrane; salting out such as ammonium sulfate precipitation; chromatography using various columns such as gel filtration, ion exchange chromatography, and affinity chromatography; alone or in appropriate combination, to isolate or purify proteins such as Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and/or Gag p24 or an immunogenic fragment thereof.
• the lipid complex of the present disclosure has a structure in which at least one type of nucleic acid is encapsulated in a lipid.
• the lipid complex is a lipid particle, which has a structure in which the nucleic acid is encapsulated within a small lipid particle.
• the lipid complex is a lipid nanoparticle (LNP).
• the LNP has a structure in which the nucleic acid is encapsulated in a particle composed of lipids and having an average nano-sized particle size (e.g., about 1 nm to 1000 nm).
• the lipid constituting the lipid complex comprises (i) a cationic lipid and (ii) at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol.
• lipid complex comprising (i) and (ii) include a complex of a nucleic acid and a membrane (reverse micelle) consisting of a single lipid layer (single molecule), a complex of a nucleic acid and a liposome, a complex of a nucleic acid and a micelle, etc.
• the cationic lipid is not particularly limited.
• cationic lipids described in International Publication No. 2015/105131, International Publication No. 2016/104580, International Publication No. 2017/222016, and International Publication No. 2019/131580 can be used.
• the cationic lipid may be in the form of a salt, a hydrate, or a solvate.
• An example of the cationic lipid according to one embodiment is a compound represented by the following formula (I) or a pharma- ceutically acceptable salt thereof.
• L1 and L2 each independently represent an alkylene group having 3 to 10 carbon atoms (e.g., 3 to 8 carbon atoms).
• L1 and L2 each independently represent a linear alkylene group having 3 to 10 carbon atoms (e.g., 3 to 8 carbon atoms).
• R 1 and R 2 each independently represent an alkyl group having 4 to 22 carbon atoms or an alkenyl group having 4 to 22 carbon atoms.
• X1 represents a single bond or -CO-O-.
• ring P represents any one of the following formulae (P-1) to (P-6). In one embodiment, ring P represents any one of the following formulae (P-1), (P-2), (P-4), (P-5), and (P-6). In one embodiment, ring P represents the following formula (P-1) or (P-6). In one embodiment, ring P represents the following formula (P-1). In the above formulas (P-1), (P-2), (P-3), (P-4), (P-5), and (P-6), R 3 represents an alkyl group having 1 to 3 carbon atoms.
• cationic lipids include, for example, 1-oxo-1-(undecane-5-yloxy)nonadecan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxonadecan-10-yl-1-methylpiperidine-4-carboxylate, 1-oxo-1-(undecane-5-yloxy)heptadecan-8-yl-1-methylpiperidine-4-carboxylate, 2 ...
• the cationic lipid is 1-((2-butyloctyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxononadecan-10-yl-1-methylpiperidine-4-carboxylate, 2- ⁇ 9-oxo-9-[(3-pentyloctyl)oxy]nonyl ⁇ dodecyl 1-methylpiperidine-4-carboxylate, 1-(2-octyl It contains at least one selected from (3S)-2- ⁇ 9-oxo-9-[(3-pentyloctyl)oxy]nonyl ⁇ dodecyl 1-methylpyrrolidine-3-carboxylate, (3R)-2- ⁇ 9-oxo-9-[(3-pentyloctyl)oxy]nonyl ⁇ dodecyl 1-methylpyrrolidine-3-carboxylate, or pharma-
• the cationic lipid is 2- ⁇ 9-oxo-9-[(3-pentyloctyl)oxy]nonyl ⁇ dodecyl 1-methylpiperidine-4-carboxylate or a pharma- ceutically acceptable salt thereof.
• the structure of 2- ⁇ 9-oxo-9-[(3-pentyloctyl)oxy]nonyl ⁇ dodecyl 1-methylpiperidine-4-carboxylate is shown below.
• the cationic lipids can be used alone or in combination of two or more types.
• Neutral lipids examples include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), palmitoyloleoylphosphatidylcholine (POPC), stearyloleoylphosphatidylcholine (SOPC), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DMPC), dioleo ...
• DOPE dioleoylphosphatidylethanolamine
• POPE phosphatidylethanolamine
• POPC dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclo
• phospholipids such as dioleoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), diarachidoyl phosphatidylcholine (DAPC), dibehenoyl phosphatidylcholine (DBPC), dilignoyl phosphatidylcholine (DLPC), dioleoyl phosphatidylcholine (DOPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylglycerol (DSPG), dioleyl phosphatidylserine (DOPS), sphingomyelin, and the like; ceramide (Cer), and the like.
• DPPC dioleoyl phosphatidylcholine
• DSPC distearoyl phosphatidylcholine
• the neutral lipid comprises at least one selected from DOPE, HSPC, DPPC, DSPC, or DAPC. In certain embodiments, the neutral lipid comprises DPPC.
• Neutral lipids can be used alone or in combination of two or more types.
• the polyethylene glycol-modified lipid is not particularly limited, but examples thereof include PEG2000-DMG (PEG2000-dimyristylglycerol), PEG2000-DPG (PEG2000-dipalmitoylglycerol), PEG2000-DSG (PEG2000-distearoylglycerol), PEG5000-DMG (PEG5000-dimyristylglycerol), PEG5000-DPG (PEG5000-dipalmitoylglycerol), PEG5000-DSG (PEG5000-distearoyl glycerol), PEG-cDMA (N-[(methoxypoly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxylpropyl-3-amine), PEG-C-DOMG (R-3-[( ⁇ -methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxylpropyl-3-
• the polyethylene glycol-modified lipid comprises at least one selected from PEG2000-DMG, PEG2000-DPG, PEG2000-DSG, PEG-cDMA, or PEG-C-DOMG. In certain embodiments, the polyethylene glycol-modified lipid comprises PEG2000-DMG.
