Mutant polypeptides of human interleukin-4 receptor and agents for inducing differentiation or polarization into M2 macrophages

Abstract

To provide variant polypeptides of human interleukin 4 receptor, and inducers for differentiation or polarization to M2 macrophage.SOLUTION: Disclosed is a polypeptide derived from human interleukin 4 receptor with addition, deletion or substitution of one or several amino acids in an amino acid sequence of a specific sequence. Preferably, isoleucine at position 242 is substituted with asparagine in the amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a mutant IL-4 receptor polypeptide derived from human interleukin-4 receptor, a polynucleotide encoding the mutant IL-4 receptor polypeptide, an expression vector containing the polynucleotide, macrophages or macrophage precursor cells isolated from a living organism containing the polynucleotide or expression vector, macrophages or macrophage precursor cells isolated from a living organism containing the polypeptide, an agent for preventing or treating inflammatory diseases, neurodegenerative diseases, or ischemic diseases, comprising the macrophages or macrophage precursor cells and a pharmacologically acceptable additive, an agent for inducing differentiation or polarization of macrophages or macrophage precursor cells isolated from a living organism into M2 macrophages, and a method for inducing differentiation or polarization of macrophages or macrophage precursor cells isolated from a living organism into M2 macrophages, which comprises the polynucleotide or the expression vector as an active ingredient. [Background technology]

[0002] There are various types of inflammatory, neurodegenerative, and ischemic diseases, and although drugs have been developed to treat each disease, there are still many diseases for which sufficient efficacy can not be expected. Macrophages, which are immune cells, are present in the lesions of these inflammatory, neurodegenerative, and ischemic diseases and are thought to be involved in the pathogenesis of these diseases.

[0003] Recent advances in macrophage research have revealed that macrophages are divided into M1 macrophages, which induce inflammation, and M2 macrophages, which suppress inflammation and repair tissue. M2 macrophages have been shown to have beneficial functions for the body, such as suppressing excessive inflammation and phagocytosing β-amyloid proteins, which are seen in Alzheimer's disease (see Non-Patent Documents 1 and 2). Furthermore, the ratio of M1 to M2 macrophages becomes more prevalent with age, and an M1 / M2 imbalance is thought to be one of the causes of aging (see Non-Patent Document 3). Against this background, various methods for activating M2 macrophages are underway. Specifically, the main method for activating M2 macrophages is the systemic administration of humoral factors or low-molecular-weight compounds, but there has been no progress toward clinical application (see Non-Patent Document 4). This is because humoral factors used to activate M2 macrophages can affect B lymphocytes and increase IgE production, potentially exacerbating allergic diseases, potentially posing a risk to cells other than macrophages. Furthermore, humoral factors cannot pass through the blood-brain barrier, which means they have the problem of not being sufficiently effective against central inflammatory and neurodegenerative diseases such as Alzheimer's disease.

[0004] Furthermore, because inflammatory sites are rich in physiologically active substances that activate M1 macrophages, it has been suggested that M2 macrophages that were once activated by systemic administration of humoral factors may repolarize to M1 macrophages at the actual inflammatory site.

[0005] Based on the above, there is a need to develop substances that do not rely on humoral factors, are less likely to polarize to M1 macrophages at the site of inflammation, and activate M2 macrophages.

[0006] Incidentally, IL-4 receptor mutations have been reported in hematologic malignant lymphoma, bladder cancer, and brain tumors (see Non-Patent Documents 5 to 7). However, the relationship between such IL-4 receptor mutations and macrophage differentiation or polarization is unknown. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Abbas Shapouri-Moghaddam et al., Journal of Cellular Physiology Volume 233, Issue 9 September (2018):P6425-6440 [Non-patent document 2] LeahZuroff et al., Cell. Mol. Life Sci. (2017) 74:P2167-2201 [Non-patent document 3] Laren Becker et al., Gut. 2018 May; 67(5): P827-836. [Non-patent document 4] Ying Bi et al., Neural Plasticity Volume 2019, Article ID 6724903:P21 [Non-patent document 5] Elena Vigano et al., Blood 2018; 131(18):P2036-2046 [Non-patent document 6] Guo G et al., Nat Genet. 2013;45(12): 1459-1463 [Non-Patent Document 7] Frattini V et al., Nat Genet. 2013;45(10):1141-1149 Summary of the Invention [Problem to be solved by the invention]

[0008] Previous attempts to increase M2 macrophages have involved the systemic administration of humoral factors and compounds. However, systemic administration poses challenges, such as the high local concentration of humoral factors that induce M1 macrophages at the site of inflammation, resulting in low efficacy relative to the dose administered. Furthermore, systemic administration can potentially act on other cells, exacerbating immune disorders such as asthma. Furthermore, while M2 macrophages are expected to be effective in treating central nervous system degenerative diseases such as Alzheimer's disease, these humoral factors and compounds cannot cross the blood-brain barrier, making these approaches unsuitable for the treatment of these conditions. Furthermore, when IL-4 is used to activate M2 macrophages, its many effects, such as proliferation of immature T cells and enhanced expression of mast cells, may result in effects other than activating M2 macrophages. Therefore, an object of the present invention is to provide a polypeptide that is involved in differentiation or polarization into M2 macrophages independently of IL-4, and to provide an agent that induces differentiation or polarization into M2 macrophages. [Means for solving the problem]

[0009] The inventors focused on human interleukin-4 receptor (IL-4R) and investigated whether introducing a mutant IL-4R into macrophages would activate them into M2 macrophages. As a result, they succeeded in differentiating or polarizing macrophages into M2 macrophages by expressing a mutant IL-4R in macrophages or macrophage precursor cells, thereby completing the present invention.

