WO2021161997A1 - Modified human interleukin-4 receptor polypeptide and induction agent for differentiation or polarization into m2 macrophages - Google Patents

Modified human interleukin-4 receptor polypeptide and induction agent for differentiation or polarization into m2 macrophages Download PDF

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WO2021161997A1
WO2021161997A1 PCT/JP2021/004780 JP2021004780W WO2021161997A1 WO 2021161997 A1 WO2021161997 A1 WO 2021161997A1 JP 2021004780 W JP2021004780 W JP 2021004780W WO 2021161997 A1 WO2021161997 A1 WO 2021161997A1
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amino acid
polypeptide
seq
macrophages
macrophage
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PCT/JP2021/004780
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French (fr)
Japanese (ja)
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昭彦 田口
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国立大学法人山口大学
昭彦 田口
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Priority claimed from JP2021018256A external-priority patent/JP2021126113A/en
Application filed by 国立大学法人山口大学, 昭彦 田口 filed Critical 国立大学法人山口大学
Publication of WO2021161997A1 publication Critical patent/WO2021161997A1/en

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    • 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
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention includes a mutant polypeptide of IL-4 receptor derived from human interleukin 4 receptor, a polynucleotide encoding the mutant polypeptide of the IL-4 receptor, an expression vector containing the polynucleotide, and the like. , Macrophages or macrophage precursor cells isolated from the living body containing the polynucleotide or expression vector, macrophages or macrophage precursor cells isolated from the living body containing the polypeptide, or pharmacologically with the macrophages or macrophage precursor cells.
  • the present invention relates to an agent for inducing differentiation or polarization into M2 macrophages, and a method for inducing differentiation or polarization of macrophages or macrophage precursor cells isolated from a living body into M2 macrophages.
  • Macrophages which are immune cells, are present in the lesions of these inflammatory diseases, divine neurodegenerative diseases, and ischemic diseases, and are considered to be involved in pathogenesis.
  • macrophages include M1 macrophages that induce inflammation and M2 macrophages that are responsible for inflammation suppression and tissue repair. It has been found that M2 macrophages have beneficial functions for living organisms, such as sinking excessive inflammation and phagocytosing ⁇ -amyloid protein and the like found in Alzheimer's disease (see Non-Patent Documents 1 and 2). In addition, the ratio of M1 and M2 macrophages becomes predominant in M1 with aging, and it is considered that the M1 / M2 imbalance promotes aging (see Non-Patent Document 3). against this background, research is underway to activate M2 macrophages by various methods.
  • the main method is to systemically administer a humoral factor or a small molecule compound to activate M2 macrophages, but there is currently no trend toward clinical application (see Non-Patent Document 4).
  • a humoral factor for activating to M2 macrophages acts on B lymphocytes and increases IgE production, which causes exacerbation of allergic diseases, and there is a risk of giving to cells other than macrophages.
  • humoral factors cannot cross the blood-brain barrier, they have problems such as not being able to exert sufficient effects on central inflammatory and neurodegenerative diseases such as Alzheimer's disease. rice field.
  • M2 macrophages that were once activated by systemic administration of humoral factors are repolarized into M1 macrophages in the actual inflamed site. It is suggested that there is a possibility.
  • M2 macrophages are also expected to be effective for degenerative diseases of the central nervous system such as Alzheimer's disease, but since humoral factors and compounds cannot cross the blood-brain barrier, these methods are used to treat the central nervous system such as Alzheimer's disease. The problem was that it could not be used for.
  • an object of the present invention is to provide a polypeptide involved in differentiation or polarization into M2 macrophages without depending on IL-4, or to provide a differentiation or polarization inducer into M2 macrophages.
  • the inventor focused on the human interleukin-4 receptor, and proceeded with the study, thinking that if a mutant of the human interleukin-4 receptor was introduced into the macrophage, it would be activated as an M2 macrophage.
  • the present invention was completed by succeeding in differentiation or polarization into M2 macrophages by expressing a mutant of human interleukin-4 receptor in macrophages or macrophage progenitor cells.
  • the present invention is as follows.
  • [1] A polypeptide derived from the human interleukin-4 receptor, wherein one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1.
  • [2] The polypeptide according to the above [1], which is the following (1-1) or (1-2).
  • (1-1) A polypeptide derived from the human interleucine 4 receptor, in the amino acid sequence shown in SEQ ID NO: 1, aspartic acid at position 37, tyrosine at position 62, asparagin at position 78, and phenylalanine at position 115.
  • SEQ ID NO: 1 Aspartic acid at position 37, tyrosine at position 62, asparagin at position 78, phenylalanine at position 115, arginine at position 176, arginine at position 200, isoleucine at position 242, and isoleucine at position 251.
  • the polypeptide according to [2] above, wherein the polypeptide substituted with cysteine and / or lysine at position 308 is any of the following (2-1) to (2-9).
  • Macrophages or macrophage progenitor cells isolated from living organisms characterized in that the polynucleotide described in [6] above or the expression vector described in [7] above is introduced into macrophages or macrophage progenitor cells isolated from living organisms.
  • a method for inducing differentiation or polarization from M2 macrophages is provided.
  • the differentiation or polarization inducing agent for M2 macrophages of the present invention makes it possible to differentiate or polarize macrophages or macrophage progenitor cells isolated from living organisms into M2 macrophages.
  • Example 1 a polynucleotide encoding an I242N mutation (isoleucine at position 242 replaced with asparagine) human IL-4 receptor was introduced into RAW264.7 cells or THP-1 cells to express IL-4 receptor. This is the result of the investigation.
  • the polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into RAW264.7 cells or THP-1 cells, and the expression of Arg1 or CD206 was examined.
  • Example 3 the polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into RAW264.7 cells, and LPS was allowed to act to examine the expression of TNF ⁇ or Arg1.
  • Example 4 a polynucleotide encoding a mutant human IL-4 receptor other than I242N was introduced into RAW264.7 cells, and the expression of Arg1 was examined.
  • Example 5 the polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into a bone marrow-derived macrophage (BMDM), and the expression of Arg1 was examined. This is the result of introducing a polynucleotide encoding the I242N mutant human IL-4 receptor into RAW264.7 cells and examining the expression of IL-10 in Example 6.
  • BMDM bone marrow-derived macrophage
  • a polypeptide derived from the "human interleukin-4 receptor" of the present invention in which one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1 (hereinafter, "mutation of the present invention).
  • the human interleukin-4 receptor (human IL-4 receptor) in "IL-4R polypeptide") is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and in NCBI, Reference No. It is registered as NP_000409.1.
  • polypeptide in which one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1" in the above-mentioned mutant IL-4R polypeptide is preferably "in the amino acid sequence shown in SEQ ID NO: 1.”
  • amino acids constituting the extracellular region or transmembrane region, in which one or several amino acids are added, deleted or substituted more preferably “amino acids constituting the transmembrane region in the amino acid sequence shown in SEQ ID NO: 1".
  • a polypeptide to which one or several amino acids have been added, deleted or substituted can be mentioned.
  • the extracellular space or transmembrane region can be predicted using a known extracellular space or transmembrane region prediction algorithm.
  • the 1st to 232nd regions are considered to be extracellular regions, and the 233rd to 256th regions are considered to be transmembrane regions.
  • the positions where one or several amino acids are added, deleted or substituted in the above "amino acids constituting the extracellular region" are aspartic acid at position 37, tyrosine at position 62, and position 78 in the amino acid sequence shown in SEQ ID NO: 1.
  • isoleucine at position 242 and cysteine at position 251 can be mentioned, and isoleucine at position 242 is preferable. Can be listed in.
  • the neutral non-polar amino acids include glycine (G), alanine (A), valine (V), proline (P), leucine (L), isoleucine (I), phenylalanine (F), and methionine ( It means M) or tryptophan (W).
  • the neutral polar amino acid means asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), or cysteine (C).
  • the acidic amino acid means aspartic acid (D) and glutamic acid (E).
  • polypeptide in which one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1 preferably "in the amino acid sequence shown in SEQ ID NO: 1, one or several amino acids are substituted".
  • Polypeptides ".
  • any of the following (2-1) to (2-9) can be mentioned.
  • More preferably, any of the following (2-1') to (2-9') can be mentioned, and more preferably, a polypeptide in which isoleucine at the 242nd position is replaced with asparagine can be preferably mentioned. Not limited to.
  • the "1 or several amino acids” is, for example, in the range of 1 to 30, preferably in the range of 1 to 20, more preferably in the range of 1 to 15, and further preferably in the range of 1. It means an amino acid in the range of ⁇ 10, more preferably in the range of 1 to 5, still more preferably in the range of 1 to 3, more preferably 1 to 2, and most preferably 1 amino acid.
  • THP-1 cells stimulated with Holball 12-millistart 13-acetate (PMA) or RAW264.7 cells humans are compared with the case where the polypeptide is not expressed.
  • polypeptides that increase the expression of M2 macrophage markers THP-1 cells or RAW264.7 cells stimulated with PMA expressing the mutant IL-4R polypeptide and the mutant IL-4R polypeptide are expressed. The expression of M2 macrophage markers was examined by culturing THP-1 cells or RAW264.7 cells stimulated with non-PMA, and compared with the case where the mutant IL-4R polypeptide was not expressed, the mutant IL-4R.
  • M2 macrophage markers in THP-1 cells or RAW264.7 cells stimulated with PMA expressing the polypeptide is increased, preferably 2 times or more, more preferably 5 times or more, still more preferably 10 times or more.
  • Polypeptides can be mentioned.
  • examples of the human M2 macrophage marker include CD206, CD163, IL-27RA, CD367, F13A1, and CCL22.
  • THP-1 cells differentiate into macrophages by stimulating with PMA.
  • polynucleotide encoding the mutant IL-4R polypeptide (hereinafter, also referred to as the" polynucleotide ")" of the present invention is not particularly limited as long as it is a polynucleotide encoding the mutant IL-4R polypeptide.
  • the codon selected to encode the amino acid may be appropriately optimized depending on the type of host cell to be expressed.
  • the expression vector in the "expression vector containing the Polynucleotide of the present invention may be linear or cyclic, and may be a viral vector or a non-viral vector such as a plasmid, depending on a transposon. It may be a vector.
  • such a vector may contain a control sequence such as a promoter or a terminator, or a selectable marker sequence such as a drug resistance gene or a reporter gene.
  • virus vector examples include a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated virus vector, and a lentiviral vector can be preferably mentioned. If a lentiviral vector or a retroviral vector is used, the transgene is incorporated into the genome of the host cell, so that it can be stably expressed for a long period of time. When expression in a short period of time is preferable, an adenovirus vector or an adeno-associated virus vector that does not integrate into the genome is preferable.
  • the promoter examples include a lentivirus LTR promoter, an SV40 initial promoter, a cytomegalovirus promoter, a virus-derived promoter such as the thymidine kinase promoter of simple herpesvirus, a phosphoglycerate kinase (PGK) promoter, an Xist promoter, and a ⁇ -actin promoter.
  • a lentivirus LTR promoter an SV40 initial promoter
  • a cytomegalovirus promoter examples include a virus-derived promoter such as the thymidine kinase promoter of simple herpesvirus, a phosphoglycerate kinase (PGK) promoter, an Xist promoter, and a ⁇ -actin promoter.
  • PGK phosphoglycerate kinase
  • Xist promoter Xist promoter
  • ⁇ -actin promoter Mammalian-derived promoters such as RNA polyme
  • a tetracycline-responsive promoter induced by tetracycline an Mx1 promoter induced by interleukin, a CD68 promoter having a promoter activity in macrophages, an M-CSF promoter and the like may be used.
  • the "macrophages” in the “macrophages or macrophage progenitor cells isolated from the living body containing the polynucleotide or expression vector of the present invention" are referred to as M1 macrophages. Two phenotypes of M2 macrophages are included.
  • macrophage progenitor cells include cells capable of differentiating into at least M1 macrophages or M2 macrophages, and include CD14-positive (CD14 + ) cells, inactive macrophages (M0 macrophages), monocytes, monocytes, and bone marrow.
  • the M2 macrophages are also called anti-inflammatory macrophages, and among macrophages that are CD14-positive cells, cells expressing CD206, CD163, IL-27RA, CD367, F13A1, or CCL22 in humans, and arginase 1 in mice.
  • Cells expressing (Arg1), CD206, iNOS, CD163 or CD204 can be mentioned.
  • the mutant IL-4R macrophage A and the mutant IL-4R macrophage B described later also include cultures obtained by culturing them.
  • the monocytes are blood cells in the circulating blood and can differentiate into macrophages when they leave the bloodstream and enter connective tissue. Examples of the origin of the monocytes include mammals such as humans, monkeys, and mice.
  • Peripheral blood mononuclear cells (PBMCs) are separated from peripheral blood whole blood collected from a patient by a known method such as centrifugation, and the monocytes are purified by immunomagnetic bead method or the like as necessary. Can be used.
  • PBMCs Peripheral blood mononuclear cells
  • Such monocytes can be activated and differentiated into macrophages by culturing in the presence of cytokines such as M-CSF.
  • the monocytes can also be produced from bone marrow fluid, ES cells, iPS cells and the like using a known method.
  • containing the polynucleotide may mean that the polynucleotide is contained in a macrophage or a macrophage precursor cell separated from a living body, and for example, the polynucleotide is contained in a state of being incorporated into an expression vector.
  • the polynucleotide is contained in a state of being integrated into the genome of a macrophage or a macrophage precursor cell isolated from a living body.
  • Examples of the method for producing the mutant IL-4R macrophage A include a method of introducing the polynucleotide or the expression vector into macrophages or macrophage progenitor cells isolated from a living body. Alternatively, it can also be produced by introducing the polynucleotide or the present expression vector into somatic cells such as fibroblasts isolated from the living body, and then establishing iPS cells from such somatic cells.
  • the method for introducing the polynucleotide or the expression vector into macrophages or macrophage progenitor cells isolated from the living body is not particularly limited, but is limited to a virus infection method, a calcium phosphate method, a lipofection method, a microinjection method, an electroporation method, etc. A known method of the above can be mentioned, and a method of introducing by a virus infection method can be preferably mentioned.
  • mutant IL-4R macrophage A is a genome of a macrophage or a macrophage progenitor cell isolated from a living body so that the polynucleotide can be expressed using a known gene editing technique under the control of an appropriate promoter. It may be produced by incorporating it into.
  • Known gene editing techniques include endonucleases such as zinc finger nucleases, TALENs (transcriptional activation-like effector nucleases), CRISPR (Crustered Regularly Interspaced Short Palindromic Repeat) -Cas9 system, and CRISPR-Cas3 systems. ..
  • the polynucleotide in question functionally linked downstream of the appropriate promoter (that is, can be expressed under the control of the promoter).
  • Examples thereof include a method of incorporating a polynucleotide having a coding sequence linked thereto into a non-coding region of the cell genome, a method of incorporating the polynucleotide into the downstream of an endogenous promoter of the cell genome, and the like.
  • the "macrophages or macrophage progenitor cells isolated from the living body containing the mutant IL-4R polypeptide" include macrophages isolated from the living body.
  • the macrophage progenitor cell contains the mutant IL-4R polypeptide, and the extracellular region of the mutant IL-4R polypeptide is arranged outside the macrophage or the macrophage progenitor cell. preferable.
  • the polynucleotide or the expression vector of the present invention is introduced into the macrophage or macrophage progenitor cell isolated from the living body as described above, and the macrophage or macrophage progenitor cell isolated from the living body is introduced.
  • Examples thereof include a method for expressing the mutant IL-4R polypeptide.
  • a method of directly editing an endogenous IL-4 receptor gene into a mutant IL-4 receptor gene sequence encoding the mutant IL-4R polypeptide by using the above-mentioned known gene editing technique will be mentioned. be able to.
  • the macrophage or macrophage progenitor cell obtained by the above method may be subsequently subjected to cell recovery, separation and purification by a known method.
  • the cells When the polynucleotide or the expression vector is introduced into the macrophage progenitor cell, the cells may be cultured after the polynucleotide or the expression vector is introduced to separate or purify the macrophage.
  • macrophage progenitor cells are cultured using a petri dish, the macrophage progenitor cells and macrophages are separated or purified by utilizing the property that when they differentiate into macrophages, they adhere to the petri dish and other cells are contained in the supernatant. can do.
  • a prophylactic or therapeutic agent for an inflammatory disease, a neurodegenerative disease, or an ischemic disease which comprises the "mutated IL-4R macrophages and a pharmacologically acceptable additive" of the present invention (hereinafter, "the inflammatory disease, the neurodegenerative disease”. Or, also referred to as a “preventive or therapeutic agent for ischemic diseases"), the present invention is not particularly limited as long as the mutant IL-4R macrophage contains a pharmacologically acceptable additive.
  • Such pharmacologically acceptable additives include saline, buffered saline, cell culture medium, dextrose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants. Buffers and preservatives, isotonic agents, fillers, and lubricants can be mentioned.
  • Examples of the inflammatory disease, neurodegenerative disease, or ischemic disease include diseases in which macrophages are involved in pathogenesis.