• the polyethylene glycol-modified lipid may be used alone or in combination of two or more kinds.
• the polyethylene glycol-modified lipid may have a methoxylated PEG terminal (MPEG; methoxypolyethylene glycol).
• MPEG methoxylated PEG terminal
• PEG2000-DMG includes MPEG2000-DMG.
• Sterols include, but are not limited to, cholesterol, dihydrocholesterol, lanosterol, ⁇ -sitosterol, campesterol, stigmasterol, brassicasterol, ergocastrol, fucosterol, 3 ⁇ -[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), etc.
• the sterol comprises at least one selected from cholesterol, dihydrocholesterol, lanosterol, or ⁇ -sitosterol. In certain embodiments, the sterol comprises cholesterol.
• Sterols can be used alone or in combination of two or more types.
• composition/mixture ratio The lipid content in the lipid complex and the blending ratio of each lipid component constituting the lipid are not particularly limited.
• the lipid complexes contain, for example, 50-99.99% by weight, such as 70-99.99% by weight, such as 90-99% by weight, of the lipid component based on the total weight of the lipid complex.
• the lipid complex contains, for example, 10-99 mol %, 20-90 mol %, or 30-70 mol % of the above-mentioned cationic lipids based on the total lipids contained in the lipid complex.
• the lipid complexes may contain, for example, 0-50 mol %, 0-40 mol %, or 0-30 mol % neutral lipids based on the total lipids contained in the lipid complexes.
• the lipid complex may contain, for example, 0-30 mol %, 0-20 mol %, or 0-10 mol % polyethylene glycol-modified lipid based on the total lipid contained in the lipid complex.
• the lipid complex may contain, for example, 0 to 90 mol %, 10 to 80 mol %, or 20 to 50 mol % of sterol based on the total lipid contained in the lipid complex.
• the content of nucleic acid in the lipid complex is not particularly limited.
• the lipid complexes contain, for example, 0.01-50% by weight, such as 0.1-30% by weight, such as 1-10% by weight, of nucleic acid based on the total weight of the lipid complex.
• the lipid complex comprises 0.01-50% by weight of nucleic acid and 50-99.99% by weight of lipid, based on the total weight of the lipid complex, and comprises 30-70 mol% of cationic lipid, 0-30 mol% of neutral lipid, 0-10 mol% of polyethylene glycol-modified lipid, and 20-50 mol% of sterol, based on the total lipid contained in the lipid complex.
• the lipid complex comprises 0.01-50% by weight of nucleic acid and 50-99.99% by weight of lipid, based on the total weight of the lipid complex, and comprises 30-70 mol% of cationic lipid, 1-30 mol% of neutral lipid, 1-10 mol% of polyethylene glycol-modified lipid, and 20-50 mol% of sterol, based on the total lipid contained in the lipid complex.
• the combination of lipid components in the lipid complex is not particularly limited.
• the lipids comprise a combination of cationic lipids, neutral lipids, and sterols.
• the lipid comprises the combination of the above-mentioned cationic lipid, neutral lipid, polyethylene glycol-modified lipid and sterol.It has been reported that the cationic lipid is necessary for the encapsulation of nucleic acid and for the efficient delivery of nucleic acid into target cells, and the polyethylene glycol-modified lipid is necessary for the inhibition of particle aggregation.Furthermore, in addition to these two types of lipid, the simultaneous presence of four types of lipid including neutral lipid and sterol can form stable particles with nucleic acid encapsulated therein.
• the lipid complex is composed of cationic lipid/neutral lipid/polyethylene glycol-modified lipid/sterol as lipids, and the molar ratio of the lipids may be, for example, 10-99/0-50/0-30/0-90, 20-90/0-40/0-20/10-80, or 30-70/0-30/0-10/20-50.
• the molar ratio of cationic lipid/neutral lipid/polyethylene glycol-modified lipid/sterol in the lipid complex may be 10-99/1-50/1-30/1-90, 20-90/1-40/1-20/10-80, or 30-70/1-30/1-10/20-50.
• the molar ratio of cationic lipid/neutral lipid/polyethylene glycol-modified lipid/sterol in the lipid complex is 47/11/1.5/40.5.
• the lipid includes 2- ⁇ 9-oxo-9-[(3-pentyloctyl)oxy]nonyl ⁇ dodecyl 1-methylpiperidine-4-carboxylate as a cationic lipid, DPPC as a neutral lipid, (M)PEG2000-DMG as a polyethylene glycol-modified lipid, and cholesterol as a sterol.
• the "average particle size" of the lipid complexes herein refers to the Z-average particle size, which is measured by dynamic light scattering.
• the average particle size (Z-average) of the lipid complexes can be, for example, 10-1000 nm, for example, 30-500 nm, for example, 30-200 nm.
• the encapsulation rate of the nucleic acid in the lipid complex was measured, for example, using Quant-iT RiboGreen RNA Reagent (Invitrogen, Cat#R11491). Specifically, the nucleic acid concentration (A) measured by dilution with RNase Free Water is defined as the nucleic acid present in the lipid complex external solution, and the nucleic acid concentration (B) measured by dilution with 1% Triton X-100 is defined as the total nucleic acid concentration in the formulation, and the encapsulation rate can be calculated.
• A nucleic acid concentration measured by dilution with RNase Free Water
• B nucleic acid concentration measured by dilution with 1% Triton X-100
• Inclusion rate (%) 100 - (A/B) x 100
• the encapsulation rate (%) of the nucleic acid in the lipid complexes calculated by the above method is, for example, greater than 80%, 85% or 90%. In one embodiment, the encapsulation rate (%) of the nucleic acid in the lipid complexes is greater than 90%.
• the polydispersity index (PDI) is less than 0.3 (particularly less than 0.2, especially less than 0.1).
• Methods for encapsulating effective molecules into lipid complexes include, for example, reverse phase evaporation, Zwitterion (NaCl) hydration, cationic core hydration, and a method using ethanol and calcium (see also Biomembr., 1468, 239-252 (2000)).
