Immunomodulator and pharmaceutical composition for preventing or treating aging-related diseases

Abstract

This immune inducer contains, as an active ingredient, a peptide (a) or (b) below or an expression vector having a structural gene including a region encoding the peptide. (a) A peptide comprising the amino acid sequence represented by SEQ ID NO: 1 or 2; or (b) a peptide having immunity induction activity and comprising an amino acid sequence including a sequence in which in the amino acid sequence represented by SEQ ID NO: 1 or 2, one or more amino acids are deleted, substituted, or added. This pharmaceutical composition for preventing or treating aging-related diseases contains the immune inducer and an adjuvant.

Description

Technical Field

[0001] The present invention relates to an immune inducer and a pharmaceutical composition for preventing or treating aging-related diseases. This application claims priority based on Japanese Patent Application No. 2019-139031 filed in Japan on July 29, 2019, and the content thereof is incorporated herein by reference.

Background Art

[0002] Accumulation of senescent cells in various tissues has been reported to have a pathological role in age-related diseases. Direct inhibition of cell senescence regulators (e.g., p53, p21, etc.) prevents the onset of age-related pathologies, but there is a risk of increasing the incidence of cancer. Recently, it has been reported that removal of senescent cells (senolysis) reversibly improves the pathological aging phenotype of aged mice without leading to cancer development. Based on the analysis of transcriptome data from senescent vascular endothelial cells, the inventors identified Transmembrane glycoprotein nonmetastatic melanoma protein B (GPNMB) protein as a senescent cell-specific molecule having a transmembrane domain, and clarified that the expression of GPNMB protein is upregulated in vascular cells of patients with atherosclerosis and mice (see, for example, Patent Document 1).

[0003] On the other hand, it has been reported that GPNMB protein is overexpressed in most human malignant melanomas, breast cancers, and brain tumors, and immune inducers containing a peptide fragment of GPNMB protein as an active ingredient for the treatment or prevention of cancer by immunotherapy (see, for example, Patent Document 2), and antibodies that specifically bind to GPNMB protein (see, for example, Patent Document 3) have been developed.

[0004] However, the epitope of GPNMB protein effective for the purpose of removing senescent cells is not known.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0006] The present invention has been made in view of the above circumstances, and provides an immune inducer effective for preventing or treating aging-related diseases and a pharmaceutical composition for preventing or treating aging-related diseases containing the immune inducer.

Means for Solving the Problems

[0007] As a result of intensive studies to achieve the above object, the inventors have found that regions at positions 63 to 71 or 150 to 159 in the amino acid sequence of human GPNMB protein are epitopes effective for removing senescent cells, and that senescent cells can be removed and phenotypes related to cellular senescence can be improved by administering a peptide consisting of the amino acid sequence of the region, thus completing the present invention.

[0008] That is, the present invention includes the following aspects. The immune inducer according to the first aspect of the present invention contains, as an active ingredient, any one of the following peptides (a) or (b), or an expression vector comprising a structural gene containing a region encoding the peptide. (a) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; (b) A peptide consisting of an amino acid sequence containing a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2 and having immune induction activity

[0009] The peptide may consist of the amino acid sequence shown in any of SEQ ID NOs: 3 to 6. The immunomodulator according to the first aspect may contain the peptide as an active ingredient. The immunomodulator according to the first aspect may further have a carrier protein bound to the peptide. The immunomodulator according to the first aspect may be used for removing senescent cells.

[0010] The pharmaceutical composition for preventing or treating an aging-related disease according to the second aspect of the present invention contains an immunomodulator and an adjuvant.

Advantages of the Invention

[0011] The immunomodulator of the above aspect is effective for preventing or treating an aging-related disease.

Brief Description of the Drawings

[0012]

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Modes for Carrying Out the Invention

[0013] Hereinafter, an immunomodulator according to an embodiment of the present invention will be described in detail.

[0014] ≪Immunomodulator≫ The immunomodulator of this embodiment contains, as an active ingredient, a peptide consisting of the region at positions 63 to 71 or 150 to 159 in the amino acid sequence of human or mouse GPNMB protein. Such an immunomodulator is also called a "peptide vaccine". In this specification, "containing as an active ingredient" means containing a therapeutically effective amount of a peptide or an expression vector having a structural gene containing a region encoding the peptide.

[0015] GPNMB is a single-pass transmembrane protein, and a part of it is cleaved and known to be secreted into the blood. As shown in the examples described later, the inventors have clarified that senescent cells can be removed by administering a peptide consisting of the region at positions 63 to 71 or 150 to 159 in the amino acid sequence of mouse GPNMB protein. In addition, the GPNMB protein is highly conserved in all mammals. Therefore, from such findings, a peptide consisting of a region corresponding to the region at positions 63 to 71 or 150 to 159 in the amino acid sequence of mouse GPNMB protein can be used as a prophylactic or therapeutic agent for aging-related diseases. The RefSeq accession number of human GPNMB protein is NP_001005340. The RefSeq accession number of mouse GPNMB protein is NP_444340. The amino acid sequence of human GPNMB protein is shown in SEQ ID NO: 7. The amino acid sequence of mouse GPNMB protein is shown in SEQ ID NO: 8. The region corresponding to the region at positions 63 to 71 or 150 to 159 in the amino acid sequence of mouse GPNMB protein of mammals other than mouse can be examined using a known multiple alignment tool.

[0016] In this specification, "senescent cells" mean cells that show an increase in the expression level of senescence markers compared to normal cells. Examples of senescence markers include senescence-associated acidic β-galactosidase, P53, P16 INK4a , P21 CIP1 , etc. Senescent cells are characterized by irreversible growth arrest in the G1 phase, and are formed by the suppression of genes that promote cell cycle progression and the increased expression of P53, P16 INK4a , P21 CIP1 . It is known that senescent cells can be cells that have stopped dividing but remain metabolically active. Non-dividing cells can survive for several weeks, but cannot proliferate and replicate DNA despite the presence of sufficient space, nutrients, and growth factors in the medium. Therefore, even if a physiological stimulus is applied to senescent cells, they cannot be stimulated to proliferate, so this arrest of division is essentially permanent. Senescent cells can be cells that have stopped dividing but remain metabolically active. Non-dividing cells can survive for several weeks, but cannot proliferate and replicate DNA despite the presence of sufficient space, nutrients, and growth factors in the medium. Therefore, even if a physiological stimulus is applied to senescent cells, they cannot be stimulated to proliferate, so this arrest of division is essentially permanent. Senescent cells can differ from non-senescent cells in one or more of the following aspects: 1) Senescent cells have stopped proliferating and cannot be stimulated to re-enter the cell cycle by physiological mitogens; 2) Senescent cells are resistant to apoptotic cell death; 3) Senescent cells acquire altered differentiation functions.

[0017] Senescent cells can be caused by replicative cell senescence, premature cell senescence, or cell senescence induced by treatment, etc. Senescent cells caused by replicative cell senescence may have undergone multiple cell divisions, for example, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more cell divisions. Senescent cells caused by premature cell senescence can be induced by, but are not limited to, ultraviolet rays, reactive oxygen species, environmental toxins, smoking, ionizing radiation, chromatin structure distortion, excessive mitogenic signaling, etc. In certain embodiments, premature cell senescence can be induced by ionizing radiation. In another specific embodiment, premature cell senescence can be induced by ectopic transfection using the Ras protein. Senescent cells caused by cell senescence induced by treatment can be induced by radiotherapy, chemotherapy, DNA damage therapy, etc.