  • the inflammatory diseases include autoimmune diseases, autoimmune diseases, collagen diseases, ulcerative colitis, Crohn's diseases, autoimmune hepatitis, encephalitis, myocarditis, nephritis, and chronic inflammation predominantly M1 macrophages that increases with aging. Dermatitis, photoaging of the skin, ophthalmitis, pancreatitis, enteritis, diabetes, arteriosclerosis such as atherosclerosis, vasculitis, parasite infection, allergic disease, implant-to-host disease (GVHD) can.
  • autoimmune diseases include autoimmune diseases, autoimmune diseases, collagen diseases, ulcerative colitis, Crohn's diseases, autoimmune hepatitis, encephalitis, myocarditis, nephritis, and chronic inflammation predominantly M1 macrophages that increases with aging. Dermatitis, photoaging of the skin, ophthalmitis,
  • neurodegenerative diseases include Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyloidosis, Parkinson's disease, amyotrophic lateral sclerosis, and spinocerebellar degeneration.
  • ischemic disease ischemia-reperfusion disorder can be mentioned, and stroke or myocardial infarction can be mentioned.
  • the prophylactic or therapeutic agent for inflammatory disease, neurodegenerative disease, or ischemic disease is administered to a subject in need of treatment for inflammatory disease, neurodegenerative disease, or ischemic disease by a method known to those skilled in the art.
  • Intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intra-articular, intrasacral, intracranial, intrathecal, And submucosal (medullary fluid) injections can be mentioned.
  • the amount of the mutant IL-4R macrophages contained in the prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease to be administered is the type, location, and severity of the inflammatory disease, neurodegenerative disease, or ischemic disease. It can be appropriately adjusted according to the age, weight, condition, etc. of the subject to be treated, but preferably 1 ⁇ 10 4 to 1 ⁇ 10 10 pieces, preferably 1 ⁇ 10 5 to 1 ⁇ 10 per administration. Nine , more preferably 1 ⁇ 10 6 to 1 ⁇ 10 8 can be mentioned.
  • the prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease to be administered is 4 times a day, 3 times, 2 times or 1 time, every 1 day, every 2 days, every 3 days, every 4 days. Independent administration every 5, 5 days, once a week, every 7 days, every 8 days, every 9 days, twice a week, twice a month, once a month, every 1 month, or every 2 months be able to.
  • the target for administration of the prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease and the macrophages used for producing the prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease.
  • it may be the same as or different from the donor of macrophage precursor cells. That is, the donor and the administration subject may be the same or disagreeable.
  • the administration target is a human
  • the macrophages or macrophage precursor cells used for producing a prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease are selected from the patient himself / herself as the administration target.
  • the collected autologous cells may be used, or the allogeneic cells collected from another person may be used.
  • allogeneic cells for example, ⁇ 2 microglobulin ( ⁇ 2M), human leukocyte antigen (HLA), etc. may be modified by a known method such as genome editing technology in order to reduce immune rejection. ..
  • “Differentiation or polarization inducer from macrophage or macrophage progenitor cell isolated from living body to M2 macrophage containing the polynucleotide or expression vector of the present invention as an active ingredient” (hereinafter, “differentiation or induction of polarization to M2 macrophage”
  • the agent also referred to as
  • the agent is not particularly limited as long as the polynucleotide or the expression vector is used as an active ingredient, and is introduced into macrophages or macrophage progenitor cells isolated from a living body by the above-mentioned known method, and is determined as necessary.
  • macrophages or macrophage progenitor cells By culturing in the medium of, macrophages or macrophage progenitor cells can be differentiated or polarized into M2 macrophages.
  • the acquisition of different properties or functions in cells from macrophage precursor cells other than M0 macrophages to becoming M0 macrophages is differentiated, that M0 macrophages become M1 macrophages or M2 macrophages, and that M1 macrophages become M2.
  • Becoming a macrophage or changing from an M2 macrophage to an M1 macrophage is called polarization.
  • M2 macrophage differentiation or polarization inducer may contain the above-mentioned pharmacologically acceptable additive.
  • the polynucleotide may be incorporated into the expression vector.
  • the present polynucleotide or the present expression vector may be introduced into macrophages or macrophage progenitor cells isolated from the living body and cultured in a predetermined medium as needed.
  • the present polynucleotide or the present expression vector may be the present mutant IL-4R macrophages. Can be introduced into macrophages or macrophage progenitor cells by the same method as described in the method for producing.
  • macrophages or macrophage precursor cells isolated from a living body containing the polynucleotide or the expression vector can be used for the prevention or treatment of inflammatory diseases, neurodegenerative diseases, or ischemic diseases. Used as a prophylactic or therapeutic agent for inflammatory, neurodegenerative, or ischemic diseases, and 2) prophylactic or therapeutic agents for inflammatory, neurodegenerative, or ischemic diseases, characterized by administration to patients in need.
  • Macrophages or macrophages precursor cells isolated from the living body containing the polynucleotide or the expression vector for the purpose of 3) macrophages or macrophages precursor cells isolated from the living body containing the polynucleotide or the expression vector.
  • prophylactic or therapeutic agents for inflammatory diseases, neurodegenerative diseases, or ischemic diseases can be mentioned for use in the preparation of prophylactic or therapeutic agents for inflammatory diseases, neurodegenerative diseases, or ischemic diseases.
  • the polynucleotide or the present expression vector for use as a differentiation or polarization inducing agent for M2 macrophages and 2) the present polynucleotide or the present expression vector to M2 macrophages.
  • Use in the preparation of differentiation or polarization inducers can be mentioned.
  • kits for producing a differentiation or polarization inducing agent for M2 macrophages which comprises the above-mentioned polynucleotide or the present expression vector, can be mentioned, and the above-mentioned kit includes the above-mentioned present invention.
  • the present invention is not particularly limited as long as it has a polynucleotide or the present expression vector, and a description for producing a differentiation or polarization inducer into M2 macrophages and an expression vector containing the present polynucleotide are introduced into macrophages or macrophage progenitor cells. It may contain a reagent used to do so.
  • Example 1 (Preparation of cell line expressing human IL-4 receptor)
  • the mutant amino acid sequence of the human IL-4 receptor in which isoleucine at position 242 is replaced with asparagine (I242N) (I242N mutant human IL-4 receptor: SEQ ID NO: 3) was produced by outsourcing to Vector Builder.
  • THP-1 cells purchased from JCRB Cell Bank, National Institute of Pharmaceutical Sciences, Health and Nutrition
  • control cells cells were prepared in which a polynucleotide encoding the red fluorescent protein mCherry or green fluorescent protein (GFP) was introduced in the same manner as above in place of the I242N mutant human IL-4 receptor.
  • GFP green fluorescent protein
  • RAW264.7 cells are DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (Fetal Bovine Serum), and THP-1 cells are THP medium (2 mM Glutamine, 10). the% FBS-containing RPMI1640 (Roswell Park Memorial Institute medium, Inc.)), 37 °C, were cultured in CO 2 5%. THP-1 cells were cultured in THP medium containing 250 nM phorbol 12-myristate 13-acetylate (PMA) for 24 hours, differentiated into macrophages, and then in THP medium without PMA. The cells were cultured for another 2 days.
  • PMA phorbol 12-myristate 13-acetylate
  • RNA is isolated by Trizol reagent (Invitrogen), cDNA is synthesized, and expression of IL-4 receptor and human GAPDH or mouse Gapdh (internal standard) is confirmed by real-time PCR method. bottom.
  • the primers shown in Table 1 below were used.
  • the hIL-4R forward primer set forth in SEQ ID NO: 4 and the hIL-4R reverse primer set forth in SEQ ID NO: 5 are used to obtain the amino acid sequence of the human IL-4 receptor shown in wild-type SEQ ID NO: 1 and the human shown in SEQ ID NO: 2. Any of the mutated amino acid sequences of the IL-4 receptor (I242N mutated human IL-4) can be amplified.
  • FIG. 1 The results are shown in Fig. 1.
  • the upper row shows RAW264.7 cells (mCherry RAW264.7 cells) into which a polynucleotide encoding mCherry was introduced and RAW264.7 cells (mut_IL-) into which a polynucleotide encoding an I242N mutant human IL-4 receptor was introduced. This is the result of 4R RAW264.7 cells).
  • the lower row shows THP-1 cells (GFP THP-1 cells) into which a polynucleotide encoding a GFP was introduced and THP-1 cells (GFP THP-1 cells) into which a polynucleotide encoding an I242N mutant human IL-4 receptor was introduced.
  • the vertical axis is the relative mutant human IL-4 receptor expression level when the IL-4 receptor expression level in the control mCherry RAW264.7 cells or GFP THP-1 cells is 1.
  • the expression level was corrected by human GAPDH or mouse Gapdh, and tested by Unpaired T-test.
  • human IL-4 receptor was expressed in mut_IL-4R RAW264.7 cells, and I242N mutant human IL-4 receptor introduced by lentivirus was expressed. ..
  • the expression of human IL-4 receptor was increased as compared with the control, and the I242N mutant human IL-4 receptor introduced by lentivirus was increased. It was confirmed that the body was expressed.
  • Example 2 (Expression of M2 macrophage marker Arg1 or CD206 in cells expressing the I242N mutant human IL-4 receptor) The differentiation or polarization of the cells prepared in Example 1 into M2 macrophages was examined by the expression of Arg1 which is a mouse M2 macrophage marker and CD206 which is a human M2 macrophage marker.
  • the specific method is as follows.
  • a polynucleotide encoding a wild-type human IL-4 receptor was introduced into RAW264.7 cells to prepare WT_IL-4R RAW264.7 cells.
  • the expression of Arg1 and the expression of CD206 were examined by real-time PCR.
  • mCherry RAW264.7 cells, mut_IL-4R RAW264.7 cells and WT_IL-4R RAW264.7 cells were cultured in RAW medium, and mCherry THP-1 cells and mut_IL-4R THP-1 cells were cultured in THP cells ( Contains PMA for the first 24 hours of the 3 days).
  • the primers shown in Table 2 below were used.
  • Fig. 2 The results are shown in Fig. 2.
  • the expression level was corrected by human GAPDH or mouse Gapdh, RAW264.7 cells were tested by 1-way ANOVA, and THP-1 cells were tested by Unpaired T-test.
  • Arg1 was the control mCherry RAW264.7 cells and WT_IL-4R RAW264.7 cells, but in the mut_IL-4R RAW264.7 cells, Arg1 was the control mCherry RAW264.7. It was 59 times more expressed than the cells and 28 times more than the WT_IL-4R RAW264.7 cells.
  • CD206 was low in the control GFP THP-1 cells, but the expression of CD206 in the mut_IL-4R THP-1 cells was 13 times higher than that in the control cells. Therefore, it was revealed that macrophage progenitor cells differentiate and / or polarize into M2 macrophages by expressing the I242N mutant human IL-4 receptor.
  • Example 3 (Suppression of M1 macrophage induction in cells expressing the I242N mutant human IL-4 receptor) Whether M2 macrophages polarized from mut_IL-4R RAW264.7 cells are maintained as M2 macrophages without being polarized to M1 macrophages when lipopolysaccharide (LPS), which is a humoral factor that induces M1 macrophages, is allowed to act. Whether or not it was examined by the expression of tumor necrosis factor (TNF ⁇ ), which is an Arg1 and M1 macrophage marker.
  • LPS lipopolysaccharide
  • TNF ⁇ tumor necrosis factor
  • the results are shown in Fig. 3.
  • the expression level was corrected by human GAPDH or mouse Gapdh, and tested by Unpaired T-test.
  • the expression of the M1 macrophage marker TNF ⁇ is increased by acting on LPS in the control mCherry RAW264.7 cells, but even if it is allowed to act on LPS in mut_IL-4R RAW264.7 cells.
  • the expression of TNF ⁇ was hardly increased.
  • the expression of Arg1 of the M2 macrophage marker was not significantly different from that of Basal (control without LPS), which did not act on LPS, and no decrease in Arg1 expression was observed. rice field.
  • Example 4 (Polarization to M2 macrophages in human IL-4 receptors other than I242N)
  • the mutation at position 242 shown in SEQ ID NO: 2 was used, but from macrophage progenitor cells to M2 macrophages in control GFP and mutations other than positions 242 of (1) to (10) below.
  • the differentiation and / or polarization effect of Agr1 was examined by the expression of Agr1.
  • Expression of the receptor, R200W mutant human IL-4 receptor, I242D mutant human IL-4 receptor, C251W mutant human IL-4 receptor, and K308N mutant human IL-4 receptor increased the expression of Arg1. rice field. That is, it was clarified that even if the mutant IL-4R polypeptide other than the I242N mutant human IL-4 receptor is contained, it can be differentiated or polarized into M2 macrophages.
  • Mouse bone marrow cells were collected by a known method and then cultured in a culture medium containing M-CSF (20 ng / ml) for 5 days to differentiate into bone marrow-derived macrophages (BMDM). Then, in the presence of M-CSF (20 ng / ml), as in Example 1, a polynucleotide or control (polynucleotide encoding GFP) encoding the I242N mutant human IL-4 receptor was used with a lentivirus. The BMDM was introduced into the above BMDM, and after culturing for 48 hours, the BMDM was collected and stored.
  • Each BMDM was stimulated with IL-4 (100 pg / ml) for 24 hours to polarize it into M2 macrophages, and then cultured as it was for 1 week, and the expression of Arg1 which is an M2 macrophage marker was analyzed by real-time PCR.
  • the horizontal axis is the number of days of culture (Day 0 is defined after stimulation with IL-4 for 24 hours), and the vertical axis is the relative expression level of Arg1 when the expression level of Arg1 on the control Day 0 is 1.
  • BMDM (cells) polarized into M2 macrophages only by control IL-4 stimulation reduced the expression of Arg1 on Day 3, whereas cells expressing the I242N mutant human IL-4 receptor were Day 7 (1 week). Later), the expression of Arg1 continued. Therefore, it was clarified that if the mutant IL-4R polypeptide is contained, it can be differentiated or polarized into M2 macrophages and then maintained as M2 macrophages for at least one week. In the conventional method, when M2 macrophages enter the body, they lose their function within a few days, so that the therapeutic effect is limited.
  • the M2 macrophage produced in the present invention can maintain its function as an M2 macrophage for a long period of time, it is expected to have a high therapeutic effect on inflammatory diseases, myoneurodegenerative diseases, and ischemic diseases.
  • Example 6 secretion of IL-10
  • IL-10 which is a potent anti-inflammatory cytokine
  • a polynucleotide encoding an I242N mutant human IL-4 receptor or control polynucleotide encoding GFP
  • the present invention can be used as a biopharmacy for inflammatory diseases, wound healing, neurodegenerative diseases, and ischemic diseases.

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Abstract

The present invention addresses the problem of providing: a polypeptide that contributes to polarization into M2 macrophages independent of IL-4; and an induction agent for differentiation or polarization into M2 macrophages.　A polypeptide derived from the human interleukin-4 receptor, the polypeptide being the result of modifying the amino acid sequence given by SEQ ID NO:1 by adding, deleting, or substituting one or more amino acids, preferably by substituting the 242nd isoleucine of the amino acid sequence given by SEQ ID NO:1 with asparagine.

Description

ヒトインターロイキン４受容体の変異ポリペプチド、及びＭ２マクロファージへの分化又は分極誘導剤Mutant polypeptide of human interleukin-4 receptor and differentiation or polarization inducer to M2 macrophages
　本発明は、ヒトインターロイキン４受容体に由来するＩＬ－４受容体の変異ポリペプチドや、前記ＩＬ－４受容体の変異ポリペプチドをコードするポリヌクレオチドや、前記ポリヌクレオチドを含有する発現ベクターや、前記ポリヌクレオチド又は発現ベクターを含有する生体から分離されたマクロファージ又はマクロファージ前駆細胞や、前記ポリペプチドを含有する生体から分離されたマクロファージ又はマクロファージ前駆細胞や、前記マクロファージ又はマクロファージ前駆細胞と薬理学的に許容される添加剤を含む、炎症疾患、神経変性疾患若しくは虚血性疾患の予防又は治療剤や、前記ポリヌクレオチド又は前記発現ベクターを有効成分とする、生体から分離されたマクロファージ又はマクロファージ前駆細胞からＭ２マクロファージへの分化又は分極誘導剤や、生体から分離されたマクロファージ又はマクロファージ前駆細胞からＭ２マクロファージへの分化又は分極誘導方法に関する。 The present invention includes a mutant polypeptide of IL-4 receptor derived from human interleukin 4 receptor, a polynucleotide encoding the mutant polypeptide of the IL-4 receptor, an expression vector containing the polynucleotide, and the like. , Macrophages or macrophage precursor cells isolated from the living body containing the polynucleotide or expression vector, macrophages or macrophage precursor cells isolated from the living body containing the polypeptide, or pharmacologically with the macrophages or macrophage precursor cells. From macrophages or macrophage precursor cells isolated from living organisms, including prophylactic or therapeutic agents for inflammatory diseases, neurodegenerative diseases or ischemic diseases, and macrophages or macrophage precursor cells containing the polynucleotide or the expression vector as an active ingredient. The present invention relates to an agent for inducing differentiation or polarization into M2 macrophages, and a method for inducing differentiation or polarization of macrophages or macrophage precursor cells isolated from a living body into M2 macrophages.
