WO2008065752A1 - Immunotherapeutic agent containing dirna as active ingredient - Google Patents

Immunotherapeutic agent containing dirna as active ingredient Download PDF

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Publication number
WO2008065752A1
WO2008065752A1 PCT/JP2007/001315 JP2007001315W WO2008065752A1 WO 2008065752 A1 WO2008065752 A1 WO 2008065752A1 JP 2007001315 W JP2007001315 W JP 2007001315W WO 2008065752 A1 WO2008065752 A1 WO 2008065752A1
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Prior art keywords
dirna
strain
immunotherapeutic agent
seq
measles virus
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PCT/JP2007/001315
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French (fr)
Japanese (ja)
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Tsukasa Seya
Misako Matsumoto
Masashi Shingai
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National University Corporation Hokkaido University
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Priority to JP2008546877A priority Critical patent/JPWO2008065752A1/en
Publication of WO2008065752A1 publication Critical patent/WO2008065752A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
    • C12N2760/18432—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • Immunotherapy drugs containing di RNA as an active ingredient containing di RNA as an active ingredient
  • the present invention relates to an immunotherapy drug comprising as an active ingredient a diRNA having a measles virus-derived RNA sequence, and a tumor immunotherapy composition comprising the diRNA and a cell wall skeleton of a Gram-positive bacterium.
  • One of the cancer treatment methods is so-called cancer immunotherapy that induces cancer regression by enhancing the immunity of the patient's own cancer cells.
  • the method of administering a peptide vaccine as a cancer antigen is the mainstream, but according to Rosenberg et al. (Non-Patent Document 1), the effectiveness of this method is said to be 2.5 ⁇ 1 ⁇ 2. .
  • Patent Document 1 As a method for increasing the effective rate of this cancer immunotherapy, a method has been proposed in which an adjuvant that activates dendritic cells is simultaneously administered simultaneously with the administration of a cancer antigen (Patent Document 1).
  • the adjuvant disclosed in Patent Document 1 is a cell wall skeleton (hereinafter referred to as BGG and GWS) of the gram-positive bacterium Mycobacterium mupovis Calmette and Guerin, and immature dendritic cells are matured dendritic cells.
  • BGG and GWS cell wall skeleton
  • immature dendritic cells are matured dendritic cells.
  • GTL cytotoxic T lymphocytes
  • BGG— GWS has the advantage of improving the 5-year survival rate by more than 10% by actually administering it to cancer patients and showing few side effects.
  • BGG-GWS is a ligand that activates TLR (Toll Like Receptor) 2 and 4. This fact is based on the fact that BGG- GWS is an adjuvant that has neither the ability to induce type I interferon (referred to as type I interferon I FN-S) or the ability to enhance NK activity in human dendritic cells. Matches.
  • GTL induced by single administration of BGG-GWS cannot kill cancers with low MHG class I protein expression levels. Therefore, although it has better anti-cancer effect than the peptide vaccine of Rosenberg et al. Is considered difficult.
  • Patent Document 1 W001 / 048154
  • Non-Patent Document 1 Rosenberg et al., Nat. Med., 2004, 10, 9, 909-92, p. 5
  • the present invention provides an immunotherapeutic agent that exhibits a further excellent anticancer effect by using it instead of or at the same time as BGG-GWS, and particularly usable for immunotherapy of cancer. It is an object of the present invention to provide a substance exhibiting an immunostimulatory action useful for immunotherapy of cancer, particularly a substance that induces the production of IFNyS and / or enhances NK activity.
  • the present inventors have surprisingly confirmed the presence of a substance having a desired activity in measles virus, particularly acclimated measles virus strain and measles virus vaccine strain, and the substance is diRNA. Ascertaining that it is (also called defective interference RNA, defective interfering, or loop RNA), the following inventions were completed.
  • An immunotherapeutic agent comprising, as an active ingredient, diRNA having a measles virus-derived RNA sequence.
  • the measles virus is an acclimated virus strain or a vaccine strain, (1) or (
  • the immunotherapeutic agent according to 2).
  • the acclimated virus strain is an Edmonston strain, a Nagahata strain, or an lchinose_V strain.
  • diRNA comprising any RNA sequence represented by SEQ ID NOs: 9 to 15
  • a composition for tumor immunotherapy comprising the immunotherapeutic agent according to any one of (1) to (7) and a cell wall skeleton of a Gram-positive bacterium.
  • composition for tumor immunotherapy according to (8), wherein the cell wall skeleton of the Gram-positive bacterium is BGG-GWS.
  • Medicament for promoting interferon expression and / or enhancing NK activity comprising diRNA having measles virus-derived RNA sequence as an active ingredient (1 1) Measles virus acclimatized The medicament according to (10), which is a virus strain or a vaccine strain.
  • diR whose diRNA is composed of any RNA sequence shown in SEQ ID NOs: 9 to 15
  • the immunotherapeutic agent of the present invention can enhance the immune defense power of chicks against foreign substances such as bacteria or neoplastic tumors such as cancer, and can be used as an anticancer agent or a tumor immunotherapeutic agent. is there. Furthermore, by using the immunotherapeutic agent of the present invention in combination with BGG-GWS, a pharmaceutical composition (tumor immunity therapeutic composition) effective for immunotherapy of cancer can be obtained.
  • the diRNA according to the present invention does not show toxicity even when administered subcutaneously (100 ⁇ g) to mice and humans and can be a safe pharmaceutical.
  • FIG. 1 is an electrophoretogram showing the detection results of diRNA of each measles virus strain.
  • FIG. 2 shows a schematic diagram of p (+) MV Dl GFP plasmid.
  • FIG. 3 shows a schematic diagram of diRNA-IGV-G.
  • FIG. 4 shows the amount of fluorescence of luciferase from HEK cells into which di RNA-IGV-G has been introduced.
  • FIG. 5 shows the regression effect of transplanted cancer-bearing cancer by administration of diRNA-IGV-G.
  • FIG. 6 It shows the increase in cytotoxic activity of NK cells by [ ⁇ --6 administration (_ ⁇ _)]. The letter indicates the control.
  • the diRNA is a double-stranded RNA having a stem-and-loop structure, as contained in, for example, measles virus, in particular, acclimatized measles virus or measles virus strain.
  • measles virus vaccine strains diRNA is a contaminant and has been reported to be a non-infectious RNA produced during viral replication (Robert et al., Cell, 1981, 26, 145-154).
  • DiRNA has been confirmed to exist in measles virus vaccine strains and acclimated measles virus, but is considered to be a completely unnecessary substance for replication of measles virus. It is a substance that has received little attention for its effects.
  • measles virus particularly measles vaccine virus
  • the present invention is an invention based on the fact that a substance that enhances immunity against cancer exists in such measles virus vaccine, and that the substance is confirmed to be diRNA.
  • the diRNA has a combination of either a trailer sequence and a leader sequence, or a trailer sequence and a sequence complementary to the trailer sequence, and has a stem-and-loop structure formed thereby.
  • RNA A trailer sequence is present at the 5 'end of the virus genome and a leader sequence is present at the 3' end, each of which is a specific sequence essential for transcription, replication, etc. of the measles virus genome.
  • the trailer sequence and the leader sequence are almost complementary sequences, and when they are aligned, a diRNA stem-and-loop structure is formed.
  • DiRNAs having a trailer sequence and a complementary sequence of the trailer sequence are also By ringing, a stem and loop structure is formed.
  • the viral genomes of the habituated measles virus strains Edmonston (ED) strain, lchi nose-V (ICV) strain, and vaccine strains Schwarz strain and Tanabe strain are the RNA shown in SEQ ID NO: 1. It has a trailer array consisting of an array.
  • the Nagahata (NV) strain, a habituated measles virus strain has a trailer sequence consisting of the RNA sequence shown in SEQ ID NO: 2 in which only the 15th base of the RNA sequence shown in SEQ ID NO: 1 is mutated from guanine to adenine. Have.
  • the IGV strain which is an acclimated measles virus strain, has a leader sequence consisting of the RNA sequence shown in SEQ ID NO: 3
  • the Edmonston (ED) strain is a leader sequence consisting of the RNA sequence shown in SEQ ID NO: 4.
  • the Schwarz strain which is a vaccine strain, has a leader sequence consisting of the RNA sequence shown in SEQ ID NO: 5, respectively.
  • Each leader sequence shown in SEQ ID NOs: 3 to 5 has the same RNA sequence except that the 26th, 42nd, 50th and 50th bases are different.
  • Di RNA derived from measles virus has a variation in RNA sequence and number of bases, and includes a trailer sequence and a leader sequence, or a trailer sequence and a sequence complementary to the trailer. It also has the structural feature of forming a & loop structure. Therefore, the present invention is not limited to diRNA containing any of the specific RNA sequences shown in SEQ ID NOs: 1 to 5 above, but includes measles virus-derived RNA, which retains the stem-and-loop structure, and is As long as the function of enhancing immunity against cancer is maintained, diRNAs comprising RNA sequences in which a plurality of, preferably several to several tens of bases are substituted, deleted, added, etc. in the above RNA sequence are also included.
