Method and kit for assisting in determination of malignant pancreatic cystic tumor

Abstract

The present invention provides: a simple, non-invasive detection method for assisting in determination of whether a pancreatic cystic tumor is benign or malignant; and a detection kit for use in said method.　Specifically, the present invention is: a method for assisting in the determination of a malignant pancreatic cystic tumor, the method including a step for measuring, in vitro, the amount of APOA2-AT protein and / or APOA2-ATQ protein present in a body fluid sample from a subject who has a pancreatic cystic tumor and assisting in the determination of whether the pancreatic cystic tumor is benign or malignant on the basis of the amount; and a kit for assisting in the determination of a malignant pancreatic cystic tumor, the kit being for measuring the amount of APOA2-AT protein and / or APOA2-ATQ protein, and being usable in the aforementioned method.

Description

Method and kit for assisting in the diagnosis of malignant pancreatic cystic tumors

[0001] The present invention relates to a method for detecting malignant pancreatic cystic tumors, which comprises measuring the amount of an APOA2 protein variant in a body fluid sample from a subject suffering from pancreatic cystic tumors, and a kit for detecting malignant pancreatic cystic tumors, which comprises an anti-APOA2 antibody.

[0002] Pancreatic cystic tumors are lesions that have sac-like structures (cysts) filled with fluid within the tumor tissue, and in recent years, with advances in imaging technology, the frequency of incidental detection in routine medical care has increased. Pancreatic cystic tumors progress slowly and are often asymptomatic, but over time they can become malignant and develop into pancreatic cancer, so regular follow-up examinations are necessary to determine whether pancreatic cystic tumors are benign or malignant.

[0003] Various imaging techniques, such as CT (computed tomography), MRI (magnetic resonance imaging), MRCP (magnetic resonance cholangiopancreatography), and EUS (endoscopic ultrasound), are used to identify pancreatic cystic tumors, but determining whether a pancreatic cystic tumor is benign or malignant remains difficult even with the latest imaging techniques (Non-Patent Documents 1 and 2). EUS is considered effective in detecting malignant lesions, but has problems such as the results being dependent on the skill of the examiner and being relatively invasive. Furthermore, a test method has been attempted in which the tumor marker CEA in cyst fluid is detected by EUS-FNA (Endoscopic Ultrasound-Fine Needle Aspiration), but although high CEA values ​​are considered effective in distinguishing between non-mucinous and mucinous lesions, they are not considered useful for distinguishing between malignant and benign lesions. Similarly, cytology, which detects cancer cells in cyst fluid using EUS-FNA, is said to be a test method that can provide a definitive diagnosis of malignant lesions, but is not recommended in Japan due to the risk of peritoneal dissemination due to leakage of cyst fluid. Another method is cytology using pancreatic juice collected during endoscopic retrograde cholangiopancreatography (ERCP), which is effective in detecting malignant lesions but carries the risk of developing pancreatitis at the time of diagnosis (Non-Patent Documents 1 and 2). Currently, in the medical field, there is a need for the development of new testing techniques that can accurately determine whether pancreatic cystic tumors are benign or malignant using a non-invasive method.

[0004] APOA2 (Apolipoprotein A2, or Apolipoprotein A-II) protein (GenBank Accession No. NP_001634.1) is a member of the apolipoprotein family that constitutes serum lipoproteins. More than 10 types of apolipoproteins have been identified, and their main functions include stabilizing lipoprotein structure, activating enzymes involved in lipoprotein metabolism, and acting as ligands for lipoprotein receptors present on cell surfaces. APOA2 protein is synthesized in liver tissue as a 100-amino acid precursor containing a signal peptide, and the mature form present in blood consists of 77 amino acids. Mature APOA2 protein has a glutamine residue (Q) at its amino terminus (N-terminus), a threonine residue (T) at the 76th position from the N-terminus, and a glutamine residue (Q) at the 77th position (C-terminus). It is one of the apolipoproteins that constitute high-density lipoproteins (HDL). It has also been reported that APOA2 protein includes variants with different masses, such as APOA2-ATQ protein, which is a full-length APOA2 protein; APOA2-AT protein, which is an APOA2 protein lacking a glutamine residue (Q) at the C-terminus; and APOA2-A protein, which is an APOA2 protein lacking a glutamine-threonine residue (QT) at the C-terminus (Non-Patent Document 3).

[0005] According to analysis based on the three-dimensional structure data (PDB ID: 1L6L) of the APOA2 protein registered in the Protein Data Bank (PDB; http: / / www.rcsb.org / pdb / home.do), the APOA2 protein is believed to form a dimer via a disulfide bond (SS bond) via a cysteine ​​residue on the N-terminus. Therefore, it is known that the APOA2 protein exists in blood as dimers of various molecular weights resulting from combinations of the three variants. Known dimers include a dimer consisting of the full-length APOA2-ATQ protein (APOA2-ATQ / ATQ protein dimer), a dimer of APOA2-ATQ protein and APOA2-AT protein (APOA2-ATQ / AT protein dimer), a dimer of APOA2-AT protein (APOA2-AT / AT protein dimer), a dimer of APOA2-AT protein and APOA2-A protein (APOA2-AT / A protein dimer), and a dimer consisting of APOA2-A protein (APOA2-A / A protein dimer).

[0006] It is known that the various APOA2 protein dimers exhibit quantitative variations in the blood of pancreatic cancer patients compared to healthy individuals. In particular, the APOA2-ATQ / AT protein dimer was identified as a protein with a molecular weight of 17,253 ± 9 (m / z) by mass spectrometry, and its abundance was significantly reduced in pancreatic cancer patients compared to healthy individuals. It has also been revealed that the use of this protein dimer as a pancreatic cancer marker enables the detection of pancreatic cancer with high accuracy, with an AUC (area under the curve) value of 0.85 or higher (Patent Document 1, Non-Patent Document 3). It is also known that by measuring the amounts of the two variants constituting the various APOA2 protein dimers by ELISA (Enzyme-Linked Immunosorbent Assay) and combining the measurement results, various pancreatic tumors, including pancreatic cancer and pancreatic cystic tumors, can be detected with accuracy equal to or higher than that achieved by mass spectrometry. (Patent Document 2, Non-Patent Document 4)

[0007] International Publication No. WO 2006 / 098087 International Publication No. WO 2015 / 050107

[0008] International Pancreas Society Working Group (Representative: Masao Tanaka), IPMN International Clinical Practice Guidelines 2017 Edition International Pancreas Society Working Group (Representative: Masao Tanaka), IPMN / MCN International Clinical Practice Guidelines 2012 Edition Honda K. et al., 2012, PLoS One, Vol. 7, e46908 Honda K. et al., 2015, Sci Rep. Vol. 5, 15921

[0009] Until now, the testing methods used to identify malignant pancreatic cystic tumors have had problems such as being highly invasive and difficult to master the testing techniques.

[0010] An object of the present invention is to provide a simple and non-invasive method for assisting in the diagnosis of malignant pancreatic cystic tumors, which is targeted at the blood of patients with pancreatic cystic tumors, and a kit for assisting in the diagnosis of malignant pancreatic cystic tumors.

[0011] In order to solve the above problems, the present inventors first investigated whether two APOA2 protein variants (APOA2-AT protein and APOA2-ATQ protein), which have conventionally been considered effective in aiding in the diagnosis of pancreatic cancer and pancreatic cystic tumors, are effective in distinguishing between benign and malignant pancreatic cystic tumors. As a result, they found that the blood concentrations of APOA2-AT protein are lower and the blood concentrations of APOA2-ATQ protein are higher in malignant pancreatic cystic tumors compared to benign pancreatic cystic tumors. Based on this finding, they discovered that it is possible to determine whether a pancreatic cystic tumor is benign or malignant based on the presence of the above two APOA2 protein variants in a body fluid sample, and have thus completed the present invention.

[0012] The present invention includes the following: (1) A method for assisting in the determination of malignant pancreatic cystic tumor in a subject suffering from pancreatic cystic tumor, the method comprising the step of measuring in vitro the amount of APOA2-AT protein, which is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 30 at its carboxyl terminus, or / and APOA2-ATQ protein, which is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 31 at its carboxyl terminus, present in a body fluid sample obtained from the subject, and assisting in the determination of whether the subject's pancreatic cystic tumor is benign or malignant based on the amount. (2) The method according to (1), wherein the pancreatic cystic tumor is intraductal papillary mucinous neoplasm (IPMN) or mucinous cystic neoplasm (MCN). (3) The method according to (1) or (2), which aids in the determination of a pancreatic cystic tumor as malignant when the amount of APOA2-AT protein present in a body fluid sample obtained from the subject is less than the amount of APOA2-AT protein present in a body fluid sample obtained from a subject with a known benign pancreatic cystic tumor. (4) The method according to (3), which aids in the determination of a pancreatic cystic tumor as malignant when the amount of APOA2-AT protein present in a body fluid sample obtained from the subject is less than a cutoff value set based on the amount of APOA2-AT protein present in a body fluid sample obtained from a subject with a known benign pancreatic cystic tumor or the amount of APOA2-AT protein present in a sample obtained from a subject with a known malignant pancreatic cystic tumor. (5) The method according to any one of (1) to (4), which aids in the determination of a pancreatic cystic tumor as malignant when the amount of APOA2-ATQ protein present in a body fluid sample obtained from the subject is greater than the amount of APOA2-ATQ protein present in a body fluid sample from a subject with a known benign pancreatic cystic tumor. (6) The method according to (5), which aids in the determination of a pancreatic cystic tumor as malignant when the amount of APOA2-ATQ protein present in a body fluid sample obtained from the subject is greater than a cutoff value set based on the amount of APOA2-ATQ protein present in a body fluid sample obtained from a subject with a known benign pancreatic cystic tumor or the amount of APOA2-ATQ protein present in a sample obtained from a subject with a known malignant pancreatic cystic tumor.(7) The method according to any one of (1) to (6), wherein the body fluid sample is blood, plasma, or serum. (8) The method according to any one of (1) to (7), wherein the subject's prevalence of pancreatic cystic tumor has been determined in advance by an imaging test and / or the following steps (A) to (C): (A) a first step of measuring the amount of APOA2-ATQ protein in the sample using an anti-APOA2-ATQ terminal antibody that specifically binds to the carboxyl-terminal region of the APOA2-ATQ protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and an anti-APOA2-ATQ non-terminal antibody that binds to an amino acid sequence other than the carboxyl-terminal region; (B) a second step of measuring the amount of APOA2-AT protein in the sample using an anti-APOA2-ATQ terminal antibody that specifically binds to the carboxyl-terminal region of the APOA2-AT protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and an anti-APOA2-AT non-terminal antibody that binds to an amino acid sequence other than the carboxyl-terminal region; (C) a third step for determining the presence or absence of pancreatic cystic tumor, in which a discriminant value obtained by inputting the measured value of the APOA2-ATQ protein amount obtained in the first step and the measured value of the APOA2-AT protein amount obtained in the second step into a predetermined discriminant, and comparing the obtained discriminant value with the discriminant value of a known healthy subject. (9) A kit for assisting in the diagnosis of malignant pancreatic cystic tumor, for measuring in vitro the amount of APOA2-AT protein, which is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 30 at its carboxyl terminus, or / and APOA2-ATQ protein, which is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 31 at its carboxyl terminus, contained in a body fluid sample obtained from a subject. (10) The kit according to (9), comprising an anti-APOA2-AT terminus antibody or a binding fragment thereof, and / or an anti-APOA2-ATQ terminus antibody or a binding fragment thereof.(11) The kit according to (10), wherein the anti-APOA2-ATQ terminal antibody or binding fragment thereof specifically binds to the carboxyl-terminal region of the APOA2-ATQ protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and the heavy chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, or 10, 11, and 12, respectively, and the light chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, or 13, 14, and 15, respectively. (12) The kit according to (10), comprising an anti-APOA2-AT terminus monoclonal antibody or a binding fragment thereof, which specifically binds to the carboxyl-terminal region of the APOA2-AT protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and in which the heavy chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 33, 34, and 35, 39, 40, and 41, or 45, 46, and 47, respectively, and the light chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 36, 37, and 38, 42, 43, and 44, or 48, 49, and 50, respectively. (13) The kit according to any one of (10) to (12), further comprising an anti-APOA2 non-terminal monoclonal antibody that specifically binds to an amino acid sequence other than the carboxyl-terminal region of the APOA2-AT protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or the APOA2-ATQ protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and that has heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, or 22, 23, and 24, respectively, and light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 19, 20, and 21, or 25, 26, and 27, respectively.(14) An anti-APOA2-AT terminal monoclonal antibody or a binding fragment thereof that specifically binds to the carboxyl-terminal region of the APOA2-AT protein consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the heavy chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 33, 34, and 35, 39, 40, and 41, or 45, 46, and 47, respectively, and the light chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 36, 37, and 38, 42, 43, and 44, or 48, 49, and 50, respectively. This specification includes the disclosures of Japanese Patent Application No. 2021-049931, from which the present application claims priority.