[0010] That is, the present invention is as follows. [1] A polypeptide derived from human interleukin-4 receptor, in which one or several amino acids have been added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1. [2] The polypeptide according to [1] above, which is (1-1) or (1-2) below. (1-1) A polypeptide derived from human interleukin-4 receptor, which has, in the amino acid sequence shown in SEQ ID NO: 1, a substitution of aspartic acid at position 37, tyrosine at position 62, asparagine at position 78, phenylalanine at position 115, asparagine at position 176, arginine at position 200, isoleucine at position 242, cysteine ​​at position 251, and / or lysine at position 308; (1-2) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, in which one or more amino acids have been added, deleted, or substituted at amino acids other than the 37th, 62nd, 78th, 115th, 176th, 200th, 242nd, 251st, and / or 308th amino acids substituted in (1-1) above, and which, when expressed in THP-1 cells or RAW264.7 cells stimulated with phorbol 12-myristate 13-acetate, increases the expression of a human M2 macrophage marker compared to when the polypeptide is not expressed. [3] The polypeptide according to [2] above, wherein in the amino acid sequence shown in SEQ ID NO: 1, the polypeptide in which aspartic acid at position 37, tyrosine at position 62, asparagine at position 78, phenylalanine at position 115, asparagine at position 176, arginine at position 200, isoleucine at position 242, cysteine ​​at position 251, and / or lysine at position 308 is substituted is any one of the following (2-1) to (2-9): (2-1) a polypeptide in which the aspartic acid at position 37 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with a neutral polar amino acid; (2-2) a polypeptide in which the tyrosine at position 62 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with a neutral polar amino acid; (2-3) a polypeptide in which asparagine at position 78 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with a neutral polar amino acid; (2-4) A polypeptide in which phenylalanine at position 115 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with a neutral nonpolar amino acid; (2-5) a polypeptide in which asparagine at position 176 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with a neutral polar amino acid; (2-6) a polypeptide in which arginine at position 200 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with a neutral nonpolar amino acid; (2-7) a polypeptide in which the isoleucine at position 242 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with an acidic amino acid or a neutral nonpolar amino acid; (2-8) A polypeptide in which the cysteine ​​at position 251 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with a neutral nonpolar amino acid; (2-9) a polypeptide in which the lysine at position 308 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with a neutral polar amino acid; [4] The polypeptide according to [2] above, which is a polypeptide derived from human interleukin-4 receptor, characterized in that the isoleucine at position 242 in the amino acid sequence shown in SEQ ID NO: 1 is substituted. [5] The polypeptide according to [4] above, which is a polypeptide derived from human interleukin-4 receptor, characterized in that the 242nd isoleucine in the amino acid sequence shown in SEQ ID NO: 1 is substituted with asparagine. [6] A polynucleotide encoding the polypeptide according to any one of [1] to [5] above. [7] An expression vector containing the polynucleotide described in [6] above. [8] A macrophage or macrophage precursor cell isolated from a living organism, which contains the polynucleotide described in [6] above or the expression vector described in [7] above. [9] A macrophage or macrophage precursor cell isolated from a living organism, which contains the polypeptide according to any one of [1] to [5] above.

[10] A preventive or therapeutic agent for inflammatory diseases, neurodegenerative diseases, or ischemic diseases, comprising the macrophage or macrophage precursor cells according to [8] or [9] above and a pharmacologically acceptable additive.

[11] An agent for inducing differentiation or polarization of macrophages or macrophage precursor cells isolated from a living body into M2 macrophages, the agent comprising the polynucleotide according to [6] above or the expression vector according to [7] above as an active ingredient.

[12] A method for inducing differentiation or polarization of macrophages or macrophage precursor cells isolated from a living organism into M2 macrophages, comprising introducing the polynucleotide described in [6] above or the expression vector described in [7] above into macrophages or macrophage precursor cells isolated from a living organism. [Effects of the Invention]

[0011] The agent for inducing differentiation or polarization into M2 macrophages of the present invention makes it possible to differentiate or polarize macrophages or macrophage precursor cells isolated from a living body into M2 macrophages. [Brief explanation of the drawings]

[0012] [Figure 1] In Example 1, a polynucleotide encoding the human IL-4 receptor with the I242N mutation (isoleucine at position 242 replaced with asparagine) was introduced into RAW264.7 cells or THP-1 cells, and the expression of the IL-4 receptor was examined. [Figure 2] In Example 2, a 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. [Figure 3] 1 shows the results of Example 3, in which a polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into RAW264.7 cells, and the cells were exposed to LPS to examine the expression of TNFα or Arg1. [Figure 4] In Example 4, polynucleotides encoding mutant human IL-4 receptors other than I242N were introduced into RAW264.7 cells, and the expression of Arg1 was examined. [Figure 5]In Example 5, a polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into bone marrow-derived macrophages (BMDMs), and the expression of Arg1 was examined. [Figure 6] In Example 6, a polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into RAW264.7 cells, and the expression of IL-10 was examined. DETAILED DESCRIPTION OF THE INVENTION

[0013] The human interleukin-4 receptor (human IL-4 receptor) in the "polypeptide derived from human interleukin-4 receptor, in which one or more amino acids have been added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1 (hereinafter also referred to as the "present mutant IL-4R polypeptide")" of the present invention is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and is registered in NCBI under Reference No. NP_000409.1.

[0014] The "polypeptide in which one or more amino acids have been added, deleted, or substituted in the amino acid sequence of SEQ ID NO: 1" in the present mutant IL-4R polypeptide preferably refers to a "polypeptide in which one or more amino acids have been added, deleted, or substituted in the amino acids constituting the extracellular domain or transmembrane domain in the amino acid sequence of SEQ ID NO: 1," and more preferably refers to a "polypeptide in which one or more amino acids have been added, deleted, or substituted in the amino acids constituting the transmembrane domain in the amino acid sequence of SEQ ID NO: 1." The extracellular domain or transmembrane domain can be predicted using a known extracellular domain or transmembrane domain prediction algorithm. In the amino acid sequence of SEQ ID NO: 1, the extracellular domain is considered to be located at positions 1 to 232, and the transmembrane domain is considered to be located at positions 233 to 256. Examples of positions in the "amino acids constituting the extracellular domain" where one or more amino acids are added, deleted, or substituted include aspartic acid at position 37, tyrosine at position 62, asparagine at position 78, phenylalanine at position 115, asparagine at position 176, and arginine at position 200 in the amino acid sequence of SEQ ID NO: 1. Among the above-mentioned "amino acids constituting the transmembrane domain," positions at which one or more amino acids are added, deleted, or substituted include isoleucine at position 242 and cysteine ​​at position 251, with isoleucine at position 242 being preferred.