  • the di RNA according to the present invention is conventionally known as reported to Robert et al. According to the method of Maniatis et al. (Molecular Cloning, 1989, Go Id Spring Harbor Laboratory Press), it can be isolated and purified from measles virus, particularly acclimated measles virus or measles virus vaccine strains widely supplied to the parties concerned. Acclimatized measles virus strains ED, NV, and IG-V can be obtained from the National Institute of Infectious Diseases. Schwarz and Tanabe strains are available from Takeda Pharmaceutical Co., Ltd. and Tanabe Seiyaku Co., Ltd., respectively.
  • diRNA obtained from the above strains or diRNA obtained by artificially recombining part of the RNA sequence based on them can be produced using a general genetic recombination technique.
  • an appropriate expression vector incorporating a DNA containing a base sequence complementary to any RNA sequence shown in SEQ ID NOs: 1 to 5 is expressed in a host cell to produce diRNA, Collect it.
  • RNA may be produced in vitro by collecting a DNA containing a base sequence complementary to any of the RNA sequences shown in SEQ ID NOs: 1 to 5 in a cage shape and recovering it.
  • gene expression systems such as expression vectors and promoters that can be used in the present invention, the types of recombinant host cells, in vitro transcription systems, and RNA recovery methods. Any available method can be used.
  • the composition for tumor immunotherapy of the present invention comprises an immunotherapeutic drug containing the above-described diRNA as an active ingredient and a cell wall skeleton of Gram-positive bacteria.
  • the cell wall skeleton of a Gram-positive bacterium is an insoluble residue obtained through a refinement process such as denucleation, protein removal, and degreasing after physically pulverizing the Gram-positive bacterium, and is described in detail in Patent Document 1.
  • Examples of cell wall skeletons of Gram-positive bacteria in the present invention include cell wall skeletons such as Mycobacterium bacteria, Nocardia bacteria, Corynebacterium bacteria, and the like. (BGG—GWS) is preferred.
  • the composition for tumor immunotherapy of the present invention comprising the cell wall skeleton of Gram-positive bacteria and diRNA derived from measles virus is a type of cancer, in particular, an MHG class I protein. Regardless of current level, it has the effect of reducing cancer.
  • the composition for tumor immunotherapy of the present invention comprises various excipients, carriers, and other pharmaceutical compositions depending on the administration route to the living body, dosage form, etc. It can be adjusted by blending other ingredients.
  • the administration route of the composition for tumor immunotherapy of the present invention is not particularly limited, but oral administration, subcutaneous administration, vascular administration, and transmucosal administration are preferable.
  • the diRNA according to the present invention has a function of inducing IFN-; 3 expression to human cells and enhancing the NK activity of NK cells as described above. It can also be used as a medicament for promoting S expression and / or enhancing NK activity.
  • measles virus vaccines are known to weaken the immunity of living organisms, but diRNA contained in measles virus vaccines promotes the expression of IFN-3; It was an unexpected finding that it had a function to enhance immune defense ability.
  • the pharmaceutical for promoting the expression of interferon containing diRNA and / or enhancing NK activity depends on the administration route to the living body and the dosage form in addition to diRNA as an active ingredient.
  • Various excipients, carriers, other pharmaceutical ingredients and other ingredients can be blended and adjusted. There are no particular restrictions on the route of administration, but oral administration, subcutaneous administration, and vascular administration are preferred.
  • P1 5′-TATAAGCTTACCAGACAAAGCTGGGAATAGAAACTTCG-3 ′ (SEQ ID NO: 6)
  • P2 5′-CGAAGATATTCTGGTGTAAGTCTAGTA-3 ′ (SEQ ID NO: 7) Primers for the control reaction indicating that the PGR reaction is in progress used P1 and P3. [0040] P3: 5'-TTTATCCAGAATCTCAARTCCGG-3 '(SEQ ID NO: 8)
  • RNA-ED Sequence
  • diRNA_ED, diRNA-ICV, diRNA-SH, diRNA-TAU diRNA_TA2, diRNA_TA3 include the trailer sequence shown in SEQ ID NO: 1 and a sequence complementary to the trailer sequence, and diRNA-NV in SEQ ID NO: 2. The trailer sequence shown and the sequence complementary to the trailer sequence were included.
  • a plasmid (Takeda et al., Viro, 2000, 74, 6643-6647) having a genomic sequence of the IG-V strain incorporated with a DNA encoding a GFP fluorescent protein p (+ ) MV323GFP plasmid (Singai et al., J. I country unol., 2005, Vol. 175, No. 5, pp.
  • 3252-3261 is a saddle type, and a leader sequence (SEQ ID NO: 3) and trailer from the plasmid
  • the plasmid p (+) MV DI GFP from which the genomic sequence of the IG-V strain excluding the sequence (SEQ ID NO: 1) was deleted was converted to PGR using primers (P4, P5) consisting of the following base sequences: (Fig. 2).
  • P4 5′-GCCATCGATTATTACTTGTACAGCTCGTCC-3 ′ (SEQ ID NO: 16)
  • p (+) MV DI GFP is a cocoon type, and one leader sequence is one GFP—one trailer sequence in the opposite direction to the ⁇ type.
  • p (-) MV DI GFP was prepared by the PGR method using the following two sets of primers-(P6 and P7, and P8 and P9).
  • P6 5′-GGCCGGCATGGTCCCAGCCTCCTCGCT-3 ′ (SEQ ID NO: 1 8)
  • P7 5'-TATAGTGAGTCGTATTACGCGCGCTT-3 '(SEQ ID NO: 1 9)
  • P8 5'-ACCAAACAAAGTTGGGTAAGGATAGATCAATCAATGATCAT-3' (SEQ ID NO: 2)
  • diRNA-IGV-G (which has a stem-and-loop structure in which the region encoding GFP fluorescent protein is looped over Figure 3) was prepared in vitro.
  • a plSRE_Luc plasmid (Stratagene) that incorporates a nucleotide sequence that codes for luciferase fluorescent protein downstream of the nucleotide sequence that codes for IFN-stimulated response element (ISRE), a transcription factor that induces IFN production.
  • ISRE IFN-stimulated response element
  • mice G57BL / 6 inbred mice were transplanted with B16 melanoma cells (distributed from the Osaka Prefectural Center for Adult Diseases) 1 x 10 6 cel ls, and changes in tumor size over time were measured (Fig. 5). ) Since B16 melanoma cells do not express MHG class I molecules, activation of killer T cells is not induced, and only by the activity of NK cells enhanced by interferon. The mammary tumor regresses.
  • BGG-GWS 25 g / 0.1 m prepared by the method described in Patent Document 1 MALP-2 ( Oncologics Inc., Nagoya Japan) B16 melanoma cell cancer antigen EP with physiological saline containing di RNA-1 CV-G 10 g / ml as an adjuvant.
  • TRP2_pep8 distributed by Rosenberg S.
  • 0.1 mg was administered intraperitoneally three times every other week before transplantation of B16 melanoma cells and three times every other week after transplantation (indicated by the arrow in FIG. 5).
  • diRNA-IGV-G was administered, a marked cancer regression effect (proliferation inhibitory effect) was observed.
  • Lymphocytes were extracted using Lympho I yte-M (Cederlane Laboratory) from the three types of mice administered with the adjuvant in Example 3, and then negative for NK preparation using MACS beads (Mi Itenyi Biotec) NK cells were obtained by selection. NK cells and B16 melanoma cells were co-cultured using RPM-1640 medium, and the cytotoxic activity of NK cells was measured by 5 iCr-re lease assay. As shown in Fig. 6, NK cells derived from mice treated with diRNA-IGV-G showed a marked increase in cytotoxic activity compared to NK cells derived from mice administered with saline (control). It was.

Abstract

It is intended to provide a substance exerting an immunostimulatory action useful for immunotherapy for cancer, particularly a substance which induces the production of IFN-β and/or enhances NK activity in cells associated with human immunity. The invention provides an immunotherapeutic agent containing as an active ingredient, diRNA including RNA derived from measles virus, and an immunotherapeutic composition for tumors containing the immunotherapeutic agent and a Gram-positive bacterial cell wall structure. The immunotherapeutic agent of the invention can be an extremely safe drug which can enhance a human immune defense ability against foreign substances such as bacteria or neoplastic tumors such as cancer.

