Prorenin receptor peptide, conjugate, and pharmaceutical composition

Abstract

Provided is a prorenin receptor peptide having enhanced ability to induce an antibody against a prorenin receptor.　The prorenin receptor peptide of the present disclosure includes the following polypeptide (P1), (P2) or (P3): (P1) a polypeptide comprising an amino acid sequence represented by any of SEQ ID NOS: 1-3; (P2) a polypeptide comprising an amino acid sequence obtained by deleting, substituting or adding 1-3 amino acids in an amino acid sequence represented by any of SEQ ID NOS: 1-3; and (P3) a polynucleotide comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence represented by any of SEQ ID NOS: 1-3.

Description

Prorenin receptor peptides, conjugates, and pharmaceutical compositions

[0001] The present invention relates to prorenin receptor peptides, conjugates, and pharmaceutical compositions.

[0002] Abnormalities in the Wnt / β-catenin pathway can cause various diseases. Abnormalities in the Wnt / β-catenin pathway can lead to, for example, tumors (benign tumors) such as familial adenomatous polyposis coli, osteoporosis, and the like (Non-Patent Documents 1 to 4). In recent years, attempts have been made to treat diseases caused by abnormalities in the Wnt / β-catenin pathway by blocking Wnt / β-catenin.

[0003] Caldwell, GM, et al. "Wnt signaling in adenomas of familial adenomatous polyposis patients." British journal of cancer 103.6 (2010): 910-917. Obrador‐Hevia, Antonia, et al. "Oncogenic KRAS is not necessary for Wnt signaling activation in APC‐associated FAP adenomas." The Journal of pathology 221.1 (2010): 57-67.Zhong, Zhendong A et al. “Regulation of Wnt receptor activity: Implications for therapeutic development in colon cancer.” The Journal of biological chemistry vol. 296 (2021): 100782. doi:10.1016 / j.jbc.2021.100782Phull, Manjinder Singh et al. “A perspective on medicinal chemistry approaches towards adenomatous polyposis coli and Wnt signal based colorectal cancer inhibitors.” European journal of medicinal chemistry vol. 212 (2021): 113149. doi:10.1016 / j.ejmech.2020.113149

[0004] However, the Wnt / β-catenin pathway has various physiological activities in the body, and therefore, blocking the Wnt / β-catenin pathway also inhibits these various physiological activities in the body, making it difficult to use it as a therapeutic target.

[0005] The present inventors have discovered that the prorenin receptor (PR) contributes to activation of the Wnt / β-catenin pathway and that the Wnt / β-catenin pathway can be inhibited by using antibodies against the PRR. The inventors then came up with the idea of ​​using PRR peptides to treat diseases caused by the Wnt / β-catenin pathway. Furthermore, they investigated whether an antitumor effect could be achieved in a cancer-bearing mouse model using the peptide (PRR0) spanning positions 200 to 213 of the PRR. However, no sufficient antitumor effect was achieved in mice administered with PRR0. This was presumed to be due to insufficient induction of antibodies against the PRR.

[0006] Therefore, an object of the present disclosure is to provide a PRR peptide with enhanced ability to induce antibodies against the prorenin receptor.

[0007] In order to achieve the above-mentioned object, the prorenin receptor peptides of the present disclosure include the following polypeptides (P1), (P2), or (P3): (P1) a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3; (P2) a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, in which 1 to 3 amino acids have been deleted, substituted, or added; (P3) a polynucleotide consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 3.

[0008] The conjugate of the present disclosure comprises an antigenic peptide and a carrier protein, wherein the antigenic peptide is bound to the carrier protein, and the antigenic peptide comprises a prorenin receptor peptide of the present disclosure.

[0009] The nucleic acids of the present disclosure encode the prorenin receptor peptides of the present disclosure.

[0010] An expression vector of the present disclosure comprises a nucleic acid of the present disclosure.

[0011] Pharmaceutical compositions of the present disclosure comprise a prorenin receptor peptide, conjugate, nucleic acid, and / or expression vector of the present disclosure and a pharmaceutically acceptable carrier.

[0012] The PRR peptides of the present disclosure have enhanced ability to induce antibodies against the prorenin receptor.

[0013] FIG. 1 is a graph showing the antibody titer of prorenin receptor vaccine PRR0 in Example 1. FIG. 2 is a photograph showing a mouse 10 weeks after the first vaccination with prorenin receptor vaccine PRR0 in Example 1. FIG. 3 is a graph showing the antibody titers of prorenin receptor vaccines PRR1 to 4 in Example 1. FIG. 4 is a graph showing the antitumor effect of prorenin receptor vaccines PRR1 to 4 in Example 1. FIG. 5 is a graph showing the antitumor effect of prorenin receptor vaccines PRR2 and PRR3 in Example 1. FIG. 6 is a photograph of β-catenin staining in colon tissue in Example 1. FIG. 7 is a graph showing the antibody titers of prorenin receptor vaccines PRR2 and PRR3 under short-term observation in Example 1. FIG. 8 is a graph showing the antibody titers of prorenin receptor vaccines PRR2 and PRR3 under long-term observation in Example 1. FIG. 9 is a graph showing the antibody titer of CRM197-PRR2 in mice in Example 2. FIG. 10 is a graph showing the antibody titer of CRM197-PRR2 in cynomolgus monkeys in Example 2. FIG. 11 is a graph showing β-catenin activity in HEK293 cells in Example 3. FIG. 12 is a graph showing the inhibitory effect on polyp formation in adenomatous polyposis model mice in Example 3. FIG. 13 is a graph showing the inhibitory effect on polyp formation in adenomatous polyposis model mice in Example 3. FIG. 14 is a photograph of staining of ATP6ap2 or active β-catenin in the intestine in Example 3. FIG. 15 is a graph showing the results of side effects in adenomatous polyposis model mice and normal mice in Example 3. FIG. 16 is a graph showing the survival rate of adenomatous polyposis model mice in Example 3. FIG. 17 is a graph showing the antibody titer of vaccine PRR2 in Example 4. FIG. 18 is a graph showing weight shift due to PRR2 in Example 4.

[0014] The present invention will be described below with reference to examples. In the following description, the descriptions of the inventions can be mutually incorporated unless otherwise specified.

[0015] <Definitions> As used herein, the terms "protein" and "polypeptide" refer to a peptide polymer composed of unmodified (naturally occurring), modified, and / or artificial amino acids. The polymer may be, for example, linear, branched, or cyclic.

[0016] As used herein, the term "prorenin receptor" refers to a single-pass transmembrane protein that has been identified as a component of the renin-angiotensin system and functions as a receptor for prorenin. The prorenin receptor is also called ATP6AP2. The prorenin receptor is presumed to indirectly enhance the activity of the Wnt / β-catenin system.

[0017] As used herein, "conjugate" refers to a compound in which two or more peptides with different amino acid sequences are covalently bonded. The peptides with different amino acid sequences are, for example, antigenic peptides, carrier peptides, or carrier proteins. The antigenic peptides are, for example, peptides that aim to induce antibodies by eliciting an immune response and inducing activation of lymphocytes, etc. The carrier peptides and carrier proteins are, for example, peptides or proteins that confer immunogenicity to the antigenic peptide, or peptides or proteins that enhance the immunogenicity of the antigenic peptide. The carrier peptides and carrier proteins can also be referred to as, for example, peptide carriers and protein carriers.

[0018] As used herein, the term "Wnt / β-catenin pathway" refers to a signal transduction pathway in which Wnt activates β-catenin via the Wnt receptor (Frizzled). The Wnt / β-catenin pathway contributes to the induction of cancers such as colon cancer through the abnormal accumulation of β-catenin and the inactivation of gene expression of Wnt inhibitory factors.

[0019] As used herein, "inhibition of activity" means that the activity of a target is changed to an inhibited state, or that the activity is in an inhibited state. When the "inhibition of activity" is used in combination with a specific protein or a signal pathway mediated by the specific protein, the "inhibition of activity" means that the function of the specific protein or the signal pathway mediated by the specific protein is changed to an inhibited state, or that the function is in an inhibited state.

[0020] As used herein, "sarcopenia or frailty" refers to a disease caused by impaired function of muscle tissue.

[0021] As used herein, "positive" means that a higher signal or the like is detected by an analytical method such as flow cytometry that utilizes an antigen-antibody reaction, compared to a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen. Also, as used herein, "negative" means that a signal or the like that is equal to or lower than a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen is detected.

[0022] As used herein, "treatment" means therapeutic treatment and / or prophylactic treatment. As used herein, "treatment" means treating, curing, preventing, suppressing, ameliorating, or improving a disease, pathology, or disorder, or halting, inhibiting, reducing, or delaying the progression of a disease, pathology, or disorder. As used herein, "prevention" means reducing the likelihood of developing a disease or pathology, or delaying the onset of a disease or pathology. The "treatment" may be, for example, treatment of a subject (patient) who develops the target disease, or treatment of an animal model of the target disease.

[0023] As used herein, the term "subject" refers to an animal or a cell, tissue, or organ derived from an animal, and is used to particularly include humans. The term "animal" refers to both humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions.

[0024] As used herein, "nucleic acid" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. When "nucleic acid" is used in combination with a specific protein, the "nucleic acid" refers to a polymer of nucleotides that encodes the amino acid sequence of the protein. Examples of the nucleic acid include genomic DNA, cDNA, and mRNA. The nucleic acid may be, for example, single-stranded or double-stranded. The nucleic acid can be interchangeably referred to as a "polynucleotide" or a "nucleic acid molecule."

[0025] As used herein, the term "host" refers to a cell and / or an individual into which exogenous nucleic acid is introduced. When the host is a cell, the host may also be referred to as a host cell.

[0026] As used herein, the terms "vector" and "expression vector" refer to a recombinant plasmid or virus containing a nucleic acid that is delivered to a host or host cell in vitro or in vivo. The term "vector" and "expression vector" includes viral vectors and non-viral vectors.

[0027] As used herein, the term "transformant" refers to a host into which exogenous nucleic acid has been introduced.

[0028] As used herein, "isolated" or "isolated" means identified and separated and / or recovered from components of its natural state. The "isolation" or "isolated" can be achieved, for example, by at least one purification step.

[0029] The present disclosure will be described below using examples, but the present disclosure is not limited to the following examples and can be implemented with any modifications. Furthermore, the descriptions in this disclosure can be mutually incorporated unless otherwise specified. In this specification, when the expression "~" is used, it is used to mean including the numerical or physical values ​​before and after it. In addition, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0030] <Prorenin Receptor Polypeptides> In one aspect, the present disclosure provides polypeptides with enhanced ability to induce antibodies against the prorenin receptor. The prorenin receptor peptides of the present disclosure include the following polypeptides (P1), (P2), or (P3): (P1) a polypeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 3; (P2) a polypeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 3, in which one or several amino acids have been deleted, substituted, or added; (P3) a polypeptide consisting of an amino acid sequence having 70% or more identity to the amino acid sequence of any of SEQ ID NOs: 1 to 3.

