Pancreatic cancer detection method and detection reagent

Abstract

The present invention addresses the problem of providing a method for detecting pancreatic cancers and a reagent usable in the method. The method for detecting pancreatic cancers is characterized by measuring the amount of TFPI2 in body fluid sampled from a subject. Furthermore, an antibody that specifically recognizes TFPI2 processing polypeptide and intact TFPI2 is included in the reagent for detecting pancreatic cancers.

Description

Technical Field

[0001] The present invention relates to a method for detecting pancreatic cancer and a detection reagent for measuring Tissue Factor Pathway Inhibitor 2 (TFPI2).

Background Art

[0002] Approximately 450,000 new cases of pancreatic cancer occur worldwide and approximately 40,000 new cases occur in Japan each year (GLOBOCAN 2018). The 5-year relative survival rate of pancreatic cancer is 7.9% for men and 7.5% for women, and the 10-year relative survival rate is 4.6% for men and 4.8% for women, making it the cancer type with the worst prognosis.

[0003] CA19-9, a representative pancreatic cancer marker, has a high positive rate in pancreatic cancer, bile duct cancer, etc., and is useful for evaluating the efficacy of chemotherapy and monitoring recurrence. However, there are problems such as (1) a low positive rate in the early stage, (2) positive results in colorectal cancer, lung cancer, breast cancer, etc., (3) undetectable in patients with Lewis negative blood type, and (4) elevation in cases of biliary obstruction, cholangitis, liver cirrhosis, diabetes, etc. Other pancreatic cancer biomarkers include DUPAN-2, SPan-1, etc. DUPAN-2 is useful for cases of false negative CA19-9 due to Lewis type blood group antigen negativity, but the detection performance of any of these markers for early pancreatic cancer is low, and the development of new biomarkers for pancreatic cancer diagnosis is eagerly desired.

[0004] It has been confirmed that the blood level of apolipoprotein A2 (apoA2) isoform is decreased in patients with early pancreatic cancer compared to healthy subjects, and it has been reported that it may be able to detect stage I and stage II pancreatic cancer with higher accuracy than CA19-9 (Non-Patent Document 1), and large-scale clinical studies are underway for clinical application. Tissue factor pathway inhibitor 2 (TFPI2) is the same protein as placental protein 5 (PP5) and is a placenta-derived serine protease inhibitor containing three Kunitz-type protease inhibitor domains. TFPI2 is specifically produced by clear cell cancer cell lines in ovarian cancer cell lines, and gene expression in ovarian cancer patient tissues was shown to be specifically enhanced only in clear cell cancer patients (Patent Document 1), and a method for detecting ovarian clear cell cancer by measuring TFPI2 in blood was disclosed (Patent Documents 2 and 3, Non-Patent Documents 2 and 3). In addition, a method for detecting renal cancer by measuring the amount of TFPI2 in a specimen has been disclosed (Patent Document 4).

[0005] It has been revealed that TFPI2 is methylated at the TFPI2 DNA promoter in many cancer types, and clinical research as a gene diagnosis target using methylation modification as an index is underway (Non-Patent Documents 4 to 7). In pancreatic cancer, as a result of examining the methylation modification of the TFPI2 DNA promoter in various pancreatic cancer cell lines, it has been shown that the cell lines are completely methylated, partially methylated or non-methylated, and the TFPI2 DNA promoter is also a mixture of methylation and non-methylation in pancreatic cancer tissues (Non-Patent Document 8). However, until now, it has been unclear whether TFPI2 protein in body fluids can be applied to the detection of pancreatic cancer.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0007] [Non-Patent Document 1] Honda, K., et al., Scientific reports, 5, 15921. [Non-Patent Document 2] J. Proteome Res., 2013, 12 (10), pp 4340-4350 [Non-Patent Document 3] PloS one 11.10 (2016): e0165609. [Non-Patent Document 4] Takada, H., et al., Cancer genetics and cytogenetics, 197(1), 16(2010) [Non-Patent Document 5] Hibi, K., et al., Anticancer research, 30(4), 1205(2010) [Non-Patent Document 6] Sun, F. K., et al., Digestive diseases and sciences, 58(4), 1010(2013) [Non-Patent Document 7] Nigro, C. L., et al., Journal of Investigative Dermatology, 133(5), 1278(2013). [Non-Patent Document 8] Sato,N., et al., Oncogene, 24.5,(2005):850. [Summary of the Invention] [Problems to be Solved by the Invention]

[0008] An object of the present invention is to provide a method for detecting pancreatic cancer and a reagent that can be used in the method. [Means for Solving the Problems]