[0015] As used herein, the term "neutral nonpolar amino acid" refers to glycine (G), alanine (A), valine (V), proline (P), leucine (L), isoleucine (I), phenylalanine (F), methionine (M), or tryptophan (W). The term "neutral polar amino acid" refers to asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), or cysteine ​​(C). The term "acidic amino acid" refers to aspartic acid (D) or glutamic acid (E).

[0016] 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 includes "a polypeptide in which one or several amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 1." Such a "polypeptide in which one or several amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 1" preferably includes any of the following (2-1) to (2-9), more preferably any of the following (2-1') to (2-9'), and even more preferably includes, but is not limited to, a polypeptide in which isoleucine at position 242 is substituted with asparagine.

[0017] (2-1) substitution of aspartic acid at position 37 with a neutral polar amino acid; (2-2) substitution of tyrosine at position 62 with a neutral polar amino acid; (2-3) substitution of asparagine at position 78 with a neutral polar amino acid; (2-4) substitution of phenylalanine at position 115 with a neutral nonpolar 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 nonpolar amino acid; (2-7) substitution of isoleucine at position 242 with an acidic amino acid or a neutral nonpolar amino acid; (2-8) substitution of cysteine ​​at position 251 with a neutral nonpolar amino acid; (2-9) substitution of lysine at position 308 with a neutral polar amino acid;

[0018] (2-1') substitution of aspartic acid with asparagine at position 37; (2-2') substitution of tyrosine with cysteine ​​at position 62; (2-3') Substitution of asparagine with tyrosine at position 78; (2-4') substitution of phenylalanine with leucine at position 115; (2-5') Substitution of asparagine to serine at position 176; (2-6') substitution of arginine with tryptophan at position 200; (2-7') substitution of isoleucine with asparagine or aspartic acid at position 242; (2-8') substitution of cysteine ​​at position 251 with tryptophan; (2-9') Lysine to asparagine substitution at position 308;

[0019] As used herein, "one or several amino acids" means, for example, in the range of 1 to 30 amino acids, preferably in the range of 1 to 20 amino acids, more preferably in the range of 1 to 15 amino acids, even more preferably in the range of 1 to 10 amino acids, more preferably in the range of 1 to 5 amino acids, even more preferably in the range of 1 to 3 amino acids, more preferably 1 or 2 amino acids, and most preferably 1 amino acid.

[0020] As used herein, "a polypeptide that, when expressed in THP-1 cells or RAW264.7 cells stimulated with phorbol 12-myristate 13-acetate (PMA), increases the expression of human M2 macrophage markers compared to cells in which the polypeptide is not expressed" refers to a polypeptide that increases, preferably by at least two-fold, more preferably at least five-fold, and even more preferably at least ten-fold, the expression of M2 macrophage markers in THP-1 cells or RAW264.7 cells stimulated with PMA that express the mutant IL-4R polypeptide compared to cells in which the mutant IL-4R polypeptide is not expressed, when the expression of M2 macrophage markers is examined in THP-1 cells or RAW264.7 cells stimulated with PMA that express the mutant IL-4R polypeptide. Examples of human M2 macrophage markers include CD206, CD163, IL-27RA, CD367, F13A1, and CCL22. THP-1 cells differentiate into macrophages upon stimulation with PMA.

[0021] The "polynucleotide encoding the mutant IL-4R polypeptide of the present invention (hereinafter also referred to as "the polynucleotide")" is not particularly limited as long as it is a polynucleotide that encodes the mutant IL-4R polypeptide of the present invention, and the codons selected to encode amino acids may be optimized as appropriate depending on the type of host cell in which the polypeptide is to be expressed.

[0022] The expression vector of the present invention, "an expression vector containing the present polynucleotide" (hereinafter also referred to as "the present expression vector"), may be linear or circular, and may be a viral vector, a non-viral vector such as a plasmid, or a transposon-based vector. Furthermore, such a vector may contain a regulatory sequence such as a promoter or terminator, or a selection marker sequence such as a drug resistance gene or a reporter gene.

[0023] Examples of the viral vector include lentiviral vectors, retroviral vectors, adenoviral vectors, and adeno-associated viral vectors, with lentiviral vectors being preferred. When a lentiviral vector or retroviral vector is used, the transgene is incorporated into the genome of the host cell, enabling long-term and stable expression. When short-term expression is preferred, adenoviral vectors and adeno-associated viral vectors that do not integrate into the genome are preferred.

[0024] Examples of the promoter include virus-derived promoters such as the lentivirus LTR promoter, SV40 early promoter, cytomegalovirus promoter, and herpes simplex virus thymidine kinase promoter, as well as mammalian-derived promoters such as the phosphoglycerate kinase (PGK) promoter, Xist promoter, β-actin promoter, RNA polymerase II promoter, CAG promoter, and EF1A. Also usable are tetracycline-responsive promoters induced by tetracycline, Mx1 promoters induced by interleukin, CD68 promoters that have promoter activity in macrophages, and M-CSF promoters.

[0025] The "macrophage" in the "macrophage or macrophage precursor cell isolated from a living body containing the present polynucleotide or the present expression vector" (hereinafter also referred to as "the present mutant IL-4R macrophage A") of the present invention includes two phenotypes, M1 macrophage and M2 macrophage. Furthermore, the "macrophage precursor cell" includes cells that can differentiate into at least M1 macrophage or M2 macrophage, and are CD14 positive (CD14 + Examples of such macrophages include non-activated macrophages (M0 macrophages), monocytes, monoblasts, bone marrow mononuclear cells, peripheral blood mononuclear cells, ES cells, and iPS cells. M2 macrophages are also called anti-inflammatory macrophages, and are CD14-positive macrophages. Examples of such macrophages include cells that express CD206, CD163, IL-27RA, CD367, F13A1, or CCL22 in humans, and cells that express arginase 1 (Arg1), CD206, iNOS, CD163, or CD204 in mice. The present mutant IL-4R macrophage A and the present mutant IL-4R macrophage B described below also encompass cultures obtained by culturing them.