[0018] The senescent cells targeted by the present invention can generally be eukaryotic cells. Examples of senescent cells include, but are not limited to: mammary epithelial cells, keratinocytes, cardiomyocytes, chondrocytes, endothelial cells (large blood vessels), endothelial cells (microvessels), epithelial cells, fibroblasts, dermal papilla cells, hepatocytes, melanocytes, osteoblasts, adipose progenitor cells, immune system cells, skeletal muscle cells, smooth muscle cells, adipocytes, neurons, glial cells, contractile cells, exocrine epithelial cells, extracellular matrix cells, hormone-secreting cells, keratinized epithelial cells, pancreatic islet cells, lens cells, mesenchymal stem cells, pancreatic adenocarcinoma cells, small intestine Paneth cells, hematopoietic system cells, nervous system cells, cells supporting sensory organs and peripheral nerve cells, wet stratified barrier epithelial cells.

[0019] Furthermore, the senescent cells targeted by the present invention can also be found in renewable tissues including the vascular system, hematopoietic system, epithelial organs, and stroma. Senescent cells can also be found at sites of aging and chronic pathological conditions associated with aging (e.g., osteoarthritis, atherosclerosis). Furthermore, senescent cells can be associated with benign neoplastic lesions and benign prostatic hyperplasia. In one embodiment, senescent cells can be found in normal tissues after DNA damage therapy. In another specific embodiment, senescent cells can be found at sites of pathological conditions associated with aging.

[0020] The number of senescent cells in various organs and tissues usually increases with age. The accumulation of senescent cells can further promote aging and deterioration under aging-related diseases. For example, the accumulation of senescent cells in aging tissues can contribute to age-related tissue dysfunction, decreased regenerative capacity, and diseases. In one embodiment, the aging tissue with accumulated senescent cells lacks the ability to respond to stress that requires proliferation, thereby resulting in a reduction in health associated with aging.

[0021] In addition, in this specification, "removal of senescent cells" means removing senescent cells from tissues, organs, etc., or killing senescent cells. It is particularly preferable that cells that are not senescent cells (hereinafter referred to as "non-senescent cells") are not significantly killed and that senescent cells are selectively or specifically killed.

[0022] In addition, in this specification, "aging-related diseases" may include any disease or condition that is mediated, in whole or in part, by inducing or maintaining a non-proliferative or senescent state in cells or cell populations in a subject. Aging-related diseases may include atrophy or fibrosis of tissues or organs where symptoms of the disease state cannot be visually observed, or visible disease states such as degenerative diseases and hypofunction disorders.

[0023] Examples of aging-related diseases include, for example, Alzheimer's disease, Parkinson's disease, cataract, macular degeneration, glaucoma, atherosclerosis, acute coronary syndrome, myocardial infarction, heart failure, diabetes, stroke, hypertension, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, coronary artery disease, cerebrovascular disease, periodontal disease, atrophy and fibrosis in various tissues, brain or heart injury, treatment-related myelodysplastic syndrome, etc. In addition, aging-related diseases may also include Hutchinson-Gilford progeria syndrome, Werner syndrome, Cockayne syndrome, xeroderma pigmentosum, ataxia telangiectasia, Fanconi anemia, neurogenic anemia, etc.

[0024] As further examples of aging-related diseases, there are cardiovascular diseases such as angina pectoris, aortic aneurysm, arrhythmia, cerebral aneurysm, diastolic heart failure, myocardial fibrosis, cardiomyopathy, carotid artery disease, coronary thrombosis, endocarditis, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, etc.; inflammatory or autoimmune diseases such as intervertebral disc herniation, oral mucositis, erythema, interstitial cystitis, scleroderma, alopecia, etc.; neurodegenerative diseases such as dementia, Huntington's disease, motor nerve dysfunction, memory decline associated with aging, depression, mood disorders, etc.; metabolic diseases such as metabolic syndrome, etc.; lung diseases such as decline in lung function associated with aging, asthma, bronchiectasis, cystic fibrosis, emphysema, etc.; gastrointestinal diseases such as Barrett's esophagus, etc.; diseases associated with aging such as liver fibrosis, muscle fatigue, oral mucosal fibrosis, pancreatic fibrosis, benign prostatic hyperplasia (BPH), sleep disorders, etc.; reproductive disorders such as menopause, decreased egg supply, decreased sperm survival rate, decreased reproductive ability, decreased libido, erectile dysfunction, excitement, etc.; skin diseases such as atopic dermatitis, skin erythema, cutaneous lymphoma, dysesthesia, eczema, eosinophilic dermatitis, fibrous proliferation of the skin, hyperpigmentation, immunobullous disease, nevus, pemphigus vulgaris, pruritus, psoriasis, rash, reactive neutrophilic dermatosis, wrinkles, urticaria, etc.; post-transplant renal fibrosis; carotid thrombosis, etc.

[0025] Furthermore, preferred examples of aging-related diseases include cardiovascular diseases such as heart failure, arteriosclerosis, myocardial infarction, diabetes, atherosclerotic cerebrovascular diseases, hypertension, etc.; cerebrovascular diseases such as cerebral infarction, cerebral hemorrhage, etc.; metabolic diseases such as dyslipidemia, etc.; respiratory diseases such as pulmonary fibrosis, emphysema, etc.; musculoskeletal syndromes such as skeletal muscle atrophy (sarcopenia), osteoarthritis, etc.; geriatric syndromes such as dementia, frailty, etc.; chronic kidney disease; eye diseases such as cataract, glaucoma, age-related macular degeneration, presbyopia, etc.; age-related alopecia; age-related hearing loss; pain such as low back pain and joint pain associated with aging, etc.; skin diseases such as seborrheic dermatitis and pruritus, etc.; liver diseases such as fatty liver, non-alcoholic steatohepatitis (NASH), liver cirrhosis, etc.; bone diseases such as osteoporosis and osteoarthritis, etc.; progeria such as Hutchinson-Gilford progeria syndrome, Werner syndrome, Cockayne syndrome, Rothmund-Thomson syndrome, etc.

[0026] The accumulation of senescent cells is known to promote pathological conditions such as senescence-related diseases. Therefore, by administering the immune inducer of the present embodiment to activate the immune system in the body of the administration subject and remove senescent cells, it is possible to prevent or treat diseases in which improvement of the pathological condition is expected.

[0027] Also, as shown in the examples described later, the inventors have clarified that by administering a peptide consisting of the region at positions 63 to 71 or 150 to 159 in the amino acid sequence of the mouse GPNMB protein, atherosclerotic plaques are reduced. Therefore, the above peptide is particularly suitable as a prophylactic or therapeutic agent for atherosclerosis.

[0028] Specifically, the immune inducer of the present embodiment contains the following peptide (a) as an active ingredient. (a) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2

[0029] The amino acid sequence shown in SEQ ID NO: 1 in (a) above is the amino acid sequence at positions 63 to 71 in the amino acid sequence of the human or mouse GPNMB protein. The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence at positions 150 to 159 in the amino acid sequence of the human or mouse GPNMB protein.

[0030] SEQ ID NO: 1: XRGDXRWKB SEQ ID NO: 2: XSXHXXFPDX

[0031] In addition, the X located at the first position from the left in SEQ ID NO: 1 is arginine or lysine, the X located at the fifth position from the left is glycine or methionine, and the B at the ninth position from the left is aspartic acid or asparagine. That is, the amino acid sequence shown in SEQ ID NO: 1 can be the following amino acid sequence. SEQ ID NO: 3 is the amino acid sequence of the region at positions 63 to 71 in the amino acid sequence of the human GPNMB protein. SEQ ID NO: 4 is the amino acid sequence of the region at positions 63 to 71 in the amino acid sequence of the mouse GPNMB protein.

