WO2021193081A1 - Type 1 herpes simplex virus - Google Patents

Type 1 herpes simplex virus Download PDF

Info

Publication number
WO2021193081A1
WO2021193081A1 PCT/JP2021/009720 JP2021009720W WO2021193081A1 WO 2021193081 A1 WO2021193081 A1 WO 2021193081A1 JP 2021009720 W JP2021009720 W JP 2021009720W WO 2021193081 A1 WO2021193081 A1 WO 2021193081A1
Authority
WO
WIPO (PCT)
Prior art keywords
type
virus
herpes simplex
rev
simplex virus
Prior art date
Application number
PCT/JP2021/009720
Other languages
French (fr)
Japanese (ja)
Inventor
英樹 粕谷
吉則 直江
繁 松村
イブラヒム ラガブ ナシー エイッサ
Original Assignee
国立大学法人東海国立大学機構
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人東海国立大学機構 filed Critical 国立大学法人東海国立大学機構
Priority to JP2022509899A priority Critical patent/JPWO2021193081A1/ja
Publication of WO2021193081A1 publication Critical patent/WO2021193081A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/763—Herpes virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/869—Herpesviral vectors
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention relates to type 1 herpes simplex virus and the like.
  • Malignant tumor (cancer) (hereinafter sometimes referred to simply as "tumor”) is the number one factor in the mortality rate of Japanese people, but if early treatment by early diagnosis is possible, the mortality rate. Is said to be able to be significantly reduced.
  • treatments for tumors include surgical excision, radiation, and treatment with anticancer agents. Further, in recent years, tumor treatment using an oncolytic virus that specifically proliferates in tumor cells is also known.
  • C-REV is an oncolytic herpes simplex virus (HSV), a mutant virus that selectively destroys tumors and exerts antitumor effects.
  • HSV herpes simplex virus
  • the virus spreads from the tumor destroyed by the virus infection, and the released cancer antigen causes activation of tumor-specific lymphocytes. This activated lymphocyte has strong tumor immunity.
  • Non-Patent Document 1 when C-REV is administered to a tumor, it is applied not only to the tumor cells actually infected with C-REV, but also to the tumor cells surrounding the tumor cells destroyed by C-REV, and to the tumor cells that have distantly metastasized. Is also known to exert an antitumor effect by tumor-specific lymphocytes rather than virus infection (Non-Patent Document 1).
  • An object of the present invention is to provide a virus having a higher antitumor effect.
  • the present inventor has found that a type 1 herpes simplex virus containing an IL21 expression cassette in the viral genome can solve the above problems.
  • the present inventor has completed the present invention as a result of further research based on this finding. That is, the present invention includes the following aspects.
  • Item 1 Type 1 herpes simplex virus containing an IL21 expression cassette in the viral genome.
  • Item 2. The type 1 herpes simplex virus according to Item 1, which contains an expression cassette of CCL19 in the viral genome.
  • Item 3. Item 2.
  • Item 4. Item 2. The type 1 herpes simplex virus according to Item 2 or 3, wherein all the expression cassettes are controlled by a single promoter.
  • Item 5. Item 4. The type 1 herpes simplex virus according to any one of Items 1 to 4, which has cancer cell selectivity.
  • Item 6. Item 2.
  • Item 7. A drug comprising the type 1 herpes simplex virus according to any one of Items 1 to 6.
  • Item 8. Item 2. The drug according to Item 7, which is a drug.
  • Item 9. Item 2. The agent according to Item 7, which is an anticancer agent.
  • Item A A method for treating cancer, which comprises administering the type 1 herpes simplex virus according to any one of Items 1 to 6 to an animal.
  • Item B Item 1. The type 1 herpes simplex virus according to any one of Items 1 to 6 for use in the treatment of cancer.
  • Item C Use of the type 1 herpes simplex virus according to any one of Items 1 to 6 for the production of an anticancer drug.
  • Item D Use of the type 1 herpes simplex virus according to any one of Items 1 to 6 for the treatment of cancer.
  • the present invention it is possible to provide a virus having a higher antitumor effect. Furthermore, the virus of the present invention can exert an antitumor effect on a tumor distant from the administration site.
  • FIG. 1 It is a schematic diagram which shows the structure of the cytokine expression cassette in the cytokine-mounted C-REV (Example 1), and the position where the cytokine expression cassette is mounted in the viral genome.
  • the results of Elisa measurement of cytokine expression levels are shown (Example 1).
  • the vertical axis shows the IL21 concentration, and in the horizontal axis, MOCK indicates the case of non-virus infection, and the others indicate the virus species infecting the cells.
  • the results of tumor size measurement in the antitumor effect evaluation test 1 (Example 2) are shown.
  • the vertical axis shows the tumor size, and the horizontal axis shows the number of days elapsed since the start of virus administration.
  • the graph on the left shows the tumor size on the administration side, and the graph on the right shows the tumor size on the non-administration side.
  • Control indicates the case of non-administration of virus, and the others indicate the type of virus administered.
  • the results of body weight measurement in the antitumor effect evaluation test 1 (Example 2) are shown.
  • the vertical axis shows the body weight, and the horizontal axis shows the number of days elapsed from the start of virus administration.
  • Control indicates the case of non-administration of virus, and the others indicate the type of virus administered.
  • the results of tumor size measurement in the antitumor effect evaluation test 2 (Example 3) are shown.
  • the vertical axis shows the tumor size
  • the horizontal axis shows the number of days elapsed since the start of virus administration.
  • the graph on the left shows the tumor size on the administration side
  • the graph on the right shows the tumor size on the non-administration side.
  • Control indicates the case of non-administration of virus, and the others indicate the type of virus administered.
  • the results of body weight measurement in the antitumor effect evaluation test 2 (Example 3) are shown.
  • the vertical axis shows the body weight
  • the horizontal axis shows the number of days elapsed from the start of virus administration.
  • Control indicates the case of non-administration of virus, and the others indicate the type of virus administered.
  • the results of tumor size measurement in the antitumor effect evaluation test 3 are shown.
  • the vertical axis shows the tumor size, and the horizontal axis shows the number of days elapsed since the start of virus administration.
  • the graph on the left shows the tumor size on the administration side, and the graph on the right shows the tumor size on the non-administration side.
  • Control indicates the case of non-administration of virus, and the others indicate the type of virus administered.
  • the results of body weight measurement in the antitumor effect evaluation test 3 (Example 4) are shown.
  • the vertical axis shows the body weight, and the horizontal axis shows the number of days elapsed from the start of virus administration.
  • Control indicates the case of non-administration of virus, and the others indicate the type of virus administered.
  • the results of tumor size measurement in the antitumor effect evaluation test 4 (Example 5) are shown.
  • the vertical axis shows the tumor size, and the horizontal axis shows the number of days since virus administration.
  • the graph on the left shows the tumor size on the administration side, and the graph on the right shows the tumor size on the non-administration side.
  • Control indicates the case of non-administration of virus, and the others indicate the type of virus administered.
  • the serum IL-21 measurement result in the safety test (Example 6) is shown.
  • the vertical axis shows the IL-21 concentration
  • the horizontal axis shows the number of days elapsed since virus administration.
  • Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. It is a figure which represented the content of FIG. 10 by a heat map.
  • the results of serum CCL19 measurement in the safety test (Example 6) are shown.
  • the vertical axis shows the CCL19 concentration, and the horizontal axis shows the number of days elapsed since virus administration.
  • Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. It is a figure which represented the content of FIG. 12 by a heat map.
  • the serum IL-7 measurement result in the safety test (Example 6) is shown.
  • the vertical axis shows the IL-7 concentration
  • the horizontal axis shows the number of days elapsed since virus administration.
  • Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. It is a figure which represented the content of FIG. 14 by a heat map.
  • the serum IL-15 measurement result in the safety test (Example 6) is shown.
  • the vertical axis shows the IL-15 concentration, and the horizontal axis shows the number of days elapsed since virus administration.
  • Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. It is a figure which represented the content of FIG. 16 by a heat map.
  • the "identity" of an amino acid sequence refers to the degree of coincidence of two or more comparable amino acid sequences with respect to each other. Therefore, the higher the match between two amino acid sequences, the higher the identity or similarity of those sequences.
  • the level of amino acid sequence identity is determined, for example, using FASTA, a sequence analysis tool, using default parameters.
  • FASTA a sequence analysis tool
  • the algorithm BLAST by Karlin and Altschul Karlin S, Altschul SF. “Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes” Proc Natl Acad Sci USA.
  • substitution between amino acid residues having a basic side chain such as lysine, arginine, and histidine is a conservative substitution.
  • Other amino acid residues with acidic side chains such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine and tryptophan; amino acid residues with ⁇ -branched side chains such as threonine, valine and isoleucine; with aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine Substitution between amino acid residues is also a conservative substitution.
  • polynucleotides such as DNA and RNA may be subjected to known chemical modifications as illustrated below.
  • Substituting the phosphate residue (phosphate) of each nucleotide with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate to prevent degradation by hydrolytic enzymes such as nucleases. Can be done.
  • the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is changed to -OR (R is, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC (NH) NH2, It may be replaced with CH2CONHCH3, CH2CH2CN, etc.).
  • R is, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC (NH) NH2, It may be replaced with CH2CONHCH3, CH2CH2CN, etc.
  • the base moiety pyrimidine, purine
  • examples thereof include those in which the phosphoric acid moiety and the hydroxyl moiety are modified with biotin, an amino group, a lower alkylamine group, an acetyl group and the like, but the present invention is not limited thereto.
  • BNA LNA
  • the "coding sequence” is a base sequence encoding an amino acid sequence of a protein, and is not particularly limited as long as it is.
  • the present invention relates to a type 1 herpes simplex virus (sometimes referred to herein as "the virus of the present invention"), which comprises an IL21 expression cassette in the viral genome. This will be described below.
  • Type 1 herpes simplex virus is mainly composed of core protein, viral genome (DNA), capsid, segment protein, and envelope.
  • Type 1 herpes simplex virus invades the cells of the host animal, replicates within the cells, and eventually destroys / lyses the infected cells. Therefore, type 1 herpes simplex virus can exhibit oncolytic virus by infecting cancer cells.
  • Type 1 herpes simplex virus preferably has cancer cell selectivity.
  • the cancer cell selectivity include a property of specifically proliferating in cancer cells, a property of infecting cancer cells specifically, and a property of exhibiting cytolysis specifically in cancer cells. Among these, the property of specifically proliferating in cancer cells is preferable.
  • the type 1 herpes simplex virus having cancer cell selectivity may be either a naturally isolated virus strain or an artificially modified (for example, introduced with a heterologous gene) virus strain.
  • the type 1 herpes simplex virus having cancer cell selectivity is preferably Canerpaturev (C-REV: former name: HF10).
  • C-REV is an attenuated mutant of herpes simplex virus type 1 that carries the functional ⁇ 1 34.5 gene without artificial modification, and its antitumor effect can be obtained by local administration to the tumor. Be done.
  • C-REV is provided by Takara Bio Inc. for joint research purposes.
  • C-REV is WO2002 / 092826 International Pamphlet, Hepato-Gastroenterology 2003; 50: 961-966, Microbes Infect. 2007; 1-8, Current Gene Therapy. 2008 Jun; 8 (3): It is a simple herpes virus reported in 208-21 or Front Oncol. 2017; 7,149, etc.
  • type 1 herpes simplex virus having cancer cell selectivity examples include talimogene laherparepvec (T-VEC: Immunotherapy. 2015, 7, (6): 611-9.).
  • T-VEC is an oncolytic virus derived from type 1 herpes simplex virus carrying the GM-CSF gene, and has been approved as a drug by the US Food and Drug Administration.
  • G47 ⁇ is also an oncolytic virus produced by modifying the gene of type 1 herpes simplex virus, and its composition has also been reported (Proc Natl Acad Sci U S A. 2001, 98 (11): 6396). -401).
  • JS1 / 34.5- / 47- / mGM-CSF is a type 1 herpes simplex virus that lacks ICP34.5 and ICP47 and has a gene encoding mGM-CSF inserted, and has strong oncolytic virus. It has been reported (Gene Therapy 2003,10 (4); 292-303).
  • type 1 herpes simplex virus having cancer cell selectivity in addition to the above, those obtained by screening, those obtained by gene modification, etc. can be used.
  • a part of the endogenous gene is deleted (in the present specification, "deficiency” includes inactivation of the gene, for example, loss of function due to a frame shift).
  • deficiency includes inactivation of the gene, for example, loss of function due to a frame shift.
  • some endogenous genes eg UL56, UL43, UL49.5, UL55, etc.
  • the virus of the present invention is a mutant virus in the above-mentioned "type 1 herpes simplex virus" containing an IL21 expression cassette in the viral genome.
  • IL21 is one of the cytokines expressed in various animals.
  • the species from which IL21 is derived is not particularly limited.
  • the organism is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
  • human-derived IL21 includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (NCBI Accession Number: NP_068575) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (NCBI Accession Number: NP_068575). NCBI Accession Number: NP_001193935), these mature proteins (for example, proteins obtained by cleaving signal peptides) and the like can be mentioned.
  • mouse-derived IL21 (NCBI Gene ID: 60505), a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 (NCBI Accession Number: NP_001277970) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (NCBI Accession Number:) NP_068554), these mature proteins (for example, proteins obtained by cleaving signal peptides) and the like can be mentioned.
  • IL21 may have mutations such as amino acid substitutions, deletions, additions, and insertions with respect to wild-type IL21 as long as the properties as cytokines are not significantly reduced. Mutations include preferably substitutions, more preferably conservative substitutions. When IL21 has an amino acid mutation, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% with respect to the amino acid sequence of wild-type IL21. It has the above identity.
  • IL21 include the protein described in (a) below and the protein described in (b) below: (A) A protein consisting of a wild-type IL21 amino acid sequence (for example, the amino acid sequence shown in any of SEQ ID NOs: 1 to 4 or a mature sequence thereof), and (b) A wild-type IL21 amino acid sequence (for example, any of SEQ ID NOs: 1 to 4). At least one selected from the group consisting of proteins consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in (1) or its mature sequence) can be mentioned.
  • the identity is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more.
  • the number of amino acids mutated in (b) above is, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 2.
  • IL21 may be added with a protein or peptide such as a known protein tag or signal sequence.
  • a protein tag such as a known protein tag or signal sequence.
  • the protein tag include His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like.
  • the IL21 expression cassette is not particularly limited as long as it is a polynucleotide capable of expressing IL21 in the cell.
  • Typical examples of IL21 expression cassettes include promoters and polynucleotides containing the IL21 coding sequence placed under the control of the promoter. Further, if necessary, it may have another sequence such as a polyadenylation signal sequence, a reporter protein (for example, a fluorescent protein, etc.) coding sequence and the like.
  • the promoter contained in the IL21 expression cassette is not particularly limited, and can be appropriately selected according to the target cancer cells.
  • various pol II promoters can be used.
  • the polII promoter is not particularly limited, but for example, CMV promoter, EF1 promoter, SV40 promoter, MMTV promoter, MSCV promoter, hTERT promoter, ⁇ -actin promoter, CAG promoter, CEA promoter, AFP promoter, tyrosinase promoter, albumin promoter, etc.
  • Examples include the HSVUL29 promoter and the HSVUL39 promoter.
  • the reporter protein examples include a luminescent (color-developing) protein that reacts with a specific substrate to emit (color) the substrate, a fluorescent protein that fluoresces due to excitation light, and the like, and is not particularly limited.
  • luminescent (color-developing) proteins include luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, ⁇ -glucuronidase, etc.
  • fluorescent proteins examples include GFP, Azami-Green, ZsGreen, GFP2, HyPer, Sirius, BFP, etc.
  • Examples include CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, Jred, KillerRed, Cherry, HcRed, mPlum and the like.
  • the virus of the present invention preferably contains an expression cassette of CCL19 in the viral genome in the above-mentioned "type 1 herpes simplex virus".
  • CCL19 (C-C motif chemokine ligand 19) is one of the cytokines expressed in various animals.
  • the species from which CCL19 is derived is not particularly limited.
  • the organism is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
  • the amino acid sequences of various CCL19 are known. For example, it can be obtained from a known database (for example, NCBI: https://www.ncbi.nlm.nih.gov/). Specifically, for example, as human-derived CCL19 (NCBI Gene ID: 6363), a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 (NCBI Accession Number: NP_006265) and this mature protein (for example, a signal peptide are cleaved). Protein) and the like.
  • NCBI Gene ID: 6363 a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 (NCBI Accession Number: NP_006265) and this mature protein (for example, a signal peptide are cleaved). Protein
  • mouse-derived CCL19 (NCBI Gene ID: 24047) includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (NCBI Accession Number: NP_036018), this mature protein (for example, a protein obtained by cleaving a signal peptide), and the like. Can be mentioned.
  • CCL19 may have mutations such as amino acid substitutions, deletions, additions, and insertions with respect to wild-type CCL19 as long as the properties as cytokines are not significantly reduced. Mutations include preferably substitutions, more preferably conservative substitutions. When CCL19 has an amino acid mutation, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% with respect to the amino acid sequence of wild-type CCL19. It has the above identity.
  • CCL19 examples include the protein described in (c) below and the protein described in (d) below:
  • C A protein consisting of a wild-type CCL19 amino acid sequence (for example, the amino acid sequence shown in any of SEQ ID NOs: 5 to 6 or a mature sequence thereof), and (d) a wild-type CCL19 amino acid sequence (for example, any of SEQ ID NOs: 5 to 6).
  • At least one selected from the group consisting of proteins consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in (1) or its mature sequence can be mentioned.
  • the identity is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more.
  • the number of amino acids mutated in (d) above is, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 2.
  • CCL19 may be added with a protein or peptide such as a known protein tag or signal sequence.
  • a protein tag such as a known protein tag or signal sequence.
  • the protein tag include His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like.
  • the CCL19 expression cassette is the same as the IL21 expression cassette.
  • the virus of the present invention preferably contains at least one expression cassette selected from the group consisting of an IL7 expression cassette and an IL15 expression cassette in the virus genome.
  • a CCL19 expression cassette and at least one expression cassette selected from the group consisting of an IL7 expression cassette and an IL15 expression cassette are included.
  • the expression cassette of CCL19 and the expression cassette of IL7 may be included. preferable.