Description

明 細 書  Specification
d i RNAを有効成分とする免疫治療用薬剤  Immunotherapy drugs containing di RNA as an active ingredient
技術分野  Technical field
[0001 ] 本発明は、 麻疹ウィルス由来の RNA配列を有する d i RNAを有効成分とする免 疫治療用薬剤、 ならびに当該 d i RNAとグラム陽性菌の細胞壁骨格とを含む腫瘍 免疫治療用組成物に関する。  [0001] The present invention relates to an immunotherapy drug comprising as an active ingredient a diRNA having a measles virus-derived RNA sequence, and a tumor immunotherapy composition comprising the diRNA and a cell wall skeleton of a Gram-positive bacterium.
背景技術  Background art
[0002] 癌の治療法の一つに、 患者自身の癌細胞に対する免疫力を亢進させて癌の 退縮を誘導する、 いわゆる癌免疫療法がある。 この療法において、 癌抗原と してペプチドワクチンを投与する方法が主流であるが、 Rosenbergら （非特許 文献 1 ) によれば、 この方法の有効率は 2. 5<½に留まるとされている。  [0002] One of the cancer treatment methods is so-called cancer immunotherapy that induces cancer regression by enhancing the immunity of the patient's own cancer cells. In this therapy, the method of administering a peptide vaccine as a cancer antigen is the mainstream, but according to Rosenberg et al. (Non-Patent Document 1), the effectiveness of this method is said to be 2.5 <½. .
[0003] この癌免疫療法の有効率を上げる方法として、 癌抗原の投与と同時に、 樹 状細胞を活性化させるアジュバントを同時投与する方法が提唱されている （ 特許文献 1 ) 。 特許文献 1が開示するアジュバントは、 グラム陽性菌である マイコバクテリゥムポビスのカルメット一ゲラン菌の細胞壁骨格 （以下、 BGG 一 GWSとする） であり、 未成熟樹状細胞を成熟樹状細胞に成熟化させる機能を 有しており、 その結果、 細胞障害 Tリンパ球 （GTL) が誘導され、 癌に対する 免疫が賦活化される。  [0003] As a method for increasing the effective rate of this cancer immunotherapy, a method has been proposed in which an adjuvant that activates dendritic cells is simultaneously administered simultaneously with the administration of a cancer antigen (Patent Document 1). The adjuvant disclosed in Patent Document 1 is a cell wall skeleton (hereinafter referred to as BGG and GWS) of the gram-positive bacterium Mycobacterium mupovis Calmette and Guerin, and immature dendritic cells are matured dendritic cells. As a result, cytotoxic T lymphocytes (GTL) are induced, and immunity against cancer is activated.
[0004] BGG— GWSは、 実際に癌患者に投与することで 5年生存率を 10%以上向上さ せる他、 殆ど副作用を示さないという利点を有する。 しかしその後の研究に より、 BGG—GWSは TLR (To l l L i ke Receptor) 2及び 4を活性化するリガンド であることが明らかとなった。 この事実は、 BGG— GWSがヒト樹状細胞に対す る I型インタ一フエロン （I型 i nterferon I FN- Sとする） 誘導能ならびに NK 活性を増強させる能力のいずれも有しないアジュバントである事実と一致す る。 また、 BGG— GWSの単独投与により誘導される GTLは、 MHGクラス I蛋白質の 発現レベルが低い癌を殺傷することができない。 そのため、 Rosenbergらのぺ プチドワクチンと比べて抗癌作用は優れるものの、 癌の完全退縮を行うこと は難しいと考えられる。 [0004] BGG— GWS has the advantage of improving the 5-year survival rate by more than 10% by actually administering it to cancer patients and showing few side effects. However, subsequent studies revealed that BGG-GWS is a ligand that activates TLR (Toll Like Receptor) 2 and 4. This fact is based on the fact that BGG- GWS is an adjuvant that has neither the ability to induce type I interferon (referred to as type I interferon I FN-S) or the ability to enhance NK activity in human dendritic cells. Matches. In addition, GTL induced by single administration of BGG-GWS cannot kill cancers with low MHG class I protein expression levels. Therefore, although it has better anti-cancer effect than the peptide vaccine of Rosenberg et al. Is considered difficult.
特許文献 1 ： W001/048154  Patent Document 1: W001 / 048154
非特許文献 1 ： Rosenbergら、 Nat. Med. 、 2004年、 第 10巻、 第 9号、 第 909-92 5頁  Non-Patent Document 1: Rosenberg et al., Nat. Med., 2004, 10, 9, 909-92, p. 5
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0005] 本発明は、 BGG— GWSに代わり、 あるいはこれと同時に利用することによつ てさらに優れた抗癌作用を示す、 特に癌の免疫療法に利用可能な免疫治療用 薬剤を提供すること、 ならびに癌の免疫療法に有用な免疫賦活作用を示す物 質、 特に IFNySの産生を誘導し、 及び/又は NK活性を増強させる物質を提供す ることを課題とする。  [0005] The present invention provides an immunotherapeutic agent that exhibits a further excellent anticancer effect by using it instead of or at the same time as BGG-GWS, and particularly usable for immunotherapy of cancer. It is an object of the present invention to provide a substance exhibiting an immunostimulatory action useful for immunotherapy of cancer, particularly a substance that induces the production of IFNyS and / or enhances NK activity.
[0006] 本発明者らは、 まったく意外なことに、 麻疹ウィルス、 特に馴化麻疹ウイ ルス株ならびに麻疹ウィルスワクチン株の中に所望の活性を有する物質の存 在を確認し、 さらにその物質が diRNA (defective interference RNA、 defect ive interfering, またはループ型 RNAとも呼ばれる） であることを突き止め 、 下記の各発明を完成した。  [0006] The present inventors have surprisingly confirmed the presence of a substance having a desired activity in measles virus, particularly acclimated measles virus strain and measles virus vaccine strain, and the substance is diRNA. Ascertaining that it is (also called defective interference RNA, defective interfering, or loop RNA), the following inventions were completed.
[0007] ( 1 ) 麻疹ウィルス由来の RNA配列を有する diRNAを有効成分とする免疫治療 用薬剤。  [0007] (1) An immunotherapeutic agent comprising, as an active ingredient, diRNA having a measles virus-derived RNA sequence.
[0008] (2) 腫瘍免疫治療に用いるための （1 ) に記載の免疫治療用薬剤。  [0008] (2) The immunotherapeutic agent according to (1) for use in tumor immunotherapy.
[0009] (3) 麻疹ウィルスが馴化ウィルス株又はワクチン株である、 （1 ) 又は （[0009] (3) The measles virus is an acclimated virus strain or a vaccine strain, (1) or (
2) に記載の免疫治療用薬剤。 The immunotherapeutic agent according to 2).
[0010] (4) 馴化ウィルス株が Edmonston株、 Nagahata株又は lchinose_V株である、 [0010] (4) The acclimated virus strain is an Edmonston strain, a Nagahata strain, or an lchinose_V strain.
(3) に記載の免疫治療用薬剤。  The immunotherapeutic drug according to (3).
[0011] (5) ワクチン株が Schwarz株又は Tanabe株である、 （3) に記載の免疫治療 用薬剤。 [0011] (5) The immunotherapeutic agent according to (3), wherein the vaccine strain is a Schwarz strain or a Tanabe strain.
[0012] (6) diRNAが配列番号 1〜5に示されるいずれかの RNA配列を有する diRNAで ある、 （1 ) 又は （2) に記載の免疫治療用薬剤。  [0012] (6) The immunotherapeutic agent according to (1) or (2), wherein the diRNA is a diRNA having any one of the RNA sequences shown in SEQ ID NOs: 1 to 5.
[0013] (7) diRNAが配列番号 9〜 1 5に示されるいずれかの RNA配列からなる diRNA である、 （6) に記載の免疫治療用薬剤。 [0013] (7) diRNA comprising any RNA sequence represented by SEQ ID NOs: 9 to 15 The immunotherapeutic agent according to (6), wherein
[0014] (8) (1 ) 〜 （7) のいずれかに記載の免疫治療用薬剤とグラム陽性菌の 細胞壁骨格とを含む腫瘍免疫治療用組成物。 [0014] (8) A composition for tumor immunotherapy comprising the immunotherapeutic agent according to any one of (1) to (7) and a cell wall skeleton of a Gram-positive bacterium.
[0015] (9) グラム陽性菌の細胞壁骨格が BGG-GWSである、 （8) に記載の腫瘍免疫 治療用組成物。 [0015] (9) The composition for tumor immunotherapy according to (8), wherein the cell wall skeleton of the Gram-positive bacterium is BGG-GWS.
[0016] ( 1 0) 麻疹ウィルス由来の RNA配列を有する diRNAを有効成分とする、 イン タ一フヱロンの発現を促進させる及び/又は NK活性を増強させるための医薬 ( 1 1 ) 麻疹ウィルスが馴化ウィルス株又はワクチン株である、 （1 0) に 記載の医薬。  [0016] (1 0) Medicament for promoting interferon expression and / or enhancing NK activity, comprising diRNA having measles virus-derived RNA sequence as an active ingredient (1 1) Measles virus acclimatized The medicament according to (10), which is a virus strain or a vaccine strain.
