Method and reagent for detecting bone metastasis of cancer

Abstract

The subject of the present invention is to provide a method for detecting bone metastasis of cancer (excluding castration-resistant prostate cancer) with simplicity and high precision and a reagent that can be used for the aforementioned method. By measuring the amount of complete growth differentiation factor 15 (GDF15) propeptide, the amount of GDF15 propeptide fragment, or the total amount of complete GDF15 propeptide and GDF15 propeptide fragment in the specimen, bone metastasis of cancer (excluding castration-resistant prostate cancer) is detected. As the above-mentioned method for detecting bone metastasis of cancer, a method for detecting bone metastasis of prostate cancer, kidney cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, bladder cancer, colorectal cancer, melanoma, myeloma or lymphoma of non-castration-resistant prostate cancer is included. Furthermore, the detection reagent for bone metastasis of cancer (excluding castration-resistant prostate cancer) contains an antibody that specifically recognizes the GDF15 propeptide.

Description

Technical Field

[0001] The present invention relates to a method and a detection reagent for detecting bone metastasis of a cancer to be measured (excluding castration-resistant prostate cancer (CRPC)) using the propeptide of the growth and differentiation factor 15 (GDF15) protein and its degradation products in the blood as indicators. Background Art

[0002] As cancer progresses, cancer cells become metastatic, and cancer cells released from cancerous tissue can be transported to various parts of the body through blood vessels. The lungs, liver, lymph nodes rich in growth factors, and bone matrix with high blood flow are considered to be easy targets for cancer metastasis. If cancer metastasizes to the bone, it is called metastatic bone tumor, and skeletal-related events (Skeletal Related Events) such as bone pain, spinal cord compression, pathological fractures, surgery for bone metastases, radiotherapy, and hypercalcemia may occur, which are known to have a negative impact on quality of life and prognosis.

[0003] When bone metastasis is suspected, the first step is to evaluate the risk of fracture and select treatment methods through simple X-rays and computed tomography. 18 F-fluorodeoxyglucose-positron emission tomography (F-FDG-PET), positron emission tomography-computed tomography (PET), and magnetic resonance imaging (MRI) are highly useful in diagnosing bone metastases and are recommended in the 2015 Guidelines for the Diagnosis and Treatment of Bone Metastases. Each method has its advantages and disadvantages, and these imaging diagnoses are used based on the type of cancer and the nature of the bone metastasis to detect bone metastases.

[0004] As in vitro diagnostic markers, the bone metabolism markers listed in Table 1 have been reported to be useful for monitoring bone metastasis treatment. However, bone metabolism markers have not been proven to be direct predictors of treatment efficacy, and their use in routine diagnosis and treatment is not recommended in domestic and international guidelines.

[0005] While diagnostic imaging is highly useful in diagnosing bone metastases, it also faces challenges such as variability between interpreters, concomitant exposure, and the lack of widespread use of equipment (whole-body MRI). Therefore, the discovery of markers that can easily and accurately detect bone metastases and the development of detection methods are desired.

[0006] [Table 1]

[0007] Table 1

[0008]

[0009] GDF15 is a protein that belongs to the same family as macrophage inhibitory cytokine 1 (MIC-1) and nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1), and belongs to the TGF-β family. GDF15 is expressed as preproGDF15, which contains a secretion signal and a propeptide. The secretion signal is cleaved, resulting in proGDF15, which is secreted extracellularly. ProGDF15 is stored in the extracellular matrix via the propeptide. When the propeptide forms a dimer, GDF15 is cleaved by furin-like proteases and released into the blood (Non-Patent Document 1). The molecular weight of full-length proGDF15 is reported to be approximately 40,000, while mature GDF15 is fractionated at a molecular weight of approximately 15,000 (Non-Patent Document 2).

[0010] Increased levels of mature GDF15 in the blood have been confirmed in various cancers, including pancreatic and colorectal cancers (Non-Patent Documents 3-8), and it has also been reported to serve as a prognostic indicator associated with metastasis and malignancy in prostate cancer (Non-Patent Documents 9-12).

[0011] It has been reported that the blood content of GDF15 propeptide increases in the case of CRPC, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, and small cell lung cancer (Patent Documents 1-2). Patent Document 1 states that no high correlation was observed between the complete GDF15 propeptide and the indicators of bone metastasis, EOD and BSI. However, on the other hand, it is also recorded that malignant progression such as bone metastasis was observed when the complete GDF15 propeptide was high, suggesting that it may be a marker for the malignant progression of CRPC. Thus, contradictory records were found in Patent Document 1, and the relationship between GDF15 propeptide and bone metastasis is unclear.

[0012] The GDF15 propeptide (hereinafter also referred to as "GDPP") is a 165-residue polypeptide located at the N-terminus of pre-GDF15. More specifically, the GDF15 propeptide herein refers to a sequence comprising at least a leucine at position 30 to an arginine at position 194, following a signal peptide from an initiator methionine to an alanine at position 29, in the amino acid sequence (SEQ ID NO: 2) based on the human GDF15 cDNA (GeneBank Accession No.: NM_004864) shown in SEQ ID NO: 1, or an amino acid sequence having at least 80% identity to the aforementioned sequence.

[0013] Prior art literature

[0014] Patent Literature

[0015] Patent Document 1: International Publication No. 2017 / 150314 Pamphlet

[0016] Patent Document 2: Japanese Patent Application Publication No. 2019-045486

[0017] Non-patent literature

[0018] Non-patent literature 1: Prostate Cancer Prostatic Dis. 2012; 15(4): 320-328

[0019] Non-patent document 2: Cancer Res. 2005; 65(6): 2330-2336

[0020] Non-patent literature 3: BMC Cancer. 2014; 14: 578-588

[0021] Non-patent literature 4: Biochemical Pharmacology. 2013; 85: 597-606

[0022] Non-patent literature 5: Clin Cancer Res. 2009; 15(21): 6658-6664

[0023] Non-patent literature 6: Clin Cancer Res. 2011; 17: 4825-4833

[0024] Non-patent literature 7: Clin Cancer Res. 2003; 9: 2642-2650

[0025] Non-patent literature 8: Clin Cancer Res. 2006; 12: 442-446

[0026] Non-patent literature 9: Cancer Epidemiol Biomarkers Prev. 2007; 16(3): 532-537

[0027] Non-patent literature 10: Anticancer Research. 2016; 36: 1973-1978

[0028] Non-patent literature 11: Anticancer Research. 2017; 37(3): 1501-1505

[0029] Non-patent literature 12: Technology in Cancer Research & Treatment. 17: 1-7 Summary of the Invention

[0030] Problems to be solved by the invention

[0031] An object of the present invention is to provide a method for detecting bone metastasis of cancer simply and with high accuracy, and a reagent that can be used in the method.