• the lipid complexes encapsulating the nucleic acid of the present disclosure can be prepared by the methods known in the art as described above.
• the lipid complex can be prepared by mixing a lipid solution containing, for example, a cationic lipid and at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols with an acidic buffer containing nucleic acid. This method produces a lipid complex whose interior is filled with nucleic acid and a lipid core.
• Solvents for dissolving lipids include polar organic solvents such as alcohols, which may be, for example, ethanol, isopropanol, chloroform, or t-butanol.
• polar organic solvents such as alcohols, which may be, for example, ethanol, isopropanol, chloroform, or t-butanol.
• acidic buffers that dissolve nucleic acids include sulfate buffers, phosphate buffers, phthalate buffers, tartrate buffers, citrate buffers, formate buffers, oxalate buffers, and acetate buffers.
• the lipid complex can be produced by a method comprising, for example, a step (a) of mixing an aqueous solution containing a polar organic solvent, which contains a cationic lipid and at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols, with an aqueous solution containing nucleic acid to obtain a mixed solution, and a step (b) of reducing the content of the polar organic solvent in the mixed solution.
• the electrostatic interaction between water-soluble nucleic acid and cationic lipid, and the hydrophobic interaction between lipids, can form a lipid complex in which nucleic acid is encapsulated in a particle composed of lipid.
• a lipid complex in which nucleic acid is encapsulated in a particle composed of lipid.
• the solubility of the lipid component which includes cationic lipid and at least one lipid selected from the group consisting of neutral lipid, polyethylene glycol-modified lipid, and sterol, in the aqueous solution containing polar organic solvent can be changed, thereby forming a lipid complex.
• the polar organic solvent can be alcohol such as ethanol.
• step (a) (I) the above-mentioned cationic lipid and (II) at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols are dissolved in a polar organic solvent-containing aqueous solution, and (III) an aqueous solution containing nucleic acid is mixed to obtain a mixed solution.
• concentration of the polar organic solvent in the polar organic solvent-containing aqueous solution is not particularly limited as long as it satisfies the condition for dissolving the lipid molecules even after mixing with the aqueous solution of nucleic acid.
• the concentration of the polar organic solvent in the polar organic solvent-containing aqueous solution in step (a) can be 0.1 to 60% by weight.
• the aqueous solution containing nucleic acid can be obtained, for example, by dissolving nucleic acid in an acidic buffer.
• step (b) the content of the polar organic solvent is reduced by adding water or the like to the above-mentioned mixed solution. This allows a lipid complex to be formed. In order to efficiently form a lipid complex, it is preferable to rapidly reduce the content of the polar organic solvent.
• the concentration of the polar organic solvent in the final polar organic solvent-containing aqueous solution in step (b) can be 0 to 5% by weight.
• the mixture obtained in step (a) may be subjected to dialysis to remove the polar organic solvent and replace the solvent with a pharma- ceutically acceptable medium.
• the content of the polar organic solvent in the solution is reduced during the dialysis process, which allows the lipid complex to be formed. According to the production method of this embodiment, a lipid complex in which nucleic acid is efficiently encapsulated inside the lipid particle can be obtained.
• compositions comprising the lipid complex of the present disclosure.
• the present disclosure provides a use of the lipid complex for producing a pharmaceutical composition.
• the pharmaceutical composition may contain the lipid complex described above, a pharma- ceutical acceptable carrier, and optionally other additives.
• Examples of pharma- ceutically acceptable carriers include pharma-ceutically acceptable vehicles, suspending agents, solubilizing agents, preservatives, fillers, bulking agents, binders, wetting agents, disintegrants, lubricants, dispersants, flavorings, stabilizers, isotonicity agents, preservatives, adsorption inhibitors, surfactants, diluents, pH adjusters, soothing agents, buffers, sulfur-containing reducing agents, antioxidants, etc., which are added appropriately to the extent that they do not interfere with the effects of the present disclosure.
• Pharmaceutically acceptable media include sterile water; physiological saline; isotonic solutions containing adjuvants such as glucose, D-sorbitol, D-mannose, D-mannitol, and sodium chloride; and buffer solutions such as phosphate buffer, citrate buffer, and acetate buffer.
• the suspending agent is not particularly limited, and examples thereof include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, powdered tragacanth, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, and the like.
• the solubilizing agent is not particularly limited, and examples thereof include alcohols such as ethanol, propylene glycol, and polyethylene glycol, polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil fatty acid ethyl esters.
• the stabilizer is not particularly limited, and examples include dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, etc.
• the isotonicity agent is not particularly limited, and examples include D-mannitol, sorbitol, etc.
• the preservative is not particularly limited, and examples thereof include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.
• the adsorption inhibitor is not particularly limited, and examples thereof include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, hydrogenated castor oil, polyethylene glycol, etc.
• the sulfur-containing reducing agent is not particularly limited, and examples thereof include those having a sulfhydryl group, such as N-acetylcysteine, N-acetylhomocysteine, thioxanthate, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.
• a sulfhydryl group such as N-acetylcysteine, N-acetylhomocysteine, thioxanthate, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon
• the antioxidant is not particularly limited, and examples thereof include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, ⁇ -tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.
• EDTA sodium ethylenediaminetetraacetate
• additives include, for example, sugars such as sucrose, glucose, sorbitol, lactose, etc.; amino acids such as glutamine, glutamic acid, monosodium glutamate, histidine, etc.; organic salts such as citric acid (e.g. sodium citrate, potassium citrate), phosphoric acid, acetic acid (e.g. sodium acetate), lactic acid, carbonic acid, tartaric acid, etc.; inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate, etc.
• sugars such as sucrose, glucose, sorbitol, lactose, etc.
• amino acids such as glutamine, glutamic acid, monosodium glutamate, histidine, etc.