[0009] As a result of intensive studies, the present inventors have found that the blood TFPI2 level is significantly increased in pancreatic cancer patients compared to healthy individuals, and have conceived that pancreatic cancer can be detected with high specificity by TFPI2, thus completing the present invention. That is, the present invention includes the following aspects. [1] A method for detecting pancreatic cancer, comprising measuring the amount of TFPI2 in a collected body fluid. [2] The method according to [1], wherein pancreatic cancer is detected when the measured value of the amount of TFPI2 exceeds a preset reference value. [3] The method according to [1] or [2], wherein the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2. [4] The method according to any one of [1] to [3], wherein the measurement of the amount of TFPI2 is performed by an antigen-antibody reaction using an antibody that binds to an antigenic determinant within the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue of the amino acid sequence of SEQ ID NO: 1. [5] The method according to [4], wherein the antibody is an antibody that recognizes the knotted domain 1 of TFPI2. [6] The method according to any one of [1] to [5], wherein the measurement is performed using a mass spectrometry method. [7] The method according to any one of [1] to [6], further comprising measuring the amount of a pancreatic cancer marker and / or a pancreatic enzyme other than TFPI2 in the body fluid. [8] A reagent for detecting pancreatic cancer, comprising an antibody that binds to an antigenic determinant within the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue of the amino acid sequence shown in SEQ ID NO: 1. [Advantages of the Invention]

[0010] The present invention provides a method for simply and highly accurately detecting pancreatic cancer, and a reagent that can be used in the method. [Brief Description of the Drawings]

[0011]

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Mode for Carrying Out the Invention

[0012] <1> Method for Detecting Pancreatic Cancer of the Present Invention A first aspect of the present invention is a method for detecting pancreatic cancer, which includes measuring the amount of TFPI2 in a sample. This method is based on the fact that the presence of TFPI2 increases in biological samples of pancreatic cancer patients such as blood compared to healthy individuals. The measurement of the amount of TFPI2 in a sample is usually performed in vitro. By this method, pancreatic cancer can be detected with high accuracy as shown in the examples described later. In particular, compared with existing pancreatic cancer markers, the method of the present invention has excellent detection performance for non-invasive pancreatic cancer (stages 1 to 3), and especially has excellent detection performance for early-stage pancreatic cancer (stage 1). Note that the method of the present invention includes up to the stage of detecting pancreatic cancer and does not include the final judgment act regarding the diagnosis of pancreatic cancer. A doctor refers to the detection results and the like by the method of the present invention to diagnose pancreatic cancer or establish a treatment policy.

[0013] TFPI2 measured in the present invention is not particularly limited and may be, for example, intact TFPI2 (hereinafter also referred to as "I-TFPI2"), a TFPI2 processing polypeptide (hereinafter also referred to as "NT-TFPI2"), or both of them. SEQ ID NO: 1 shows the amino acid sequence based on the cDNA of human TFPI2. In SEQ ID NO: 1, from the start methionine to glycine at the 22nd residue is a signal peptide. "Intact TFPI2" refers to a peptide represented by the 23rd residue to the 235th residue of the amino acid sequence of SEQ ID NO: 1.

[0014] In addition, as described in Patent Document 3, "NT-TFPI2" refers to a peptide fragment containing Knotted domain 1 located on the N-terminal side of intact TFPI2. More specifically, NT-TFPI2 is a peptide containing at least the sequence from the 23rd residue aspartic acid to the 131st residue histidine or the 130th residue cysteine of the amino acid sequence of SEQ ID NO: 1, or a peptide containing an amino acid sequence having 80% or more identity with the above sequence. The identity is preferably 90% or more, more preferably 95% or more. Further, this polypeptide may be a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the above sequence. Note that several preferably refers to 2 to 20, more preferably 2 to 10, and even more preferably 2 to 5. In addition, other peptide fragments may be present on both sides of the above sequence, but it is preferably free of the antigenic determinant of the antibody that recognizes Knotted domain 3 of TFPI2.

[0015] The specimen (test sample) in the present invention is a body fluid collected from a subject (test subject), and examples include blood components such as whole blood, blood cells, serum, and plasma, urine, cerebrospinal fluid, ascites, etc. The subject (test subject) usually includes a person suspected of having pancreatic cancer, a patient diagnosed with pancreatic cancer, etc., but is not particularly limited. Using body fluids such as blood components and urine as specimens is preferable because it can be performed simply and non-invasively. Considering the ease of specimen collection and the versatility for other test items, it is particularly preferable to use blood components as specimens. The dilution factor of the specimen may be appropriately selected according to the type and state of the specimen to be used from undiluted to 100-fold dilution.