　炎症疾患、神経変性疾患、及び虚血性疾患には様々な疾患が存在し、それぞれに応じた薬剤が開発されているが、未だ十分な効果が期待できない疾患も少なくない。こうした炎症性疾患、神神経変性疾患、及び虚血性疾患の病巣には免疫細胞であるマクロファージが存在し、病態形成に関与していると考えられている。 There are various diseases such as inflammatory diseases, neurodegenerative diseases, and ischemic diseases, and drugs have been developed according to each, but there are still many diseases for which sufficient effects cannot be expected. Macrophages, which are immune cells, are present in the lesions of these inflammatory diseases, divine neurodegenerative diseases, and ischemic diseases, and are considered to be involved in pathogenesis.
　近年マクロファージの研究が進み、マクロファージには炎症を惹起するＭ１マクロファージと炎症抑制や組織修復を担うＭ２マクロファージが存在することが分かってきた。Ｍ２マクロファージは過剰な炎症を沈め、アルツハイマーで見られるβアミロイド蛋白質等を貪食する作用等、生体にとって有益な働きを持っていることが分かってきている（非特許文献１、２参照）。また、Ｍ１とＭ２マクロファージの割合は加齢とともにＭ１優位となり、Ｍ１／Ｍ２バランス不均衡が老化を進める一因とも考えられている(非特許文献３参照)。このような背景からさまざまな方法でＭ２マクロファージを活性化させる研究が進んでいる。具体的には液性因子や低分子化合物を全身投与しＭ２マクロファージを活性化させる手法が主体であるが、臨床応用に向けた流れは今のところない（非特許文献４参照）。その理由として、たとえば活性化させてＭ２マクロファージとするための液性因子はＢリンパ球に作用し、ＩｇＥの産生を高めることでアレルギー疾患の悪化を引き起こす等、マクロファージ以外の細胞へ与える危険性が知られている。さらに液性因子では血液脳関門（ｂｌｏｏｄ－ｂｒａｉｎ　ｂａｒｒｉｅｒ）を通過することができないため、アルツハイマー病をはじめとする中枢の炎症・神経変性疾患には十分な効果を発揮できない等の課題を有していた。 In recent years, research on macrophages has progressed, and it has become clear that macrophages include M1 macrophages that induce inflammation and M2 macrophages that are responsible for inflammation suppression and tissue repair. It has been found that M2 macrophages have beneficial functions for living organisms, such as sinking excessive inflammation and phagocytosing β-amyloid protein and the like found in Alzheimer's disease (see Non-Patent Documents 1 and 2). In addition, the ratio of M1 and M2 macrophages becomes predominant in M1 with aging, and it is considered that the M1 / M2 imbalance promotes aging (see Non-Patent Document 3). Against this background, research is underway to activate M2 macrophages by various methods. Specifically, the main method is to systemically administer a humoral factor or a small molecule compound to activate M2 macrophages, but there is currently no trend toward clinical application (see Non-Patent Document 4). The reason is that, for example, a humoral factor for activating to M2 macrophages acts on B lymphocytes and increases IgE production, which causes exacerbation of allergic diseases, and there is a risk of giving to cells other than macrophages. Are known. Furthermore, since humoral factors cannot cross the blood-brain barrier, they have problems such as not being able to exert sufficient effects on central inflammatory and neurodegenerative diseases such as Alzheimer's disease. rice field.
　さらに炎症部位においては、Ｍ１マクロファージを活性化させる生理活性物質が豊富なため、液性因子の全身投与によって一度は活性化されたＭ２マクロファージも、実際の炎症部位においてはＭ１マクロファージに再分極している可能性が示唆されている。 Furthermore, since the inflamed site is rich in bioactive substances that activate M1 macrophages, M2 macrophages that were once activated by systemic administration of humoral factors are repolarized into M1 macrophages in the actual inflamed site. It is suggested that there is a possibility.
　以上より、液性因子に頼らず、炎症部位でＭ１マクロファージへと分極しにくく、Ｍ２マクロファージを活性化する物質の開発が求められている。 From the above, it is required to develop a substance that does not rely on humoral factors, is less likely to polarize to M1 macrophages at the inflamed site, and activates M2 macrophages.
　ところで、これまで血液悪性リンパ腫、膀胱癌又は脳腫瘍において、ＩＬ－４受容体の変異体が報告されている（非特許文献５～７参照）。しかしながら、かかるＩＬ－４受容体の変異とマクロファージの分化又は分極との関係は知られていない。 By the way, variants of IL-4 receptor have been reported in hematological malignant lymphoma, bladder cancer or brain tumor (see Non-Patent Documents 5 to 7). However, the relationship between such IL-4 receptor mutations and macrophage differentiation or polarization is unknown.
　これまで、Ｍ２マクロファージを増やす試みとしては、液性因子や化合物を全身に投与する方法が試みられていた。しかしながら、全身投与の問題点として、実際の炎症部位では、Ｍ１マクロファージへ誘導する液性因子の局所濃度が高く、投与量の割に効果が低いことや、他の細胞へ作用して、喘息等の免疫疾患を悪化させる危険性があった。また、Ｍ２マクロファージはアルツハイマー病等の中枢神経の変性疾患への効果も期待されているが、液性因子や化合物では血液脳関門を通過できないため、こうした手法ではアルツハイマー病等の中枢神経系の治療に用いることができない等が課題であった。さらに、Ｍ２マクロファージの活性化にＩＬ－４を用いた場合には、ＩＬ－４がマクロファージ以外へと作用することによる未熟Ｔ細胞の増殖、マスト細胞の増殖等の発現増強等、多くの作用を有していることからＭ２マクロファージの活性化以外の作用をもたらすという問題もあった。そこで、本発明の課題は、ＩＬ－４に依存せずにＭ２マクロファージへの分化又は分極に関与するポリペプチドを提供することや、Ｍ２マクロファージへの分化又は分極誘導剤を提供することにある。 Until now, as an attempt to increase M2 macrophages, a method of systemically administering a humoral factor or compound has been attempted. However, as a problem of systemic administration, in the actual inflammatory site, the local concentration of the humoral factor that induces M1 macrophages is high, the effect is low for the dose, and it acts on other cells to cause asthma, etc. There was a risk of exacerbating immune disorders. In addition, M2 macrophages are also expected to be effective for degenerative diseases of the central nervous system such as Alzheimer's disease, but since humoral factors and compounds cannot cross the blood-brain barrier, these methods are used to treat the central nervous system such as Alzheimer's disease. The problem was that it could not be used for. Furthermore, when IL-4 is used to activate M2 macrophages, it exerts many actions such as proliferation of immature T cells and enhancement of expression of mast cell proliferation due to the action of IL-4 on other than macrophages. Since it has, there is also a problem that it brings about an action other than activation of M2 macrophages. Therefore, an object of the present invention is to provide a polypeptide involved in differentiation or polarization into M2 macrophages without depending on IL-4, or to provide a differentiation or polarization inducer into M2 macrophages.
　発明者はヒトインターロイキン４受容体に着目し、マクロファージに対してヒトインターロイキン４受容体の変異体を導入すれば、Ｍ２マクロファージとして活性化されるのではないかと考え検討を進めた。その結果、マクロファージ又はマクロファージ前駆細胞においてヒトインターロイキン４受容体の変異体を発現させることでＭ２マクロファージへの分化又は分極に成功し、本発明を完成した。 The inventor focused on the human interleukin-4 receptor, and proceeded with the study, thinking that if a mutant of the human interleukin-4 receptor was introduced into the macrophage, it would be activated as an M2 macrophage. As a result, the present invention was completed by succeeding in differentiation or polarization into M2 macrophages by expressing a mutant of human interleukin-4 receptor in macrophages or macrophage progenitor cells.
　すなわち、本発明は、以下のとおりである。
〔１〕ヒトインターロイキン４受容体に由来するポリペプチドであって、配列番号１に示すアミノ酸配列において、１又は数個のアミノ酸が付加、欠失若しくは置換したポリペプチド。
〔２〕以下の（１－１）又は（１－２）である、上記〔１〕記載のポリペプチド。
（１－１）ヒトインターロイキン４受容体に由来するポリペプチドであって、配列番号１に示すアミノ酸配列において、３７番目のアスパラギン酸、６２番目のチロシン、７８番目のアスパラギン、１１５番目のフェニルアラニン、１７６番目のアスパラギン、２００番目のアルギニン、２４２番目のイソロイシン、２５１番目のシステイン、及び／又は３０８番目のリシンが置換したポリペプチド；
（１－２）配列番号１に示すアミノ酸配列において、上記（１－１）において置換した３７番目、６２番目、７８番目、１１５番目、１７６番目、２００番目、２４２番目、２５１番目及び／又は３０８番目以外のアミノ酸において１又は数個のアミノ酸が付加、欠失若しくは置換したポリペプチドを含み、かつ、ホルボール１２－ミリスタート１３－アセタートで刺激したＴＨＰ－１細胞、若しくはＲＡＷ２６４．７細胞で発現した場合に、当該ポリペプチドを発現していない場合と比較してヒトＭ２マクロファージマーカーの発現を増加するポリペプチド。
〔３〕配列番号１に示すアミノ酸配列において、３７番目のアスパラギン酸、６２番目のチロシン、７８番目のアスパラギン、１１５番目のフェニルアラニン、１７６番目のアスパラギン、２００番目のアルギニン、２４２番目のイソロイシン、２５１番目のシステイン、及び／又は３０８番目のリシンが置換したポリペプチドが、以下の（２－１）～（２－９）のいずれかである、上記〔２〕記載のポリペプチド。
（２－１）配列番号１に示すアミノ酸配列において、３７番目におけるアスパラギン酸が中性極性アミノ酸へ置換したポリペプチド；
（２－２）配列番号１に示すアミノ酸配列において、６２番目におけるチロシンが中性極性アミノ酸へ置換したポリペプチド；
（２－３）配列番号１に示すアミノ酸配列において、７８番目におけるアスパラギンが中性極性アミノ酸へ置換したポリペプチド；
（２－４）配列番号１に示すアミノ酸配列において、１１５番目におけるフェニルアラニンが中性非極性アミノ酸へ置換したポリペプチド；
（２－５）配列番号１に示すアミノ酸配列において、１７６番目におけるアスパラギンが中性極性アミノ酸へ置換したポリペプチド；
（２－６）配列番号１に示すアミノ酸配列において、２００番目におけるアルギニンが中性非極性アミノ酸へ置換したポリペプチド；
（２－７）配列番号１に示すアミノ酸配列において、２４２番目におけるイソロイシンが酸性アミノ酸又は中性非極性アミノ酸へ置換したポリペプチド；
（２－８）配列番号１に示すアミノ酸配列において、２５１番目におけるシステインが中性非極性アミノ酸へ置換したポリペプチド；
（２－９）配列番号１に示すアミノ酸配列において、３０８番目におけるリシンが中性極性アミノ酸へ置換したポリペプチド；
〔４〕ヒトインターロイキン４受容体に由来するポリペプチドであって、配列番号１に示すアミノ酸配列において、２４２番目のイソロイシンが置換したポリペプチドであることを特徴とする上記〔２〕記載のポリペプチド。
〔５〕ヒトインターロイキン４受容体に由来するポリペプチドであって、配列番号１に示すアミノ酸配列において、２４２番目のイソロイシンがアスパラギンへ置換したポリペプチドであることを特徴とする上記〔４〕記載のポリペプチド。
〔６〕上記〔１〕～〔５〕のいずれか記載のポリペプチドをコードするポリヌクレオチド。
〔７〕上記〔６〕記載のポリヌクレオチドを含有する発現ベクター。
〔８〕上記〔６〕記載のポリヌクレオチド又は上記〔７〕記載の発現ベクターを含有する、生体から分離されたマクロファージ又はマクロファージ前駆細胞。
〔９〕上記〔１〕～〔５〕のいずれか記載のポリペプチドを含有する、生体から分離されたマクロファージ又はマクロファージ前駆細胞。
〔１０〕上記〔８〕又は〔９〕記載のマクロファージ又はマクロファージ前駆細胞と薬理学的に許容される添加剤を含む、炎症疾患、神経変性疾患若しくは虚血性疾患の予防又は治療剤。
〔１１〕上記〔６〕記載のポリヌクレオチド又は上記〔７〕記載の発現ベクターを有効成分とする、生体から分離されたマクロファージ又はマクロファージ前駆細胞からＭ２マクロファージへの分化又は分極誘導剤。
〔１２〕上記〔６〕記載のポリヌクレオチド又は上記〔７〕記載の発現ベクターを生体から分離されたマクロファージ又はマクロファージ前駆細胞に導入することを特徴とする、生体から分離されたマクロファージ又はマクロファージ前駆細胞からＭ２マクロファージへの分化又は分極誘導方法。 That is, the present invention is as follows.
[1] A polypeptide derived from the human interleukin-4 receptor, wherein one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1.
[2] The polypeptide according to the above [1], which is the following (1-1) or (1-2).
(1-1) A polypeptide derived from the human interleucine 4 receptor, in the amino acid sequence shown in SEQ ID NO: 1, aspartic acid at position 37, tyrosine at position 62, asparagin at position 78, and phenylalanine at position 115. Polypeptide substituted with aspartic acid at position 176, arginine at position 200, isoleucine at position 242, cysteine at position 251 and / or lysine at position 308;
(1-2) In the amino acid sequence shown in SEQ ID NO: 1, the 37th, 62nd, 78th, 115th, 176th, 200th, 242nd, 251st and / or 308 substituted in the above (1-1). It was expressed in THP-1 cells or RAW264.7 cells containing a polypeptide in which one or several amino acids were added, deleted or substituted in a non-th amino acid and stimulated with Holbol 12-millistart 13-acetate. A polypeptide that, in some cases, increases the expression of human M2 macrophage markers as compared to the case where the polypeptide is not expressed.
[3] In the amino acid sequence shown in SEQ ID NO: 1, aspartic acid at position 37, tyrosine at position 62, asparagin at position 78, phenylalanine at position 115, arginine at position 176, arginine at position 200, isoleucine at position 242, and isoleucine at position 251. The polypeptide according to [2] above, wherein the polypeptide substituted with cysteine and / or lysine at position 308 is any of the following (2-1) to (2-9).
(2-1) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which aspartic acid at position 37 is replaced with a neutral polar amino acid;
(2-2) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which tyrosine at position 62 is replaced with a neutral polar amino acid;
(2-3) In the amino acid sequence shown in SEQ ID NO: 1, the polypeptide in which asparagine at position 78 is replaced with a neutral polar amino acid;
(2-4) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which phenylalanine at position 115 is replaced with a neutral non-polar amino acid;
(2-5) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which asparagine at position 176 is replaced with a neutral polar amino acid;
(2-6) In the amino acid sequence shown in SEQ ID NO: 1, the polypeptide in which arginine at position 200 is replaced with a neutral non-polar amino acid;
(2-7) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which isoleucine at position 242 is replaced with an acidic amino acid or a neutral non-polar amino acid;
(2-8) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which cysteine at position 251 is replaced with a neutral non-polar amino acid;
(2-9) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which lysine at position 308 is replaced with a neutral polar amino acid;
[4] The polypeptide according to the above [2], which is a polypeptide derived from the human interleukin-4 receptor and is a polypeptide substituted with isoleucine at position 242 in the amino acid sequence shown in SEQ ID NO: 1. peptide.
[5] The above-mentioned [4], which is a polypeptide derived from the human interleukin-4 receptor, wherein the isoleucine at position 242 is replaced with asparagine in the amino acid sequence shown in SEQ ID NO: 1. Polypeptide.
[6] A polynucleotide encoding the polypeptide according to any one of the above [1] to [5].
[7] An expression vector containing the polynucleotide according to [6] above.
[8] Macrophages or macrophage progenitor cells isolated from a living body containing the polynucleotide described in [6] above or the expression vector described in [7] above.
[9] Macrophage or macrophage progenitor cell isolated from a living body containing the polypeptide according to any one of the above [1] to [5].
[10] A prophylactic or therapeutic agent for an inflammatory disease, a neurodegenerative disease or an ischemic disease, which comprises the macrophage or macrophage progenitor cell according to the above [8] or [9] and a pharmacologically acceptable additive.
[11] An agent for inducing differentiation or polarization of macrophages or macrophage progenitor cells isolated from a living body into M2 macrophages, which comprises the polynucleotide described in [6] above or the expression vector described in [7] above as an active ingredient.
[12] Macrophages or macrophage progenitor cells isolated from living organisms, characterized in that the polynucleotide described in [6] above or the expression vector described in [7] above is introduced into macrophages or macrophage progenitor cells isolated from living organisms. A method for inducing differentiation or polarization from M2 macrophages.
　本発明のＭ２マクロファージへの分化又は分極誘導剤により、生体から分離されたマクロファージ又はマクロファージ前駆細胞をＭ２マクロファージへと分化又は分極することが可能となる。 The differentiation or polarization inducing agent for M2 macrophages of the present invention makes it possible to differentiate or polarize macrophages or macrophage progenitor cells isolated from living organisms into M2 macrophages.