[0031] As a result of extensive research, the present inventors have discovered that antibodies against prorenin receptors (PRRs) exhibit anti-tumor effects and the like by regulating the activity of the Wnt / β-catenin pathway. Further research led the present inventors to discover that administering a peptide having a specific amino acid sequence in a PRR can induce antibodies against PRR in vivo, thereby achieving anti-tumor effects and muscle mass loss suppression effects similar to those of antibodies against the PRR, and thus completing the present disclosure. It is presumed that the PRR peptides of the present disclosure exhibit anti-tumor effects and muscle mass loss suppression effects by inducing the production of antibodies that indirectly regulate the activity of the Wnt / β-catenin pathway in a living body to which the PRR peptide is administered. However, this presumption does not limit the present disclosure in any way. Therefore, the PRR peptides of the present disclosure can be suitably used, for example, in the treatment of tumors (e.g., benign tumors such as familial adenomatous polyposis) and sarcopenia or frailty.

[0032] The origin of the prorenin receptor is not particularly limited and can be appropriately determined depending on, for example, the type of subject. Examples of the origin include humans and non-human animals other than humans. Examples of the non-human animals include mammals such as mice, rats, dogs, monkeys, rabbits, sheep, and horses. For prorenin receptors derived from various animals, reference can be made to information registered in existing databases. Specific examples of human prorenin receptors include, as cDNA, the region from bases 103 to 1155 (including a stop codon) in the following nucleotide sequence (SEQ ID NO: 4) registered under NCBI accession number NM_005765, and as a protein, the following amino acid sequence (SEQ ID NO: 5) registered under NCBI accession number NP_005756. The nucleotide sequence of SEQ ID NO: 4 encodes the amino acid sequence of SEQ ID NO: 5.

[0033]

[0034] Prorenin receptor (SEQ ID NO: 5) MAVFVVLLALVAGVLGNEFSILKSPGSVVFRNGNWPIPGERIPDVAALSMGFSVKEDLSWPGLAVGNLFHRPRATVMVMVKGVNKLALPPGSVISYPLENAVPFSLDSVANSIHSLFSEETPVVLQLAPSEERVYMVGKANSVFEDLSVTLRQLRKRLFQENSVLSSLPLNSHSRNNEVDLLFLSELQVLHDISSLLSRHKHLAKDHSPDLYSLELAGLDEIGKRYGEDSEQFRDASKILVDALQKFADDMYSLYGGNAVVELVTVKSFDTSLIRKTRTILEAKQAKNPASPYNLAYKYNFEYSVVFNMVLWIMIALALAVIITSYNIWNMDPGYDSIIYRMTNQKIRMD

[0035] In (P1), the polypeptides consisting of the amino acid sequences of SEQ ID NOs: 1 to 3 are polypeptides derived from the human prorenin receptor. The amino acid sequence of SEQ ID NO: 1 corresponds to the amino acid sequence of positions 198 to 211 in the amino acid sequence of the human prorenin receptor (SEQ ID NO: 5). The amino acid sequence of SEQ ID NO: 2 corresponds to the amino acid sequence of positions 223 to 236 in the amino acid sequence of the human prorenin receptor (SEQ ID NO: 5). The amino acid sequence of SEQ ID NO: 3 corresponds to the amino acid sequence of positions 221 to 230 in the amino acid sequence of the human prorenin receptor (SEQ ID NO: 5).

[0036] PRR1 peptide (SEQ ID NO: 1) SRHKHLAKDHSPDL PRR2 peptide (SEQ ID NO: 2) GKRYGEDSEQFRDA PRR3 peptide (SEQ ID NO: 3) EIGKRYGEDS

[0037] In the polypeptide (P2), "one or several" preferably refers to a range in which the polypeptide (P2) can induce the production of antibodies against PRR when conjugated with a carrier protein. "One or several" refers to, for example, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (P1). In the present disclosure, a numerical range of the number discloses, for example, all positive integers within that range (the same applies hereinafter).

[0038] In the polypeptide (P2), the substitution is preferably a conservative substitution. The conservative substitution refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Examples of the conservative substitution include substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine; substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; substitutions between amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitutions between amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitutions between amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and substitutions between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0039] In (P3), the "identity" is preferably within a range in which the polypeptide of (P3) can induce the production of antibodies against PRR when conjugated with, for example, a carrier protein. The "identity" is, for example, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the amino acid sequence of (P1). The "identity" can be calculated, for example, using default parameters in the homology algorithm BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ) of the National Center for Biotechnology Information (NCBI) (the same applies hereinafter).

[0040] The PRR peptide may include, for example, a linker amino acid or a linker peptide. The linker amino acid or linker peptide is an additional amino acid or peptide for linking to a carrier protein or a carrier peptide at the N-terminus and / or C-terminus. The PRR peptide may also be modified at the N-terminus, C-terminus, amino acid backbone, and / or amino acid side chain. The linker amino acid is, for example, cysteine.

[0041] The PRR peptides of the present disclosure can be used, for example, in combination with a carrier peptide or carrier protein to induce antibodies against the PRR.

[0042] In another aspect, the present disclosure provides a conjugate having an enhanced ability to induce antibodies against the prorenin receptor, the conjugate comprising an antigenic peptide and a carrier protein, wherein the antigenic peptide is bound to the carrier protein, and the antigenic peptide comprises the prorenin receptor peptide.

[0043] In the conjugate, the carrier protein may be any protein capable of imparting or enhancing the immunogenicity of the PRR peptide. Examples of the carrier protein include diphtheria toxin (DT) or a diphtheria toxin mutant, tetanus toxin (TT) or tetanus toxin fragment C, keyhole limpet hemocyanin (KLH), or functional equivalents thereof. The functional equivalent is a mutant in which a mutation has been introduced into the amino acid sequence to the extent that the functional equivalent retains the function of the carrier protein.

[0044] The diphtheria toxin mutant may be, for example, a detoxified diphtheria toxin CRM. １９７ , CRM １９７ Examples include the A chain of (CN103495161), CRM176, CRM228, CRM45 (Uchida et al. (1973) J. Biol. Chem. 218:3838-3844), CRM9, CRM102, CRM103, CRM107, and the like.

[0045] The carrier protein is preferably a CRM, since it can be suitably used as, for example, a vaccine composition. １９７The CRM １９７ Examples of the protein include a protein in which glycine at position 52 of the following amino acid sequence (SEQ ID NO: 6) registered under Uniprot accession number Q5PY51 has been substituted with glutamic acid, or a functional equivalent thereof.

[0046] CRM １９７ (SEQ ID NO: 6) MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVT GTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTV EDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0047] In the conjugate, the antigen peptide and the carrier protein are covalently bound to each other, and the binding position of the antigen peptide to the carrier protein is, for example, the N-terminus, C-terminus, and / or an amino acid side chain of the antigen peptide, preferably the N-terminus (amino group) and / or C-terminus (carboxyl group).

[0048] The binding site of the carrier protein to the antigen peptide is, for example, the N-terminus, C-terminus, and / or amino acid side chain of the carrier protein, preferably the amino acid side chain, such as the side chain of a cysteine ​​residue (thiol group), the side chain of a lysine residue, arginine residue, glutamine residue, or serine residue (amino group), or the side chain of glutamic acid or aspartic acid (carboxyl group).

[0049] In the conjugate, the antigenic peptide and the carrier protein are directly or indirectly bound. Direct binding means that the antigenic peptide is directly bound to an amino acid of the carrier protein. On the other hand, indirect binding means that the antigenic peptide is bound to the carrier protein via a linker peptide (peptide linker).

[0050] In the conjugate, the antigenic peptide is preferably conjugated to, for example, a cysteine ​​residue of the carrier protein. The bond between the antigenic peptide and the carrier protein can be set, for example, based on the reactive group of the antigenic peptide and the reactive group of the carrier protein used for the bond. The antigenic peptide and the carrier protein can be conjugated, for example, by directly reacting the reactive group of the antigenic peptide with the reactive group of the carrier protein, or by using a crosslinker to react the crosslinker with the antigenic peptide and the carrier protein. As a specific example, when the amino group of the antigenic peptide is conjugated to the side chain (thiol group) of the cysteine ​​residue of the carrier protein, the antigenic peptide and the carrier protein can be conjugated by crosslinking using a crosslinker having different reactive groups, more specifically, a crosslinker having a reactive group reactive with an amino group and a reactive group reactive with a thiol group. The crosslinking agent is, for example, N-ε-malemidocaproyl-oxysuccinimide ester (EMCS), N-ε-maleimidocaproyl-oxysulfosuccinimide ester (Sulfo-EMCS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), sulfosuccinimidyl Examples include 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC), N-α-maleimidoacet-oxysuccinimide ester (AMAS), N-β-maleimidopropyl-oxysuccinimide ester (BMPS), and N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS).

[0051] In the conjugate, the ratio of the carrier protein to the antigen peptide (molecular ratio, carrier protein:antigen peptide) is, for example, 1:1 to 1:100, or 1:1 to 1:10.

[0052] Preferably, the conjugate, when administered to a subject, is capable of inducing the formation of an antibody capable of inhibiting the activity of the Wnt / β-catenin pathway in a Wnt / β-catenin pathway activity assay. The Wnt / β-catenin pathway activity assay is a system for evaluating the degree of β-catenin pathway activation in vitro using plasma or serum from a subject administered with the conjugate and reporter cells, for example, according to Example 3(1) described below. The reporter cells are cells in which a reporter (e.g., luciferase) is detected upon activation of the β-catenin pathway.

[0053] The induction ability can be measured, for example, by administering (inoculating) the analyte conjugate (analyte) to a subject and evaluating serum or plasma derived from the subject using a Wnt / β-catenin pathway activity assay. Specifically, the analyte is optionally mixed with an immunostimulant and administered to the subject, for example, as in Examples 1 and 2 described below. The administration is preferably two times. Next, for example, 2 to 4 weeks after the final administration, blood is collected from the subject and serum or plasma is isolated. The induction ability can then be evaluated, for example, by performing the Wnt / β-catenin pathway activity assay using the serum or plasma and comparing it with the Wnt / β-catenin pathway activity of plasma or serum derived from a subject administered with physiological saline (control).

[0054] The conjugate has the ability to induce the formation of antibodies capable of inhibiting Wnt / β-catenin pathway activity by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, for example, when the Wnt / β-catenin pathway activity of the control is taken as the standard (100%) in the Wnt / β-catenin pathway activity assay.