[0032] Solutions for solving problems

[0033] In order to solve the above-mentioned problems, the present inventors conducted in-depth research and obtained the following findings: In cases of bone metastasis of prostate cancer (excluding CRPC), kidney cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, bladder cancer, colorectal cancer, melanoma, myeloma or lymphoma, according to the immunoassay method using antibodies that recognize GDF15 propeptide, the GDF15 propeptide in the blood of bone metastasis cases of these cancers was shown to be increased compared with healthy subjects and non-bone metastasis cases of various cancers. GDF15 propeptide can serve as a marker for detecting bone metastasis of cancer (excluding CRPC), and the present invention was completed.

[0034] That is, the present invention is as follows.

[0035] [1] A method for detecting bone metastasis of cancer, comprising measuring the amount of intact growth differentiation factor 15 (GDF15) propeptide in a subject, wherein the cancer does not include castration-resistant prostate cancer.

[0036] [2] A method for detecting bone metastasis of cancer, comprising measuring the amount of a GDF15 propeptide fragment in a subject, wherein the cancer does not include castration-resistant prostate cancer.

[0037] [3] A method for detecting bone metastasis of cancer, comprising measuring the total amount of intact GDF15 propeptide and GDF15 propeptide fragments in a subject, wherein the cancer does not include castration-resistant prostate cancer.

[0038] [4] The method according to [2] or [3], wherein the GDF15 propeptide fragment comprises the GDF15 propeptide fragment described in (A) and / or (B) below,

[0039] (A) GDF15 propeptide fragment with the following characteristics.

[0040] An amino acid sequence comprising lysine at residue 58 to at least aspartic acid at residue 167 of the GDF15 amino acid sequence shown in SEQ ID NO: 2, or a sequence having 80% or greater identity thereto.

[0041] (B) GDF15 propeptide fragment with the following characteristics.

[0042] An amino acid sequence comprising glutamic acid at residue 74 to at least aspartic acid at residue 167 of the GDF15 amino acid sequence shown in SEQ ID NO: 2, or a sequence having 80% or greater identity thereto.

[0043] [5] The method according to any one of [1] to [4], which detects bone metastasis of prostate cancer, kidney cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, bladder cancer, colorectal cancer, melanoma, myeloma or lymphoma of non-castration-resistant prostate cancer.

[0044] [6] The method according to any one of [1] to [5], wherein the measurement is performed using an antigen-antibody reaction using an antibody that recognizes the GDF15 propeptide.

[0045] [7] The method according to any one of [1] to [5], wherein the measurement is performed using mass spectrometry.

[0046] [8] A reagent for detecting bone metastasis of cancer, comprising an antibody that specifically recognizes the GDF15 propeptide, wherein the cancer does not include castration-resistant prostate cancer.

[0047] Effects of the Invention

[0048] According to the present invention, a method for detecting bone metastasis of cancer (excluding CRPC) with ease and high accuracy, and a reagent that can be used in the method are provided.

[0049] Furthermore, the reagent of the present invention is used to detect the propeptide of GDF15, a member of the TFG-β family. This may reflect the degree of osteoclast activation by cytokines released from cancer cells. This is hypothesized to reflect the therapeutic efficacy of existing bone-modifying agents. Therefore, the reagent of the present invention can also serve as a companion diagnostic for the treatment of bone metastasis in cancer (excluding CRPC). BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Graph showing box plots of the measured values ​​of various markers in the serum of benign (biopsy negative), prostate cancer (no bone metastasis), and prostate cancer metastatic bone tumors.

[0051] Figure 2 The graph shows the results of receiver operating characteristic (ROC) curve analysis of various markers in the serum of prostate cancer (no bone metastasis) and prostate cancer metastatic bone tumors, or benign (biopsy negative) and prostate cancer metastatic bone tumors.

[0052] Figure 3The graph shows the results of ROC curve analysis of various markers using serum and plasma samples collected simultaneously from prostate cancer (without bone metastasis) and prostate cancer metastatic bone tumors.

[0053] Figure 4 The diagram shows box plots of various markers in the serum of renal cancer (without bone metastasis) and renal cancer metastatic bone tumors.

[0054] Figure 5 The graph shows the results of ROC curve analysis of various markers in the serum of renal cancer (without bone metastasis) and renal cancer metastatic bone tumor.

[0055] Figure 6 Graphs showing box plots of various markers in the plasma of healthy individuals, patients with metastatic bone tumors from lung cancer, or patients with metastatic bone tumors from breast cancer.

[0056] Figure 7 The graph shows the results of ROC curve analysis of various markers in the plasma of healthy individuals and those with metastatic bone tumors from lung cancer or breast cancer.

[0057] Figure 8 It is a diagram showing box plots of various markers in the serum of cases without bone metastasis and cases with metastatic bone tumors in CRPC.

[0058] Figure 9 The diagram shows box plots of various markers in the serum of prostate cancer other than CRPC, cases without bone metastasis and cases with metastatic bone tumors.

[0059] Figure 10 The graph shows the results of ROC curve analysis of various markers in the serum of prostate cancer cases without bone metastasis and metastatic bone tumor cases that are not CRPC.

[0060] Figure 11 The diagram shows box plots of various markers classified by specimen for lung cancer (no metastasis), lung cancer (metastasis other than bone), and lung cancer metastatic bone tumor.

[0061] Figure 12 The graph shows the results of ROC curve analysis of various markers for each sample classification in lung cancer cases without bone metastasis and cases with metastatic bone tumor.

[0062] Figure 13 The diagram shows box plots of various markers classified by specimen for breast cancer (no metastasis), breast cancer (metastasis other than bone), and breast cancer metastatic bone tumor.

[0063] Figure 14The graph shows the results of ROC curve analysis of various markers for specimen classification in breast cancer cases without bone metastasis and cases with metastatic bone tumor.

[0064] Figure 15 Graphs showing box plots of various markers in plasma of healthy individuals, thyroid cancer, pancreatic cancer, bladder cancer, colorectal cancer, melanoma, myeloma, and metastatic bone tumors of lymphoma. DETAILED DESCRIPTION

[0065] <1> Method for detecting bone metastasis of cancer (excluding CRPC) of the present invention

[0066] The first embodiment of the present invention is a method for detecting bone metastasis of cancer (excluding CRPC), which includes measuring the amount of GDF15 propeptide in a specimen. This method is based on the characteristic presence of GDF15 propeptide in biological samples such as blood from patients with bone metastasis of cancer (excluding CRPC) compared to specimens from healthy individuals and patients without bone metastasis. The amount of GDF15 propeptide in a specimen is typically measured in vitro.

[0067] As shown in the examples described below, this method can detect bone metastasis of cancer (excluding CRPC) with higher sensitivity and specificity than when measuring a conventionally known bone metabolism marker (ALP).

[0068] It should be noted that the method of the present invention includes methods up to the stage of detecting bone metastasis of cancer (excluding CRPC) and does not include final judgment related to the diagnosis of bone metastasis. Doctors diagnose bone metastasis or formulate treatment guidelines based on the test results of the method of the present invention.