  • IL7 (interleukin 7) is one of the cytokines expressed in various animals.
  • the species from which IL7 is derived is not particularly limited.
  • the organism is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
  • IL7 amino acid sequences are known. For example, it can be obtained from a known database (for example, NCBI: https://www.ncbi.nlm.nih.gov/). Specifically, for example, as human-derived IL7 (NCBI Gene ID: 3574), a protein consisting of the amino acid sequence shown in SEQ ID NO: 7 (NCBI Accession Number: NP_000871) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (NCBI Accession Number: NP_000871).
  • NCBI Accession Number: NP_001186815 protein consisting of the amino acid sequence shown in SEQ ID NO: 9 (NCBI Accession Number: NP_001186816), protein consisting of the amino acid sequence shown in SEQ ID NO: 10 (NCBI Accession Number: NP_001186817), these Examples include mature proteins (for example, proteins obtained by cleaving a signal peptide).
  • mouse-derived IL7 (NCBI Gene ID: 16196), a protein consisting of the amino acid sequence shown in SEQ ID NO: 11 (NCBI Accession Number: NP_001300817) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 12 (NCBI Accession Number: NP_001300818), a protein consisting of the amino acid sequence shown in SEQ ID NO: 13 (NCBI Accession Number: NP_001300819), a protein consisting of the amino acid sequence shown in SEQ ID NO: 14 (NCBI Accession Number: NP_032397), these mature proteins (for example, A protein obtained by cleaving a signal peptide) and the like.
  • NCBI Gene ID: 16196 a protein consisting of the amino acid sequence shown in SEQ ID NO: 11 (NCBI Accession Number: NP_001300817) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 12 (NCBI Accession Number: NP_001300818), a protein consisting of the
  • IL7 may have mutations such as amino acid substitutions, deletions, additions, and insertions with respect to wild-type IL7 as long as the properties as cytokines are not significantly reduced. Mutations include preferably substitutions, more preferably conservative substitutions. When IL7 has an amino acid mutation, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% with respect to the amino acid sequence of wild-type IL7. It has the above identity.
  • IL7 include the protein described in (e) below and the protein described in (f) below: (E) A protein consisting of a wild-type IL7 amino acid sequence (for example, the amino acid sequence shown in any of SEQ ID NOs: 7 to 14 or a mature sequence thereof), and (f) A wild-type IL7 amino acid sequence (for example, any of SEQ ID NOs: 7 to 14). At least one selected from the group consisting of proteins consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in (1) or its mature sequence) can be mentioned.
  • the identity is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more.
  • the number of amino acids mutated in (f) above is, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 2.
  • IL7 may be added with a protein or peptide such as a known protein tag or signal sequence.
  • a protein tag such as a known protein tag or signal sequence.
  • the protein tag include His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like.
  • the IL7 expression cassette is the same as the IL21 expression cassette.
  • IL15 is one of the cytokines expressed in various animals.
  • the species from which IL15 is derived is not particularly limited.
  • the organism is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
  • the amino acid sequences of various IL15 are known. For example, it can be obtained from a known database (for example, NCBI: https://www.ncbi.nlm.nih.gov/). Specifically, for example, as human-derived IL15 (NCBI Gene ID: 3600), a protein consisting of the amino acid sequence shown in SEQ ID NO: 15 (NCBI Accession Number: NP_000576) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 16 (NCBI Accession Number: NP_751915), these mature proteins (for example, proteins obtained by cleaving a signal peptide) and the like can be mentioned.
  • NCBI Gene ID: 3600 a protein consisting of the amino acid sequence shown in SEQ ID NO: 15
  • NP_751915 a protein consisting of the amino acid sequence shown in SEQ ID NO: 16
  • these mature proteins for example, proteins obtained by cleaving a signal peptide
  • mouse-derived IL15 (NCBI Gene ID: 16168), a protein consisting of the amino acid sequence shown in SEQ ID NO: 17 (NCBI Accession Number: NP_001241676) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 18 (NCBI Accession Number NP_ 032383)
  • These mature proteins for example, proteins obtained by cleaving a signal peptide
  • IL15 may have mutations such as amino acid substitutions, deletions, additions, and insertions with respect to wild-type IL15 as long as the properties as cytokines are not significantly reduced. Mutations include preferably substitutions, more preferably conservative substitutions. When IL15 has an amino acid mutation, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% with respect to the amino acid sequence of wild-type IL15. It has the above identity.
  • IL15 include the protein described in (g) below and the protein described in (h) below: (G) A protein consisting of a wild-type IL15 amino acid sequence (for example, the amino acid sequence shown in any of SEQ ID NOs: 15 to 18 or a mature sequence thereof), and (h) A wild-type IL15 amino acid sequence (for example, any of SEQ ID NOs: 15 to 18). At least one selected from the group consisting of proteins consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in (1) or its mature sequence) can be mentioned.
  • the identity is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more.
  • the number of amino acids mutated in (h) above is, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 2.
  • IL15 may be added with a protein or peptide such as a known protein tag or signal sequence.
  • a protein tag such as a known protein tag or signal sequence.
  • the protein tag include His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like.
  • the IL15 expression cassette is the same as the IL21 expression cassette.
  • each cytokine gene expression is independent.
  • a plurality of expression cassettes controlled by a promoter may be used, or an expression cassette in which a plurality of gene expressions are controlled by a single promoter may be used.
  • each expression cassette does not have a promoter, but the IL21 expression cassette and the CCL19 expression cassette typically have a promoter.
  • a polynucleotide containing the IL21 coding sequence and the CCL19 coding sequence placed under the control of the promoter more specifically, from the 5'side, the promoter, the IL21 coding sequence, the CCL19 coding sequence (or The promoter, the coding sequence of CCL19, and the coding sequence of IL21) can be the polynucleotides arranged in this order.
  • self-cleaving peptide means a peptide sequence with cleavage activity that occurs between two amino acid residues within the peptide sequence itself.
  • self-cleaving peptides include 2A peptides and 2A-like peptides. For example, in 2A peptides or 2A-like peptides, cleavage occurs between glycine residues and proline residues on these peptides.
  • the ribosome skipping mechanism prevents the formation of normal peptide bonds between glycine residues and proline residues during translation, and does not affect downstream translation.
  • the ribosome skip mechanism is known in the art and is used for the expression of multiple proteins encoded by a single molecule of messenger RNA (mRNA).
  • mRNA messenger RNA
  • the self-cleaving peptide used in the present invention can be obtained from a viral 2A peptide or a 2A-like peptide having an equivalent function.
  • 2A peptide derived from foot-and-mouth disease virus (FMDV)
  • 2A peptide E2A
  • E2A horse rhinitis A virus
  • P2A Porcine teschovirus
  • TaV Thosea signavirus
  • the above-mentioned expression cassette is included in the locus of the deficient endogenous gene. This makes it possible to reduce the influence of cis elements in the viral genome on transcription from the expression cassette, and facilitates control of transcription from the expression cassette based on the configuration of the expression cassette.
  • the virus of the present invention can be easily produced according to a known genetic engineering method. For example, it can be prepared by using PCR, restriction enzyme cleavage, DNA ligation technology, or the like.
  • the virus of the present invention has oncolytic properties. Therefore, the virus of the present invention is more specifically effective as an active ingredient of a drug, a reagent, etc. (sometimes referred to as "the drug of the present invention” in the present specification), more specifically, an anticancer agent, etc. It can be used as an ingredient.
  • the agent of the present invention is not particularly limited as long as it contains the virus of the present invention, and may further contain other components if necessary.
  • the other components are not particularly limited as long as they are pharmaceutically acceptable components.
  • Other components include additives as well as components having a pharmacological action. Additives include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, etc. Examples include a chelating agent.
  • the drug of the present invention can be used as a drug for cancer treatment (anticancer drug).
  • the agent of the present invention can be used in combination with other anticancer agents.
  • the other anticancer agent is not particularly limited, and various anticancer agents can be used.
  • the anticancer agent include alkylating agents, antimetabolites, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum preparations, molecular targeting agents, hormone agents, biological preparations and the like, which are preferable.
  • examples include antimetabolites, antibiotic anticancer agents, platinum preparations and the like.
  • alkylating agent examples include cyclophosphamide, iphosphamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ranimustine.
  • Antimetabolites include, for example, enocitabine, carmofur, capecitabine, tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, gemcitabine, cytarabine, cytarabine ocphosphate, nelarabine, fluorouracil, fludalabine, pemetrexed.
  • Examples include cladribine, doxifluridine, hydroxycarbamide, mercaptopurine and the like.
  • Examples of the microtubule inhibitor include alkaloid anticancer agents such as vincristine and taxane anticancer agents such as docetaxel and paclitaxel.
  • Antibiotics Anticancer agents include, for example, mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, acralubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amurubicin, dinostatin stimalamar and the like.
  • topoisomerase inhibitor examples include CPT-11 having a topoisomerase I inhibitory action, irinotecan, nogitecan, and etoposide and sobzoxane having a topoisomerase II inhibitory action.
  • platinum preparation examples include cisplatin, nedaplatin, oxaliplatin, carboplatin and the like.
  • Hormonal agents include, for example, dexamethasone, finasteride, tamoxifen, astrosol, exemestane, ethinyl estradiol, chlormadinone, goseleline, bicalutamide, flutamide, bredonizolone, leuprorelin, letrozole, estramustine, tremiphen, fosfestrol, mitotan, Examples thereof include methyltestosterone, medroxyprogesterone, and mepitiostane.
  • Biopharmaceuticals include, for example, interferon ⁇ , ⁇ and ⁇ , interleukin 2, ubenimex, dried BCG and the like.
  • Molecular-targeted drugs include, for example, rituximab, alemtuzumab, trastuzumab, cetuximab, panitummab, imatinib, dasatinib, nirotinib, gefitinib, elrotinib, temsirolimus, bebashizumab, temshirolimus, bebashizumab, vEGF trap , Ibritumomab tiuxetan, tamibarotene, tretinoin, etc.
  • human epithelial growth factor receptor 2 inhibitor In addition to the molecular-targeted drugs specified here, human epithelial growth factor receptor 2 inhibitor, epithelial growth factor receptor inhibitor, Bcr-Abl tyrosine kinase inhibitor, epithelial growth factor tyrosine kinase inhibitor, mTOR inhibition Agents, inhibitors targeting angiogenesis such as vascular endothelial growth factor receptor 2 inhibitors ( ⁇ -VEGFR-2 antibody), various tyrosine kinase inhibitors such as MAP kinase inhibitors, inhibitors targeting cytokines, Molecular-targeted drugs such as proteasome inhibitors, antibody-anticancer drug formulations, etc. can also be included. These inhibitors also include antibodies.
  • the agent of the present invention is used in combination with an oncolytic virus different from the virus of the present invention, or a drug (cell medicine) containing cells such as cytotoxic T cells, natural killer cells, and antigen-presenting cells as active ingredients. You can also do it.
  • the cell medicine include artificially modified cells, for example, T cells (CAR-T) into which a chimeric antigen receptor gene has been introduced and T cells (TCR-T) into which a T cell receptor gene has been introduced.
  • the usage mode of the drug of the present invention is not particularly limited, and an appropriate usage mode can be adopted according to the type of the drug.
  • the agent of the present invention can be used, for example, in vitro (for example, added to the medium of cultured cells) or in vivo (for example, administered to an animal). You can also.
  • the application target of the agent of the present invention is not particularly limited, and examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
  • examples of cells include animal cells and the like.
  • the type of cell is not particularly limited, and for example, blood cell, hematopoietic stem cell / precursor cell, sperm (sperm, egg), fibroblast, epithelial cell, vascular endothelial cell, nerve cell, hepatocyte, keratin-producing cell, muscle cell. , Epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells and the like.
  • the target cancer is not particularly limited, and for example, ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, urinary tract. Cancer, colon cancer, skin cancer, malignant melanoma, osteosarcoma, squamous epithelial cancer of the head and neck, gastric cancer, prostate cancer, breast cancer, lung cancer, colon cancer, lymphoma, liver cancer, mesenteric cancer, black Tumors, stellate cell tumors, oligodendroglioma, meningeal tumors, neurofibromas, glioblastomas, coat cell tumors, nerve sheath tumors, neurofibrosarcoma, neuromyelblastoma, fibrosarcoma, squamous epithelium Examples include cell cancer, neuroendoblast cell cancer, thyroid tumor, pituitary tumor, epidermoid cancer and the like. Among these, solid cancer is preferable.
  • the agent of the present invention can be used in any dosage form, for example, tablets (including medially disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.), rounds, granules, fine granules, powders, etc.
  • Oral formulation forms such as hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), jelly, and injection formulations (eg, drip injections (eg, intravenous drip)).
  • intravenous injections intramuscular injections, subcutaneous injections, intradermal injections
  • external preparations eg, ointments, paps, lotions
  • suppository inhalants e.g., ointments, paps, lotions
  • suppository inhalants e.g., ophthalmic agents, eye ointments, dots
  • Parenteral preparation forms such as nasal preparations, ear drops, and liposome preparations can be taken.
  • the route of administration of the agent of the present invention is not particularly limited as long as a desired effect can be obtained, and is locally administered to tumor tissue; oral administration, tube feeding, enema administration, etc.
  • Parenteral administration such as arterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration can be mentioned.
  • the agent of the present invention is locally administered to tumor tissue as an injection.
  • the liquid medium for preparing the injection is not particularly limited as long as it is a liquid solvent that does not affect the living body, and a known medicinal liquid medium can be used. In addition, it can contain various components that are permitted for use in injections.
  • the number of viruses of the present invention in the agent of the present invention depends on the mode of use, the target of application, the state of the target of application, and the like, and is not limited.
  • the number of viruses is, for example, 10 3 or more, 2 ⁇ 10 3 or more, 5 ⁇ 10 3 or more, 10 4 or more, 2 ⁇ 10 4 or more, 5 ⁇ 10 4 or more, 10 5 or more, 2 ⁇ 10 5 or more, 5 ⁇ 10 5 or more, 10 6 or more, 2 ⁇ 10 6 or more, 5 ⁇ 10 6 or more, 10 7 or more, 2 ⁇ 10 7 or more, 5 ⁇ 10 7 or more, 10 8 or more, 2 ⁇ 10 8 or more, 5 ⁇ 10 8 or more, 10 9 or more, 2 ⁇ 10 9 or more, 5 ⁇ 10 9 or more, 10 10 or more.
  • the upper limit is not particularly limited, but is, for example, 10 12 or less and 10 11 or less.
  • the administration schedule is not particularly limited, and can be, for example, only once, or twice or more (for example, 2 to 10 times) at a frequency of once a day to once a month. It can be appropriately adjusted according to the usage mode, application target, state of application target, and the like.
  • Example 1 Cytokine-loaded C-REV production
  • a cytokine gene was introduced into the UL43 locus where a gene defect (function loss due to frameshift) was observed in C-REV.
  • the expression of the cytokine gene was regulated by the CMV promoter, and the green fluorescent protein (Green) was also expressed via IRES.
  • a plasmid was constructed at both ends of a cytokine expression cassette transcriptionally regulated by the CMV promoter, sandwiched in the same base sequence as the UL43 gene of 500 bases each.
  • the cytokines to be introduced are mouse IL7 (SEQ ID NO: 19), mouse IL15 (SEQ ID NO: 20), mouse IL21 (SEQ ID NO: 21), and mouse CCL19 (SEQ ID NO: 22).
  • FIG. 1 shows a schematic diagram showing the configuration of the cytokine expression cassette and the position of the cytokine expression cassette in the viral genome.
  • Cytokine production from cytokine-loaded C-REV was confirmed as follows. In vitro, mouse squamous cell carcinoma cell SCC7 was infected with 1 MOI of various cytokine-loaded C-REVs, and 24 hours later, the expression level of cytokines was measured by RT-qPCR method and ELISA method. As a result, it was confirmed that the loaded cytokine was expressed. A part of the result is shown in FIG.
  • Example 2 Antitumor effect evaluation test 1 Mouse pancreatic cancer cell Pan02 is transplanted subcutaneously into mice, and virus (C-REV or cytokine-loaded C-REV (C-REV-IL7CCL19IL21 or C-REV-IL15CCL19IL21)) is administered. The antitumor effect was examined. Specifically, it is as follows. One tumor was transplanted subcutaneously into the bilateral abdomen of the mouse with a tumor transplant needle (bilateral abdominal transplant model). When the size of the tumor reached 100 mm 3 , grouping was performed and administration of the virus was started.
  • virus C-REV or cytokine-loaded C-REV (C-REV-IL7CCL19IL21 or C-REV-IL15CCL19IL21)
  • the antitumor effect was examined. Specifically, it is as follows. One tumor was transplanted subcutaneously into the bilateral abdomen of the mouse with a tumor transplant needle (bilateral abdominal transplant model). When the size of the tumor reached 100 mm 3
  • C-REV or cytokine-carrying C-REV 5x10 5 pfu was administered into one tumor 3 times every 3 days (Day 0, 3, 6). Twice a week, the tumor size and body weight on the treated side and the non-treated side were measured to evaluate the antitumor effect.
  • the tumor growth was also suppressed in the tumors on the non-administered side, but the effects of C-REV-IL7CCL19IL21 and C-REV-IL15CCL19IL21 were clearly stronger than those of C-REV. In addition, the effects of C-REV-IL7CCL19IL21 and C-REV-IL15CCL19IL21 were equivalent. There was no difference in body weight of the treated mice between the groups.
  • Example 3 Antitumor effect evaluation test 2 The test was carried out in the same manner as in Example 2 except that C-REV-IL21, C-REV-IL7IL21 and C-REV-IL7CCL19IL21 were used as viruses.
  • Example 4 Antitumor effect evaluation test 3 C-REV-GMCSF and C-REV-IL7CCL19IL21 were used as viruses and tested in the same manner as in Example 2 except that the administration schedule was 4 times every 2 days (Day 0, 2, 4, 6). ..
  • Example 5 Antitumor effect evaluation test 4 C-REV-IL7CCL19IL21 and C-REV-IL15CCL19IL21 were used as viruses, and the test was carried out in the same manner as in Example 2 except that the virus was administered only once (Day 0, dose: 1x10 7 pfu).
  • Example 6 Antitumor effect evaluation test 5 The test was carried out in the same manner as in Example 5 except that mouse squamous cell carcinoma cell SCC7 was used as the cell to be transplanted into the mouse. As a result, it was found that C-REV-IL7CCL19IL21 and C-REV-IL15CCL19IL21 also exert an antitumor effect on squamous cell carcinoma cells.
  • Example 7 Safety evaluation test Cytokine-loaded C-REV was administered, and the blood expression of the loaded cytokine was measured. Specifically, for tumor-transplanted mice obtained in the same manner as in Example 2 except that the tumor was transplanted only subcutaneously on one side of the abdomen of the mouse, a virus (C-REV or cytokine-loaded C-REV (C-)) was used. REV-IL7CCL19IL21 or C-REV-IL15CCL19IL21)) 1x10 7 pfu was administered intratumorally only once (Day 0), 1 day (Day 1), 3 days (Day 3), and 7 days (Day 7) after administration. ), The concentration of each cytokine in the serum was measured by the ELISA method.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Provided is a virus with a stronger antineoplastic effect.　A type 1 herpes simplex virus that includes an IL21 expression cassette within the virus genome.