[0026] Monocytes are blood cells present in circulating blood and can differentiate into macrophages when they leave the bloodstream and enter connective tissue. The monocytes can be derived from mammals such as humans, monkeys, and mice. Peripheral blood mononuclear cells (PBMCs) can be isolated from whole peripheral blood collected from a patient using known techniques such as centrifugation, and then purified, if necessary, using immunomagnetic bead techniques. These monocytes can be activated and differentiated into macrophages by culturing them in the presence of cytokines such as M-CSF. Furthermore, monocytes can also be produced from bone marrow fluid, ES cells, iPS cells, and the like using known techniques.

[0027] The above phrase "containing the polynucleotide of the present invention" means that the polynucleotide of the present invention is contained in macrophages or macrophage precursor cells isolated from a living organism. For example, the polynucleotide of the present invention may be contained in a state where it is incorporated into an expression vector, or in a state where it is incorporated into the genome of macrophages or macrophage precursor cells isolated from a living organism.

[0028] The mutant IL-4R macrophage A can be produced by introducing the polynucleotide or expression vector into macrophages or macrophage precursor cells isolated from a living organism. Alternatively, the mutant IL-4R macrophage A can be produced by introducing the polynucleotide or expression vector into somatic cells such as fibroblasts isolated from a living organism, and then establishing iPS cells from such somatic cells. The method for introducing the polynucleotide or expression vector into macrophages or macrophage precursor cells isolated from a living organism is not particularly limited, and includes known methods such as viral infection, calcium phosphate precipitation, lipofection, microinjection, and electroporation, with viral infection being a preferred example.

[0029] Alternatively, the mutant IL-4R macrophage A may be prepared by incorporating the polynucleotide into the genome of macrophages or macrophage progenitor cells isolated from a living organism using known gene editing techniques so that the polynucleotide can be expressed under the control of an appropriate promoter. Examples of known gene editing techniques include techniques using endonucleases such as zinc finger nucleases, TALENs (transcription activation-like effector nucleases), the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 system, and the CRISPR-Cas3 system. Methods for incorporating the polynucleotide into a cellular genome so that it can be expressed under the control of an appropriate promoter include incorporating the polynucleotide operably linked downstream of a suitable promoter (i.e., a polynucleotide linked to a coding sequence so that it can be expressed under the control of the promoter) into a non-coding region of the cellular genome, or incorporating the polynucleotide downstream of an endogenous promoter in the cellular genome.

[0030] The "macrophages or macrophage precursor cells isolated from a living body containing the present mutant IL-4R polypeptide" (hereinafter also referred to as "the present mutant IL-4R macrophage B") of the present invention are sufficient if they are macrophages or macrophage precursor cells isolated from a living body and contain the present mutant IL-4R polypeptide, and it is preferable that the extracellular domain of the present mutant IL-4R polypeptide is located extracellularly on the macrophage or macrophage precursor cell.

[0031] Methods for producing the mutant IL-4R macrophage B include, as described above, a method in which the polynucleotide or the expression vector is introduced into macrophages or macrophage precursor cells isolated from a living body, and the mutant IL-4R polypeptide is expressed in the macrophages or macrophage precursor cells isolated from a living body. Another method includes a method in which the endogenous IL-4 receptor gene is directly edited using the known gene editing technology described above to form a mutant IL-4 receptor gene sequence encoding the mutant IL-4R polypeptide.

[0032] The macrophages or macrophage precursor cells obtained by the above method may subsequently be recovered, separated, and purified by known methods. When the present polynucleotide or the present expression vector is introduced into macrophage precursor cells, the cells may be cultured after the introduction of the present polynucleotide or the present expression vector, and the macrophages may be separated or purified. When macrophage precursor cells are cultured in a petri dish, the macrophages differentiate into macrophages and adhere to the dish, while other cells are contained in the supernatant. Taking advantage of this property, macrophage precursor cells and macrophages can be separated or purified.

[0033] The "agent for preventing or treating inflammatory diseases, neurodegenerative diseases, or ischemic diseases, comprising the present mutant IL-4R macrophages and a pharmacologically acceptable additive (hereinafter also referred to as the "agent for preventing or treating inflammatory diseases, neurodegenerative diseases, or ischemic diseases") of the present invention is not particularly limited, as long as it contains the present mutant IL-4R macrophages and a pharmacologically acceptable additive. Examples of 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, isotonicity agents, fillers, and lubricants.

[0034] Examples of the inflammatory, neurodegenerative, or ischemic disease include diseases in which macrophages are involved in the pathogenesis. Examples of the inflammatory diseases include autoinflammatory diseases, autoimmune diseases, collagen diseases, ulcerative colitis, Crohn's disease, autoimmune hepatitis, encephalitis, myocarditis, nephritis, M1 macrophage-dominated chronic inflammation that increases with aging, dermatitis, skin photoaging, ophthalmitis, pancreatitis, enteritis, diabetes, arteriosclerosis such as atherosclerosis, vasculitis, parasitic infections, allergic diseases, and graft-versus-host disease (GVHD). Examples of the neurodegenerative diseases include Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyloidosis, Parkinson's disease, amyotrophic lateral sclerosis, and spinocerebellar degeneration. Examples of the ischemic diseases include ischemia-reperfusion injury, such as stroke or myocardial infarction.

[0035] The agent for preventing or treating an inflammatory disease, neurodegenerative disease, or ischemic disease can be administered to a subject in need of treatment for an inflammatory disease, neurodegenerative disease, or ischemic disease using methods known to those skilled in the art, including intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intraarticular, intrasynovial, intracranial, intrathecal, and subarachnoid (spinal fluid) injection.

[0036] The amount of the mutant IL-4R macrophages contained in the agent for preventing or treating an inflammatory disease, neurodegenerative disease, or ischemic disease to be administered can be adjusted appropriately depending on the type, location, and severity of the inflammatory disease, neurodegenerative disease, or ischemic disease, as well as the age, weight, and condition of the subject to be treated. Preferably, the amount of the mutant IL-4R macrophages contained in the agent for preventing or treating an inflammatory disease, neurodegenerative disease, or ischemic disease to be administered is 1×10 4 ~1×10 10 pieces, preferably 1 x 10 5 ~1×10 9 pieces, more preferably 1×10 6 ~1×10 8 I can list some examples.

[0037] The prophylactic or therapeutic agent for inflammatory diseases, neurodegenerative diseases, or ischemic diseases may be administered independently four times, three times, twice or once a day, every other day, every third day, every fourth day, every fifth day, once a week, every seventh day, every eighth day, every ninth day, twice a week, twice a month, once a month, every other month, or every two months.