実施例１において、Ｉ２４２Ｎ変異（２４２位のイソロイシンをアスパラギンに置換）ヒトＩＬ－４受容体をコードするポリヌクレオチドをＲＡＷ２６４．７細胞又はＴＨＰ－１細胞に導入してＩＬ－４受容体の発現を調べた結果である。In Example 1, a polynucleotide encoding an I242N mutation (isoleucine at position 242 replaced with asparagine) human IL-4 receptor was introduced into RAW264.7 cells or THP-1 cells to express IL-4 receptor. This is the result of the investigation. 実施例２において、Ｉ２４２Ｎ変異ヒトＩＬ－４受容体をコードするポリヌクレオチドをＲＡＷ２６４．７細胞又はＴＨＰ－１細胞に導入して、Ａｒｇ１又はＣＤ２０６の発現を調べた結果である。In Example 2, the polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into RAW264.7 cells or THP-1 cells, and the expression of Arg1 or CD206 was examined. 実施例３において、Ｉ２４２Ｎ変異ヒトＩＬ－４受容体をコードするポリヌクレオチドをＲＡＷ２６４．７細胞に導入し、ＬＰＳを作用させてＴＮＦα又はＡｒｇ１の発現を調べた結果である。In Example 3, the polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into RAW264.7 cells, and LPS was allowed to act to examine the expression of TNFα or Arg1. 実施例４において、Ｉ２４２Ｎ以外の変異ヒトＩＬ－４受容体をコードするポリヌクレオチドをＲＡＷ２６４．７細胞に導入して、Ａｒｇ１の発現を調べた結果である。In Example 4, a polynucleotide encoding a mutant human IL-4 receptor other than I242N was introduced into RAW264.7 cells, and the expression of Arg1 was examined. 実施例５において、Ｉ２４２Ｎ変異ヒトＩＬ－４受容体をコードするポリヌクレオチドを骨髄由来マクロファージ（ＢＭＤＭ）に導入し、Ａｒｇ１の発現を調べた結果である。In Example 5, the polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into a bone marrow-derived macrophage (BMDM), and the expression of Arg1 was examined. 実施例６において、Ｉ２４２Ｎ変異ヒトＩＬ－４受容体をコードするポリヌクレオチドをＲＡＷ２６４．７細胞に導入し、ＩＬ－１０の発現を調べた結果である。This is the result of introducing a polynucleotide encoding the I242N mutant human IL-4 receptor into RAW264.7 cells and examining the expression of IL-10 in Example 6.
　本発明の「ヒトインターロイキン４受容体に由来するポリペプチドであって、配列番号１に示すアミノ酸配列において、１又は数個のアミノ酸が付加、欠失若しくは置換したポリペプチド（以下、「本件変異ＩＬ－４Ｒポリペプチド」ともいう）」におけるヒトインターロイキン４受容体（ヒトＩＬ－４受容体）は、配列番号１に示すアミノ酸配列からなるポリペプチドであり、ＮＣＢＩにおいてＲｅｆｅｒｅｎｃｅ　Ｎｏ．ＮＰ＿０００４０９．１として登録されている。 A polypeptide derived from the "human interleukin-4 receptor" of the present invention in which one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1 (hereinafter, "mutation of the present invention". The human interleukin-4 receptor (human IL-4 receptor) in "IL-4R polypeptide") is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and in NCBI, Reference No. It is registered as NP_000409.1.
　上記本件変異ＩＬ－４Ｒポリペプチドにおける「配列番号１に示すアミノ酸配列において、１又は数個のアミノ酸が付加、欠失若しくは置換したポリペプチド」としては、好ましくは「配列番号１に示すアミノ酸配列における細胞外領域又は膜貫通領域を構成するアミノ酸において、１又は数個のアミノ酸が付加、欠失若しくは置換したポリペプチド」、より好ましくは「配列番号１に示すアミノ酸配列における膜貫通領域を構成するアミノ酸において、１又は数個のアミノ酸が付加、欠失若しくは置換したポリペプチド」を挙げることができる。細胞外領域又は膜貫通領域は、公知の細胞外領域又は膜貫通領域予測アルゴリズムを用いて予測することができる。なお、配列番号１に示すアミノ酸配列において、１番目から２３２番目が細胞外領域、２３３番目から２５６番目が膜貫通領域と考えられる。上記「細胞外領域を構成するアミノ酸」において１又は数個のアミノ酸が付加、欠失若しくは置換する位置としては、配列番号１に示すアミノ酸配列における３７番目のアスパラギン酸、６２番目のチロシン、７８番目のアスパラギン、１１５番目のフェニルアラニン、１７６番目のアスパラギン、及び２００番目のアルギニンを挙げることができる。上記「膜貫通領域を構成するアミノ酸」において１又は数個のアミノ酸が付加、欠失若しくは置換する位置としては、２４２番目のイソロイシン及び２５１番目のシステインを挙げることができ、２４２番目のイソロイシンを好適に挙げることができる。 The "polypeptide in which one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1" in the above-mentioned mutant IL-4R polypeptide is preferably "in the amino acid sequence shown in SEQ ID NO: 1." "Amino acids constituting the extracellular region or transmembrane region, in which one or several amino acids are added, deleted or substituted", more preferably "amino acids constituting the transmembrane region in the amino acid sequence shown in SEQ ID NO: 1". In, a polypeptide to which one or several amino acids have been added, deleted or substituted ”can be mentioned. The extracellular space or transmembrane region can be predicted using a known extracellular space or transmembrane region prediction algorithm. In the amino acid sequence shown in SEQ ID NO: 1, the 1st to 232nd regions are considered to be extracellular regions, and the 233rd to 256th regions are considered to be transmembrane regions. The positions where one or several amino acids are added, deleted or substituted in the above "amino acids constituting the extracellular region" are aspartic acid at position 37, tyrosine at position 62, and position 78 in the amino acid sequence shown in SEQ ID NO: 1. Aspartic acid, 115th phenylalanine, 176th aspartic acid, and 200th arginine. As the position where one or several amino acids are added, deleted or substituted in the above "amino acid constituting the transmembrane region", isoleucine at position 242 and cysteine at position 251 can be mentioned, and isoleucine at position 242 is preferable. Can be listed in.
　本明細書において、中性非極性アミノ酸とは、グリシン（Ｇ）、アラニン（Ａ）、バリン（Ｖ）、プロリン（Ｐ）、ロイシン（Ｌ）、イソロイシン（Ｉ）、フェニルアラニン（Ｆ）、メチオニン（Ｍ）、又はトリプトファン（Ｗ）を意味する。また、中性極性アミノ酸とは、アスパラギン（Ｎ）、グルタミン（Ｑ）、セリン（Ｓ）、スレオニン（Ｔ）、チロシン（Ｙ）、又はシステイン（Ｃ）を意味する。さらに、酸性アミノ酸とは、アスパラギン酸（Ｄ）、グルタミン酸（Ｅ）を意味する。 In the present specification, the neutral non-polar amino acids include glycine (G), alanine (A), valine (V), proline (P), leucine (L), isoleucine (I), phenylalanine (F), and methionine ( It means M) or tryptophan (W). The neutral polar amino acid means asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), or cysteine (C). Further, the acidic amino acid means aspartic acid (D) and glutamic acid (E).
　上記「配列番号１に示すアミノ酸配列において、１又は数個のアミノ酸が付加、欠失若しくは置換したポリペプチド」として、好ましくは「配列番号１に示すアミノ酸配列において、１又は数個のアミノ酸が置換したポリペプチド」を挙げることができる。かかる「配列番号１に示すアミノ酸配列において、１又は数個のアミノ酸が置換したポリペプチド」としては、好ましくは以下の（２－１）～（２－９）のいずれかを挙げることができ、より好ましくは以下の（２－１’）～（２－９’）のいずれかを挙げることができ、さらに好ましくは２４２番目におけるイソロイシンがアスパラギンへ置換したポリペプチドを好適に挙げることができるがこれらに限定されない。 As the above-mentioned "polypeptide in which one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1", preferably "in the amino acid sequence shown in SEQ ID NO: 1, one or several amino acids are substituted". Polypeptides ". As the "polypeptide in which one or several amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 1", preferably any of the following (2-1) to (2-9) can be mentioned. More preferably, any of the following (2-1') to (2-9') can be mentioned, and more preferably, a polypeptide in which isoleucine at the 242nd position is replaced with asparagine can be preferably mentioned. Not limited to.
（２－１）３７番目におけるアスパラギン酸の中性極性アミノ酸への置換；
（２－２）６２番目におけるチロシンの中性極性アミノ酸への置換；
（２－３）７８番目におけるアスパラギンの中性極性アミノ酸への置換；
（２－４）１１５番目におけるフェニルアラニンの中性非極性アミノ酸への置換；
（２－５）１７６番目におけるアスパラギンの中性極性アミノ酸への置換；
（２－６）２００番目におけるアルギニンの中性非極性アミノ酸への置換；
（２－７）２４２番目におけるイソロイシンの酸性アミノ酸又は中性非極性アミノ酸への置換；
（２－８）２５１番目におけるシステインの中性非極性アミノ酸への置換；
（２－９）３０８番目におけるリシンの中性極性アミノ酸への置換； (2-1) Substitution of aspartic acid at position 37 with a neutral polar amino acid;
(2-2) Substitution of tyrosine with neutral polar amino acid at position 62;
(2-3) Substitution of asparagine at position 78 with neutral polar amino acid;
(2-4) Substitution of phenylalanine at position 115 with a neutral non-polar amino acid;
(2-5) Substitution of asparagine at position 176 with a neutral polar amino acid;
(2-6) Substitution of arginine at position 200 with a neutral non-polar amino acid;
(2-7) Substitution of isoleucine at position 242 with an acidic or neutral non-polar amino acid;
(2-8) Substitution of cysteine at position 251 with a neutral non-polar amino acid;
(2-9) Substitution of lysine at position 308 with a neutral polar amino acid;
（２－１’）３７番目におけるアスパラギン酸のアスパラギンへの置換；
（２－２’）６２番目におけるチロシンのシステインへの置換；
（２－３’）７８番目におけるアスパラギンのチロシンへの置換；
（２－４’）１１５番目におけるフェニルアラニンのロイシンへの置換；
（２－５’）１７６番目におけるアスパラギンのセリンへの置換；
（２－６’）２００番目におけるアルギニンのトリプトファンへの置換；
（２－７’）２４２番目におけるイソロイシンのアスパラギン又はアスパラギン酸への置換；
（２－８’）２５１番目におけるシステインのトリプトファンへの置換；
（２－９’）３０８番目におけるリシンのアスパラギンへの置換； (2-1') Substitution of aspartic acid for asparagine at position 37;
(2-2') Substitution of tyrosine with cysteine at position 62;
(2-3') Substitution of asparagine to tyrosine at position 78;
(2-4') Replacement of phenylalanine with leucine at position 115;
(2-5') Substitution of asparagine with serine at position 176;
(2-6') Substitution of arginine for tryptophan at position 200;
(2-7') Substitution of isoleucine for asparagine or aspartic acid at position 242;
(2-8') Replacement of cysteine with tryptophan at position 251;
(2-9') Substitution of lysine for asparagine at position 308;
　本明細書において、「１又は数個のアミノ酸」としては、例えば１～３０個の範囲内、好ましくは１～２０個の範囲内、より好ましくは１～１５個の範囲内、さらに好ましくは１～１０個の範囲内、より好ましくは１～５個の範囲内、さらに好ましくは１～３個の範囲内、より好ましくは１～２個、最も好ましくは１個のアミノ酸を意味する。 In the present specification, the "1 or several amino acids" is, for example, in the range of 1 to 30, preferably in the range of 1 to 20, more preferably in the range of 1 to 15, and further preferably in the range of 1. It means an amino acid in the range of ~ 10, more preferably in the range of 1 to 5, still more preferably in the range of 1 to 3, more preferably 1 to 2, and most preferably 1 amino acid.
　本明細書において、「ホルボール１２－ミリスタート１３－アセタート（ＰＭＡ）で刺激したＴＨＰ－１細胞、若しくはＲＡＷ２６４．７細胞で発現した場合に、当該ポリペプチドを発現していない場合と比較してヒトＭ２マクロファージマーカーの発現を増加するポリペプチド」としては、本件変異ＩＬ－４Ｒポリペプチドを発現するＰＭＡで刺激したＴＨＰ－１細胞、若しくはＲＡＷ２６４．７細胞と、本件変異ＩＬ－４Ｒポリペプチドを発現していないＰＭＡで刺激したＴＨＰ－１細胞、若しくはＲＡＷ２６４．７細胞を培養してＭ２マクロファージマーカーの発現を調べ、本件変異ＩＬ－４Ｒポリペプチドを発現していない場合と比較して本件変異ＩＬ－４Ｒポリペプチドを発現するＰＭＡで刺激したＴＨＰ－１細胞、若しくはＲＡＷ２６４．７細胞におけるＭ２マクロファージマーカーの発現が増加、好ましくは２倍以上、より好ましくは５倍以上、さらに好ましくは１０倍以上増加となるポリペプチドを挙げることができる。ここで、ヒトＭ２マクロファージマーカーとしては、ＣＤ２０６、ＣＤ１６３、ＩＬ－２７ＲＡ、ＣＤ３６７、Ｆ１３Ａ１、又はＣＣＬ２２を挙げることができる。なお、ＴＨＰ－１細胞はＰＭＡで刺激することによってマクロファージに分化する。 In the present specification, "when expressed in THP-1 cells stimulated with Holball 12-millistart 13-acetate (PMA) or RAW264.7 cells, humans are compared with the case where the polypeptide is not expressed. As "polypeptides that increase the expression of M2 macrophage markers", THP-1 cells or RAW264.7 cells stimulated with PMA expressing the mutant IL-4R polypeptide and the mutant IL-4R polypeptide are expressed. The expression of M2 macrophage markers was examined by culturing THP-1 cells or RAW264.7 cells stimulated with non-PMA, and compared with the case where the mutant IL-4R polypeptide was not expressed, the mutant IL-4R. The expression of M2 macrophage markers in THP-1 cells or RAW264.7 cells stimulated with PMA expressing the polypeptide is increased, preferably 2 times or more, more preferably 5 times or more, still more preferably 10 times or more. Polypeptides can be mentioned. Here, examples of the human M2 macrophage marker include CD206, CD163, IL-27RA, CD367, F13A1, and CCL22. In addition, THP-1 cells differentiate into macrophages by stimulating with PMA.
　本発明の「本件変異ＩＬ－４Ｒポリペプチドをコードするポリヌクレオチド（以下、「本件ポリヌクレオチド」ともいう）」は、本件変異ＩＬ－４Ｒポリペプチドをコードするポリヌクレオチドであれば特に制限されず、アミノ酸をコードするために選択されるコドンは、発現させる宿主細胞の種類に応じて適宜最適化されてもよい。 The "polynucleotide encoding the mutant IL-4R polypeptide (hereinafter, also referred to as the" polynucleotide ")" of the present invention is not particularly limited as long as it is a polynucleotide encoding the mutant IL-4R polypeptide. The codon selected to encode the amino acid may be appropriately optimized depending on the type of host cell to be expressed.
　本発明の「本件ポリヌクレオチドを含有する発現ベクター（以下、「本件発現ベクター」ともいう）における発現ベクターとしては直鎖状でも環状でもよく、ウイルスベクターでも、プラスミド等の非ウイルスベクターでも、トランスポゾンによるベクターでもよい。また、かかるベクターには、プロモーターやターミネーター等の制御配列や、薬剤耐性遺伝子、レポーター遺伝子等の選択マーカー配列を含有していてもよい。 The expression vector in the "expression vector containing the Polynucleotide of the present invention (hereinafter, also referred to as the" expression vector ") may be linear or cyclic, and may be a viral vector or a non-viral vector such as a plasmid, depending on a transposon. It may be a vector. In addition, such a vector may contain a control sequence such as a promoter or a terminator, or a selectable marker sequence such as a drug resistance gene or a reporter gene.
　前記ウイルスベクターとしては、レンチウイルスベクター、レトロウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクターを挙げることができ、レンチウイルスベクターを好適に挙げることができる。なお、レンチウイルスベクターやレトロウイルスベクターを用いれば、導入遺伝子はホスト細胞のゲノムへ取り込まれるため、長期間かつ安定に発現することが可能となる。短期間での発現が好ましい際には、ゲノムへの組み込みのないアデノウイルスベクター、アデノ随伴ウイルスベクターが好適である。 Examples of the virus vector include a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated virus vector, and a lentiviral vector can be preferably mentioned. If a lentiviral vector or a retroviral vector is used, the transgene is incorporated into the genome of the host cell, so that it can be stably expressed for a long period of time. When expression in a short period of time is preferable, an adenovirus vector or an adeno-associated virus vector that does not integrate into the genome is preferable.