[0016] In the method for detecting pancreatic cancer of the present invention, a method for detecting TFPI2 and a method for detecting other pancreatic cancer markers and / or pancreatic enzymes may be used in combination. The combination method is not particularly limited. As an example of the combination method of the method for detecting TFPI2 and the method for detecting other pancreatic cancer markers and / or pancreatic enzymes in the method for detecting pancreatic cancer of the present invention, (A) A method for detecting pancreatic cancer by simultaneously (in parallel) or separately performing a method for detecting TFPI2 and a method for detecting other pancreatic cancer markers and / or pancreatic enzymes on a specimen to be measured. (B) A method for detecting pancreatic cancer by first applying a method for detecting TFPI2 to a specimen to be measured, and then, for the specimen determined to be negative as a result, detecting other pancreatic cancer markers and / or pancreatic enzymes. (C) A method for detecting pancreatic cancer by first applying a method for detecting other pancreatic cancer markers and / or pancreatic enzymes to a specimen to be measured, and then, for the specimen determined to be negative as a result, detecting TFPI2. Among these, the methods of (B) or (C) are preferable in that there is no waste of reagents used in detection. The method of (A) is more preferable in that pancreatic cancer can be detected promptly. Here, detecting TFPI2, other pancreatic cancer markers, or pancreatic enzymes includes measuring their amounts.

[0017] The other pancreatic cancer markers to be detected here may be appropriately selected from, for example, conventionally known markers. As an example, cancer antigen 19-9 (CA19-9), human pancreatic cancer-related antigen (SPan-1), human cancer-related carbohydrate antigen (DUPAN-2), cancer antigen 50 (CA50), and carcinoembryonic antigen (CEA) can be mentioned. Also, the pancreatic enzymes to be detected here may be appropriately selected from, for example, conventionally known markers. As an example, amylase (pancreatic amylase), elastase, lipase, and trypsin can be mentioned. Among these, CA19-9, which is the most widely used as a pancreatic cancer marker in particular, is preferable as the pancreatic cancer marker used here in that its clinical usefulness has been established. Also, the tumor markers or pancreatic enzymes detected here may be only one kind, or two kinds or more. From the viewpoint of being able to detect early pancreatic cancer, it is preferable to detect the combination of TFPI2 and CA19-9 or SPan-1. On the other hand, from the viewpoint of being able to detect pancreatic cancer that cannot be detected by other markers, in addition to the combination of TFPI2 and CA19-9 or SPan-1, it is more preferable to detect in combination with DUPAN-2.

[0018] In addition, the collection time of the specimen in the present invention is not particularly limited. For example, it may be at any time from before surgery when a precise examination is performed due to suspicion of pancreatic cancer based on clinical symptoms such as jaundice or imaging diagnosis, to the follow-up observation time after a definite diagnosis of pancreatic cancer by biopsy or postoperative pathological examination. Specimens collected at any stage, such as before and after a definite diagnosis, before and after the start of treatment, etc., can be subjected to the method of the present invention.

[0019] In the detection method of the present invention, when the amount of TFPI2 obtained by measurement exceeds a preset reference value (Cutoff value), it is preferably determined that pancreatic cancer has been detected. Here, the amount of TFPI2 may be any of the intact TFPI2 amount, the NT-TFPI2 amount, or the total of the intact TFPI2 amount and the NT-TFPI2 amount, but the total of the intact TFPI2 amount and the NT-TFPI2 amount is more preferable from the viewpoint of achieving both ease of measurement and sufficient sensitivity and specificity.

[0020] The reference value used for determination may be either a measured value or a converted concentration value. The converted concentration value refers to a value converted from a measured value based on a calibration curve prepared using TFPI2 as a standard sample. The reference value (Cutoff value) for determining pancreatic cancer can be appropriately set to a measured value that exhibits optimal sensitivity and specificity by receiver operating characteristic (ROC) curve analysis by measuring healthy individuals and those with pancreatic cancer respectively. For example, the reference value (Cutoff value) of TFPI2 may be set to 200 pg / mL as shown in the examples described later, but it is not limited thereto.

[0021] Hereinafter, the measurement method of TFPI2 will be described. In the present invention, the amount of NT-TFPI2 or the amount of intact TFPI2 in a specimen may be measured individually, or the values may be summed to obtain a total amount. Alternatively, measurement may be performed using a measurement system capable of measuring the total amount of NT-TFPI2 and intact TFPI2 in a specimen at once. Or, as will be described later, the amount of NT-TFPI2 may be indirectly measured from the total amount obtained by both measurements and the measured amount of intact TFPI2 alone.