[0038] The recipient of the present agent for preventing or treating inflammatory diseases, neurodegenerative diseases, or ischemic diseases and the donor of the macrophages or macrophage precursor cells used to produce the agent for preventing or treating inflammatory diseases, neurodegenerative diseases, or ischemic diseases may be the same or different. That is, the donor and the recipient may be identical or mismatched. Furthermore, when the recipient is a human, the macrophages or macrophage precursor cells used to produce the agent for preventing or treating inflammatory diseases, neurodegenerative diseases, or ischemic diseases may be autologous cells collected from the patient themselves or allogeneic cells collected from another person. When allogeneic cells are used, for example, β2-microglobulin (β2M) or human leukocyte antigens (HLA) may be modified by known techniques such as genome editing to reduce immune rejection.

[0039] The "agent for inducing differentiation or polarization of macrophages or macrophage precursor cells isolated from a living organism into M2 macrophages, which contains the present polynucleotide or the present expression vector as an active ingredient (hereinafter also referred to as the "agent for inducing differentiation or polarization into M2 macrophages") of the present invention is not particularly limited as long as it contains the present polynucleotide or the present expression vector as an active ingredient. By introducing the polynucleotide or the present expression vector into macrophages or macrophage precursor cells isolated from a living organism using the above-mentioned known method and culturing them in a predetermined medium as necessary, it becomes possible to differentiate or polarize macrophages or macrophage precursor cells into M2 macrophages. In this specification, differentiation refers to the acquisition of different properties or functions in cells from macrophage precursor cells other than M0 macrophages to M0 macrophages, and polarization refers to the transformation of M0 macrophages into M1 or M2 macrophages, the transformation of M1 macrophages into M2 macrophages, or the transformation of M2 macrophages into M1 macrophages. For example, when myeloid stem cells develop into monocytes, M0 macrophages, and then M2 macrophages, the process leading to M0 macrophages is called differentiation, and the process leading from M0 macrophages to M2 macrophages is called polarization. The agent for inducing differentiation or polarization into M2 macrophages may contain the above-mentioned pharmacologically acceptable additive. The agent for inducing differentiation or polarization into M2 macrophages may also contain the above-mentioned expression vector containing the present polynucleotide.

[0040] The "method for inducing differentiation or polarization of macrophages or macrophage precursor cells isolated from a living organism into M2 macrophages, which comprises introducing the present polynucleotide into macrophages or macrophage precursor cells isolated from a living organism" of the present invention involves introducing the present polynucleotide or the present expression vector into macrophages or macrophage precursor cells isolated from a living organism and culturing them in a predetermined medium as necessary, and the present polynucleotide or the present expression vector can be introduced into macrophages or macrophage precursor cells by the same method as described for the present method for producing mutant IL-4R macrophages.

[0041] Another aspect 1 of the present invention includes: 1) the prevention or treatment of inflammatory diseases, neurodegenerative diseases, or ischemic diseases, characterized by administering to a patient in need thereof macrophages or macrophage precursor cells isolated from a living organism containing the present polynucleotide or the present expression vector; 2) macrophages or macrophage precursor cells isolated from a living organism containing the present polynucleotide or the present expression vector for use as a preventive or therapeutic agent for inflammatory diseases, neurodegenerative diseases, or ischemic diseases; and 3) the use of macrophages or macrophage precursor cells isolated from a living organism containing the present polynucleotide or the present expression vector in the preparation of a preventive or therapeutic agent for inflammatory diseases, neurodegenerative diseases, or ischemic diseases.

[0042] Another aspect 2 of the present invention includes 1) the present polynucleotide or the present expression vector for use as an agent for inducing differentiation or polarization into M2 macrophages, and 2) use of the present polynucleotide or the present expression vector in the preparation of an agent for inducing differentiation or polarization into M2 macrophages.

[0043] Another aspect 3 of the present invention is a kit for producing an agent for inducing differentiation or polarization into M2 macrophages, which comprises the present polynucleotide or the present expression vector. Such a kit is not particularly limited as long as it comprises the present polynucleotide or the present expression vector, and may also include instructions for producing the agent for inducing differentiation or polarization into M2 macrophages, and reagents used for introducing the expression vector containing the present polynucleotide into macrophages or macrophage progenitor cells.

[0044] The present invention will be described in more detail below with reference to examples. The examples are not limiting.

[0045] [Example 1] (Creation of a cell line expressing human IL-4 receptor) A polynucleotide (SEQ ID NO: 3) encoding a mutant amino acid sequence of the human IL-4 receptor (I242N mutant human IL-4 receptor: SEQ ID NO: 2), in which the isoleucine at position 242 in the amino acid sequence of the human IL-4 receptor (SEQ ID NO: 1) was replaced with asparagine (I242N), was constructed by Vector Builder. The polynucleotide encoding the I242N mutant human IL-4 receptor was then transfected using lentivirus into RAW264.7 cells (n = 4, manufactured by KAC) an immortalized mouse macrophage cell line, or THP-1 cells (n = 3, purchased from the JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition), a human monocyte-derived cell line that differentiates into macrophages upon activation. Control cells were prepared in the same manner as above, but transfected with polynucleotides encoding the red fluorescent protein mCherry or green fluorescent protein (GFP) instead of the I242N mutant human IL-4 receptor.

[0046] The above cells transfected with each polynucleotide were cultured at 37°C and 5% CO2 in RAW medium (Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS (Fetal Bovine Serum)) for RAW264.7 cells, and THP-1 cells in THP medium (RPMI 1640 (Roswell Park Memorial Institute) containing 2 mM glutamine and 10% FBS) for THP-1 cells. THP-1 cells were cultured in THP medium containing 250 nM phorbol 12-myristate 13-acetate (PMA) for 24 hours to differentiate into macrophages, and then cultured in THP medium without PMA for an additional 2 days. The cells were then harvested, total RNA was isolated using Trizol reagent (Invitrogen), and cDNA was synthesized. Expression of the IL-4 receptor and human GAPDH or mouse Gapdh (internal standard) was then confirmed by real-time PCR. The following primers were used in real-time PCR. The hIL-4R forward primer shown in SEQ ID NO: 4 and the hIL-4R reverse primer shown in SEQ ID NO: 5 were capable of amplifying both the wild-type human IL-4 receptor amino acid sequence shown in SEQ ID NO: 1 and the mutant human IL-4 receptor amino acid sequence shown in SEQ ID NO: 2 (I242N mutant human IL-4).