[0013] According to the present invention, simply by measuring the concentration of an APOA2 protein variant contained in a body fluid sample obtained from a subject suffering from a pancreatic cystic tumor, it is possible to assist in a simple and non-invasive method of determining whether the subject has a benign or malignant pancreatic cystic tumor.

[0014] This figure shows the measurement of the binding activity of anti-APOA2-AT terminus monoclonal antibodies clones 4C6-1, 5D9-3, and 6B4-2 to various variants of the APOA2 protein. This figure shows the measurement of APOA2-AT protein by sandwich ELISA using three types of anti-APOA2-AT terminus monoclonal antibodies (4C6-1, 5D9-3, or 6B4-2) or an anti-APOA2-AT terminus polyclonal antibody and an anti-APOA2 non-terminus monoclonal antibody. This figure shows the measured concentrations of the pancreatic tumor marker CA19-9 in the plasma of 15 cases of benign intraductal papillary mucinous neoplasm (IPMN) and 7 cases of malignant IPMN, and plots the results. This figure shows the APOA2-AT protein concentrations measured in the plasma of 15 benign IPMN cases and 7 malignant IPMN cases by sandwich ELISA using a polyclonal antibody that specifically recognizes the amino acid sequence in the carboxyl (C)-terminal region of the APOA2-AT protein (anti-APOA2-AT terminal polyclonal antibody) and an antibody that specifically recognizes an amino acid sequence other than the C-terminal region (anti-APOA2 protein non-terminal antibody). This figure shows the APOA2-ATQ protein concentrations measured in the plasma of 15 benign IPMN cases and 7 malignant IPMN cases by sandwich ELISA using a monoclonal antibody that specifically recognizes the amino acid sequence in the C-terminal region of the APOA2-AT protein (anti-APOA2-ATQ terminal monoclonal antibody) and an antibody that specifically recognizes an amino acid sequence other than the C-terminal region (anti-APOA2 protein non-terminal antibody). This figure shows ROC curves showing the discrimination performance between benign IPMN15 cases and malignant IPMN7 cases using the pancreatic tumor marker CA19-9. (A) This figure shows ROC curves showing the discrimination performance between benign IPMN15 cases and malignant IPMN7 cases using the APOA2-AT protein. (B) This figure shows ROC curves showing the discrimination performance between benign IPMN15 cases and malignant IPMN7 cases using the APOA2-ATQ protein.

[0015] The measurement subject of the present invention is a subject suffering from a pancreatic cystic tumor. As used herein, "pancreatic cystic tumor" refers to any tumor formed in the pancreas and exhibiting the form of a cyst containing fluid, mucus, etc. Specifically, this includes intraductal papillary mucinous neoplasm (IPMN), pancreatic mucinous cystic neoplasm (MCN), and pancreatic serous cystic neoplasm (SCN).

[0016] As used herein, the term "malignant pancreatic cystic tumor" refers to a tumor derived from a pancreatic cystic tumor and has a poor prognosis, and can be determined by the presence of high-grade dysplasia (HGD), invasive carcinoma, or mural nodules. Furthermore, as used herein, "malignant pancreatic cystic tumor" also includes conventional invasive pancreatic ductal carcinoma (concomitant cancer) that forms in the pancreas of a patient with a pancreatic cystic tumor and that forms at a site different from the pancreatic cystic tumor.

[0017] Although the effectiveness of a blood tumor marker (CA19-9) in predicting such malignant pancreatic cystic tumors has been reported, a definitive diagnosis requires detailed examinations such as pancreatic juice cytology under ERCP or needle biopsy under endoscopic ultrasound (EUS-FNA) (Non-Patent Document 1). If a diagnosis is made that a patient has a "malignant pancreatic cystic tumor," treatment by resection is considered depending on the location and extent of the lesion.

[0018] As used herein, a "benign pancreatic cystic tumor" refers to a tumor that is low-grade dysplasia, localized to the site where it developed, and does not invade surrounding tissues, or if there is infiltration, the extent of the infiltration is localized.

[0019] 1. Method for Assisting in the Diagnosis of Malignant Pancreatic Cystic Tumor Using Anti-APOA2 Protein Terminal Antibody The method of the present invention aims to detect malignant pancreatic cystic tumor in a subject. The method of the present invention assists in the diagnosis of malignant pancreatic cystic tumor in a subject suffering from pancreatic cystic tumor, and comprises the steps of measuring in vitro the amounts of APOA2-AT protein, which is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 30 at its carboxyl terminus, and / or APOA2-ATQ protein, which is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 31 at its carboxyl terminus, present in a body fluid sample obtained from the subject, and assisting in the diagnosis of whether the subject's pancreatic cystic tumor is benign or malignant based on the amounts measured. While there are no particular limitations on the means for measuring APOA2-AT protein and / or APOA2-ATQ protein, in a first aspect of the method of the present invention, an anti-APOA2 protein terminal antibody is used in the measurement of APOA2-AT protein and / or APOA2-ATQ protein. Hereinafter, the anti-APOA2 protein terminal antibody that can be used in the first aspect of the present invention will be described.

[0020] 1-1. Anti-APOA2 Protein Terminal Antibody As used herein, the term "APOA2 protein" refers to APOA2 proteins of various biological species, preferably human-derived APOA2 protein (GenBank Accession No. NP_001634.1), and specifically includes variants of the human-derived wild-type APOA2 protein shown in SEQ ID NO: 1, 2, or 3, as well as naturally occurring mutants and fragments thereof. Furthermore, as used herein, APOA2 protein refers to a protein in which the 6 amino acids at its carboxyl terminal have the amino acid sequence shown in any of SEQ ID NOs: 30 to 32.

[0021] As used herein, the term "variant" refers to a different molecular form of the APOA2 protein that may be present in human or animal plasma, serum, or other body fluid samples. For example, this refers to an APOA2 protein having a different structure in the C-terminal region of the APOA2 protein, or a natural mutant thereof. Specifically, for example, an APOA2-AT protein in which the amino acid sequence of the C-terminal region shown in SEQ ID NO: 1 ends with AT, an APOA2-ATQ protein in which the amino acid sequence of the C-terminal region shown in SEQ ID NO: 2 ends with ATQ, or an APOA2-A protein in which the amino acid sequence of the C-terminal region shown in SEQ ID NO: 3 ends with A corresponds to a variant of the APOA2 protein.

[0022] As used herein, the term "carboxyl-terminal region (often referred to herein as "C-terminal region")" refers to a region consisting of 6 to 25 amino acids, preferably 8 to 20 amino acids or 10 to 17 amino acids, including the carboxyl-terminal (C-terminal) amino acid and several contiguous amino acids surrounding it in the amino acid sequence. In the present invention, the C-terminal region specifically refers to a region containing any of the amino acid sequences of SEQ ID NOs: 30 to 32.

[0023] As used herein, the term "natural variant" refers to a variant occurring in nature, such as a variant in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3, or a variant having 90% or more, 92% or more, or 94% or more, preferably 95% or more, more preferably 97% or more, and even more preferably 98% or more, or 99% or more identity with the amino acid sequence. "Identity" refers to the percentage (%) of identical amino acid residues in one amino acid sequence relative to the total number of amino acid residues (including gaps) in the other amino acid sequence when the two amino acid sequences are aligned with or without introducing gaps to maximize identity. "Several" refers to an integer between 2 and 10, for example, between 2 and 7, between 2 and 5, between 2 and 4, or between 2 and 3. Specific examples of natural variants include variants based on polymorphisms such as SNPs (single nucleotide polymorphisms) and splice variants. The substitution is preferably a conservative amino acid substitution. This is because conservative amino acid substitutions can have substantially the same structure or properties as the APOA2 protein having the amino acid sequence. Conservative amino acids refer to the relationship between amino acids classified in the same amino acid group. Known amino acid groups include nonpolar amino acids (glycine, alanine, phenylalanine, valine, leucine, isoleucine, methionine, proline, tryptophan), polar amino acids (amino acids other than nonpolar amino acids), charged amino acids (acidic amino acids (aspartic acid, glutamic acid) and basic amino acids (arginine, histidine, lysine)), uncharged amino acids (amino acids other than charged amino acids), aromatic amino acids (phenylalanine, tryptophan, tyrosine), branched-chain amino acids (leucine, isoleucine, valine), and aliphatic amino acids (glycine, alanine, leucine, isoleucine, valine).

[0024] The "fragments thereof" refer to various APOA2 protein variants and fragments of APOA2 protein variants and mutants thereof that include the C-terminal regions of these naturally occurring mutants. Specifically, this includes protease digests of various APOA2 protein variants and mutants thereof.

[0025] The method of the present invention can use an anti-APOA2 protein-end antibody, including an anti-APOA2-AT-end antibody or / and an anti-APOA2-AT-Q-end antibody. In addition to the anti-APOA2 protein-end antibody, the method of the present invention can also use an anti-APOA2 protein-non-end antibody that does not bind to the C-terminus of APOA2.

[0026] An "anti-APOA2-AT terminal antibody" refers to an antibody or a binding fragment thereof that can specifically recognize and bind to an epitope present in the C-terminal region of the APOA2-AT protein, specifically a region comprising the amino acid sequence set forth in SEQ ID NO: 30. "Specifically recognize and bind" means that the antibody has no or very weak cross-reactivity with other APOA2 protein variants, and therefore cannot recognize or bind to other APOA2 protein variants, or barely does so. Specifically, the antibody specifically binds to the C-terminal region of the APOA2-AT protein but does not bind to the C-terminal region of the APOA2-ATQ protein or the C-terminal region of the APOA2-A protein. On the other hand, an "anti-APOA2-ATQ terminal antibody" refers to an antibody or a fragment thereof that can specifically recognize and bind to an epitope present in the C-terminal region of the APOA2-ATQ protein, specifically a region comprising the amino acid sequence set forth in SEQ ID NO: 31. Specifically, it refers to an antibody that specifically binds to the C-terminal region of the APOA2-ATQ protein but does not bind to the C-terminal region of the APOA2-AT protein or the C-terminal region of the APOA2-A protein. Such an antibody may be either a polyclonal antibody or a monoclonal antibody or a binding fragment thereof. Monoclonal antibodies are preferred for enabling mass production and achieving a uniform effect.

[0027] An "anti-APOA2 protein non-terminal antibody" refers to an anti-APOA2 antibody that recognizes and binds to an epitope present in a region other than the C-terminal region in the full-length amino acid sequence of an APOA2 protein variant. In other words, the epitopes recognized by anti-APOA2 protein non-terminal antibodies and anti-APOA2 protein terminal antibodies are completely different. Anti-APOA2 protein non-terminal antibodies are also referred to as "non-terminal" antibodies, which is a convenient name for anti-APOA2 protein terminal antibodies. Therefore, there are no particular limitations on the epitope present outside the C-terminal region, and antibodies that recognize and bind to epitopes present at the N-terminus may also be included. In this specification, an antibody simply referred to as an "anti-APOA2 antibody" refers to an antibody that encompasses both anti-APOA2 protein terminal antibodies and anti-APOA2 protein non-terminal antibodies.