[0055] The conjugate is used, for example, to treat a tumor. The tumor may be a benign tumor or a malignant tumor (cancer). The tumor may be, for example, a prorenin receptor-positive tumor. The tumor may be, for example, a colon cancer, pancreatic cancer, or brain tumor. The tumor may be, for example, a benign tumor or familial adenomatous polyposis.

[0056] The conjugate can be suitably used for treating, for example, familial adenomatous polyposis accompanied by complications, such as gastrointestinal benign multiple tumor prevalence disease, Gardner syndrome, Turcot syndrome, and / or desmoid prevalence syndrome.

[0057] The conjugate of the present disclosure may be used, for example, in vitro or in vivo. The conjugate of the present disclosure may be used, for example, as a research reagent or as a pharmaceutical. In the former case, the conjugate of the present disclosure may also be referred to as a test reagent or test kit.

[0058] The subject to which the conjugate of the present disclosure is administered is not particularly limited. When the conjugate of the present disclosure is used in vivo, the subject (subject to administration) can be, for example, the above-mentioned examples. When the conjugate of the present disclosure is used in vitro, the subject to administration can be, for example, a cell, a tissue, an organ, etc. Examples of the cell include a cell collected from a living body, a cultured cell, etc., and examples of the tissue or organ include a tissue (biological tissue) or an organ, etc. collected from a living body. Examples of the cell include immune cells such as T cells, B cells, NK cells, dendritic cells, etc.

[0059] When the conjugate of the present disclosure is used in vivo, the subject may be a healthy individual without a tumor, a person who may have a tumor, or a patient with a tumor, but preferably a subject for whom treatment is desired. Furthermore, when the subject is suffering from familial adenomatous polyposis, the subject may be a patient without a complication of familial adenomatous polyposis, a person who may have a complication of familial adenomatous polyposis, or a patient with a complication of familial adenomatous polyposis, but preferably a subject for whom treatment is desired. Furthermore, the subject may be a healthy individual without sarcopenia or frailty, a person who may have sarcopenia or frailty, or a patient with sarcopenia or frailty, but preferably a subject for whom treatment is desired.

[0060] The conditions for use (administration conditions) of the conjugate of the present disclosure are not particularly limited, and the administration form, administration timing, dosage, etc. can be appropriately set depending on, for example, the type of active ingredient in the conjugate (protein, peptide, VLP, virus, nucleic acid, etc.), the type of subject to administration, etc.

[0061] Examples of methods for administering the conjugate of the present disclosure include intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, and intravenous administration. However, intramuscular administration or subcutaneous administration is preferred because it allows safe and stable administration regardless of the skill of the administerer.

[0062] The dose of the conjugate of the present disclosure may be an amount that, when administered to a subject, is capable of inducing antibodies against the prorenin receptor compared to a non-administered subject, i.e., an effective dose (therapeutically effective dose). The dose can be appropriately determined depending on, for example, the age, weight, symptoms, etc. of the subject.

[0063] The conjugate of the present disclosure may be administered once or multiple times. The multiple times may be, for example, two, three, four, five, or more times. The number of administrations may be appropriately determined while confirming the therapeutic effect on the subject. When administering multiple times, the administration interval may be appropriately determined while confirming the therapeutic effect on the subject, and may be, for example, once a day, once a week, once every two weeks, once a month, once every three months, or once every six months.

[0064] The conjugate of the present disclosure can prevent or alleviate at least one symptom caused by a tumor in a subject to which it is administered. Examples of symptoms of tumor onset include general fatigue, loss of appetite, and weight loss. Examples of symptoms of familial adenomatous polyposis include an increase in the number of polyps with a diameter of 5 mm or more that require endoscopic removal. The conjugate of the present disclosure can prevent or alleviate at least one symptom associated with a tumor. The alleviation of the symptom can be evaluated, for example, subjectively or objectively, and specific examples include self-assessment by the subject; physician's assessment; quality of life (QOL) assessment; evaluation of delayed progression of tumor onset symptoms, or reduction in the severity of tumor onset symptoms. The objective evaluation can be performed using animals or humans.

[0065] When the conjugate of the present disclosure is used in vivo, the subject to which it is administered may be a healthy individual not suffering from sarcopenia or frailty, a person who may be suffering from sarcopenia or frailty, or a patient suffering from sarcopenia or frailty.

[0066] The conjugate of the present disclosure can prevent or alleviate at least one symptom caused by sarcopenia or frailty in a subject to which it is administered. Examples of symptoms of sarcopenia or frailty include a decrease in body weight, skeletal muscle mass, muscle strength (grip strength), or physical function (walking speed). The conjugate of the present disclosure can prevent or alleviate at least one symptom associated with sarcopenia or frailty. The alleviation of the symptom can be evaluated, for example, subjectively or objectively, and specific examples include self-assessment by the subject to which it is administered; physician's assessment; quality of life (QOL) assessment; evaluation of delay in progression of sarcopenia or frailty symptoms, or reduction in the severity of sarcopenia or frailty symptoms. The objective evaluation can be performed using animals or humans.

[0067] The conjugate of the present disclosure contains the prorenin receptor peptide and is therefore capable of inducing antibodies against PRR, making it suitable for use in the treatment of cancer and sarcopenia or frailty.

[0068] <Nucleic Acid> In another aspect, the present disclosure provides a nucleic acid encoding the PRR peptide. The nucleic acid of the present disclosure encodes the prorenin receptor peptide of the present disclosure.

[0069] The nucleic acid may be composed of deoxynucleotide residues, ribonucleotide residues, or both. The nucleic acid may also be composed of natural nucleic acid residues, unnatural nucleic acid residues, or both. Specific examples of the nucleic acid include DNA, RNA, and / or DNA / RNA composed of natural and / or unnatural nucleic acid residues. Examples of the unnatural nucleic acid residue include modified nucleotide residues or modified ribonucleotide residues in which the base, sugar residue, or sugar phosphate backbone of the nucleotide residue is modified. When the sugar residue is modified, examples of the unnatural nucleic acid residue include cEt (constrained ethyl bicyclic nucleic acid, manufactured by Ionis Pharmaceuticals), LNA (trademark), Locked Nucleic Acid, and ENA (registered trademark, 2'-O,4'-C-Ethylenebridged Nucleic Acid). The nucleic acid may have, for example, a 5' cap at the 5' end.

[0070] The nucleic acid may be a single-stranded or double-stranded nucleic acid molecule.

[0071] The nucleic acid of the present disclosure can be designed based on the amino acid sequence of the PRR peptide of the present disclosure by substituting corresponding codons. The base sequence of the nucleic acid of the present disclosure may be, for example, codon-optimized.

[0072] In the present disclosure, various nucleic acids and prorenin receptor peptides can be synthesized, for example, by genetic engineering techniques or organic synthesis techniques, and can also be referred to as synthetic DNA such as cDNA, or synthetic RNA.

[0073] The nucleic acid of the present disclosure encodes the PRR peptide and can therefore be suitably used for synthesizing the PRR peptide.Furthermore, since the nucleic acid of the present disclosure encodes the PRR peptide, when used as an active ingredient in a vaccine, it can induce antibodies against the PRR.

[0074] <Expression Vector> In another aspect, the present disclosure provides an expression vector that can be used to synthesize or express a prorenin receptor peptide. The expression vector of the present disclosure comprises the nucleic acid of the present disclosure. Using the expression vector of the present disclosure, the prorenin receptor peptide of the present disclosure can be suitably produced by genetic engineering techniques.

[0075] The expression vector of the present disclosure is, for example, an expression vector in which the nucleic acid of the present disclosure is inserted. The expression vector can also be said to be, for example, a vector in which the nucleic acid is operably linked. The expression vector refers to, for example, a nucleic acid molecule that can transport an inserted gene into a target such as a cell.

[0076] The expression vector is not particularly limited in its configuration, as long as it contains a polynucleotide encoding a prorenin receptor peptide so as to be able to express the prorenin receptor peptide encoded by the polynucleotide of the nucleic acid of the present disclosure.

[0077] The expression vector can be prepared, for example, by inserting a polynucleotide encoding a prorenin receptor peptide, i.e., the nucleic acid of the present disclosure, into a backbone vector (hereinafter also referred to as a "basic vector"). The type of expression vector is not particularly limited and can be appropriately determined, for example, depending on the type of host. Specifically, when the expression vector is synthesized by genetic engineering techniques, the expression vector is first synthesized by, for example, designing and synthesizing a nucleic acid encoding the prorenin receptor peptide. The design and synthesis can be performed, for example, by PCR using a vector containing the nucleic acid encoding the prorenin receptor peptide as a template and primers designed to synthesize the desired nucleic acid region. The resulting nucleic acid can then be ligated to an appropriate vector to obtain a recombinant vector for protein expression (expression vector), and this recombinant vector can be introduced into a host so that the target gene can be expressed to obtain a transformant (Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory Press (2012))).

[0078] The host used for transformation is not particularly limited as long as it can express the target nucleic acid, and examples thereof include non-human hosts such as microorganisms, animal cells, insect cells, or cultured cells thereof, isolated human cells or cultured cells thereof, and mammalian cells. When the expression vector is administered to a subject, the host is, for example, the cells to be administered. Examples of prokaryotes include bacteria such as Escherichia genus bacteria (e.g., Escherichia coli) and Pseudomonas genus bacteria (e.g., Pseudomonas putida). Examples of eukaryotes include yeasts such as Saccharomyces cerevisiae. Examples of animal cells include COS cells and CHO cells, and examples of insect cells include Sf9 and Sf21.

[0079] The expression vector may be a viral vector or a non-viral vector. Examples of the viral vector include baculovirus; poxviruses such as vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoon pox virus, and swinepox virus; adenoviruses such as canine adenovirus; adeno-associated virus; herpes virus; and retrovirus. When transforming a host using the heat shock method, the expression vector may be, for example, a binary vector. Examples of the expression vector include pETDuet-1, pQE-80L, and pUCP26Km. When transforming bacteria such as Escherichia coli, examples of the expression vector include the pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, and pUC8. When transforming yeast, examples of the expression vector include pYepSec1, pMFa, pYES2, etc. When transforming insect cells, examples of the expression vector include pAc, pVL, etc. When transforming mammalian cells, examples of the expression vector include pCDM8, pMT2PC, etc.

[0080] The expression vector preferably contains a regulatory sequence that regulates the expression of the polynucleotide encoding the prorenin receptor peptide and the expression of the prorenin receptor peptide of the present disclosure encoded by the polynucleotide encoding the prorenin receptor peptide. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, and an origin of replication (ori). The location of the regulatory sequence in the expression vector is not particularly limited. The regulatory sequence in the expression vector may be located, for example, so long as it is capable of functionally regulating the expression of the polynucleotide encoding the prorenin receptor peptide and the expression of the polypeptide encoded thereby, and may be located according to known methods. The regulatory sequence may be, for example, a sequence already present in the expression vector, or an additional regulatory sequence may be inserted into the expression vector, or the regulatory sequence present in the basic vector may be replaced with another regulatory sequence.