Description

１型単純ヘルペスウイルス Type 1 herpes simplex virus
　本発明は、1型単純ヘルペスウイルス等に関する。 The present invention relates to type 1 herpes simplex virus and the like.
　悪性腫瘍（癌）（以下、単に「腫瘍」と記載することがある。）は日本人の死亡率の第一位の要因であるが、早期診断による早期治療が可能であれば、その死亡率を著しく減少させることができると言われている。腫瘍の治療は、手術による切除、放射線照射、抗癌剤による治療等が知られている。また、近年、腫瘍細胞において特異的に増殖する腫瘍溶解性ウイルスを用いた腫瘍治療も知られている。 Malignant tumor (cancer) (hereinafter sometimes referred to simply as "tumor") is the number one factor in the mortality rate of Japanese people, but if early treatment by early diagnosis is possible, the mortality rate. Is said to be able to be significantly reduced. Known treatments for tumors include surgical excision, radiation, and treatment with anticancer agents. Further, in recent years, tumor treatment using an oncolytic virus that specifically proliferates in tumor cells is also known.
　腫瘍溶解性ウイルスとしては、アデノウイルスやその他のウイルス種に由来する複数のウイルスが知られているが、例えば、Canerpaturev（以下、「C-REV」と記載することがある。）が挙げられる。C-REVは、腫瘍溶解性単純ヘルペスウイルス（HSV）であり、腫瘍を選択的に破壊し、抗腫瘍効果を発揮する変異ウイルスである。さらに、ウイルス感染によって破壊された腫瘍からウイルスが拡散するとともに、放出される癌抗原により、腫瘍特異的リンパ球の活性化が起きる。この活性化リンパ球は強い腫瘍免疫を有する。そのため、腫瘍にC-REVを投与すると、実際にC-REVが感染した腫瘍細胞のみでなく、C-REVにより破壊された腫瘍細胞の周囲の腫瘍細胞、さらには遠隔転移した腫瘍細胞に対してもウイルス感染ではなく腫瘍特異的リンパ球による抗腫瘍効果が発揮されることが知られている（非特許文献１）。 As the oncolytic virus, a plurality of viruses derived from adenovirus and other virus species are known, and examples thereof include Canerpaturev (hereinafter, may be referred to as "C-REV"). C-REV is an oncolytic herpes simplex virus (HSV), a mutant virus that selectively destroys tumors and exerts antitumor effects. In addition, the virus spreads from the tumor destroyed by the virus infection, and the released cancer antigen causes activation of tumor-specific lymphocytes. This activated lymphocyte has strong tumor immunity. Therefore, when C-REV is administered to a tumor, it is applied not only to the tumor cells actually infected with C-REV, but also to the tumor cells surrounding the tumor cells destroyed by C-REV, and to the tumor cells that have distantly metastasized. Is also known to exert an antitumor effect by tumor-specific lymphocytes rather than virus infection (Non-Patent Document 1).
　本発明は、抗腫瘍効果がより高いウイルスを提供することを課題とする。 An object of the present invention is to provide a virus having a higher antitumor effect.
　本発明者は鋭意研究を進めた結果、ウイルスゲノム内にIL21の発現カセットを含む、1型単純ヘルペスウイルス、であれば、上記課題を解決できることを見出した。本発明者はこの知見に基づいてさらに研究を進めた結果、本発明を完成させた。即ち、本発明は、下記の態様を包含する。 As a result of diligent research, the present inventor has found that a type 1 herpes simplex virus containing an IL21 expression cassette in the viral genome can solve the above problems. The present inventor has completed the present invention as a result of further research based on this finding. That is, the present invention includes the following aspects.
　項１．　ウイルスゲノム内にIL21の発現カセットを含む、1型単純ヘルペスウイルス．
　項２．　ウイルスゲノム内にCCL19の発現カセットを含む、項１に記載の1型単純ヘルペスウイルス．
　項３．　ウイルスゲノム内にIL7の発現カセット及びIL15の発現カセットからなる群より選択される少なくとも1種の発現カセットを含む、項２に記載の1型単純ヘルペスウイルス．
　項４．　全ての前記発現カセットが単一プロモーターで制御される、項２又は３に記載の1型単純ヘルペスウイルス．
　項５．　がん細胞選択性を有する、項１～４のいずれかに記載の1型単純ヘルペスウイルス．
　項６．　内在性遺伝子が欠損しており、且つ欠損した前記内在性遺伝子の遺伝子座に前記発現カセットが含まれる、項１～５のいずれかに記載の1型単純ヘルペスウイルス．
　項７．　項１～６のいずれかに記載の1型単純ヘルペスウイルスを含む、薬剤．
　項８．　医薬である、項７に記載の薬剤．
　項９．　抗がん剤である、項７に記載の薬剤。 Item 1. Type 1 herpes simplex virus containing an IL21 expression cassette in the viral genome.
Item 2. Item 2. The type 1 herpes simplex virus according to Item 1, which contains an expression cassette of CCL19 in the viral genome.
Item 3. Item 2. The type 1 herpes simplex virus according to Item 2, which contains at least one expression cassette selected from the group consisting of an IL7 expression cassette and an IL15 expression cassette in the viral genome.
Item 4. Item 2. The type 1 herpes simplex virus according to Item 2 or 3, wherein all the expression cassettes are controlled by a single promoter.
Item 5. Item 4. The type 1 herpes simplex virus according to any one of Items 1 to 4, which has cancer cell selectivity.
Item 6. Item 2. The type 1 herpes simplex virus according to any one of Items 1 to 5, wherein the endogenous gene is deficient and the expression cassette is contained at the locus of the deficient endogenous gene.
Item 7. A drug comprising the type 1 herpes simplex virus according to any one of Items 1 to 6.
Item 8. Item 2. The drug according to Item 7, which is a drug.
Item 9. Item 2. The agent according to Item 7, which is an anticancer agent.
　項Ａ．　項１～６のいずれかに記載の1型単純ヘルペスウイルスを動物に投与することを含む、がんを治療する方法．
　項Ｂ．　がんの治療における使用のための、項１～６のいずれかに記載の1型単純ヘルペスウイルス．
　項Ｃ．　項１～６のいずれかに記載の1型単純ヘルペスウイルスの、抗がん剤の製造のための使用．
　項Ｄ．項１～６のいずれかに記載の1型単純ヘルペスウイルスの、がんの治療のための使用。 Item A. A method for treating cancer, which comprises administering the type 1 herpes simplex virus according to any one of Items 1 to 6 to an animal.
Item B. Item 1. The type 1 herpes simplex virus according to any one of Items 1 to 6 for use in the treatment of cancer.
Item C. Use of the type 1 herpes simplex virus according to any one of Items 1 to 6 for the production of an anticancer drug.
Item D. Use of the type 1 herpes simplex virus according to any one of Items 1 to 6 for the treatment of cancer.
　本発明によれば、抗腫瘍効果がより高いウイルスを提供することができる。さらに、本発明のウイルスは、投与部位から遠隔に存在する腫瘍に対しても抗腫瘍効果を発揮することが可能である。 According to the present invention, it is possible to provide a virus having a higher antitumor effect. Furthermore, the virus of the present invention can exert an antitumor effect on a tumor distant from the administration site.
サイトカイン搭載C-REV（実施例1）における、サイトカイン発現カセットの構成及びウイルスゲノムにおけるサイトカイン発現カセット搭載位置を表す模式図である。It is a schematic diagram which shows the structure of the cytokine expression cassette in the cytokine-mounted C-REV (Example 1), and the position where the cytokine expression cassette is mounted in the viral genome. サイトカインの発現量のELISAによる測定結果を示す（実施例1）。縦軸はIL21濃度を示し、横軸中、MOCKはウイルス非感染の場合を示し、その他は細胞に感染させたウイルス種を示す。The results of Elisa measurement of cytokine expression levels are shown (Example 1). The vertical axis shows the IL21 concentration, and in the horizontal axis, MOCK indicates the case of non-virus infection, and the others indicate the virus species infecting the cells. 抗腫瘍効果評価試験1（実施例2）における腫瘍サイズ測定の結果を示す。縦軸は腫瘍サイズを示し、横軸はウイルス投与開始からの経過日数を示す。左側のグラフは投与側の腫瘍サイズを示し、右側のグラフは非投与側の腫瘍サイズを示す。凡例中、Controlはウイルス非投与の場合を示し、その他は投与したウイルス種を示す。The results of tumor size measurement in the antitumor effect evaluation test 1 (Example 2) are shown. The vertical axis shows the tumor size, and the horizontal axis shows the number of days elapsed since the start of virus administration. The graph on the left shows the tumor size on the administration side, and the graph on the right shows the tumor size on the non-administration side. In the legend, Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. 抗腫瘍効果評価試験1（実施例2）における体重測定の結果を示す。縦軸は体重を示し、横軸はウイルス投与開始からの経過日数を示す。凡例中、Controlはウイルス非投与の場合を示し、その他は投与したウイルス種を示す。The results of body weight measurement in the antitumor effect evaluation test 1 (Example 2) are shown. The vertical axis shows the body weight, and the horizontal axis shows the number of days elapsed from the start of virus administration. In the legend, Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. 抗腫瘍効果評価試験2（実施例3）における腫瘍サイズ測定の結果を示す。縦軸は腫瘍サイズを示し、横軸はウイルス投与開始からの経過日数を示す。左側のグラフは投与側の腫瘍サイズを示し、右側のグラフは非投与側の腫瘍サイズを示す。凡例中、Controlはウイルス非投与の場合を示し、その他は投与したウイルス種を示す。The results of tumor size measurement in the antitumor effect evaluation test 2 (Example 3) are shown. The vertical axis shows the tumor size, and the horizontal axis shows the number of days elapsed since the start of virus administration. The graph on the left shows the tumor size on the administration side, and the graph on the right shows the tumor size on the non-administration side. In the legend, Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. 抗腫瘍効果評価試験2（実施例3）における体重測定の結果を示す。縦軸は体重を示し、横軸はウイルス投与開始からの経過日数を示す。凡例中、Controlはウイルス非投与の場合を示し、その他は投与したウイルス種を示す。The results of body weight measurement in the antitumor effect evaluation test 2 (Example 3) are shown. The vertical axis shows the body weight, and the horizontal axis shows the number of days elapsed from the start of virus administration. In the legend, Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. 抗腫瘍効果評価試験3（実施例4）における腫瘍サイズ測定の結果を示す。縦軸は腫瘍サイズを示し、横軸はウイルス投与開始からの経過日数を示す。左側のグラフは投与側の腫瘍サイズを示し、右側のグラフは非投与側の腫瘍サイズを示す。凡例中、Controlはウイルス非投与の場合を示し、その他は投与したウイルス種を示す。The results of tumor size measurement in the antitumor effect evaluation test 3 (Example 4) are shown. The vertical axis shows the tumor size, and the horizontal axis shows the number of days elapsed since the start of virus administration. The graph on the left shows the tumor size on the administration side, and the graph on the right shows the tumor size on the non-administration side. In the legend, Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. 抗腫瘍効果評価試験3（実施例4）における体重測定の結果を示す。縦軸は体重を示し、横軸はウイルス投与開始からの経過日数を示す。凡例中、Controlはウイルス非投与の場合を示し、その他は投与したウイルス種を示す。The results of body weight measurement in the antitumor effect evaluation test 3 (Example 4) are shown. The vertical axis shows the body weight, and the horizontal axis shows the number of days elapsed from the start of virus administration. In the legend, Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. 抗腫瘍効果評価試験4（実施例5）における腫瘍サイズ測定の結果を示す。縦軸は腫瘍サイズを示し、横軸はウイルス投与からの経過日数を示す。左側のグラフは投与側の腫瘍サイズを示し、右側のグラフは非投与側の腫瘍サイズを示す。凡例中、Controlはウイルス非投与の場合を示し、その他は投与したウイルス種を示す。The results of tumor size measurement in the antitumor effect evaluation test 4 (Example 5) are shown. The vertical axis shows the tumor size, and the horizontal axis shows the number of days since virus administration. The graph on the left shows the tumor size on the administration side, and the graph on the right shows the tumor size on the non-administration side. In the legend, Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. 安全性試験（実施例6）における血清中IL-21測定結果を示す。縦軸は、IL-21濃度を示し、横軸はウイルス投与からの経過日数を示す。凡例中、Controlはウイルス非投与の場合を示し、その他は投与したウイルス種を示す。The serum IL-21 measurement result in the safety test (Example 6) is shown. The vertical axis shows the IL-21 concentration, and the horizontal axis shows the number of days elapsed since virus administration. In the legend, Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. 図１０の内容をヒートマップで表した図である。It is a figure which represented the content of FIG. 10 by a heat map. 安全性試験（実施例6）における血清中CCL19測定結果を示す。縦軸は、CCL19濃度を示し、横軸はウイルス投与からの経過日数を示す。凡例中、Controlはウイルス非投与の場合を示し、その他は投与したウイルス種を示す。The results of serum CCL19 measurement in the safety test (Example 6) are shown. The vertical axis shows the CCL19 concentration, and the horizontal axis shows the number of days elapsed since virus administration. In the legend, Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. 図１２の内容をヒートマップで表した図である。It is a figure which represented the content of FIG. 12 by a heat map. 安全性試験（実施例6）における血清中IL-7測定結果を示す。縦軸は、IL-7濃度を示し、横軸はウイルス投与からの経過日数を示す。凡例中、Controlはウイルス非投与の場合を示し、その他は投与したウイルス種を示す。The serum IL-7 measurement result in the safety test (Example 6) is shown. The vertical axis shows the IL-7 concentration, and the horizontal axis shows the number of days elapsed since virus administration. In the legend, Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. 図１４の内容をヒートマップで表した図である。It is a figure which represented the content of FIG. 14 by a heat map. 安全性試験（実施例6）における血清中IL-15測定結果を示す。縦軸は、IL-15濃度を示し、横軸はウイルス投与からの経過日数を示す。凡例中、Controlはウイルス非投与の場合を示し、その他は投与したウイルス種を示す。The serum IL-15 measurement result in the safety test (Example 6) is shown. The vertical axis shows the IL-15 concentration, and the horizontal axis shows the number of days elapsed since virus administration. In the legend, Control indicates the case of non-administration of virus, and the others indicate the type of virus administered. 図１６の内容をヒートマップで表した図である。It is a figure which represented the content of FIG. 16 by a heat map.
　1．定義
　本明細書中において、「含有」及び「含む」なる表現については、「含有」、「含む」、「実質的にからなる」及び「のみからなる」という概念を含む。 1. 1. Definitions In the present specification, the expressions "contains" and "contains" include the concepts of "contains", "contains", "substantially consists" and "consists of only".
　本明細書において、アミノ酸配列の「同一性」とは、2以上の対比可能なアミノ酸配列の、お互いに対するアミノ酸配列の一致の程度をいう。従って、ある2つのアミノ酸配列の一致性が高いほど、それらの配列の同一性又は類似性は高い。アミノ酸配列の同一性のレベルは、例えば、配列分析用ツールであるFASTAを用い、デフォルトパラメータを用いて決定される。若しくは、Karlin及びAltschulによるアルゴリズムBLAST（Karlin S， Altschul SF．“Methods for assessing the statistical significance of molecular sequence features by using general scoringschemes”Proc Natl Acad Sci USA．87:2264－2268（1990）、KarlinS，Altschul SF．“Applications and statistics for multiple high－scoring segments in molecular sequences．”Proc Natl Acad Sci USA．90:5873－7（1993））を用いて決定できる。このようなBLASTのアルゴリズムに基づいたBLASTXと呼ばれるプログラムが開発されている。これらの解析方法の具体的な手法は公知であり、National Center of Biotechnology Information（NCBI）のウェブサイト（http://www.ncbi.nlm.nih.gov/）を参照すればよい。また、塩基配列の「同一性」も上記に準じて定義される。　本明細書中において、「保存的置換」とは、アミノ酸残基が類似の側鎖を有するアミノ酸残基に置換されることを意味する。例えば、リジン、アルギニン、ヒスチジンといった塩基性側鎖を有するアミノ酸残基同士で置換されることが、保存的な置換にあたる。その他、アスパラギン酸、グルタミン酸といった酸性側鎖を有するアミノ酸残基；グリシン、アスパラギン、グルタミン、セリン、スレオニン、チロシン、システインといった非帯電性極性側鎖を有するアミノ酸残基；アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファンといった非極性側鎖を有するアミノ酸残基；スレオニン、バリン、イソロイシンといったβ－分枝側鎖を有するアミノ酸残基；チロシン、フェニルアラニン、トリプトファン、ヒスチジンといった芳香族側鎖を有するアミノ酸残基同士での置換も同様に、保存的な置換にあたる。 In the present specification, the "identity" of an amino acid sequence refers to the degree of coincidence of two or more comparable amino acid sequences with respect to each other. Therefore, the higher the match between two amino acid sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence identity is determined, for example, using FASTA, a sequence analysis tool, using default parameters. Alternatively, the algorithm BLAST by Karlin and Altschul (Karlin S, Altschul SF. “Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes” Proc Natl Acad Sci USA. 87: 2264-2268 (1990), It can be determined using “Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc Natl Acad Sci USA. 90: 5873-7 (1993)). A program called BLASTX based on such a BLAST algorithm has been developed. Specific methods for these analysis methods are known, and the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) can be referred to. In addition, the "identity" of the base sequence is also defined according to the above. In the present specification, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having a basic side chain such as lysine, arginine, and histidine is a conservative substitution. Other amino acid residues with acidic side chains such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine and tryptophan; amino acid residues with β-branched side chains such as threonine, valine and isoleucine; with aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine Substitution between amino acid residues is also a conservative substitution.
　本明細書において、DNA、RNAなどのポリヌクレオチドには、次に例示するように、公知の化学修飾が施されていてもよい。ヌクレアーゼなどの加水分解酵素による分解を防ぐために、各ヌクレオチドのリン酸残基（ホスフェート）を、例えば、ホスホロチオエート（PS）、メチルホスホネート、ホスホロジチオネート等の化学修飾リン酸残基に置換することができる。また、各リボヌクレオチドの糖（リボース）の2位の水酸基を、-OR（Rは、例えばCH3（2´-O-Me）、CH2CH2OCH3（2´-O-MOE）、CH2CH2NHC(NH)NH2、CH2CONHCH3、CH2CH2CN等を示す）に置換してもよい。さらに、塩基部分（ピリミジン、プリン）に化学修飾を施してもよく、例えば、ピリミジン塩基の5位へのメチル基やカチオン性官能基の導入、あるいは2位のカルボニル基のチオカルボニルへの置換などが挙げられる。さらには、リン酸部分やヒドロキシル部分が、例えば、ビオチン、アミノ基、低級アルキルアミン基、アセチル基等で修飾されたものなどを挙げることができるが、これに限定されない。また、ヌクレオチドの糖部の2´酸素と4´炭素を架橋することにより、糖部のコンフォーメーションをN型に固定したものであるBNA（LNA）等もまた、好ましく用いられ得る。　本明細書中において、「コード配列」とは、タンパク質のアミノ酸配列をコードする塩基配列であり、その限りにおいて特に制限されない。 In the present specification, polynucleotides such as DNA and RNA may be subjected to known chemical modifications as illustrated below. Substituting the phosphate residue (phosphate) of each nucleotide with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate to prevent degradation by hydrolytic enzymes such as nucleases. Can be done. In addition, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is changed to -OR (R is, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC (NH) NH2, It may be replaced with CH2CONHCH3, CH2CH2CN, etc.). Further, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Can be mentioned. Further, examples thereof include those in which the phosphoric acid moiety and the hydroxyl moiety are modified with biotin, an amino group, a lower alkylamine group, an acetyl group and the like, but the present invention is not limited thereto. Further, BNA (LNA) or the like in which the formation of the sugar portion is fixed to N type by cross-linking the 2'oxygen and 4'carbon of the sugar part of the nucleotide can also be preferably used. In the present specification, the "coding sequence" is a base sequence encoding an amino acid sequence of a protein, and is not particularly limited as long as it is.
　2．ウイルス
　本発明は、その一態様において、ウイルスゲノム内にIL21の発現カセットを含む、1型単純ヘルペスウイルス（本明細書において、「本発明のウイルス」と示すこともある。）に関する。以下、これについて説明する。 2. Virus In one aspect of the invention, the present invention relates to a type 1 herpes simplex virus (sometimes referred to herein as "the virus of the present invention"), which comprises an IL21 expression cassette in the viral genome. This will be described below.