[0047] [Table 1]

[0048] The results are shown in Figure 1. The upper panel of Figure 1 shows the results for RAW264.7 cells transfected with a polynucleotide encoding mCherry (mCherry RAW264.7 cells) and RAW264.7 cells transfected with a polynucleotide encoding the I242N mutant human IL-4 receptor (mut_IL-4R RAW264.7 cells). The lower panel of Figure 1 shows the results for THP-1 cells transfected with a polynucleotide encoding GFP (GFP THP-1 cells) and THP-1 cells transfected with a polynucleotide encoding the I242N mutant human IL-4 receptor (mut_IL-4R THP-1 cells). The vertical axis represents the relative expression level of mutant human IL-4 receptor, with the expression level in control mCherry RAW264.7 cells or GFP THP-1 cells set at 1. Expression levels were normalized with human GAPDH or mouse Gapdh and tested using an unpaired t-test.

[0049] As shown in the upper panel of Figure 1, human IL-4 receptor was expressed in mut_IL-4R RAW264.7 cells, confirming the expression of the lentiviral-transduced I242N mutant human IL-4 receptor. Furthermore, as shown in the lower panel of Figure 1, human IL-4 receptor expression was increased in mut_IL-4R THP-1 cells compared to the control, confirming the expression of the lentiviral-transduced I242N mutant human IL-4 receptor.

[0050] Example 2 (Expression of M2 macrophage markers Arg1 and CD206 in cells expressing 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, a mouse M2 macrophage marker, and CD206, a human M2 macrophage marker. The specific method is as follows.

[0051] As in Example 1, WT_IL-4R RAW264.7 cells were generated by introducing a polynucleotide encoding wild-type human IL-4 receptor into RAW264.7 cells. Next, the four types of cells generated in Example 1 (mCherry RAW264.7 cells (n = 4), mut_IL-4R RAW264.7 cells (n = 4), mCherry THP-1 cells (n = 3), and mut_IL-4R THP-1 cells (n = 3)) and WT_IL-4R RAW264.7 cells (n = 4) were cultured at 37°C and 5% CO for 3 days, and the cells were harvested and RNA was extracted. Next, Arg1 expression and CD206 expression 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, while mCherry THP-1 cells and mut_IL-4R THP-1 cells were cultured in THP cells (containing PMA for the first 24 hours of the three-day period). The following primers were used for real-time PCR.

[0052] [Table 2]

[0053] The results are shown in Figure 2. The expression levels were normalized with human GAPDH or mouse Gapdh, and the results were tested by 1-way ANOVA for RAW264.7 cells and by Unpaiered T-test for THP-1 cells.

[0054] As shown in Figure 2, Arg1 expression was low in control mCherry RAW264.7 cells and WT_IL-4R RAW264.7 cells, whereas Arg1 expression was 59-fold higher in mut_IL-4R RAW264.7 cells than in control mCherry RAW264.7 cells and 28-fold higher than in WT_IL-4R RAW264.7 cells. Similarly, CD206 expression was low in control GFP THP-1 cells, whereas CD206 expression was 13-fold higher in mut_IL-4R THP-1 cells than in the control cells. Therefore, expression of the I242N mutant human IL-4 receptor induced differentiation and / or polarization of macrophage precursor cells into M2 macrophages.

[0055] [Example 3] (Suppression of M1 macrophage induction in cells expressing I242N mutant human IL-4 receptor) When lipopolysaccharide (LPS), a humoral factor that induces M1 macrophages, was applied to mut_IL-4R RAW264.7 cells, we investigated whether M2 macrophages polarized from the cells remained as M2 macrophages without polarizing to M1 macrophages by measuring the expression of Arg1 and tumor necrosis factor (TNFα), an M1 macrophage marker.

[0056] mut_IL-4R RAW264.7 cells (n=4) and control mCherry RAW264.7 cells (n=4) were cultured in RAW medium containing LPS (1 ng / mL LPS or LPS-free (Basal) as a control) at 37°C and 5% CO for 3 days. Cells were harvested and RNA was extracted. Expression of TNFα and Arg1 was then examined by real-time PCR. The following primers were used for real-time PCR:

[0057] [Table 3]

[0058] The results are shown in Figure 3. The expression levels were normalized with human GAPDH or mouse Gapdh and tested by unpaiered t-test.

[0059] As is clear from Figure 3, in the control mCherry RAW264.7 cells, exposure to LPS increased the expression of the M1 macrophage marker TNFα, but in mut_IL-4R RAW264.7 cells, exposure to LPS hardly increased TNFα expression. Furthermore, in mut_IL-4R RAW264.7 cells, the expression of the M2 macrophage marker Arg1 was not significantly different from that of basal cells (control without LPS), and no decrease in Arg1 expression was observed.

[0060] The results in Figures 2 and 3 demonstrate that expression of the I242N mutant human IL-4 receptor leads to differentiation and / or polarization of macrophage precursor cells into M2 macrophages, or polarizes M1 macrophages into M2 macrophages, and that even in the presence of humoral factors that induce M1 macrophages, macrophages can be maintained as M2 macrophages without polarization into M1 macrophages.

[0061] [Example 4] (Polarization of M2 macrophages by 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 the differentiation and / or polarization of macrophage precursor cells to M2 macrophages in the control GFP and the following mutations (1) to (10) other than position 242 were examined by Agr1 expression. Agr1 expression was performed in the same manner as in Example 2. Specifically, polynucleotides encoding GFP or the following (1) to (10) were prepared using the PrimeSTAR® Mutagenesis Basal Kit (TAKARA Corporation), and each was introduced into RAW264.7 cells (n=3). The cells were cultured in RAW medium at 37°C and 25% CO for 3 days. The cells were then harvested, RNA was extracted, and Arg1 expression was examined by real-time PCR.