[0081] <Transformant and method for producing the transformant> In another aspect, the present disclosure provides a transformant capable of producing a prorenin receptor peptide and a method for producing the same. The transformant of the present disclosure comprises a nucleic acid encoding the prorenin receptor peptide of the present disclosure. The transformant of the present disclosure is characterized by comprising a nucleic acid encoding the prorenin receptor peptide of the present disclosure, and other configurations and conditions are not particularly limited. The transformant of the present disclosure can be used to suitably produce the prorenin receptor peptide of the present disclosure.

[0082] The method for producing a transformant of the present disclosure includes the step of introducing the nucleic acid of the present disclosure into a host. The method for producing a transformant of the present disclosure is characterized by introducing the nucleic acid of the present disclosure into the host, and other steps and conditions are not particularly limited. The transformant can be produced by the method for producing a transformant of the present disclosure. The transformant and its production method of the present disclosure can be produced by reference to the explanations of the methods for producing the prorenin receptor peptide, conjugate, nucleic acid, and expression vector of the present disclosure.

[0083] In the transformant of the present disclosure, the nucleic acid encoding the prorenin receptor peptide can be the same as that described above for the nucleic acid encoding the prorenin receptor peptide of the present disclosure. The nucleic acid of the present disclosure may be an expression vector of the present disclosure.

[0084] In the transformant of the present disclosure, the nucleic acid of the present disclosure is present as an exogenous molecule. Therefore, the transformant of the present disclosure can be produced, for example, by introducing the nucleic acid of the present disclosure into the host.

[0085] The method for introducing the nucleic acid is not particularly limited and can be performed by a known method. The nucleic acid may be introduced, for example, via the expression vector. The method for introducing the expression vector into the host is not particularly limited and can be performed by a known method. The introduction method can be appropriately selected depending on, for example, the type of the host. Examples of the introduction method include introduction using a gene gun such as a particle gun, the calcium phosphate method, the polyethylene glycol method, lipofection using liposomes, electroporation, ultrasonic nucleic acid introduction, DEAE-dextran method, direct injection using microglass tubes or the like, the hydrodynamic method, the cationic liposome method, a method using an introduction adjuvant, and a method mediated by Agrobacterium. Examples of the liposome include lipofectamine and cationic liposome, and examples of the introduction adjuvant include atelocollagen, nanoparticles, and polymers. When the host is a microorganism, a method mediated by yeast or the like is preferred. The prorenin receptor peptide polynucleotide of the present disclosure may be introduced into the host, for example, via an expression vector of the present disclosure.

[0086] <Method for producing PRR peptide and conjugate> In another aspect, the present disclosure provides a method for producing a prorenin receptor peptide. The method for producing a prorenin receptor peptide of the present disclosure includes an expression step of expressing the prorenin receptor peptide of the present disclosure. The method for producing a prorenin receptor peptide of the present disclosure is characterized by including the expression step, and other steps and conditions are not particularly limited. The method for producing a prorenin receptor peptide of the present disclosure allows the prorenin receptor peptide of the present disclosure to be produced.

[0087] The prorenin receptor peptide may be produced by a genetic engineering technique using a host such as yeast, or may be produced in a cell-free system, and methods well known to those skilled in the art can be used. For example, WO 2016 / 017037 (U.S. Patent Application Publication No. 2016 / 0202251) can be referred to for the production of the polypeptide.

[0088] When the prorenin receptor peptide is produced by the genetic engineering technique, the production method of the present disclosure may include preparing a transformant containing the nucleic acid or expression vector of the present disclosure prior to the expression step. In this case, the expression step may include, for example, a culture step of culturing the transformant and an isolation step of isolating the prorenin receptor peptide from the culture. In the culture step, the culture conditions can be appropriately set depending on the type of host used to produce the transformant. The culture may be a culture supernatant, a transformant such as cultured cells or cultured bacterial cells, or a processed or disrupted product thereof.

[0089] If the prorenin receptor peptide is produced within the host after the culture, the production method of the present disclosure includes an isolation step, for example, by disrupting the host to isolate the prorenin receptor peptide. Alternatively, if the prorenin receptor peptide is produced or secreted outside the host, the production method of the present disclosure includes, for example, using the culture medium as is or removing the host by centrifugation or the like. The production method of the present disclosure then can isolate or purify the prorenin receptor peptide by, for example, using a general biochemical method used for isolating and purifying proteins, specifically, concentration using an ultrafiltration membrane; salting out such as ammonium sulfate precipitation; or chromatography using various columns such as gel filtration, ion exchange chromatography, and affinity chromatography, either alone or in appropriate combination.

[0090] In the production method of the present disclosure, the prorenin receptor peptide may be obtained by in vitro translation using a cell-free synthesis system. In this case, the production method of the present disclosure may be carried out using RNA encoding the prorenin receptor peptide as a template, or using DNA encoding the prorenin receptor peptide as a template (transcription and translation). A commercially available system can be used as the cell-free synthesis system, specifically, the Expressway™ system (Invitrogen). After the translation, the production method of the present disclosure can isolate or purify the prorenin receptor peptide using, for example, common biochemical methods used for protein isolation and purification, specifically, concentration using an ultrafiltration membrane; salting out such as ammonium sulfate precipitation; chromatography using various columns such as gel filtration, ion exchange chromatography, and affinity chromatography, either alone or in combination as appropriate. The isolation or purification can be carried out, for example, in the same manner as when using the transformant.

[0091] The prorenin receptor peptide obtained by the production method of the present disclosure may be used, for example, as a crude purified product as is, or as a partially purified product, or as a single purified product.

[0092] In the production method of the present disclosure, the obtained prorenin receptor peptide may be powdered by, for example, freeze-drying, vacuum drying, spray drying, etc. In this case, in the production method of the present disclosure, for example, the prorenin receptor peptide may be dissolved in advance in a buffer solution such as acetate buffer, phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or GOOD buffer (e.g., PIPES, MES, MOPS, etc.).

[0093] The production method of the present disclosure may further comprise conjugating the obtained PRR peptide to the carrier protein, which can be determined based on the substituent (reactive group) at the binding position between the PRR peptide and the carrier protein.

[0094] <Pharmaceutical Composition> The present disclosure provides a pharmaceutical composition capable of inducing antibodies against PRR. The pharmaceutical composition of the present disclosure is a pharmaceutical composition comprising the prorenin receptor peptide, conjugate, nucleic acid, and / or expression vector of the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present disclosure is characterized by comprising the prorenin receptor peptide, conjugate, nucleic acid, and / or expression vector (hereinafter collectively referred to as the "active ingredient") as an active ingredient, and other configurations and conditions are not particularly limited. Because the pharmaceutical composition of the present disclosure comprises the prorenin receptor peptide or conjugate, or a nucleic acid encoding them, as an active ingredient, it may exhibit antitumor and / or anti-muscle loss effects when used as an active ingredient in a vaccine. The description of the conjugate can be applied to the pharmaceutical composition of the present disclosure.

[0095] For example, when administered to a subject, the pharmaceutical composition of the present disclosure can induce antibodies against the prorenin receptor peptide or conjugate, or a nucleic acid encoding the same. Therefore, the pharmaceutical composition of the present disclosure can also be referred to as a vaccine, vaccine composition, or vaccine formulation.

[0096] The pharmaceutical composition of the present disclosure contains a pharmaceutically acceptable carrier, which may be a suspending agent, solubilizing agent, stabilizer, isotonicity agent, preservative, anti-adsorption agent, surfactant, diluent, vehicle, pH adjuster, soothing agent, buffer, sulfur-containing reducing agent, antioxidant, or the like, for administering the active ingredient, and may be appropriately added within a range that does not impair the effects of the present disclosure.

[0097] The pharmaceutical composition of the present invention may further comprise, for example, an immunostimulant. Examples of the immunostimulant include toll-like receptor stimulants such as aluminum hydroxide, aluminum phosphate, aluminum chloride, complete Freund's adjuvant, incomplete Freund's adjuvant, CpG oligonucleotide, Poly I:C, and lipopolysaccharide (LPS), as well as cytokines, lymphokines, and chemokines. Examples of the cytokines and lymphokines include interferons such as interferon (IFN-γ); inflammatory cytokines such as TNF-α; interleukins such as IL-1, IL-2, IL-3, IL-4, IL-12, and IL-13; growth factors such as granulocyte-macrophage (GM-CSF) colony-stimulating factor (GM-CSF) and granulocyte-colony-stimulating factor (G-CSF); Flt3 ligand; B7-1; B7-2; and the like.

[0098] <Tumor Treatment Method> In another aspect, the present disclosure provides a tumor treatment method, which comprises administering to a subject a prorenin receptor peptide, conjugate, nucleic acid, expression vector, and / or pharmaceutical composition (hereinafter collectively referred to as "active ingredient") of the present disclosure.

[0099] The tumor treatment method of the present disclosure includes, for example, a step of administering the active ingredient to the subject. The administration conditions in the administration step can be the same as those described for the conjugate of the present disclosure. The administration may be in vitro or in vivo.

[0100] The tumor treatment method of the present disclosure includes the prorenin receptor peptide, conjugate, or nucleic acid encoding the same as described above as an active ingredient, and therefore can induce the production of antibodies against the prorenin receptor when administered to a subject, thereby suppressing tumor growth.

[0101] <Method for treating sarcopenia or frailty> In another aspect, the present disclosure provides a method for treating sarcopenia or frailty. The method for treatment of a subject of the present disclosure comprises administering to the subject a prorenin receptor peptide, conjugate, nucleic acid, expression vector, and / or pharmaceutical composition (hereinafter collectively referred to as "active ingredient") of the present disclosure. The method for treatment of the present disclosure is characterized by the use of a prorenin receptor peptide, conjugate, nucleic acid, or expression vector as an active ingredient, and other steps and conditions are not particularly limited.

[0102] The method for treating sarcopenia or frailty of the present disclosure includes, for example, administering the active ingredient to the subject. The administration conditions in the administration step can be the same as those described for the conjugate of the present disclosure. The administration may be in vitro or in vivo.

[0103] The method for treating sarcopenia or frailty disclosed herein includes the prorenin receptor peptide, conjugate, or nucleic acid encoding the same as described above as an active ingredient, and thus can induce the production of antibodies against the prorenin receptor when administered to a subject, thereby suppressing sarcopenia or frailty.