　1型単純ヘルペスウイルス（Herpes simplex virus type 1 (HSV-1)＝学名：Human herpesvirus 1 (HHV-1)）は、単純ヘルペスの原因ウイルスであり、100Kbp以上の直鎖状2本鎖DNAをゲノムとして保有するDNAウイルスである。1型単純ヘルペスウイルスは、主に、コアタンパク質、ウイルスゲノム（DNA）、カプシド、テグメントタンパク質、及びエンベロープから構成される。1型単純ヘルペスウイルスは、宿主動物の細胞内に侵入して、細胞内で複製され、最終的に感染細胞を破壊／溶解する。このため、1型単純ヘルペスウイルスはがん細胞に感染することにより、腫瘍溶解性を発揮することができる。腫瘍溶解により周囲に拡散した1型単純ヘルペスウイルス、及び／又は腫瘍溶解により生成されたがん抗原の抗原提示による免疫刺激に基づいて、近傍のみならず遠隔に存在する腫瘍に対しても腫瘍溶解性を発揮することができる。 Type 1 herpes simplex virus (Herpes simplex virus type 1 (HSV-1) = scientific name: Human herpesvirus 1 (HHV-1)) is the causative virus of herpes simplex, and has a genome of linear double-stranded DNA of 100 Kbp or more. It is a DNA virus that is carried as. Type 1 herpes simplex virus is mainly composed of core protein, viral genome (DNA), capsid, segment protein, and envelope. Type 1 herpes simplex virus invades the cells of the host animal, replicates within the cells, and eventually destroys / lyses the infected cells. Therefore, type 1 herpes simplex virus can exhibit oncolytic virus by infecting cancer cells. Oncolytic tumor lysis not only in the vicinity but also in distant tumors based on immunostimulation by antigen presentation of type 1 herpes simplex virus spread to the surroundings by oncolytic virus and / or cancer antigen generated by oncolytic virus Can exert sex.
　1型単純ヘルペスウイルスは、がん細胞選択性を有することが好ましい。がん細胞選択性としては、例えば、がん細胞において特異的に増殖する性質、がん細胞特異的に感染する性質、がん細胞特異的に細胞溶解性を発揮する性質等が挙げられる。これらの中でも、好ましくはがん細胞において特異的に増殖する性質が挙げられる。 Type 1 herpes simplex virus preferably has cancer cell selectivity. Examples of the cancer cell selectivity include a property of specifically proliferating in cancer cells, a property of infecting cancer cells specifically, and a property of exhibiting cytolysis specifically in cancer cells. Among these, the property of specifically proliferating in cancer cells is preferable.
　がん細胞選択性を有する1型単純ヘルペスウイルスは、天然から単離されたウイルス株、人為的に改変された（例えば異種遺伝子を導入された）ウイルス株のいずれでもよい。 The type 1 herpes simplex virus having cancer cell selectivity may be either a naturally isolated virus strain or an artificially modified (for example, introduced with a heterologous gene) virus strain.
　がん細胞選択性を有する1型単純ヘルペスウイルスとしては、好ましくはCanerpaturev（C-REV：旧称：HF10）が挙げられる。C-REVは、人為的な改変が行われていない、機能的なγ1 34．5遺伝子を保持した単純ヘルペスウイルス1型の弱毒化変異株で、腫瘍局所に投与することによって抗腫瘍作用が得られる。C-REVは、タカラバイオ株式会社から共同研究目的に供与されている。本明細書中C-REVとは、WO2002／092826号国際公開パンフレット、Hepato-Gastroenterology 2003；50：961-966、Microbes Infect. 2007;1-8、Current Gene Therapy. 2008 Jun;8(3):208-21、あるいは、Front Oncol. 2017;7,149等で報告されている単純ヘルペスウイルスである。 The type 1 herpes simplex virus having cancer cell selectivity is preferably Canerpaturev (C-REV: former name: HF10). C-REV is an attenuated mutant of herpes simplex virus type 1 that carries the functional γ1 34.5 gene without artificial modification, and its antitumor effect can be obtained by local administration to the tumor. Be done. C-REV is provided by Takara Bio Inc. for joint research purposes. In this specification, C-REV is WO2002 / 092826 International Pamphlet, Hepato-Gastroenterology 2003; 50: 961-966, Microbes Infect. 2007; 1-8, Current Gene Therapy. 2008 Jun; 8 (3): It is a simple herpes virus reported in 208-21 or Front Oncol. 2017; 7,149, etc.
　がん細胞選択性を有する1型単純ヘルペスウイルスとしては、その他にも、例えばtalimogene laherparepvec（T-VEC:Immunotherapy. 2015, 7, (6): 611-9.）が挙げられる。T-VECは、GM-CSF遺伝子が搭載された1型単純ヘルペスウイルス由来の腫瘍溶解性ウイルスであり、米国食品医薬品局により医薬として承認されている。また、G47Δも1型単純ヘルペスウイルスの遺伝子を改変して作製された腫瘍溶解性ウイルスであり、その構成についても報告されている（Proc Natl Acad Sci U S A. 2001, 98 (11):6396-401）。JS1／34.5-／47-／mGM-CSFは、ICP34.5ならびにICP47を欠損し、かつmGM-CSFをコードする遺伝子が挿入された1型の単純ヘルペスウイルスであり、強い腫瘍溶解性を有すると報告されている（Gene Therapy 2003,10(4);292-303）。 Other examples of type 1 herpes simplex virus having cancer cell selectivity include talimogene laherparepvec (T-VEC: Immunotherapy. 2015, 7, (6): 611-9.). T-VEC is an oncolytic virus derived from type 1 herpes simplex virus carrying the GM-CSF gene, and has been approved as a drug by the US Food and Drug Administration. In addition, G47Δ is also an oncolytic virus produced by modifying the gene of type 1 herpes simplex virus, and its composition has also been reported (Proc Natl Acad Sci U S A. 2001, 98 (11): 6396). -401). JS1 / 34.5- / 47- / mGM-CSF is a type 1 herpes simplex virus that lacks ICP34.5 and ICP47 and has a gene encoding mGM-CSF inserted, and has strong oncolytic virus. It has been reported (Gene Therapy 2003,10 (4); 292-303).
　がん細胞選択性を有する1型単純ヘルペスウイルスとしては、上記以外にも、スクリーニングにより得られたもの、遺伝子改変により得られたもの等を使用することができる。 As the type 1 herpes simplex virus having cancer cell selectivity, in addition to the above, those obtained by screening, those obtained by gene modification, etc. can be used.
　1型単純ヘルペスウイルスは、その一態様において、内在性遺伝子の一部が欠損（本明細書において、「欠損」には、遺伝子の不活化、例えばフレームシフトによる機能喪失も包含される）している。例えば、がん細胞選択性の獲得のために、内在性遺伝子の一部（例えば、UL56、UL43、UL49.5、UL55等）が欠損している。 In one aspect of the type 1 herpes simplex virus, a part of the endogenous gene is deleted (in the present specification, "deficiency" includes inactivation of the gene, for example, loss of function due to a frame shift). There is. For example, some endogenous genes (eg UL56, UL43, UL49.5, UL55, etc.) are deficient in order to acquire cancer cell selectivity.
　本発明のウイルスは、上記した「1型単純ヘルペスウイルス」において、ウイルスゲノム内にIL21の発現カセットを含む、変異型ウイルスである。 The virus of the present invention is a mutant virus in the above-mentioned "type 1 herpes simplex virus" containing an IL21 expression cassette in the viral genome.
　IL21（インターロイキン21）は、各種動物において発現する、サイトカインの1種である。IL21の由来生物種は特に制限されない。該生物種としては、特に制限されず、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ、ブタ、ウマ、ウシ、ヒツジ、ヤギ、シカなどの種々の哺乳類動物が挙げられる。 IL21 (interleukin 21) is one of the cytokines expressed in various animals. The species from which IL21 is derived is not particularly limited. The organism is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
　種々のIL21のアミノ酸配列は公知である。例えば、公知のデータベース（例えば、NCBI：https://www.ncbi.nlm.nih.gov/）等から入手することができる。具体的には、例えばヒト由来IL21（NCBI Gene ID: 59067）としては、配列番号1に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_068575）、配列番号2に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 001193935）、これらの成熟タンパク質（例えばシグナルペプチドが切断されてなるタンパク質）等が挙げられる。例えばマウス由来IL21（NCBI Gene ID: 60505）としては、配列番号3に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 001277970）、配列番号4に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 068554）、これらの成熟タンパク質（例えばシグナルペプチドが切断されてなるタンパク質）等が挙げられる。 The amino acid sequences of various IL21 are known. For example, it can be obtained from a known database (for example, NCBI: https://www.ncbi.nlm.nih.gov/). Specifically, for example, human-derived IL21 (NCBI Gene ID: 59067) includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (NCBI Accession Number: NP_068575) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (NCBI Accession Number: NP_068575). NCBI Accession Number: NP_001193935), these mature proteins (for example, proteins obtained by cleaving signal peptides) and the like can be mentioned. For example, as mouse-derived IL21 (NCBI Gene ID: 60505), a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 (NCBI Accession Number: NP_001277970) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (NCBI Accession Number:) NP_068554), these mature proteins (for example, proteins obtained by cleaving signal peptides) and the like can be mentioned.
　IL21は、サイトカインとしての性質が著しく低減しない限りにおいて、野生型のIL21に対してアミノ酸の置換、欠失、付加、挿入等の変異を有するものであってもよい。変異としては、好ましくは置換、より好ましくは保存的置換が挙げられる。IL21がアミノ酸変異を有する場合、野生型のIL21のアミノ酸配列に対して、例えば85％以上、好ましくは90％以上、より好ましくは95％以上、さらに好ましくは98％以上、よりさらに好ましくは99％以上の同一性を有する。 IL21 may have mutations such as amino acid substitutions, deletions, additions, and insertions with respect to wild-type IL21 as long as the properties as cytokines are not significantly reduced. Mutations include preferably substitutions, more preferably conservative substitutions. When IL21 has an amino acid mutation, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% with respect to the amino acid sequence of wild-type IL21. It has the above identity.
　IL21の好ましい具体例としては、下記（a）に記載するタンパク質及び下記（b）に記載するタンパク質：
　（a）野生型IL21アミノ酸配列（例えば配列番号1～4のいずれかに示されるアミノ酸配列又はその成熟配列）からなるタンパク質、及び
　（b）野生型IL21アミノ酸配列（例えば配列番号1～4のいずれかに示されるアミノ酸配列又はその成熟配列）と85％以上の同一性を有するアミノ酸配列からなるタンパク質からなる群より選択される少なくとも1種が挙げられる。 Preferred specific examples of IL21 include the protein described in (a) below and the protein described in (b) below:
(A) A protein consisting of a wild-type IL21 amino acid sequence (for example, the amino acid sequence shown in any of SEQ ID NOs: 1 to 4 or a mature sequence thereof), and (b) A wild-type IL21 amino acid sequence (for example, any of SEQ ID NOs: 1 to 4). At least one selected from the group consisting of proteins consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in (1) or its mature sequence) can be mentioned.
　上記（b）において、同一性は、好ましくは90％以上、より好ましくは95％以上、さらに好ましくは98％以上である。また、上記（b）において変異しているアミノ酸数は、例えば1～10個、好ましくは1～5個、より好ましくは1～2個である。 In (b) above, the identity is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more. The number of amino acids mutated in (b) above is, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 2.
　IL21は、公知のタンパク質タグ、シグナル配列等のタンパク質又はペプチドが付加されたものであってもよい。タンパク質タグとしては、例えばHisタグ、FLAGタグ、Haloタグ、MBPタグ、HAタグ、Mycタグ、V5タグ、PAタグ等が挙げられる。 IL21 may be added with a protein or peptide such as a known protein tag or signal sequence. Examples of the protein tag include His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like.
　IL21の発現カセットは、細胞内でIL21を発現可能なポリヌクレオチドである限り特に制限されない。IL21の発現カセットの典型例としては、プロモーター、及びそのプロモーターの制御下に配置されたIL21のコード配列を含むポリヌクレオチドが挙げられる。また、必要に応じて他の配列、例えばポリアデニル化シグナル配列、レポータータンパク質（例えば、蛍光タンパク質等）コード配列等を有していてもよい。 The IL21 expression cassette is not particularly limited as long as it is a polynucleotide capable of expressing IL21 in the cell. Typical examples of IL21 expression cassettes include promoters and polynucleotides containing the IL21 coding sequence placed under the control of the promoter. Further, if necessary, it may have another sequence such as a polyadenylation signal sequence, a reporter protein (for example, a fluorescent protein, etc.) coding sequence and the like.
　IL21の発現カセットに含まれるプロモーターとしては、特に制限されず、対象がん細胞に応じて適宜選択することも可能である。プロモーターとしては、例えばpol II系プロモーターを各種使用することができる。pol II系プロモーターとしては、特に制限されないが、例えばCMVプロモーター、EF1プロモーター、SV40プロモーター、MMTVプロモーター、MSCVプロモーター、hTERTプロモーター、βアクチンプロモーター、CAGプロモーター、CEAプロモーター、AFPプロモーター、チロシナーゼプロモーター、アルブミンプロモーター、HSV UL29プロモーター、HSV UL39プロモーター等が挙げられる。 The promoter contained in the IL21 expression cassette is not particularly limited, and can be appropriately selected according to the target cancer cells. As the promoter, for example, various pol II promoters can be used. The polII promoter is not particularly limited, but for example, CMV promoter, EF1 promoter, SV40 promoter, MMTV promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, CEA promoter, AFP promoter, tyrosinase promoter, albumin promoter, etc. Examples include the HSVUL29 promoter and the HSVUL39 promoter.
　レポータータンパク質としては、特定の基質と反応して前記基質を発光（発色）させる発光（発色）タンパク質、或いは励起光によって蛍光を発する蛍光タンパク質等が例示され、特に限定されない。発光（発色）タンパク質としては、例えばルシフェラーゼ、βガラクトシダーゼ、クロラムフェニコールアセチルトランスフェラーゼ、βグルクロニダーゼ等が挙げられ、蛍光タンパク質としては、例えばGFP、Azami-Green、ZsGreen、GFP2、HyPer、Sirius、BFP、CFP、Turquoise、Cyan、TFP1、YFP、Venus、ZsYellow、Banana、KusabiraOrange、RFP、DsRed、AsRed、Strawberry、Jred、KillerRed、Cherry、HcRed、mPlum等が挙げられる。 Examples of the reporter protein include a luminescent (color-developing) protein that reacts with a specific substrate to emit (color) the substrate, a fluorescent protein that fluoresces due to excitation light, and the like, and is not particularly limited. Examples of luminescent (color-developing) proteins include luciferase, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase, etc., and examples of fluorescent proteins include GFP, Azami-Green, ZsGreen, GFP2, HyPer, Sirius, BFP, etc. Examples include CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, Jred, KillerRed, Cherry, HcRed, mPlum and the like.
　本発明のウイルスは、上記した「1型単純ヘルペスウイルス」において、ウイルスゲノム内にCCL19の発現カセットを含むことが好ましい。 The virus of the present invention preferably contains an expression cassette of CCL19 in the viral genome in the above-mentioned "type 1 herpes simplex virus".
　CCL19（C-Cモチーフ ケモカインリガンド19）は、各種動物において発現する、サイトカインの1種である。CCL19の由来生物種は特に制限されない。該生物種としては、特に制限されず、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ、ブタ、ウマ、ウシ、ヒツジ、ヤギ、シカなどの種々の哺乳類動物が挙げられる。 CCL19 (C-C motif chemokine ligand 19) is one of the cytokines expressed in various animals. The species from which CCL19 is derived is not particularly limited. The organism is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
　種々のCCL19のアミノ酸配列は公知である。例えば、公知のデータベース（例えば、NCBI：https://www.ncbi.nlm.nih.gov/）等から入手することができる。具体的には、例えばヒト由来CCL19（NCBI Gene ID: 6363）としては、配列番号5に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_006265）、この成熟タンパク質（例えばシグナルペプチドが切断されてなるタンパク質）等が挙げられる。例えばマウス由来CCL19（NCBI Gene ID: 24047）としては、配列番号6に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 036018）、この成熟タンパク質（例えばシグナルペプチドが切断されてなるタンパク質）等が挙げられる。 The amino acid sequences of various CCL19 are known. For example, it can be obtained from a known database (for example, NCBI: https://www.ncbi.nlm.nih.gov/). Specifically, for example, as human-derived CCL19 (NCBI Gene ID: 6363), a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 (NCBI Accession Number: NP_006265) and this mature protein (for example, a signal peptide are cleaved). Protein) and the like. For example, mouse-derived CCL19 (NCBI Gene ID: 24047) includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (NCBI Accession Number: NP_036018), this mature protein (for example, a protein obtained by cleaving a signal peptide), and the like. Can be mentioned.
　CCL19は、サイトカインとしての性質が著しく低減しない限りにおいて、野生型のCCL19に対してアミノ酸の置換、欠失、付加、挿入等の変異を有するものであってもよい。変異としては、好ましくは置換、より好ましくは保存的置換が挙げられる。CCL19がアミノ酸変異を有する場合、野生型のCCL19のアミノ酸配列に対して、例えば85％以上、好ましくは90％以上、より好ましくは95％以上、さらに好ましくは98％以上、よりさらに好ましくは99％以上の同一性を有する。 CCL19 may have mutations such as amino acid substitutions, deletions, additions, and insertions with respect to wild-type CCL19 as long as the properties as cytokines are not significantly reduced. Mutations include preferably substitutions, more preferably conservative substitutions. When CCL19 has an amino acid mutation, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% with respect to the amino acid sequence of wild-type CCL19. It has the above identity.
　CCL19の好ましい具体例としては、下記（c）に記載するタンパク質及び下記（d）に記載するタンパク質：
　（c）野生型CCL19アミノ酸配列（例えば配列番号5～6のいずれかに示されるアミノ酸配列又はその成熟配列）からなるタンパク質、及び
　（d）野生型CCL19アミノ酸配列（例えば配列番号5～6のいずれかに示されるアミノ酸配列又はその成熟配列）と85％以上の同一性を有するアミノ酸配列からなるタンパク質からなる群より選択される少なくとも1種が挙げられる。 Preferred specific examples of CCL19 include the protein described in (c) below and the protein described in (d) below:
(C) A protein consisting of a wild-type CCL19 amino acid sequence (for example, the amino acid sequence shown in any of SEQ ID NOs: 5 to 6 or a mature sequence thereof), and (d) a wild-type CCL19 amino acid sequence (for example, any of SEQ ID NOs: 5 to 6). At least one selected from the group consisting of proteins consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in (1) or its mature sequence) can be mentioned.
　上記（d）において、同一性は、好ましくは90％以上、より好ましくは95％以上、さらに好ましくは98％以上である。また、上記（d）において変異しているアミノ酸数は、例えば1～10個、好ましくは1～5個、より好ましくは1～2個である。 In (d) above, the identity is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more. The number of amino acids mutated in (d) above is, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 2.
　CCL19は、公知のタンパク質タグ、シグナル配列等のタンパク質又はペプチドが付加されたものであってもよい。タンパク質タグとしては、例えばHisタグ、FLAGタグ、Haloタグ、MBPタグ、HAタグ、Mycタグ、V5タグ、PAタグ等が挙げられる。 CCL19 may be added with a protein or peptide such as a known protein tag or signal sequence. Examples of the protein tag include His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like.
　CCL19の発現カセットについてはIL21の発現カセットと同様である。 The CCL19 expression cassette is the same as the IL21 expression cassette.
　本発明のウイルスは、ウイルスゲノム内にIL7の発現カセット及びIL15の発現カセットからなる群より選択される少なくとも1種の発現カセットを含むことが好ましい。本発明の好ましい一態様においては、CCL19の発現カセットと、IL7の発現カセット及びIL15の発現カセットからなる群より選択される少なくとも1種の発現カセットとを含む。本発明の一態様においては、抗腫瘍効果の観点（特に、本発明のウイルスの投与領域から離れた領域における抗腫瘍効果の観点）から、CCL19の発現カセットと及びIL7の発現カセットを含むことが好ましい。 The virus of the present invention preferably contains at least one expression cassette selected from the group consisting of an IL7 expression cassette and an IL15 expression cassette in the virus genome. In a preferred embodiment of the present invention, a CCL19 expression cassette and at least one expression cassette selected from the group consisting of an IL7 expression cassette and an IL15 expression cassette are included. In one aspect of the present invention, from the viewpoint of antitumor effect (particularly, from the viewpoint of antitumor effect in a region away from the administration region of the virus of the present invention), the expression cassette of CCL19 and the expression cassette of IL7 may be included. preferable.
　IL7（インターロイキン7）は、各種動物において発現する、サイトカインの1種である。IL7の由来生物種は特に制限されない。該生物種としては、特に制限されず、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ、ブタ、ウマ、ウシ、ヒツジ、ヤギ、シカなどの種々の哺乳類動物が挙げられる。 IL7 (interleukin 7) is one of the cytokines expressed in various animals. The species from which IL7 is derived is not particularly limited. The organism is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