[0032] Sequence number 3: KRGDMRWKN Sequence number 4: RRGDGRWKD

[0033] In addition, X located at the first position from the left in SEQ ID NO: 2 is arginine or glutamine, X located at the third position from the left is glutamine or histidine, X located at the fifth position from the left is leucine or asparagine, X located at the sixth position from the left is arginine or valine, and X located at the tenth position from the left is arginine or glycine. That is, the amino acid sequence shown in SEQ ID NO: 2 may be the following amino acid sequence. Note that SEQ ID NO: 5 is the amino acid sequence of the region at positions 150-159 in the amino acid sequence of human GPNMB protein. SEQ ID NO: 6 is the amino acid sequence of the region at positions 150-159 in the amino acid sequence of mouse GPNMB protein.

[0034] Sequence number 5: QSHHNVFPDG Sequence number 6: RSQHLRFPDR

[0035] Among them, it is preferable that the peptide contained in the immunopotentiator of the present embodiment is a peptide consisting of the amino acid sequence shown in any of SEQ ID NOS: 3 to 6.

[0036] The immunopotentiator of the present embodiment may contain the following peptide (b) as a peptide functionally equivalent to the peptide of (a) above. (b) A peptide consisting of an amino acid sequence containing a sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1 or 2 and having immunopotentiating activity

[0037] Here, the number of amino acids that may be deleted, substituted, or added is preferably 1 or more and 2 or less, and more preferably 1.

[0038] In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residues that may be deleted are preferably exemplified by X located at the 1st position from the left (N-terminal amino acid residue) or B located at the 9th position from the left (C-terminal amino acid residue) in SEQ ID NO: 1. In the amino acid sequence shown in SEQ ID NO: 1, arbitrary amino acid residues may be added to its N-terminus or C-terminus, but as the amino acid residues added to the N-terminus or C-terminus, it is preferable that other amino acid residues having a side chain chemically similar to the amino acid residue located at the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO: 1 are added. Other amino acid residues having a chemically similar side chain will be described later.

[0039] In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residues that may be substituted are preferably exemplified by X located at the 1st position from the left, X located at the 5th position from the left, or B located at the 9th position from the left in SEQ ID NO: 1. These amino acid residues may be substituted with any amino acid residues, but it is preferable that they are substituted with other amino acid residues having a side chain chemically similar to the amino acid residue to be substituted. Other amino acid residues having a chemically similar side chain will be described later.

[0040] Also, in the amino acid sequence shown in SEQ ID NO: 2, the amino acid residues that may be deleted are preferably exemplified by X located at the 1st position from the left (N-terminal amino acid residue) or X located at the 10th position from the left (C-terminal amino acid residue) in SEQ ID NO: 2. In the amino acid sequence shown in SEQ ID NO: 2, arbitrary amino acid residues may be added to its N-terminus or C-terminus, but as the amino acid residues added to the N-terminus or C-terminus, it is preferable that other amino acid residues having a side chain chemically similar to the amino acid residue located at the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO: 2 are added. Other amino acid residues having a chemically similar side chain will be described later.

[0041] In the amino acid sequence shown in SEQ ID NO:2, preferred examples of the amino acid residue that may be substituted include X located at the 1st position from the left, X located at the 3rd position from the left, X located at the 5th position from the left, X located at the 6th position from the left, or X located at the 10th position from the left. These amino acid residues may be substituted with any amino acid residue, but are preferably substituted with another amino acid residue having a chemically similar side chain to the substituting amino acid residue. Other amino acid residues having chemically similar side chains will be described later.

[0042] In the present specification, "substitution" means substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the peptide contained in the immunomodulator of the present embodiment belongs. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine and histidine), and among neutral amino acids, amino acids having a hydrocarbon chain (glycine, alanine, valine, leucine, isoleucine and proline), amino acids having a hydroxy group (serine and threonine), amino acids containing sulfur (cysteine and methionine), amino acids having an amide group (asparagine and glutamine), amino acids having an imino group (proline), amino acids having an aromatic group (phenylalanine, tyrosine and tryptophan), etc. can be classified. Generally possible amino acid substitutions include, for example, alanine / serine, valine / isoleucine, aspartic acid / glutamic acid, threonine / serine, alanine / glycine, alanine / threonine, serine / asparagine, alanine / valine, serine / glycine, tyrosine / phenylalanine, alanine / proline, lysine / arginine, aspartic acid / asparagine, leucine / isoleucine, leucine / valine, alanine / glutamic acid, aspartic acid / glycine, etc.

[0043] Furthermore, the peptide in (b) above has immunogenic activity. As used herein, "immunogenic activity" means the ability to promote the production of antibodies that specifically bind to GPNMB. Specifically, as shown in the examples described below, it means that the antibody titer when the peptide is administered is significantly higher than that when the control peptide is administered. The antibody titer can be measured using the method described in the examples below.

[0044] The peptide contained in the immunogen of this embodiment is predicted to be a sequence that is difficult to be presented on the major histocompatibility complex (MHC) from algorithm analysis. Therefore, antibodies that specifically bind to the above peptide usually do not exist in vivo. However, by administering the immunogen of this embodiment to the target animal, antibodies that specifically bind to the above peptide can be produced in the target animal's body with high efficiency.

[0045] Also, the mechanism of immunogenic induction and removal of senescent cells in the body of the administration target by the immunogen of this embodiment is presumed to be as follows. By administering the immunogen of this embodiment, the production of antibodies that specifically bind to GPNMB is promoted, and the produced anti-GPNMB antibodies bind to cells expressing GPNMB and act as neutralizing antibodies to suppress the proliferation of cells expressing GPNMB (neutralizing effect). Alternatively, as shown in the examples described below, the produced anti-GPNMB antibodies bind to cells expressing GPNMB, inducing macrophages and natural killer cells (NK cells) to kill the cells to which the antibodies are bound (antibody-dependent cellular cytotoxicity: ADCC activity). Alternatively, the produced anti-GPNMB antibodies bind to cells expressing GPNMB, activating the complement system, causing a series of reactions on the cell surface, and destroying the cells (complement-dependent cytotoxicity: CDC activity). It should be noted that the above mechanism is only a speculation, and even if the desired effect is obtained by a mechanism different from the above mechanism, it is included in the technical scope.

[0046] The peptide of (a) or (b) above may consist of L - amino acids, D - amino acids, or a combination thereof. L - amino acids are naturally occurring amino acids, and D - amino acids are those in which the chirality of the L - amino acid residue is inverted. Further, it may be chemically modified to amplify the immune induction activity or optimize other physical properties.

[0047] In addition, the immune inducer of the present embodiment may contain, as an active ingredient, an expression vector having a structural gene containing a region encoding the above peptide. Such an expression vector containing a structural gene encoding the above peptide in vivo is also called a "gene vaccine". The immune inducer of the present embodiment may be a peptide vaccine or a gene vaccine, but is preferably a peptide vaccine.

[0048] The Genbank accession number of the human Gpnmb gene is NM_001005340. The Genbank accession number of the mouse Gpnmb gene is NM_053110. The nucleotide sequence of human Gpnmb cDNA is shown in SEQ ID NO: 9. The nucleotide sequence of mouse Gpnmb cDNA is shown in SEQ ID NO: 10. By using a vector containing the nucleotide sequence of the region encoding the amino acid residues at positions 63 - 71 or 150 - 159 in the amino acid sequence of the human GPNMB protein among the nucleotide sequences shown in SEQ ID NO: 9, or the nucleotide sequence of the region encoding the amino acid residues at positions 63 - 71 or 150 - 159 in the amino acid sequence of the mouse GPNMB protein among the nucleotide sequences shown in SEQ ID NO: 10, the above peptide can be expressed in vivo.