[0104] <Use of Pharmaceutical Compositions> The present disclosure relates to a prorenin receptor peptide, conjugate, nucleic acid, expression vector, and / or pharmaceutical composition of the present disclosure, or use thereof, for use in treating a tumor. The present disclosure relates to a prorenin receptor peptide, conjugate, nucleic acid, expression vector, and / or pharmaceutical composition of the present disclosure, or use thereof, for the manufacture of a medicament for treating a tumor. The present disclosure relates to a prorenin receptor peptide, conjugate, nucleic acid, expression vector, and / or pharmaceutical composition of the present disclosure, or use thereof, for use in the treatment of sarcopenia or frailty. The present disclosure relates to a prorenin receptor peptide, conjugate, nucleic acid, expression vector, and / or pharmaceutical composition of the present disclosure, or use thereof, for the manufacture of a medicament for treating sarcopenia or frailty.

[0105] The present invention will be described in detail below using examples, but the present disclosure is not limited to the embodiments described in the examples.

[0106] [Example 1] The PRR peptide of the present disclosure was confirmed to have the ability to induce antibodies against PRR and to have an anti-tumor effect.

[0107] (1) Preparation of Prorenin Receptor Peptide Antigens Peptide fragments of the human prorenin receptor protein (SEQ ID NO: 5) were prepared as follows: PRR1 peptide (PRR1, SEQ ID NO: 1) at amino acids 198-211, PRR2 peptide (PRR2, SEQ ID NO: 2), PRR3 peptide (PRR3, SEQ ID NO: 3), PRR4 peptide (PRR4, SEQ ID NO: 7) at amino acids 224-233, and, as a comparative example, PRR0 peptide (PRR0, SEQ ID NO: 8) at amino acids 200-213. Conch protein (keyhole limbet hemocyanin: KLH) was linked to the N-terminus of each polypeptide as a carrier protein by a known method to obtain PRR0-Cg, PRR1-Cg, PRR2-Cg, PRR3-Cg, and PRR4-Cg, corresponding to polypeptides 0-4. Each antigen was adjusted to 1 mg / ml. For the linkage, a cross-linking agent (EMCS) was used to cross-link the amino group of the peptide with the thiol group of the side chain of the cysteine ​​residue of the KLH to prepare a conjugate.

[0108] PRR4 peptide (SEQ ID NO: 7) KRYGEDSEQ PRR0 peptide (SEQ ID NO: 8) HKHLAKDHSPDLYS

[0109] (2) Measurement of PRR0 antibody titer and examination of antitumor effect Antibodies against PRR0 exhibit antitumor effects in vivo. Therefore, mice (Balb / c mice, 7 weeks old) were inoculated with the PRR0-Cg as a prorenin receptor vaccine, and the antibody titer against PRR (hereinafter referred to as "antibody titer") and the antitumor effect were measured. Specifically, the mice were inoculated with 5 μg or 20 μg (1 mg / ml) of PRR0-Cg obtained in Example 1(1) above together with an adjuvant (Freund's Complete Adjuvant, Cat No. 014-09541, manufactured by WAKO) (n=4 each). Serum was collected two and four weeks after the inoculation. Five weeks after the first inoculation, 1 × 10 ６Mouse colon cancer cells, CT26, were transplanted into the mice. The antibody titer in the serum was measured by absorbance at 450 nm. The effect on tumors was also evaluated 10 weeks after the first inoculation. As a negative control, the same procedure was used except that mice (n=2) were inoculated with the adjuvant alone. These results are shown in Figures 1 and 2.

[0110] Figure 1 is a graph showing the antibody titer of the prorenin receptor vaccine PRR0. In Figure 1, the horizontal axis represents the serum dilution rate, and the vertical axis represents absorbance. As shown in Figure 1, in the negative control mice, no increase in antibody titer was observed 2 weeks and 4 weeks after the first administration. Furthermore, in the mice inoculated with PRR0-Cg, a tendency for an increase in antibody titer was observed 2 weeks and 4 weeks after the first administration compared to the negative control, but no significant increase was observed.

[0111] Figure 2 is a photograph showing mice 10 weeks after the first inoculation with the prorenin receptor vaccine PRR0-Cg (5 weeks after transplantation of mouse colon cancer cells CT26: Day 35). The upper row of Figure 2 shows negative control mice, the middle row shows mice inoculated with 5 μg of PRR0-Cg, and the lower row shows mice inoculated with 20 μg of PRR0-Cg. As shown in Figure 2, no antitumor effect was observed regardless of the dose of PRR0-Cg inoculated. From the above, it was found that although antibodies against PRR0 exhibit antitumor effects, their epitope, PRR0, does not function as a prorenin receptor vaccine.

[0112] (3) Measurement of antibody titers of PRR1 to PRR4 and examination of antitumor effects Next, the conjugates were inoculated into mice (Balb / c, 7 weeks old) as prorenin receptor vaccines, and antibody titers were measured and antitumor effects were examined. Specifically, 50 μg of PRR1-Cg, PRR2-Cg, PRR3-Cg, or PRR4-Cg prepared at 1 mg / ml as obtained in Example 1(1) was inoculated into the mice together with an adjuvant (Freud's Complete Adjuvant) (PRR1-Cg, 2-Cg, and 4-Cg: n=9, PRR3-Cg: n=8). Two weeks after the first inoculation, the mice were again inoculated with 50 μg of PRR1-Cg, PRR2-Cg, PRR3-Cg, or PRR4-Cg together with an adjuvant (Freund's Incomplete Adjuvant). ５ Mouse colon cancer cells, CT26, were transplanted into the mice. Seven weeks after the first inoculation, serum was collected from the mice. The antibody titer in the serum was measured as the half-maximum absorbance at 450 nm. Furthermore, tumor size in the mice was measured 3, 4, 5, 6, and 7 weeks after the first inoculation. The same procedure was followed, except that mice were inoculated with adjuvant alone (n=7) as a negative control. These results are shown in Figures 3 and 4.

[0113] FIG. 3 is a graph showing antibody titers 7 weeks after the first administration (5 weeks after the second administration) of prorenin receptor vaccines PRR1 to 4. In FIG. 3, the horizontal axis indicates the type of immunogen, and the vertical axis indicates half-maximum absorbance at 450 nm. As shown in FIG. 3, it was found that antibody titers were elevated in mice inoculated with PRR1-Cg, PRR2-Cg, and PRR3-Cg. Furthermore, it was found that antibody titers were significantly elevated in mice inoculated with PRR1-Cg and PRR2-Cg. On the other hand, no increase in antibody titer was observed in mice inoculated with PRR4-Cg. From the above, it was found that the PRR1 peptide, PRR2 peptide, and PRR3 peptide of the present disclosure have the function of inducing antibodies against PRR.

[0114] Figure 4 is a graph showing the antitumor effects of the prorenin receptor vaccines PRR1 to 4. In Figure 4, the horizontal axis represents the number of days after CT26 transplantation, and the vertical axis represents the tumor size (mm ３ ) are shown. As shown in Figure 4, it was found that the tumor size was smaller in mice inoculated with PRR1-Cg, PRR2-Cg, or PRR3-Cg compared to mice inoculated with adjuvant alone. On the other hand, no change in tumor size was observed in mice inoculated with PRR4-Cg compared to mice inoculated with adjuvant alone. From the above, it was found that the PRR1 peptide, PRR2 peptide, and PRR3 peptide of the present disclosure function as a prorenin receptor vaccine against colorectal cancer. Furthermore, since the PRR1 peptide, PRR2 peptide, and PRR3 peptide can induce antibodies against PRR, it was presumed that the PRR1 peptide, PRR2 peptide, and PRR3 peptide exhibit anti-tumor effects via the antibodies induced by these antigens.

[0115] (4) Examination of the antitumor effect of PRR2 and PRR3 PRR2-Cg and PRR3-Cg, which showed antitumor effects, were inoculated into mice (Balb / c, 6 weeks old) to re-examine their antitumor effects. Specifically, 50 μg (1 mg / ml) of PRR2-Cg or PRR3-Cg obtained in Example 1(1) above was inoculated into the mice together with an adjuvant (Freund's Complete Adjuvant) (n=10 for each). Two weeks after the first inoculation, the mice were again inoculated with 50 μg of PRR2-Cg or PRR3-Cg together with an adjuvant (Freund's Incomplete Adjuvant). Three weeks after the first inoculation, 1 × 10 ５ Mouse colon cancer cells, CT26, were subcutaneously implanted into the mice. Tumor size was measured 14, 17, 21, 24, and 28 days after implantation of the CT26 cells. A negative control was performed in the same manner, except that mice were inoculated with adjuvant alone (n=10). The results are shown in Figure 5.

[0116] 5 is a graph showing the antitumor effects of the prorenin receptor vaccines PRR2 and PRR3. In FIG. 5, the horizontal axis represents the number of days after CT26 transplantation, and the vertical axis represents tumor size (mm ３ ) are shown. As shown in Figure 5, the tumor size was recapitulated as being smaller in mice inoculated with PRR2-Cg or PRR3-Cg compared to mice inoculated with adjuvant alone. From the above, it was found that the PRR2 peptide and PRR3 peptide of the present disclosure function as prorenin receptor vaccines against colorectal cancer.

[0117] (5) Examination of the Antitumor Effect of PRR2 and PRR3 by Histological Staining β-catenin accumulation is observed in the majority of colon cancers. We investigated whether administration of PRR2-Cg or PRR3-Cg, which demonstrated antitumor effects, reduced β-catenin accumulation in the colon by immunohistochemical staining. Specifically, tumor tissues were collected from mice administered with PRR2-Cg or PRR3-Cg using the same method as in Example 1(3) above. As a negative control, mice were inoculated with saline alone instead of the peptide antigen (n=5). Tumor tissues were collected using the same method. After collection, the colon tissues were fixed using 4% paraformaldehyde. After fixation, the colon tissues were embedded in paraffin, and 2-μm-thick paraffin-embedded sections were prepared. The resulting paraffin-embedded sections were subjected to immunohistochemistry for β-catenin. For β-catenin staining, anti-β-catenin antibody (300-fold dilution, Cat. No. 05-665, Merck Millipore) was used as the primary antibody, and rabbit serum was used as a negative control. The results are shown in Figure 6.

[0118] FIG. 6 is a photograph of staining of activated β-catenin in colon tissue. In FIG. 6, the upper photograph shows a stained image of the colon tissue of a negative control, the lower left photograph shows a stained image of the colon tissue of a PRR2-Cg-inoculated mouse, and the lower right photograph shows a stained image of the colon tissue of a PRR3-Cg-inoculated mouse. As shown in FIG. 6, little expression of activated β-catenin was observed in the colon tissue of mice administered with PRR2-Cg or PRR3-Cg. From the above, it was found that administration of the PRR2 peptide or PRR3 peptide of the present disclosure induces antibodies against PRR, thereby suppressing activation of the Wnt / β-catenin pathway.