[0022] In the method of the present invention, the method for measuring the amount of NT-TFPI2 and / or the amount of intact TFPI2 is not particularly limited. For example, methods using an antigen-antibody reaction using an antibody that recognizes NT-TFPI2 and / or intact TFPI2, and methods using a mass spectrometry method can be exemplified. (a) A competitive method using a labeled measurement target and an antibody that recognizes the measurement target, wherein the labeled measurement target and the measurement target contained in the specimen competitively bind to the antibody. (b) A method using surface plasmon resonance in which a specimen is brought into contact with a chip on which an antibody that recognizes the measurement target is immobilized, and a signal depending on the binding between the antibody and the measurement target is detected. (c) A fluorescence polarization immunoassay using an antibody that recognizes a fluorescence-labeled measurement target, wherein the fluorescence polarization increases when the antibody binds to the measurement target. (d) A sandwich method using two types of antibodies that recognize the measurement target and have different antigenic determinants (one of which is a labeled antibody), and forming a complex of the two antibodies and the measurement target. (e) A method in which the measurement target in a specimen is concentrated with an antibody that recognizes the measurement target as a pretreatment, and then the polypeptide of the binding protein is detected using a mass spectrometer or the like. (d) and (e) are simple and highly versatile, but in processing multiple specimens, the method of (d) is more preferable in that the technology regarding reagents and equipment is well established.

[0023] Specific examples of the method for measuring the amount of NT-TFPI2 and / or the amount of intact TFPI2 using an antigen-antibody reaction include the following. (A) A method for measuring the total amount of NT-TFPI2 and intact TFPI2 using an antibody that recognizes both NT-TFPI2 and intact TFPI2 (NT+I-TFPI2 measurement system). The antibody that recognizes both NT-TFPI2 and intact TFPI2 is preferably an antibody that binds to an antigenic determinant within the region from the 23rd residue aspartic acid to the 131st residue histidine or the 130th residue cysteine of the TFPI2 amino acid sequence represented by SEQ ID NO: 1, and more preferably an antibody having an antigenic determinant in the knotted domain 1 of TFPI2. When the sandwich method described above is used in this method, usually, two different types of the antibodies are used.

[0024] (B) A method for measuring the amount of intact TFPI2 alone using an antibody that does not recognize NT-TFPI2 but recognizes intact TFPI2 (I-TFPI2 measurement system). The antibody that does not recognize NT-TFPI2 but recognizes intact TFPI2 is preferably an antibody having an antigenic determinant in the knotted domain 3 of TFPI2. When the sandwich method described above is used in this method, usually, two different types of the antibodies are used, at least one of which is an antibody that does not recognize NT-TFPI2 but recognizes intact TFPI2, and the other may be an antibody that does not recognize NT-TFPI2 but recognizes intact TFPI2 or an antibody that recognizes both NT-TFPI2 and intact TFPI2.

[0025] (C) A method for calculating the amount of NT-TFPI2 alone by subtracting the amount of intact TFPI2 alone measured by the I-TFPI2 measurement system of (B) from the total amount of NT-TFPI2 and intact TFPI2 measured by the NT+I-TFPI2 measurement system of (A). (D) A method for measuring the amount of NT-TFPI2 alone using an antibody that does not recognize intact TFPI2 but recognizes NT-TFPI2. The antibody that does not recognize intact TFPI2 but recognizes NT-TFPI2 includes, for example, an antibody that specifically recognizes the peptide sequence of the C-terminal portion of NT-TFPI2. When the above-described sandwich method is used, for example, the antibody is used as a solid-phase antibody, and an antibody having an antigenic determinant in Kunitz domain 1 is used as a detection antibody.

[0026] In the method for detecting pancreatic cancer of the present invention, the amount of NT-TFPI2 alone measured by the method (C) or (D) described above may be used as a criterion for determination. However, the total amount of NT-TFPI2 and intact TFPI2 measured by the method (A) is used as a criterion for determination. Sufficient sensitivity and specificity can be obtained, and since antibodies are easily obtained and the measurement is simple in one step, the latter is more preferable.

[0027] Antibodies that recognize NT-TFPI2 and / or intact TFPI2 can be obtained by immunizing animals with, for example, NT-TFPI2 polypeptide or protein, oligopeptides consisting of partial regions of intact TFPI2 polypeptide or TFPI2 protein, polynucleotides encoding intact or partial regions of NT-TFPI2 polypeptide or TFPI2 protein, etc. as immunogens. The protein, oligopeptide, or polypeptide does not reflect the three-dimensional structure of TFPI2 in vivo, or its structure may change during the preparation process. Therefore, the obtained antibody may not have high specificity and binding affinity for the desired TFPI2 in vivo, and even if a measurement system is constructed using this antibody, it may not be possible to accurately quantify the TFPI2 concentration contained in the sample as a result.

[0028] On the one hand, by using an expression vector containing a polynucleotide encoding an intact or partial region of a TFPI2 polypeptide or an intact TFPI2 protein as an immunogen, an intact or partial region of a TFPI2 polypeptide or an intact TFPI2 protein is expressed in the body of an immunized animal to induce an immune response, so that an antibody having high specificity and binding ability (i.e., high affinity) for TFPI2 in a sample can be obtained, which is more preferable.