[0069] Typically, the subject (animal subject) for detecting bone metastasis is a human.

[0070] In this embodiment, the GDF15 propeptide to be measured includes: an intact GDF15 propeptide (hereinafter also referred to as "iGDPP") comprising the amino acid sequence from leucine at position 30 to arginine at position 194 of the GDF15 amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having 80% or greater identity with the aforementioned sequence; and GDF15 propeptide fragments. The intact GDF15 propeptide refers to an unprocessed (undegraded) GDF15 propeptide. GDF15 propeptide fragments include: dNT57-GDPP (a peptide comprising the amino acid sequence from residues 58 to 167 of the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having 80% or greater identity with the aforementioned sequence), dNT73-GDPP (a peptide comprising the amino acid sequence from residues 74 to 167 of the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having 80% or greater identity with the aforementioned sequence), and other peptide fragments. Other peptide fragments are not particularly limited as long as they are processed peptide fragments of the GDF15 propeptide, but preferably include a portion of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 80% or greater identity with the aforementioned sequence.

[0071] In the detection method of the present invention, the method for measuring the amount of GDF15 propeptide is not particularly limited, and examples thereof include methods utilizing an antigen-antibody reaction using an antibody that specifically recognizes GDF15 propeptide and methods utilizing mass spectrometry.

[0072] Specific examples of the measurement method utilizing an antigen-antibody reaction using an antibody that specifically recognizes the GDF15 propeptide include the following methods.

[0073] (a) A competitive method using antibodies that specifically recognize a labeled analyte and a target, and utilizing the competitive binding of the labeled analyte and the target contained in the sample to the antibodies.

[0074] (b) A surface plasmon resonance method is used in which a sample is brought into contact with a chip on which an antibody that specifically recognizes the target is immobilized, and a signal dependent on the binding of the antibody to the target is detected.

[0075] (c) Fluorescence polarization immunoassay using a fluorescently labeled antibody that specifically recognizes the analyte and utilizing an increase in the degree of fluorescence polarization caused by the binding of the antibody to the analyte.

[0076] (d) A sandwich method using two antibodies (one of which is a labeled antibody) that specifically recognize the target of measurement and have different epitopes, and forming a three-component complex of the two antibodies and the target of measurement.

[0077] (e) A method in which the analyte in the specimen is concentrated as a pretreatment using an antibody that specifically recognizes the analyte, and then the protein-binding polypeptide is detected using a mass spectrometer or the like.

[0078] Methods (d) and (e) are simple and highly versatile, but method (d) is more preferable for processing a large number of samples because the technologies related to reagents and devices are well established.

[0079] As antibodies that specifically recognize the GDF15 propeptide, antibodies that specifically recognize the N-terminal region of the GDF15 propeptide, for example, antibodies that bind to an antigenic determinant within the region from leucine at residue 30 to arginine at residue 57 of SEQ ID NO: 2, can be preferably used for measuring the amount of iGDPP. Furthermore, antibodies that specifically recognize the C-terminal region of the GDF propeptide, for example, antibodies that bind to an antigenic determinant within the region from glutamic acid at residue 74 to arginine at residue 196 of SEQ ID NO: 2, can be preferably used for measuring the total amount of iGDPP and GDPP fragments (total GDPP, hereinafter also referred to as "tGDPP").

[0080] Antibodies that specifically recognize the GDF15 propeptide can be obtained by immunizing animals with the GDF15 propeptide itself, an oligopeptide consisting of a partial region of the GDF15 propeptide, or a polynucleotide encoding the entire preGDF15 protein or a partial region thereof as an immunogen.

[0081] The animal used for immunization is not particularly limited as long as it has the ability to produce antibodies, and may be mammals commonly used for immunization such as mice, rats, and rabbits, or poultry such as chickens.

[0082] It should be noted that when the GDF15 propeptide itself or an oligopeptide composed of a partial region of the GDF15 propeptide is used as an immunogen, its structure may change during the preparation of the aforementioned protein or oligopeptide. Therefore, the resulting antibody may not have high specificity and binding ability for the desired antigen, and as a result, it may become impossible to accurately quantify the amount of GDF15 propeptide contained in the subject to be tested. On the other hand, when an expression vector containing a polynucleotide encoding the entire protein or partial region of the pre-GDF15 protein is used as an immunogen, the entire protein or partial region of the GDF15 propeptide protein that is consistent with the introduction is expressed in the body of the immunized animal without structural changes. Therefore, antibodies with high specificity and binding ability (i.e., high affinity) for the GDF15 propeptide in the subject to be tested can be obtained, and are therefore preferred.

[0083] The antibody that specifically recognizes the GDF15 propeptide may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.

[0084] The establishment of hybridoma cells that produce antibodies that specifically recognize the GDF15 propeptide can be carried out by appropriately selecting from established technical methods. As an example, B cells are collected from an animal immunized using the aforementioned method, and these B cells are fused with myeloma cells by electrofusion or in the presence of polyethylene glycol. Hybridomas that produce the desired antibody are selected using HAT medium, and the selected hybridoma cells are monoclonalized using the limiting dilution method. This allows the establishment of hybridoma cells that produce monoclonal antibodies that specifically recognize the GDF15 propeptide.

[0085] Selection of an antibody specifically recognizing GDF15 propeptide, for example, a monoclonal antibody specifically recognizing GDF15 propeptide, used in the method of detecting bone metastasis of cancer (excluding CRPC) of the present invention can be based on its affinity for GPI (glycosyl phosphatidyl inositol)-anchored GDF15 propeptide or secreted GDF15 propeptide derived from a host expression system.

[0086] It should be noted that the aforementioned host is not particularly limited, and those skilled in the art can appropriately select from microbial cells such as Escherichia coli and yeast, insect cells, and animal cells commonly used for protein expression. Preferred hosts are mammalian cells that can express a protein having a structure close to that of the native GDF15 propeptide through post-translational modifications such as disulfide bonds or sugar chain additions. Examples of mammalian cells include the long-standing human fetal kidney (HEK) 293T cell line, monkey kidney COS-7 cell line, Chinese hamster ovary (CHO) cell line, and cancer cells isolated from humans.

[0087] Regarding the purification of antibodies used in the method for detecting bone metastasis of cancer (excluding CRPC) of the present invention, it is sufficient to appropriately select from established methods. As an example, after culturing antibody-producing hybridoma cells established using the aforementioned method, the culture supernatant is recovered, and after the antibodies are concentrated by ammonium sulfate precipitation as needed, the antibodies can be purified by affinity chromatography and / or ion exchange chromatography using a carrier immobilized with protein A, protein G, or protein L.

[0088] The labeled antibody used in the antigen-antibody reaction using the sandwich method can be labeled with an enzyme such as peroxidase or alkaline phosphatase, or the antibody purified using the above method. This labeling can also be performed using an established technique.