[0028] The methods of the present invention can use anti-APOA2 protein non-terminal antibodies. Preferably, anti-APOA2 protein non-terminal antibodies have approximately the same binding activity to an APOA2 protein having a specific C-terminal sequence (e.g., APOA2-AT) as to an APOA2 protein having a different C-terminal sequence (e.g., APOA2-ATQ), and do not inhibit the binding of the anti-APOA2 protein non-terminal antibody to the C-terminal region. Specifically, for example, an "anti-APOA2-AT non-terminal antibody" that binds to an amino acid sequence other than the C-terminal region of the APOA2-AT protein shown in SEQ ID NO: 1 and an "anti-APOA2-ATQ non-terminal antibody" that binds to an amino acid sequence other than the C-terminal region of the APOA2-ATQ protein shown in SEQ ID NO: 2 have equivalent binding activity to the APOA2 protein, and neither antibody inhibits the binding of the anti-APOA2-AT terminal antibody or the anti-APOA2-ATQ terminal antibody to the C-terminal region of the APOA2 protein. The anti-APOA2 protein non-terminal antibody may be either a polyclonal antibody or a monoclonal antibody or a binding fragment thereof. Monoclonal antibodies are preferred for the purpose of enabling mass production and achieving uniform effects.

[0029] As used herein, the term "monoclonal antibody" refers to an antibody that comprises a single immunoglobulin or its framework region (hereinafter referred to as "FR") and complementarity determining region (hereinafter referred to as "CDR"), and that is capable of specifically recognizing and binding to a specific antigen (epitope).

[0030] A typical immunoglobulin molecule is composed of a tetramer of two pairs of polypeptide chains, called heavy and light chains, interconnected by disulfide bonds. The heavy chain consists of an N-terminal heavy chain variable region (H chain V region: hereinafter referred to as "VH") and a C-terminal heavy chain constant region (H chain C region: hereinafter referred to as "CH"). The light chain consists of an N-terminal light chain variable region (L chain V region: hereinafter referred to as "VL") and a C-terminal light chain constant region (L chain C region: hereinafter referred to as "CL"). Of these, VH and VL are particularly important in that they contribute to the binding specificity of the antibody. Both VH and VL consist of approximately 110 amino acid residues and contain three CDRs (CDR1, CDR2, CDR3) that are directly involved in antigen-binding specificity and four FRs (FR1, FR2, FR3, FR4) that function as the framework structure of the variable region. CDRs are known to form a complementary three-dimensional structure with antigen molecules and determine antibody specificity (E.A. Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Vol. 1, eds. 5, NIH publication). While the amino acid sequences of constant regions are nearly constant among antibodies within a species, the amino acid sequences of CDRs are highly variable among antibodies, and therefore are also called hypervariable regions. In the variable region, the CDRs and FRs are arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In an immunoglobulin molecule, VL and VH dimerize relative to each other to form an antigen-binding site. Immunoglobulins are known to be classified into the following classes: IgG, IgM, IgA, IgE, and IgD, and the antibodies used in the method of the present invention may be of any class, preferably IgG.

[0031] The anti-APOA2-ATQ terminal monoclonal antibody of the present invention specifically binds to the C-terminal region of the APOA2-ATQ protein shown in SEQ ID NO: 2, but does not exhibit binding affinity to the APOA2-AT protein shown in SEQ ID NO: 1 or the APOA2-A protein shown in SEQ ID NO: 3. Specific examples of such antibodies include anti-APOA2-ATQ terminal monoclonal antibody clones designated 7F2 and 6G2. The 7F2 clone has a heavy chain in which CDR1 consists of the sequence shown in SEQ ID NO: 4, CDR2 consists of the sequence shown in SEQ ID NO: 5, and CDR3 consists of the sequence shown in SEQ ID NO: 6, and a light chain in which CDR1 consists of the sequence shown in SEQ ID NO: 7, CDR2 consists of the sequence shown in SEQ ID NO: 8, and CDR3 consists of the sequence shown in SEQ ID NO: 9. In addition, the 6G2 clone has a heavy chain in which CDR1 has the sequence shown in SEQ ID NO: 10, CDR2 has the sequence shown in SEQ ID NO: 11, and CDR3 has the sequence shown in SEQ ID NO: 12, and a light chain in which CDR1 has the sequence shown in SEQ ID NO: 13, CDR2 has the sequence shown in SEQ ID NO: 14, and CDR3 has the sequence shown in SEQ ID NO: 15.

[0032] The anti-APOA2-AT terminus monoclonal antibody of the present invention specifically binds to the C-terminal region of the APOA2-AT protein shown in SEQ ID NO: 1, but does not exhibit binding affinity to the APOA2-ATQ protein shown in SEQ ID NO: 2 or the APOA2-A protein shown in SEQ ID NO: 3. Specific examples of such antibodies include anti-APOA2-AT terminus monoclonal antibody clones designated 4C6-1, 5D9-3, and 6B4-2. The 4C6-1 clone has a heavy chain in which CDR1 consists of the sequence shown in SEQ ID NO: 33, CDR2 consists of the sequence shown in SEQ ID NO: 34, and CDR3 consists of the sequence shown in SEQ ID NO: 35, and a light chain in which CDR1 consists of the sequence shown in SEQ ID NO: 36, CDR2 consists of the sequence shown in SEQ ID NO: 37, and CDR3 consists of the sequence shown in SEQ ID NO: 38. In addition, the 5D9-3 clone has a heavy chain in which CDR1 has the sequence shown in SEQ ID NO: 39, CDR2 has the sequence shown in SEQ ID NO: 40, and CDR3 has the sequence shown in SEQ ID NO: 41, and a light chain in which CDR1 has the sequence shown in SEQ ID NO: 42, CDR2 has the sequence shown in SEQ ID NO: 43, and CDR3 has the sequence shown in SEQ ID NO: 44. In addition, the 6B4-2 clone has a heavy chain in which CDR1 has the sequence shown in SEQ ID NO: 45, CDR2 has the sequence shown in SEQ ID NO: 46, and CDR3 has the sequence shown in SEQ ID NO: 47, and a light chain in which CDR1 has the sequence shown in SEQ ID NO: 48, CDR2 has the sequence shown in SEQ ID NO: 49, and CDR3 has the sequence shown in SEQ ID NO: 50.

[0033] The anti-APOA2 protein non-terminal antibodies of the present invention preferably have equivalent binding activity to variants of the APOA2 protein shown in any of SEQ ID NOS: 1 to 3. Specific examples include anti-APOA2 antibody clones designated MAB1 and MAB2. In the MAB1 clone, the heavy chain CDR1 consists of the amino acid sequence shown in SEQ ID NOS: 16, 17, and 18, respectively, and the light chain CDR1 consists of the amino acid sequence shown in SEQ ID NOS: 19, 20, and 21, respectively. Furthermore, in the MAB2 clone, CDR1 in the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 22, CDR2 consists of the amino acid sequence shown in SEQ ID NO: 23, and CDR3 consists of the amino acid sequence shown in SEQ ID NO: 24, and in the light chain, CDR1 consists of the amino acid sequence shown in SEQ ID NO: 25, CDR2 consists of the amino acid sequence shown in SEQ ID NO: 26, and CDR3 consists of the amino acid sequence shown in SEQ ID NO: 27. Furthermore, as the anti-APOA2 protein non-terminal antibody, the anti-APOA2-ATQ non-terminal antibody or the anti-APOA2-AT non-terminal antibody can be used.

[0034] The "binding fragment thereof" in the above-mentioned "polyclonal antibody and monoclonal antibody or binding fragment thereof" refers to a partial fragment of a polyclonal antibody or monoclonal antibody having an epitope in the C-terminal region or a region other than the C-terminus of the APOA2 protein, and refers to a polypeptide chain or complex thereof having substantially the same antigen-specific binding activity as the antibody. For example, this refers to an antibody portion containing at least one antigen-binding site, i.e., a polypeptide chain or complex thereof having at least one pair of VL and VH. Specific examples include the numerous well-characterized antibody fragments generated by cleaving immunoglobulins with various peptidases. More specific examples include Fab, F(ab') ２Fab is a fragment generated by cleaving an IgG molecule with papain at the N-terminal side of the disulfide bond in the hinge region, and is composed of a polypeptide consisting of CH1 adjacent to VH, of the three domains (CH1, CH2, CH3) that make up VH and CH, and a light chain. F(ab') ２ Fab' is a dimer of Fab' generated by pepsin cleavage of an IgG molecule at the C-terminal side of the disulfide bond in the hinge region. Fab' has a slightly longer heavy chain than Fab due to the length of the hinge region, but has substantially the same structure as Fab (Fundamental Immunology, Paul ed., 3rd ed., 1993). Fab' is a dimer of F(ab'). ２ can be obtained by reducing the antibody fragment under mild conditions to cleave the disulfide bonds in the hinge region. All of these antibody fragments contain an antigen-binding site and have the ability to specifically bind to an antigen (i.e., a specific variant of the APOA2 protein in the present invention).

[0035] The antibody-binding fragments used in the methods of the present invention may be synthesized chemically or by recombinant DNA techniques. Examples include antibody fragments newly synthesized using recombinant DNA techniques. Specifically, but not limited to, monomeric polypeptide molecules in which one or more VLs and one or more VHs of the antibodies used in the methods of the present invention are artificially linked via a linker peptide or the like having an appropriate length and sequence, or multimeric polypeptides thereof. Examples of such polypeptides include synthetic antibodies such as single-chain Fvs (scFvs: single chain fragment of variable region) (see Pierce catalog and Handbook, 1994-1995, Pierce Chemical Co., Rockford, IL), diabodies, triabodies, and tetrabodies. In immunoglobulin molecules, VL and VH are usually located on separate polypeptide chains (L and H chains). Single-chain Fvs are synthetic antibody fragments in which these variable regions are linked by a sufficiently long, flexible linker, resulting in a single polypeptide chain containing VL and VH. In single-chain Fvs, both variable regions can self-assemble to form a functional antigen-binding site. Single-chain Fvs can be obtained by incorporating recombinant DNA encoding them into a phage genome using known techniques and expressing it. Diabodies are molecules based on the dimeric structure of single-chain Fvs (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA, 90:6444-6448). For example, when the linker length is shorter than about 12 amino acid residues, the two variable regions in a single-chain Fv cannot self-assemble, but by forming a diabody, i.e., by allowing two single-chain Fvs to interact, the VL of one Fv chain can assemble with the VH of the other Fv chain to form two functional antigen-binding sites (Marvin et al., 2005, Acta Pharmacol. Sin., 26:649-658).Furthermore, by adding a cysteine ​​residue to the C-terminus of the single-chain Fv, disulfide bonding between the two Fv chains becomes possible, allowing the formation of stable diabodies (Alafsen et al., 2004, Prot. Engr. Des. Sel., 17:21-27). Thus, diabodies are bivalent antibody fragments, but each antigen-binding site does not necessarily bind to the same epitope; they may have bispecificity, recognizing and specifically binding to different epitopes. Triabodies and tetrabodies, like diabodies, have trimer and tetramer structures based on the single-chain Fv structure. These are trivalent and tetravalent antibody fragments, respectively, and may be multispecific antibodies. Furthermore, antibody fragments used in the methods of the present invention include antibody fragments identified using a phage display library (see, for example, McCafferty et al., 1990, Nature, Vol. 348, 522-554) and that have antigen-binding ability. See also, for example, Kuby, J., Immunology, 3rd Ed., 1998, W. H. Freeman & Co., New York.

[0036] In the present invention, anti-APOA2 antibodies can be modified. Modifications herein include both functional modifications (e.g., glycosylation) necessary for the anti-APOA2-ATQ-terminus antibody to have specific binding activity to a particular variant of the APOA2 protein, as well as labels necessary for detecting the antibody used in the methods of the present invention. Examples of antibody labels include fluorescent dyes (FITC, rhodamine, Texas Red, Cy3, Cy5), fluorescent proteins (e.g., PE, APC, GFP), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, glucose oxidase), biotin, or (strept)avidin. Furthermore, the glycosylation of the antibody may be modified to adjust the affinity of the antibody for the antigen. Such modifications can be achieved, for example, by changing one or more glycosylation sites within the antibody sequence. More specifically, for example, one or more amino acid substitutions can be introduced into the amino acid sequence constituting one or more glycosylation sites in the FR to remove the glycosylation sites, thereby eliminating the glycosylation at the sites. Such deglycosylation is effective in increasing the affinity of an antibody for an antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861).