[0062] (1) A mutant amino acid sequence of human IL-4 receptor in which aspartic acid at position 37 in the amino acid sequence of human IL-4 receptor is replaced with asparagine (D37N mutant human IL-4 receptor: SEQ ID NO: 16). (2) A mutant amino acid sequence of the human IL-4 receptor in which the tyrosine at position 62 in the amino acid sequence of the human IL-4 receptor is replaced with cysteine ​​(Y62C mutant human IL-4 receptor: SEQ ID NO: 17). (3) A mutant amino acid sequence of the human IL-4 receptor in which asparagine at position 78 in the amino acid sequence of the human IL-4 receptor is substituted with tyrosine (N78Y mutant human IL-4 receptor: SEQ ID NO: 18). (4) A mutant amino acid sequence of human IL-4 receptor in which phenylalanine at position 115 in the amino acid sequence of human IL-4 receptor is replaced with leucine (F115L mutant human IL-4 receptor: SEQ ID NO: 19). (5) A mutant amino acid sequence of human IL-4 receptor in which asparagine at position 176 in the amino acid sequence of human IL-4 receptor is substituted with serine (N176S mutant human IL-4 receptor: SEQ ID NO: 20). (6) A mutant amino acid sequence of human IL-4 receptor in which arginine at position 200 in the amino acid sequence of human IL-4 receptor is replaced with tryptophan (R200W mutant human IL-4 receptor: SEQ ID NO: 21). (7) A mutant amino acid sequence of human IL-4 receptor in which isoleucine at position 242 in the amino acid sequence of human IL-4 receptor is substituted with aspartic acid (I242D mutant human IL-4 receptor: SEQ ID NO: 22). (8) A mutant amino acid sequence of human IL-4 receptor in which cysteine ​​at position 251 in the amino acid sequence of human IL-4 receptor is replaced with tryptophan (C251W mutant human IL-4 receptor: SEQ ID NO: 23). (9) A mutant amino acid sequence of human IL-4 receptor in which lysine at position 308 in the amino acid sequence of human IL-4 receptor is substituted with asparagine (K308N mutant human IL-4 receptor: SEQ ID NO: 24).

[0063] The results are shown in Figure 4. The expression levels were corrected for human GAPDH or mouse Gapdh and tested by unpaiered t-test.

[0064] As shown in Figure 4, expression of the 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 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 Arg1 expression. In other words, it was revealed that differentiation or polarization into M2 macrophages is possible even when cells contain the mutant IL-4R polypeptides of the present invention other than the I242N mutant human IL-4 receptor.

[0065] [Example 5] (Persistence of M2 macrophages) We investigated how long cells expressing the I242N mutant human IL-4 receptor could maintain the characteristics of M2 macrophages using Arg1 expression as an indicator.

[0066] Mouse bone marrow cells were collected using a known method and cultured for 5 days in a culture medium supplemented with M-CSF (20 ng / ml) to differentiate into bone marrow-derived macrophages (BMDMs). Subsequently, in the presence of M-CSF (20 ng / ml), a polynucleotide encoding the I242N mutant human IL-4 receptor or a control (a polynucleotide encoding GFP) was transfected into the BMDMs using lentivirus, as in Example 1. After 48 hours of culture, the BMDMs were harvested and stored. Each BMDM was stimulated with IL-4 (100 pg / ml) for 24 hours to polarize them into M2 macrophages, then cultured for one week. The expression of the M2 macrophage marker Arg1 was analyzed by real-time PCR. The results are shown in Figure 5. The horizontal axis represents the number of days in culture (day 0 after 24 hours of stimulation with IL-4 is defined as day 0), and the vertical axis represents the relative expression level of Arg1, where the expression level of Arg1 in the control on day 0 is defined as 1. Expression levels were normalized using mouse Gapdh (n = 3).

[0067] In BMDMs (cells) polarized to M2 macrophages by control IL-4 stimulation alone, Arg1 expression decreased by Day 3, whereas cells expressing the I242N mutant human IL-4 receptor continued to express Arg1 even on Day 7 (1 week later). Therefore, it was demonstrated that the inclusion of the present mutant IL-4R polypeptide allows differentiation or polarization into M2 macrophages, which can then be maintained as M2 macrophages for at least 1 week. Conventional methods have limited therapeutic efficacy because M2 macrophages lose their function within a few days after in vivo administration. In contrast, the M2 macrophages produced by the present invention can maintain their M2 macrophage function for a long period of time, and are therefore expected to have a high therapeutic effect against inflammatory diseases, neurodegenerative diseases, and ischemic diseases.

[0068] [Example 6] (IL-10 secretion) To confirm the anti-inflammatory effect of M2 macrophages expressing the present mutant IL-4R polypeptide, we examined the secretion of IL-10, a potent anti-inflammatory cytokine. As in Example 1, a polynucleotide encoding the I242N mutant human IL-4 receptor or a control (a polynucleotide encoding GFP) was introduced into RAW264.7 cells using a lentivirus to generate a stable-expressing cell line. Each cell line was then seeded onto a culture dish and cultured for 24 hours. After the culture, the supernatant was collected, and the IL-10 contained in the supernatant was analyzed by ELISA. An unpaintered T-test (N=3) was performed. The results are shown in Figure 6.

[0069] As is clear from Figure 6, when the mutant IL-4R polypeptide was contained, IL-10 expression was increased by more than four times compared to the control, confirming that M2 macrophages containing the mutant IL-4R polypeptide have anti-inflammatory activity. [Industrial Applicability]

[0070] The present invention can be used as a biopharmaceutical for inflammatory diseases, wound healing, neurodegenerative diseases, and ischemic diseases.