　種々のIL7のアミノ酸配列は公知である。例えば、公知のデータベース（例えば、NCBI：https://www.ncbi.nlm.nih.gov/）等から入手することができる。具体的には、例えばヒト由来IL7（NCBI Gene ID: 3574）としては、配列番号7に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_000871）、配列番号8に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 001186815）、配列番号9に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 001186816）、配列番号10に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 001186817）、これらの成熟タンパク質（例えばシグナルペプチドが切断されてなるタンパク質）等が挙げられる。例えばマウス由来IL7（NCBI Gene ID: 16196）としては、配列番号11に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 001300817）、配列番号12に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 001300818）、配列番号13に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 001300819）、配列番号14に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 032397）、これらの成熟タンパク質（例えばシグナルペプチドが切断されてなるタンパク質）等が挙げられる。 Various IL7 amino acid sequences are known. For example, it can be obtained from a known database (for example, NCBI: https://www.ncbi.nlm.nih.gov/). Specifically, for example, as human-derived IL7 (NCBI Gene ID: 3574), a protein consisting of the amino acid sequence shown in SEQ ID NO: 7 (NCBI Accession Number: NP_000871) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (NCBI Accession Number: NP_000871). NCBI Accession Number: NP_001186815), protein consisting of the amino acid sequence shown in SEQ ID NO: 9 (NCBI Accession Number: NP_001186816), protein consisting of the amino acid sequence shown in SEQ ID NO: 10 (NCBI Accession Number: NP_001186817), these Examples include mature proteins (for example, proteins obtained by cleaving a signal peptide). For example, as mouse-derived IL7 (NCBI Gene ID: 16196), a protein consisting of the amino acid sequence shown in SEQ ID NO: 11 (NCBI Accession Number: NP_001300817) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 12 (NCBI Accession Number: NP_001300818), a protein consisting of the amino acid sequence shown in SEQ ID NO: 13 (NCBI Accession Number: NP_001300819), a protein consisting of the amino acid sequence shown in SEQ ID NO: 14 (NCBI Accession Number: NP_032397), these mature proteins (for example, A protein obtained by cleaving a signal peptide) and the like.
　IL7は、サイトカインとしての性質が著しく低減しない限りにおいて、野生型のIL7に対してアミノ酸の置換、欠失、付加、挿入等の変異を有するものであってもよい。変異としては、好ましくは置換、より好ましくは保存的置換が挙げられる。IL7がアミノ酸変異を有する場合、野生型のIL7のアミノ酸配列に対して、例えば85％以上、好ましくは90％以上、より好ましくは95％以上、さらに好ましくは98％以上、よりさらに好ましくは99％以上の同一性を有する。 IL7 may have mutations such as amino acid substitutions, deletions, additions, and insertions with respect to wild-type IL7 as long as the properties as cytokines are not significantly reduced. Mutations include preferably substitutions, more preferably conservative substitutions. When IL7 has an amino acid mutation, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% with respect to the amino acid sequence of wild-type IL7. It has the above identity.
　IL7の好ましい具体例としては、下記（e）に記載するタンパク質及び下記（f）に記載するタンパク質：
　（e）野生型IL7アミノ酸配列（例えば配列番号7～14のいずれかに示されるアミノ酸配列又はその成熟配列）からなるタンパク質、及び
　（f）野生型IL7アミノ酸配列（例えば配列番号7～14のいずれかに示されるアミノ酸配列又はその成熟配列）と85％以上の同一性を有するアミノ酸配列からなるタンパク質からなる群より選択される少なくとも1種が挙げられる。 Preferred specific examples of IL7 include the protein described in (e) below and the protein described in (f) below:
(E) A protein consisting of a wild-type IL7 amino acid sequence (for example, the amino acid sequence shown in any of SEQ ID NOs: 7 to 14 or a mature sequence thereof), and (f) A wild-type IL7 amino acid sequence (for example, any of SEQ ID NOs: 7 to 14). At least one selected from the group consisting of proteins consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in (1) or its mature sequence) can be mentioned.
　上記（f）において、同一性は、好ましくは90％以上、より好ましくは95％以上、さらに好ましくは98％以上である。また、上記（f）において変異しているアミノ酸数は、例えば1～10個、好ましくは1～5個、より好ましくは1～2個である。 In the above (f), the identity is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more. The number of amino acids mutated in (f) above is, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 2.
　IL7は、公知のタンパク質タグ、シグナル配列等のタンパク質又はペプチドが付加されたものであってもよい。タンパク質タグとしては、例えばHisタグ、FLAGタグ、Haloタグ、MBPタグ、HAタグ、Mycタグ、V5タグ、PAタグ等が挙げられる。 IL7 may be added with a protein or peptide such as a known protein tag or signal sequence. Examples of the protein tag include His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like.
　IL7の発現カセットについてはIL21の発現カセットと同様である。 The IL7 expression cassette is the same as the IL21 expression cassette.
　IL15（インターロイキン15）は、各種動物において発現する、サイトカインの1種である。IL15の由来生物種は特に制限されない。該生物種としては、特に制限されず、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ、ブタ、ウマ、ウシ、ヒツジ、ヤギ、シカなどの種々の哺乳類動物が挙げられる。 IL15 (interleukin 15) is one of the cytokines expressed in various animals. The species from which IL15 is derived is not particularly limited. The organism is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
　種々のIL15のアミノ酸配列は公知である。例えば、公知のデータベース（例えば、NCBI：https://www.ncbi.nlm.nih.gov/）等から入手することができる。具体的には、例えばヒト由来IL15（NCBI Gene ID: 3600）としては、配列番号15に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 000576）、配列番号16に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_751915）、、これらの成熟タンパク質（例えばシグナルペプチドが切断されてなるタンパク質）等が挙げられる。例えばマウス由来IL15（NCBI Gene ID: 16168）としては、配列番号17に示されるアミノ酸配列からなるタンパク質（NCBI Accession Number：NP_ 001241676）、配列番号18に示されるアミノ酸配列からなるタンパク質（NCBI Accession NumberNP_ 032383）、これらの成熟タンパク質（例えばシグナルペプチドが切断されてなるタンパク質）等が挙げられる。 The amino acid sequences of various IL15 are known. For example, it can be obtained from a known database (for example, NCBI: https://www.ncbi.nlm.nih.gov/). Specifically, for example, as human-derived IL15 (NCBI Gene ID: 3600), a protein consisting of the amino acid sequence shown in SEQ ID NO: 15 (NCBI Accession Number: NP_000576) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 16 (NCBI Accession Number: NP_751915), these mature proteins (for example, proteins obtained by cleaving a signal peptide) and the like can be mentioned. For example, as mouse-derived IL15 (NCBI Gene ID: 16168), a protein consisting of the amino acid sequence shown in SEQ ID NO: 17 (NCBI Accession Number: NP_001241676) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 18 (NCBI Accession Number NP_ 032383) ), These mature proteins (for example, proteins obtained by cleaving a signal peptide) and the like.
　IL15は、サイトカインとしての性質が著しく低減しない限りにおいて、野生型のIL15に対してアミノ酸の置換、欠失、付加、挿入等の変異を有するものであってもよい。変異としては、好ましくは置換、より好ましくは保存的置換が挙げられる。IL15がアミノ酸変異を有する場合、野生型のIL15のアミノ酸配列に対して、例えば85％以上、好ましくは90％以上、より好ましくは95％以上、さらに好ましくは98％以上、よりさらに好ましくは99％以上の同一性を有する。 IL15 may have mutations such as amino acid substitutions, deletions, additions, and insertions with respect to wild-type IL15 as long as the properties as cytokines are not significantly reduced. Mutations include preferably substitutions, more preferably conservative substitutions. When IL15 has an amino acid mutation, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% with respect to the amino acid sequence of wild-type IL15. It has the above identity.
　IL15の好ましい具体例としては、下記（g）に記載するタンパク質及び下記（h）に記載するタンパク質：
　（g）野生型IL15アミノ酸配列（例えば配列番号15～18のいずれかに示されるアミノ酸配列又はその成熟配列）からなるタンパク質、及び
　（h）野生型IL15アミノ酸配列（例えば配列番号15～18のいずれかに示されるアミノ酸配列又はその成熟配列）と85％以上の同一性を有するアミノ酸配列からなるタンパク質からなる群より選択される少なくとも1種が挙げられる。 Preferred specific examples of IL15 include the protein described in (g) below and the protein described in (h) below:
(G) A protein consisting of a wild-type IL15 amino acid sequence (for example, the amino acid sequence shown in any of SEQ ID NOs: 15 to 18 or a mature sequence thereof), and (h) A wild-type IL15 amino acid sequence (for example, any of SEQ ID NOs: 15 to 18). At least one selected from the group consisting of proteins consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in (1) or its mature sequence) can be mentioned.
　上記（h）において、同一性は、好ましくは90％以上、より好ましくは95％以上、さらに好ましくは98％以上である。また、上記（h）において変異しているアミノ酸数は、例えば1～10個、好ましくは1～5個、より好ましくは1～2個である。 In the above (h), the identity is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more. The number of amino acids mutated in (h) above is, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 2.
　IL15は、公知のタンパク質タグ、シグナル配列等のタンパク質又はペプチドが付加されたものであってもよい。タンパク質タグとしては、例えばHisタグ、FLAGタグ、Haloタグ、MBPタグ、HAタグ、Mycタグ、V5タグ、PAタグ等が挙げられる。 IL15 may be added with a protein or peptide such as a known protein tag or signal sequence. Examples of the protein tag include His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like.
　IL15の発現カセットについてはIL21の発現カセットと同様である。 The IL15 expression cassette is the same as the IL21 expression cassette.
　本発明のウイルスが、IL21の発現カセットと、CCL19の発現カセット、IL7の発現カセット、及びIL15の発現カセットからなる群より選択される少なくとも1種とを含む場合、各サイトカイン遺伝子発現がそれぞれ独立したプロモーターによって制御される複数の発現カセットを用いても、複数の遺伝子発現が単一プロモーターで制御された発現カセットを用いてもよい。例えば、本発明のウイルスが、IL21の発現カセットとCCL19の発現カセットを含む場合、各発現カセットそれぞれがプロモーターを有するのではなく、IL21の発現カセット及びCCL19の発現カセットは、典型的には、プロモーター、及びそのプロモーターの制御下に配置されたIL21のコード配列及びCCL19のコード配列を含むポリヌクレオチド、より具体的には、5’側から、プロモーター、IL21のコード配列、CCL19のコード配列（或いは、プロモーター、CCL19のコード配列、IL21のコード配列）がこの順で配置されたポリヌクレオチドとすることができる。 When the virus of the present invention contains an IL21 expression cassette and at least one selected from the group consisting of a CCL19 expression cassette, an IL7 expression cassette, and an IL15 expression cassette, each cytokine gene expression is independent. A plurality of expression cassettes controlled by a promoter may be used, or an expression cassette in which a plurality of gene expressions are controlled by a single promoter may be used. For example, when the virus of the present invention contains an IL21 expression cassette and a CCL19 expression cassette, each expression cassette does not have a promoter, but the IL21 expression cassette and the CCL19 expression cassette typically have a promoter. , And a polynucleotide containing the IL21 coding sequence and the CCL19 coding sequence placed under the control of the promoter, more specifically, from the 5'side, the promoter, the IL21 coding sequence, the CCL19 coding sequence (or The promoter, the coding sequence of CCL19, and the coding sequence of IL21) can be the polynucleotides arranged in this order.
　この場合、サイトカインのコード配列間に、内部リボソーム進入部位（internal ribosomal entry site；IRES）または自己切断ペプチドドメインのコード配列を含むことが好ましい。「自己切断ペプチド」とは、ペプチド配列自体の中の2つのアミノ酸残基の間に生じる切断活性を伴うペプチド配列を意味する。自己切断ペプチドとしては、例えば2Aペプチド又は2A様ペプチドが例示される。例えば2Aペプチド又は2A様ペプチドでは、切断はこれらのペプチド上のグリシン残基とプロリン残基との間で生じる。これは翻訳の間に、グリシン残基とプロリン残基との間の正常なペプチド結合の形成が行われない「リボソームスキップ機構」によって生じ、下流の翻訳には影響することはない。リボソームスキップ機構は当分野において知られており、そして1分子のメッセンジャーRNA（mRNA）によりコードされる複数のタンパク質の発現のために使用される。本発明で使用する自己切断ペプチドは、ウイルスの2Aペプチド又はそれと同等の機能を有する2A様ペプチドから得ることが可能である。例えば、口蹄疫ウイルス（FMDV）由来の2Aペプチド（F2A）、ウマ鼻炎Aウイルス（ERAV）由来の2Aペプチド（E2A）、Porcine teschovirus（PTV－1）由来の2Aペプチド（P2A）及びThosea asigna virus（TaV）由来の2Aペプチド（T2A）から成る群から選択することができる。自己切断ペプチドドメインは、その活性が著しく損なわれない限り、適宜変異が導入されていてもよい。 In this case, it is preferable to include the coding sequence of the internal ribosome entry site (IRES) or the self-cleaving peptide domain between the cytokine coding sequences. "Self-cleaving peptide" means a peptide sequence with cleavage activity that occurs between two amino acid residues within the peptide sequence itself. Examples of self-cleaving peptides include 2A peptides and 2A-like peptides. For example, in 2A peptides or 2A-like peptides, cleavage occurs between glycine residues and proline residues on these peptides. This is caused by the "ribosome skipping mechanism", which prevents the formation of normal peptide bonds between glycine residues and proline residues during translation, and does not affect downstream translation. The ribosome skip mechanism is known in the art and is used for the expression of multiple proteins encoded by a single molecule of messenger RNA (mRNA). The self-cleaving peptide used in the present invention can be obtained from a viral 2A peptide or a 2A-like peptide having an equivalent function. For example, 2A peptide (F2A) derived from foot-and-mouth disease virus (FMDV), 2A peptide (E2A) derived from horse rhinitis A virus (ERAV), 2A peptide (P2A) derived from Porcine teschovirus (PTV-1), and Thosea signavirus (TaV). ) Derived from the group consisting of 2A peptides (T2A). Mutations may be appropriately introduced into the self-cleaving peptide domain as long as its activity is not significantly impaired.
　1型単純ヘルペスウイルス固有の内在性遺伝子が欠損している場合、本発明の好ましい一態様においては、欠損した内在性遺伝子の遺伝子座に上記した発現カセットが含まれる。これにより、ウイルスゲノム中のシスエレメントが発現カセットからの転写に与える影響を低減させることができ、発現カセットからの転写を発現カセットの構成に基づいて制御することが容易になる。 When the endogenous gene unique to type 1 herpes simplex virus is deficient, in a preferred embodiment of the present invention, the above-mentioned expression cassette is included in the locus of the deficient endogenous gene. This makes it possible to reduce the influence of cis elements in the viral genome on transcription from the expression cassette, and facilitates control of transcription from the expression cassette based on the configuration of the expression cassette.
　本発明のウイルスは、公知の遺伝子工学的手法に従って容易に作製することができる。例えば、PCR、制限酵素切断、DNA連結技術等を利用して作製することができる。 The virus of the present invention can be easily produced according to a known genetic engineering method. For example, it can be prepared by using PCR, restriction enzyme cleavage, DNA ligation technology, or the like.
　3．用途
　本発明のウイルスは、腫瘍溶解性を有する。このため、本発明のウイルスは、医薬、試薬等（本明細書において、「本発明の薬剤」と示すこともある。）の有効成分として、より具体的には、抗がん剤等の有効成分としての利用が可能である。 3. 3. Uses The virus of the present invention has oncolytic properties. Therefore, the virus of the present invention is more specifically effective as an active ingredient of a drug, a reagent, etc. (sometimes referred to as "the drug of the present invention" in the present specification), more specifically, an anticancer agent, etc. It can be used as an ingredient.
　本発明の薬剤は、本発明のウイルスを含有する限りにおいて特に制限されず、必要に応じてさらに他の成分を含んでいてもよい。他の成分としては、薬学的に許容される成分であれば特に限定されるものではない。他の成分としては、薬理作用を有する成分のほか、添加剤も含まれる。添加剤としては、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤等が挙げられる。 The agent of the present invention is not particularly limited as long as it contains the virus of the present invention, and may further contain other components if necessary. The other components are not particularly limited as long as they are pharmaceutically acceptable components. Other components include additives as well as components having a pharmacological action. Additives include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, etc. Examples include a chelating agent.
　本発明の薬剤はがん治療用の医薬（抗がん剤）として使用することができる。この場合、本発明の薬剤は、他の抗がん剤と併用することができる。これにより、より向上した効果を発揮することも可能である。他の抗がん剤としては、特に制限されず、各種抗がん剤を用いることができる。抗がん剤としては、例えばアルキル化剤、代謝拮抗剤、微小管阻害剤、抗生物質抗がん剤、トポイソメラーゼ阻害剤、白金製剤、分子標的薬、ホルモン剤、生物製剤などが挙げられ、好ましくは代謝拮抗剤、抗生物質抗がん剤、白金製剤等が挙げられる。アルキル化剤としては、例えばシクロホスファミド、イホスファミド、ニトロソウレア、ダカルバジン、テモゾロミド、ニムスチン、ブスルファン、メルファラン、プロカルバジン、ラニムスチンなどが挙げられる。代謝拮抗剤としては、例えば、エノシタビン、カルモフール、カペシタビン、テガフール、テガフール・ウラシル、テガフール・ギメラシル・オテラシルカリウム、ゲムシタビン、シタラビン、シタラビンオクホスファート、ネララビン、フルオロウラシル、フルダラビン、ペメトレキセド、ペントスタチン、メトトレキサート、クラドリビン、ドキシフルリジン、ヒドロキシカルバミド、メルカプトプリンなどが挙げられる。微小管阻害剤としては、例えば、ビンクリスチンなどのアルカロイド系抗がん剤、ドセタキセル、パクリタキセルなどのタキサン系抗がん剤が挙げられる。抗生物質抗がん剤としては、例えば、マイトマイシンC、ドキソルビシン、エピルビシン、ダウノルビシン、ブレオマイシン、アクチノマイシンD、アクラルビシン、イダルビシン、ピラルビシン、ペプロマイシン、ミトキサントロン、アムルビシン、ジノスタチンスチマラマーなどが挙げられる。トポイソメラーゼ阻害剤としてはトポイソメラーゼI阻害作用を有するCPT-11、イリノテカン、ノギテカン、トポイソメラーゼII阻害作用をもつエトポシド、ソブゾキサンが挙げられる。白金製剤としては、例えば、シスプラチン、ネダプラチン、オキサリプラチン、カルボプラチンなどが挙げられる。ホルモン剤としては、例えば、デキサメタゾン、フィナステリド、タモキシフェン、アストロゾール、エキセメスタン、エチニルエストラジオール、クロルマジノン、ゴセレリン、ビカルタミド、フルタミド、ブレドニゾロン、リュープロレリン、レトロゾール、エストラムスチン、トレミフェン、ホスフェストロール、ミトタン、メチルテストステロン、メドロキシプロゲステロン、メピチオスタンなどが挙げられる。生物製剤としては、例えば、インターフェロンα、βおよびγ、インターロイキン2、ウベニメクス、乾燥BCGなどが挙げられる。分子標的薬としては、例えば、リツキシマブ、アレムツズマブ、トラスツズマブ、セツキシマブ、パニツムマブ、イマチニブ、ダサチニブ、ニロチニブ、ゲフィチニブ、エルロチニブ、テムシロリムス、ベバシズマブ、VEGF trap、スニチニブ、ソラフェニブ、トシツズマブ、ボルテゾミブ、ゲムツズマブ・オゾガマイシン、イブリツモマブ・オゾガマイシン、イブリツモマブチウキセタン、タミバロテン、トレチノインなどが挙げられる。ここに特定する分子標的薬以外にも、ヒト上皮性増殖因子受容体2阻害剤、上皮性増殖因子受容体阻害剤、Bcr-Ablチロシンキナーゼ阻害剤、上皮性増殖因子チロシンキナーゼ阻害剤、mTOR阻害剤、血管内皮増殖因子受容体2阻害剤（α-VEGFR-2抗体）などの血管新生を標的にした阻害剤、MAPキナーゼ阻害剤などの各種チロシンキナーゼ阻害剤、サイトカインを標的とした阻害剤、プロテアソーム阻害剤、抗体－抗がん剤配合体などの分子標的薬なども含めることができる。これら阻害剤には抗体も含む。さらに、本発明の薬剤は本発明のウイルスとは異なる腫瘍溶解性ウイルスや、細胞傷害性Ｔ細胞、ナチュラルキラー細胞、抗原提示細胞のような細胞を有効成分とする医薬（細胞医薬）と併用することもできる。前記の細胞医薬には、人為的に改変した細胞、例えばキメラ抗原受容体遺伝子を導入したＴ細胞（ＣＡＲ－Ｔ）やＴ細胞受容体遺伝子を導入したＴ細胞（ＴＣＲ－Ｔ）が挙げられる。 The drug of the present invention can be used as a drug for cancer treatment (anticancer drug). In this case, the agent of the present invention can be used in combination with other anticancer agents. As a result, it is possible to exert a further improved effect. The other anticancer agent is not particularly limited, and various anticancer agents can be used. Examples of the anticancer agent include alkylating agents, antimetabolites, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum preparations, molecular targeting agents, hormone agents, biological preparations and the like, which are preferable. Examples include antimetabolites, antibiotic anticancer agents, platinum preparations and the like. Examples of the alkylating agent include cyclophosphamide, iphosphamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ranimustine. Antimetabolites include, for example, enocitabine, carmofur, capecitabine, tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, gemcitabine, cytarabine, cytarabine ocphosphate, nelarabine, fluorouracil, fludalabine, pemetrexed. Examples include cladribine, doxifluridine, hydroxycarbamide, mercaptopurine and the like. Examples of the microtubule inhibitor include alkaloid anticancer agents such as vincristine and taxane anticancer agents such as docetaxel and paclitaxel. Antibiotics Anticancer agents include, for example, mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, acralubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amurubicin, dinostatin stimalamar and the like. Examples of the topoisomerase inhibitor include CPT-11 having a topoisomerase I inhibitory action, irinotecan, nogitecan, and etoposide and sobzoxane having a topoisomerase II inhibitory action. Examples of the platinum preparation include cisplatin, nedaplatin, oxaliplatin, carboplatin and the like. Hormonal agents include, for example, dexamethasone, finasteride, tamoxifen, astrosol, exemestane, ethinyl estradiol, chlormadinone, goseleline, bicalutamide, flutamide, bredonizolone, leuprorelin, letrozole, estramustine, tremiphen, fosfestrol, mitotan, Examples thereof include methyltestosterone, medroxyprogesterone, and mepitiostane. Biopharmaceuticals include, for example, interferon α, β and γ, interleukin 2, ubenimex, dried BCG and the like. Molecular-targeted drugs include, for example, rituximab, alemtuzumab, trastuzumab, cetuximab, panitummab, imatinib, dasatinib, nirotinib, gefitinib, elrotinib, temsirolimus, bebashizumab, temshirolimus, bebashizumab, vEGF trap , Ibritumomab tiuxetan, tamibarotene, tretinoin, etc. In addition to the molecular-targeted drugs specified here, human epithelial growth factor receptor 2 inhibitor, epithelial growth factor receptor inhibitor, Bcr-Abl tyrosine kinase inhibitor, epithelial growth factor tyrosine kinase inhibitor, mTOR inhibition Agents, inhibitors targeting angiogenesis such as vascular endothelial growth factor receptor 2 inhibitors (α-VEGFR-2 antibody), various tyrosine kinase inhibitors such as MAP kinase inhibitors, inhibitors targeting cytokines, Molecular-targeted drugs such as proteasome inhibitors, antibody-anticancer drug formulations, etc. can also be included. These inhibitors also include antibodies. Furthermore, the agent of the present invention is used in combination with an oncolytic virus different from the virus of the present invention, or a drug (cell medicine) containing cells such as cytotoxic T cells, natural killer cells, and antigen-presenting cells as active ingredients. You can also do it. Examples of the cell medicine include artificially modified cells, for example, T cells (CAR-T) into which a chimeric antigen receptor gene has been introduced and T cells (TCR-T) into which a T cell receptor gene has been introduced.