[0089] The following specifically describes the method for detecting GDF15 propeptide using mass spectrometry in the detection method of the present invention.

[0090] When the specimen is blood, the pretreatment step is preferably to remove proteins such as albumin, immunoglobulins, and transferrin contained in large quantities in the blood using Agilent Human 14, etc., and then further fractionate by ion exchange, gel filtration, or reverse phase HPLC.

[0091] The determination can be performed by tandem mass spectrometry (MS / MS), liquid chromatography / tandem mass spectrometry (LC / MS / MS), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF / MS), surface enhanced laser desorption ionization mass spectrometry (SELDI-MS), etc.

[0092] In the detection method of the present invention, it is preferred that bone metastasis of cancer (excluding CRPC) be determined to have been detected when the amount of GDF15 propeptide obtained by measurement exceeds a reference value (cutoff value) calculated from a control.

[0093] The amount of GDF15 propeptide used in the determination may be either a measured value or a converted concentration value. The converted concentration value refers to a value converted from the measured value based on a calibration curve prepared using GDF15 propeptide as a standard sample. The concentration of the standard sample may be determined by converting the measured value based on a calibration curve of a standard peptide using mass spectrometry.

[0094] For the benchmark value (cutoff value), the non-bone metastasis cancer patient specimens and the bone metastasis cancer patient specimens (excluding CRPC) can be measured separately, and the measurement value showing the best sensitivity and specificity can be appropriately set through receiver operating characteristic (ROC) curve analysis.

[0095] The method for detecting bone metastasis of the present invention can be used for a method for treating metastatic bone tumors. That is, according to the present invention, a method for treating metastatic bone tumors in a patient is provided, the method comprising the following steps:

[0096] (i) a step of identifying a person whose GDF15 propeptide level exceeds a predetermined reference value as a patient; and

[0097] (ii) a step of administering treatment to the patient identified above.

[0098] In the identification in the aforementioned step (i), the amount of the GDF15 propeptide can be measured using an antibody that specifically recognizes the GDF15 propeptide, or by mass spectrometry.

[0099] Examples of the treatment in step (ii) include surgical treatment, drug therapy, and radiotherapy, but are not particularly limited.

[0100] <2> Reagent for detecting bone metastasis of cancer (excluding CRPC) of the present invention

[0101] A second embodiment of the present invention is a reagent for detecting bone metastasis of cancer (excluding CRPC), comprising an antibody that recognizes the GDF15 propeptide. The antibody typically binds to an antigenic determinant within the region from leucine at residue 30 to arginine at residue 196 of proGDF15 as shown in SEQ ID NO: 2.

[0102] In this embodiment, the GDF15 propeptide recognized by the antibody includes the entire GDF15 propeptide and / or GDF15 propeptide fragments. GDF15 propeptide fragments include dNT57-GDPP, dNT73-GDPP, and other peptide fragments. The description of these propeptides and propeptide fragments is the same as that of the first embodiment described above.

[0103] When the reagent of the present invention is used in the sandwich method, it is necessary to include two antibodies having different epitopes as the antibodies.

[0104] The detection reagent of the present invention may further include a detection reagent for a bone metabolism marker containing an antibody that specifically recognizes a bone metabolism marker. Examples of bone metabolism markers include those listed in Table 1.

[0105] The antibody contained in the reagent of the present invention may be the antibody itself, or may be labeled or immobilized on a solid phase.

[0106] The reagent of the present invention will be specifically described below when used in the two-step sandwich method, which is one embodiment of the sandwich method. However, the present invention is not limited thereto.

[0107] First, the reagent of the present invention can be prepared by the methods shown in the following (I) to (III).

[0108] (I) First, antibody 1 of the two antibodies (hereinafter referred to as "antibody 1" and "antibody 2") used in the sandwich method, which specifically recognize the GDF15 propeptide and have different epitopes, is bound to a support capable of B / F (bound / free) separation, such as an immunoplate or magnetic particles. The binding method may be physical binding using a hydrophobic bond or chemical binding using a linking reagent that cross-links the two substances.

[0109] (II) After the aforementioned antibody 1 is bound to the carrier, the carrier surface is blocked with bovine serum albumin, skim milk, a commercially available blocking agent for immunoassays, or the like to prevent nonspecific binding, thereby preparing a primary reagent.

[0110] (III) Labeling the other antibody, i.e., Antibody 2, and preparing a solution containing the resulting labeled antibody as a secondary reagent. Preferred substances for labeling Antibody 2 include enzymes such as peroxidase and alkaline phosphatase, fluorescent substances, chemiluminescent substances, radioactive isotopes, and substances that can be detected by a detection device; or substances that specifically bind to another substance, such as avidin and biotin. Furthermore, the secondary reagent solution is preferably a buffer solution that effectively supports antigen-antibody reactions, such as phosphate buffered saline or Tris-HCl buffer.

[0111] The reagent of the present invention thus prepared can be freeze-dried as needed.

[0112] In the case of the one-step sandwich method, antibody 1 is prepared and bound to a support and blocked in the same manner as in (I) to (II) above, and a buffer containing labeled antibody 2 is further added to the antibody-immobilized support to prepare the reagent.

[0113] Next, when the reagent obtained by the above method is used to detect and measure the GDF15 propeptide by a two-step sandwich method, the following methods (IV) to (VI) can be used.

[0114] (IV) The primary reagent prepared in (II) is brought into contact with the sample at a certain temperature and for a certain period of time. The reaction conditions may be a temperature range of 4°C to 40°C for 5 to 180 minutes.

[0115] (V) Unreacted substances are removed by B / F separation, and then the mixture is contacted with the secondary reagent prepared in (III) at a certain temperature and for a certain period of time to form a sandwich complex. The reaction conditions are preferably a temperature range of 4°C to 40°C for 5 to 180 minutes.

[0116] (VI) Unreacted substances were removed by B / F separation, the labeled substance of the labeled antibody was quantified, and the concentration of human GDF15 propeptide in the specimen was quantified using a calibration curve prepared using a GDF15 propeptide solution of known concentration as a standard.

[0117] The amount of reagent components, such as antibodies, contained in the detection reagent can be appropriately set depending on various conditions, such as the amount of the specimen, the type of specimen, the type of reagent, and the detection method. Specifically, for example, as described below, when measuring the amount of GDF15 propeptide using a sandwich method using 50 μL of 2.5-fold diluted serum or plasma as the specimen, the amount of the antibody bound to the carrier can be 100 ng to 1000 μg, and the amount of the labeled antibody can be 2 ng to 20 μg, relative to the reaction system in which 50 μL of the specimen reacts with the antibody.