• organic salts such as citric acid (e.g. sodium citrate, potassium citrate), phosphoric acid, acetic acid (e.g. sodium acetate), lactic acid, carbonic acid, tartaric
• the pharmaceutical composition may be formulated in a variety of dosage forms.
• an injection may be used as a dosage form of the pharmaceutical composition.
• the pharmaceutical composition may be in a powder state from which the solvent has been removed by freeze-drying or the like, or may be in a liquid state.
• the pharmaceutical composition of some embodiments is a powder composition containing the lipid complex of the above-mentioned embodiment.
• the powder composition may be prepared by removing the solvent from a liquid composition (dispersion) containing the lipid complex, for example, by filtration, centrifugation, etc., or by freeze-drying the dispersion.
• the pharmaceutical composition When the pharmaceutical composition is in a powder state, it can be suspended or dissolved in a pharma- ceutical acceptable medium before use and used as an injection.
• composition of one embodiment of the present disclosure is a liquid composition comprising the lipid complex of the above-mentioned embodiment and a pharma- ceutically acceptable medium.
• a liquid composition comprising the lipid complex of the above-mentioned embodiment and a pharma- ceutically acceptable medium.
• the subject to which the pharmaceutical composition of the present disclosure is administered is not limited, and it can be applied to various animals.
• the pharmaceutical composition of the present disclosure can be applied to, for example, humans or non-human mammals (monkeys, mice, rats, rabbits, cows, horses, goats, etc.), preferably humans, and experimental animals in clinical trials, screening, and experiments.
• the conditions for use (administration conditions) of the pharmaceutical composition of the present disclosure are not particularly limited, and the administration form, administration timing, dosage, etc. can be appropriately set depending on, for example, the type of active ingredient in the pharmaceutical composition, the type of subject to administration, etc.
• the method of administering the pharmaceutical composition of the present disclosure to a subject is not limited, and can be appropriately determined by a person skilled in the art (e.g., a physician) depending on the subject's health condition, the severity of the disease, the type of concomitant drug, etc.
• the number of times of administration of the pharmaceutical composition is one or more times.
• the number of times is, for example, two, three, four, five or more times.
• the number of times of administration may be appropriately determined while confirming the preventive effect on the subject of administration.
• the administration interval can be appropriately determined while confirming the preventive effect on the subject of administration, and examples thereof include once a day, once a week, once every two weeks, once a month, once every three months, once every six months, etc.
• Administration of the pharmaceutical composition of the present disclosure to a subject is not particularly limited, and may be parenteral administration, such as intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, intradermal administration, and intravenous administration.
• parenteral administration such as intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, intradermal administration, and intravenous administration.
• intramuscular or subcutaneous administration is preferred, as it can be administered safely and stably regardless of the skill of the administerer.
• the dosage of the pharmaceutical composition varies depending on the subject, target organ, symptoms, and administration method.
• the pharmaceutical composition of the present disclosure may be administered in an amount sufficient to induce an immune response against HTLV-1, i.e., an effective dose, depending on the administration form.
• the dosage may be appropriately determined depending on, for example, the age, body weight, symptoms, etc. of the subject.
• the dosage of the pharmaceutical composition may be, for example, 0.01 mg to 100 mg per kg of the subject's body weight, 0.1 mg to 50 mg per kg, or 0.3 mg to 10 mg per kg of the subject's body weight.
• the pharmaceutical composition of the present disclosure may be used, for example, in vitro or in vivo.
• the pharmaceutical composition of the present disclosure may be used, for example, as a research reagent or as a pharmaceutical. In the former case, the pharmaceutical composition of the present disclosure may also be referred to as a test reagent or test kit.
• the subject of administration of the pharmaceutical composition of the present disclosure is not particularly limited.
• the subject of administration can be, for example, a cell, a tissue, an organ, etc.
• the cell can be, for example, a cell or a cultured cell taken from a living body
• the tissue or organ can be, for example, a tissue (living tissue) or an organ taken from a living body, etc.
• the cell can be, for example, an immune cell such as a T cell, a B cell, a NK cell, or a dendritic cell, etc.
• the pharmaceutical composition of the present disclosure is used in vivo, the above-mentioned examples can be used for the subject (administration subject), for example.
• the pharmaceutical composition of the present disclosure when administered to a subject, can induce an immune response against HTLV-1 (particularly, cellular immunity against HTLV-1, particularly cellular immunity by cytotoxic T cells). For this reason, the pharmaceutical composition of the present disclosure can also be called a vaccine, vaccine composition, or vaccine formulation.
• the vaccine, vaccine composition, or vaccine formulation of the present disclosure can prevent the onset of HTLV-1-related diseases (e.g., ATL onset) by activating cellular immunity and suppressing the increase of infected cells.
• the pharmaceutical composition of the present disclosure can efficiently release the nucleic acid into the cytoplasm by administering it in the form of a lipid complex in which the nucleic acid is encapsulated.
• a pharmaceutical composition for inducing an immune response against HTLV-1 (particularly, cellular immunity against HTLV-1, particularly cellular immunity by cytotoxic T cells) is provided.
• a method for inducing an immune response against HTLV-1 comprising the step of administering the pharmaceutical composition to a subject in need thereof.
• the pharmaceutical composition of the present disclosure may be useful for the prevention and/or treatment of HTLV-1-associated diseases, since it may induce an immune response against HTLV-1. That is, according to some aspects of the present disclosure, a pharmaceutical composition for use in the prevention and/or treatment of HTLV-1-associated diseases is provided. According to other aspects of the present disclosure, a method for preventing and/or treating HTLV-1-associated diseases is provided, the method comprising administering the pharmaceutical composition to a subject in need thereof. According to other aspects of the present disclosure, a use of the lipid complex or pharmaceutical composition for use in the prevention and/or treatment of HTLV-1-associated diseases is provided.