[0017] ( 1 2) 馴化ウィルス株が Edmonston株、 Nagahata株又は lchinose_V株である [0017] (1 2) Acclimatized virus strain is Edmonston strain, Nagahata strain or lchinose_V strain
、 ( 1 1 ) に記載の医薬。 (1) The medicine according to (1).
[0018] ( 1 3) ワクチン株が Schwarz株又は Tanabe株である、 （1 1 ) に記載の医薬 [0018] (1 3) The medicament according to (1 1), wherein the vaccine strain is a Schwarz strain or a Tanabe strain.
[0019] ( 1 4) diRNAが配列番号 1〜5に示されるいずれかの RNA配列を有する diRNA である、 （1 0) に記載の医薬。 [0019] (14) The medicament according to (10), wherein the diRNA is a diRNA having any one of the RNA sequences shown in SEQ ID NOs: 1 to 5.
[0020] ( 1 5) diRNAが配列番号 9〜 1 5に示されるいずれかの RNA配列からなる diR[0020] (15) diR whose diRNA is composed of any RNA sequence shown in SEQ ID NOs: 9 to 15
NAである、 （1 4) に記載の医薬。 The medicament according to (1 4), which is NA.
発明の効果  The invention's effect
[0021] 本発明の免疫治療用薬剤は、 細菌等の異物あるいは癌などの新生腫瘍等に 対してのヒ卜の免疫防御力を高めることが出来、 抗癌剤あるいは腫瘍免疫療 法剤として利用可能である。 また、 本発明の免疫治療用薬剤と BGG-GWSとを組 み合わせて使用することにより、 癌の免疫治療に有効な医薬組成物 （腫瘍免 疫治療用組成物） となり得る。 また本発明にかかる diRNAは、 マウス、 ヒ トに 皮下投与 （100 ^g) しても毒性は見られず、 安全な医薬となり得る。  [0021] The immunotherapeutic agent of the present invention can enhance the immune defense power of chicks against foreign substances such as bacteria or neoplastic tumors such as cancer, and can be used as an anticancer agent or a tumor immunotherapeutic agent. is there. Furthermore, by using the immunotherapeutic agent of the present invention in combination with BGG-GWS, a pharmaceutical composition (tumor immunity therapeutic composition) effective for immunotherapy of cancer can be obtained. In addition, the diRNA according to the present invention does not show toxicity even when administered subcutaneously (100 ^ g) to mice and humans and can be a safe pharmaceutical.
図面の簡単な説明  Brief Description of Drawings
[0022] [図 1]各麻疹ウィルス株の diRNAの検出結果を示す電気泳動写真である。  [0022] FIG. 1 is an electrophoretogram showing the detection results of diRNA of each measles virus strain.
[図 2]p(+)MV Dl GFPプラスミ ドの概略図を示す。  FIG. 2 shows a schematic diagram of p (+) MV Dl GFP plasmid.
[図 3]diRNA-IGV-Gの概略図を示す。 [図 4]di RNA-IGV-Gを導入した HEK細胞のルシフヱラーゼの蛍光量を示す。 FIG. 3 shows a schematic diagram of diRNA-IGV-G. FIG. 4 shows the amount of fluorescence of luciferase from HEK cells into which di RNA-IGV-G has been introduced.
[図 5]diRNA-IGV-Gの投与による移植担癌の退縮効果を示す。  FIG. 5 shows the regression effect of transplanted cancer-bearing cancer by administration of diRNA-IGV-G.
[図6] [^^- -6の投与 （_〇_) による NK細胞の細胞傷害活性の増加を示 す。 書はコントロールを示す。  [Fig. 6] It shows the increase in cytotoxic activity of NK cells by [^^--6 administration (_〇_)]. The letter indicates the control.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0023] diRNAは、 例えば麻疹ウィルス、 特に馴化麻疹ウィルス又は麻疹ウィルスヮ クチン株の中に含まれている様な、 ステム &ループ構造を有する 2重鎖 RNAで ある。 麻疹ウィルスワクチン株においては、 diRNAは夾雑物であり、 ウィルス の複製過程に生じる非感染性の RNAであると報告されている （Robertら、 Cell 、 1981年、 第 26巻、 第 145-154頁） 。 diRNAは、 麻疹ウィルスワクチン株並び に馴化麻疹ウィルスにおいてその存在が確認されているが、 麻疹ウィルスの 複製には全く不要の物質と考えられており、 ウィルスの複製、 増殖における 役割や被感染宿主に対する影響等について、 殆ど注目されることのなかった 物質である。 [0023] The diRNA is a double-stranded RNA having a stem-and-loop structure, as contained in, for example, measles virus, in particular, acclimatized measles virus or measles virus strain. In measles virus vaccine strains, diRNA is a contaminant and has been reported to be a non-infectious RNA produced during viral replication (Robert et al., Cell, 1981, 26, 145-154). ) DiRNA has been confirmed to exist in measles virus vaccine strains and acclimated measles virus, but is considered to be a completely unnecessary substance for replication of measles virus. It is a substance that has received little attention for its effects.
[0024] また、 麻疹ウィルス、 特に麻疹ワクチンウィルスは、 投与された個体に対 して麻疹ウィルスの感染に対する免疫能を与えるが、 逆に個体の免疫力自体 は低下することが報告されている (Hahm Bら、 Virology, 2004年、 第 323巻、 第 2号、 第 292-302頁） 。 本発明は、 全く意外にも、 かかる麻疹ウィルスワク チンの中に癌に対する免疫力を高める物質が存在し、 さらに該物質が diRNAで あることが確認されたことに基づく発明である。  [0024] In addition, measles virus, particularly measles vaccine virus, has given immunity against measles virus infection to administered individuals, but conversely, immunity of individuals has been reported to decrease ( Hahm B et al., Virology, 2004, 323, 2, 292-302). The present invention is an invention based on the fact that a substance that enhances immunity against cancer exists in such measles virus vaccine, and that the substance is confirmed to be diRNA.
[0025] diRNAは、 トレイラ一配列及びリーダ一配列、 又はトレイラ一配列及び該ト レイラー配列に相補的な配列のどちらかの組み合わせを有し、 これによつて 形成されるステム &ループ構造を有する RNAである。 トレイラ一配列はウィル スゲノムの 5' 末端部分に存在し、 リーダー配列は 3'末端部分に存在し、 それ ぞれ麻疹ウィルスゲノムの転写、 複製等に必須の特異的配列である。 トレィ ラー配列とリーダ一配列はほぼ相補的な配列となっており、 これらがァ二一 リングすることによって、 diRNAのステム &ループ構造が形成される。 トレィ ラー配列と該トレイラ一配列の相補的配列を有する diRNAも、 これらがァ二一 リングすることによってステム &ル一プ構造が形成される。 [0025] The diRNA has a combination of either a trailer sequence and a leader sequence, or a trailer sequence and a sequence complementary to the trailer sequence, and has a stem-and-loop structure formed thereby. RNA. A trailer sequence is present at the 5 'end of the virus genome and a leader sequence is present at the 3' end, each of which is a specific sequence essential for transcription, replication, etc. of the measles virus genome. The trailer sequence and the leader sequence are almost complementary sequences, and when they are aligned, a diRNA stem-and-loop structure is formed. DiRNAs having a trailer sequence and a complementary sequence of the trailer sequence are also By ringing, a stem and loop structure is formed.
[0026] 馴化麻疹ウィルス株である Edmonston (ED)株、 l ch i nose-V ( I C-V) 株、 及び ワクチン株である Schwarz株、 Tanabe株のウィルスゲノムは、 配列番号 1に示 される RNA配列からなるトレイラ一配列を有している。 また馴化麻疹ウィルス 株である Nagahata (NV) 株は、 配列番号 1に示される RNA配列の 1 5塩基目の みがグァニンからァデニンへ変異した配列番号 2に示される RNA配列からなる トレイラ一配列を有する。 また、 馴化麻疹ウィルス株である I G-V株は配列番 号 3に示される RNA配列からなるリーダ一配列を、 Edmonston (ED)株は配列番 号 4に示される RNA配列からなるリ一ダ一配列を、 ワクチン株である Schwa r z 株は配列番号 5に示される RNA配列からなるリーダ一配列をそれぞれ有してい る。 なお、 配列番号 3〜5に示される各リーダ一配列は、 2 6番目、 4 2番 目、 5 0番目の塩基が異なる以外は、 同一の RNA配列となっている。  [0026] The viral genomes of the habituated measles virus strains Edmonston (ED) strain, lchi nose-V (ICV) strain, and vaccine strains Schwarz strain and Tanabe strain are the RNA shown in SEQ ID NO: 1. It has a trailer array consisting of an array. The Nagahata (NV) strain, a habituated measles virus strain, has a trailer sequence consisting of the RNA sequence shown in SEQ ID NO: 2 in which only the 15th base of the RNA sequence shown in SEQ ID NO: 1 is mutated from guanine to adenine. Have. In addition, the IGV strain, which is an acclimated measles virus strain, has a leader sequence consisting of the RNA sequence shown in SEQ ID NO: 3, and the Edmonston (ED) strain is a leader sequence consisting of the RNA sequence shown in SEQ ID NO: 4. The Schwarz strain, which is a vaccine strain, has a leader sequence consisting of the RNA sequence shown in SEQ ID NO: 5, respectively. Each leader sequence shown in SEQ ID NOs: 3 to 5 has the same RNA sequence except that the 26th, 42nd, 50th and 50th bases are different.