[0118] The present invention's reagent for detecting bone metastasis of cancer (excluding CRPC) can be used for both manual and automated immunodiagnostic testing. Automated immunodiagnostic testing is particularly preferred because it allows for detection without being affected by endogenous measurement-inhibiting factors or competing enzymes within the specimen and allows for rapid quantification of the concentrations of GDF15 propeptide and bone metabolism markers in the specimen.

[0119] Another embodiment of the second aspect of the present invention is use of an antibody that specifically recognizes the GDF15 propeptide for the manufacture of a reagent for detecting bone metastasis of cancer (excluding CRPC).

[0120] Another embodiment of the second aspect of the present invention is the use of an antibody that specifically recognizes the GDF15 propeptide for detecting bone metastasis of cancer (excluding CRPC).

[0121] The body to be examined (tested sample) that becomes the object of the method for detecting bone metastasis of cancer (wherein, excluding CRPC) of the present invention and the detection reagent of the present invention is usually collected by cancer patients. More specifically, can list out patient collectors of prostate cancer, kidney cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, bladder cancer, colorectal cancer, melanoma, myeloma or lymphoma of non-castration resistant prostate cancer, but are not limited thereto. Among these, preferably by patient collectors of prostate cancer, kidney cancer, lung cancer, breast cancer of non-castration resistant prostate cancer, further preferably by patient collectors of kidney cancer, lung cancer, breast cancer.

[0122] That is, in the method of the present invention, the bone metastasis to be detected is preferably bone metastasis of prostate cancer such as non-castration-resistant prostate cancer, kidney cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, bladder cancer, colorectal cancer, melanoma, myeloma, or lymphoma.

[0123] Examples of the test subjects of the present invention include whole blood, blood cells, serum, plasma, and other blood components, cell or tissue extracts, urine, cerebrospinal fluid, etc. The use of body fluids such as blood components and urine as test subjects allows for simple and non-invasive detection of bone metastasis of cancer (excluding CRPC), and is therefore preferred. Taking into account the ease of sample collection and the versatility of use in other test items, the use of blood components as test subjects is particularly preferred.

[0124] In the present invention, when the specimen is tested, a sample such as blood collected from a patient may be used directly, or a sample that has undergone appropriate treatment such as dilution or the addition of an anticoagulant may be used. When diluting the specimen, the dilution factor can be selected appropriately, ranging from no dilution to a 100-fold dilution, depending on the type and condition of the specimen. For example, in the case of serum or plasma, 50 μL of a 2.5-fold diluted specimen can be used.

[0125] In addition, the period for collecting the specimen of the present invention can be any period after the diagnosis of cancer, without special limitation. It can be the early or advanced stage of cancer. In addition, it can be after any treatment such as surgical therapy, drug therapy, radiotherapy, etc., or it can be during the observation period after a certain treatment. The specimen collected at any stage can be provided for the method of the present invention.

[0126] Example

[0127] Although examples are shown below in order to specifically explain the present invention, these examples are merely examples of the present invention, and the present invention is not limited to the examples.

[0128] <Example 1> Preparation of GDF15 propeptide assay reagent

[0129] Based on the description in Patent Document 2, two GDPP assay reagents were prepared for use. One assay detected intact GDPP (iGDPP) using a combination of an antibody recognizing the N-terminal region of GDPP (TS-GDPP02) and an antibody recognizing the C-terminal region (TS-GDPP04). The other assay detected both iGDPP and N-terminally deleted GDF15 propeptide fragments (including dNT57-GDPP and dNT73-GDPP) using a combination of antibodies recognizing the C-terminal region (TS-GDPP04 and TS-GDPP08). The value detected using the latter assay was defined as total GDPP (tGDPP).

[0130] <Example 2> Performance of Determining Metastatic Bone Tumors in Prostate Cancer Serum Samples

[0131] Details of the serum sample group (54 cases in total) used in this example are shown in Table 2. All samples were collected according to the same protocol at the Department of Urology, Osaka University, and provided with informed consent and approval from the Clinical Research Review Committee of Osaka University.

[0132] [Table 2]

[0133] Table 2

[0134] Number of cases Benign (biopsy negative) 14 Prostate cancer (no bone metastasis) 22 Prostate cancer metastatic bone tumor 18

[0135] In this study, alkaline phosphatase (ALP), prostate-specific antigen (PSA), iGDPP, tGDPP and GDF15 were measured to compare the performance of judging metastatic bone tumors in prostate cancer. For PSA, iGDPP and tGDPP, the measurement values ​​were calculated using dedicated measurement reagents and a fully automatic enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation), and for GDF15, a commercially available ELISA kit (R&D company). ALP refers to the measurement value at the time of insurance diagnosis and treatment closest to blood sampling. The box diagrams of various measurement values ​​are shown in Figure 1 The results of the Mann-Whitney U test for significant differences are shown in Table 3.

[0136] [Table 3]

[0137] Table 3

[0138]

[0139] Significant differences were observed in the performance of prostate cancer metastatic bone tumors among the four markers, excluding ALP, with iGDPP and tGDPP showing the largest P values. Significant differences were observed in the performance of prostate cancer metastatic bone tumors between benign biopsy-negative cases and iGDPP, tGDPP, and GDF15, with tGDPP showing the largest P value.

[0140] Next, the performance of determining metastatic bone tumors in prostate cancer was analyzed using a receiver operating characteristic (ROC) curve. Figure 2 The AUC (Area Under the Curve, area under the ROC curve) is shown in Table 4.

[0141] [Table 4]

[0142] Table 4

[0143]

[0144] iGDPP and tGDPP were shown to be superior to other markers including ALP in terms of the performance in determining metastatic bone tumors in prostate cancer and in determining benign biopsy-negative cases and metastatic bone tumors in prostate cancer.

[0145] <Example 3> Performance of Detecting Metastatic Bone Tumors in Prostate Cancer Serum / Plasma Samples

[0146] Using the cases of serum and plasma samples collected simultaneously from the prostate cancer serum samples analyzed in Example 2 (9 cases of prostate cancer (no bone metastasis) and 16 cases of prostate cancer metastatic bone tumors), the diagnostic performance of various markers between serum and plasma was compared by Mann-Whitney U test or ROC analysis. The P value and AUC of the U test are shown in Table 5, and the ROC analysis is shown in Figure 3 .

[0147] [Table 5]

[0148] Table 5

[0149]

[0150] No significant differences in the diagnostic performance of GDF15 and PSA were observed between serum and plasma. However, the diagnostic performance of iGDPP was significantly improved in plasma compared to serum. Previous studies have shown that GDPP is susceptible to degradation by proteases and other agents, suggesting that plasma is the preferred method for iGDPP detection.

[0151] <Example 4> Performance of Detecting Metastatic Bone Tumors in Renal Cancer Serum Samples

[0152] The details of the serum sample group (29 cases in total) used in this example are shown in Table 6. All samples were collected according to the same protocol at the Department of Urology, Osaka University, and provided with informed consent and approval from the Clinical Research Review Committee of Osaka University.