　本発明の薬剤の使用態様は、特に制限されず、その種類に応じて適切な使用態様を採ることができる。本発明の薬剤は、その用途に応じて、例えばin vitroで使用する（例えば、培養細胞の培地に添加する。）こともできるし、in vivoで使用する（例えば、動物に投与する。）こともできる。 The usage mode of the drug of the present invention is not particularly limited, and an appropriate usage mode can be adopted according to the type of the drug. Depending on its use, the agent of the present invention can be used, for example, in vitro (for example, added to the medium of cultured cells) or in vivo (for example, administered to an animal). You can also.
　本発明の薬剤の適用対象は特に限定されないが、哺乳動物では、例えば、ヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ、ブタ、ウマ、ウシ、ヒツジ、ヤギ、シカ等が挙げられる。また、細胞としては、動物細胞等が挙げられる。細胞の種類も特に制限されず、例えば血液細胞、造血幹細胞・前駆細胞、配偶子（精子、卵子）、線維芽細胞、上皮細胞、血管内皮細胞、神経細胞、肝細胞、ケラチン生成細胞、筋細胞、表皮細胞、内分泌細胞、ES細胞、iPS細胞、組織幹細胞、がん細胞等が挙げられる。 The application target of the agent of the present invention is not particularly limited, and examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. In addition, examples of cells include animal cells and the like. The type of cell is not particularly limited, and for example, blood cell, hematopoietic stem cell / precursor cell, sperm (sperm, egg), fibroblast, epithelial cell, vascular endothelial cell, nerve cell, hepatocyte, keratin-producing cell, muscle cell. , Epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells and the like.
　本発明の薬剤を抗がん剤として用いる場合、及びがん細胞に用いる場合、対象がんとしては、特に制限されず、例えば卵巣がん、肝臓がん、膵臓がん、膀胱がん、尿道がん、大腸がん、皮膚がん、悪性メラノーマ、骨肉腫、頭頚部の扁平上皮がん、胃がん、前立腺がん、乳がん、肺がん、結腸がん、リンパ腫、肝がん、中皮腫、黒色腫、星状細胞腫、乏突起神経膠腫、髄膜腫、神経線維腫、神経膠芽細胞腫、上衣細胞腫、神経鞘腫、神経線維肉腫、神経髄芽細胞腫、線維肉腫、扁平上皮細胞がん、神経外胚葉細胞がん、甲状腺腫瘍、下垂体腫瘍、類表皮がん腫等が挙げられる。これらの中でも、固形がんが好ましい。 When the agent of the present invention is used as an anticancer agent or for cancer cells, the target cancer is not particularly limited, and for example, ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, urinary tract. Cancer, colon cancer, skin cancer, malignant melanoma, osteosarcoma, squamous epithelial cancer of the head and neck, gastric cancer, prostate cancer, breast cancer, lung cancer, colon cancer, lymphoma, liver cancer, mesenteric cancer, black Tumors, stellate cell tumors, oligodendroglioma, meningeal tumors, neurofibromas, glioblastomas, coat cell tumors, nerve sheath tumors, neurofibrosarcoma, neuromyelblastoma, fibrosarcoma, squamous epithelium Examples include cell cancer, neuroendoblast cell cancer, thyroid tumor, pituitary tumor, epidermoid cancer and the like. Among these, solid cancer is preferable.
　本発明の薬剤は、任意の剤形、例えば錠剤（口腔内側崩壊錠、咀嚼可能錠、発泡錠、トローチ剤、ゼリー状ドロップ剤などを含む）、丸剤、顆粒剤、細粒剤、散剤、硬カプセル剤、軟カプセル剤、ドライシロップ剤、液剤（ドリンク剤、懸濁剤、シロップ剤を含む）、ゼリー剤などの経口製剤形態や、注射用製剤（例えば、点滴注射剤（例えば点滴静注用製剤等）、静脈注射剤、筋肉注射剤、皮下注射剤、皮内注射剤）、外用剤（例えば、軟膏剤、パップ剤、ローション剤）、坐剤吸入剤、眼剤、眼軟膏剤、点鼻剤、点耳剤、リポソーム剤等の非経口製剤形態を採ることができる。 The agent of the present invention can be used in any dosage form, for example, tablets (including medially disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.), rounds, granules, fine granules, powders, etc. Oral formulation forms such as hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), jelly, and injection formulations (eg, drip injections (eg, intravenous drip)). Pharmaceuticals, etc.), intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (eg, ointments, paps, lotions), suppository inhalants, ophthalmic agents, eye ointments, dots Parenteral preparation forms such as nasal preparations, ear drops, and liposome preparations can be taken.
　本発明の薬剤の投与経路としては、所望の効果が得られる限り特に制限されず、腫瘍組織への局所投与；　経口投与、経管栄養、注腸投与等の経腸投与；　経静脈投与、経動脈投与、筋肉内投与、心臓内投与、皮下投与、皮内投与、腹腔内投与等の非経口投与等が挙げられる。 The route of administration of the agent of the present invention is not particularly limited as long as a desired effect can be obtained, and is locally administered to tumor tissue; oral administration, tube feeding, enema administration, etc. Parenteral administration such as arterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration can be mentioned.
　本発明の好適な態様において、本発明の薬剤は注射剤として腫瘍組織に局所投与される。注射剤を調製するための液体媒体としては、生体に影響のない液体溶媒であれば特に限定はなく、公知の医薬用の液体媒体を使用することができる。さらに、注射剤への使用が許容されている各種の成分を含有させることができる。 In a preferred embodiment of the present invention, the agent of the present invention is locally administered to tumor tissue as an injection. The liquid medium for preparing the injection is not particularly limited as long as it is a liquid solvent that does not affect the living body, and a known medicinal liquid medium can be used. In addition, it can contain various components that are permitted for use in injections.
　本発明の薬剤中の本発明のウイルス数は、使用態様、適用対象、適用対象の状態等に左右されるものであり、限定はされない。該ウイルス数は、例えば、103以上、2×103以上、5×103以上、104以上、2×104以上、5×104以上、105以上、2×105以上、5×105以上、106以上、2×106以上、5×106以上、107以上、2×107以上、5×107以上、108以上、2×108以上、5×108以上、109以上、2×109以上、5×109以上、1010以上である。上限は、特に制限されないが、例えば1012以下、1011以下である。 The number of viruses of the present invention in the agent of the present invention depends on the mode of use, the target of application, the state of the target of application, and the like, and is not limited. The number of viruses is, for example, 10 3 or more, 2 × 10 3 or more, 5 × 10 3 or more, 10 4 or more, 2 × 10 4 or more, 5 × 10 4 or more, 10 5 or more, 2 × 10 5 or more, 5 × 10 5 or more, 10 6 or more, 2 × 10 6 or more, 5 × 10 6 or more, 10 7 or more, 2 × 10 7 or more, 5 × 10 7 or more, 10 8 or more, 2 × 10 8 or more, 5 × 10 8 or more, 10 9 or more, 2 × 10 9 or more, 5 × 10 9 or more, 10 10 or more. The upper limit is not particularly limited, but is, for example, 10 12 or less and 10 11 or less.
　投与スケジュールは特に制限されず、例えば1回のみ、或いは例えば1日～1ヶ月に1回の頻度で2回以上（例えば2～10回）とすることができる。使用態様、適用対象、適用対象の状態等に応じて適宜調節することができる。 The administration schedule is not particularly limited, and can be, for example, only once, or twice or more (for example, 2 to 10 times) at a frequency of once a day to once a month. It can be appropriately adjusted according to the usage mode, application target, state of application target, and the like.
　以下に、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例によって限定されるものではない。 Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
　実施例1．サイトカイン搭載C-REV作製
　C-REVにおいて遺伝子欠損（フレームシフトによる機能喪失）が見られるUL43遺伝子座にサイトカイン遺伝子を導入した。サイトカイン遺伝子はCMVプロモーターにより発現を制御し、IRESを介して緑色蛍光タンパク質（Green）も発現するようにした。CMVプロモーターにより転写制御されるサイトカイン発現カセットの両端に各500塩基のUL43遺伝子と同じ塩基配列に挟まれるプラスミドを構築した。導入するサイトカインはマウスIL7（配列番号19）、マウスIL15（配列番号20）、マウスIL21（配列番号21）、マウスCCL19（配列番号22）とし、複数のサイトカインを搭載する場合は自己切断型2a配列により結合した。本実施例において、IL21搭載C-REV (C-REV-IL21)；　IL7及びIL21搭載C-REV (C-REV-IL7IL21)；　IL7、CCL19、及びIL21搭載C-REV (C-REV-IL7CCL19IL21)；　及びIL15、CCL19、及びIL21搭載C-REV (C-REV-IL15CCL19IL21)の4種類のサイトカイン搭載C-REVを作製した。さらに、抗腫瘍効果の比較対象の為にGM-CSF搭載C-REV (C-REV-GMCSF)も作製した。図１に、サイトカイン発現カセットの構成及びウイルスゲノムにおけるサイトカイン発現カセット搭載位置を表す模式図を示す。 Example 1. Cytokine-loaded C-REV production A cytokine gene was introduced into the UL43 locus where a gene defect (function loss due to frameshift) was observed in C-REV. The expression of the cytokine gene was regulated by the CMV promoter, and the green fluorescent protein (Green) was also expressed via IRES. A plasmid was constructed at both ends of a cytokine expression cassette transcriptionally regulated by the CMV promoter, sandwiched in the same base sequence as the UL43 gene of 500 bases each. The cytokines to be introduced are mouse IL7 (SEQ ID NO: 19), mouse IL15 (SEQ ID NO: 20), mouse IL21 (SEQ ID NO: 21), and mouse CCL19 (SEQ ID NO: 22). Combined by. In this embodiment, IL21-equipped C-REV (C-REV-IL21); IL7 and IL21-equipped C-REV (C-REV-IL7IL21); IL7, CCL19, and IL21-equipped C-REV (C-REV-IL7CCL19 IL21) And, four types of cytokine-loaded C-REVs, IL15, CCL19, and IL21-loaded C-REV (C-REV-IL15CCL19IL21) were prepared. In addition, a GM-CSF-equipped C-REV (C-REV-GMCSF) was also prepared for comparison of antitumor effects. FIG. 1 shows a schematic diagram showing the configuration of the cytokine expression cassette and the position of the cytokine expression cassette in the viral genome.
　サイトカイン搭載C-REVからのサイトカイン産生は次のようにして確認した。in vitroにおいてマウス扁平上皮癌細胞SCC7に各種サイトカイン搭載C-REVを1 MOI感染させ、24時間後にRT-qPCR法及びELISA法にてサイトカインの発現量を測定した。その結果、搭載したサイトカインが発現していることが確認された。結果の一部を図２に示す。 Cytokine production from cytokine-loaded C-REV was confirmed as follows. In vitro, mouse squamous cell carcinoma cell SCC7 was infected with 1 MOI of various cytokine-loaded C-REVs, and 24 hours later, the expression level of cytokines was measured by RT-qPCR method and ELISA method. As a result, it was confirmed that the loaded cytokine was expressed. A part of the result is shown in FIG.
　実施例2．抗腫瘍効果評価試験1
　マウスの皮下にマウス膵臓癌細胞Pan02を移植し、ウイルス（C-REV又はサイトカイン搭載C-REV（C-REV-IL7CCL19IL21又はC-REV-IL15CCL19IL21））を投与し、腫瘍増殖抑制を指標にそれぞれの抗腫瘍効果を検討した。具体的には次の通りである。3 mm角に細切した腫瘍をマウスの両側側腹部皮下に腫瘍移植針によりそれぞれ１個移植した(両側側腹部移植モデル)。腫瘍の大きさが100mm3の大きさに達した時点で群分けを行い、ウイルスの投与を開始した。ウイルス（C-REV又はサイトカイン搭載C-REV）5x105pfu を一方の腫瘍内に3日毎に3回（Day　0、3、6）投与した。週2回、投与側並びに非投与側腫瘍サイズ及び体重を測定し、抗腫瘍効果を評価した。 Example 2. Antitumor effect evaluation test 1
Mouse pancreatic cancer cell Pan02 is transplanted subcutaneously into mice, and virus (C-REV or cytokine-loaded C-REV (C-REV-IL7CCL19IL21 or C-REV-IL15CCL19IL21)) is administered. The antitumor effect was examined. Specifically, it is as follows. One tumor was transplanted subcutaneously into the bilateral abdomen of the mouse with a tumor transplant needle (bilateral abdominal transplant model). When the size of the tumor reached 100 mm 3 , grouping was performed and administration of the virus was started. Virus (C-REV or cytokine-carrying C-REV) 5x10 5 pfu was administered into one tumor 3 times every 3 days ( Day 0, 3, 6). Twice a week, the tumor size and body weight on the treated side and the non-treated side were measured to evaluate the antitumor effect.
　結果を図３及び図４に示す。C-REV、C-REV-IL7CCL19IL21及びC-REV-IL15CCL19IL21を投与された腫瘍の増殖は明らかに抑制され、C-REV-IL7CCL19IL21及びC-REV-IL15CCL19IL21の効果はC-REVの効果に比べ明らかに強かった。また、C-REV-IL7CCL19IL21及びC-REV-IL15CCL19IL21の効果は同等であった。一方、非投与側の腫瘍においても、腫瘍の増殖は抑制されたが、C-REV-IL7CCL19IL21及びC-REV-IL15CCL19IL21の効果はC-REVの効果に比べ明らかに強かった。また、C-REV-IL7CCL19IL21及びC-REV-IL15CCL19IL21の効果は同等であった。投与されたマウスの体重は各群において差はなかった。 The results are shown in FIGS. 3 and 4. Tumor growth administered with C-REV, C-REV-IL7CCL19IL21 and C-REV-IL15CCL19IL21 was clearly suppressed, and the effects of C-REV-IL7CCL19IL21 and C-REV-IL15CCL19IL21 were clear compared to the effects of C-REV. Was strong. In addition, the effects of C-REV-IL7CCL19IL21 and C-REV-IL15CCL19IL21 were equivalent. On the other hand, the tumor growth was also suppressed in the tumors on the non-administered side, but the effects of C-REV-IL7CCL19IL21 and C-REV-IL15CCL19IL21 were clearly stronger than those of C-REV. In addition, the effects of C-REV-IL7CCL19IL21 and C-REV-IL15CCL19IL21 were equivalent. There was no difference in body weight of the treated mice between the groups.
　実施例3．抗腫瘍効果評価試験2
　ウイルスとして、C-REV-IL21、C-REV-IL7IL21及びC-REV-IL7CCL19IL21を使用する以外は、実施例2と同様にして試験した。 Example 3. Antitumor effect evaluation test 2
The test was carried out in the same manner as in Example 2 except that C-REV-IL21, C-REV-IL7IL21 and C-REV-IL7CCL19IL21 were used as viruses.
　結果を図５及び図６に示す。C-REV-IL21、C-REV-IL7IL21及びC-REV-IL7CCL19IL21を投与された腫瘍の増殖は明らかに抑制され、それらの効果はほぼ同等であった。一方、非投与側の腫瘍においても、腫瘍の増殖は抑制されたが、C-REV-IL7CCL19IL21の効果はC-REV-IL21、C-REV-IL7IL21の効果に比べ明らかに強かった。投与されたマウスの体重は各群において差はなかった。 The results are shown in FIGS. 5 and 6. Tumor growth treated with C-REV-IL21, C-REV-IL7IL21 and C-REV-IL7CCL19IL21 was clearly suppressed and their effects were similar. On the other hand, the tumor growth was also suppressed in the tumors on the non-administered side, but the effect of C-REV-IL7CCL19IL21 was clearly stronger than that of C-REV-IL21 and C-REV-IL7IL21. There was no difference in body weight of the treated mice between the groups.
　実施例4．抗腫瘍効果評価試験3
　ウイルスとして、C-REV-GMCSF及びC-REV-IL7CCL19IL21を使用し、投与スケジュールを2日毎に4回（Day　0、2、4、6）とする以外は、実施例2と同様にして試験した。 Example 4. Antitumor effect evaluation test 3
C-REV-GMCSF and C-REV-IL7CCL19IL21 were used as viruses and tested in the same manner as in Example 2 except that the administration schedule was 4 times every 2 days (Day 0, 2, 4, 6). ..
　結果を図７及び図８に示す。C-REV-GMCSF及びC-REV-IL7CCL19IL21を投与された腫瘍の増殖は明らかに抑制され、C-REV-IL7CCL19IL21の効果はC-REV-GMCSFの効果に比べ明らかに強かった。一方、非投与側の腫瘍においても、腫瘍の増殖は抑制されたが、C-REV-IL7CCL19IL21の効果はC-REV-GMCSFの効果に比べ明らかに強かった。 The results are shown in FIGS. 7 and 8. The growth of tumors to which C-REV-GMCSF and C-REV-IL7CCL19IL21 were administered was clearly suppressed, and the effect of C-REV-IL7CCL19IL21 was clearly stronger than that of C-REV-GMCSF. On the other hand, the tumor growth was also suppressed in the tumors on the non-administered side, but the effect of C-REV-IL7CCL19IL21 was clearly stronger than that of C-REV-GMCSF.
　実施例5．抗腫瘍効果評価試験4
　ウイルスとしてC-REV-IL7CCL19IL21及びC-REV-IL15CCL19IL21を使用し、ウイルス投与を1回のみ（Day 0、投与量：1x107pfu）とする以外は、実施例2と同様にして試験した。 Example 5. Antitumor effect evaluation test 4
C-REV-IL7CCL19IL21 and C-REV-IL15CCL19IL21 were used as viruses, and the test was carried out in the same manner as in Example 2 except that the virus was administered only once (Day 0, dose: 1x10 7 pfu).
　結果を図９に示す。非投与側の腫瘍において、C-REV-IL7CCL19IL21の方がC-REV-IL15CCL19IL21よりも抗腫瘍効果が強かった。 The results are shown in Fig. 9. Among the tumors on the non-administered side, C-REV-IL7CCL19IL21 had a stronger antitumor effect than C-REV-IL15CCL19IL21.
　実施例6．抗腫瘍効果評価試験5
　マウスに移植する細胞としてマウス扁平上皮癌細胞SCC7を使用する以外は、実施例5と同様にして試験した。その結果、C-REV-IL7CCL19IL21及びC-REV-IL15CCL19IL21は、扁平上皮癌細胞に対しても抗腫瘍効果を発揮することが分かった。 Example 6. Antitumor effect evaluation test 5
The test was carried out in the same manner as in Example 5 except that mouse squamous cell carcinoma cell SCC7 was used as the cell to be transplanted into the mouse. As a result, it was found that C-REV-IL7CCL19IL21 and C-REV-IL15CCL19IL21 also exert an antitumor effect on squamous cell carcinoma cells.
　実施例7．安全性評価試験
　サイトカイン搭載C-REVを投与し、搭載されたサイトカインの血中発現を測定した。具体的には、マウスの片側側腹部皮下にのみ腫瘍を移植する以外は実施例2と同様にして得られた腫瘍移植マウスに対して、ウイルス（C-REV又はサイトカイン搭載C-REV（C-REV-IL7CCL19IL21又はC-REV-IL15CCL19IL21））1x107pfuを腫瘍内に1回のみ投与し（Day 0）、投与から1日後（Day 1）、3日後（Day 3）、及び7日後（Day 7）に、ELISA法により血清中の各サイトカイン濃度を測定した。 Example 7. Safety evaluation test Cytokine-loaded C-REV was administered, and the blood expression of the loaded cytokine was measured. Specifically, for tumor-transplanted mice obtained in the same manner as in Example 2 except that the tumor was transplanted only subcutaneously on one side of the abdomen of the mouse, a virus (C-REV or cytokine-loaded C-REV (C-)) was used. REV-IL7CCL19IL21 or C-REV-IL15CCL19IL21)) 1x10 7 pfu was administered intratumorally only once (Day 0), 1 day (Day 1), 3 days (Day 3), and 7 days (Day 7) after administration. ), The concentration of each cytokine in the serum was measured by the ELISA method.
　結果を図１０～１７に示す。サイトカイン搭載C-REVを投与しても、C-REV（サイトカイン非搭載ウイルス）に比べて、サイトカインの血清中発現量は増加しなかった。 The results are shown in FIGS. 10 to 17. Administration of cytokine-loaded C-REV did not increase the serum expression level of cytokines as compared with C-REV (cytokine-free virus).