[0037] 1-2. Preparation of Immunogen In producing anti-APOA2 protein terminal antibodies in the present invention, an APOA2 protein variant is prepared as an immunogen (antigen). APOA2 protein variants that can be used as immunogens in the present invention include, for example, APOA2 proteins having the amino acid sequences set forth in SEQ ID NOS: 1 to 3, mutants thereof, or polypeptide fragments thereof, as well as fusion polypeptides of these proteins with other peptides (e.g., signal peptides, labeling peptides, etc.). APOA2 protein variants as immunogens can be synthesized, for example, using the amino acid sequence information of SEQ ID NOS: 1 to 3 by methods known in the art, such as solid-phase peptide synthesis. For example, they can be prepared by the following method.

[0038] The APOA2 protein variant may be any of a natural APOA2 protein, a recombinant APOA2 protein, and a synthetic APOA2 protein that has been chemically synthesized in whole or in part. For example, the APOA2 protein variant used as an antigen to obtain an antibody that binds to the C-terminus of the APOA2 protein (anti-APOA2 protein terminal antibody) may be any APOA2 protein variant derived from the natural APOA2 protein, recombinant APOA2 protein, or synthetic APOA2 protein, as long as it contains an amino acid sequence of 6 or more consecutive amino acids in the C-terminal region.

[0039] Native APOA2 protein can be recovered from samples, including body fluid samples such as blood (including plasma and serum), or from the culture supernatant of cultured cells using known protein separation and purification techniques, such as gel filtration, ion exchange chromatography, and affinity chromatography.

[0040] The recombinant APOA2 protein can be expressed in microorganisms, insect cells, or animal cells into which DNA encoding the protein has been introduced, and then recovered from the cells using known protein separation and purification techniques.

[0041] The synthetic APOA2 protein can be synthesized by a method known in the art, such as solid-phase peptide synthesis, using, for example, published amino acid sequence information for the APOA2 protein. This synthetic APOA2 protein may be linked to a carrier protein such as KLH (keyhole limpet hemocyanin), OVA (ovalbumin), or BSA (bovine serum albumin).

[0042] When preparing anti-APOA2 protein terminal antibodies, even when a fragment of an APOA2 protein variant is used as the immunogen, any of a natural APOA2 protein fragment, a recombinant APOA2 protein fragment, and a synthetic APOA2 protein fragment may be used. For example, an APOA2 protein fragment that can be used as an antigen is an oligopeptide or polypeptide containing 6 or more, preferably 10 or more, preferably 18 or more, and more preferably 30 or more consecutive amino acid residues, including the C-terminus, of the amino acid sequence shown in any of SEQ ID NOs: 1 to 3. For example, a peptide containing the amino acid sequence shown in SEQ ID NO: 28 or 29 can be used.

[0043] When a fragment of a native APOA2 protein is used as an immunogen, for example, to prepare an anti-APOA2 protein terminal antibody, the purified APOA2 protein is first treated with an appropriate protease such as trypsin, and the peaks are separated and fractionated on a reverse-phase column. The amino acid sequences of peptides contained in each peak are then determined using a mass spectrometer. If the peptide contains a partial sequence consisting of 6 or more consecutive amino acids from the C-terminal region of the APOA2 protein shown in any of SEQ ID NOS: 1 to 3, that peptide can be used as an immunogen.

[0044] When using an amino acid partial sequence of a recombinant APOA2 protein as an immunogen, for example, to prepare an anti-APOA2 protein terminal antibody, a DNA sequence encoding a peptide (C-terminal fragment) consisting of a partial sequence of 6 or more consecutive amino acids containing the C-terminal amino acid residue of the APOA2 protein shown in any of SEQ ID NOS: 1 to 3 is first inserted into an expression vector. Next, the expression vector is introduced into various cells to express the encoded C-terminal fragment. Finally, the C-terminal fragment is extracted from the cells after expression using standard methods. The obtained C-terminal fragment can then be used as an immunogen.

[0045] Furthermore, in the present invention, when anti-APOA2 protein non-terminal antibodies are produced, the basic preparation method may be the same as the method for producing the anti-APOA2 protein terminal antibodies. However, the region of the APOA2 protein that can be used as an immunogen is a region different from the region used as an immunogen in the production of anti-APOA2 protein terminal antibodies. In other words, all or part of a region other than the C-terminal region of the APOA2 protein may be used as the immunogen. As in the case of producing anti-APOA2 protein terminal antibodies, when producing anti-APOA2 protein non-terminal antibodies, an oligopeptide or polypeptide containing amino acid residues from a region other than the C-terminal region of the APOA2 protein can be used as an antigen.

[0046] (Preparation of Recombinant APOA2 Protein) The preparation of the recombinant APOA2 protein (variant of the recombinant APOA2 protein) shown in any one of SEQ ID NOS: 1 to 3 will be described in detail below.

[0047] (a) Preparation of Polynucleotides Encoding Recombinant APOA2 Protein Variants Vectors used to express various APOA2 protein variants can be phages or plasmids capable of autonomously replicating in a host microorganism. Examples of plasmids include E. coli-derived plasmids (pET30a, pGEX6p, pUC118, pUC119, pUC18, pUC19, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.), and yeast-derived plasmids (YEp13, YEp24, YCp50, etc.). Examples of plasmids include λ phages (λgt11, λZAP, etc.). Furthermore, animal virus vectors such as vaccinia virus and insect virus vectors such as baculovirus can also be used.

[0048] To insert a polynucleotide encoding a variant of the APOA2 protein into the above-mentioned vector, for example, the purified polynucleotide is cleaved with an appropriate restriction enzyme and then ligated using DNA ligase or the like into a vector that has been cleaved with the corresponding appropriate restriction enzyme.

[0049] (b) Introduction of APOA2 protein variant expression vector into host The obtained APOA2 protein variant expression vector is introduced into a host capable of expressing the expression vector to obtain a transformant capable of expressing an APOA2 protein variant (variant-expressing transformant). The host used is not particularly limited, as long as it is a host suitable for the vector used and capable of expressing an APOA2 protein variant. For example, bacteria (e.g., Escherichia coli, Bacillus subtilis, etc.), yeast, insect cells, animal cells (COS cells, CHO cells (Journal of Immunology, 1998, Vol. 160, 3393-3402)), etc. are preferably used. The method for introducing the vector into bacteria is not particularly limited, as long as it is a known method for introducing the vector into bacteria. Examples of such techniques include heat shock, calcium ion, and electroporation. These techniques are all well known in the art and are described in various publications. See, for example, Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. For transformation of animal cells, lipofectin method (PNAS, 1989, Vol. 86, 6077; PNAS, 1987, Vol. 84, 7413), electroporation method, calcium phosphate method (Virology, 1973, Vol. 52, 456-467), DEAE-Dextran method, etc. are preferably used.

[0050] When a bacterium is used as a host, it is preferable that the APOA2 protein variant expression vector is capable of autonomous replication in the bacterium and is composed of a promoter sequence, a ribosome binding sequence, a DNA sequence encoding the APOA2 protein variant, and a transcription termination sequence. It may also contain a gene encoding a regulatory factor that controls the promoter. Any promoter may be used as long as it can function in a host such as Escherichia coli.

[0051] When eukaryotic cells such as yeast, animal cells, or insect cells are used as hosts, transformants expressing APOA2 protein variants can be obtained similarly according to techniques known in the art. APOA2 protein variant expression vectors used in eukaryotic cells may, in addition to a promoter sequence and a DNA sequence encoding an APOA2 protein variant, optionally be linked with cis elements such as enhancers, splicing signals (donor site, acceptor site, branch point, etc.), poly(A) addition signals, selection marker sequences, ribosome binding sequences (SD sequences), etc.

[0052] (c) Culturing the Variant-Expressing Transformant and Expression of Recombinant APOA2 Protein Variant Next, the variant-expressing transformant prepared above is cultured. The method for culturing the variant-expressing transformant in a medium is carried out according to a conventional method used for culturing a host. For example, when a bacterium is used as the host, the medium is not particularly limited as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that the bacterium can utilize and allows the bacterium to grow and proliferate. Either a natural medium or a synthetic medium can be used. A more specific example is LB medium, but this is not limiting. Furthermore, to selectively culture the variant-expressing transformant, antibiotics such as ampicillin or tetracycline may be added to the medium as needed. Culturing is usually carried out under aerobic conditions, such as aeration and agitation culture, at 37°C for 6 to 24 hours. During the culture period, it is preferable to maintain the pH at approximately neutral. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. When the variant-expressing transformant is an animal cell such as a CHO cell, 1 × 10 cells are cultured in a DMEM medium manufactured by Life Technologies (now Thermo Fisher Scientific). ５ Host cells are inoculated so that the total number of cells / mL is 100, and the cells are cultured in a 5% CO2 incubator at 37°C. During culture, antibiotics such as ampicillin or tetracycline may be added to the medium as needed.

[0053] When the APOA2 protein variant expression vector is an inducible protein expression vector containing a protein expression control system (e.g., when the host is a bacterium, this corresponds to a repressor gene and operator, etc.), the variant-expressing transformant must be subjected to a predetermined treatment to induce expression of the APOA2 protein variant. The expression induction method varies depending on the protein expression control system contained in the vector, so an induction treatment appropriate for that system may be performed. For example, the most commonly used protein expression control system in inducible protein expression vectors that use a bacterium as a host is a system consisting of a lac repressor gene and a lac operator. This system can be induced to express the desired APOA2 protein variant in a transformant harboring an APOA2 protein expression vector containing this system by adding an appropriate amount of IPTG (e.g., a final concentration of 1 mM) to the medium.

[0054] (d) Extraction and / or Recovery of Recombinant APOA2 Protein Variant After culturing, if an APOA2 protein variant is produced intracellularly or intracellularly, the target protein can be extracted by recovering and disrupting the bacterial cells or cells. Alternatively, if the APOA2 protein variant is secreted extracellularly or extracellularly, the culture medium can be used as is, or the bacterial cells or cells can be removed by centrifugation or the like, and the supernatant can be used. Subsequently, the APOA2 protein variant can be isolated and purified from the culture by using a common protein purification method, such as ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combinations. Whether or not an APOA2 protein variant has been obtained can be confirmed by SDS-polyacrylamide gel electrophoresis or the like.

[0055] 1-3. Preparation of anti-APOA2 monoclonal antibodies 1-3-1. Method for preparing anti-APOA2 monoclonal antibodies and hybridomas Hybridomas that produce the anti-APOA2 monoclonal antibodies of the present invention can be prepared by the method described below. However, the method is not limited to this, and the antibodies can also be prepared by any other method known in the art.

[0056] (1) Method for Producing Anti-APOA2 Monoclonal Antibodies To produce an anti-APOA2 protein terminal monoclonal antibody that specifically binds to the C-terminal region of any one of the APOA2 proteins shown in SEQ ID NO: 1, 2, or 3, among the amino acid sequences constituting the APOA2 protein, a monoclonal antibody is produced using an APOA2 protein variant or a peptide containing the C-terminal region of an APOA2 protein variant as an immunogen, and then antibodies that bind only to a specific APOA2 protein variant may be screened using the APOA2 protein itself or a peptide containing the C-terminal region of an APOA2 protein variant shown in any one of SEQ ID NOs: 1 to 3. For example, anti-APOA2-ATQ terminal monoclonal antibodies can be screened using as an index whether they specifically bind to the C-terminal region of the APOA2-ATQ protein shown in SEQ ID NO: 2 (a region containing the amino acid sequence of SEQ ID NO: 31) and do not or barely bind to the APOA2 protein variant shown in SEQ ID NO: 1 or 3. Furthermore, anti-APOA2-AT terminal monoclonal antibodies can be screened using as an index the ability to specifically bind to the C-terminal region of the APOA2-AT protein shown in SEQ ID NO: 1 (a region containing the amino acid sequence of SEQ ID NO: 30) and not to bind or hardly bind to variants of the APOA2 protein shown in SEQ ID NO: 2 or 3.