[0049] Specific examples of the structural gene containing the region encoding the above peptide include, for example, a structural gene containing a region consisting of the nucleotide sequence shown in any of SEQ ID NOs: 11 to 14, or a region having 80% or more, for example 85% or more, for example 90% or more, for example 95% or more identity with the nucleotide sequence shown in any of SEQ ID NOs: 11 to 14 and encoding a peptide having immune-inducing activity, and the like. The nucleotide sequence shown in SEQ ID NO: 11 is the nucleotide sequence of the region encoding the peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 above. The nucleotide sequence shown in SEQ ID NO: 12 is the nucleotide sequence of the region encoding the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 above. The nucleotide sequence shown in SEQ ID NO: 13 is the nucleotide sequence of the region encoding the peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 above. The nucleotide sequence shown in SEQ ID NO: 14 is the nucleotide sequence of the region encoding the peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 above.

[0050] Here, the sequence identity of the control nucleotide sequence with respect to the reference nucleotide sequence can be determined, for example, as follows. First, the reference nucleotide sequence and the target nucleotide sequence are aligned. Here, each nucleotide sequence may include gaps so that the sequence identity is maximized. Subsequently, in the reference nucleotide sequence and the target nucleotide sequence, the number of nucleotides that match is calculated, and the sequence identity can be determined according to the following formula.

[0051] "Sequence identity (%)" = [Number of matching nucleotides] / [Total number of nucleotides in the target nucleotide sequence] × 100

[0052] The structural gene containing the region encoding the above peptide may have other components added upstream or downstream of the region encoding the above peptide, as long as the expression and immune-inducing activity of the above peptide are not inhibited.

[0053] The vector used in the immunopotentiator of the present embodiment is not particularly limited as long as it can be expressed in animal cells (preferably mammalian cells) to be administered, and may be a plasmid vector or a viral vector, and vectors known in the field of gene vaccines can be used. As a method using a viral vector, for example, there is a method of incorporating and introducing a structural gene containing a region encoding the above peptide into an RNA virus or DNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, etc. Among them, methods using retrovirus, adenovirus, adeno-associated virus or vaccinia virus are particularly preferred. As these vectors, commercially available products can preferably be used. For example, a gene vaccine can be obtained by inserting a structural gene containing a region encoding the above peptide into the multiple cloning site of a commercially available vector. The structural gene containing the region encoding the above peptide can be easily prepared using known genetic engineering techniques. In addition, the incorporation of the structural gene into the vector can be carried out using methods well known to those skilled in the art.

[0054] In addition, the vector used in the immunopotentiator of the present embodiment may have, for example, a promoter, a multiple cloning site, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an origin of replication, etc., as long as it does not inhibit the expression and immunopotentiating activity of the above peptide.

[0055] <Other components> The peptide contained in the immunization inducer of the present embodiment is preferably used in the form of a peptide-carrier protein complex in which a carrier protein is physically or chemically bound directly or via a linker or the like in order to enhance immunogenicity. Specific binding methods include, for example, coordination bonds, covalent bonds, hydrogen bonds, hydrophobic interactions, physical adsorption, etc., and any known binding, linker, and binding method can be adopted. Also, the binding position between the peptide and the carrier protein can be appropriately selected as needed. Furthermore, even in the case where there is no physical or chemical bond, those in which the movement of one is restricted by the three-dimensional structure and they can move together are also included in the bound state in the present embodiment.

[0056] A carrier protein is generally a substance that binds to a molecule (hapten) that has no immunogenicity due to its small molecular weight and imparts immunogenicity. Examples of carrier proteins include, but are not limited to, bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), keyhole limpet hemocyanin (KLH), thyroglobulin (TG), immunoglobulins, etc. Among them, keyhole limpet hemocyanin (KLH) is preferred.

[0057] Also, the immunization inducer of the present embodiment can be used as a pharmaceutical composition in combination with an adjuvant. That is, in one embodiment, the present invention provides a pharmaceutical composition for preventing or treating an aging-related disease, containing the above immunization inducer and an adjuvant. Thereby, the immunization-inducing activity of the immunization inducer can be enhanced, and the removal of aging cells can be promoted. Therefore, for example, when used as a pharmaceutical composition for preventing or treating an aging-related disease, the preventive or therapeutic effect of the aging-related disease can be further enhanced.

[0058] As the adjuvant, those used in ordinary vaccine preparations can be used. Specifically, as the adjuvant, for example, alum, CpG oligodeoxynucleotide, dsRNA, Montanide, Cervarix, incomplete Freund's adjuvant (Sigma-Aldrich, F5506), complete Freund's adjuvant (Sigma-Aldrich, F5881), TiterMax Gold (TiterMax) etc. can be mentioned. Incidentally, the incomplete Freund's adjuvant (IFA) contains paraffin oil and a surfactant, and the complete Freund's adjuvant (CFA) is obtained by adding heat-killed Mycobacterium butyricum to IFA. Also, TiterMax Gold is an adjuvant composed of a water-in-oil emulsion of a nonionic copolymer having a hydrophilic region made of polyoxyethylene and a hydrophobic region made of polyoxypropylene. Among them, CpG oligodeoxynucleotide is preferable because it can induce Th1-type helper T cells.

[0059] In addition, the immunopotentiator of the present embodiment can be used as a pharmaceutical composition in combination with a pharmaceutically acceptable carrier, and it is preferable to use it as a pharmaceutical composition in combination with the above adjuvant and a pharmaceutically acceptable carrier. That is, in one embodiment, the present invention provides a pharmaceutical composition for preventing or treating an aging-related disease, containing the above immunopotentiator, adjuvant and pharmaceutically acceptable carrier.

[0060] The pharmaceutical composition is preferably in a dosage form for parenteral use such as an injection or a topical skin preparation. More specifically, as the topical skin preparation, dosage forms such as an ointment and a patch can be mentioned.

[0061] As a pharmaceutically acceptable carrier, those usually used in the formulation of pharmaceutical compositions can be used without particular limitation. Specific examples include, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic; excipients such as starch, crystalline cellulose; swelling agents such as alginic acid; solvents for injections such as water, ethanol, glycerin; adhesives such as rubber-based adhesives, silicone-based adhesives, and the like.

[0062] The pharmaceutical composition may contain additives. Examples of additives include lubricants such as calcium stearate, magnesium stearate; sweeteners such as sucrose, lactose, saccharin, maltitol; flavoring agents such as peppermint, perilla oil; stabilizers such as benzyl alcohol, phenol; buffers such as phosphates, sodium acetate; solubilizing agents such as benzyl benzoate, benzyl alcohol; antioxidants; preservatives, and the like.

[0063] The pharmaceutical composition may be used in combination with at least one selected from the group consisting of a therapeutic agent having an effect of removing senescent cells other than the above immunomodulator (hereinafter sometimes abbreviated as "senescent cell-removing agent") and therapeutic agents for other diseases. The above immunomodulator and other drugs may be in the same formulation or in separate formulations. Also, each formulation may be administered by the same administration route or by separate administration routes. Furthermore, each formulation may be administered simultaneously, sequentially, or separately with a certain time or period in between. In one embodiment, the immunomodulator and other drugs may be in a kit containing them.

[0064] <Administration method> The administration targets include, but are not limited to, for example, humans, monkeys, dogs, cows, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, hamsters, and their cells. Among them, mammals or mammalian cells are preferred, and humans or human cells are particularly preferred.