[0027] 麻疹ウィルス由来の d i RNAは、 RNA配列や塩基数のバリエーションを有する —方で、 トレイラ一配列及びリーダ一配列、 又はトレイラ一配列及び該トレ イラ一と相補的な配列を含み、 ステム &ループ構造を形成するという構造的 な特徴も有している。 従って本発明は、 上記配列番号 1〜5に示される具体 的な RNA配列のいずれかを含む d i RNAに限定されず、 麻疹ウィルス由来の RNAを 含み、 ステム &ループ構造が保持され、 かつ生体の癌に対する免疫力を高め る機能を維持する限りにおいて、 上記 RNA配列において複数の、 好ましくは数 個から数十の塩基が置換、 欠失、 付加等された RNA配列からなる d i RNAも含む  [0027] Di RNA derived from measles virus has a variation in RNA sequence and number of bases, and includes a trailer sequence and a leader sequence, or a trailer sequence and a sequence complementary to the trailer. It also has the structural feature of forming a & loop structure. Therefore, the present invention is not limited to diRNA containing any of the specific RNA sequences shown in SEQ ID NOs: 1 to 5 above, but includes measles virus-derived RNA, which retains the stem-and-loop structure, and is As long as the function of enhancing immunity against cancer is maintained, diRNAs comprising RNA sequences in which a plurality of, preferably several to several tens of bases are substituted, deleted, added, etc. in the above RNA sequence are also included.
[0028] 後述する実施例において示すように、 d i RNAを含む本発明の免疫治療用薬剤 をインビトロでヒト細胞に加えると細胞からのインタ一フエロンの産生が誘 導され、 また移植担癌マウスに d i RNAを含む本発明の免疫治療用薬剤を腹腔投 与すると、 NK細胞の細胞傷害活性による明らかな癌の退縮が観察された。 そ の一方、 投与された d i RNAによる感染症その他の有害事象は全く観察されなか つた。 [0028] As shown in the examples described later, when the immunotherapeutic agent of the present invention containing diRNA is added to human cells in vitro, the production of interferon from the cells is induced, and transplanted tumor-bearing mice are also induced. When the immunotherapeutic agent of the present invention containing diRNA was intraperitoneally administered, a clear cancer regression due to the cytotoxic activity of NK cells was observed. On the other hand, no infection or other adverse events due to the administered di RNA were observed.
[0029] 本発明にかかる d i RNAは Robertらに報告されるように従来公知であり、 医療 関係者に広く供給されている麻疹ウィルス、 特に馴化麻疹ウィルス又は麻疹 ウィルスワクチン株から Maniatisらの方法 （Molecular Cloning, 1989年、 Go Id Spring Harbor Laboratory Press) に準じて分離、 精製することができる 。 馴化麻疹ウィルス株である ED株、 NV株、 IG-V株は、 それぞれ国立感染症研 究所より入手することができる。 また麻疹ウィルスヮクチン株である Schwar z 株、 Tanabe株は、 それぞれ武田薬品工業株式会社、 田辺製薬株式会社より入 手可能である。 [0029] The di RNA according to the present invention is conventionally known as reported to Robert et al. According to the method of Maniatis et al. (Molecular Cloning, 1989, Go Id Spring Harbor Laboratory Press), it can be isolated and purified from measles virus, particularly acclimated measles virus or measles virus vaccine strains widely supplied to the parties concerned. Acclimatized measles virus strains ED, NV, and IG-V can be obtained from the National Institute of Infectious Diseases. Schwarz and Tanabe strains are available from Takeda Pharmaceutical Co., Ltd. and Tanabe Seiyaku Co., Ltd., respectively.
[0030] また、 上記の株より得られる diRNAあるいはそれらを基に RNA配列の一部を 人為的に組み換えてなる diRNAは、 一般的な遺伝子組み換え手法を用いて製造 することもできる。 遺伝子組み換え手法による製造は、 例えば配列番号 1〜 5に示されるいずれかの RNA配列に相補的な塩基配列を含む DNAを組み込んだ 適当な発現ベクターを宿主細胞内で発現させて diRNAを生産させ、 回収すれば よい。 あるいはインビトロで配列番号 1〜 5に示されるいずれかの RNA配列に 相補的な塩基配列を含む DNAを錶型にして RNAを生産し、 回収すればよい。 本 発明で利用可能な発現ベクターやプロモーター等の遺伝子発現系の種類、 さ らには組み換え宿主細胞の種類、 インビトロ転写系、 さらには RNAの回収方法 などには格別の制限はなく、 当業者が利用可能なあらゆる手法を用いること ができる。  [0030] In addition, diRNA obtained from the above strains or diRNA obtained by artificially recombining part of the RNA sequence based on them can be produced using a general genetic recombination technique. In the production by genetic recombination, for example, an appropriate expression vector incorporating a DNA containing a base sequence complementary to any RNA sequence shown in SEQ ID NOs: 1 to 5 is expressed in a host cell to produce diRNA, Collect it. Alternatively, RNA may be produced in vitro by collecting a DNA containing a base sequence complementary to any of the RNA sequences shown in SEQ ID NOs: 1 to 5 in a cage shape and recovering it. There are no particular limitations on the types of gene expression systems such as expression vectors and promoters that can be used in the present invention, the types of recombinant host cells, in vitro transcription systems, and RNA recovery methods. Any available method can be used.
[0031] 本発明の腫瘍免疫治療用組成物は、 上記の diRNAを有効成分とする免疫治療 用薬剤とグラム陽性菌の細胞壁骨格とを含む。 グラム陽性菌の細胞壁骨格は 、 グラム陽性菌を物理的に粉砕した後、 除核酸、 除タンパク、 脱脂などの精 製工程を経て得られる不溶性残渣であり、 特許文献 1に詳しく記載されてい る。  [0031] The composition for tumor immunotherapy of the present invention comprises an immunotherapeutic drug containing the above-described diRNA as an active ingredient and a cell wall skeleton of Gram-positive bacteria. The cell wall skeleton of a Gram-positive bacterium is an insoluble residue obtained through a refinement process such as denucleation, protein removal, and degreasing after physically pulverizing the Gram-positive bacterium, and is described in detail in Patent Document 1.
[0032] 本発明におけるグラム陽性菌の細胞壁骨格としては、 マイコバクテリア属 細菌、 ノカルディア属細菌、 コリネバクテリア属細菌等の細胞壁骨格が挙げ られるが、 特にマイコバクテリア属ゥシ型結核菌の細胞壁骨格 （BGG— GWS) が好ましい。 このグラム陽性菌の細胞壁骨格と麻疹ウイルス由来の d i RNAを含 む本発明の腫瘍免疫治療用組成物は、 癌の種類、 特に MHGクラス I蛋白質の発 現の高低によらずに、 癌を退縮させる効果を有する。 [0032] Examples of cell wall skeletons of Gram-positive bacteria in the present invention include cell wall skeletons such as Mycobacterium bacteria, Nocardia bacteria, Corynebacterium bacteria, and the like. (BGG—GWS) is preferred. The composition for tumor immunotherapy of the present invention comprising the cell wall skeleton of Gram-positive bacteria and diRNA derived from measles virus is a type of cancer, in particular, an MHG class I protein. Regardless of current level, it has the effect of reducing cancer.
[0033] 本発明の腫瘍免疫治療用組成物は、 グラム陽性菌の細胞壁骨格と diRNAの他 に、 生体への投与経路、 剤型などに応じた種々の賦形剤、 担体、 他の医薬成 分そのほかの成分を配合して調整することができる。 本発明の腫瘍免疫治療 用組成物の投与経路には格別の制限は無いが、 経口投与ならびに皮下投与、 血管投与、 経粘膜投与が好ましい。  [0033] In addition to the cell wall skeleton of gram-positive bacteria and diRNA, the composition for tumor immunotherapy of the present invention comprises various excipients, carriers, and other pharmaceutical compositions depending on the administration route to the living body, dosage form, etc. It can be adjusted by blending other ingredients. The administration route of the composition for tumor immunotherapy of the present invention is not particularly limited, but oral administration, subcutaneous administration, vascular administration, and transmucosal administration are preferable.