Claims (13)

  1. ウイルスゲノム内にIL21の発現カセットを含む、1型単純ヘルペスウイルス。 Type 1 herpes simplex virus containing an IL21 expression cassette within the viral genome.
  2. ウイルスゲノム内にCCL19の発現カセットを含む、請求項１に記載の1型単純ヘルペスウイルス。 The type 1 herpes simplex virus according to claim 1, which comprises an expression cassette of CCL19 in the viral genome.
  3. ウイルスゲノム内にIL7の発現カセット及びIL15の発現カセットからなる群より選択される少なくとも1種の発現カセットを含む、請求項２に記載の1型単純ヘルペスウイルス。 The type 1 herpes simplex virus according to claim 2, wherein the virus genome contains at least one expression cassette selected from the group consisting of an IL7 expression cassette and an IL15 expression cassette.
  4. 全ての前記発現カセットが単一プロモーターで制御される、請求項２又は３に記載の1型単純ヘルペスウイルス。 The type 1 herpes simplex virus according to claim 2 or 3, wherein all the expression cassettes are controlled by a single promoter.
  5. がん細胞選択性を有する、請求項１～４のいずれかに記載の1型単純ヘルペスウイルス。 The type 1 herpes simplex virus according to any one of claims 1 to 4, which has cancer cell selectivity.
  6. 内在性遺伝子が欠損しており、且つ欠損した前記内在性遺伝子の遺伝子座に前記発現カセットが含まれる、請求項１～５のいずれかに記載の1型単純ヘルペスウイルス。 The type 1 herpes simplex virus according to any one of claims 1 to 5, wherein the endogenous gene is deficient and the expression cassette is contained in the locus of the deficient endogenous gene.
  7. 請求項１～６のいずれかに記載の1型単純ヘルペスウイルスを含む、薬剤。 An agent comprising the type 1 herpes simplex virus according to any one of claims 1 to 6.
  8. 医薬である、請求項７に記載の薬剤。 The drug according to claim 7, which is a drug.
  9. 抗がん剤である、請求項７に記載の薬剤。 The agent according to claim 7, which is an anticancer agent.
  10. 請求項１～６のいずれかに記載の1型単純ヘルペスウイルスを動物に投与することを含む、がんを治療する方法。 A method for treating cancer, which comprises administering to an animal the type 1 herpes simplex virus according to any one of claims 1 to 6.
  11. がんの治療における使用のための、請求項１～６のいずれかに記載の1型単純ヘルペスウイルス。 The type 1 herpes simplex virus according to any one of claims 1 to 6, for use in the treatment of cancer.
  12. 請求項１～６のいずれかに記載の1型単純ヘルペスウイルスの、抗がん剤の製造のための使用。 Use of the type 1 herpes simplex virus according to any one of claims 1 to 6 for the production of an anticancer agent.
  13. 請求項１～６のいずれかに記載の1型単純ヘルペスウイルスの、がんの治療のための使用。 Use of the type 1 herpes simplex virus according to any one of claims 1 to 6 for the treatment of cancer.
PCT/JP2021/009720 2020-03-23 2021-03-11 Type 1 herpes simplex virus WO2021193081A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2022509899A JPWO2021193081A1 (en) 2020-03-23 2021-03-11