[0029] The animal used for immunization is not particularly limited as long as it has the ability to produce antibodies, and may be a mammal usually used for immunization such as a mouse, a rat, a rabbit, or a bird such as a chicken.

[0030] Furthermore, TFPI1, which is known as a homologue of TFPI2, also exists in the blood. Therefore, it is desirable to use an antibody that does not cross-react with TFPI1 and specifically recognizes only TFPI2.

[0031] Another aspect of the present invention is the use of a reagent for measuring the amount of TFPI2 in the manufacture of a reagent for detecting pancreatic cancer. Here, the reagent for measuring the amount of TFPI2 is preferably a reagent for measuring the total amount of TFPI2 processing polypeptide and intact TFPI2. The reagent for measuring the amount of TFPI2 is preferably an antibody that binds to an antigenic determinant within the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue of the amino acid sequence of SEQ ID NO: 1, and more preferably an antibody that recognizes the knotted domain 1 of TFPI2.

[0032] Therefore, the present invention can also be said to be the use of an antibody that binds to an antigenic determinant within the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue of the amino acid sequence shown in SEQ ID NO: 1 in the manufacture of a reagent for detecting pancreatic cancer.

[0033] Furthermore, the present invention can also refer to the use of an antibody that binds to an antigenic determinant within the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue of the amino acid sequence shown in SEQ ID NO:1 in the detection of pancreatic cancer.

[0034] The antibody that recognizes TFPI2 may be a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred.

[0035] The establishment of hybridoma cells that produce an antibody that recognizes TFPI2 can be appropriately selected from established techniques. As an example, B cells are collected from an animal immunized by the method described above, the B cells and myeloma cells are fused electrically or in the presence of polyethylene glycol, hybridoma cells that produce the desired antibody are selected using HAT medium, and the selected hybridoma cells are monocloned by the limiting dilution method to establish hybridoma cells that produce a monoclonal antibody that recognizes TFPI2.

[0036] The selection of the monoclonal antibody that recognizes TFPI2 used in the present invention can be performed, for example, based on the affinity for GPI (glycosylphosphatidylinositol) anchor type TFPI2 or secreted TFPI2 derived from the host expression system.

[0037] The 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 that are commonly used for protein expression. However, it is preferable to use mammalian cells as the host, which can express a protein having a structure close to that of natural type TFPI2 by post-translational modifications such as disulfide bond formation or sugar chain addition. Examples of mammalian cells include the conventionally used human embryonic kidney-derived cell (HEK) 293T cell line, monkey kidney cell COS7 line, Chinese hamster ovary (CHO) cell, or cancer cells isolated from humans.

[0038] The purification of the antibody used in the present invention may be appropriately selected from established methods. As an example, after culturing hybridoma cells that produce the antibody established by the method described above, the culture supernatant is recovered, and after concentrating the antibody by ammonium sulfate precipitation if necessary, affinity chromatography and / or ion exchange chromatography using a carrier immobilized with Protein A, Protein G, or Protein L, etc. can be used to purify the antibody.

[0039] In addition, the labeled antibody used when performing the antigen-antibody reaction by the sandwich method described above may be obtained by labeling the antibody purified by the method described above with an enzyme such as peroxidase or alkaline phosphatase, and the labeling may also be performed using a method with a well-established technique.

[0040] In the method of the present invention, the method for measuring the amount of TFPI2 using mass spectrometry will be specifically described below. When the sample is blood, as a pretreatment step, major proteins such as albumin, immunoglobulin, and transferrin, which are abundant in blood, are removed using Agilent Human 14 or the like, and then it is preferably further fractionated by ion exchange, gel filtration, or reverse-phase HPLC or the like. Alternatively, it is also possible to specifically recover only TFPI2 by an immunological technique using an anti-TFPI2 antibody.

[0041] The measurement 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), or the like.

[0042] <2>Reagent for detecting pancreatic cancer of the present invention When the reagent of the present invention is used in the sandwich method described above, it is preferable to include two types of antibodies having different antigenic determinants as the antibody. The antibody contained in the reagent of the present invention may be the antibody itself, may be labeled, or may be immobilized on a solid phase.

[0043] Regarding the case of using the reagent of the present invention in the two-step sandwich method which is one embodiment of the sandwich method described above, it will be specifically described below. However, the present invention is not limited thereto. First, the reagent of the present invention can be produced by the methods shown in the following (I) to (III). (I) First, among two types of antibodies (hereinafter referred to as "antibody 1" and "antibody 2") that recognize TFPI2 and have different antigenic determinants, which are used in the sandwich method, antibody 1 is bound to a carrier capable of separating B / F (Bound / Free) such as an immunoplate or magnetic particles. The binding method may be a physical bond using a hydrophobic bond, or a chemical bond using a linker reagent capable of crosslinking between two substances.