Claims

1. Macrophage precursor cells isolated from a living organism containing the following polypeptides (2-1') to (2-9') derived from the human interleukin 4 (human IL-4) receptor: (2-1') substitution of aspartic acid at position 37 with asparagine in the amino acid sequence shown in SEQ ID NO: 1; (2-2') substitution of tyrosine with cysteine ​​at position 62 in the amino acid sequence shown in SEQ ID NO: 1; (2-3') substitution of asparagine with tyrosine at position 78 in the amino acid sequence set forth in SEQ ID NO: 1; (2-4') substitution of phenylalanine with leucine at position 115 in the amino acid sequence shown in SEQ ID NO: 1; (2-5') substitution of asparagine with serine at position 176 in the amino acid sequence set forth in SEQ ID NO: 1; (2-6') substitution of arginine with tryptophan at position 200 in the amino acid sequence shown in SEQ ID NO: 1; (2-7') substitution of isoleucine at position 242 with asparagine or aspartic acid in the amino acid sequence shown in SEQ ID NO: 1; (2-8') substitution of cysteine ​​at position 251 with tryptophan in the amino acid sequence shown in SEQ ID NO: 1; (2-9') Substitution of lysine at position 308 in the amino acid sequence shown in SEQ ID NO: 1 with asparagine.

2. Macrophage precursor cells isolated from a living organism containing a polynucleotide encoding the following polypeptides (2-1') to (2-9') derived from the human interleukin-4 receptor: (2-1') substitution of aspartic acid at position 37 with asparagine in the amino acid sequence shown in SEQ ID NO: 1; (2-2') substitution of tyrosine with cysteine ​​at position 62 in the amino acid sequence shown in SEQ ID NO: 1; (2-3') substitution of asparagine with tyrosine at position 78 in the amino acid sequence set forth in SEQ ID NO: 1; (2-4') substitution of phenylalanine with leucine at position 115 in the amino acid sequence shown in SEQ ID NO: 1; (2-5') substitution of asparagine with serine at position 176 in the amino acid sequence set forth in SEQ ID NO: 1; (2-6') substitution of arginine with tryptophan at position 200 in the amino acid sequence shown in SEQ ID NO: 1; (2-7') substitution of isoleucine at position 242 with asparagine or aspartic acid in the amino acid sequence shown in SEQ ID NO: 1; (2-8') substitution of cysteine ​​at position 251 with tryptophan in the amino acid sequence shown in SEQ ID NO: 1; (2-9') Substitution of lysine at position 308 in the amino acid sequence shown in SEQ ID NO: 1 with asparagine.

3. Macrophage precursor cells isolated from a living organism, which contain an expression vector containing a polynucleotide encoding the following polypeptides (2-1') to (2-9') derived from human interleukin-4 receptor: (2-1') substitution of aspartic acid at position 37 with asparagine in the amino acid sequence shown in SEQ ID NO: 1; (2-2') substitution of tyrosine with cysteine ​​at position 62 in the amino acid sequence shown in SEQ ID NO: 1; (2-3') substitution of asparagine with tyrosine at position 78 in the amino acid sequence set forth in SEQ ID NO: 1; (2-4') substitution of phenylalanine with leucine at position 115 in the amino acid sequence shown in SEQ ID NO: 1; (2-5') substitution of asparagine with serine at position 176 in the amino acid sequence set forth in SEQ ID NO: 1; (2-6') substitution of arginine with tryptophan at position 200 in the amino acid sequence shown in SEQ ID NO: 1; (2-7') substitution of isoleucine at position 242 with asparagine or aspartic acid in the amino acid sequence shown in SEQ ID NO: 1; (2-8') substitution of cysteine ​​at position 251 with tryptophan in the amino acid sequence shown in SEQ ID NO: 1; (2-9') Substitution of lysine at position 308 in the amino acid sequence shown in SEQ ID NO: 1 with asparagine.

4. A preventive or therapeutic agent for inflammatory diseases, neurodegenerative diseases, or ischemic diseases, comprising the macrophage precursor cells according to any one of claims 1 to 3 and a pharmacologically acceptable additive.

5. An agent for inducing differentiation and / or polarization of macrophage precursor cells isolated from a living body into M2 macrophages, comprising, as an active ingredient, an expression vector containing a polynucleotide encoding the following polypeptides (2-1') to (2-9') derived from human interleukin-4 receptor: (2-1') substitution of aspartic acid at position 37 with asparagine in the amino acid sequence shown in SEQ ID NO: 1; (2-2') substitution of tyrosine with cysteine ​​at position 62 in the amino acid sequence shown in SEQ ID NO: 1; (2-3') substitution of asparagine with tyrosine at position 78 in the amino acid sequence set forth in SEQ ID NO: 1; (2-4') substitution of phenylalanine with leucine at position 115 in the amino acid sequence shown in SEQ ID NO: 1; (2-5') substitution of asparagine with serine at position 176 in the amino acid sequence set forth in SEQ ID NO: 1; (2-6') substitution of arginine with tryptophan at position 200 in the amino acid sequence shown in SEQ ID NO: 1; (2-7') substitution of isoleucine at position 242 with asparagine or aspartic acid in the amino acid sequence shown in SEQ ID NO: 1; (2-8') substitution of cysteine ​​at position 251 with tryptophan in the amino acid sequence shown in SEQ ID NO: 1; (2-9') Substitution of lysine at position 308 in the amino acid sequence shown in SEQ ID NO: 1 with asparagine.

6. A method for inducing differentiation and / or polarization of macrophage precursor cells isolated from a living body into M2 macrophages, comprising introducing into macrophage precursor cells isolated from a living body an expression vector containing a polynucleotide encoding the following polypeptides (2-1') to (2-9') derived from human interleukin-4 receptor: (2-1') substitution of aspartic acid at position 37 with asparagine in the amino acid sequence shown in SEQ ID NO: 1; (2-2') substitution of tyrosine with cysteine ​​at position 62 in the amino acid sequence shown in SEQ ID NO: 1; (2-3') substitution of asparagine with tyrosine at position 78 in the amino acid sequence set forth in SEQ ID NO: 1; (2-4') substitution of phenylalanine with leucine at position 115 in the amino acid sequence shown in SEQ ID NO: 1; (2-5') substitution of asparagine with serine at position 176 in the amino acid sequence set forth in SEQ ID NO: 1; (2-6') substitution of arginine with tryptophan at position 200 in the amino acid sequence shown in SEQ ID NO: 1; (2-7') substitution of isoleucine at position 242 with asparagine or aspartic acid in the amino acid sequence shown in SEQ ID NO: 1; (2-8') substitution of cysteine ​​at position 251 with tryptophan in the amino acid sequence shown in SEQ ID NO: 1; (2-9') Substitution of lysine at position 308 in the amino acid sequence shown in SEQ ID NO: 1 with asparagine.

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