[0057] Furthermore, to produce anti-APOA2 protein non-terminal antibodies that recognize amino acids other than those in the C-terminal region of the APOA2 protein, monoclonal antibodies are produced using an APOA2 protein variant or a peptide containing a partial sequence as an immunogen, and then the antibodies can be screened using as an indicator whether the binding activity is comparable when compared to an APOA2 protein variant shown in any of SEQ ID NOs: 1 to 3 or a peptide with a different C-terminus.

[0058] (2) Preparation of Cells Producing Anti-APOA2 Protein Terminal Antibody The recombinant APOA2 protein, which is the immunogen obtained in 1-2 above, is dissolved in a buffer solution to prepare an immunogen solution. At this time, an adjuvant may be added, if necessary, to ensure effective immunization. Examples of adjuvants include commercially available Freund's complete adjuvant (FCA) and incomplete Freund's adjuvant (FIA), and these may be used alone or in combination.

[0059] Next, the prepared immunogen solution is administered to a mammal, such as a rat, mouse (e.g., an inbred BALB / c mouse), or rabbit, for immunization. Methods for administering the immunogen include, but are not limited to, subcutaneous injection using FIA or FCA, intraperitoneal injection using FIA, or intravenous injection using 0.15 M sodium chloride. The single dose of immunogen is determined appropriately depending on the type of animal to be immunized, the route of administration, etc., but is approximately 50 to 200 μg per animal. The immunization interval is not particularly limited; after the initial immunization, booster immunizations are administered 2 to 6 times, preferably 3 to 4 times, at intervals of several days to several weeks, preferably 1 to 4 weeks. After the initial immunization, the antibody titer in the serum of the immunized animal is measured using ELISA (Enzyme-Linked Immunosorbent Assay) or other methods. If the antibody titer shows a sufficient increase, the immunogen is injected intravenously or intraperitoneally as the final immunization. Then, antibody-producing cells are collected 2 to 5 days, preferably 3 days, after the final immunization.

[0060] 1-3-2. Method for Producing Hybridomas Producing Anti-APOA2 Monoclonal Antibodies (1) Recovery of Antibody-Producing Cells from Immunized Animals and Cell Fusion Hybridomas producing monoclonal antibodies that specifically recognize a specific region of the APOA2 protein can be produced by cell fusion of antibody-producing cells obtained from an immunized animal with myeloma cells. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, with spleen cells or local lymph node cells being preferred. Commonly available established cell lines derived from mice or the like can be used as myeloma cells to be fused with antibody-producing cells. The cell line used preferably has drug selectivity, cannot survive in HAT selection medium (containing hypoxanthine, aminopterin, and thymine) in the unfused state, and can grow only when fused with antibody-producing cells. Furthermore, the established cell line is preferably derived from an animal of the same species as the immunized animal. Specific examples of myeloma cells include hypoxanthine guanine phosphoribosyl transferase (HGPRT)-deficient cell lines derived from BALB / c mice, such as P3X62-Ag.8 strain (ATCC TIB9), P3X63-Ag.8.U1 strain (JCRB9085), P3 / NSI / 1-Ag4-1 strain (JCRB0009), P3x63Ag8.653 strain (JCRB0028), and SP2 / 0-Ag14 strain (JCRB0029).

[0061] To fuse the above-mentioned myeloma cells with antibody-producing cells, the antibody-producing cells and myeloma cells are mixed in a ratio of approximately 1:1 to 20:1 in an animal cell culture medium such as serum-free DMEM or RPMI 1640 medium, and the fusion reaction is carried out in the presence of a cell fusion accelerator. Examples of the cell fusion accelerator include polyethylene glycol with an average molecular weight of 1,500 to 4,000 Da, at a concentration of approximately 10 to 80%. In some cases, an adjuvant such as dimethyl sulfoxide may be used in combination to enhance fusion efficiency. Furthermore, antibody-producing cells and myeloma cells can also be fused using a commercially available cell fusion device that utilizes electrical stimulation (e.g., electroporation) (Nature, 1977, Vol. 266, pp. 550-552).

[0062] (2) Selection of the desired hybridomas Hybridomas that produce the desired anti-APOA2 monoclonal antibodies can be selected from the cells after cell fusion treatment, for example, by the following method: The cell suspension is appropriately diluted with, for example, RPMI 1640 medium containing fetal bovine serum, and then plated at 2 × 10 cells per well on a 96-well microtiter plate. ６ Cells are seeded at approximately 1000 cells / well, and selective medium is added to each well. Thereafter, the selective medium is replaced as appropriate and culture is continued. The culture temperature is 20 to 40°C, preferably about 37°C. When the myeloma cells are HGPRT-deficient or thymidine kinase (TK)-deficient, selective medium (HAT medium) containing hypoxanthine, aminopterin, and thymidine can be used to selectively grow and proliferate only hybridomas between antibody-producing cells and myeloma cells. Therefore, cells that begin to grow in the selective medium about 10 days after the start of culture can be selected as hybridomas.

[0063] For hybridomas selected in HAT medium, the produced antibodies are first screened using as an index the binding activity to various variants of the APOA2 protein represented by any of SEQ ID NOS: 1 to 3. Antibodies with binding activity are then tested for cross-reactivity, and acceptable antibodies are selected. "Acceptable (cross-reactive)" means that the cross-reactivity is negligible for the intended use of the antibody. For example, in the case of a monoclonal antibody to be used in immunological assays, if the signal intensity due to cross-reaction in the final assay system is reduced from the background level to less than 1% of the signal intensity due to the specific reaction, it can be considered that the antibody does not actually cross-react.

[0064] To confirm the reaction specificity of APOA2 protein to a specific variant, for example, ELISA can be used. In ELISA, a microplate is prepared in which various APOA2 protein variants or fragments thereof serving as antigens are immobilized in different wells, and a sample of the hybridoma culture supernatant appropriately diluted is added to the wells and allowed to react. After sufficient reaction, the wells are washed, and a labeled secondary antibody against immunoglobulin is added to further react. The wells are washed again, and finally, measurement is performed using the labeled secondary antibody bound to the wells, thereby quantitatively determining the binding activity of the antibody present in the culture supernatant to the antigen. For example, to prepare an anti-APOA2 protein terminal monoclonal antibody, specificity can be determined by using as an indicator that the antibody exhibits binding activity only to the C-terminal region of a specific APOA2 protein variant and does not exhibit cross-reactivity with other APOA2 protein variants. Furthermore, to prepare an anti-APOA2 protein non-terminal monoclonal antibody, an antibody is selected based on the following indicators: it exhibits similar binding affinity to all variants of the APOA2 protein with different C-termini; and the prepared antibody does not inhibit the binding of the anti-APOA2 protein terminal monoclonal antibody to the C-terminal region.

[0065] Hybridomas can also be selected using recombinant DNA technology. First, mRNA is extracted from the group of hybridomas obtained according to the method described above. mRNA extraction can be performed using methods known in the art. Next, cDNA from the mRNA is obtained using an oligo dT primer or random primers. Using this cDNA as a template, PCR is performed using a primer set containing the nucleotide sequence of a signal sequence upstream of the gene encoding the variable region and the nucleotide sequence of the constant region. The resulting amplified product is inserted into an appropriate cloning vector and cloned to obtain a library of variable region genes for antibodies produced by the hybridomas. As a more specific, non-limiting example, PCR can be performed using Mouse Ig Primer provided by Merck Millipore, and the amplified product (mouse immunoglobulin variable region cDNA) can be cloned by inserting it into the EcoRI site of ZERO BLUNT PCR TOPO Vector provided by Life Technologies (now Thermo Fisher Scientific), and the resulting vectors can be used as a gene library encoding variable region amino acid sequences. Next, probes can be designed based on the amino acid sequences of the variable regions or each CDR disclosed in the present invention, and positive clones can be screened from the library to select hybridomas that produce antibodies to be used in the methods of the present invention.

[0066] (3) Antibody Production Using Hybridomas The hybridomas of the present invention can be used for antibody production by forming ascites in mice. Specifically, the hybridomas can be inoculated into the peritoneal cavity of nude mice or mice derived from the cells used as the fusion partner in producing the hybridomas, and the ascites fluid can be appropriately collected to recover antibody-containing ascites fluid. More specifically, hybridomas using SP2 / 0 cells as the fusion partner can be inoculated into the peritoneal cavity of BALB / c mice 10 days after inoculation with pristane to recover antibody-containing ascites fluid.

[0067] The hybridoma of the present invention can be used for antibody production by culturing it in an appropriate medium. Specifically, the hybridoma can be cultured at 1 × 10 in Hybridoma SFM medium manufactured by Life Technologies (now Thermo Fisher Scientific). ５ Hybridomas are inoculated so that the concentration is 1000 cells / mL, and cultured in a 5% CO 2 incubator at 37° C. until the hybridomas die, thereby obtaining a culture supernatant containing antibodies, although this is not a limitation.

[0068] (4) Methods for Producing Recombinant Anti-APOA2 Monoclonal Antibodies or Fragments Thereof by Recombinant DNA Manipulation The antibodies used in the present invention can also be obtained by recombinant DNA manipulation using a cDNA sequence encoding the amino acid sequence of the antibody.

[0069] Using a nucleotide sequence encoding the amino acid sequence of the variable region of an antibody derived from an anti-APOA2 monoclonal antibody-producing hybridoma, for example, an anti-APOA2 protein-terminal monoclonal antibody-producing hybridoma obtained by the method described above in "1-3-2(2)," the nucleotide sequences of the VH and VL can be linked to a nucleotide sequence encoding any human CL and human CH, respectively, and the respective polynucleotides can be incorporated into an appropriate expression vector and introduced into a host cell for expression as a complete immunoglobulin molecule. Alternatively, using CDR-grafted antibody technology, polynucleotides encoding the amino acid sequence of each CDR sequence from the amino acid sequence of the variable region of an antibody derived from an anti-APOA2 protein-terminal monoclonal antibody-producing hybridoma obtained by the method described above in "1-3-2(2)," can be ligated in a predetermined order to a polynucleotide encoding the amino acid sequence of the human FR sequence, which can then be incorporated into an appropriate expression vector and introduced into a host cell for expression as a complete immunoglobulin molecule. In this case, it is convenient to express the heavy chain and light chain in the same host cell so that they can be produced as a heavy / light chain dimer. Specifically, for example, cells can be co-transformed with a light chain expression vector and a heavy chain expression vector, and the antibody of the present invention can be obtained from the transformed cells. Alternatively, polynucleotides encoding the amino acid sequences of the variable regions can be directly incorporated into an appropriate expression vector, introduced into host cells, and then expressed as fragments of immunoglobulin molecules. Alternatively, as described above, polynucleotides encoding VL and VH, or light and heavy chains, each containing the amino acid sequences can be linked with an appropriate linker and incorporated into a phage to form a single-chain Fv, or a synthetic antibody fragment such as a diabody. Alternatively, specific antibodies can be obtained by artificially shuffling genes encoding heavy and light chains to express diversified single-chain Fv antibodies as phage fusion proteins using the recently developed phage display antibody technology (Brinkmann et al., 1995, J. Immunol. Methods, 182, 41-50; International Publication Nos. WO 97 / 13844 and WO 90-02809), which utilizes genetic engineering techniques to express recombinant antibodies on the surface of phages.

[0070] The preparation of a polynucleotide encoding a recombinant anti-APOA2 antibody or a fragment thereof, a vector incorporating the polynucleotide, and a method for introducing the vector into a host can be carried out using recombinant DNA techniques known in the art. The desired recombinant anti-APOA2 protein antibody or a fragment thereof can be obtained from the culture medium of transformed cells or from within the cells.