[0034] また本発明にかかる diRNAは、 上記の通りヒト細胞に対して I FN-；3発現を誘 導し、 また NK細胞の NK活性を増強させる機能を有していることから、 IFN-;S の発現を促進させる及び/又は NK活性を増強させるための医薬として利用す ることも可能である。 前述の通り、 麻疹ウィルスワクチンは生体の免疫力を 弱めることが知られているが、 麻疹ウィルスワクチンに含まれる diRNAが、 生 体に対して I FN- ;3の発現を促進させる等によって生体の免疫防御能を高める 機能を有していることは意外な知見であった。  [0034] The diRNA according to the present invention has a function of inducing IFN-; 3 expression to human cells and enhancing the NK activity of NK cells as described above. It can also be used as a medicament for promoting S expression and / or enhancing NK activity. As described above, measles virus vaccines are known to weaken the immunity of living organisms, but diRNA contained in measles virus vaccines promotes the expression of IFN-3; It was an unexpected finding that it had a function to enhance immune defense ability.
[0035] 本発明にかかる diRNAを含むインターフェロンの発現を促進させる及び/又 は NK活性を増強させるための医薬は、 有効成分である diRNAの他に、 生体への 投与経路、 剤型に応じた種々の賦形剤、 担体、 他の医薬成分その他の成分を 配合して調整することができる。 投与経路にも格別の制限はないが、 経口投 与ならびに皮下投与、 血管投与が好ましい。  [0035] The pharmaceutical for promoting the expression of interferon containing diRNA and / or enhancing NK activity according to the present invention depends on the administration route to the living body and the dosage form in addition to diRNA as an active ingredient. Various excipients, carriers, other pharmaceutical ingredients and other ingredients can be blended and adjusted. There are no particular restrictions on the route of administration, but oral administration, subcutaneous administration, and vascular administration are preferred.
[0036] 以下、 実施例を通じてさらに本発明を詳細に説明するが、 本発明はかかる 実施例に限定されるものではない。  Hereinafter, the present invention will be described in more detail through examples, but the present invention is not limited to such examples.
実施例  Example
[0037] <実施例 1 > diRNAの確認、 生産  [0037] <Example 1> Confirmation and production of diRNA
( 1 ) 表 1に示される任意の麻疹ウィルス株から QIAamp Viral RNA Mini kit (QIAGEN社）を用いて の全 RNAを抽出し、 70°Cにて 5分熱処理した後、 氷上 にて 2分冷却した。 ほ 1 ] (1) Extract total RNA from any measles virus strain shown in Table 1 using the QIAamp Viral RNA Mini kit (QIAGEN), heat-treat at 70 ° C for 5 minutes, and cool on ice for 2 minutes did. 1
Figure imgf000010_0001
Figure imgf000010_0001
[0038] 冷却後、 MMRV- reverse transcr i ptase (Promega社）を用いて 90分間 37°Cに て逆転写反応を行った。 逆転写後、 作製された cDNAを錶型として PGRを行い、 PGR産物の有無を電気泳動にて確認した。 以下に PGRに使用したプライマ一配 列 (PU P2) を示す。 [0038] After cooling, a reverse transcription reaction was performed at 37 ° C for 90 minutes using MMRV-reverse transcriptase (Promega). After reverse transcription, PGR was performed using the prepared cDNA as a cage, and the presence or absence of the PGR product was confirmed by electrophoresis. The primer sequence (PU P2) used for PGR is shown below.
[0039] P1 ： 5' -TATAAGCTTACCAGACAAAGCTGGGAATAGAAACTTCG-3' (配列番号 6 ) [0039] P1: 5′-TATAAGCTTACCAGACAAAGCTGGGAATAGAAACTTCG-3 ′ (SEQ ID NO: 6)
P2： 5' -CGAAGATATTCTGGTGTAAGTCTAGTA-3 ' (配列番号 7 ) PGR反応が進行していることを示すコント口一ル反応用のプライマ一は上記 P1と P3を用いた。 [0040] P3： 5' -TTTATCCAGAATCTCAARTCCGG-3' (配列番号 8) P2: 5′-CGAAGATATTCTGGTGTAAGTCTAGTA-3 ′ (SEQ ID NO: 7) Primers for the control reaction indicating that the PGR reaction is in progress used P1 and P3. [0040] P3: 5'-TTTATCCAGAATCTCAARTCCGG-3 '(SEQ ID NO: 8)
その結果、 図 1に示すように ED株、 NV株、 IG-V株、 Schwarz株で各 1種類、 Tanabe株で 3種類の PGR産物が確認された。 各 PGR産物をそれぞれ pGR blant v ector (Invitrogen社）に揷入し、 BigDye (登録商標) Terminator v3.1 Cycl e Sequence Kit (Applied Biosystems社）を用いて RNA配列を決定した (diRNA -ED：配列番号 9、 d i RNA-NV：配列番号 1 0、 diRNA-ICV：配列番号 1 1、 diR NA-SH：配列番号 1 2、 d i RNA-TA1 ：配列番号 1 3、 d i RNA-TA2：配列番号 1 4 、 d i RNA-TA3：配列番号 1 5) 。  As a result, as shown in Fig. 1, one type each of ED strain, NV strain, IG-V strain and Schwarz strain, and three types of PGR products were confirmed in Tanabe strain. Each PGR product was inserted into pGR blant v ector (Invitrogen) and the RNA sequence was determined using BigDye (registered trademark) Terminator v3.1 Cycle Sequence Kit (Applied Biosystems) (diRNA-ED: Sequence) No. 9, diRNA-NV: SEQ ID NO: 10, diRNA-ICV: SEQ ID NO: 1, diRNA-SH: SEQ ID NO: 1, diRNA-TA1: SEQ ID NO: 13, diRNA-TA2: SEQ ID NO: 1 4, diRNA-TA3: SEQ ID NO: 15).
[0041] diRNA_ED、 diRNA-ICV, diRNA- SH、 diRNA-TAU diRNA_TA2、 diRNA_TA3は配 列番号 1に示されるトレイラー配列及び該トレイラー配列に相補的な配列を 含み、 di RNA-NVは配列番号 2に示されるトレイラ一配列及び該トレイラ一配 列に相補的な配列を含んでいた。  [0041] diRNA_ED, diRNA-ICV, diRNA-SH, diRNA-TAU diRNA_TA2, diRNA_TA3 include the trailer sequence shown in SEQ ID NO: 1 and a sequence complementary to the trailer sequence, and diRNA-NV in SEQ ID NO: 2. The trailer sequence shown and the sequence complementary to the trailer sequence were included.
[0042] (2) IG-V株のゲノム配列を有するプラスミ ド （Takedaら、 Viroし 2000 年、 第 74巻、 第 6643-6647頁） に GFP蛍光蛋白質をコードする DNAを組み込んだ p(+)MV323GFPプラスミ ド （Singaiら、 J. I國 unol.、 2005年、 第 175巻 5号、 第 3252-3261頁） を錶型とし、 同プラスミ ドからリーダ一配列 （配列番号 3) 及 びトレイラー配列 （配列番号 1 ) を除く IG-V株のゲノム配列を欠失させたプ ラスミ ド p(+)MV DI GFPを、 下記の塩基配列からなるプライマ一 （P4、 P5) を 用いて、 PGR法によって作製した（図 2)。  [0042] (2) A plasmid (Takeda et al., Viro, 2000, 74, 6643-6647) having a genomic sequence of the IG-V strain incorporated with a DNA encoding a GFP fluorescent protein p (+ ) MV323GFP plasmid (Singai et al., J. I country unol., 2005, Vol. 175, No. 5, pp. 3252-3261) is a saddle type, and a leader sequence (SEQ ID NO: 3) and trailer from the plasmid The plasmid p (+) MV DI GFP from which the genomic sequence of the IG-V strain excluding the sequence (SEQ ID NO: 1) was deleted was converted to PGR using primers (P4, P5) consisting of the following base sequences: (Fig. 2).
[0043] P4： 5' -GCCATCGATTATTACTTGTACAGCTCGTCC-3' (配列番号 1 6 )  [0043] P4: 5′-GCCATCGATTATTACTTGTACAGCTCGTCC-3 ′ (SEQ ID NO: 16)
P5： 5' -GGATCGATTAAGGATTAATTGGTTGAACTCC-3' (配列番号 1 7 ) 次に、 p (+) MV DI GFPを錶型とし、 リーダ一配列一 GFP—トレイラ一配列を 錶型とは逆向きに有するプラスミ ド p (-) MV DI GFPを、 下記の 2組のプライマ - (P6及び P7、 ならびに P8及び P9) を用いて、 PGR法によって作製した。  P5: 5'-GGATCGATTAAGGATTAATTGGTTGAACTCC-3 '(SEQ ID NO: 17) Next, p (+) MV DI GFP is a cocoon type, and one leader sequence is one GFP—one trailer sequence in the opposite direction to the 錶 type. p (-) MV DI GFP was prepared by the PGR method using the following two sets of primers-(P6 and P7, and P8 and P9).