• Subjects in need may be subjects infected with HTLV-1, subjects judged to be at high risk of being infected with HTLV-1, subjects who may be infected with HTLV-1, or healthy individuals not infected with HTLV-1. Subjects in need may also be subjects judged to be at high risk of developing or recurring an HTLV-1-related disease, and subjects who have developed an HTLV-1-related disease.
• the pharmaceutical composition of the present disclosure may prevent or reduce at least one symptom resulting from HTLV-1 infection in a subject to which the composition is administered.
• a symptom of HTLV-1 infection is the onset of ATL.
• the prevention of the symptom may be evaluated subjectively or objectively, for example, by self-assessment by the subject to which the composition is administered; a physician's evaluation; a quality of life (QOL) evaluation; an evaluation of the onset of ATL or delay in the progression of ATL symptoms, or a reduction in the severity of ATL symptoms.
• the objective evaluation may be an evaluation by an animal or a human.
• the nucleic acid is mRNA
• the pharmaceutical composition can be used to induce an immune response against HTLV-1 (particularly, cellular immunity against HTLV-1, particularly cellular immunity by cytotoxic T cells).
• the pharmaceutical composition in this form can be referred to as an mRNA vaccine, an mRNA vaccine composition, or an mRNA vaccine preparation.
• the pharmaceutical composition can be used for the prevention and/or treatment of HTLV-1-associated diseases.
• the pharmaceutical composition is a vaccine.
• the nucleic acid is mRNA
• the pharmaceutical composition can be utilized as an mRNA vaccine.
• the pharmaceutical composition may be utilized as an mRNA vaccine for the prevention of HTLV-1 associated diseases.
• the pharmaceutical composition of the present disclosure is used for the prevention and/or treatment of ATL. In certain embodiments, the pharmaceutical composition of the present disclosure is used for the prevention of onset, aggravation, or recurrence of ATL.
• the nucleic acid comprises a nucleic acid comprising a polypeptide encoding at least one selected from Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and Gag p24 or an immunogenic fragment thereof.
• Gag a structural protein of HTLV-1
• cytotoxic T cells against Gag are present in patients who do not suffer from recurrent ATL, and it has been confirmed that an effective immune response against HTLV-1 can be induced by using HTLV-1 Gag as an antigen.
• the present inventors have found that a cellular immune response of T cells can be induced by using a pharmaceutical composition containing HTLV-1 Gag p15, p19, and p24 as active ingredients. Therefore, the pharmaceutical composition of the present disclosure is capable of inducing an immune response against, for example, HTLV-1.
• the pharmaceutical composition of the present disclosure is a vaccine for use in, for example, preventing the onset of ATL, the aggravation of ATL, or the recurrence of ATL.
• the subject of administration may be a patient infected with HTLV-1 who has not developed ATL, a patient who has developed ATL, or a patient who has gone into remission from ATL.
• the subject of administration is preferably a patient infected with HTLV-1 who is desired to prevent the onset of ATL or the recurrence of ATL.
• the subject of administration may also be a patient who has undergone bone marrow transplantation or hematopoietic stem cell transplantation after developing ATL.
• a method for preventing the onset, aggravation, or recurrence of ATL comprising the step of administering the pharmaceutical composition of the present disclosure to a subject in need thereof.
• the preventive method of this embodiment is characterized by administering to a subject a pharmaceutical composition containing the lipid complex of the present disclosure, and other configurations and conditions are not particularly limited.
• the preventive method of this embodiment contains, as an active ingredient, a nucleic acid containing a polypeptide encoding at least one selected from Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and Gag p24 or an immunogenic fragment thereof, it is possible to induce an immune response against HTLV-1 when treated in a subject.
• this form of prevention method can induce, for example, an immune response against HTLV-1, it can also be referred to as, for example, a vaccination method against HTLV-1, a method for inducing protective immunity, a method for stimulating an immune response, or a method for inducing an immune response.
• the active ingredient is administered to a subject, and by inducing an immune response in the subject, it is possible to confer immunity against HTLV-1 to the subject.
• an immune response against Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and/or Gag p24 or an immunogenic fragment thereof is induced in the subject, and a cellular immune response by T cells or the like against Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and/or Gag p24 or an immunogenic fragment thereof occurs.
• the administration step is carried out once or multiple times.
• the present disclosure provides for the use of a pharmaceutical composition of the present disclosure for use in a method of prevention against the onset of ATL, the progression of ATL, or the recurrence of ATL.
• the present disclosure is the use of a lipid complex or pharmaceutical composition of the present disclosure for use in the manufacture of a vaccine for use in suppressing the onset of ATL, the progression of ATL, or the recurrence of ATL.
• room temperature generally refers to about 10°C to about 35°C.
• Example 1 Preparation of LNPs encapsulating mRNA
• a plasmid was synthesized having a DNA fragment in which a T7 promoter sequence (TAATACGACTCACTATA: SEQ ID NO: 18), a 5'UTR sequence (AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA: SEQ ID NO: 19), a KOZAK sequence (GCCACC: SEQ ID NO: 20), each antigen sequence (Gag antigen, Tax antigen, HBZ antigen), and a 3'UTR sequence (TAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGG: SEQ ID NO: 21) were linked in this order.
• 1 ⁇ g of the plasmid was dissolved in nuclease-free water (210 ⁇ L), to which Q5 Hot Start High-Fidelity 2 ⁇ Master Mix (250 ⁇ L, NEB# M0494L), 10 ⁇ M sense primer (20 ⁇ L), and 10 ⁇ M poly-T-containing antisense primer (20 ⁇ L) were added. After incubation at 95° C. for 1 minute, 35 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 3 minutes were carried out, and further incubation was continued at 72° C. for 5 minutes, thereby amplifying the template DNA by PCR.
• the mixture was purified using a NucleoSpin Gel and PCR Clean Up Kit (TaKaRa #U0609A) to obtain template DNAs having the antigen sequences of interest (SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24).