[0071] Examples of immunoglobulin expression vectors that can be used include, but are not limited to, plasmids, phagemids, cosmids, and viral vectors (e.g., SV40 virus-based vectors, EB virus-based vectors, and BPV-based vectors). For example, the BCMGS Neo vector, a type of BPV-based vector, is a desirable vector that efficiently expresses foreign genes when transformed into COS7 cells or the like (Hajime Karasuyama, "Bovine Papillomavirus Vectors," in Masami Muramatsu and Hiroto Okayama (eds.), Experimental Medicine Supplement: Genetic Engineering Handbook, 1991, Yodosha, pp. 297-299).

[0072] In addition to a polynucleotide encoding an antibody or a fragment thereof, the vector can include control elements (e.g., promoter, enhancer, terminator, polyadenylation site, splicing site) necessary for expressing the antibody or fragment thereof, or a selectable marker, if necessary.

[0073] As hosts for transformation, in addition to the hosts described above in "1-2. Preparation of immunogens," SP2 / 0 (mouse myeloma) cells (European Journal of Cancer Research Preview (1996) Vol. 5, 512-519; Cancer Research (1990) Vol. 50, 1495-1502) are preferably used.

[0074] In the present invention, host cells containing a vector that expresses an antibody or a fragment thereof can be cultured according to a conventional method to produce an antibody in the culture supernatant or within the host cells. Specifically, when CHO cells are used as hosts, 1 x 10 cells are cultured in a DMEM medium manufactured by Life Technologies (now Thermo Fisher Scientific). ５ The host cells were inoculated at 100 cells / mL and incubated at 37°C in 5% CO ２ By culturing in an incubator, a culture supernatant containing an antibody can be obtained. Furthermore, for example, when Escherichia coli is used as the host cell, the host cell is inoculated into a general medium used for culturing Escherichia coli, such as LB medium, and cultured, and protein expression can be induced, thereby producing an antibody in the culture supernatant or within the host cells.

[0075] When the antibody or fragment thereof that is the expression product contains a constant region, it can be purified and recovered from the culture supernatant or cell lysate using a protein A column, a protein G column, an anti-immunoglobulin antibody affinity column, or the like. On the other hand, when the antibody or fragment thereof is expressed in a state consisting only of variable regions and not containing a constant region, the above purification method cannot be applied, and other appropriate purification methods are applied. For example, if the antibody or fragment is expressed in a structure in which a tag sequence advantageous for purification, such as a histidine tag, is fused to its C-terminus, it can be purified by affinity chromatography using the corresponding ligand. When the antibody or fragment is not a fusion protein with a tag, it can be purified using standard protein purification methods such as ammonium sulfate precipitation, ion exchange chromatography, reverse-phase chromatography, gel filtration chromatography, and hydroxyapatite chromatography.

[0076] In addition, to confirm the specificity of the monoclonal antibody or fragment thereof used in the present invention to a particular variant or fragment thereof of the APOA2 protein, it is preferable to verify its cross-reactivity with other variants before use, as described above. For example, the antigens for which cross-reactivity should be confirmed for the anti-APOA2-ATQ protein terminal monoclonal antibody or fragment thereof of the present invention are the APOA2-AT protein and the APOA2-A protein.

[0077] In addition to the above proteins, it is more preferable to confirm the cross-reactivity of the antibodies or fragments thereof used in the present invention with other proteins that share a partial structure with the APOA2 protein variant. Cross-reactivity can be confirmed, for example, by using an ELISA method using the APOA2-ATQ protein as the antigen. Cross-reactivity can be confirmed by observing the competitive state between the antibody whose reaction specificity is to be tested, i.e., the anti-APOA2 protein terminal antibody, and its fragment, and the APOA2 protein variant, if another antigen protein whose cross-reactivity is to be confirmed is present in the same site. Such a method of confirming cross-reactivity, which utilizes the principle of competitive inhibition, allows for rapid screening, since it is not necessary to prepare reaction systems for all antigens.

[0078] 1-3-3. Confirmation of the structure of the APOA2 protein domain recognized by the obtained anti-APOA2 protein terminal monoclonal antibody The type of APOA2 protein variant specifically recognized by the obtained anti-APOA2 monoclonal antibody can be determined by preparing various APOA2 protein variant genes using a PCR reaction or the like based on the gene for the protein, and analyzing the binding affinity between the various APOA2 protein variants obtained from the genes and the monoclonal antibody.

[0079] Specifically, in the case of anti-APOA2 protein terminal monoclonal antibodies, the method is as follows. First, the full-length APOA2 gene or fragments of various lengths in which 6 or 9 bases, including the stop codon, are deleted from the 5'-end of the APOA2 gene from the stop codon are prepared, and expression vectors are constructed into which these fragments are inserted. The method for preparing gene fragments with such deletion mutations is described in "Continued Biochemical Experiment Course, Vol. 1, Genetic Research Methods II, pp. 289-305, edited by the Japanese Biochemical Society." Next, various APOA2 protein variants are prepared by the above-mentioned method from host cells into which the respective APOA2 protein variant expression vectors have been introduced. Subsequently, the binding ability of anti-APOA2 protein monoclonal antibodies to various APOA2 protein variants is evaluated by ELISA using these proteins as antigens. If binding is observed only to a specific variant and no or little binding is observed to other variants, the monoclonal antibody can be determined to be a terminal monoclonal antibody that specifically binds only to a specific APOA2 protein variant.

[0080] The APOA2 protein variant recognized by the obtained anti-APOA2 protein terminal monoclonal antibody can also be confirmed by the following method.

[0081] First, sequence peptides of the C-terminal regions of various APOA2 protein variants are solid-phase synthesized using known methods. Subsequently, the binding ability of anti-APOA2 protein terminal monoclonal antibodies to various peptides is evaluated by ELISA using these peptides as antigens. If binding ability of the anti-APOA2 protein monoclonal antibody is found only to a specific C-terminal region sequence peptide, the monoclonal antibody can be determined to be an anti-APOA2 protein terminal monoclonal antibody that specifically binds only to a specific APOA2 protein variant.

[0082] 1-4. Preparation of anti-APOA2 polyclonal antibodies Anti-APOA2 polyclonal antibodies can be prepared by methods known in the art. As an example, a method for obtaining an anti-APOA2 protein terminal antibody that specifically binds only to a specific variant of the APOA2 protein is specifically described below.

[0083] 1-4-1. Obtaining Antisera Anti-APOA2 protein terminal polyclonal antibodies can be produced in the same manner as in the method for producing anti-APOA2 antibody-producing cells described in 1-3-1(2). The antigen used can be a C-terminal fragment of a specific APOA2 protein variant sequence having a length of at least 6 amino acids, such as the peptide shown in SEQ ID NO: 28 or 29. Antisera containing polyclonal antibodies that recognize the APOA2 protein can be collected from the blood of the immunized animal 2 to 5 days, preferably 3 days, after the final immunization.

[0084] 1-4-2. Purification of anti-APOA2 terminal polyclonal antibodies (1) Preparation of peptide-immobilized columns Affinity columns are prepared on which the C-terminal region peptide of APOA2 protein and the peptide obtained by adding an amide group to the C-terminus of the C-terminal region peptide of APOA2 protein are immobilized. Detailed methods are described in "Anti-Peptide Antibody Experimental Protocol," 2nd Edition, Shujunsha. Supports for affinity columns include those capable of binding functional groups on the support to amino groups on peptides, such as formyl-cellulofine and CNBr agarose, or those capable of binding to cysteine ​​residues in the peptide sequence via maleimide groups covalently bound to the support. Furthermore, the length of the immobilized peptide is 6 or more amino acids, preferably 10 or more amino acids, preferably 18 or more amino acids, and more preferably 30 or more amino acids, as long as it includes the C-terminus of the APOA2 protein.

[0085] (2) Antibody Purification Anti-APOA2 protein terminal polyclonal antibodies can be purified from the antiserum using a peptide-immobilized affinity column. For example, the antiserum is diluted with an appropriate buffer, and the IgG antibodies contained in the antiserum are adsorbed onto an affinity column onto which a C-terminal region peptide of the APOA2 protein is immobilized, and the adsorbed fraction is collected. Subsequently, immunoglobulins that exhibit binding to regions other than the C-terminal region of the peptide are adsorbed and removed using an affinity column onto which a C-terminally amidated APOA2 protein peptide is immobilized. Finally, the non-adsorbed fraction is obtained as an anti-APOA2 protein terminal polyclonal antibody that specifically recognizes a specific APOA2 protein variant.

[0086] 2. Steps of the diagnostic aid method for determining whether a subject has a malignant pancreatic cystic tumor A second aspect of the method of the present invention relates to steps of a diagnostic aid method for determining in vitro whether a subject has a malignant pancreatic cystic tumor. The present invention is characterized by measuring the amount of APOA2-AT protein or APOA2-ATQ protein present in a body fluid sample obtained from a subject with a pancreatic cystic tumor, and aiding in the determination of whether the pancreatic cystic tumor is benign or malignant based on the amount.

[0087] The method of the present invention includes a step of measuring a marker for assisting in the diagnosis of malignant pancreatic cystic tumor, and a step of assisting in the diagnosis of whether a pancreatic cystic tumor is malignant or benign. Each step will be described in detail below.

[0088] 2-1. Step of measuring a marker for assisting the diagnosis of malignant pancreatic cystic tumor The "step of measuring a marker for assisting the diagnosis of malignant pancreatic cystic tumor" is a step of measuring in vitro the amount of the marker for assisting the diagnosis of malignant pancreatic cystic tumor of the present invention, i.e., the APOA2-AT protein or APOA2-ATQ protein, or a fragment thereof, present in a body fluid sample derived from a subject.

[0089] As used herein, the term "subject" refers to an individual who has been diagnosed with a pancreatic cystic tumor and for whom a determination of whether the pancreatic cystic tumor is benign or malignant is required. The presence of a pancreatic cystic tumor can be determined using imaging techniques such as CT, MRI, MRCP, and EUS. It is also known that a method for detecting pancreatic tumors that combines the blood concentrations of APOA2-AT protein and APOA2-ATQ protein can detect pancreatic cancer and benign pancreatic tumors, including pancreatic cystic tumors, with high sensitivity (Patent Document 2, Non-Patent Document 4). Such existing tumor markers may also be used alone or in combination with imaging tests. Specific examples of methods for detecting pancreatic tumors using markers include methods comprising the following steps: A method for detecting pancreatic cancer or benign pancreatic tumors by measuring the amount of an APOA2 protein variant in a body fluid sample from a subject, the method comprising the steps of (A) to (C): (A) a first step of measuring the amount of APOA2-ATQ protein in the sample using an anti-APOA2-ATQ terminal antibody that specifically binds to the carboxyl-terminal region of the APOA2-ATQ protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and an anti-APOA2-ATQ non-terminal antibody that binds to an amino acid sequence other than the carboxyl-terminal region; (B) a second step of measuring the amount of APOA2-AT protein in the sample using an anti-APOA2-AT terminal antibody that specifically binds to the carboxyl-terminal region of the APOA2-AT protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and an anti-APOA2-AT non-terminal antibody that binds to an amino acid sequence other than the carboxyl-terminal region; and (C) a third step for determining the presence or absence of pancreatic cystic tumor, in which a discriminant value obtained by inputting the measured value of the APOA2-ATQ protein amount obtained in the first step and the measured value of the APOA2-AT protein amount obtained in the second step into a predetermined discriminant formula is compared with a discriminant value of a known healthy subject. The determination of whether a subject is suffering from pancreatic cystic tumor may be carried out by an imaging test and the above-mentioned detection method, either alone or in combination.

[0090] Examples of individuals herein include vertebrates, preferably mammals, such as primates (humans, monkeys, chimpanzees, orangutans, gorillas, etc.), rodents (mice, rats, guinea pigs, etc.), and ungulates (cows, horses, sheep, goats, etc.), more preferably humans. Herein, when the subject is a human, the subject will hereinafter be referred to as a "subject."