[0044] (II) After binding the antibody 1 to the carrier, in order to avoid non-specific binding, the surface of the carrier is blocked with bovine serum albumin, skim milk, a commercially available blocking agent for immunoassay, a chemically synthesized polymer for suppressing protein adsorption, etc. to obtain a primary reagent.

[0045] (III) The other antibody 2 is labeled, and a solution containing the obtained labeled antibody is prepared as a secondary reagent. As the substance to label antibody 2, substances detectable by a detection device such as enzymes such as peroxidase and alkaline phosphatase, fluorescent substances, chemiluminescent substances, radioisotopes, or substances having a specific binding partner such as avidin to biotin are preferable. Also, as the solution of the secondary reagent, a buffer solution in which an antigen-antibody reaction can be performed well, for example, a phosphate buffer solution, a Tris-HCl buffer solution, etc. are preferable. The reagent of the present invention thus produced may be freeze-dried as necessary. In the case of the one-step sandwich method, a product obtained by binding antibody 1 to a carrier and performing a blocking treatment in the same manner as (I) to (II) described above may be prepared, and a reagent may be prepared by further adding a buffer solution containing labeled antibody 2 to the antibody-immobilized carrier.

[0046] Next, in order to detect and measure TFPI2 by the two-step sandwich method using the reagent obtained by the method described above, it may be performed by the methods shown in (IV) to (VI) below. (IV) The primary reagent prepared in (II) and the specimen are brought into contact with each other for a certain period of time under a certain temperature. The reaction conditions may be such that the reaction is carried out at a temperature in the range of 4°C to 40°C for 5 minutes to 180 minutes. (V) Unreacted substances are removed by B / F separation, and then the secondary reagent prepared in (III) is brought into contact with the separated substances for a certain period of time under a certain temperature to form a sandwich complex. The reaction conditions may be such that the reaction is carried out at a temperature in the range of 4°C to 40°C for 5 minutes to 180 minutes. (VI) Unreacted substances are removed by B / F separation, the labeling substance of the labeled antibody is quantified, and human TFPI2 in the specimen is quantified by a calibration curve prepared using a TFPI2 solution with a known concentration as a standard.

[0047] The amount of reagent components such as antibodies contained in the reagent of the present invention may be appropriately set according to various conditions such as the amount of the specimen, the type of the specimen, the type of the reagent, and the measurement method. Specifically, for example, when measuring the amount of TFPI2 by the sandwich method using 20 μL of serum or plasma as a specimen as described below, the amount of antibody bound to the carrier per reaction system for reacting 20 μL of the specimen with the antibody may be 100 ng to 1000 μg, and the amount of the labeled antibody may be 2 ng to 20 μg.

[0048] The reagent of the present invention can also be used for measurement by a manual method and can also be used for measurement using an automated immunoassay device. In particular, measurement using an automated immunoassay device is preferable because it can perform measurement without being affected by endogenous measurement interfering factors and competing enzymes contained in the specimen, and can quantify TFPI2 in the specimen in a short time.

[0049] The method for detecting pancreatic cancer of the present invention can be applied to the method for treating pancreatic cancer. That is, according to the present invention, there is provided a method for treating pancreatic cancer in a subject, comprising: (i) a step of identifying a subject as one in which the measured value of the amount of TFPI2 exceeds a preset reference value; and (ii) a step of treating the subject identified as one in which the measured value of the amount of TFPI2 exceeds a preset reference value. A method is provided that includes these steps.

[0050] In a preferred embodiment of the method for treating pancreatic cancer, the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2. In addition, in the identification of step (i), the measurement of the amount of TFPI2 is preferably performed by an antigen-antibody reaction using an antibody that binds to an antigenic determinant within the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue of the amino acid sequence of SEQ ID NO: 1. More preferably, the antibody is an antibody that recognizes the knotted domain 1 of TFPI2.

[0051] In addition, in the identification of step (i), the measurement of the amount of TFPI2 may be performed using a mass spectrometry method. Examples of the treatment in step (ii) include surgical resection, drug therapy, radiation therapy, etc. Examples of the drug include tyrosine kinase inhibitors, mToR inhibitors, immune checkpoint inhibitors, etc., but are not particularly limited.

Example

[0052] Examples are shown below to specifically illustrate the present invention, but these examples show an example of the present invention, and the present invention is not limited to the examples.