[0065] The administration route is preferably a parenteral administration route such as subcutaneous administration, transdermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, intraarterial administration.

[0066] The dosage of the immune inducer varies depending on the symptoms, age, gender, etc. of the subject to be administered, and cannot be determined unconditionally. However, it is not particularly limited as long as it can induce an immune response in the living body of the subject to be administered. For example, it is about 0.1 μg or more and 100 mg or less, preferably about 1 μg or more and 10 mg or less, in terms of the mass of the peptide (preferably a peptide-carrier protein complex) per administration. The administration of the immune inducer may be a single administration or multiple administrations. In the case of multiple administrations, for example, it can be administered 2 or 3 times at intervals of 2 weeks or 1 month, and then at intervals of half a year or 1 year, once every time.

[0067] ≪Other Embodiments≫ In one embodiment, the present invention provides a method for preventing or treating an aging-related disease, which includes administering an effective amount of an expression vector containing any one of the peptides of (a) or (b) above, or a structural gene containing a region encoding the peptide, to a patient in need of treatment. The same aging-related diseases as those described above can be used.

[0068] In one embodiment, the present invention provides a method for preventing or treating an aging-related disease, which includes administering an effective amount of a peptide-carrier protein complex composed of any one of the peptides of (a) or (b) above and a carrier protein bound to the peptide, to a patient in need of treatment. The same carrier proteins as those described above can be used.

[0069] In one embodiment, the present invention provides a method for preventing or treating an aging-related disease, which includes administering an effective amount of a pharmaceutical composition containing any one of the peptides of (a) or (b) above, or an expression vector containing a structural gene containing a region encoding the peptide, and an adjuvant, to a patient in need of treatment. The same adjuvants as those described above can be used.

[0070] In one embodiment, the present invention provides a method for preventing or treating an aging-related disease, which comprises administering an effective amount of a pharmaceutical composition containing a peptide-carrier protein complex composed of any one of the peptides of (a) or (b) above and a carrier protein bound to the peptide, and an adjuvant, to a patient in need of treatment.

[0071] In one embodiment, the present invention provides an expression vector containing any one of the peptides of (a) or (b) above for preventing or treating an aging-related disease, or a structural gene containing a region encoding the peptide.

[0072] In one embodiment, the present invention provides a peptide-carrier protein complex composed of any one of the peptides of (a) or (b) above for preventing or treating an aging-related disease and a carrier protein bound to the peptide.

[0073] In one embodiment, the present invention provides a pharmaceutical composition for preventing or treating an aging-related disease, which contains an expression vector containing any one of the peptides of (a) or (b) above, or a structural gene containing a region encoding the peptide, and an adjuvant.

[0074] In one embodiment, the present invention provides a pharmaceutical composition for preventing or treating an aging-related disease, which contains a peptide-carrier protein complex composed of any one of the peptides of (a) or (b) above and a carrier protein bound to the peptide, and an adjuvant.

[0075] In one embodiment, the present invention provides the use of an expression vector containing any one of the peptides of (a) or (b) above, or a structural gene containing a region encoding the peptide, for manufacturing a medicament for preventing or treating an aging-related disease.

[0076] In one embodiment, the present invention provides the use of a peptide-carrier protein complex comprising any one of the peptides of (a) or (b) above for producing a prophylactic or therapeutic agent for an aging-related disease and a carrier protein bound to the peptide.

[0077] In one embodiment, the present invention provides the use of any one of the peptides of (a) or (b) above, or an expression vector containing a structural gene containing a region encoding the peptide, and an adjuvant for producing a prophylactic or therapeutic agent for an aging-related disease.

Example

[0078] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[0079] (Animal model) All animal experiments were reviewed by the Animal Health Research Institute Review Committee of Niigata University and approved by the President of Niigata University. C57BL / 6 mice were purchased from SLC Japan (Shizuoka, Japan). In addition, the mice used in the following tests were maintained on a high-fat diet (HFD, CLEA Japan) or a normal diet (NC) until 4 to 12 weeks of age, and maintained until 4 to 16 weeks of age for peptide inoculation, unless otherwise specified.

[0080] In addition, apolipoprotein E-deficient mice (ApoE KO mice) (ApoE- / -; C57BL / 6 background) used in the following tests were obtained from the Jackson Laboratory. Note that ApoE KO mice exhibit spontaneous atherosclerotic lesions even under normal diet breeding.

[0081] ApoE / Gpnmb-DTR mice (C57BL / 6 background) were generated by mating ApoE KO mice with Gpnmb-DTR mice described below. ApoE / Gpnmb-DTR mice were fed an HFD from 4 weeks of age, subjected to DT treatment at 12 and 14 weeks of age, and analyzed at 16 weeks of age.

[0082] In addition, the DTR (human HB-EGF I117V / L148V) and luciferase used in the following tests were expressed at the p19 Arf locus in a genetically modified mouse model (hereinafter abbreviated as "p19 Arf -DTR-Luc mouse") (C57BL / 6 background) was prepared using the method described in Reference 1 (Hashimoto M et al., “Elimination of p19 ARF -expressing cells enhances pulmonary function in mice.”, JCI Insight, Vol. 1, Issu 12: e87732, 2016.).

[0083] In addition, Zmpste24-deficient (KO) mice (a model of Hutchinson-Gilford progeria syndrome, MGI: 2158363) were used as an early aging mouse model. The generation and genotyping of Zmpste24 KO mice were performed according to a previous report (http: / / www.informatics.jax.org / allele / MGI:2158363).

[0084] (Statistical analysis) In addition, the statistical analysis of the test data shown below was performed using SPSS software (version 24 or 25). All data were obtained from different biological replicates and are shown as box-and-whisker plots indicating the range of the data (whiskers), the 25th and 75th percentiles (box), and the median (solid line) or mean ± standard error. Outliers and abnormal values were excluded by box plot analysis. Differences between groups were tested using two-sided Student's t-test or two-way analysis of variance (two-way ANOVA), followed by Tukey's multiple comparison test for comparisons between two or more groups, or repeated measures analysis using Tukey's multiple comparison test for comparisons over time. Survival curves were drawn by the Kaplan–Meier method and compared by the log-rank test. In all analyses, P < 0.05 was considered statistically significant.

[0085] [Reference Example 1] 1. Establishment of a genetically modified mouse capable of removing GPNMB protein-positive cells The inventors considered that the GPNMB protein could be a target molecule for senolytic therapy, and established a genetically modified mouse model (hereinafter abbreviated as "Gpnmb-DTR mouse" or "Gpnmb-DTR-Luc mice") in which DTR (human HB-EGF I117V / L148V) and luciferase were expressed at the Gpnmb locus.

[0086] First, a BAC clone (RP23-284O3, ~200 kb) containing the complete 21.7 kb mouse Gpnmb genomic sequence with an additional 97 kb of 5'-flanking region and 80 kb of 3'-flanking region was purchased from Advanced Geno Techs. A chimeric Gpnmb-DTR-2A-luciferase BAC transgenic construct (hereinafter sometimes abbreviated as "BAC transgenic construct") containing the configuration shown below was recombinantly prepared (see Figure 1A). Composition of the construct: tandem cassette - 2A peptide gene - luciferase reporter gene - Gpnmb gene of human heparin - binding epidermal growth factor - like growth factor (human HB - EGF) with DTR - generating I117V / L148V mutation

[0087] Specifically, the DTR - 2A - luciferase gene was transferred into the Gpnmb BAC clone using the Red / ET counter - selection BAC modification kit (Gene Bridges) to generate it. The obtained BAC transgenic construct was used to generate Gpnmb - DTR mice by pronuclear injection into C57BL / 6 mouse embryos. That the BAC transgenic construct had genetically modified was evaluated by Southern blotting of tail DNA digested with Hinc I and 32 probing with a P - labeled luciferase gene fragment. Twenty lines of Gpnmb - DTR mice were obtained, and for analysis, two lines with high copy numbers (~10 copies, lines 2 - 2 and 5 - 4) were selected. Two independent mouse lines were confirmed to exhibit similar phenotypes. To create a diet - induced obesity model mouse, unless otherwise specified, Gpnmb - DTR mice were maintained on an HFD from 4 to 16 weeks of age.