[0153] [Table 6]

[0154] Table 6

[0155] Number of cases Kidney cancer (no bone metastasis) 9 Renal cell carcinoma metastatic bone tumor 20

[0156] In this study, ALP, iGDPP, tGDPP and GDF15 were measured, and the performance of judging metastatic bone tumors in renal cancer serum specimens was compared. For iGDPP and tGDPP, the measurement values ​​were calculated using dedicated measurement reagents and a fully automatic enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation), and for GDF15, the measurement values ​​were calculated using a commercially available ELISA kit (R&D company). ALP refers to the measurement value at the time of the insurance diagnosis closest to the blood draw. The box diagrams of various measurement values ​​are shown in Figure 4 , the ROC analysis is shown in Figure 5 The results of the Mann-Whitney U test for significant difference and the AUC based on ROC analysis are shown in Table 7.

[0157] [Table 7]

[0158] Table 7

[0159]

[0160] ALP was shown to have low performance in determining metastatic bone tumors in renal cancer, similar to prostate cancer. On the other hand, iGDPP and GDF15 were shown to have good performance in determining renal cancer serum samples.

[0161] <Example 5> Performance of determining metastatic bone tumors in lung cancer and breast cancer plasma samples

[0162] Details of the plasma sample group (29 cases in total) used in this example are shown in Table 8. Healthy human serum samples were purchased from Bioreclamation IVT, and various cancer serum samples were purchased from PROMEDDX. The product documentation of each company clearly stated that the samples were collected using protocols approved by the ethics committee.

[0163] [Table 8]

[0164] Table 8

[0165] Number of cases Healthy people 16 Lung cancer metastatic bone tumor 8 Breast cancer metastatic bone tumor 5

[0166] In this study, iGDPP, tGDPP, and GDF15 were measured and their performance in judging bone tumors in healthy individuals and those with metastatic lung cancer or breast cancer was compared. For iGDPP and tGDPP, the measurement values ​​were calculated using dedicated measurement reagents and a fully automatic enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation), and for GDF15, a commercially available ELISA kit (manufactured by R&D Co., Ltd.). The box plots of various measurement values ​​are shown in Figure 1. Figure 6 The results of the Mann-Whitney U test for significant differences are shown in Table 9.

[0167] [Table 9]

[0168] Table 9

[0169]

[0170] iGDPP showed the best performance in determining metastatic bone tumors from lung cancer or breast cancer. Relatively good performance was also observed for GDF15, but elevated blood levels in some healthy individuals may reduce its performance.

[0171] Next, the performance of determining lung cancer metastasis bone tumor or breast cancer metastasis bone tumor was analyzed by ROC curve analysis. The analysis results are shown in Figure 7 , and AUC is shown in Table 10.

[0172] [Table 10]

[0173] Table 10

[0174]

[0175] iGDPP showed the best performance in determining lung cancer metastasis to bone tumors or breast cancer metastasis to bone tumors.

[0176] The above examples of the present invention demonstrate that GDPP propeptide has significantly higher performance in determining metastatic bone tumors than ALP, a bone metastasis marker. Furthermore, using plasma as a sample, iGDPP demonstrates superior performance in determining metastatic bone tumors compared to GDF15.

[0177] <Example 6> Performance of Detecting Metastatic Bone Tumors in Serum Samples of Non-castration-resistant Prostate Cancer (CRPC) and Prostate Cancer (Non-CRPC)

[0178] Table 11 shows the details of the classification of the prostate cancer serum specimen group used in Example 2 (40 cases in total) according to CRPC / non-CRPC and the absence / presence of metastatic bone tumors.

[0179] [Table 11]

[0180] Table 11

[0181]

[0182] In this study, iGDPP, tGDPP, GDF15 and alkaline phosphatase (ALP) were measured to compare the performance of judging metastatic bone tumors in the CRPC group and the non-CRPC group. For iGDPP and tGDPP, the measurement values ​​were calculated using dedicated measurement reagents and a fully automatic enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation), and for GDF15, a commercially available ELISA kit (R&D company). ALP refers to the measurement value at the time of the insurance diagnosis closest to blood sampling. The box diagrams of various measurement values ​​of the CRPC group are shown in Figure 8 , the box plots of various measured values ​​of the non-CRPC group are shown in Figure 9 The results of the Mann-Whitney U test for significant differences are shown in Table 12.

[0183] [Table 12]

[0184] Table 12

[0185]

[0186] No significant differences were observed for any marker in the CRPC group, but significant differences were observed for iGDPP and tGDPP in the non-CRPC group.

[0187] Next, the performance of determining metastatic bone tumors in the non-CRPC group was analyzed by receiver operating characteristic (ROC) curve analysis. Figure 10 The AUC (Area Under the Curve, area under the ROC curve) is shown in Table 13.

[0188] [Table 13]

[0189] Table 13

[0190]

[0191] Regarding the judgment performance of metastatic bone tumors in the non-CRPC group, iGDPP and tGDPP showed superiority compared with other markers including ALP.

[0192] Example 7: Performance of Determining Metastatic Bone Tumors in Serum and Plasma Samples of Lung Cancer

[0193] Details of the serum and plasma specimens used in this example (a total of 27 cases) are shown in Table 14 (the plasma specimens for metastatic bone tumors were the same as those in Example 5). The specimens used were purchased from Bioreclamation IVT / PROMEDDX, and the accompanying product materials clearly state that they were collected using a protocol approved by the ethics committee.

[0194] [Table 14]

[0195] Table 14

[0196]

[0197] In this study, iGDPP and tGDPP were measured and their performance in determining metastatic bone tumors in lung cancer was compared. iGDPP and tGDPP were calculated using dedicated assay reagents and the fully automatic enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation). The box plots of various measured values ​​are shown in Figure 11 The results of the Mann-Whitney U test for significant differences are shown in Table 15.

[0198] [Table 15]

[0199] Table 15

[0200]

[0201] No significant differences were observed in serum samples, but significant differences were observed in both iGDPP and tGDPP in plasma samples. Since the evaluation was not based on serum / plasma samples from the same individual, generalizations cannot be made. However, the difference in diagnostic performance between serum and plasma is thought to be due to the effects of protease degradation on the stability of the sample.

[0202] Next, the performance of determining metastatic bone tumors in lung cancer was analyzed by receiver operating characteristic (ROC) curve analysis. Figure 12 The AUC (Area Under the Curve, area under the ROC curve) is shown in Table 16.

[0203] [Table 16]

[0204] Table 16

[0205]

[0206] The results showed that the measurement of iGDPP or tGDPP in plasma samples has excellent diagnostic performance for bone tumors metastasized from lung cancer.

[0207] <Example 8> Performance of Detecting Metastatic Bone Tumors in Serum and Plasma Samples of Breast Cancer

[0208] Details of the serum and plasma specimens used in this example (a total of 28 cases) are shown in Table 17 (the plasma specimens for metastatic bone tumors were the same as those in Example 5). The specimens used were purchased from Bioreclamation IVT / PROMEDDX, and the accompanying product materials clearly state that they were collected using a protocol approved by the ethics committee.