[0053] <Example 1> Preparation of TFPI2 Measurement Reagent According to the method of Patent Document 3, a TFPI2 measurement reagent was prepared as follows using a TFPI2 antibody obtained by the DNA immunization method. (1) Anti-TFPI2 monoclonal antibody (TS-TF04) was physically adsorbed onto a water-insoluble ferrite-containing carrier at room temperature for 24 hours at a concentration of 100 ng / carrier, and then blocking was performed at 53 °C for 4 hours with 100 mM Tris buffer (pH 8.0) containing 1% BSA to prepare an anti-TFPI2 antibody-immobilized carrier. (2) Anti-TFPI2 monoclonal antibody (TS-TF01) was labeled with an alkaline phosphatase labeling kit (manufactured by Dojindo Laboratories) to prepare an alkaline phosphatase-labeled anti-TFPI2 antibody. (3) Twelve antibody-immobilized carriers prepared in (1) were placed in a magnetically permeable container (volume 1.2 mL), and then 100 μL of a buffer solution (Tris buffer containing 3% BSA, pH 8.0) containing 1 μg / mL of the alkaline phosphatase-labeled antibody prepared in (2) was added, and lyophilization was carried out to prepare a TFPI2 measurement reagent. The prepared TFPI2 measurement reagent was hermetically sealed under nitrogen filling and stored at 4 °C until measurement.

[0054] <Example 2> Evaluation of Clinical Specimens The breakdown of the clinical specimens used in this example is shown in Table 1. Among 241 cases of healthy human sera (102 males and 139 females), 1 case of serum from a patient with pancreatic cystic tumor (benign pancreatic tumor) (male), and 23 cases of serum from pancreatic cancer patients (17 males and 6 females), specimens from cases excluding male healthy individuals were collected at Yokohama City University under the same protocol, and were provided with the consent of informed consent and the approval of the Yokohama City University Ethics Committee. Male healthy individuals used in-house volunteer specimens with the consent of informed consent and the approval of the Tosoh Corporation In-house Ethics Committee.

[0055]

Table 1

[0056] The evaluation apparatus used was a fully automated enzyme immunoassay apparatus AIA-2000 (manufactured by Tosoh Corporation: Manufacturing and Sales Notification Number 13B3X90002000009). The measurement of TFPI2 by the fully automated enzyme immunoassay apparatus AIA-2000 was performed according to the following procedure. (1) Automatically dispense 20 μL of the sample and 100 μL of the diluent containing the surfactant into the container containing the TFPI2 measurement reagent prepared in Example 1. (2) Conduct an antigen-antibody reaction at a constant temperature of 37 °C for 10 minutes. (3) After B / F separation, perform washing 8 times with the buffer containing the surfactant. (4) Add 4-methylumbelliferyl phosphate, and use the generation concentration of 4-methylumbelliferone by alkaline phosphatase per unit time as the measured value (TFPI2 intensity, nmol / (L·s)).

[0057] A calibration curve was created using commercially available TFPI2 recombinant protein (R&D) as a standard, and the TFPI2 concentration in the sample was calculated. The blood CA19-9 values of patients with pancreatic benign tumors and pancreatic cancer were cited as the measured values described in the electronic medical record closest to the blood sampling date.

[0058] The BoxPlot of blood TFPI2 values in healthy individuals, pancreatic cancer patients, and pancreatic benign tumor patients is shown in Figure 1, and the ROC analysis results of healthy individuals and pancreatic cancer patients are shown in Figure 2. TFPI2 showed significantly higher values in pancreatic cancer patients compared to healthy individuals (Mann-Whitney U test, p < 0.0001), and it was revealed that TFPI2 showed low values similar to those of healthy individuals in pancreatic benign tumor patients. The area under the curve (AUC) calculated from the ROC curve of TFPI2 was 0.9230. From these results, it was shown that TFPI2 has good pancreatic cancer detection performance.

[0059] <Example 3> Comparison of TFPI2 in Pancreatic Cancer Onset Sites and Gender Differences Regarding the clinical specimens in Example 2, the BoxPlot of blood TFPI2 values with pancreatic cancer patients classified by pancreatic head, body, and tail as the pancreatic cancer onset sites is shown in Figure 3, and the BoxPlot of blood TFPI2 values with pancreatic cancer patients classified by gender difference is shown in Figure 4. No inter-group differences were observed in blood TFPI2 values according to the pancreatic cancer onset site and gender difference.

[0060] <Example 4> Complementarity of TFPI2 and CA19-9 in Pancreatic Cancer Detection For the clinical specimens of Example 2, the positive rates are shown in Table 2 when the cut-off values are set to 200 pg / mL at which the Youden index (specificity + sensitivity - 1) reaches the maximum value for TFPI2 according to the ROC analysis results of Example 2, and 37 U / mL which is used in the clinical field for CA19-9. Although the positive rate of TFPI2 alone is inferior to that of CA19-9, it has been shown that the positive rate increases by combining CA19-9 and TFPI2. CA19-9 has the problem of false negatives in patients with Lewis blood group antigen negative, but improvement in the positive rate of pancreatic cancer patients is expected by simultaneously (in parallel) measuring TFPI2.