　上記プロモーターとしては、レンチウイルスのＬＴＲプロモーター、ＳＶ４０初期プロモーター、サイトメガロウイルスプロモーター、単純ヘルペスウイルスのチミジンキナーゼプロモーター等のウイルス由来プロモーター、ホスホグリセリン酸キナーゼ（ＰＧＫ）プロモーター、Ｘｉｓｔプロモーター、β－アクチンプロモーター、ＲＮＡポリメラーゼＩＩプロモーター、ＣＡＧプロモーター、ＥＦ1Ａ等の哺乳類由来プロモーターを挙げることができる。また、テトラサイクリンによって誘導されるテトラサイクリン応答型プロモーター、インターロイキンによって誘導されるＭｘ１プロモーターや、マクロファージでプロモーター活性を有するＣＤ６８プロモーター、Ｍ-ＣＳＦプロモーター等を用いてもよい。 Examples of the promoter include a lentivirus LTR promoter, an SV40 initial promoter, a cytomegalovirus promoter, a virus-derived promoter such as the thymidine kinase promoter of simple herpesvirus, a phosphoglycerate kinase (PGK) promoter, an Xist promoter, and a β-actin promoter. Mammalian-derived promoters such as RNA polymerase II promoter, CAG promoter, and EF1A can be mentioned. Further, a tetracycline-responsive promoter induced by tetracycline, an Mx1 promoter induced by interleukin, a CD68 promoter having a promoter activity in macrophages, an M-CSF promoter and the like may be used.
　本発明の「本件ポリヌクレオチド又は本件発現ベクターを含有する、生体から分離されたマクロファージ又はマクロファージ前駆細胞」（以下、「本件変異ＩＬ－４ＲマクロファージＡ」ともいう）における「マクロファージ」にはＭ１マクロファージとＭ２マクロファージの二つのフェノタイプが含まれる。また、「マクロファージ前駆細胞」には、少なくともＭ１マクロファージ又はＭ２マクロファージに分化可能な細胞が含まれ、ＣＤ１４陽性（ＣＤ１４＋）細胞、非活性マクロファージ（Ｍ０マクロファージ）、単球、単芽球、骨髄単核球、末梢血単核球、ＥＳ細胞、ｉＰＳ細胞を挙げることができる。前記Ｍ２マクロファージは、抗炎症性マクロファージとも呼ばれ、ＣＤ１４陽性の細胞であるマクロファージのうち、ヒトにおいてはＣＤ２０６、ＣＤ１６３、ＩＬ－２７ＲＡ、ＣＤ３６７、Ｆ１３Ａ１、又はＣＣＬ２２を発現する細胞、マウスにおいてはアルギナーゼ１（Ａｒｇ１）、ＣＤ２０６、ｉＮＯＳ、ＣＤ１６３又はＣＤ２０４を発現する細胞を挙げることができる。なお、本件変異ＩＬ－４ＲマクロファージＡ及び後述の本件変異ＩＬ－４ＲマクロファージＢには、それらを培養して得られた培養物も包含する。 The "macrophages" in the "macrophages or macrophage progenitor cells isolated from the living body containing the polynucleotide or expression vector of the present invention" (hereinafter, also referred to as "mutant IL-4R macrophages A") are referred to as M1 macrophages. Two phenotypes of M2 macrophages are included. In addition, "macrophage progenitor cells" include cells capable of differentiating into at least M1 macrophages or M2 macrophages, and include CD14-positive (CD14 + ) cells, inactive macrophages (M0 macrophages), monocytes, monocytes, and bone marrow. Examples thereof include nuclear spheres, peripheral blood mononuclear cells, ES cells, and iPS cells. The M2 macrophages are also called anti-inflammatory macrophages, and among macrophages that are CD14-positive cells, cells expressing CD206, CD163, IL-27RA, CD367, F13A1, or CCL22 in humans, and arginase 1 in mice. Cells expressing (Arg1), CD206, iNOS, CD163 or CD204 can be mentioned. The mutant IL-4R macrophage A and the mutant IL-4R macrophage B described later also include cultures obtained by culturing them.
　上記単球は循環血液中の血球細胞であり、血流から離れて結合組織に入るとマクロファージに分化しうる。上記単球の由来としては、ヒト、サル、マウス等の哺乳動物を挙げることができる。上記単球は、患者から採血した末梢血全血から遠心分離等の公知の手法を用いて末梢血単核球（ＰＢＭＣｓ）を分離し、必要に応じて免疫磁気ビーズ法等により精製した単球を用いることができる。かかる単球は、Ｍ－ＣＳＦ等のサイトカインの存在下で培養することで活性化させてマクロファージへと分化させることができる。また上記単球は、公知の手法を用いて骨髄液、ＥＳ細胞、ｉＰＳ細胞等から作製することもできる。 The monocytes are blood cells in the circulating blood and can differentiate into macrophages when they leave the bloodstream and enter connective tissue. Examples of the origin of the monocytes include mammals such as humans, monkeys, and mice. Peripheral blood mononuclear cells (PBMCs) are separated from peripheral blood whole blood collected from a patient by a known method such as centrifugation, and the monocytes are purified by immunomagnetic bead method or the like as necessary. Can be used. Such monocytes can be activated and differentiated into macrophages by culturing in the presence of cytokines such as M-CSF. The monocytes can also be produced from bone marrow fluid, ES cells, iPS cells and the like using a known method.
　上記「本件ポリヌクレオチドを含有する」とは、生体から分離されたマクロファージ又はマクロファージ前駆細胞内に本件ポリヌクレオチドを含有していればよく、例えば本件ポリヌクレオチドが発現ベクターに組み込まれた状態で含有することや、本件ポリヌクレオチドが生体から分離されたマクロファージ又はマクロファージ前駆細胞のゲノムに組み込まれた状態で含有することを挙げることができる。 The above-mentioned "containing the polynucleotide" may mean that the polynucleotide is contained in a macrophage or a macrophage precursor cell separated from a living body, and for example, the polynucleotide is contained in a state of being incorporated into an expression vector. In addition, it can be mentioned that the polynucleotide is contained in a state of being integrated into the genome of a macrophage or a macrophage precursor cell isolated from a living body.
　本件変異ＩＬ－４ＲマクロファージＡを作製する方法としては、本件ポリヌクレオチド又は本件発現ベクターを、生体から分離されたマクロファージ又はマクロファージ前駆細胞に導入する方法を挙げることができる。あるいは、生体内から分離された線維芽細胞等の体細胞に本件ポリヌクレオチド又は本件発現ベクターを導入し、その後かかる体細胞からｉＰＳ細胞を樹立することによっても製造することができる。本件ポリヌクレオチド又は本件発現ベクターを生体から分離されたマクロファージ又はマクロファージ前駆細胞に導入する方法としては特に制限されないが、ウイルス感染法、カルシウムリン酸法、リポフェクション法、マイクロインジェクション法、エレクトロポレーション法等の公知の方法を挙げることができ、ウイルス感染法により導入する方法を好適に挙げることができる。 Examples of the method for producing the mutant IL-4R macrophage A include a method of introducing the polynucleotide or the expression vector into macrophages or macrophage progenitor cells isolated from a living body. Alternatively, it can also be produced by introducing the polynucleotide or the present expression vector into somatic cells such as fibroblasts isolated from the living body, and then establishing iPS cells from such somatic cells. The method for introducing the polynucleotide or the expression vector into macrophages or macrophage progenitor cells isolated from the living body is not particularly limited, but is limited to a virus infection method, a calcium phosphate method, a lipofection method, a microinjection method, an electroporation method, etc. A known method of the above can be mentioned, and a method of introducing by a virus infection method can be preferably mentioned.
　また、本件変異ＩＬ－４ＲマクロファージＡは、本件ポリヌクレオチドを、公知の遺伝子編集技術を用いて、適切なプロモーターの制御下で発現可能なように、生体から分離されたマクロファージ又はマクロファージ前駆細胞のゲノムに組み込むことによって作製してもよい。公知の遺伝子編集技術としては、ジンクフィンガーヌクレアーゼ、ＴＡＬＥＮ（転写活性化様エフェクターヌクレアーゼ）、ＣＲＩＳＰＲ（Ｃｌｕｓｔｅｒｅｄ  Ｒｅｇｕｌａｒｌｙ  Ｉｎｔｅｒｓｐａｃｅｄ  Ｓｈｏｒｔ  Ｐａｌｉｎｄｒｏｍｉｃ  Ｒｅｐｅａｔ）－Ｃａｓ９システム、又はＣＲＩＳＰＲ－Ｃａｓ３システム等のエンドヌクレアーゼを用いる技術が挙げられる。適切なプロモーターの制御下で発現可能なようにポリヌクレオチドを細胞ゲノムに組み込む方法としては、適切なプロモーターの下流に機能的に連結した本件ポリヌクレオチド（即ち、当該プロモーターの制御下で発現可能なようにコード配列を連結したポリヌクレオチド）を、細胞ゲノムの非コード領域等に組み込む方法や本件ポリヌクレオチドを、細胞ゲノムの内在性プロモーターの下流に組み込む方法等が挙げられる。 In addition, the mutant IL-4R macrophage A is a genome of a macrophage or a macrophage progenitor cell isolated from a living body so that the polynucleotide can be expressed using a known gene editing technique under the control of an appropriate promoter. It may be produced by incorporating it into. Known gene editing techniques include endonucleases such as zinc finger nucleases, TALENs (transcriptional activation-like effector nucleases), CRISPR (Crustered Regularly Interspaced Short Palindromic Repeat) -Cas9 system, and CRISPR-Cas3 systems. .. As a method of incorporating a polynucleotide into the cell genome so that it can be expressed under the control of an appropriate promoter, the polynucleotide in question functionally linked downstream of the appropriate promoter (that is, can be expressed under the control of the promoter). Examples thereof include a method of incorporating a polynucleotide having a coding sequence linked thereto into a non-coding region of the cell genome, a method of incorporating the polynucleotide into the downstream of an endogenous promoter of the cell genome, and the like.
　本発明の「本件変異ＩＬ－４Ｒポリペプチドを含有する、生体から分離されたマクロファージ又はマクロファージ前駆細胞」（以下、「本件変異ＩＬ－４ＲマクロファージＢ」ともいう）としては、生体から分離されたマクロファージ又はマクロファージ前駆細胞に本件変異ＩＬ－４Ｒポリペプチド含有していればよく、本件変異ＩＬ－４Ｒポリペプチドの細胞外領域がマクロファージ又はマクロファージ前駆細胞の細胞外に配置されるようになっていることが好ましい。 The "macrophages or macrophage progenitor cells isolated from the living body containing the mutant IL-4R polypeptide" (hereinafter, also referred to as "mutated IL-4R macrophages B") of the present invention include macrophages isolated from the living body. Alternatively, it is sufficient that the macrophage progenitor cell contains the mutant IL-4R polypeptide, and the extracellular region of the mutant IL-4R polypeptide is arranged outside the macrophage or the macrophage progenitor cell. preferable.
　本件変異ＩＬ－４ＲマクロファージＢを作製する方法としては、上述のように本件ポリヌクレオチド又は本件発現ベクターを生体から分離されたマクロファージ又はマクロファージ前駆細胞に導入し、生体から分離されたマクロファージ又はマクロファージ前駆細胞内で本件変異ＩＬ－４Ｒポリペプチドを発現させる方法を挙げることができる。また、上述の公知の遺伝子編集技術を用いて、内因性のＩＬ－４受容体遺伝子を本件変異ＩＬ－４Ｒポリペプチドをコードする変異ＩＬ－４受容体遺伝子配列へと直接編集する方法等を挙げることができる。 As a method for producing the mutant IL-4R macrophage B, the polynucleotide or the expression vector of the present invention is introduced into the macrophage or macrophage progenitor cell isolated from the living body as described above, and the macrophage or macrophage progenitor cell isolated from the living body is introduced. Examples thereof include a method for expressing the mutant IL-4R polypeptide. In addition, a method of directly editing an endogenous IL-4 receptor gene into a mutant IL-4 receptor gene sequence encoding the mutant IL-4R polypeptide by using the above-mentioned known gene editing technique will be mentioned. be able to.
　上記方法で得られたマクロファージ又はマクロファージ前駆細胞は、引き続き、公知の方法による細胞回収、分離、精製を行ってもよい。なお、マクロファージ前駆細胞に本件ポリヌクレオチド又は本件発現ベクターを導入した場合には、本件ポリヌクレオチド又は本件発現ベクターを導入後に細胞を培養して、マクロファージを分離、又は精製してもよい。また、シャーレを用いてマクロファージ前駆細胞を培養した場合には、マクロファージに分化するとシャーレに付着して他の細胞は上清に含まれるという性質を利用してマクロファージ前駆細胞とマクロファージを分離、又は精製することができる。 The macrophage or macrophage progenitor cell obtained by the above method may be subsequently subjected to cell recovery, separation and purification by a known method. When the polynucleotide or the expression vector is introduced into the macrophage progenitor cell, the cells may be cultured after the polynucleotide or the expression vector is introduced to separate or purify the macrophage. In addition, when macrophage progenitor cells are cultured using a petri dish, the macrophage progenitor cells and macrophages are separated or purified by utilizing the property that when they differentiate into macrophages, they adhere to the petri dish and other cells are contained in the supernatant. can do.
　本発明の「本件変異ＩＬ－４Ｒマクロファージと薬理学的に許容される添加剤を含む、炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療剤（以下、「本件炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療剤」ともいう）としては、本件変異ＩＬ－４Ｒマクロファージに薬理学的に許容される添加剤を含んでいれば特に制限されない。かかる薬理学的に許容される添加剤としては、生理食塩水、緩衝生理食塩水、細胞培養培地、デキストロース、注射用水、グリセロール、エタノール及びこれらの組合せ、安定剤、可溶化剤及び界面活性剤、緩衝剤及び防腐剤、等張化剤、充填剤、並びに潤滑剤を挙げることができる。 A prophylactic or therapeutic agent for an inflammatory disease, a neurodegenerative disease, or an ischemic disease, which comprises the "mutated IL-4R macrophages and a pharmacologically acceptable additive" of the present invention (hereinafter, "the inflammatory disease, the neurodegenerative disease". Or, also referred to as a "preventive or therapeutic agent for ischemic diseases"), the present invention is not particularly limited as long as the mutant IL-4R macrophage contains a pharmacologically acceptable additive. Such pharmacologically acceptable additives include saline, buffered saline, cell culture medium, dextrose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants. Buffers and preservatives, isotonic agents, fillers, and lubricants can be mentioned.
　上記炎症疾患、神経変性疾患、若しくは虚血性疾患としては、マクロファージが病態形成に関与する疾患を挙げることができる。上記炎症疾患としては、自己炎症性疾患、自己免疫疾患、膠原病、潰瘍性大腸炎、クローン病、自己免疫性肝炎、脳炎、心筋炎、腎炎、老化に伴い増加するＭ１マクロファージ優位の慢性炎症、皮膚炎、皮膚の光老化、眼炎、膵炎、腸炎、糖尿病、アテローム性動脈硬化等の動脈硬化症、血管炎、寄生虫感染症、アレルギー疾患、移植片対宿主病（ＧＶＨＤ）を挙げることができる。また、上記神経変性疾患としては、アルツハイマー病、クロイツフェルトヤコブ病、多発性硬化症、アミロイドーシス、パーキンソン病、筋萎縮性側索硬化症、脊髄小脳変性症を挙げることができる。さらに、上記虚血性疾患としては、虚血再灌流障害を挙げることができ、脳卒中又は心筋梗塞を挙げることができる。 Examples of the inflammatory disease, neurodegenerative disease, or ischemic disease include diseases in which macrophages are involved in pathogenesis. The inflammatory diseases include autoimmune diseases, autoimmune diseases, collagen diseases, ulcerative colitis, Crohn's diseases, autoimmune hepatitis, encephalitis, myocarditis, nephritis, and chronic inflammation predominantly M1 macrophages that increases with aging. Dermatitis, photoaging of the skin, ophthalmitis, pancreatitis, enteritis, diabetes, arteriosclerosis such as atherosclerosis, vasculitis, parasite infection, allergic disease, implant-to-host disease (GVHD) can. Examples of the neurodegenerative diseases include Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyloidosis, Parkinson's disease, amyotrophic lateral sclerosis, and spinocerebellar degeneration. Further, as the ischemic disease, ischemia-reperfusion disorder can be mentioned, and stroke or myocardial infarction can be mentioned.
　本件炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療剤は、当業者に既知の方法を用いて、炎症疾患、神経変性疾患、若しくは虚血性疾患の治療を必要とする被験体に投与することができ、投与方法としては、静脈内、腫瘍内、皮内、皮下、筋肉内、腹腔内、動脈内、髄内、心臓内、関節内、滑液嚢内、頭蓋内、髄腔内、及びくも膜下（髄液）への注射を挙げることができる。 The prophylactic or therapeutic agent for inflammatory disease, neurodegenerative disease, or ischemic disease is administered to a subject in need of treatment for inflammatory disease, neurodegenerative disease, or ischemic disease by a method known to those skilled in the art. Intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intra-articular, intrasacral, intracranial, intrathecal, And submucosal (medullary fluid) injections can be mentioned.
　投与する本件炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療剤に含まれる本件変異ＩＬ－４Ｒマクロファージの量は、炎症疾患、神経変性疾患、若しくは虚血性疾患の種類、位置、重症度、治療を受ける被験体の年齢、体重及び状態等に応じて適宜調整できるが、好ましくは、一回の投与において１×１０４～１×１０１０個、好ましくは１×１０５～１×１０９個、より好ましくは１×１０６～１×１０８個を挙げることができる。 The amount of the mutant IL-4R macrophages contained in the prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease to be administered is the type, location, and severity of the inflammatory disease, neurodegenerative disease, or ischemic disease. It can be appropriately adjusted according to the age, weight, condition, etc. of the subject to be treated, but preferably 1 × 10 4 to 1 × 10 10 pieces, preferably 1 × 10 5 to 1 × 10 per administration. Nine , more preferably 1 × 10 6 to 1 × 10 8 can be mentioned.