• mRNA was prepared by in vitro transcription (IVT). 300 ⁇ g/mL template DNA (80 ⁇ L), 100mM CleanCap AG (32 ⁇ L, TriLink catalog #N-7113), 100mM ATP (40 ⁇ L, TriLink catalog #N-1510), 100mM CTP (40 ⁇ L, TriLink catalog #N-1511), 100mM GTP (40 ⁇ L, TriLink catalog #N-1512), 100mM N1-methyl- ⁇ -Uridine-5'-Triphosphate (40 ⁇ L), UltraPure DNase/RNase-Free Distilled Water (405.6 ⁇ L, Thermo Fisher catalog #10977015), T7 Transcription 10 ⁇ buffer (80 ⁇ L), RNase inhibitor (20 ⁇ L, NEB, catalog #M0314L), Yeast Inorganic pyrophosphatase (16 ⁇ L, NEB, catalog #M2403L), and T7 RNA Polymerase (6.4 ⁇ L, Roche catalog #08140669
• RNase-Free DNase I 24 ⁇ L, TaKaRa catalog #2270A was added and incubated for 30 minutes at 37° C. Then, 10 ⁇ phosphatase buffer (96 ⁇ L, NEB, catalog #B0289S) and Antarctic phosphatase (48 ⁇ L, NEB, catalog #M0289L) were added and incubated for 30 minutes at 37° C. The mixture was mixed with 8M LiCl solution (484 ⁇ L, Sigma-Aldrich catalog catalog #L7026), left to stand at ⁇ 20° C. for 1 hour or more, centrifuged (4° C., 15,000 rpm, 30 minutes), and the supernatant was discarded.
• 8M LiCl solution 484 ⁇ L, Sigma-Aldrich catalog catalog #L7026
• mRNAs SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 9 expressing proteins having each amino acid sequence (SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 8) were obtained.
• a cap structure and a polyA sequence were added to each mRNA.
• the cap structure was the structure shown below as CleanCap (Cap1) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).
• ⁇ SEQ ID NO:1> Amino acid sequence of Gag MGQIFSRSASPIPRPPRGLAAHHWLNFLQAAYRLEPGPSSYDFHQLKKFLKIALETPVWICPINYSLLASLLPKGYPGRVNEILHILIQTQAQIPSRPAPPPPSSPT HDPPDSDPQIPPPYVEPTAPQVLPVMHPHGAPPNHRPWQMKDLQAIKQEVSQAAPGSPQFMQTIRLAVQQFDPTAKDLQDLLQYLCSSLVASLHHQQLDSLISEAET RGITGYNPLAGPLRVQANNPQQQGLRREYQQLWLAAFAALPGSAKDPSWASILQGLEEPYHAFVERLNIALDNGLPEGTPKDPILRSLAYSNANKECQKLLQARGHT NSPLGDMLRACQTWTPKDKTKVLVVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWKRDCPRLKPTIPEPEED
• ⁇ SEQ ID NO:8> Amino acid sequence of HBZ MAASGLFRCLPVSCPEDLLVEELVDGLLSLEEELKDKEEEEAVLDGLLSLEEESRGRLRRGPPGEKAPPRGETHRDRQRRAEEKRKRKKEREKEEEKQIAEYL KRKEEEKARRRRRAEKKAADVARRKQEEQERRERKWRQGAEKAKQHSARKEKMQELGIDGYTRQLEGEVESLEAERRKLLQEKEDLMGEVNYWQGRLEAMWLQ
• the ratio of mRNA to lipid was set to a weight ratio of 0.05, and the mRNA dilution solution and the lipid solution were mixed at a flow rate of 3:1 in volume ratio to obtain Lipid Nanoparticles (LNP).
• LNP Lipid Nanoparticles
• the obtained LNP aqueous solution was dialyzed using a Float-A-Lyzer G2 (SPECTRUM, 100K MWCO) to replace the external solution with PBS, and then with a sucrose solution. After concentration, filtration sterilization was performed, and the formulation quality of the LNP was evaluated.
• SPECTRUM Float-A-Lyzer G2
• lipid nanoparticles were obtained in which each of the mRNA encoding the Gag protein or its immunogenic fragment, the mRNA encoding the Tax protein or its immunogenic fragment, and the mRNA encoding the HBZ protein or its immunogenic fragment of the present disclosure was encapsulated in lipid. It was confirmed that the encapsulation rate of all LNPs was higher than 90%, indicating a high encapsulation rate of mRNA.
• the polydispersity index (PDI) was also less than 0.05.
• mice Examination of cellular immune responses in mice
• the mice were euthanized and the spleens were removed.
• the spleens were crushed on a cell strainer using a plunger of a sterile syringe and washed with 10 ml of RPMI to collect a cell suspension.
• the cell suspension was centrifuged, the supernatant was removed, and hemolysis was performed with 1 ml of NH 4 Cl for 2 minutes. The reaction was then stopped with 9 ml of RPMI and centrifuged. The cell pellet was resuspended in 20 ml of RPMI with 10% FBS.
• Gag-3 p19-1 (p19 1-50 )
• Gag-4 p19-2 (p19 51-100 )
• Gag-5 p19-3 (p19 101-121 )
• Gag p24 peptides 206 peptides were pooled as Gag-6 (p24-1 (p24 1-51 ), Gag-7 (p24-2 (p24 52-103 )), Gag-8 (p24-3 (p24 104-154 )), and Gag-9 (p24-4 (p24 155-206 )).
• HBZ was pooled as 198 peptides, HBZ-1 (HBZ 1-50 ), HBZ-2 (HBZ 51-100 ), HBZ-3 (HBZ 101-150 ), and HBZ-4 (HBZ 151-198 ).
• the ELISpot assay was performed using MABTECH's ELISpot Flex: IFN- ⁇ , Mouse-ALP, as follows.