[0088] 2. Analysis of Gpnmb - dependent luciferase activity Gpnmb-DTR mice that were bred by being fed a normal diet or a HFD for 4 weeks, and Gpnmb-DTR mice (16 weeks old) that were bred by being fed a HFD for 4 weeks and then, while continuing to be fed a HFD, were intraperitoneally injected with DT (50 μg / kg body weight) twice at 2-week intervals (12 weeks old and 14 weeks old) were subjected to in vivo luciferase imaging analysis using an IVIS imaging system (Perkin Elmer). The mice were shaved on the ventral side, anesthetized with isoflurane (Wako Pure Chemicals Industries), and intraperitoneally injected with luciferin (150 mg / kg body weight) according to the manufacturer's instructions (Vivo-Glo; Promega). Luciferase activity was monitored 5 minutes after luciferin administration. To quantify the luciferase activity, the signals were analyzed using Living Image software (Perkin Elmer). The results are shown in Fig. 1B. In Fig. 1B, "Before" refers to Gpnmb-DTR mice (6 weeks old) before being fed a HFD, "HFD" refers to Gpnmb-DTR mice (12 weeks old) that were fed a HFD from 6 weeks old and bred for 6 weeks, and "After DT" refers to Gpnmb-DTR mice (16 weeks old) that were bred by being fed a HFD for 4 weeks and then, while continuing to be fed a HFD, were intraperitoneally injected with DT (50 μg / kg body weight) twice at 2-week intervals (12 weeks old and 14 weeks old).

[0089] From Fig. 1B, when a HFD was administered to Gpnmb-DTR mice, a strong luciferase signal was detected in the abdomen, and the abdominal luciferase activity was attenuated by the administration of DT. That is, in the obtained Gpnmb-DTR mice, cells expressing the GPNMB protein (hereinafter sometimes referred to as "GPNMB-positive cells") could be detected by monitoring the luciferase activity, and the GPNMB-positive cells could be removed by the administration of diphtheria toxin (DT).

[0090] 3. SA-β-gal staining The aging-related acidic β-galactosidase (SA-β-gal) activity in adipose tissue was tested. Briefly, adipose tissue newly isolated from each Gpnmb-DTR mouse was incubated in a β-gal staining solution (containing 1 mg / ml of 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal), 5 mmol / L of potassium ferrocyanide, 150 mmol / L of sodium chloride, 2 mmol / L of magnesium chloride, 0.01% of sodium deoxycholate, and 0.02% of Nonidet-40) at 37 °C for 2 hours. Thereafter, the stained adipose tissue was photographed (see Figure 1C). The SA-β-gal stainability was quantified using Image-J software (see Figure 1D). In Figures 1C and 1D, "DTR+" refers to Gpnmb-DTR mice (16 weeks old) that were fed a HFD from 4 weeks of age and then, while continuing to be fed a HFD, were intraperitoneally injected with DT (50 μg / kg body weight) twice at 2-week intervals (12 weeks and 14 weeks of age). "DTR-" refers to the littermate mice (16 weeks old, control wild type) of Gpnmb-DTR mice that were fed a HFD from 4 weeks of age and then, while continuing to be fed a HFD, were intraperitoneally injected with DT (50 μg / kg body weight) twice at 2-week intervals (12 weeks and 14 weeks of age). For "DTR+" and "DTR-", unless otherwise specified hereinafter, the same mouse groups are shown. In Figure 1C, the scale bar is 5 mm.

[0091] 4. RNA analysis Total RNA (1 μg) was isolated from adipose tissue samples using RNA-Bee (TEL-TEST Inc.). Real-time PCR (qPCR) was performed using a Light Cycler 480 (Roche) equipped with a Universal Probe Library and LightCycler 480 Probe Master (Roche) according to the manufacturer's instructions. The primers and their sequences are shown in Table 1 below. Rplp0 was used as an internal control. The results are shown in Figure 1E.

[0092]

Table 1

[0093] 5. Metabolic test Mice were individually housed starting one week before the test. On the day of the glucose tolerance test (GTT), the mice were fasted for 6 hours and then intraperitoneally injected with glucose at a dose of 1 g / kg (body weight) in the early afternoon. For the insulin tolerance test (ITT), human insulin was intraperitoneally administered to the mice (1 U / kg (body weight)). After administration of glucose or insulin, tail vein blood was collected at 0, 15, 30, 60, and 120 minutes, and blood glucose levels were measured using a glucose analyzer (Sanwa Chemical Research Institute). The results are shown in Figure 1F.

[0094] From Figures 1C to 1E, in DTR+ Gpnmb-DTR mice (16 weeks old), a decrease in SA-β-gal activity and downregulation of the expression of Gpnmb, Cdkn1a, and Cdkn2a were shown, and the aging-like phenotype of visceral adipose tissue was improved. Also, from Figure 1F, it was shown that the removal of GPNMB-positive cells resulted in a significant improvement in metabolic abnormalities induced by HFD intake.

[0095] 6. Detection of atherosclerotic plaques Atherosclerotic plaques were detected by Oil Red O staining. Briefly, adventitial fat was removed by dissection to open the entire aorta, and the aorta was flat-fixed at room temperature in 4 v / v% paraformaldehyde for 12 hours. Subsequently, the fixed aorta was immersed in 60 v / v% isopropyl alcohol for 1 minute for washing. Then, the aorta was incubated in a 0.5% Oil Red O (Sigma-Aldrich) solution (diluted with 60 v / v% isopropyl alcohol) at 37 °C for 15 minutes. After staining, the samples were immersed in a 60 v / v% isopropyl alcohol solution for a short time and then washed with double-distilled water. Atheromatous atherosclerotic lesions were stained red with Oil Red O (see Figure 1G). The Oil Red O staining area was quantified for the plaque area using Image-J software (see Figure 1H). In Figures 1G and 1H, "DTR+" refers to ApoE / Gpnmb-DTR mice (16 weeks old) that were fed a HFD from 4 weeks of age and then, while continuing to be fed a HFD, were intraperitoneally injected with DT (50 μg / kg body weight) twice at 2-week intervals (12 weeks and 14 weeks of age), and "DTR-" refers to littermate mice (16 weeks old, control wild type) of ApoE / Gpnmb-DTR mice that were fed a HFD from 4 weeks of age and then, while continuing to be fed a HFD, were intraperitoneally injected with DT (50 μg / kg body weight) twice at 2-week intervals (12 weeks and 14 weeks of age).

[0096] From Figures 1G and 1H, in DTR+ ApoE / Gpnmb-DTR mice, the removal of GPNMB-positive cells significantly reduced atherosclerotic plaques. This suggests that the GPNMB protein may be a useful target for senescent cell death induction therapy.