　投与する本件炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療剤は、１日４回、３回、２回又は１回、１日おき、２日おき、３日おき、４日おき、５日おき、週１回、７日おき、８日おき、９日おき、週２回、月２回、月１回、１カ月おき、又は２カ月おきに独立して投与する方法を挙げることができる。 The prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease to be administered is 4 times a day, 3 times, 2 times or 1 time, every 1 day, every 2 days, every 3 days, every 4 days. Independent administration every 5, 5 days, once a week, every 7 days, every 8 days, every 9 days, twice a week, twice a month, once a month, every 1 month, or every 2 months be able to.
　なお、本件炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療剤の投与対象と、本件炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療剤を製造するために使用されるマクロファージ又はマクロファージ前駆細胞のドナーとは、同じであっても異なってもよい。すなわち、ドナーと投与対象は一致しても不一致でもよい。さらに、投与対象がヒトの場合において、本件炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療剤を製造するために使用されるマクロファージ又はマクロファージ前駆細胞としては、投与対象としての患者本人から採取した自家細胞を用いても、他人から採取した他家細胞を用いてもよい。なお、他家細胞を用いる場合には、免疫拒絶反応を低減するために、例えばβ２ミクログロブリン（β２Ｍ）やヒト白血球抗原（ＨＬＡ）等をゲノム編集技術等の公知の手法により改変してもよい。 It should be noted that the target for administration of the prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease, and the macrophages used for producing the prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease. Alternatively, it may be the same as or different from the donor of macrophage precursor cells. That is, the donor and the administration subject may be the same or disagreeable. Furthermore, when the administration target is a human, the macrophages or macrophage precursor cells used for producing a prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease are selected from the patient himself / herself as the administration target. The collected autologous cells may be used, or the allogeneic cells collected from another person may be used. When allogeneic cells are used, for example, β2 microglobulin (β2M), human leukocyte antigen (HLA), etc. may be modified by a known method such as genome editing technology in order to reduce immune rejection. ..
　本発明の「本件ポリヌクレオチド又は本件発現ベクターを有効成分とする、生体から分離されたマクロファージ又はマクロファージ前駆細胞からＭ２マクロファージへの分化又は分極誘導剤（以下、「本件Ｍ２マクロファージへの分化又は分極誘導剤」ともいう）としては、本件ポリヌクレオチド又は本件発現ベクターを有効成分としていれば特に制限されず、生体から分離されたマクロファージ又はマクロファージ前駆細胞に上記公知の方法で導入し、必要に応じて所定の培地で培養することによって、マクロファージ又はマクロファージ前駆細胞をＭ２マクロファージへ分化又は分極することが可能となる。なお、本明細書においてＭ０マクロファージ以外のマクロファージ前駆細胞からＭ０マクロファージになるまでの細胞における異なった性質又は機能の獲得のことを分化、Ｍ０マクロファージがＭ１マクロファージ若しくはＭ２マクロファージになること、Ｍ１マクロファージからＭ２マクロファージになること又はＭ２マクロファージからＭ１マクロファージになることを分極という。例えば、骨髄性幹細胞から単球、Ｍ０マクロファージを介してＭ２マクロファージとなる場合には、Ｍ０マクロファージになるまでが分化、Ｍ０マクロファージからＭ２マクロファージになるまでを分極という。本件Ｍ２マクロファージへの分化又は分極誘導剤は上記薬理学的に許容される添加剤を含んでもよい。かかる本件Ｍ２マクロファージへの分化又は分極誘導剤は、本件ポリヌクレオチドが上記発現ベクターに組み込まれていてもよい。 "Differentiation or polarization inducer from macrophage or macrophage progenitor cell isolated from living body to M2 macrophage containing the polynucleotide or expression vector of the present invention as an active ingredient" (hereinafter, "differentiation or induction of polarization to M2 macrophage" The agent (also referred to as) is not particularly limited as long as the polynucleotide or the expression vector is used as an active ingredient, and is introduced into macrophages or macrophage progenitor cells isolated from a living body by the above-mentioned known method, and is determined as necessary. By culturing in the medium of, macrophages or macrophage progenitor cells can be differentiated or polarized into M2 macrophages. In the present specification, the acquisition of different properties or functions in cells from macrophage precursor cells other than M0 macrophages to becoming M0 macrophages is differentiated, that M0 macrophages become M1 macrophages or M2 macrophages, and that M1 macrophages become M2. Becoming a macrophage or changing from an M2 macrophage to an M1 macrophage is called polarization. For example, when bone marrow stem cells become M2 macrophages via monocytes and M0 macrophages, differentiation is called until they become M0 macrophages, and polarization is called from M0 macrophages to M2 macrophages. The present M2 macrophage differentiation or polarization inducer may contain the above-mentioned pharmacologically acceptable additive. In the differentiation or polarization inducer for the M2 macrophages, the polynucleotide may be incorporated into the expression vector.
　本発明の「本件ポリヌクレオチドを生体から分離されたマクロファージ又はマクロファージ前駆細胞に導入することを特徴とする、生体から分離されたマクロファージ又はマクロファージ前駆細胞からＭ２マクロファージへの分化又は分極誘導方法」としては、本件ポリヌクレオチド又は本件発現ベクターを生体から分離されたマクロファージ又はマクロファージ前駆細胞に導入し、必要に応じて所定の培地で培養すればよく、本件ポリヌクレオチド又は本件発現ベクターは本件変異ＩＬ－４Ｒマクロファージを作製する方法に記載と同様の方法でマクロファージ又はマクロファージ前駆細胞に導入することが可能である。 The "method for inducing differentiation or polarization of macrophages or macrophage progenitor cells isolated from living organisms into M2 macrophages, which comprises introducing the polynucleotide into macrophages or macrophage progenitor cells isolated from living organisms" of the present invention. , The present polynucleotide or the present expression vector may be introduced into macrophages or macrophage progenitor cells isolated from the living body and cultured in a predetermined medium as needed. The present polynucleotide or the present expression vector may be the present mutant IL-4R macrophages. Can be introduced into macrophages or macrophage progenitor cells by the same method as described in the method for producing.
　本発明の別の態様１として、１）本件ポリヌクレオチド又は本件発現ベクターを含有する、生体から分離されたマクロファージ又はマクロファージ前駆細胞を、炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療に必要とする患者に投与することを特徴とする、炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療や、２）炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療剤として使用するための、本件ポリヌクレオチド又は本件発現ベクターを含有する、生体から分離されたマクロファージ又はマクロファージ前駆細胞や、３）本件ポリヌクレオチド又は本件発現ベクターを含有する、生体から分離されたマクロファージ又はマクロファージ前駆細胞の、炎症疾患、神経変性疾患、若しくは虚血性疾患の予防又は治療剤の調製における使用を挙げることができる。 As another aspect 1 of the present invention, 1) macrophages or macrophage precursor cells isolated from a living body containing the polynucleotide or the expression vector can be used for the prevention or treatment of inflammatory diseases, neurodegenerative diseases, or ischemic diseases. Used as a prophylactic or therapeutic agent for inflammatory, neurodegenerative, or ischemic diseases, and 2) prophylactic or therapeutic agents for inflammatory, neurodegenerative, or ischemic diseases, characterized by administration to patients in need. Macrophages or macrophages precursor cells isolated from the living body containing the polynucleotide or the expression vector for the purpose of 3) macrophages or macrophages precursor cells isolated from the living body containing the polynucleotide or the expression vector. Can be mentioned for use in the preparation of prophylactic or therapeutic agents for inflammatory diseases, neurodegenerative diseases, or ischemic diseases.
　本発明の別の態様２として、１）Ｍ２マクロファージへの分化又は分極誘導剤として使用するための、本件ポリヌクレオチド又は本件発現ベクターや、２）本件ポリヌクレオチド又は本件発現ベクターの、Ｍ２マクロファージへの分化又は分極誘導剤の調製における使用を挙げることができる。 As another aspect 2 of the present invention, 1) the polynucleotide or the present expression vector for use as a differentiation or polarization inducing agent for M2 macrophages, and 2) the present polynucleotide or the present expression vector to M2 macrophages. Use in the preparation of differentiation or polarization inducers can be mentioned.
　本発明の別の態様３として、上記本件ポリヌクレオチド又は本件発現ベクターを備えている、Ｍ２マクロファージへの分化又は分極誘導剤を作製するためのキットを挙げることができ、かかるキットとしては、上記本件ポリヌクレオチド又は本件発現ベクターを備えていれば特に制限されず、Ｍ２マクロファージへの分化又は分極誘導剤を作製するための説明書や、本件ポリヌクレオチドを含有する発現ベクターをマクロファージ又はマクロファージ前駆細胞に導入するために用いる試薬を含んでいてもよい。 As another aspect 3 of the present invention, a kit for producing a differentiation or polarization inducing agent for M2 macrophages, which comprises the above-mentioned polynucleotide or the present expression vector, can be mentioned, and the above-mentioned kit includes the above-mentioned present invention. The present invention is not particularly limited as long as it has a polynucleotide or the present expression vector, and a description for producing a differentiation or polarization inducer into M2 macrophages and an expression vector containing the present polynucleotide are introduced into macrophages or macrophage progenitor cells. It may contain a reagent used to do so.
　以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.
［実施例１］（ヒトＩＬ－４受容体を発現する細胞株の作製）
　配列番号１に示すヒトＩＬ－４受容体のアミノ酸配列において、２４２番目のイソロイシンをアスパラギンに置換（Ｉ２４２Ｎ）したヒトＩＬ－４受容体の変異アミノ酸配列（Ｉ２４２Ｎ変異ヒトＩＬ－４受容体：配列番号２）をコードするポリヌクレオチド（配列番号３）をＶｅｃｔｏｒ　ｂｕｉｌｄｅｒ社に委託して作製した。次に、作製したＩ２４２Ｎ変異ヒトＩＬ－４受容体をコードする上記ポリヌクレオチドを、レンチウイルスを用いてマウスマクロファージ不死化細胞株であるＲＡＷ２６４．７細胞（ｎ＝４：ケー・エー・シー社製）又はヒト単球由来の細胞株であり、活性化するとマクロファージに分化するＴＨＰ－１細胞（ｎ＝３国立研究開発法人医薬基盤・健康・栄養研究所　ＪＣＲＢ細胞バンクより購入）に導入した。コントロールの細胞として、Ｉ２４２Ｎ変異ヒトＩＬ－４受容体の代わりに赤色蛍光蛋白質であるｍＣｈｅｒｒｙ又は緑色蛍光タンパク質（ＧＦＰ）をコードするポリヌクレオチドを上記と同様に導入した細胞を作製した。 [Example 1] (Preparation of cell line expressing human IL-4 receptor)
In the amino acid sequence of the human IL-4 receptor shown in SEQ ID NO: 1, the mutant amino acid sequence of the human IL-4 receptor in which isoleucine at position 242 is replaced with asparagine (I242N) (I242N mutant human IL-4 receptor: SEQ ID NO: The polynucleotide encoding 2) (SEQ ID NO: 3) was produced by outsourcing to Vector Builder. Next, the prepared polynucleotide encoding the I242N mutant human IL-4 receptor was subjected to RAW264.7 cells (n = 4: KAC), which is a mouse macrophage immortalized cell line, using lentivirus. ) Or a human monocyte-derived cell line, which was introduced into THP-1 cells (n = 3 purchased from JCRB Cell Bank, National Institute of Pharmaceutical Sciences, Health and Nutrition) that differentiate into macrophages when activated. As control cells, cells were prepared in which a polynucleotide encoding the red fluorescent protein mCherry or green fluorescent protein (GFP) was introduced in the same manner as above in place of the I242N mutant human IL-4 receptor.
　それぞれのポリヌクレオチドを導入した上記細胞を、ＲＡＷ２６４．７細胞はＲＡＷ培地（１０％ＦＢＳ(Fetal Bovine Serum)含有のＤＭＥＭ(Dulbecco’s Modified Eagle Medium))、ＴＨＰ－１細胞はＴＨＰ培地（２ｍＭ　Ｇｌｕｔａｍｉｎｅ、１０％ＦＢＳ含有のＲＰＭＩ１６４０(Roswell Park Memorial Institute medium社)）により、３７℃、ＣＯ２　５％で培養した。ＴＨＰ－１細胞は２５０ｎＭ　ホルボール１２－ミリスタート１３－アセタート（Ｐｈｏｒｂｏｌ　１２－ｍｙｒｉｓｔａｔｅ　１３－ａｃｅｔａｔｅ：ＰＭＡ）を含むＴＨＰ培地で２４時間培養し、マクロファージへと分化させたのち、ＰＭＡを除いたＴＨＰ培地でさらに２日間培養した。その後、それぞれ細胞を回収し、トータルＲＮＡをＴｒｉｚｏｌ試薬 (Invitrogen社)により単離し、ｃＤＮＡを合成した後、ＩＬ－４受容体及びヒトＧＡＰＤＨ又はマウスＧａｐｄｈ(内部標準)の発現をリアルタイムＰＣＲ法によって確認した。リアルタイムＰＣＲ法においては、以下の表１に記載のプライマーを用いた。なお、配列番号４に記載のｈＩＬ－４Ｒ　フォワードプライマー及び配列番号５に記載のｈＩＬ－４Ｒ　リバースプライマーによって野生型の配列番号１に示すヒトＩＬ－４受容体のアミノ酸配列及び配列番号２に示すヒトＩＬ－４受容体の変異アミノ酸配列（Ｉ２４２Ｎ変異ヒトＩＬ－４）のいずれも増幅可能である。 RAW264.7 cells are DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (Fetal Bovine Serum), and THP-1 cells are THP medium (2 mM Glutamine, 10). the% FBS-containing RPMI1640 (Roswell Park Memorial Institute medium, Inc.)), 37 ℃, were cultured in CO 2 5%. THP-1 cells were cultured in THP medium containing 250 nM phorbol 12-myristate 13-acetylate (PMA) for 24 hours, differentiated into macrophages, and then in THP medium without PMA. The cells were cultured for another 2 days. Then, each cell is collected, total RNA is isolated by Trizol reagent (Invitrogen), cDNA is synthesized, and expression of IL-4 receptor and human GAPDH or mouse Gapdh (internal standard) is confirmed by real-time PCR method. bottom. In the real-time PCR method, the primers shown in Table 1 below were used. The hIL-4R forward primer set forth in SEQ ID NO: 4 and the hIL-4R reverse primer set forth in SEQ ID NO: 5 are used to obtain the amino acid sequence of the human IL-4 receptor shown in wild-type SEQ ID NO: 1 and the human shown in SEQ ID NO: 2. Any of the mutated amino acid sequences of the IL-4 receptor (I242N mutated human IL-4) can be amplified.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　結果を図１に示す。図１中、上段がｍＣｈｅｒｒｙをコードするポリヌクレオチドを導入したＲＡＷ２６４．７細胞（ｍＣｈｅｒｒｙ　ＲＡＷ２６４．７細胞）及びＩ２４２Ｎ変異ヒトＩＬ－４受容体をコードするポリヌクレオチドを導入したＲＡＷ２６４．７細胞（ｍｕｔ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞）の結果である。また、図１中、下段がＧＦＰをコードするポリヌクレオチドを導入したＴＨＰ－１細胞（ＧＦＰ　ＴＨＰ－１細胞）及びＩ２４２Ｎ変異ヒトＩＬ－４受容体をコードするポリヌクレオチドを導入したＴＨＰ－１細胞（ｍｕｔ＿ＩＬ－４Ｒ　ＴＨＰ－１細胞）の結果である。また、縦軸はコントロールのｍＣｈｅｒｒｙ　ＲＡＷ２６４．７細胞又はＧＦＰ　ＴＨＰ－１細胞におけるＩＬ－４受容体発現量を１とした場合の相対変異ヒトＩＬ－４受容体発現量である。発現量はヒトＧＡＰＤＨ又はマウスＧａｐｄｈで補正し、Ｕｎｐａｉｅｒｄ　Ｔ－ｔｅｓｔにて検定した。 The results are shown in Fig. 1. In FIG. 1, the upper row shows RAW264.7 cells (mCherry RAW264.7 cells) into which a polynucleotide encoding mCherry was introduced and RAW264.7 cells (mut_IL-) into which a polynucleotide encoding an I242N mutant human IL-4 receptor was introduced. This is the result of 4R RAW264.7 cells). In addition, in FIG. 1, the lower row shows THP-1 cells (GFP THP-1 cells) into which a polynucleotide encoding a GFP was introduced and THP-1 cells (GFP THP-1 cells) into which a polynucleotide encoding an I242N mutant human IL-4 receptor was introduced. This is the result of mut_IL-4R THP-1 cells). The vertical axis is the relative mutant human IL-4 receptor expression level when the IL-4 receptor expression level in the control mCherry RAW264.7 cells or GFP THP-1 cells is 1. The expression level was corrected by human GAPDH or mouse Gapdh, and tested by Unpaired T-test.