• Millipore's MAIPS4510 assay plate was treated with coating antibody, and prepared mouse splenocytes were seeded at 106 cells/well. After stimulation with 1 ⁇ M of the above peptide pool for 24 hours, IFN- ⁇ -producing cells were stained with the detection antibody. The stained spots were analyzed using CTL's ImmunoSpot S6 ENTRY Analyzer, and the number of spots was counted.
• Figures 1 to 3 show the results showing the number of spots in each peptide pool.
• Figure 1 shows the results for the Gag peptide pool
• Figure 2 shows the results for the peptide pool
• Figure 3 shows the results for the HBZ peptide pool.
• No peptide shows the results for the negative control (no peptide stimulation)
• Positive Control shows the results for the positive control (stimulation with the mitogens PMA/Ionomycin).
• Gag-1 to Gag-2 in Figure 1 correspond to p15-1 and p15-2
• Gag-3 to Gag-5 correspond to p19-1 to p19-3
• Gag-6 to Gag-9 correspond to p24-1 to p24-4.
• spots were formed in the peptide pools of Gag p19 (Gag-3 to Gag-5), Gag p15 (Gag-1 to Gag-2), or Gag p24 (Gag-6 to Gag-9).
• spots were formed more frequently in the peptide pools of Gag p19-1 (Gag-3), p19-2 (Gag-4), and p19-3 (Gag-5) (particularly p19-1 (Gag-3), p19-2 (Gag-4)).
• spots were formed in the Tax peptide pool. In particular, many spots were formed in the Tax-4 or Tax-6 (especially Tax-6) peptide pool. These results demonstrated that IFN- ⁇ was produced in mice immunized with LNPs encapsulating mRNA encoding the Tax protein. Furthermore, the production of IFN- ⁇ suggested that a cellular immune response was induced. As shown in Figure 3, spots were formed in the HBZ peptide pool. In particular, many spots were formed in the HBZ-1 peptide pool. These results demonstrated that IFN- ⁇ was produced in mice immunized with LNPs encapsulating mRNA encoding the HBZ protein. Furthermore, the production of IFN- ⁇ suggested that a cellular immune response was induced.
• Example 3 To analyze the efficacy of Gag mRNA vaccines in animal models, we established an experimental system using a syngeneic mouse model. EL4, a T cell lymphoma line derived from the inbred mouse C57BL/6, can form tumors when subcutaneously transplanted into wild-type C57BL/6 mice. We established an EL4 subline, EL4-Gag, that expresses the gag gene, one of the HTLV-1 antigens, by the following procedure.
• the gene sequence encoding the full-length Gag protein was amplified by RT-PCR, and inserted into the multicloning site of the PiggyBac Transposon Vector System (System Biosciences, PB-CMV-EF1-Puro) to construct a plasmid expressing the full-length Gag.
• Gene transfer into EL4 was performed by lipofection with a Gag expression PiggyBac vector and a transposase expression vector, and then EL4 (EL4-Gag), which stably expresses Gag, was established by adding puromycin to the culture medium for selection. Gag expression in EL4-Gag is confirmed by real-time PCR.
• mice are also vaccinated twice with saline using the same protocol.
• 1 ⁇ 10 6 EL4-Gag cells are subcutaneously transplanted into the mice.
• the mice are weighed, tumor engraftment is confirmed, and tumor diameter is measured twice a week.
• Tax SEQ ID NO: 4
• Env SEQ ID NO: 25
• Gag p15 SEQ ID NO: 11
• Gag p19 SEQ ID NO: 13
• Gag P24 SEQ ID NO: 14
• the antigen a vector expressing a fusion protein of various proteins and NanoLuc was prepared, and after the preparation, the vector was introduced into a 293T cell line, and a cell lysate of the cells was used.
• a 96-well plate was mixed with 107 counts of antigen, LIPS buffer, and 1 ⁇ l of the serum or plasma to prepare a total of 100 ⁇ l.
• the plate was incubated using a plate mixer at room temperature (hereinafter, about 24° C.) for 30 minutes.
• Protein A/G magnetic beads manufactured by Thermo Fisher Scientific
• LIPS buffer room temperature
• 10 ⁇ l of the magnetic beads were added to each well of the plate.
• the plate was incubated using a plate mixer at room temperature for 30 minutes.
• the plate was placed on a magnet plate and the pellets in each well were washed five times with 150-200 ⁇ l of LIPS buffer. After the washing, the LIPS buffer used for washing was removed, and 100 ⁇ l of Nano-Glo luciferase assay substrate (Promega) was added to each well. After the addition, luminescence was measured using a luminometer (Promega). These results are shown in FIG. 1.
• Tax Amino acid sequence of Tax (SEQ ID NO:4) MAHFPGGQSLLFGYPVYVFGDCVQGDWCPISGGLCSARLHRHALLATCPEHQITWDPIDGRVIGSALQFLIPRLPSFPTQRTSKTLKVLTPPITHTTPNIPPSFLQAMRKYSPFRNGYMEPTLGQHLPTLSFPDPGLRPQNLYTLWGGSVVCMYLYQLSPPITWPLLPHVIFCHP GQLGAFLTNVPYKRIEELLYKISLTTGALIILPEDCLPTTLFQPARAPVTLTAWQNGLLPFHSTLTTPGLIWTFTDGTPMISGPCPKDGQPSLVLQSSSFIFHKFQTKAYHPSFLLSHGLIQYSSFHSLHLLFEEYTNIPISLLFNEKEADDNDHEPQISPGGLEPPSEKHFRETEV
• Figure 4 is a graph showing the results of the LIPS assay.
• the horizontal axis indicates the type of subject, and the vertical axis indicates luminescence intensity.
• Figure 4 (A) shows the antibody response to Tax
• Figure 4 (B) shows the antibody response to Env
• Figure 4 (C) shows the antibody response to Gag p15
• Figure 4 (D) shows the antibody response to Gag p19
• Figure 4 (E) shows the antibody response to Gag p24.