[0044] P6： 5' -GGCCGGCATGGTCCCAGCCTCCTCGCT-3 ' (配列番号 1 8 )  [0044] P6: 5′-GGCCGGCATGGTCCCAGCCTCCTCGCT-3 ′ (SEQ ID NO: 1 8)
P7： 5' -TATAGTGAGTCGTATTACGCGCGCTT-3 ' (配列番号 1 9 ) P8： 5' -ACCAAACAAAGTTGGGTAAGGATAGATCAATCAATGATCAT-3' (配列番号 2 P7: 5'-TATAGTGAGTCGTATTACGCGCGCTT-3 '(SEQ ID NO: 1 9) P8: 5'-ACCAAACAAAGTTGGGTAAGGATAGATCAATCAATGATCAT-3' (SEQ ID NO: 2)
0) P9： 5' -ACCAGACAAAGCTGGGAATAGAAACTTCGTATTTTCAAAGT-3' (配列番号 20) P9: 5'-ACCAGACAAAGCTGGGAATAGAAACTTCGTATTTTCAAAGT-3 '(SEQ ID NO: 2
1 ) 1)
この p (-) MV Dl GFPを錶型として、 MEGAscript T7 kit (Ambion社）を用い、 GFP蛍光タンパク質をコ一ドする領域がループァゥ卜されたステム &ループ構 造を有する diRNA-IGV-G (図 3) をインビトロで調製した。  Using this p (-) MV Dl GFP as a model, using the MEGAscript T7 kit (Ambion), diRNA-IGV-G (which has a stem-and-loop structure in which the region encoding GFP fluorescent protein is looped over Figure 3) was prepared in vitro.
[0045] <実施例 2 >d i RNAによる I FN-；3の産生誘導  <Example 2> Induction of production of I FN-; 3 by di RNA
( 1 ) IFN産生を誘導する転写因子である IFN-stimulated response element (ISRE)をコ一ドする塩基配列の下流にルシフェラ一ゼ蛍光タンパク質をコ一 ドする塩基配列を組み込んだ plSRE_Lucプラスミ ド （Stratagene社） を、 Lipo feet in (QIAGEN社） を用いて HEK293細胞 (ATCC American type culture col l ection社） に導入し、 形質転換細胞を得た。  (1) A plSRE_Luc plasmid (Stratagene) that incorporates a nucleotide sequence that codes for luciferase fluorescent protein downstream of the nucleotide sequence that codes for IFN-stimulated response element (ISRE), a transcription factor that induces IFN production. Was introduced into HEK293 cells (ATCC American type culture collection) using Lipo feet in (QIAGEN) to obtain transformed cells.
[0046] (2) Dulbecco' s modified Eagle' s medium (DMEM) (Invitrogen, CA) /\ 0% FGS培地で 24時間培養した （1 ) の形質転換 HEK293細胞に、 LipofectAMINE 2000 (Invitrogen社） を用いて実施例 2で調製した diRNA-IGV-Gを導入し、 導入から 6時間後にルシフェラ一ゼの蛍光量を測定した。 また、 コントロール として、 p(+)MV323プラスミ ドのみを導入した （1 ) の形質転換 HEK293細胞と 、 熱処理によって変性した diRNA-IGV-G (denatured Dl RNA)を導入した （1 ) の形質転換 HEK293細胞とを用意し、 それぞれについてルシフヱラーゼの蛍 光量を測定した。 その結果、 未変性の diRNA-IGV-Gを導入した形質転換 HEK293 細胞において、 コントロールと比較してルシフヱラ一ゼの蛍光量が増加して いることが確認された （図 4) 。  [0046] (2) Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, CA) / \ 0% FGS medium cultured for 24 hours (1) Transform HEK293 cells with LipofectAMINE 2000 (Invitrogen) The diRNA-IGV-G prepared in Example 2 was introduced, and the fluorescence level of luciferase was measured 6 hours after the introduction. In addition, as a control, transformed HEK293 cells (1) into which only p (+) MV323 plasmid was introduced and diRNA-IGV-G (denatured Dl RNA) denatured by heat treatment were introduced (1). Cells were prepared, and the fluorescence of luciferase was measured for each cell. As a result, it was confirmed that in the transformed HEK293 cells into which native diRNA-IGV-G was introduced, the amount of luciferase fluorescence increased compared to the control (Fig. 4).
[0047] <実施例 3>diRNAによる移植担癌の退縮  [0047] <Example 3> Regression of transplanted tumor with diRNA
G57BL/6近交系マウスの側腹部に B16メラノーマ細胞 （大阪府立成人病セン ター研究所より分与） を 1 x106cel ls移植し、 腫瘍の大きさの経時的変化を 計測した （図 5) 。 なお、 B16メラノ一マ細胞は MHGクラス I分子を発現してい ないため、 キラ一 T細胞の活性化が誘導されず、 インタ一フヱロンによって増 強された NK細胞の活性によつてのみ B16メラノ一マ腫瘍が退縮する。 特許文献 1に記載されている方法によって調製した BGG-GWS 25 g/0.1 mし MALP-2 ( Oncologics社， Nagoya Japan) d i RNA- 1 CV-G 10 g/mlそれぞれ を含む生理食塩水をアジュバントとして用意し、 B16メラノ一マ細胞の癌抗原 ェピ！ プである TRP2_pep8 (Rosenberg S.より分与） 0.1 mgと共に、 B16メ ラノーマ細胞の移植前に隔週で 3回、 ならびに移植後に隔週で 3回 （図 5の 矢印の時点） 腹腔投与を行った。 この結果、 diRNA-IGV-Gを投与した場合にお いて、 顕著な癌の退縮効果 （増殖抑制効果） が認められた。 G57BL / 6 inbred mice were transplanted with B16 melanoma cells (distributed from the Osaka Prefectural Center for Adult Diseases) 1 x 10 6 cel ls, and changes in tumor size over time were measured (Fig. 5). ) Since B16 melanoma cells do not express MHG class I molecules, activation of killer T cells is not induced, and only by the activity of NK cells enhanced by interferon. The mammary tumor regresses. BGG-GWS 25 g / 0.1 m prepared by the method described in Patent Document 1 MALP-2 ( Oncologics Inc., Nagoya Japan) B16 melanoma cell cancer antigen EP with physiological saline containing di RNA-1 CV-G 10 g / ml as an adjuvant. TRP2_pep8 (distributed by Rosenberg S.) 0.1 mg was administered intraperitoneally three times every other week before transplantation of B16 melanoma cells and three times every other week after transplantation (indicated by the arrow in FIG. 5). As a result, when diRNA-IGV-G was administered, a marked cancer regression effect (proliferation inhibitory effect) was observed.
<実施例 4 >d i RNAによる NK細胞活性増強作用 <Example 4> Enhancement of NK cell activity by di RNA
実施例 3でアジュバントを投与した 3種類のマウスから、 Lympho I yte-M (Cederlane Laboratory社）を用いてリンパ球を抽出した後、 MACS beads (Mi It enyi Biotec 社）を用いた NK 調製用ネガティブセレクションにより、 NK細胞 を得た。 NK細胞と B16メラノ一マ細胞を、 RPM卜 1640培地を用いて共培養し、 5 iCr-re lease assay法による NK細胞の細胞傷害活性を測定した。 図 6に示すよ うに、 diRNA-IGV-Gを投与したマウス由来の NK細胞は、 食塩水を投与したマウ ス由来の NK細胞 （コントロール） と比較して顕著な細胞傷害活性の上昇が認 められた。 Lymphocytes were extracted using Lympho I yte-M (Cederlane Laboratory) from the three types of mice administered with the adjuvant in Example 3, and then negative for NK preparation using MACS beads (Mi Itenyi Biotec) NK cells were obtained by selection. NK cells and B16 melanoma cells were co-cultured using RPM-1640 medium, and the cytotoxic activity of NK cells was measured by 5 iCr-re lease assay. As shown in Fig. 6, NK cells derived from mice treated with diRNA-IGV-G showed a marked increase in cytotoxic activity compared to NK cells derived from mice administered with saline (control). It was.

Claims

請求の範囲 The scope of the claims
[1] 麻疹ウィルス由来の RNA配列を有する diRNAを有効成分とする免疫治療用薬剤  [1] Immunotherapeutic drug containing diRNA having measles virus-derived RNA sequence as an active ingredient
[2] 腫瘍免疫治療に用いるための請求項 1に記載の免疫治療用薬剤。 [2] The immunotherapeutic agent according to claim 1, for use in tumor immunotherapy.