　図１上段に示すように、ｍｕｔ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞ではヒトＩＬ－４受容体が発現しており、レンチウイルスで導入したＩ２４２Ｎ変異ヒトＩＬ－４受容体が発現していることを確認した。また、図１下段に示すように、ｍｕｔ＿ＩＬ－４Ｒ　ＴＨＰ－１細胞ではコントロールと比して、ヒトＩＬ－４受容体の発現が増加しており、レンチウイルスで導入したＩ２４２Ｎ変異ヒトＩＬ－４受容体が発現していることを確認した。 As shown in the upper part of FIG. 1, it was confirmed that human IL-4 receptor was expressed in mut_IL-4R RAW264.7 cells, and I242N mutant human IL-4 receptor introduced by lentivirus was expressed. .. In addition, as shown in the lower part of FIG. 1, in mut_IL-4R THP-1 cells, the expression of human IL-4 receptor was increased as compared with the control, and the I242N mutant human IL-4 receptor introduced by lentivirus was increased. It was confirmed that the body was expressed.
［実施例２］（Ｉ２４２Ｎ変異ヒトＩＬ－４受容体を発現する細胞におけるＭ２マクロファージマーカーＡｒｇ１又はＣＤ２０６の発現）
　実施例１で作製した細胞のＭ２マクロファージへの分化又は分極を、マウスのＭ２マクロファージマーカーであるＡｒｇ１やヒトのＭ２マクロファージマーカーであるＣＤ２０６の発現によって調べた。具体的な手法は以下のとおりである。 [Example 2] (Expression of M2 macrophage marker Arg1 or CD206 in cells expressing the I242N mutant human IL-4 receptor)
The differentiation or polarization of the cells prepared in Example 1 into M2 macrophages was examined by the expression of Arg1 which is a mouse M2 macrophage marker and CD206 which is a human M2 macrophage marker. The specific method is as follows.
　実施例１と同様に、野生型のヒトＩＬ－４受容体をコードするポリヌクレオチドをＲＡＷ２６４．７細胞に導入してＷＴ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞を作製した。次に、実施例１で作製したｍＣｈｅｒｒｙ　ＲＡＷ２６４．７細胞（ｎ＝４）、ｍｕｔ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞（ｎ＝４）、ｍＣｈｅｒｒｙ　ＴＨＰ－１細胞（ｎ＝３）、ｍｕｔ＿ＩＬ－４Ｒ　ＴＨＰ－１細胞（ｎ＝３）の４種類及びＷＴ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞（ｎ＝４）を３７℃、ＣＯ２　５％で３日間培養し、細胞を回収してＲＮＡを抽出した。次に、リアルタイムＰＣＲ法によってＡｒｇ１の発現及びＣＤ２０６の発現を調べた。なお、ｍＣｈｅｒｒｙ　ＲＡＷ２６４．７細胞、ｍｕｔ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞及びＷＴ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞はＲＡＷ培地により培養し、ｍＣｈｅｒｒｙ　ＴＨＰ－１細胞及びｍｕｔ＿ＩＬ－４Ｒ　ＴＨＰ－１細胞はＴＨＰ細胞で培養した（３日間のうち最初の２４時間はＰＭＡを含有）。リアルタイムＰＣＲ法においては、以下の表２に記載のプライマーを用いた。 Similar to Example 1, a polynucleotide encoding a wild-type human IL-4 receptor was introduced into RAW264.7 cells to prepare WT_IL-4R RAW264.7 cells. Next, mCherry RAW264.7 cells (n = 4), mut_IL-4R RAW264.7 cells (n = 4), mCherry THP-1 cells (n = 3), mut_IL-4R THP-1 prepared in Example 1 four and WT_IL-4R RAW264.7 cells (n = 4) a 37 ° C. cell (n = 3), were cultured for 3 days in CO 2 5%, the cells were harvested and extracts the RNA. Next, the expression of Arg1 and the expression of CD206 were examined by real-time PCR. In addition, mCherry RAW264.7 cells, mut_IL-4R RAW264.7 cells and WT_IL-4R RAW264.7 cells were cultured in RAW medium, and mCherry THP-1 cells and mut_IL-4R THP-1 cells were cultured in THP cells ( Contains PMA for the first 24 hours of the 3 days). In the real-time PCR method, the primers shown in Table 2 below were used.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　結果を図２に示す。発現量はヒトＧＡＰＤＨ又はマウスＧａｐｄｈで補正し、ＲＡＷ２６４．７細胞は１－ｗａｙ　ＡＮＯＶＡで検定し、ＴＨＰ－１細胞はＵｎｐａｉｅｒｄ　Ｔ－ｔｅｓｔで検定した。 The results are shown in Fig. 2. The expression level was corrected by human GAPDH or mouse Gapdh, RAW264.7 cells were tested by 1-way ANOVA, and THP-1 cells were tested by Unpaired T-test.
　図２に示すように、コントロールのｍＣｈｅｒｒｙ　ＲＡＷ２６４．７細胞やＷＴ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞ではＡｒｇ１の発現が低値であったが、ｍｕｔ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞ではＡｒｇ１がコントロールのｍＣｈｅｒｒｙ　ＲＡＷ２６４．７細胞と比べて５９倍、ＷＴ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞と比べて２８倍発現していた。同様に、コントロールのＧＦＰ　ＴＨＰ－１細胞ではＣＤ２０６の発現が低値であったが、ｍｕｔ＿ＩＬ－４Ｒ　ＴＨＰ－１細胞ではＣＤ２０６が上記コントロールの細胞と比較して１３倍も発現していた。したがって、Ｉ２４２Ｎ変異ヒトＩＬ－４受容体を発現させることによって、マクロファージ前駆細胞がＭ２マクロファージへ分化及び／又は分極することが明らかとなった。 As shown in FIG. 2, the expression of Arg1 was low in the control mCherry RAW264.7 cells and WT_IL-4R RAW264.7 cells, but in the mut_IL-4R RAW264.7 cells, Arg1 was the control mCherry RAW264.7. It was 59 times more expressed than the cells and 28 times more than the WT_IL-4R RAW264.7 cells. Similarly, the expression of CD206 was low in the control GFP THP-1 cells, but the expression of CD206 in the mut_IL-4R THP-1 cells was 13 times higher than that in the control cells. Therefore, it was revealed that macrophage progenitor cells differentiate and / or polarize into M2 macrophages by expressing the I242N mutant human IL-4 receptor.
［実施例３］（Ｉ２４２Ｎ変異ヒトＩＬ－４受容体を発現する細胞におけるＭ１マクロファージ誘導の抑制）
　Ｍ１マクロファージへ誘導する液性因子であるリポポリサッカライド（ＬＰＳ）を作用させた場合に、ｍｕｔ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞から分極したＭ２マクロファージがＭ１マクロファージへ分極せずにＭ２マクロファージとして維持しているかどうかをＡｒｇ１及びＭ１マクロファージマーカーである腫瘍壊死因子（ＴＮＦα）の発現によって調べた。具体的な手法は以下のとおりである。 [Example 3] (Suppression of M1 macrophage induction in cells expressing the I242N mutant human IL-4 receptor)
Whether M2 macrophages polarized from mut_IL-4R RAW264.7 cells are maintained as M2 macrophages without being polarized to M1 macrophages when lipopolysaccharide (LPS), which is a humoral factor that induces M1 macrophages, is allowed to act. Whether or not it was examined by the expression of tumor necrosis factor (TNFα), which is an Arg1 and M1 macrophage marker. The specific method is as follows.
　ｍｕｔ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞（ｎ＝４）及びコントロールのｍＣｈｅｒｒｙ　ＲＡＷ２６４．７細胞（ｎ＝４）をＬＰＳ（１ｎｇ／ｍＬ含有ＲＡＷ培地又はコントロールとしてＬＰＳ未含（Ｂａｓａｌ））のＲＡＷ培地により３７℃、ＣＯ２　５％で３日間培養し、細胞を回収してＲＮＡを抽出した。次に、リアルタイムＰＣＲ法によってＴＮＦαの発現及びＡｒｇ１の発現を調べた。リアルタイムＰＣＲ法においては、以下の表３に記載のプライマーを用いた。 mut_IL-4R RAW264.7 cells (n = 4) and control mCherry RAW264.7 cells (n = 4) were mixed with LPS (1 ng / mL-containing RAW medium or control LPS-free (Basal)) RAW medium at 37 ° C. , and cultured for 3 days in CO 2 5%, the cells were harvested and extracts the RNA. Next, the expression of TNFα and the expression of Arg1 were examined by a real-time PCR method. In the real-time PCR method, the primers shown in Table 3 below were used.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　結果を図３に示す。発現量はヒトＧＡＰＤＨ又はマウスＧａｐｄｈで補正し、Ｕｎｐａｉｅｒｄ　Ｔ－ｔｅｓｔにて検定した。 The results are shown in Fig. 3. The expression level was corrected by human GAPDH or mouse Gapdh, and tested by Unpaired T-test.
　図３から明らかなように、コントロールのｍＣｈｅｒｒｙ　ＲＡＷ２６４．７細胞ではＬＰＳに作用させることによってＭ１マクロファージマーカーのＴＮＦαの発現が増加しているが、ｍｕｔ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞ではＬＰＳに作用させてもＴＮＦαの発現がほとんど増加していなかった。また、ｍｕｔ＿ＩＬ－４Ｒ　ＲＡＷ２６４．７細胞ではＬＰＳに作用させてもＭ２マクロファージマーカーのＡｒｇ１の発現がＬＰＳに作用させていないＢａｓａｌ（ＬＰＳなしのコントロール）と有意差が無く、Ａｒｇ１発現の低下を認めなかった。 As is clear from FIG. 3, the expression of the M1 macrophage marker TNFα is increased by acting on LPS in the control mCherry RAW264.7 cells, but even if it is allowed to act on LPS in mut_IL-4R RAW264.7 cells. The expression of TNFα was hardly increased. In addition, in mut_IL-4R RAW264.7 cells, the expression of Arg1 of the M2 macrophage marker was not significantly different from that of Basal (control without LPS), which did not act on LPS, and no decrease in Arg1 expression was observed. rice field.
　図２、図３の結果から、Ｉ２４２Ｎ変異ヒトＩＬ－４受容体を発現させることで、マクロファージ前駆細胞からＭ２マクロファージへ分化及び／又は分極されること、あるいはＭ１マクロファージがＭ２マクロファージへと分極されること、及びＭ１マクロファージへ誘導する液性因子の存在下でもＭ１マクロファージに分極せずにＭ２マクロファージとして維持可能であることが明らかとなった。 From the results of FIGS. 2 and 3, expression of the I242N mutant human IL-4 receptor differentiates and / or polarizes macrophage progenitor cells into M2 macrophages, or M1 macrophages are polarized into M2 macrophages. It was also clarified that it can be maintained as M2 macrophages without being polarized to M1 macrophages even in the presence of a humoral factor that induces M1 macrophages.
［実施例４］（Ｉ２４２Ｎ以外のヒトＩＬ－４受容体におけるＭ２マクロファージへの分極）
　実施例１～３では、配列番号２に示す２４２位の変異を用いたが、コントロールのＧＦＰ及び以下の（１）～（１０）の２４２位以外の他の変異におけるマクロファージ前駆細胞からＭ２マクロファージへの分化及び／又は分極作用をＡｇｒ１の発現によって調べた。Ａｇｒ１の発現は実施例２と同様の方法で行った。すなわち、ＧＦＰ又は以下の（１）～（１０）をコードするポリヌクレオチドをＰｒｉｍｅＳＴＡＲ（登録商標）　Ｍｕｔａｇｅｎｅｓｉｓ　Ｂａｓａｌ　Ｋｉｔ（TAKARA社）により作製し、それぞれＲＡＷ２６４．７細胞（ｎ＝３）に導入してＲＡＷ培地により３７℃、ＣＯ２　５％で３日間培養し、細胞を回収してＲＮＡを抽出し、リアルタイムＰＣＲ法によってＡｒｇ１の発現を調べた。 [Example 4] (Polarization to M2 macrophages in human IL-4 receptors other than I242N)
In Examples 1 to 3, the mutation at position 242 shown in SEQ ID NO: 2 was used, but from macrophage progenitor cells to M2 macrophages in control GFP and mutations other than positions 242 of (1) to (10) below. The differentiation and / or polarization effect of Agr1 was examined by the expression of Agr1. Expression of Agr1 was carried out in the same manner as in Example 2. That is, GFP or polynucleotides encoding the following (1) to (10) were prepared by PrimeSTAR (registered trademark) Mutagenesis Basal Kit (TAKARA) and introduced into RAW264.7 cells (n = 3), respectively, to RAW. 37 ° C. the culture medium, and cultured for 3 days in CO 2 5%, the cells were harvested and extracts the RNA, expression was examined Arg1 by real-time PCR method.
（１）ヒトＩＬ－４受容体のアミノ酸配列において、３７番目のアスパラギン酸をアスパラギンに置換したヒトＩＬ－４受容体の変異アミノ酸配列（Ｄ３７Ｎ変異ヒトＩＬ－４受容体：配列番号１６）
（２）ヒトＩＬ－４受容体のアミノ酸配列において、６２番目のチロシンをシステインに置換したヒトＩＬ－４受容体の変異アミノ酸配列（Ｙ６２Ｃ変異ヒトＩＬ－４受容体：配列番号１７）
（３）ヒトＩＬ－４受容体のアミノ酸配列において、７８番目のアスパラギンをチロシンに置換したヒトＩＬ－４受容体の変異アミノ酸配列（Ｎ７８Ｙ変異ヒトＩＬ－４受容体：配列番号１８）
（４）ヒトＩＬ－４受容体のアミノ酸配列において、１１５番目のフェニルアラニンをロイシンに置換したヒトＩＬ－４受容体の変異アミノ酸配列（Ｆ１１５Ｌ変異ヒトＩＬ－４受容体：配列番号１９）
（５）ヒトＩＬ－４受容体のアミノ酸配列において、１７６番目のアスパラギンをセリンに置換したヒトＩＬ－４受容体の変異アミノ酸配列（Ｎ１７６Ｓ変異ヒトＩＬ－４受容体：配列番号２０）
（６）ヒトＩＬ－４受容体のアミノ酸配列において、２００番目のアルギニンをトリプトファンに置換したヒトＩＬ－４受容体の変異アミノ酸配列（Ｒ２００Ｗ変異ヒトＩＬ－４受容体：配列番号２１）
（７）ヒトＩＬ－４受容体のアミノ酸配列において、２４２番目のイソロイシンをアスパラギン酸に置換したヒトＩＬ－４受容体の変異アミノ酸配列（Ｉ２４２Ｄ変異ヒトＩＬ－４受容体：配列番号２２）
（８）ヒトＩＬ－４受容体のアミノ酸配列において、２５１番目のシステインをトリプトファンに置換したヒトＩＬ－４受容体の変異アミノ酸配列（Ｃ２５１Ｗ変異ヒトＩＬ－４受容体：配列番号２３）
（９）ヒトＩＬ－４受容体のアミノ酸配列において、３０８番目のリシンをアスパラギンに置換したヒトＩＬ－４受容体の変異アミノ酸配列（Ｋ３０８Ｎ変異ヒトＩＬ－４受容体：配列番号２４） (1) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which the 37th aspartic acid was replaced with asparagine (D37N mutant human IL-4 receptor: SEQ ID NO: 16).
(2) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which the 62nd tyrosine was replaced with cysteine (Y62C mutant human IL-4 receptor: SEQ ID NO: 17).
(3) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which the 78th asparagine was replaced with tyrosine (N78Y mutant human IL-4 receptor: SEQ ID NO: 18).
(4) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which phenylalanine at position 115 is replaced with leucine (F115L mutant human IL-4 receptor: SEQ ID NO: 19).
(5) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which asparagine at position 176 was replaced with serine (N176S mutant human IL-4 receptor: SEQ ID NO: 20).
(6) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which the 200th arginine was replaced with tryptophan (R200W mutant human IL-4 receptor: SEQ ID NO: 21).
(7) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which isoleucine at position 242 is replaced with aspartic acid (I242D mutant human IL-4 receptor: SEQ ID NO: 22).
(8) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which the 251st cysteine is replaced with tryptophan (C251W mutant human IL-4 receptor: SEQ ID NO: 23).
(9) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which lysine at position 308 was replaced with asparagine (K308N mutant human IL-4 receptor: SEQ ID NO: 24).
　結果を図４に示す。発現量はヒトＧＡＰＤＨ又はマウスＧａｐｄｈ補正し、Ｕｎｐａｉｅｒｄ　Ｔ－ｔｅｓｔにて検定した。 The results are shown in Fig. 4. The expression level was corrected for human GAPDH or mouse Gapdh, and tested by Unpaired T-test.