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2020051489 2020-03-23
JP2020-051489 2020-03-23

Publications (1)

Publication Number Publication Date
WO2021193081A1 true WO2021193081A1 (en) 2021-09-30

Family

ID=77891707

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2021/009720 WO2021193081A1 (en) 2020-03-23 2021-03-11 Type 1 herpes simplex virus

Country Status (2)

Country Link
JP (1) JPWO2021193081A1 (en)
WO (1) WO2021193081A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019500909A (en) * 2016-01-08 2019-01-17 レプリミュン リミテッド Oncolytic virus strain
WO2019062234A1 (en) * 2017-09-26 2019-04-04 杭州康万达医药科技有限公司 Isolated recombinant oncolytic poxvirus, pharmaceutical composition, and use thereof in treatment of tumors and/or cancer
WO2019073973A1 (en) * 2017-10-10 2019-04-18 国立大学法人山口大学 Enhancer for t-cells or b-cells having memory function, malignant tumor recurrence inhibitor, and inducer for inducing memory function in t-cells or b-cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019500909A (en) * 2016-01-08 2019-01-17 レプリミュン リミテッド Oncolytic virus strain
JP2019501670A (en) * 2016-01-08 2019-01-24 レプリミュン リミテッド Modified oncolytic virus
WO2019062234A1 (en) * 2017-09-26 2019-04-04 杭州康万达医药科技有限公司 Isolated recombinant oncolytic poxvirus, pharmaceutical composition, and use thereof in treatment of tumors and/or cancer
WO2019073973A1 (en) * 2017-10-10 2019-04-18 国立大学法人山口大学 Enhancer for t-cells or b-cells having memory function, malignant tumor recurrence inhibitor, and inducer for inducing memory function in t-cells or b-cells

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CAI LINKANG, HU HAN, DUAN HAIXIAO, LI YUYING, ZOU ZONGXING, LUO KAILUN, ZHANG ZIYI, YANG JUNHAN, JIN JING, CHEN YING, KE ZONGHUANG: "The construction of a new oncolytic herpes simplex virus expressing murine interleukin‐15 with gene‐editing technology", JOURNAL OF MEDICAL VIROLOGY, JOHN WILEY & SONS, INC., US, vol. 92, no. 12, 1 December 2020 (2020-12-01), US , pages 3617 - 3627, XP055858838, ISSN: 0146-6615, DOI: 10.1002/jmv.25691 *
LI, JUN ET AL.: "Expression of CCL19 from Oncolytic Vaccinia Enhances Immunotherapeutic Potential while Maintaining Oncolytic Activity", NEOPLASIA, vol. 14, no. 12, 2012, pages 1115 - 1121, XP055330932, DOI: 10.1593/neo.121272 *
LI, YANG ET AL.: "CCL21/ IL 21-armed oncolytic adenovirus enhances antitumor activity against TERT-positive tumor cells", VIRUS RESEARCH, vol. 220, 2016, pages 172 - 178, XP029572600, DOI: 10.1016/j.virusres.2016.05.002 *
THIRUNAVUKARASU P ET AL.: "Chemokine CCL19 expressing poxvirus enhances safety and efficacy of oncolytic therapy through differential biodistribution and synergism with Cytokine-induced killer cell immunotherapy", ANNALS OF SURGICAL ONCOLOGY, vol. 18, no. 1, 2011 *

Also Published As

Publication number Publication date
JPWO2021193081A1 (en) 2021-09-30

Similar Documents

Publication Publication Date Title
JP6959258B2 (en) Modified oncolytic virus
CN101495126B (en) The purposes of herpes simplex virus type 2 mutant in Therapeutic cancer
US8076460B2 (en) Antibody and inhibitor, and transfection method or kit using them
CN112601547A (en) Treatment with oncolytic viruses
US20060052322A1 (en) Combination treatment of cancer with elicitor of gene product expression and gene-product targeting agent
AU2017405929B2 (en) Programmable oncolytic virus vaccine system and application thereof
CN111133102B (en) Method for producing recombinant viruses
CN107557337B (en) anti-ROR1 safe chimeric antigen receptor modified immune cell and application thereof
CN107475275B (en) Chimeric antigen receptor and expression gene thereof, double-antigen-regulated chimeric antigen receptor modified T cell and application thereof
CN108300699A (en) NK cells of modification and application thereof
Kottke et al. Subversion of NK-cell and TNFα immune surveillance drives tumor recurrence
WO2015148879A1 (en) Cancer immunotherapy compositions and methods
CN115552019A (en) Viral vectors for the specific expression of therapeutic proteins in myeloid and microglia cells
Kottke et al. Oncolytic virotherapy induced CSDE1 neo-antigenesis restricts VSV replication but can be targeted by immunotherapy
CN111526894B (en) Pharmaceutical composition for preventing or treating lung metastasis of cancer comprising CHI3L1 inhibitor as active ingredient
Wu et al. ING4 expressing oncolytic vaccinia virus promotes anti-tumor efficiency and synergizes with gemcitabine in pancreatic cancer
JP5887413B2 (en) Methods of reducing cancer cell radiation resistance and proliferation, metastasis and invasion by modulating TM4SF4 expression or activity in non-small cell lung cancer
US20110189237A1 (en) Therapeutic Agent for Malignant Mesothelioma and Immunostimulant
WO2021193081A1 (en) Type 1 herpes simplex virus
CN112996802A (en) Method for preparing anti-tumor arenavirus and arenavirus mutant
CN109419818B (en) Echovirus for treating tumors
CN116836283B (en) Enhanced oncolytic virus with multiple immune regulatory factors, and preparation method and application thereof
WO2024152391A1 (en) Single-stranded oligonucleotide targeting igf2bp3 protein and use thereof
RU2774677C2 (en) Rna for cancer therapy
CN116769011A (en) Retinoblastoma protein mutant and preparation method and application thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21775011

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022509899

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21775011

Country of ref document: EP

Kind code of ref document: A1