[0097] [Example 1] 1. Construction of the peptide vaccine Next, a peptide vaccine targeting the GPNMB protein was developed. Specifically, for the extracellular domain (positions 1 to 487 from the N-terminus) of the human GPNMB protein, algorithm analysis was performed using information on MHC presentation sequences (T cell epitopes) and B cell epitopes obtained from IEDB (http: / / www.iedb.org / home_v3.php, search date: May 15, 2017). As the top two peptides, a peptide consisting of the region at positions 63 to 71 (SEQ ID NO: 3: KRGDMRWKN) or the amino acid sequence at positions 150 to 159 (SEQ ID NO: 5: QSHHNVFPDG) in the amino acid sequence of the human GPNMB protein was selected. Also, through homology analysis of the human GPNMB protein and the mouse GPNMB protein, peptides consisting of the amino acid sequences shown in SEQ ID NO: 4 (RRGDGRWKD) or SEQ ID NO: 6 (RSQHLRFPDR), which correspond to the above two regions in the mouse GPNMB protein, were used in the following tests. In the following tests, the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 (RRGDGRWKD) is referred to as peptide vaccine 1, and the peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 (RSQHLRFPDR) is referred to as peptide vaccine 2.

[0098] First, the antibody titers of the above two types of peptide vaccines obtained were examined. For the induction of cytotoxic antibodies, peptide vaccine 1 or peptide vaccine 2 (20 μg) conjugated with KLH as a carrier protein and Titer Max Gold adjuvant (Titer Max USA) were emulsified by mixing in two syringes according to the manufacturer's protocol. Then, 8-week-old C57BL / 6 mice fed with HFD from 4 weeks of age were subcutaneously injected with KLH-conjugated antigen peptide (20 μg) together with Titer Max Gold adjuvant for the induction of cytotoxic antibodies. C57BL / 6 mice in the control group were subcutaneously injected with a KLH solution mixed in an equal amount with Titer Max Gold (hereinafter referred to as "control vaccine"). For the analysis of antibody titers, plasma was collected from the tail vein of each mouse immediately after administration of the peptide vaccine and at 2, 4, and 6 weeks later. The antibody titers generated by the injection of each peptide were measured by ELISA. Specifically, first, a 96-well microtiter plate (Nunc-Immuno MicroWell 96-well solid plate, Thermo Scientific) was coated with a carbonate buffer containing peptide vaccine 1 or peptide vaccine 2 conjugated to bovine serum albumin (BSA). After coating, plasma collected from the immunized mice was added to the wells and incubated overnight. Then, the plate was incubated with a secondary antibody (ECL anti-mouse IgG, horseradish peroxidase-conjugated whole sheep antibody, GE Healthcare). After reacting with the TMB substrate (Sigma-Aldrich, T0440), the optical density was measured at 450 nm (OD450) using a microtiter plate reader (iMark microplate reader, Bio-Rad). The results are shown in Figure 2A.

[0099] From Figure 2A, the antibody titer against the GPNMB protein by peptide vaccine 1 was significantly higher and that by peptide vaccine 2 was slightly higher compared to the control group administered with the control vaccine.

[0100] 2. Analysis of Gpnmb-dependent luciferase activity Luciferase activity was observed using the same method as in "2." of Reference Example 1, except that Gpnmb-DTR mice (16 weeks old) fed with HFD from 4 weeks old and administered with Peptide Vaccine 1 or Control Vaccine by subcutaneous injection at 8 weeks old were used. The results are shown in Figure 2B. In Figure 2B, "Cont vac" represents Gpnmb-DTR mice administered with the control vaccine, and "Gpnmb vac" represents Gpnmb-DTR mice administered with Peptide Vaccine 1.

[0101] 3. RNA analysis RNA analysis of Gpnmb, Cdkn1a, and Cdkn2a was performed using the same method as in "4." of Reference Example 1, except that adipose tissue collected from C57BL / 6 mice (16 weeks old) fed with HFD from 4 weeks old and administered with Peptide Vaccine 1 or Control Vaccine by subcutaneous injection at 8 weeks old was used. The results are shown in Figure 2C. In Figure 2C, "Cont vac" represents C57BL / 6 mice administered with the control vaccine, and "Gpnmb vac" represents C57BL / 6 mice administered with Peptide Vaccine 1.

[0102] 4. Western blotting analysis Adipose tissue was collected from C57BL / 6 mice (16 weeks old) that were fed a high-fat diet (HFD) starting at 4 weeks of age and received subcutaneous injection of peptide vaccine 1 or control vaccine at 8 weeks of age. Whole cell lysates were prepared using lysis buffer (containing 10 mmol / L Tris-HCl (pH 8), 140 mmol / L sodium chloride, 5 mmol / L EDTA, 0.025% NaN3, 1 w / v% Triton X-100, 1 w / v% deoxycholic acid, 0.1 w / v% SDS, 1 mmol / L phenylmethylsulfonyl fluoride (PMSF), 5 μg / mL leupeptin, 2 μg / mL aprotinin, 50 mmol / L NaF, and 1 mmol / L Na2VO3). Subsequently, whole cell lysates (40 - 50 μg) were separated by SDS-PAGE, and the proteins were transferred to a PVDF membrane (Millipore), which was incubated with a primary antibody followed by horseradish peroxidase (Jackson)-conjugated anti-rabbit immunoglobulin G (Jackson ImmunoResearch). GPNMB and β-actin were detected by enhanced chemiluminescence (Amersham). As the primary antibody, anti-GPNMB antibody (AF2330, R&D Systems) (diluted 1 / 1000) and as a control, anti-β-actin antibody (#4967, Cell Signaling) (diluted 1 / 5000) were used. As the secondary antibody, peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) (111-035-003, Jackson ImmunoResearch) (diluted 1 / 10000) was used. The results are shown in Fig. 2D. In Fig. 2D, "Cont vac" represents C57BL / 6 mice administered with the control vaccine, and "Gpnmb vac" represents C57BL / 6 mice administered with peptide vaccine 1.

[0103] From Figs. 2B to 2D, it was suggested that in Gpnmb-DTR mice and C57BL / 6 mice fed a HFD and administered with peptide vaccine 1, both the expression level of GPNMB protein and the luciferase activity related to the expression level were decreased, and the administration of peptide vaccine 1 removed GPNMB-positive cells.

[0104] 5. ADCC (Antibody-Dependent-Cellular-Cytotoxicity) Activity Analysis ADCC (Antibody-Dependent-Cellular-Cytotoxicity) was evaluated using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega). Specifically, natural killer (NK) cells were isolated from C57BL / 6 mice as effector cells using Ficoll-Paque Plus (GE Healthcare) and the EasySep Mouse NK Cell Isolation Kit (STEMCELL). Next, replicative senescent preadipocytes derived from C57BL / 6 mice were seeded into 96-well plates at a density of 10 4 cells per well. Next, purified IgG antibodies obtained from C57BL / 6 mice that had been fed a HFD from 4 weeks of age and received a subcutaneous injection of peptide vaccine 1 or control vaccine at 8 weeks of age were added to each well and incubated at 37°C for 30 minutes. After incubation, NK cells were seeded into each well such that the ratio of the number of effector cells to target cells (effector cells / target cells) was 1 / 1. The plates were then incubated at 37°C for an additional 4 hours. Next, lactate dehydrogenase released into the supernatant was detected using a microtiter plate reader (iMark Microplate Reader, Bio-Rad). To evaluate cell death, the percentage of lysed cells relative to the total number of cultured cells was calculated. The results are shown in Figure 2E. In Figure 2E, "Cont vac" represents C57BL / 6 mice administered the control vaccine, and "Gpnmb vac" represents C57BL / 6 mice administered peptide vaccine 1.