[0091] As used herein, the term "body fluid sample" refers to a biological fluid sample used to aid in the diagnosis of malignant pancreatic cystic tumor. The body fluid sample is not particularly limited as long as it is a biological fluid that may contain the marker for aiding in the diagnosis of malignant pancreatic cystic tumor of the present invention. Examples include blood, urine, lymphocyte culture supernatant, cerebrospinal fluid, digestive fluid (e.g., pancreatic juice, colonic juice, esophageal gland secretions, and saliva), sweat, ascites, nasal discharge, tears, vaginal fluid, and semen. Blood or urine is preferred. As used herein, "blood" includes plasma and serum, and whole blood is also preferred. Whole blood may be venous, arterial, or umbilical cord blood. The body fluid sample may be a combination of two or more different samples obtained from the same individual. The method for detecting malignant pancreatic cystic tumor of the present invention is highly useful as a simple detection method because it can be used to detect malignant pancreatic cystic tumors using blood or urine, which are minimally invasive.

[0092] A "body fluid sample derived from a subject" refers to a body fluid sample that has already been collected from a subject, and the act of collecting a body fluid sample itself is not included in the embodiments of the method of the present invention. A body fluid sample derived from a subject may be subjected to the method of the present invention immediately after collection from the subject, or may be refrigerated or frozen directly after collection, or after appropriate processing, and then returned to room temperature before being subjected to the method of the present invention. If the body fluid sample is blood, appropriate processing before refrigeration or freezing includes adding heparin or the like to the collected whole blood for anticoagulation treatment followed by separation as plasma, or adding a coagulation promoter or the like to the whole blood for coagulation treatment followed by separation as serum. These processing steps may be performed based on techniques known in the art.

[0093] As used herein, the term "amount of APOA2 protein variant" refers to the respective amounts of the two APOA2 protein variants present in a body fluid sample derived from a subject. This amount may be either an absolute amount or a relative amount. In the case of an absolute amount, it refers to the mass or volume of the two APOA2 protein variants contained in a given body fluid sample. In the case of a relative amount, for example, when a standard substance is used, it refers to the relative value of the measured value of the two APOA2 protein variants derived from the subject relative to the measured value of the standard substance. Examples include concentration, fluorescence intensity, absorbance, etc.

[0094] The amount of APOA2 protein variants can be measured in vitro using known methods. For example, a measurement method using a substance that can specifically bind to each of two APOA2 protein variants can be used. Alternatively, a method of directly measuring the amount of the target protein variant can be used, such as amino acid sequencing, mass spectrometry, electrophoresis, etc. Below, a method using a specific binding substance that is widely used as a protein quantification method will be exemplified, but is not intended to limit the scope of the present invention.

[0095] As used herein, the term "capable of specifically binding" means that a substance can substantially bind only to a specific variant of the APOA2 protein that is the target of the present invention. In this case, non-specific binding may occur to an extent that does not affect the detection of the specific APOA2 protein variant.

[0096] Examples of "substances capable of specific binding" include APOA2-binding proteins. More specifically, examples include "anti-APOA2 protein terminal antibodies" that recognize and bind to APOA2 protein variants that have different structures in the C-terminal region when the antigen is an APOA2 protein variant, and preferably "anti-human APOA2 protein terminal antibodies" that recognize and bind to only one of the APOA2 protein variants when the antigen is a human APOA2 protein variant comprising the amino acid sequence of SEQ ID NO: 1, 2, or 3, or binding fragments thereof. Alternatively, the antibody may be a chemically modified derivative thereof. Here, "chemically modified derivatives" include, for example, functional modifications required for the anti-APOA2 protein terminal antibody or its antibody fragment to acquire or retain specific binding activity with a specific APOA2 protein variant, or modifications for labeling required for detecting the anti-APOA2 protein terminal antibody or its binding fragment. Functional modifications are as described above.

[0097] The antibody used for measuring the APOA2 protein variant may be either a polyclonal antibody or a monoclonal antibody. To enable specific detection, a monoclonal antibody is preferred. For example, an anti-APOA2 protein terminal polyclonal antibody that specifically binds to the APOA2 protein terminal can be prepared by the above-mentioned method.

[0098] The two APOA2 protein variants can be measured by an immunological method using an anti-APOA2 antibody that binds only to a specific variant of the APOA2 protein. Any immunological method may be used as long as an anti-APOA2 antibody is used, but an ELISA method is preferred, in which an anti-APOA2 protein terminal antibody is used as a solid-phase antibody or a labeled antibody in combination with another antibody (anti-APOA2 protein non-terminal antibody) that binds to a region other than the C-terminus of the APOA2 protein. For example, the amount of APOA2-AT protein can be measured by a sandwich ELISA method using an anti-APOA2-AT terminal antibody as a labeled antibody and an anti-APOA2-AT non-terminal antibody as a solid-phase antibody. Furthermore, the amount of APOA2-ATQ protein can be measured by a sandwich ELISA method using an anti-APOA2-ATQ terminal antibody as a solid-phase antibody and an anti-APOA2-ATQ non-terminal antibody as a labeled antibody. Anti-APOA2 protein non-terminal antibodies are commercially available from Abeam, Fitzgerald, etc., and these can also be used.

[0099] 2-2. Prevalence Determination Step The "prevalence determination step" is a step of assisting in the in vitro determination of whether a pancreatic cystic tumor is benign or malignant based on the amount of protein measured in the malignant pancreatic cystic tumor determination assisting marker measurement step. The measured malignant pancreatic cystic tumor determination assisting marker, i.e., the amount of APOA2 protein variant (the amount of APOA2-AT protein and / or APOA2-ATQ protein) in a body fluid sample from a subject suffering from a pancreatic cystic tumor, is measured to assist in the determination of malignant pancreatic cystic tumor and determine whether the pancreatic cystic tumor is benign or malignant. According to this determination step, the determination may be made using only either the amount of APOA2-AT protein or the amount of APOA2-ATQ protein, but combining the two allows for a more accurate determination of whether a pancreatic cystic tumor is benign or malignant. Each determination step is described in detail below.

[0100] Whether a pancreatic cystic tumor is benign or malignant can be determined based on the amount of APOA2-AT protein. Specifically, the amount of APOA2-AT protein in a body fluid sample from a subject is measured using an anti-APOA2-AT terminal antibody that specifically binds to the C-terminal region of the APOA2-AT protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and an anti-APOA2-AT non-terminal antibody that binds to an amino acid sequence other than the C-terminal region, and if the amount of APOA2-AT in the body fluid sample from a subject suffering from a pancreatic cystic tumor is lower than that of patients with benign pancreatic cystic tumors, it can be helpful to determine whether the subject has a malignant pancreatic cystic tumor. Here, if it can be determined that the subject is suffering from a malignant pancreatic cystic tumor, by further detailed examination such as precise imaging tests using CT, EUS, MRI, etc., cytology of cyst fluid using EUS-FNA, or pancreatic juice cytology using ERCP, it is possible to definitively diagnose the subject as suffering from a malignant pancreatic cystic tumor, and determine that the subject is a pancreatic cystic tumor patient who requires treatment such as tumor resection by surgical operation.

[0101] Whether a pancreatic cystic tumor is benign or malignant can also be determined based on the amount of APOA2-ATQ protein. Specifically, the amount of APOA2-ATQ protein is measured using an anti-APOA2-ATQ terminal antibody that specifically binds to the C-terminal region of the APOA2-ATQ protein consisting of the amino acid sequence shown in SEQ ID NO: 2 and an anti-APOA2-ATQ non-terminal antibody that binds to an amino acid sequence other than the C-terminal region, and if the amount of APOA2-ATQ in a body fluid sample from a subject suffering from pancreatic cystic tumor is higher than that of a patient suffering from benign pancreatic cystic tumor, it can be determined that the subject is suffering from malignant pancreatic cystic tumor. Here too, if it can be determined that the subject is suffering from a malignant pancreatic cystic tumor, a definitive diagnosis of the subject suffering from a malignant pancreatic cystic tumor can be made by performing detailed imaging tests such as CT, EUS, or MRI, cytology of the cyst fluid using EUS-FNA, or detailed examination using ERCP, and the subject can be determined to be a pancreatic cystic tumor patient who requires treatment such as tumor removal by surgical operation.

[0102] By determining whether a pancreatic cystic tumor is malignant based solely on the amount of APOA2-AT protein, and further determining whether a pancreatic cystic tumor is malignant based on the amount of APOA2-ATQ protein, and combining the respective results, it is possible to more accurately determine whether a pancreatic cystic tumor is benign or malignant. Specifically, by narrowing down the subjects suffering from pancreatic cystic tumors to subjects whose body fluid samples contain less APOA2-AT protein than benign pancreatic cystic tumor patients and whose APOA2-ATQ protein is greater than benign pancreatic cystic tumor patients, it is possible to increase the accuracy of determining whether the subject has a malignant pancreatic cystic tumor. On the other hand, if the amount of APOA2-AT protein in a body fluid sample from a subject suffering from pancreatic cystic tumor is equal to or less than that of benign pancreatic cystic tumor patients and the amount of APOA2-ATQ protein is equal to or greater than that of benign pancreatic cystic tumor patients, it is possible to increase the accuracy of determining whether the subject has a benign pancreatic cystic tumor.

[0103] In the present invention, the amount of APOA2-AT protein in a body fluid sample from a subject can be compared with that of patients with benign pancreatic cystic tumors using a cutoff value previously set based on the amount of APOA2-AT protein in a known group of patients with benign pancreatic cystic tumors or a group of patients with malignant pancreatic cystic tumors. For example, when the measured value of the APOA2-AT protein of the subject is equal to or less than a cutoff value set based on the amount of APOA2-AT protein in a known group of patients with benign pancreatic cystic tumors, the subject can be determined to be suffering from a malignant pancreatic cystic tumor. The cutoff value can be set using standard deviation, and is the value of "mean value - 0.5 x standard deviation (SD)" of the measured values ​​of the group of patients with benign pancreatic cystic tumors, more preferably the value of "mean value - 1.0 x SD," even more preferably the value of "mean value - 2.0 x SD," and particularly preferably the value of "mean value - 3.0 x SD." Alternatively, the cutoff value may be set using the percentile method, and is preferably the 30th percentile value, more preferably the 10th percentile value, even more preferably the 5th percentile value, and particularly preferably the 1st percentile value, of the measurements in a group of patients with benign pancreatic cystic tumors. Furthermore, when the measured value of the APOA2-AT protein in a subject is equal to or lower than a cutoff value set based on the amount of APOA2-AT protein in a known group of patients with malignant pancreatic cystic tumors, the subject can also be determined to be suffering from a malignant pancreatic cystic tumor. The cutoff value can be set using standard deviation, and is preferably the "mean value + 0.5 x SD" of the measurements in a group of patients with malignant pancreatic cystic tumors, more preferably the "mean value + 1.0 x SD" value, even more preferably the "mean value + 2.0 x SD" value, and particularly preferably the "mean value + 3.0 x SD" value. Alternatively, it may be set using the percentile method, and is preferably the 70th percentile value, more preferably the 90th percentile value, even more preferably the 95th percentile value, and particularly preferably the 99th percentile value of the measurement values ​​of a group of patients with malignant pancreatic cystic tumors.

[0104] Similarly, in the present invention, when comparing the APOA2-ATQ protein level with that of benign pancreatic cystic tumor patients, a cutoff value set based on the APOA2-ATQ protein levels in a known group of benign pancreatic cystic tumor patients or malignant pancreatic cystic tumor patients can be used. For example, when the measured value of the APOA2-ATQ protein of a subject is equal to or greater than the cutoff value set based on the APOA2-ATQ protein levels in a known group of benign pancreatic cystic tumor patients, the subject can be determined to be suffering from a malignant pancreatic cystic tumor. The standard deviation can be used to set the cutoff value, and is the value of "mean value + 0.5 x standard deviation (SD)" of the measured values ​​of the benign pancreatic cystic tumor patient group, more preferably the value of "mean value + 1.0 x SD," even more preferably the value of "mean value + 2.0 x SD," and particularly preferably the value of "mean value + 3.0 x SD." Alternatively, it may be set using the percentile method, and is preferably the 70th percentile value, more preferably the 90th percentile value, even more preferably the 95th percentile value, and particularly preferably the 99th percentile value of the measurement values ​​of a group of patients with benign pancreatic cystic tumors.