[0209] [Table 17]

[0210] Table 17

[0211]

[0212] In this study, iGDPP and tGDPP were measured and their performance in determining metastatic bone tumors in breast cancer was compared. iGDPP and tGDPP were calculated using dedicated assay reagents and the fully automatic enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation). The box plots of various measured values ​​are shown in Figure 1. Figure 13 The results of the Mann-Whitney U test for significant differences are shown in Table 18.

[0213] [Table 18]

[0214] Table 18

[0215]

[0216] Regarding metastatic bone tumors from breast cancer, although there were some cases where GDPP concentrations were elevated, the number of cases was small and no statistically significant difference was observed.

[0217] Next, the performance of determining metastatic bone tumors in breast cancer was analyzed by receiver operating characteristic (ROC) curve analysis. Figure 14 The AUC (Area Under the Curve, area under the ROC curve) is shown in Table 19.

[0218] [Table 19]

[0219] Table 19

[0220]

[0221] Regarding the determination of metastatic bone tumors in breast cancer, the tGDPP measurement in plasma samples showed slightly better performance.

[0222] <Example 9> Performance of determining metastatic bone tumors in various cancer plasma specimens

[0223] The details of the plasma sample group (34 cases in total) used in this example are shown in Table 20. The samples used were purchased from Bioreclamation IVT / PROMEDDX, and the product documentation clearly stated that they were collected according to a protocol approved by the ethics committee.

[0224] [Table 20]

[0225] Table 20

[0226] Number of cases healthy 5 Thyroid cancer metastatic bone tumor 2 Pancreatic cancer metastatic bone tumor 1 Bladder cancer metastatic bone tumor 1 Colorectal cancer metastatic bone tumor 6 Melanoma metastatic bone tumors 4 Myeloma metastatic bone tumor 10 Lymphoma metastatic bone tumor 5

[0227] In this study, iGDPP and tGDPP were measured and their performance in determining metastatic bone tumors in various cancers was compared. iGDPP and tGDPP were calculated using dedicated measurement reagents and the fully automatic enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation). The box plots of various measurement values ​​are shown in Figure 15 .

[0228] Although some cancer types had a small number of cases and could not be evaluated with satisfactory significance tests, the blood levels of iGDPP and tGDPP were significantly elevated compared to healthy controls, demonstrating their potential for diagnosing metastatic bone tumors in various cancers.

[0229] Industrial applicability

[0230] According to the present invention, a method and reagent capable of detecting bone metastasis of cancer (excluding CRPC) are provided. Thus, blood diagnosis or the like can be used to easily and accurately detect whether bone metastasis occurs in cancer (excluding CRPC), which is difficult to determine using existing bone metabolism markers. As a result, the detection of bone metastasis of cancer (excluding CRPC) is simplified, making it possible to select a treatment method and determine the therapeutic effect, and therefore, it is very useful in the industry. Sequence Listing <110> National University Corporation Osaka University TOSOH corporation <120> Method and reagent for detecting bone metastasis of cancer <130> 2190137-1712 <150> JP2019-211488 <151> 2019-11-22 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 1220 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (33)..(956) <400> 1 agtcccagct cagagccgca acctgcacag cc atg ccc ggg caa gaa ctc agg 53 Met Pro Gly Gln Glu Leu Arg 1 5 acg gtg aat ggc tct cag atg ctc ctg gtg ttg ctg gtg ctc tcg tgg 101 Thr Val Asn Gly Ser Gln Met Leu Leu Val Leu Leu Val Leu Ser Trp 10 15 20 ctg ccg cat ggg ggc gcc ctg tct ctg gcc gag gcg agc cgc gca agt 149 Leu Pro His Gly Gly Ala Leu Ser Leu Ala Glu Ala Ser Arg Ala Ser 25 30 35 ttc ccg gga ccc tca gag ttg cac tcc gaa gac tcc aga ttc cga gag 197 Phe Pro Gly Pro Ser Glu Leu His Ser Glu Asp Ser Arg Phe Arg Glu 40 45 50 55 ttg cgg aaa cgc tac gag gac ctg cta acc agg ctg cgg gcc aac cag 245 Leu Arg Lys Arg Tyr Glu Asp Leu Leu Thr Arg Leu Arg Ala Asn Gln 60 65 70 agc tgg gaa gat tcg aac acc gac ctc gtc ccg gcc cct gca gtc cgg 293 Ser Trp Glu Asp Ser Asn Thr Asp Leu Val Pro Ala Pro Ala Val Arg 75 80 85 ata ctc acg cca gaa gtg cgg ctg gga tcc ggc ggc cac ctg cac ctg 341 Ile Leu Thr Pro Glu Val Arg Leu Gly Ser Gly Gly His Leu His Leu 90 95 100 cgt atc tct cgg gcc gcc ctt ccc gag ggg ctc ccc gag gcc tcc cgc 389 Arg Ile Ser Arg Ala Ala Leu Pro Glu Gly Leu Pro Glu Ala Ser Arg 105 110 115 ctt cac cgg gct ctg ttc cgg ctg tcc ccg acg gcg tca agg tcg tgg 437 Leu His Arg Ala Leu Phe Arg Leu Ser Pro Thr Ala Ser Arg Ser Trp 120 125 130 135 gac gtg aca cga ccg ctg cgg cgt cag ctc agc ctt gca aga ccc cag 485 Asp Val Thr Arg Pro Leu Arg Arg Gln Leu Ser Leu Ala Arg Pro Gln 140 145 150 gcg ccc gcg ctg cac ctg cga ctg tcg ccg ccg ccg tcg cag tcg gac 533 Ala Pro Ala Leu His Leu Arg Leu Ser Pro Pro Pro Ser Gln Ser Asp 155 160 165 caa ctg ctg gca gaa tct tcg tcc gca cgg ccc cag ctg gag ttg cac 581 Gln Leu Leu Ala Glu Ser Ser Ser Ala Arg Pro Gln Leu Glu Leu His 170 175 180 ttg cgg ccg caa gcc gcc agg ggg cgc cgc aga gcg cgt gcg cgc aac 629 Leu Arg Pro Gln Ala Ala Arg Gly Arg Arg Arg Ala Arg Ala Arg Asn 185 190 195 ggg gac cac tgt ccg ctc ggg ccc ggg cgt tgc tgc cgt ctg cac acg 677 Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr 200 205 210 215 gtc cgc gcg tcg ctg gaa gac ctg ggc tgg gcc gat tgg gtg ctg tcg 725 Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser 220 225 230 cca cgg gag gtg caa gtg acc atg tgc atc ggc gcg tgc ccg agc cag 773 Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln 235 240 245 ttc cgg gcg gca aac atg cac gcg cag atc aag acg agc ctg cac cgc 821 Phe Arg - Ala Asn Met His Only Gln Ile Lys Thr Ser Leu His Arg 250 255 260 ctg aag ccc gac acg gtg cca gcg ccc tgc tgc gtg ccc gcc agc tac 869 Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Val Pro Ala Ser Tyr 265 270 275 aat ccc atg gtg ctc att caa aag acc gac acc ggg gtg tcg ctc cag 917 Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln 280 285 290 295 acc tat gat gac ttg tta gcc aaa gac tgc cac tgc ata tgagcagtcc 966 Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile 300 305 tggtccttcc actgtgcacc tgcgcggagg acgcgacctc agttgtcctg ccctgtggaa 1026 tgggctcaag gttcctgaga cacccgattc ctgcccaaac agctgtattt atataagtct 1086 gttatttatt attaatttat tgggtgacc ttcttgggga ctcgggggct ggtctgatgg 1146 aactgtgtat ttatttaaaa ctctggtgat aaaaataaag ctgtctgaac tgttaaaaaa 1206 AAAAAAAAAAAAAAAA 1220 <210> 2 <211> 308 <212> PRT <213> Humans (Homo sapiens) <400> 2 Met Pro Gly Gln Glu Leu Arg Thr Val Asn Gly Ser Gln Met Leu Leu 1 5 10 15 Val Leu Val Leu Will Be Trp Leu Pro His Gly Gly Ala Leu Will Be Leu 20 25 30 Glu Ser Arg Ala Ser Phe Pro Gly Pro Ser Glu Leu His Ser 35 40 45 Glu Asp Ser Arg Phe Arg Glu Leu Arg Lys Arg Tyr Glu Asp Leu Leu 50 55 60 Thr Arg Leu Arg Ala Asn Gln Ser to Glu Asp Ser Asn Thr Asp Leu 65 70 75 80 Val Pro Wing Pro Wing Val Arg Ile Leu Thr Pro Glu Val Arg Leu Gly 85 90 95 Ser Gly Gly His Leu His Leu Arg With Ser Arg Ala Ala Leu Pro Glu 100 105 110 Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe Arg Leu Ser 115 120 125 Pro Thr Ala Ser Arg Ser Trp Asp Val Thr Arg Pro Leu Arg Arg Gln 130 135 140 Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala Leu His Leu Arg Leu Ser 145 150 155 160 Pro Pro Pro Ser Gln Ser Asp Gln Leu Leu Ala Glu Ser Ser Ser Ala 165 170 175 Arg Pro Gln Leu Glu Leu His Leu Arg Pro Gln Ala Ala Arg Gly Arg 180 185 190 Arg Arg Ala Arg Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly 195 200 205 Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly 210 215 220 Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys 225 230 235 240 Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln 245 250 255 Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro 260 265 270 Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr 275 280 285 Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp 290 295 300 Cys His Cys Ile 305