　図４に示されるように、Ｄ３７Ｎ変異ヒトＩＬ－４受容体、Ｙ６２Ｃ変異ヒトＩＬ－４受容体、Ｎ７８Ｙ変異ヒトＩＬ－４受容体、Ｆ１１５Ｌ変異ヒトＩＬ－４受容体、Ｎ１７６Ｓ変異ヒトＩＬ－４受容体、Ｒ２００Ｗ変異ヒトＩＬ－４受容体、Ｉ２４２Ｄ変異ヒトＩＬ－４受容体、Ｃ２５１Ｗ変異ヒトＩＬ－４受容体、Ｋ３０８Ｎ変異ヒトＩＬ－４受容体を発現させることでＡｒｇ１の発現が増加していた。すなわち、Ｉ２４２Ｎ変異ヒトＩＬ－４受容体以外の本件変異ＩＬ－４Ｒポリペプチドを含有しても、Ｍ２マクロファージへ分化又は分極できることが明らかとなった。 As shown in FIG. 4, D37N mutant human IL-4 receptor, Y62C mutant human IL-4 receptor, N78Y mutant human IL-4 receptor, F115L mutant human IL-4 receptor, N176S mutant human IL-4. Expression of the receptor, R200W mutant human IL-4 receptor, I242D mutant human IL-4 receptor, C251W mutant human IL-4 receptor, and K308N mutant human IL-4 receptor increased the expression of Arg1. rice field. That is, it was clarified that even if the mutant IL-4R polypeptide other than the I242N mutant human IL-4 receptor is contained, it can be differentiated or polarized into M2 macrophages.
［実施例５］（Ｍ２マクロファージの持続）
　Ｉ２４２Ｎ変異ヒトＩＬ－４受容体を発現する細胞において、Ｍ２マクロファージとしての特徴をどの程度の間維持できるかをＡｒｇ１の発現を指標として調べた。 [Example 5] (Persistence of M2 macrophages)
In cells expressing the I242N mutant human IL-4 receptor, the extent to which the characteristics as M2 macrophages could be maintained was investigated using the expression of Arg1 as an index.
　マウス骨髄細胞を公知の手法で採取後、Ｍ－ＣＳＦ（２０ｎｇ／ｍｌ）を添加した培養液で５日間培養し、骨髄由来マクロファージ（ＢＭＤＭ）に分化させた。その後、Ｍ－ＣＳＦ（２０ｎｇ／ｍｌ）存在下で、実施例１と同様に、Ｉ２４２Ｎ変異ヒトＩＬ－４受容体をコードするポリヌクレオチド若しくはコントロール（ＧＦＰをコードするポリヌクレオチド）を、レンチウイルスを用いて上記ＢＭＤＭへ導入し、４８時間培養後にＢＭＤＭを回収して保存した。それぞれのＢＭＤＭをＩＬ－４（１００ｐｇ／ｍｌ）で２４時間刺激してＭ２マクロファージに分極させた後に１週間そのまま培養し、Ｍ２マクロファージマーカーであるＡｒｇ１の発現をリアルタイムＰＣＲで解析した。結果を図５に示す。横軸は培養日数（ＩＬ－４で２４時間刺激した後をＤａｙ０とする）、縦軸はコントロールのＤａｙ０におけるＡｒｇ１の発現量を１とした場合のＡｒｇ１の相対発現量である。発現量はマウスＧａｐｄｈで補正した（Ｎ＝３）。 Mouse bone marrow cells were collected by a known method and then cultured in a culture medium containing M-CSF (20 ng / ml) for 5 days to differentiate into bone marrow-derived macrophages (BMDM). Then, in the presence of M-CSF (20 ng / ml), as in Example 1, a polynucleotide or control (polynucleotide encoding GFP) encoding the I242N mutant human IL-4 receptor was used with a lentivirus. The BMDM was introduced into the above BMDM, and after culturing for 48 hours, the BMDM was collected and stored. Each BMDM was stimulated with IL-4 (100 pg / ml) for 24 hours to polarize it into M2 macrophages, and then cultured as it was for 1 week, and the expression of Arg1 which is an M2 macrophage marker was analyzed by real-time PCR. The results are shown in FIG. The horizontal axis is the number of days of culture (Day 0 is defined after stimulation with IL-4 for 24 hours), and the vertical axis is the relative expression level of Arg1 when the expression level of Arg1 on the control Day 0 is 1. The expression level was corrected with mouse Gapdh (N = 3).
　コントロールのＩＬ－４刺激のみでＭ２マクロファージに分極させたＢＭＤＭ（細胞）は、Ｄａｙ３にはＡｒｇ１の発現が低下するのに対し、Ｉ２４２Ｎ変異ヒトＩＬ－４受容体を発現する細胞はＤａｙ７（１週間後）もＡｒｇ１の発現が継続していた。したがって、本件変異ＩＬ－４Ｒポリペプチドを含有すれば、Ｍ２マクロファージへ分化又は分極し、その後Ｍ２マクロファージとして少なくとも１週間は維持できることが明らかとなった。従来の方法では、Ｍ２マクロファージが体内に入ると、数日でその機能を失うため、治療効果は限定的であった。一方、本発明で作製したＭ２マクロファージは長期間Ｍ２マクロファージとしての機能を維持できるため、炎症性疾患、神神経変性疾患、及び虚血性疾患に対する高い治療効果が期待される。 BMDM (cells) polarized into M2 macrophages only by control IL-4 stimulation reduced the expression of Arg1 on Day 3, whereas cells expressing the I242N mutant human IL-4 receptor were Day 7 (1 week). Later), the expression of Arg1 continued. Therefore, it was clarified that if the mutant IL-4R polypeptide is contained, it can be differentiated or polarized into M2 macrophages and then maintained as M2 macrophages for at least one week. In the conventional method, when M2 macrophages enter the body, they lose their function within a few days, so that the therapeutic effect is limited. On the other hand, since the M2 macrophage produced in the present invention can maintain its function as an M2 macrophage for a long period of time, it is expected to have a high therapeutic effect on inflammatory diseases, myoneurodegenerative diseases, and ischemic diseases.
［実施例６］（ＩＬ－１０の分泌）
　本件変異ＩＬ－４Ｒポリペプチドを発現するＭ２マクロファージにおける抗炎症作用の確認のため、強力な抗炎症性サイトカインであるＩＬ－１０の分泌を調べた。実施例１と同様に、Ｉ２４２Ｎ変異ヒトＩＬ－４受容体若しくはコントロール（ＧＦＰをコードするポリヌクレオチド）をコードするポリヌクレオチドを、レンチウイルスを用いてＲＡＷ２６４．７細胞に導入して安定発現細胞株を作製した。次に、培養皿へそれぞれの細胞株を播種して２４時間培養した。培養後の上清を回収し、上清に含まれるＩＬ－１０をＥＬＩＳＡ法によって解析した。Ｕｎｐａｉｅｒｄ　Ｔ－ｔｅｓｔにて検定（Ｎ＝３）した。結果を図６に示す。 [Example 6] (secretion of IL-10)
In order to confirm the anti-inflammatory effect on M2 macrophages expressing the mutant IL-4R polypeptide, the secretion of IL-10, which is a potent anti-inflammatory cytokine, was examined. Similar to Example 1, a polynucleotide encoding an I242N mutant human IL-4 receptor or control (polynucleotide encoding GFP) was introduced into RAW264.7 cells using a lentivirus to obtain a stable expression cell line. Made. Next, each cell line was seeded in a culture dish and cultured for 24 hours. The supernatant after culturing was collected, and IL-10 contained in the supernatant was analyzed by the ELISA method. Tested by Unpaired T-test (N = 3). The results are shown in FIG.
　図６から明らかなように、本件変異ＩＬ－４Ｒポリペプチドを含有すれば、ＩＬ－１０の発現がコントロールと比較して４倍以上増加していた。したがって、本件変異ＩＬ－４Ｒポリペプチドを含有するＭ２マクロファージは、抗炎症作用を有することが確認された。 As is clear from FIG. 6, when the mutant IL-4R polypeptide was contained, the expression of IL-10 was increased more than 4-fold as compared with the control. Therefore, it was confirmed that the M2 macrophage containing the mutant IL-4R polypeptide has an anti-inflammatory effect.
　本発明は、炎症疾患、創傷治癒、神経変性疾患、及び虚血性疾患に対するバイオ医薬品として利用可能である。 The present invention can be used as a biopharmacy for inflammatory diseases, wound healing, neurodegenerative diseases, and ischemic diseases.

Claims (12)

  1. ヒトインターロイキン４（ヒトＩＬ－４）受容体に由来するポリペプチドであって、配列番号１に示すアミノ酸配列において、１又は数個のアミノ酸が付加、欠失若しくは置換したポリペプチド。 A polypeptide derived from the human interleukin 4 (human IL-4) receptor, wherein one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1.
  2. 以下の（１－１）又は（１－２）である、請求項１記載のポリペプチド。
    （１－１）ヒトインターロイキン４受容体に由来するポリペプチドであって、配列番号１に示すアミノ酸配列において、３７番目のアスパラギン酸、６２番目のチロシン、７８番目のアスパラギン、１１５番目のフェニルアラニン、１７６番目のアスパラギン、２００番目のアルギニン、２４２番目のイソロイシン、２５１番目のシステイン、及び／又は３０８番目のリシンが置換したポリペプチド；
    （１－２）配列番号１に示すアミノ酸配列において、上記（１－１）において置換した３７番目、６２番目、７８番目、１１５番目、１７６番目、２００番目、２４２番目、２５１番目及び／又は３０８番目以外のアミノ酸において１又は数個のアミノ酸が付加、欠失若しくは置換したポリペプチドを含み、かつ、ホルボール１２－ミリスタート１３－アセタートで刺激したＴＨＰ－１細胞、若しくはＲＡＷ２６４．７細胞で発現した場合に、当該ポリペプチドを発現していない場合と比較してヒトＭ２マクロファージマーカーの発現を増加するポリペプチド。     The polypeptide according to claim 1, which is the following (1-1) or (1-2).
    (1-1) A polypeptide derived from the human interleucine 4 receptor, in the amino acid sequence shown in SEQ ID NO: 1, aspartic acid at position 37, tyrosine at position 62, asparagin at position 78, and phenylalanine at position 115. Polypeptide substituted with aspartic acid at position 176, arginine at position 200, isoleucine at position 242, cysteine at position 251 and / or lysine at position 308;
    (1-2) In the amino acid sequence shown in SEQ ID NO: 1, the 37th, 62nd, 78th, 115th, 176th, 200th, 242nd, 251st and / or 308 substituted in the above (1-1). It was expressed in THP-1 cells or RAW264.7 cells containing a polypeptide in which one or several amino acids were added, deleted or substituted in a non-th amino acid and stimulated with Holbol 12-millistart 13-acetate. A polypeptide that, in some cases, increases the expression of human M2 macrophage markers as compared to the case where the polypeptide is not expressed.
  3. 配列番号１に示すアミノ酸配列において、３７番目のアスパラギン酸、６２番目のチロシン、７８番目のアスパラギン、１１５番目のフェニルアラニン、１７６番目のアスパラギン、２００番目のアルギニン、２４２番目のイソロイシン、２５１番目のシステイン、及び／又は３０８番目のリシンが置換したポリペプチドが、以下の（２－１）～（２－９）のいずれかである、請求項２記載のポリペプチド。
    （２－１）配列番号１に示すアミノ酸配列において、３７番目におけるアスパラギン酸が中性極性アミノ酸へ置換したポリペプチド；
    （２－２）配列番号１に示すアミノ酸配列において、６２番目におけるチロシンが中性極性アミノ酸へ置換したポリペプチド；
    （２－３）配列番号１に示すアミノ酸配列において、７８番目におけるアスパラギンが中性極性アミノ酸へ置換したポリペプチド；
    （２－４）配列番号１に示すアミノ酸配列において、１１５番目におけるフェニルアラニンが中性非極性アミノ酸へ置換したポリペプチド；
    （２－５）配列番号１に示すアミノ酸配列において、１７６番目におけるアスパラギンが中性極性アミノ酸へ置換したポリペプチド；
    （２－６）配列番号１に示すアミノ酸配列において、２００番目におけるアルギニンが中性非極性アミノ酸へ置換したポリペプチド；
    （２－７）配列番号１に示すアミノ酸配列において、２４２番目におけるイソロイシンが酸性アミノ酸又は中性非極性アミノ酸へ置換したポリペプチド；
    （２－８）配列番号１に示すアミノ酸配列において、２５１番目におけるシステインが中性非極性アミノ酸へ置換したポリペプチド；
    （２－９）配列番号１に示すアミノ酸配列において、３０８番目におけるリシンが中性極性アミノ酸へ置換したポリペプチド；     In the amino acid sequence shown in SEQ ID NO: 1, aspartic acid at position 37, tyrosine at position 62, asparagin at position 78, phenylalanine at position 115, asparaginine at position 176, arginine at position 200, isoleucine at position 242, and cysteine at position 251 ,. The polypeptide according to claim 2, wherein the polypeptide substituted with lysine at position 308 and / or is any of the following (2-1) to (2-9).
    (2-1) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which aspartic acid at position 37 is replaced with a neutral polar amino acid;
    (2-2) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which tyrosine at position 62 is replaced with a neutral polar amino acid;
    (2-3) In the amino acid sequence shown in SEQ ID NO: 1, the polypeptide in which asparagine at position 78 is replaced with a neutral polar amino acid;
    (2-4) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which phenylalanine at position 115 is replaced with a neutral non-polar amino acid;
    (2-5) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which asparagine at position 176 is replaced with a neutral polar amino acid;
    (2-6) In the amino acid sequence shown in SEQ ID NO: 1, the polypeptide in which arginine at position 200 is replaced with a neutral non-polar amino acid;
    (2-7) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which isoleucine at position 242 is replaced with an acidic amino acid or a neutral non-polar amino acid;
    (2-8) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which cysteine at position 251 is replaced with a neutral non-polar amino acid;
    (2-9) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which lysine at position 308 is replaced with a neutral polar amino acid;
  4. ヒトインターロイキン４受容体に由来するポリペプチドであって、配列番号１に示すアミノ酸配列において、２４２番目のイソロイシンが置換したポリペプチドであることを特徴とする請求項２記載のポリペプチド。 The polypeptide according to claim 2, which is a polypeptide derived from the human interleukin-4 receptor and is a polypeptide substituted with isoleucine at position 242 in the amino acid sequence shown in SEQ ID NO: 1.
  5. ヒトインターロイキン４受容体に由来するポリペプチドであって、配列番号１に示すアミノ酸配列において、２４２番目のイソロイシンがアスパラギンへ置換したポリペプチドであることを特徴とする請求項４記載のポリペプチド。 The polypeptide according to claim 4, which is a polypeptide derived from the human interleukin-4 receptor and in which isoleucine at position 242 is replaced with asparagine in the amino acid sequence shown in SEQ ID NO: 1.
  6. 請求項１～５のいずれか記載のポリペプチドをコードするポリヌクレオチド。 A polynucleotide encoding the polypeptide according to any one of claims 1 to 5.
  7. 請求項６記載のポリヌクレオチドを含有する発現ベクター。 An expression vector containing the polynucleotide according to claim 6.
  8. 請求項６記載のポリヌクレオチド又は請求項７記載の発現ベクターを含有する、生体から分離されたマクロファージ又はマクロファージ前駆細胞。 A macrophage or macrophage progenitor cell isolated from a living body containing the polynucleotide according to claim 6 or the expression vector according to claim 7.
  9. 請求項１～５のいずれか記載のポリペプチドを含有する、生体から分離されたマクロファージ又はマクロファージ前駆細胞。 A macrophage or macrophage progenitor cell isolated from a living body containing the polypeptide according to any one of claims 1 to 5.
  10. 請求項８又は９記載のマクロファージ又はマクロファージ前駆細胞と薬理学的に許容される添加剤を含む、炎症疾患、神経変性疾患若しくは虚血性疾患の予防又は治療剤。 A prophylactic or therapeutic agent for an inflammatory disease, a neurodegenerative disease or an ischemic disease, which comprises the macrophage or macrophage progenitor cell according to claim 8 or 9 and a pharmacologically acceptable additive.
  11. 請求項６記載のポリヌクレオチド又は請求項７記載の発現ベクターを有効成分とする、生体から分離されたマクロファージ又はマクロファージ前駆細胞からＭ２マクロファージへの分化又は分極誘導剤。 An agent for inducing differentiation or polarization of macrophages or macrophage progenitor cells isolated from a living body into M2 macrophages, which comprises the polynucleotide according to claim 6 or the expression vector according to claim 7 as an active ingredient.
  12. 請求項６記載のポリヌクレオチド又は請求項７記載の発現ベクターを生体から分離されたマクロファージ又はマクロファージ前駆細胞に導入することを特徴とする、生体から分離されたマクロファージ又はマクロファージ前駆細胞からＭ２マクロファージへの分化又は分極誘導方法。

          From a macrophage or macrophage progenitor cell isolated from a living body to an M2 macrophage, which comprises introducing the polynucleotide according to claim 6 or the expression vector according to claim 7 into a macrophage or macrophage progenitor cell isolated from the living body. Differentiation or polarization induction method.
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JP2002532073A (en) * 1998-12-11 2002-10-02 チルドレンズ ホスピタル メディカル センター How to determine susceptibility to asthma

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JP2002532073A (en) * 1998-12-11 2002-10-02 チルドレンズ ホスピタル メディカル センター How to determine susceptibility to asthma

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