[0105] From Figure 2E, it was confirmed that the antibody derived from mice administered peptide vaccine 1 exhibits antibody-dependent cell cytotoxicity (ADCC activity) in which the anti-GPNMB antibody contained in the antibody specifically binds to senescent preadipocytes expressing GPNMB, inducing NK cells to kill the cells to which the antibody is bound.

[0106] 6.p19 Arf Analysis of dependent luciferase activity p19 that was fed an HFD for 8 weeks and administered peptide vaccine 1 or control vaccine by subcutaneous injection at 13 weeks of age Arf Luciferase activity was observed using the same method as in "2." of Reference Example 1, except that -DTR-Luc mice (21 weeks old) were used. The results are shown in Fig. 2F. In Fig. 2F, "Cont vac" is p19 administered with the control vaccine Arf -DTR-Luc mice, and "Gpnmb vac" is p19 administered with peptide vaccine 1 Arf -DTR-Luc mice.

[0107] From Fig. 2F, it was confirmed that the administration of peptide vaccine 1 decreased the p19 Arf dependent luciferase activity in visceral adipose tissue. In mammals, it is known that the induction of the expression of the p19 Arf gene (p14 gene in humans Arf gene) inhibits the activity of the ubiquitin ligase Mdm2, suppresses the degradation of the tumor suppressor protein p53, and causes cellular senescence. Therefore, it was confirmed that the administration of peptide vaccine 1 removed senescent cells.

[0108] 7. SA-β-gal staining SA-β-gal staining was performed using the same method as in "3." of Reference Example 1, except that adipose tissue collected from C57BL / 6 mice (16 weeks old) that were fed an HFD from 4 weeks of age and administered peptide vaccine 1 or control vaccine by subcutaneous injection at 8 weeks of age was used. The results are shown in Fig. 2G. SA-β-gal staining was quantified using Image-J software (see Fig. 2H). In Fig. 2G and Fig. 2H, "Cont vac" is C57BL / 6 mice administered with the control vaccine, and "Gpnmb vac" is C57BL / 6 mice administered with peptide vaccine 1. Also, in Fig. 2G, the scale bar indicates 5 mm.

[0109] From FIGS. 2G and 2H, a decrease in SA-β-gal activity was shown by administration of peptide vaccine 1. Also, downregulation of the expression of Cdkn1a and Cdkn2a was shown in FIG. 2C above. From these results, it became clear that administration of peptide vaccine 1 improved the senescence-like phenotype of visceral adipose tissue.

[0110] 8. Metabolic test A metabolic test was conducted in the same manner as in "5." of Reference Example 1, except that C57BL / 6 mice (16 weeks old) fed with HFD from 4 weeks old and administered with peptide vaccine 1, peptide vaccine 2, or control vaccine by subcutaneous injection at 8 weeks old were used. The results are shown in FIG. 2I. In FIG. 2I, "Cont vaccine" are C57BL / 6 mice administered with the control vaccine, "Gpnmb vaccine 1" are C57BL / 6 mice administered with peptide vaccine 1, and "Gpnmb vaccine 2" are C57BL / 6 mice administered with peptide vaccine 2.

[0111] From FIG. 2I, it was shown that C57BL / 6 mice administered with peptide vaccine 1 or peptide vaccine 2 significantly improved the metabolic abnormalities induced by HFD intake as compared with C57BL / 6 mice administered with the control vaccine.

[0112] 9. Detection of atherosclerotic plaques and analysis of Gpnmb-dependent luciferase activity ApoE / Gpnmb-DTR mice (16 weeks old) were used, except that they were reared while being fed an HFD for 12 weeks from 4 weeks old to 16 weeks old, and at 8 weeks old, peptide vaccine 1 or control vaccine was administered by subcutaneous injection. Detection of atherosclerotic plaques and analysis of Gpnmb-dependent luciferase activity were performed in the same manner as in "6." and "2." of Reference Example 1 (see Figure 2J). The Oil Red O staining area was quantified using Image-J software (see Figure 2K). In Figure 2J and Figure 2K, "Cont vac" are ApoE / Gpnmb-DTR mice administered with the control vaccine, and "Gpnmb vac" are ApoE / Gpnmb-DTR mice administered with peptide vaccine 1.

[0113] From Figure 2J and Figure 2K, treatment of ApoE / Gpnmb-DTR mice with peptide vaccine 1 significantly reduced the occurrence of atheroma and decreased Gpnmb-dependent luciferase activity in the aorta.

[0114] 10. Physiological analysis C57BL / 6 mice that were reared on normal feed and at 50 weeks old, peptide vaccine 1 or control vaccine was administered by subcutaneous injection were used. The open field test was performed before and after vaccination (50 weeks old and 70 weeks old). The open field test is a test for measuring spontaneous activity in a novel environment. Each mouse was placed in the open field and the test was performed for 6 minutes. The total movement time (seconds) and average speed (cm / second) of each mouse during the test were measured. The difference in the measurement results at 70 weeks old compared to the measurement results at 50 weeks old is shown in Figure 2L. In Figure 2L, "Cont vac" are C57BL / 6 mice administered with the control vaccine, and "Gpnmb vac" are C57BL / 6 mice administered with peptide vaccine 1.

[0115] In Figure 2L, administration of peptide vaccine 1 significantly improved the performance in the open field test.

[0116] 11. Survival curve Zmpste24 KO mice were bred on a normal diet. For some of the Zmpste24 KO mice, at 10 weeks of age (about 50 weeks of age in C57BL / 6 mice), peptide vaccine 1 was administered by subcutaneous injection. Survival curves were calculated by the Kaplan-Meier method and compared by the log-rank test (see Figure 2M).

[0117] From Figure 2M, it was shown that in Zmpste24 KO mice, which are a mouse model of premature aging, even when peptide vaccine 1 was administered in middle age (about 50 weeks of age), the lifespan was extended regardless of gender.

[0118] From the above results, it was demonstrated that the elimination of GPNMB-positive cells can improve the development of atherosclerosis and metabolic dysfunction induced by HFD intake. Furthermore, the elimination of GPNMB-positive cells using peptide vaccines was able to attenuate pathological aging in old mice and extend the lifespan of premature aging mice.

[0119] Aging cells are known to become resistant to apoptosis by upregulating anti-apoptotic factors. Therefore, most conventional senolytic agents have been developed based on their ability to inhibit anti-apoptotic pathways. However, targeting the GPNMB protein could be a new strategy for senolytic therapy of age-related diseases.

[0120] It has been reported that the GPNMB protein can become a negative regulator of T lymphocyte activation by binding to syndecan 4 on T cells. Therefore, it is thought that an immunomodulator using a peptide vaccine can enhance the endogenous senolytic immune system, thereby promoting the elimination of aging cells and improving pathological aging.

Industrial Applicability

[0121] The immunomodulator of this embodiment is effective for preventing or treating aging-related diseases.

Claims

Claim 1 An immunomodulator comprising, as an active ingredient, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, or an expression vector comprising a structural gene encoding the peptide. Claim 2 The immunomodulator according to claim 1, wherein the peptide consists of the amino acid sequence shown in any of SEQ ID NOs: 3 to 6. Claim 3 The immunomodulator according to claim 1 or 2, comprising the peptide as an active ingredient. Claim 4 The immunomodulator according to any one of claims 1 to 3, wherein a carrier protein is further bound to the peptide. Claim 5 The immunomodulator according to claim 2, wherein the peptide consists of the amino acid sequence shown in SEQ ID NO: 3 or 4 and is used for removal of senescent cells. Claim 6 A pharmaceutical composition for preventing or treating aging-related diseases, comprising the immunomodulator according to any one of claims 1 to 5 and an adjuvant.

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