• cDNA was synthesized using SuperScript IV Reverse Transcriptase (Invitrogen).
• Gag cDNA was prepared using the following Gag cDNA synthesis primer.
• Tax cDNA and 18Sr RNA were synthesized using random primers.
• Gag expression was measured using Taqman real time PCR.
• Tax expression was measured using SYBR-green real time PCR.
• 18Sr RNA was quantified using Taqman Gene Expression Assays (Hs99999901_s1; Applied Biosystems). The amplification conditions were 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute, with each cycle consisting of 40 cycles. These results are shown in FIG. 5.
• Gag cDNA synthesis primer Primer 1 (SEQ ID NO: 26) 5'- TGCAGGATATGGGCC-3'.
• Primer set for Gag Primer 2 (SEQ ID NO: 27) 5'-AGTACCTTTGCTCCTCCCTC-3'
• Primer 3 (SEQ ID NO:28) 5'-TAATACCTCGGGTTTCGGCC-3'
• Probe for Gag Probe (SEQ ID NO: 29) 5'-TTCCCTCCATCACCAGCTAGATAGCCT-3' ⁇ Tax primer set
• Primer 4 (SEQ ID NO: 30) 5'-CCGCCGATCCCAAAGAA-3'
• Primer 5 (SEQ ID NO:31) 5'-CCTGTCCAAACCCTGGGAA-3'
• Figure 5 is a graph showing the results of RT-qPCR.
• the horizontal axis indicates the type of sample, and the vertical axis indicates the relative mRNA expression level.
• Figure 5 (A) shows the mRNA expression level of Gag in various cell lines
• Figure 5 (B) shows the mRNA expression level of Tax in various cell lines
• Figure 5 (C) shows the mRNA expression level of Gag in ATL case samples
• Figure 5 (D) shows the mRNA expression level of Tax in ATL case samples.
• mice anti-HTLV-1 p24 monoclonal antibody (clone 46/3.24.4, ZeptoMetrix), rabbit polyclonal antibody against HTLV-1 p24 Gag (Cat No: 5418, ABL), or mouse anti-HTLV-1 p19 Gag monoclonal antibody (Cat No: 0801003, ZeptoMetrix) were used.
• the glass cover slip was sealed with DAPI-containing In Situ Mounting Medium (Sigma-Aldrich). After the sealing, the cells were observed using a Nikon C2 confocal microscope (Nikon). These results are shown in FIG. 6.
• Figure 6 is a photograph showing the results of the proximity ligation assay.
• Figure 6 (A) shows the results for Gag p19 antibody.
• Figure 6 (B) shows the results for Gag p24 antibody.
• the upper row shows the results of DAPI staining
• the middle row shows the results of Gag p19 or Gag p24 staining
• the lower row shows the results of DAPI and Gag p19 or Gag p24 staining.
• the area surrounded by the dashed line indicated by the arrow shows the expression of Gag p19 or Gag p24.
• Gag induces cellular immune response in ATL patients who have undergone hematopoietic stem cell transplantation and are in long-term remission.
• ELISPOT assay was used. Based on the amino acid sequence of Gag (GenBank accession number AAA85841.1, SEQ ID NO: 1), overlapping peptides of 9 amino acids in length (offset: 1 amino acid) were designed. 97 peptides of Gag p15 were pooled as p15-1 (p15 1-50 ), and p15-2 (p15 51-97 ).
• 121 peptides of Gag p19 were pooled as p19-1 (p19 1-50 ), p19-2 (p19 51-100 ), and p19-3 (p19 101-121 ).
• Peripheral blood mononuclear cells PBMCs
• ELISPOT assay was performed using a human IFN- ⁇ ELISPOT kit (MABTECH). The PBMCs were seeded on an ELISPOT plate.
• Figure 7 is a photograph showing the results of the ELISPOT assay.
• the left side shows the results of stimulation with Gag p19 peptide.
• the upper and middle rows on the right side show the results of stimulation with Gag p15 peptide, and the lower row on the right side shows the results of no stimulation.
• spots were formed when stimulated with Gag p19 or Gag p15 peptide.
• Gag-induced cellular immune response was induced in mice immunized with recombinant Gag protein. Specifically, an ELISPOT assay was used. Gag p15 peptides and Gag p19 peptides were designed and pooled in the same manner as in Reference Example 1 (4). Gag p24 peptides were designed in the same manner as in Reference Example 1 (4). 206 peptides of Gag p24 were pooled as p24-1 (p24 1-51 ), p24-2 (p24 52-103 ), p24-3 (p24 104-154 ), and p24-4 (p24 155-206 ).
• mice (C57BL/6J, Jackson Laboratory Japan) were infected with HTLV-1, and 14 days later, splenocytes were collected from the mice.
• the ELISPOT assay was performed in the same manner as in Reference Example 1(4), except that splenocytes collected from the mice were used as samples.
• Figure 8 is a photograph showing the results of the ELISPOT assay.
• the top row (A) shows the results of splenocytes from mouse 1
• the middle row (B) shows the results of splenocytes from mouse 2
• the bottom row (C) shows the results of splenocytes from mouse 3.
• row 2 (Gag p15-1 and 2) shows the results of stimulation with Gag p15
• row 3 (Gag p19-3 to 5) shows the results of stimulation with Gag p19
• row 4 (Gag p24-6 to 9) shows the results of stimulation with Gag p24
• row 1 (10) shows the positive control
• row 1 (11) shows the negative control.
• spots were formed when stimulated with Gag p19, Gag p15, or Gag p24 peptides. In particular, spots were formed more frequently when stimulated with Gag p19-3 peptide.
• the lipid complex or pharmaceutical composition of the present disclosure can induce an immune response against HTLV-1. Therefore, the lipid complex and pharmaceutical composition containing the lipid complex of the present disclosure can be used, for example, in the prevention and/or treatment of HTLV-1-related diseases.

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