[0119] (6) Measurement of PRR2 and PRR3 Antibody Titers in Short-Term Observation The antibody titers of PRR2-Cg and PRR3-Cg under short-term observation were measured for each sex of mice. Specifically, 50 μg of PRR2-Cg or PRR3-Cg obtained in Example 1(1) above was inoculated into mice (C57BL / 6 mice) together with an adjuvant (Freund's Complete Adjuvant), and serum was collected (n = 10 for each). Two weeks after the first inoculation, the mice were again inoculated with 50 μg of PRR2-Cg or PRR3-Cg together with an adjuvant (Freund's Incomplete Adjuvant). Serum was collected from the mice 2, 4, and 8 weeks after the first inoculation. The antibody titers in the serum were measured as the half-maximum absorbance at 450 nm. As a negative control, the same procedure was followed except that mice were inoculated with the adjuvant alone. The results are shown in Figure 7.

[0120] Figure 7 is a graph showing antibody titers of the prorenin receptor vaccines PRR2 and PRR3 under short-term observation. In Figure 7, the upper row shows the results for male mice, and the lower row shows the results for female mice. In Figure 7, the horizontal axis indicates the type of immunogen and the number of weeks after the first inoculation, and the vertical axis indicates half-maximum absorbance at 450 nm. As shown in Figure 7, it was found that in mice inoculated with PRR2-Cg or PRR3-Cg, regardless of gender, antibody titers increased 4 weeks after the first inoculation. These results suggest that the PRR2 peptide and PRR3 peptide can induce antibodies against PRR, regardless of gender, and function as prorenin receptor vaccines.

[0121] (7) Measurement of PRR2 and PRR3 Antibody Titers in Long-Term Observation Antibody titers for PRR2-Cg and PRR3-Cg under long-term observation were measured for each sex of mice. Specifically, 50 μg of PRR2-Cg or PRR3-Cg obtained in Example 1(1) above was inoculated into mice (C57BL / 6 mice, 7 weeks old) together with an adjuvant (Freund's Complete Adjuvant), and serum was collected (n = 10 for each). Two weeks after the first inoculation, the mice were again inoculated with 50 μg of PRR2-Cg or PRR3-Cg together with an adjuvant (Freund's Incomplete Adjuvant). Serum was collected from the mice 2, 4, 8, and 89 weeks after the first inoculation (corresponding to ages of 9, 11, 15, and 96 weeks). The antibody titer in the serum was measured as the half-maximum absorbance at 450 nm. As a negative control, mice were inoculated with the adjuvant alone in the same manner. The results are shown in Figure 8.

[0122] Figure 8 is a graph showing the antibody titers of the prorenin receptor vaccines PRR2 and PRR3 under long-term observation. In Figure 8, the upper panel shows the results for female mice, and the lower panel shows the results for male mice. In Figure 8, the horizontal axis indicates the type of immunogen and the age of the mice in weeks, and the vertical axis indicates the half-maximum absorbance at 450 nm. As shown in Figure 8, it was found that in mice inoculated with PRR2-Cg, antibody titers were maintained 89 weeks after the first inoculation (96 weeks of age), regardless of gender. It was also found that in male mice inoculated with PRR3-Cg, antibody titers were maintained 89 weeks after the first inoculation (96 weeks of age). From the above, it was found that the PRR2 peptide maintains antibody titers over the long term, regardless of gender. It was also suggested that the PRR3 peptide maintains antibody titers over the long term, particularly in males.

[0123] [Example 2] For the PRR peptide of the present disclosure, CRM was added to a carrier protein. １９７ It was confirmed that even when the above-mentioned compound was used, it had the ability to induce antibodies against PRR and had an antitumor effect.

[0124] (1) Study of Carrier Protein in Mice In the conjugates of PRR2-Cg and PRR3-Cg, KLH was used as the carrier protein. Therefore, in this example, a non-toxic mutant diphtheria toxin CRM, which can be used in human clinical trials, was used as the carrier protein instead of KLH. １９７ CRM was used as a carrier protein at the N-terminus of the amino acid of the PRR2 polypeptide. １９７ were ligated by a known method to obtain CRM197-PRR2. １９７ The procedure was carried out in the same manner except that

[0125] Next, we investigated whether the CRM197-PRR2 functioned as a prorenin receptor vaccine, similar to KLH-conjugated PRR2. Specifically, the CRM197-PRR2 was inoculated into mice (C57BL / 6 mice, 7 weeks old), and antibody titers were measured. The CRM197-PRR2 was adjusted to a concentration of 7 mg / ml and then further diluted 10-fold. After the dilution, 30 μl of the diluted solution containing CRM197-PRR2 (approximately 20 μg) was inoculated into the mice together with 30 μl of adjuvant (Freund's Complete Adjuvant), and serum was collected (n=10). Two weeks after the first inoculation, the mice were again inoculated with 30 μl of the CRM197-PRR2 together with adjuvant (Freund's Incomplete Adjuvant). Serum samples were collected from the mice before the first inoculation, and two and four weeks after the first inoculation. The antibody titer in the serum was measured as the half-maximum absorbance at 450 nm. A negative control was performed in the same manner, except that mice were inoculated with adjuvant alone. The results are shown in Figure 9.

[0126] Figure 9 is a graph showing the antibody titer of CRM197-PRR2 in mice. In Figure 9, the horizontal axis represents the number of weeks since the first administration, and the vertical axis represents the half-maximum absorbance at 450 nm. As shown in Figure 9, the antibody titer did not increase in mice inoculated with adjuvant alone. On the other hand, in mice administered with CRM197-PRR2, a slight increase in antibody titer was observed two weeks after the first administration. Furthermore, in mice administered with CRM197-PRR2, it was found that the antibody titer increased four weeks after the first administration. From the above, it was found that CRM197-conjugated PRR2 increases antibody titer in mice, similar to KLH-conjugated PRR2.

[0127] (2) Study of Carrier Protein in Monkeys CRM197-PRR2 was found to be effective in increasing antibody titers in mice. Therefore, we investigated whether CRM197-PRR2 also induces antibodies against PRR in monkeys. Specifically, the CRM197-PRR2 was inoculated into cynomolgus monkeys, and the antibody titer was measured. The CRM197-PRR2 was prepared to a concentration of 7 mg / ml. After the preparation, an adjuvant (2% aluminum hydroxide) in an amount equal to the CRM197-PRR2 was added to prepare a 3.5 mg / ml concentration, yielding a preparation. Then, on days 1 and 29, the preparation was administered subcutaneously (in the lumbar region) to the dorsal surface of the cynomolgus monkeys. The administration volume was 140 μl in the low-dose group and 420 μl in the high-dose group. Serum from the cynomolgus monkeys was collected before administration and on days 29 and 57. The antibody titer in the serum was measured as the half-maximum absorbance at 450 nm. The results are shown in Figure 10.

[0128] Figure 10 is a graph showing the antibody titer of CRM197-PRR2 in cynomolgus monkeys. In Figure 10, the horizontal axis represents the inoculation group and the number of days since the first administration, and the vertical axis represents the half-maximum absorbance at 450 nm. As shown in Figure 10, it was found that the antibody titer increased over time in the low-dose group and the high-dose group. Furthermore, it was found that the antibody titer increased in a dose-dependent manner with CRM197-PRR2. From the above, it was found that antibody titer increased in cynomolgus monkeys by PRR2 conjugated with CRM197.

[0129] Example 3 The PRR peptide of the present disclosure was examined for its β-catenin suppression effect, antitumor effect, and side effects.

[0130] (1) Examination of the β-catenin-inhibitory effect of PRR2 and PRR3 PRR2-Cg, which showed antitumor effects, was examined using a luciferase reporter assay to determine whether it attenuates the activity of β-catenin activated by Wnt3a. Specifically, the pGL4.49[luc2P TCF-LEF RE Hygro] vector (see Figure 11(A) , manufactured by Promega) was expressed in cultured HEK293 cells to construct a system (reporter cells) capable of evaluating β-catenin activation in vitro. In these reporter cells, activation of the Wnt / β-catenin pathway induces luciferase expression in an activated β-catenin-dependent manner. Therefore, the activity of the Wnt / β-catenin pathway can be measured by measuring the luciferase activity of the reporter cells. The HEK293 cells were incubated with ATP6ap monoclonal antibody (Clone67_3, see WO 2018 / 084236) or purified PRR2 antibody simultaneously with Wnt3a, and then cultured at 37°C for 24 hours. After incubation, 50 μl of a luciferase assay system (Promega) containing a luciferase substrate was added, and luminescence was measured using a Centro XS3 LB 960 (Berthold). A control group was assayed in the same manner, except that neither Wnt3a nor any of the antibodies was added. These results are shown in Figure 11.

[0131] FIG. 11 is a graph showing β-catenin activity in HEK293 cells. In FIG. 11(B), the horizontal axis indicates the addition group, and the vertical axis indicates luciferase activity. As shown in FIG. 11(B), it was found that ATP6ap monoclonal antibody and purified PRR2 antibody inhibited β-catenin activity. From the above, it was found that PRR2 of the present disclosure can induce antibodies capable of inhibiting the Wnt / β-catenin pathway. Furthermore, it was suggested that PRR2 of the present disclosure inhibits cancer by inducing antibodies capable of inhibiting the Wnt / β-catenin pathway.

[0132] (2) APC ｍｉｎ／＋Examination of the Polyp Formation Inhibitory Effect of PRR2 and PRR3 in Mice PRR2-Cg and PRR3-Cg, which demonstrated antitumor effects, were examined for their ability to inhibit polyp formation in a mouse model of colon cancer (adenomatous polyposis coli). Specifically, a mouse model of colon cancer (C57BL / 6J-Apcmin / +) was administered physiological saline, PRR2-Cg, or PRR3-Cg, along with an adjuvant, using the same test protocol as in Example 1(3). The intestines (large and small intestines) were collected from the mice when they were 16 to 20 weeks old. Following collection, the intestinal polyps were analyzed. These results are shown in Figures 12 and 13.

[0133] 12 is a graph showing the inhibitory effect on polyp formation in adenomatous polyposis model mice. In FIG. 12, (A) shows a graph of the total number of polyps, and (B) shows a graph of the total polyp area (mm ２ ), and (C) shows photographs of mice inoculated with adjuvant alone, PRR2-Cg, or PRR3-Cg. In FIG. 12(A), the horizontal axis indicates the inoculation group, and the vertical axis indicates the number of polyps. In FIG. 12(B), the horizontal axis indicates the inoculation group, and the vertical axis indicates the area (mm ２ ) are shown. As shown in Figure 12, it was found that the total number of polyps and the total polyp area were reduced in the group inoculated with PRR2-Cg or PRR3-Cg compared to the group inoculated with adjuvant. From the above, it was found that the PRR2 peptide and PRR3 peptide of the present disclosure exhibit an inhibitory effect on polyp formation.