[3] 麻疹ウィルスが馴化ウィルス株又はワクチン株である、 請求項 1又は 2に記 載の免疫治療用薬剤。 [3] The immunotherapeutic agent according to claim 1 or 2, wherein the measles virus is an acclimated virus strain or a vaccine strain.
[4] 馴化ウィルス株が Edmonston株、 Nagahata株又は lchinose_V株である、 請求項 [4] The acclimated virus strain is Edmonston strain, Nagahata strain or lchinose_V strain.
3に記載の免疫治療用薬剤。 3. The immunotherapeutic agent according to 3.
[5] ワクチン株が Schwarz株又は Tanabe株である、 請求項 3に記載の免疫治療用薬 剤。 [5] The immunotherapeutic agent according to claim 3, wherein the vaccine strain is a Schwarz strain or a Tanabe strain.
[6] diRNAが配列番号 1〜 5に示されるいずれかの RNA配列を有する diRNAである、 請求項 1又は 2に記載の免疫治療用薬剤。  [6] The immunotherapeutic agent according to claim 1 or 2, wherein the diRNA is a diRNA having any one of the RNA sequences shown in SEQ ID NOs: 1 to 5.
[7] diRNAが配列番号 9〜 1 5に示されるいずれかの RNA配列からなる diRNAである[7] The diRNA is a diRNA consisting of any of the RNA sequences shown in SEQ ID NOs: 9 to 15
、 請求項 6に記載の免疫治療用薬剤。 The immunotherapeutic agent according to claim 6.
[8] 請求項 1〜 7のいずれかに記載の免疫治療用薬剤とグラム陽性菌の細胞壁骨 格とを含む腫瘍免疫治療用組成物。 [8] A composition for tumor immunotherapy comprising the immunotherapeutic agent according to any one of claims 1 to 7 and a cell wall skeleton of Gram-positive bacteria.
[9] グラム陽性菌の細胞壁骨格が BGG-GWSである、 請求項 8に記載の腫瘍免疫治療 用組成物。 [9] The composition for tumor immunotherapy according to claim 8, wherein the cell wall skeleton of the Gram-positive bacterium is BGG-GWS.
[10] 麻疹ウィルス由来の RNA配列を有する diRNAを有効成分とする、 インタ一フエ ロンの発現を促進させる及び/又は NK活性を増強させるための医薬。  [10] A medicament for promoting interferon expression and / or enhancing NK activity, comprising a diRNA having a measles virus-derived RNA sequence as an active ingredient.
[11] 麻疹ウィルスが馴化ウィルス株又はワクチン株である、 請求項 1 0に記載の 医薬。 [11] The medicament according to claim 10, wherein the measles virus is an acclimated virus strain or a vaccine strain.
[12] 馴化ウィルス株が Edmonston株、 Nagahata株又は lchinose_V株である、 請求項  [12] The acclimated virus strain is Edmonston strain, Nagahata strain or lchinose_V strain.
1 1に記載の医薬。  1 The medicament according to 1.
[13] ワクチン株が Schwarz株又は Tanabe株である、 請求項 1 1に記載の医薬。  [13] The medicament according to claim 11, wherein the vaccine strain is a Schwarz strain or a Tanabe strain.
[14] diRNAが配列番号 1〜 5に示されるいずれかの RNA配列を有する diRNAである、 請求項 1 0に記載の医薬。 [14] The medicament according to claim 10, wherein the diRNA is a diRNA having any one of the RNA sequences shown in SEQ ID NOs: 1 to 5.
[15] diRNAが配列番号 9〜 1 5に示されるいずれかの RNA配列からなる diRNAである 、 請求項 1 4に記載の医薬。 [15] The diRNA is a diRNA consisting of any one of the RNA sequences shown in SEQ ID NOs: 9 to 15 The medicine according to claim 14.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012014945A1 (en) 2010-07-28 2012-02-02 国立大学法人北海道大学 Novel nucleic acid having adjuvant activity and use thereof
WO2016088784A1 (en) * 2014-12-02 2016-06-09 国立大学法人北海道大学 Adjuvant composition
WO2017221076A1 (en) * 2016-06-24 2017-12-28 Institut Pasteur Compositions and methods comprising measles virus defective interfering particles for the prevention of infectious diseases
WO2018021400A1 (en) 2016-07-27 2018-02-01 国立大学法人北海道大学 Immune adjuvant for cancer

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001048154A1 (en) * 1999-12-28 2001-07-05 Toyoshima, Kumao Maturation-promoting agent for immature dendritic cells
JP2002507408A (en) * 1998-03-26 2002-03-12 アメリカン・サイアナミド・カンパニー Mutations responsible for attenuation of measles virus or human respiratory syncytial virus subgroup B
WO2005072088A2 (en) * 2003-12-11 2005-08-11 Sciperio, Inc. Immunotherapy compositions, method of making and method of use thereof
WO2006001122A1 (en) * 2004-06-24 2006-01-05 Dnavec Research Inc. Anticancer agent containing dendritic cell having rna virus transferred thereinto
JP2006523688A (en) * 2003-04-14 2006-10-19 フアン マレン Nucleotide vaccine composition, nucleotide and cell vaccine composition production method, vaccine composition, vaccine composition use, immune response production method, disease treatment or prevention method, kit comprising antigen presenting cells
JP2006525280A (en) * 2003-05-01 2006-11-09 アレス トレーディング ソシエテ アノニム Stable interferon liquid formulation without HSA
WO2006138435A2 (en) * 2005-06-16 2006-12-28 Mount Sinai School Of Medicine Methods for enhancing immune responses

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002507408A (en) * 1998-03-26 2002-03-12 アメリカン・サイアナミド・カンパニー Mutations responsible for attenuation of measles virus or human respiratory syncytial virus subgroup B
WO2001048154A1 (en) * 1999-12-28 2001-07-05 Toyoshima, Kumao Maturation-promoting agent for immature dendritic cells
JP2006523688A (en) * 2003-04-14 2006-10-19 フアン マレン Nucleotide vaccine composition, nucleotide and cell vaccine composition production method, vaccine composition, vaccine composition use, immune response production method, disease treatment or prevention method, kit comprising antigen presenting cells
JP2006525280A (en) * 2003-05-01 2006-11-09 アレス トレーディング ソシエテ アノニム Stable interferon liquid formulation without HSA
WO2005072088A2 (en) * 2003-12-11 2005-08-11 Sciperio, Inc. Immunotherapy compositions, method of making and method of use thereof
WO2006001122A1 (en) * 2004-06-24 2006-01-05 Dnavec Research Inc. Anticancer agent containing dendritic cell having rna virus transferred thereinto
WO2006138435A2 (en) * 2005-06-16 2006-12-28 Mount Sinai School Of Medicine Methods for enhancing immune responses

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
HOSHINO M. ET AL.: "Kodo Jakudokusho Hashika Vaccine no Kaihatsu ni Kansuru Kenkyu I. Hashika Vaccine Schwartz FF-8 Kabu no Kaihatsu to Sono Seibutsugakuteki Seijo", CLINICAL VIROLOGY, vol. 10, no. 3, 1982, pages 55 - 64 *
LAZZARINI R.A. ET AL.: "The Origins of Defective Interfering Particles of the Negative-Strand RNA Viruses", CELL, vol. 26, 1981, pages 145 - 154, XP027462809, DOI: doi:10.1016/0092-8674(81)90298-1 *
SHINGAI M. ET AL.: "Differential Type 1 IFN-Inducing Abilities of Wild-Type Versus Vaccine Strains of Measles Virus", J. IMMUNOL., vol. 179, 2007, pages 6123 - 6133 *
STRAHLE L. ET AL.: "Sendai virus defective-interfering genomes and the activation of interferon-beta", VIROLOGY, vol. 351, 2006, pages 101 - 111, XP024896474, DOI: doi:10.1016/j.virol.2006.03.022 *
TAKEDA M. ET AL.: "Hashika Virus Kenkyu no Aratana Tenkai", IGAKU NO AYUMI, vol. 205, no. 4, 2003, pages 285 - 290 *
YOUNT J.S. ET AL.: "A Novel Role for Viral-Defective Interfering Particles is Enhancing Dendritic Cell Maturation", J. IMMUNOL., vol. 177, 2006, pages 4503 - 4513 *

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* Cited by examiner, † Cited by third party
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EP3269811A1 (en) 2010-07-28 2018-01-17 National University Corporation Hokkaido University Nucleic acid having adjuvant activity and use thereof
CN103003427A (en) * 2010-07-28 2013-03-27 国立大学法人北海道大学 Novel nucleic acid having adjuvant activity and use thereof
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US11065331B2 (en) 2016-07-27 2021-07-20 Advanced Innovation Development Co. Ltd. Immune adjuvant for cancer
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