[0061]

Table 2

[0062] <Example 5> Correlation between TFPI2 and CA19-9 in Serum of Pancreatic Cancer Patients For the clinical specimens of Example 2, the correlation and correlation coefficient between TFPI2 and CA19-9 in the serum of pancreatic cancer patients are shown in Fig. 5. There was no significant correlation between TFPI2 and CA19-9 (correlation coefficient = 0.22), suggesting that each is an independent indicator.

[0063] <Example 6> Specimen Evaluation of Early Pancreatic Cancer Cases The breakdown of the clinical specimens used in this example is shown in Table 3. Twelve serum specimens of pancreatic cancer patients with relatively less advanced stages, all of which were collected at Yokohama City University under the same protocol, were provided with the consent of informed consent and the approval of the Yokohama City University Ethics Committee.

[0064]

Table 3

[0065] Using the same evaluation apparatus and evaluation method as in Example 2, the TFPI2 concentration in the specimen was calculated. The blood CA19-9, CEA, SPan-1, and DUPAN-2 values of pancreatic cancer patients were cited from the measured values described in the electronic medical record closest to the blood collection date.

[0066] <Example 7> Positive Rates of Pancreatic Cancer Markers in Early Pancreatic Cancer Detection For the clinical specimens in Example 6, the positive rates of each pancreatic cancer marker in non - progressive pancreatic cancer patients are shown in Table 4. The cut - off value of TFPI2 was set at 200 pg / mL as in Example 4, and the cut - off values of other pancreatic cancer markers were those used in clinical practice. The positive rate of non - progressive pancreatic cancer cases by TFPI2 was 75% (9 / 12 cases), indicating excellent detection performance compared with other pancreatic cancer markers. Also, when limited to early stage (Stage1), the positive rate by TFPI2 was 75% (6 / 8 cases), indicating excellent detection performance compared with other pancreatic cancer markers.

[0067]

Table 4

[0068] <Example 8> Complementarity between TFPI2 and Other Pancreatic Cancer Markers in Early Pancreatic Cancer Detection For the clinical specimens in Example 6, the correlations between TFPI2 and other pancreatic cancer markers in the sera of non - progressive pancreatic cancer patients are shown in Figures 6 - 9 (the black circles (●) in the figures indicate Stage1 cases, and the black triangles (▲) indicate Stage2,3 cases). Also, the correlation coefficients (r) between pancreatic cancer markers are shown in Table 5. No significant correlation was observed between TFPI2 and other pancreatic cancer markers (r = - 0.689 - 0.068), suggesting that they are independent indicators of each other. Generally, no correlation was observed among markers other than TFPI2, but for CA19 - 9 and SPan - 1, the correlation coefficient showed a value close to 1, indicating a high correlation.

[0069]

Table 5

[0070] Table 6 shows the positive rates when other pancreatic cancer markers are detected alone and when TFPI2 and other pancreatic cancer markers are detected in combination. It was confirmed that the positive rate is improved by combining TFPI2 with other pancreatic cancer markers, and particularly high detection performance is shown in the combination of TFPI2 and CA19-9 or SPan-1. In addition, CA19-9 and SPan-1 showed similar behavior, and in one example indicated by an arrow in each correlation diagram of Figs. 6 to 9, only DUPAN-2 was positive. From these facts, it is expected that more cases can be detected by measuring three pancreatic cancer markers, namely TFPI2 and DUPAN-2, plus CA19-9 or SPan-1, in the detection of early pancreatic cancer patients.

[0071]

Table 6

Industrial Applicability

[0072] The present invention provides a method for detecting pancreatic cancer patients by a simple body fluid test with a relatively low patient burden. This is expected to contribute to pancreatic cancer diagnosis that depends on imaging diagnosis due to the absence of effective tumor markers, and is very useful industrially.

Claims

1. A method for detecting pancreatic cancer, comprising measuring the amount of TFPI2 in a collected body fluid.

2. The method according to claim 1, wherein pancreatic cancer is detected when the measured value of the amount of TFPI2 exceeds a preset reference value.

3. The method according to claim 1 or 2, wherein the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.

4. The measurement of the amount of TFPI2 is performed by an antigen-antibody reaction using an antibody that binds to an antigenic determinant within the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue of the amino acid sequence of SEQ ID NO:

1. The method according to any one of claims 1 to 3.

5. The method according to claim 4, wherein the antibody is an antibody that recognizes the knotted domain 1 of TFPI2.

6. The method according to any one of claims 1 to 5, wherein the measurement is performed using a mass spectrometry method.

7. Furthermore, the method according to any one of claims 1 to 6, comprising measuring the amount of a pancreatic cancer marker and / or a pancreatic enzyme other than TFPI2 in the body fluid.

8. A reagent for detecting pancreatic cancer, comprising an antibody that binds to an antigenic determinant within the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue of the amino acid sequence shown in SEQ ID NO: 1.

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