[0134] 13 is a graph showing the inhibitory effect on polyp formation in adenomatous polyposis model mice. In FIG. 13, the horizontal axis represents polyp size (mm ２ ), and the vertical axis represents the number of polyps. As shown in Figure 13, compared with the adenomatous polyposis coli model mice (control) inoculated with physiological saline, inoculation with PRR2-Cg or PRR3-Cg resulted in a decrease in the number of polyps of medium size (1.1 to 2.0 mm ２ ) and large (2.1 mm ２These results suggest that the PRR2 peptide and PRR3 peptide of the present disclosure inhibit ATP6ap2, thereby suppressing polyp growth.

[0135] (3) APC by immunostaining ｍｉｎ／＋ Inhibition of β-catenin activity by PRR2 and PRR3 in mice. PRR2 and PRR3, which showed inhibitory effects on polyp formation in adenomatous polyposis coli model mice, were examined using immunohistochemistry to determine whether they also inhibited β-catenin activity. Specifically, adenomatous polyposis coli model mice (C57BL / 6J-Apcmin / +) were administered saline, PRR2-Cg, or PRR3-Cg along with an adjuvant using the same test protocol as in Example 1(3). Intestines were harvested from the mice at 16 to 20 weeks of age. Following harvest, the intestines were fixed in 4% paraformaldehyde. Following fixation, the intestines were embedded in paraffin, and 2-μm-thick paraffin-embedded sections were prepared. The resulting paraffin-embedded sections were immunostained for β-catenin. The primary antibodies used were anti-ATP6ap2 antibody (diluted 3000 times, provided by Tohoku Pharmaceutical University; see Takuo Hirose et al., "Gene expression of (pro)renin receptor is upregulated in hearts and kidneys of rats with congestive heart failure," Peptides, Volume 30, Issue 12, 2009, Pages 2316-2322) for staining ATP6ap2 (prorenin receptor: PRR), anti-active β-catenin antibody (diluted 1000 times, Cat. No. 05-665, Merck Millipore) for staining active β-catenin, and horseradish peroxidase-conjugated anti-rabbit IgG (Nichirei Biosciences) for staining active β-catenin. The results are shown in Figure 14.

[0136] Figure 14 shows photographs of ATP6ap2 or active β-catenin staining in the intestine. In Figure 14, (A) shows an image of ATP6ap2 staining, and (B) shows an image of active β-catenin staining. As shown in Figure 14(A), strong ATP6ap2 staining was observed in the intestinal polyps of mice inoculated with saline. On the other hand, ATP6ap2 staining was weakened in the intestinal polyps of mice inoculated with PRR2-Cg or PRR3-Cg. As shown in Figure 14(B), strong active β-catenin staining was observed in the intestinal nuclei of mice inoculated with saline (Control). On the other hand, active β-catenin staining was weakened in the intestinal nuclei of mice inoculated with PRR2-Cg or PRR3-Cg. From the above, it was found that the PRR2 peptide and PRR3 peptide of the present disclosure suppress the expression of ATP6ap2 and the activity of β-catenin.

[0137] (4) Investigation of Adverse Reactions Caused by PRR2 and PRR3 Adverse reactions, such as bone lesions, have been a concern with Wnt / β-catenin signaling inhibitors. Therefore, we investigated whether inoculation of PRR2 and PRR3 would cause adverse reactions, such as bone lesions. Specifically, adenomatous polyposis coli model mice (C57BL / 6J-Apcmin / +) and normal mice (C57BL / 6) were administered saline, PRR2-Cg, or PRR3-Cg along with an adjuvant using the same test protocol as in Example 1(3). Twenty-four weeks after administration, adverse reactions were confirmed in the adenomatous polyposis coli model mice. AST (liver), BUN (kidney), and bone strength were measured as indicators of adverse reactions in the adenomatous polyposis coli model mice. Furthermore, adverse reactions were confirmed in the normal mice 96 weeks after administration. As indicators of side effects in the normal mice, AST (liver), creatinine clearance (kidney), and bone strength were measured. The results are shown in Figure 15.

[0138] Figure 15 is a graph showing the results of adverse reactions in adenomatous polyposis model mice and normal mice. In Figures 15(A) to (C), the upper row shows the results for female mice, and the lower row shows the results for male mice. In Figure 15, (A) shows AST (liver) in adenomatous polyposis model mice, (B) shows BUN (kidney) in adenomatous polyposis model mice, and (C) shows bone strength in adenomatous polyposis model mice. (D) shows AST (liver) in normal mice, (E) shows creatinine clearance (kidney) in normal mice, and (F) shows bone strength in normal mice. In Figure 15(A), the horizontal axis shows the inoculation group, and the vertical axis shows AST enzyme concentration. In Figure 15(B), the horizontal axis shows the inoculation group, and the vertical axis shows BUN blood concentration. In Figure 15(C), the horizontal axis shows the inoculation group and sex, and the vertical axis shows bone strength. In Figure 15(D), the horizontal axis represents the inoculation group and sex, and the vertical axis represents the AST enzyme concentration. In Figure 15(E), the horizontal axis represents the inoculation group and sex, and the vertical axis represents the creatinine clearance value. In Figure 15(F), the horizontal axis represents the inoculation group and sex, and the vertical axis represents the bone strength. As shown in Figures 15(A) to (C), it was found that the adenomatous polyposis coli model mice inoculated with PRR2-Cg or PRR3-Cg exhibited AST enzyme concentrations similar to those of the adenomatous polyposis coli model mice inoculated with saline (control). Furthermore, the adenomatous polyposis coli model mice inoculated with PRR2-Cg or PRR3-Cg showed a decrease in BUN blood concentration and an increase or a tendency toward an increase in bone strength compared to the adenomatous polyposis coli model mice inoculated with saline. 15(D) to (F), normal model mice inoculated with PRR2-Cg or PRR3-Cg showed AST enzyme concentrations, BUN blood concentrations, and bone strength similar to those of normal mice inoculated with physiological saline. From the above, it was found that the PRR2 peptide and PRR3 peptide of the present disclosure are not accompanied by side effects and improve the decreased kidney function and bone strength caused by adenoma.

[0139] (4) Investigation of Survival Rate Improvement by PRR2 and PRR3 In adenomatous polyposis coli model mice, many deaths are observed due to intestinal obstruction and bleeding associated with adenoma growth. Therefore, we investigated whether inoculation of PRR2-Cg and PRR3-Cg would increase survival rates. Specifically, adenomatous polyposis coli model mice (C57BL / 6J-Apcmin / +) were administered physiological saline, PRR2-Cg, or PRR3-Cg, and an adjuvant using the same test protocol as in Example 1(3). The survival rates of the mice were measured for 30 weeks after administration. Mice were considered dead when they lost 10% of their body weight, and survival rates were measured. These results are shown in Figure 16.

[0140] Figure 16 is a graph showing the survival rate of adenomatous polyposis coli model mice. In Figure 16, the horizontal axis represents the number of weeks since the first inoculation, and the vertical axis represents the survival rate. As shown in Figure 16, the survival rate of female mice inoculated with PRR2-Cg or PRR3-Cg was found to be increased compared to the survival rate of mice inoculated with physiological saline (control). From the above, it was found that the PRR2 peptide and PRR3 peptide of the present disclosure are effective in improving prognosis.

[0141] [Example 4] It was confirmed that the PRR peptide of the present disclosure has an inhibitory effect on muscle loss.

[0142] The novel peptide of the present disclosure was examined for its inhibitory effect on muscle mass loss. A high-salt diet is known to cause muscle mass loss in mice. Specifically, 20 μg of CRM197-PRR2 prepared in Example 2(1) above was mixed with 30 μl of physiological saline and an adjuvant (Freund's Complete Adjuvant, Cat No. F5881, SIGMA) to prepare a PRR2 vaccine for the first administration. After preparation, the PRR2 vaccine for the first administration was inoculated into mice (C57BL / 6J mice, male, 8 weeks old). Two weeks after the administration, 20 μg of CRM197-PRR2-Cg prepared in Example 2(1) was mixed with 30 μl of saline and adjuvant (Freund's Incomplete Adjuvant, Cat. No. F5506, SIGMA) to prepare a PRR2 vaccine for the second administration. After the preparation, the mice were inoculated with the PRR2 vaccine for the second administration. Four weeks after the second administration, high-salt loading was initiated. The high-salt loading was performed by feeding the mice a high-salt diet for two weeks. Because high-salt loading causes a decrease in skeletal muscle mass in mice, the high-salt loading serves as a model for sarcopenia or frailty. Four experimental groups were used: (i) adjuvant-only vaccination + normal diet (n=10), (ii) adjuvant-only vaccination + high-salt diet (n=10), (iii) PRR2 vaccination + normal diet (n=10), and (iv) PRR2 vaccination + high-salt diet (n=10). In the adjuvant-only vaccination group, saline was used instead of the PRR2 vaccine. Serum was collected from the mice 6 weeks after the first vaccination. The antibody titer in the serum was measured by absorbance at 450 nm. These results are shown in Figures 17 and 18.

[0143] Figure 17 is a graph showing the antibody titer of the PRR2 vaccine. In Figure 17, the horizontal axis represents the experimental group, and the vertical axis represents the half-maximum absorbance at 450 nm. As shown in Figure 17, it was found that the antibody titer increased in mice inoculated with PRR2-Cg, regardless of the high salt load. On the other hand, no increase in antibody titer was observed in mice inoculated with adjuvant, regardless of the high salt load.

[0144] FIG. 18 is a graph showing the weight shift due to PRR2-Cg. In FIG. 18, (A) shows the weight shift in the control groups (i) and (ii) administered with only the adjuvant, and (B) shows the weight shift in the groups (iii) and (iv) administered with the PRR2 vaccine. In FIGS. 18(A) and (B), the horizontal axis indicates the number of days after the start of high-salt loading, and the vertical axis indicates the mouse body weight. As shown in FIG. 18, among the mice administered with only the adjuvant, the group on a high-salt diet showed a decrease in body weight compared to the group on a normal diet. On the other hand, among the mice administered with the PRR2 vaccine, the group on a high-salt diet had a body weight similar to that of the group on a normal diet. From the above, it was found that the PRR2 peptide of the present disclosure has the effect of suppressing muscle mass loss.

[0145] Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0146] This application claims priority based on Japanese Patent Application No. 2022-032709, filed March 3, 2022, the disclosure of which is incorporated herein in its entirety.