Claims

1. Use of a reagent for determining the amount of intact growth differentiation factor 15 (GDF15) propeptide in a test subject in the manufacture of a reagent for detecting bone metastasis of cancer, The complete growth differentiation factor 15 (GDF15) propeptide comprises an amino acid sequence from leucine at residue 30 to arginine at residue 194 of the GDF15 amino acid sequence shown in SEQ ID NO: 2, The reagent for detecting bone metastasis of cancer is a reagent for detecting bone metastasis of prostate cancer, kidney cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, bladder cancer, colorectal cancer, melanoma, myeloma or lymphoma.

2. Use of a reagent for measuring the amount of a GDF15 propeptide fragment in a sample in the manufacture of a reagent for detecting bone metastasis of cancer, The GDF15 propeptide fragment comprises the GDF15 propeptide fragment described in (A) and / or (B) below, (A) a GDF15 propeptide fragment comprising the amino acid sequence from lysine at residue 58 to at least aspartic acid at residue 167 of the GDF15 amino acid sequence shown in SEQ ID NO: 2, (B) a GDF15 propeptide fragment comprising an amino acid sequence from glutamic acid at residue 74 to at least aspartic acid at residue 167 of the GDF15 amino acid sequence shown in SEQ ID NO: 2, The reagent for detecting bone metastasis of cancer is a reagent for detecting bone metastasis of prostate cancer, kidney cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, bladder cancer, colorectal cancer, melanoma, myeloma or lymphoma.

3. Use of a reagent for measuring the total amount of intact GDF15 propeptide and GDF15 propeptide fragments in a sample in the manufacture of a reagent for detecting bone metastasis of cancer, The complete GDF15 propeptide comprises an amino acid sequence from leucine at residue 30 to arginine at residue 194 of the GDF15 amino acid sequence shown in SEQ ID NO:

2. The GDF15 propeptide fragment comprises the GDF15 propeptide fragment described in (A) and / or (B) below, (A) a GDF15 propeptide fragment comprising the amino acid sequence from lysine at residue 58 to at least aspartic acid at residue 167 of the GDF15 amino acid sequence shown in SEQ ID NO: 2, (B) a GDF15 propeptide fragment comprising an amino acid sequence from glutamic acid at residue 74 to at least aspartic acid at residue 167 of the GDF15 amino acid sequence shown in SEQ ID NO: 2, The reagent for detecting bone metastasis of cancer is a reagent for detecting bone metastasis of prostate cancer, kidney cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, bladder cancer, colorectal cancer, melanoma, myeloma or lymphoma.

4. The use according to any one of claims 1 to 3, wherein The measurement is performed using an antigen-antibody reaction using an antibody that recognizes GDF15 propeptide, including the entire GDF15 propeptide and GDF15 propeptide fragments.

5. The use according to any one of claims 1 to 3, wherein The determination was performed using mass spectrometry.

6. Use of an antibody that specifically recognizes GDF15 propeptide in the manufacture of a reagent for detecting bone metastasis of cancer, The GDF15 propeptide includes a complete GDF15 propeptide and a GDF15 propeptide fragment. The complete GDF15 propeptide comprises an amino acid sequence from leucine at residue 30 to arginine at residue 194 of the GDF15 amino acid sequence shown in SEQ ID NO:

2. The GDF15 propeptide fragment comprises the GDF15 propeptide fragment described in (A) and / or (B) below, (A) a GDF15 propeptide fragment comprising the amino acid sequence from lysine at residue 58 to at least aspartic acid at residue 167 of the GDF15 amino acid sequence shown in SEQ ID NO: 2, (B) a GDF15 propeptide fragment comprising an amino acid sequence from glutamic acid at residue 74 to at least aspartic acid at residue 167 of the GDF15 amino acid sequence shown in SEQ ID NO: 2, The reagent for detecting bone metastasis of cancer is a reagent for detecting bone metastasis of prostate cancer, kidney cancer, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, bladder cancer, colorectal cancer, melanoma, myeloma or lymphoma.

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