[0147] <Appendix> Some or all of the above embodiments and examples can be described as in the appendix below, but are not limited to the following. <PRR Peptides> (Appendix 1) A prorenin receptor peptide comprising the polypeptide of (P1), (P2), or (P3) below: (P1) A polypeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 3; (P2) A polypeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 3 in which 1 to 3 amino acids have been deleted, substituted, or added; (P3) A polynucleotide consisting of an amino acid sequence having 70% or more identity to the amino acid sequence of any of SEQ ID NOs: 1 to 3. <Conjugates> (Appendix 2) A conjugate comprising an antigenic peptide and a carrier protein, wherein the antigenic peptide is bound to the carrier protein, and wherein the antigenic peptide comprises the prorenin receptor peptide of Appendix 1. (Appendix 3) The conjugate of Appendix 2, wherein the carrier protein contains a cysteine ​​residue, and the antigenic peptide is bound to the cysteine ​​residue of the carrier protein. (Appendix 4) The conjugate according to appendix 2 or 3, wherein the carrier protein is diphtheria toxin or a mutant thereof, or tetanus toxin or a fragment thereof. (Appendix 5) The carrier protein is CRM １９７(Appendix 6) The conjugate of any of Appendices 2 to 5, which, when administered to a subject, induces the formation of an antibody capable of inhibiting the activity of the Wnt / β-catenin pathway in a Wnt / β-catenin pathway activity assay. (Appendix 7) The conjugate of any of Appendices 2 to 6, for use in treating a tumor. (Appendix 8) The conjugate of Appendices 7, wherein the tumor is prorenin receptor positive. (Appendix 9) The conjugate of Appendices 7 or 8, wherein the tumor is familial adenomatous polyposis. (Appendix 10) The conjugate of Appendices 9, for use in treating familial adenomatous polyposis associated with gastrointestinal benign multiple tumors, Gardner syndrome, Turcot syndrome, and / or desmoid syndrome. (Appendix 11) A conjugate according to any one of Appendices 2 to 6 for use in treating sarcopenia or frailty. <Nucleic Acid> (Appendix 12) A nucleic acid encoding the peptide according to Appendix 1. <Expression Vector> (Appendix 13) An expression vector comprising the nucleic acid according to Appendix 12. (Appendix 14) The expression vector according to Appendix 13, wherein the expression vector is a viral vector. <Pharmaceutical Composition> (Appendix 15) A pharmaceutical composition comprising the prorenin receptor peptide according to Appendix 1, the conjugate according to any one of Appendices 2 to 11, the nucleic acid according to Appendix 12, and / or the expression vector according to Appendix 13 or 14, and a pharmaceutically acceptable carrier. (Appendix 16) The pharmaceutical composition according to Appendix 15, further comprising an immunostimulant. (Appendix 17) The pharmaceutical composition according to Appendix 15 or 16, which, when administered to a subject, induces the formation of an antibody capable of inhibiting the activity of the Wnt / β-catenin pathway in an activity assay of the Wnt / β-catenin pathway. <Use for treating tumors> (Appendix 18) The pharmaceutical composition according to any of Appendixes 15 to 17, for use in treating tumors. (Appendix 19) The pharmaceutical composition according to Appendix 18, wherein the tumor is prorenin receptor positive. (Appendix 20) The pharmaceutical composition according to Appendix 18 or 19, wherein the tumor is familial adenomatous polyposis.(Appendix 21) The pharmaceutical composition according to Appendix 20, for use in the treatment of familial adenomatous polyposis complicated by gastrointestinal benign multiple tumors, Gardner syndrome, Turcot syndrome, and / or desmoid-predominant syndrome. <Use for sarcopenia or frailty> (Appendix 22) The pharmaceutical composition according to any one of Appendices 15 to 17, for use in the treatment of sarcopenia or frailty. <Transformant> (Appendix 23) A transformant comprising the prorenin receptor peptide according to Appendix 1, the nucleic acid according to Appendix 12, or the expression vector according to Appendix 13 or 14. <Production method> (Appendix 24) A method for producing a prorenin receptor peptide, comprising an expression step of expressing the nucleic acid according to Appendix 12 and / or the expression vector according to Appendix 13 or 14. (Appendix 25) The manufacturing method according to Appendix 24, wherein the expression step comprises: a culturing step of culturing the transformant according to Appendix 23; and an isolation step of isolating the prorenin receptor peptide. <Method for treating tumors> (Appendix 26) A method for treating tumors, comprising the step of administering to a subject the prorenin receptor peptide according to Appendix 1, the conjugate according to any of Appendices 2 to 10, the nucleic acid according to Appendix 12, the expression vector according to Appendix 13 or 14, and / or the pharmaceutical composition according to any of Appendices 15 to 21. <Method for treating sarcopenia or frailty> (Appendix 27) A method for treating sarcopenia or frailty, comprising the step of administering to a subject the prorenin receptor peptide according to Appendix 1, the conjugate according to any of Appendices 2 to 6 and 11, the nucleic acid according to Appendix 12, the expression vector according to Appendix 13 or 14, and / or the pharmaceutical composition according to any of Appendices 15 to 17 and 22. <Use> (Appendix 28) A prorenin receptor peptide described in Appendix 1, a conjugate described in any one of Appendices 2 to 10, a nucleic acid described in Appendix 12, an expression vector described in Appendix 13 or 14, and / or a pharmaceutical composition described in any one of Appendices 15 to 21 for use in treating tumors.(Appendix 29) A prorenin receptor peptide according to Appendix 1, a conjugate according to any one of Appendixes 2 to 6 and 11, a nucleic acid according to Appendix 12, an expression vector according to Appendix 13 or 14, and / or a pharmaceutical composition according to any one of Appendixes 15 to 17 and 22 for use in treating sarcopenia or frailty.

[0148] As described above, the present disclosure makes it possible to induce antibodies against the prorenin receptor. Furthermore, the present disclosure makes it possible to indirectly inhibit the activity of the Wnt / β-catenin pathway. Therefore, the prorenin receptor peptide of the present disclosure can treat, for example, diseases resulting from abnormalities in the Wnt / β-catenin pathway. Therefore, the present disclosure is extremely useful, for example, in the pharmaceutical field.

Claims

1. A prorenin receptor peptide consisting of the following polypeptide (P1), (P2), or (P3): (P1) a polypeptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3; (P2) A polypeptide consisting of an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted, or added in any of the amino acid sequences of SEQ ID NOs: 1 to 3; (P3) A polynucleotide consisting of an amino acid sequence having 80% or more identity to any of the amino acid sequences of SEQ ID NOs: 1 to 3.

2. A method for producing a medicament comprising an antigen peptide and a carrier protein, the antigenic peptide is conjugated to the carrier protein; A conjugate, wherein the antigenic peptide comprises the prorenin receptor peptide of claim 1.

3. the carrier protein comprises a cysteine ​​residue; The conjugate of claim 2 , wherein the antigenic peptide is attached to a cysteine ​​residue of the carrier protein.

4. The conjugate of claim 2 , wherein the carrier protein is diphtheria toxin or a mutant thereof, or tetanus toxin or a fragment thereof.

5. The carrier protein is a CRM １９７ The conjugate of claim 2, wherein

6. The conjugate of claim 2, which, when administered to a subject, induces the formation of antibodies capable of inhibiting the activity of the Wnt / β-catenin pathway in a Wnt / β-catenin pathway activity assay.

7. 3. The conjugate of claim 2 for use in treating tumors.

8. The conjugate of claim 7, wherein the tumor is prorenin receptor positive.

9. The conjugate of claim 7, wherein the tumor is familial adenomatous polyposis.

10. 10. The conjugate of claim 9 for use in the treatment of familial adenomatous polyposis associated with gastrointestinal benign multiple tumor prevalence disease, Gardner's syndrome, Turcot's syndrome, and / or desmoid prevalence syndrome.

11. 3. The conjugate of claim 2 for use in the treatment of sarcopenia or frailty.

12. A nucleic acid encoding the prorenin receptor peptide of claim 1.

13. An expression vector comprising the nucleic acid of claim 12.

14. The expression vector of claim 13 , wherein the expression vector is a viral vector.

15. A pharmaceutical composition comprising the prorenin receptor peptide of claim 1, the conjugate of claim 2, the nucleic acid of claim 12, and / or the expression vector of claim 13, and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15, further comprising an immunostimulant.

17. The pharmaceutical composition of claim 15, which, when administered to a subject, induces the formation of an antibody capable of inhibiting the activity of the Wnt / β-catenin pathway in an activity assay of the Wnt / β-catenin pathway.

18. 16. The pharmaceutical composition of claim 15 for use in treating tumors.

19. 19. The pharmaceutical composition of claim 18, wherein the tumor is prorenin receptor positive.

20. 19. The pharmaceutical composition of claim 18, wherein the tumor is familial adenomatous polyposis.

21. 21. The pharmaceutical composition of claim 20 for use in treating familial adenomatous polyposis associated with gastrointestinal benign multiple tumor prevalence, Gardner syndrome, Turcot syndrome, and / or desmoid prevalence syndrome.

22. 16. The pharmaceutical composition of claim 15 for use in treating sarcopenia or frailty.

23. A transformant comprising the prorenin receptor peptide of claim 1, the nucleic acid of claim 12, or the expression vector of claim 13.

24. A method for producing a prorenin receptor peptide, comprising an expression step of expressing the nucleic acid according to claim 12 and / or the expression vector according to claim 13.

25. The expression step comprises: a culturing step of culturing the transformant according to claim 23; isolating the prorenin receptor peptide; The method of claim 24, comprising:

26. A prorenin receptor peptide according to claim 1, a nucleic acid according to claim 12 and / or an expression vector according to claim 13 for use in treating tumors.

27. A prorenin receptor peptide according to claim 1, a nucleic acid according to claim 12, and / or an expression vector according to claim 13 for use in treating sarcopenia or frailty.

28. An antibody against a prorenin receptor peptide consisting of the following polypeptide (P1), (P2), or (P3): (P1) a polypeptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3; (P2) A polypeptide consisting of an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted, or added in any of the amino acid sequences of SEQ ID NOs: 1 to 3; (P3) A polynucleotide consisting of an amino acid sequence having 80% or more identity to any of the amino acid sequences of SEQ ID NOs: 1 to 3.

29. A method for treating a subject comprising administering to the subject a prorenin receptor peptide according to claim 1, a conjugate according to claim 2, a nucleic acid according to claim 12, and / or an expression vector according to claim 13. A method for producing or inducing antibodies against prorenin receptor peptides.

30. A prorenin receptor peptide as described in claim 1, a conjugate as described in claim 2, a nucleic acid as described in claim 12, and / or an expression vector as described in claim 13 for producing or inducing an antibody against a prorenin receptor peptide.

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