[0105] Furthermore, when the measured value of APOA2-ATQ protein in a subject is equal to or greater than a cutoff value established based on the amount of APOA2-ATQ protein in a known group of patients with malignant pancreatic cystic tumor, the subject can be determined to be suffering from malignant pancreatic cystic tumor. The cutoff value can be established using standard deviation, such as the "mean value - 0.5 x standard deviation (SD)" of the measured values ​​in the group of patients with malignant pancreatic cystic tumor, more preferably the "mean value - 1.0 x SD" value, even more preferably the "mean value - 2.0 x SD" value, and particularly preferably the "mean value - 3.0 x SD" value. Alternatively, the cutoff value may be established using the percentile method, such as the 30th percentile value, more preferably the 10th percentile value, even more preferably the 5th percentile value, and particularly preferably the 1st percentile value of the measured values ​​in the group of patients with malignant pancreatic cystic tumor.

[0106] In addition to the above method, when determining whether a subject's APOA2-AT protein amount is lower than that of benign pancreatic cystic tumor patients, statistical processing can be used, and only subjects analyzed as statistically significantly "lower" compared to the values ​​of benign pancreatic cystic tumor patients can be determined to be "lower than that of benign pancreatic cystic tumor patients." Similarly, when determining whether a subject's APOA2-ATQ protein amount is higher than that of benign pancreatic cystic tumor patients, statistical processing can be used, and only subjects analyzed as statistically significantly "higher" compared to the values ​​of benign pancreatic cystic tumor patients can be determined to be "higher than that of benign pancreatic cystic tumor patients." Statistically significant refers, for example, to a small risk factor (significance level) for the obtained value, specifically, p<0.05, p<0.01, or p<0.001. Here, "p" or "p-value" refers to the probability that an assumption will be correct by chance in a statistical test within the assumed distribution of statistics. Therefore, the smaller the "p" or "p-value," the closer the hypothesis is to the truth. "Statistically significantly different" means that there is a significant difference between the amounts of the malignant pancreatic cystic tumor determination support marker obtained from the subject and the benign pancreatic cystic tumor patient when statistically processed. When there is a statistically significant difference when compared with the benign pancreatic cystic tumor patient, the subject can be evaluated as having a malignant pancreatic cystic tumor. The statistical processing test method is not particularly limited, and any known test method capable of determining the presence or absence of significance may be used as appropriate. For example, the Student's test or multiple comparison test can be used.

[0107] The concentration of the malignant pancreatic cystic tumor determination assisting marker in a body fluid sample from a benign pancreatic cystic tumor patient or a malignant pancreatic cystic tumor patient can be measured each time the concentration of the malignant pancreatic cystic tumor determination assisting marker in a body fluid sample from a subject suffering from a pancreatic cystic tumor is measured, or a concentration of the malignant pancreatic cystic tumor determination assisting marker measured in advance can be used. In particular, if the concentrations of the malignant pancreatic cystic tumor determination assisting marker in benign pancreatic cystic tumor patients and malignant pancreatic cystic tumor patients under various physical conditions are measured in advance and the values ​​are input into a computer to create a database, then by inputting the physical conditions of the subject into the computer, the concentrations of the malignant pancreatic cystic tumor determination assisting marker in benign pancreatic cystic tumor patients or malignant pancreatic cystic tumor patients having the physical conditions most suitable for comparison with the subject can be immediately used, which is convenient.

[0108] As used herein, the term "AUC (area under the curve) value" refers to the area under a receiver operating characteristic curve (ROC curve), and is an index for measuring the accuracy of a prediction, determination, detection, or diagnostic method for dividing patients into positive and negative groups. These curves plot the value (false positive rate) obtained by subtracting the probability of a positive result in a positive patient (sensitivity) and the probability of a negative result in a negative patient (specificity) from 1 when the cutoff value of the marker is changed from a small value to a large value. The higher the AUC value, the higher the accuracy of the diagnostic method.

[0109] As used herein, "sensitivity" refers to the value of (the number of true positives) / (the number of true positives + the number of false negatives). High sensitivity reduces the risk of overlooking malignant pathology in pancreatic cystic tumor patients, leading to a comprehensive narrowing down of patients with malignant pancreatic cystic tumors.

[0110] As used herein, "specificity" means (the number of true negatives) / (the number of true negatives + the number of false positives). High specificity prevents unnecessary additional testing and treatment due to misidentification of patients with benign pancreatic cystic tumors as those with malignant pancreatic cystic tumors, thereby reducing the burden on patients and medical costs.

[0111] 3. Kit for assisting in the diagnosis of malignant pancreatic cystic tumors As used herein, the term "kit for assisting in the diagnosis of malignant pancreatic cystic tumors" refers to a kit that is used directly or indirectly to assist in the diagnosis of whether a pancreatic cystic tumor is benign or malignant. The kit of the present invention is a kit for use in the method of the present invention, and unless otherwise specified below or unless otherwise contradictory, detailed conditions such as the measurement principle are the same as those described in sections 1 and 2 above. Specifically, the kit for assisting in the diagnosis of malignant pancreatic cystic tumors of the present invention is a kit for in vitro measurement of the amount of APOA2-AT protein, which is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 30 at its carboxyl terminus, or / and APOA2-ATQ protein, which is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 31 at its carboxyl terminus, contained in a body fluid sample obtained from a subject.

[0112] The kit of the present invention is not particularly limited in its form, as long as it can detect APOA2-AT protein and / or APOA2-ATQ protein in a body fluid sample obtained from a subject, and may be, for example, a kit for immunoassays containing an antibody to an APOA2 protein variant as a component. Examples of immunoassays include enzyme immunoassays (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassays (RIA), latex agglutination, fluorescent immunoassays (FIA), chemiluminescent enzyme immunoassays (CLEIA), chemiluminescent immunoassays (CLIA), immunoturbidimetry (TIA), and immunochromatography. The kit of the present invention is particularly preferably a sandwich ELISA kit.

[0113] Specifically, the antibody against the APOA2 protein variant is an anti-APOA2 AT-terminus antibody or / and an anti-APOA2 ATQ-terminus antibody, or an antibody comprising a binding fragment thereof. In an ELISA kit, the anti-APOA2 AT-terminus antibody or the anti-APOA2 ATQ-terminus antibody may be dissolved in a buffer solution, or may be dissolved in a buffer solution and frozen or lyophilized. The antibody may also be immobilized on a solid surface such as a microwell plate or beads. Alternatively, the antibody may be labeled. In this case, in a kit containing both the anti-APOA2 AT-terminus and the anti-APOA2 ATQ-terminus antibodies, it is preferable that the two antibodies be labeled separately. The buffer solution and solid surface containing the antibodies preferably further contain a stabilizer (e.g., albumin, sugars, etc.) and a preservative (e.g., sodium azide, etc.).

[0114] The anti-APOA2-ATQ terminal antibody specifically binds to the carboxyl terminal region of the APOA2-ATQ protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and is preferably a monoclonal antibody in which the heavy chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, or 10, 11, and 12, respectively, and the light chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, or 13, 14, and 15, respectively.

[0115] The anti-APOA2-AT terminal antibody specifically binds to the carboxyl terminal region of the APOA2-AT protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and is preferably a monoclonal antibody in which the heavy chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 33, 34, and 35, 39, 40, and 41, or 45, 46, and 47, respectively, and the light chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 36, 37, and 38, 42, 43, and 44, or 48, 49, and 50, respectively.

[0116] Other components of the ELISA kit may further include antibodies, reagents, instruments, etc. necessary for detecting APOA2-AT and / or APOA2-ATQ in a body fluid sample obtained from a subject. Specific examples include a reagent solution containing an anti-APOA2 non-terminal antibody, a washing solution, and, when an enzyme label is used, a reagent solution containing an enzyme substrate. For example, when the anti-APOA2-AT terminal antibody and / or anti-APOA2-ATQ terminal antibody is immobilized on a solid phase, the anti-APOA2 non-terminal antibody is preferably labeled, and the labeled anti-APOA2 non-terminal antibody is preferably dissolved in a buffer solution to form an antibody solution.

[0117] The anti-APOA2 non-terminal antibody specifically binds to an amino acid sequence other than the carboxyl-terminal region of the APOA2-AT protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or the APOA2-ATQ protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and is preferably a monoclonal antibody in which the heavy chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, or 22, 23, and 24, respectively, and the light chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 19, 20, and 21, or 25, 26, and 27, respectively.

[0118] The kit of the present invention is not limited to an ELISA kit, and may be in other forms as described above. In ELISA and other forms, the components required for the kit are all well known to those skilled in the art.

[0119] The present invention will be explained in more detail by the following examples, but the present invention is not limited to these examples.

[0120] Example 1: Preparation of monoclonal antibodies that specifically recognize the C-terminal region of the APOA2-AT protein (a) Preparation of antibody-producing cells that recognize the C-terminal region of the APOA2-AT protein A peptide consisting of the amino acid sequence shown in SEQ ID NO: 28, which is the sequence of the C-terminal region of the APOA2-AT protein, is poorly soluble in water and has low antigenicity. Therefore, an AT peptide was synthesized in which three arginine residues were added to the N-terminus to impart hydrophilicity, and a cysteine ​​residue was further added to the N-terminus. For specificity evaluation, an ATQ peptide was synthesized in which three arginine residues were added to the N-terminus of a peptide consisting of the amino acid sequence shown in SEQ ID NO: 29, which is the sequence of the C-terminal region of the APOA2-ATQ protein.

[0121] Subsequently, KLH protein was bound to the cysteine ​​residue of the AT peptide to prepare an immunogen, which was mixed with Sigma Adjuvant System (Sigma) to give 50 to 100 μg of immunogen per mouse, and intraperitoneally administered to mice (BALB / c) five times at 2-week intervals.

[0122] Blood samples were collected periodically from the tail vein during the immunization period, and the resulting plasma was used to confirm antibody titers in the mouse blood. Proteins conjugated to bovine serum albumin were prepared in PBS at 3 μg / mL, and 50 μL of each was added to each well of an Immunoplate Maxisorp (Thermo-Fisher Scientific) and immobilized overnight. The solution was discarded, and 250 μL of Blocking One (Nacalai Tesque) was added for blocking at room temperature for at least 1 hour. After discarding the solution in the wells, the wells were washed with PBS-T (0.05% TWEEN® 20, PBS), and 50 μL of the mouse serum diluted with PBS-T was added and allowed to react for 2 hours at room temperature. After discarding the solution in the wells, the wells were washed with PBS-T, and 50 μL of Polyclonal Rabbit Anti-Mouse Immunoglobulins / HRP (GE Healthcare (now Cytiva)) diluted 5000-fold with PBS-T was added and allowed to react at room temperature for 1 hour. After washing with PBS-T, 50 μL of TMB solution (KPL) was added to carry out an enzyme reaction. 50 μL of 2N sulfuric acid solution was then added to stop the reaction, and the absorbance at 450 nm was measured. As a result, an increase in antibody titer was confirmed, so lymph nodes were removed and transplanted subcutaneously into the kidneys of immunodeficient mice (C.B.17 / SCID). Three boosts were performed, and after confirming an increase in blood antibody titer, the spleen was removed and antibody-producing cells were isolated.

[0123] (b) Recovery of antibody-producing cells from mice and cell fusion The antibody-producing cells were washed twice in RPMI 1640 medium, then mixed with Sp2 / 0 (mouse myeloma) cells and subjected to a fusion reaction using PEG 1500 (Roche Diagnostics). Subsequently, the cells were cultured in HAT medium for about one week and cloned.

[0124] (c) Selection of hybridomas producing antibodies recognizing the C-terminal region of the APOA2-AT protein Next, using the culture supernatant of hybridomas selected in HAT medium, antibodies that specifically recognize the C-terminal region of the APOA2-AT protein were selected using the difference in binding activity to the AT peptide and the ATQ peptide as an index. After adjusting the concentration of bovine serum albumin bound to the AT peptide or bovine serum albumin bound to the ATQ peptide in PBS to 3 μg / mL, 50 μL each was added to the wells of an Immunoplate Maxisorp and immobilized overnight. Screening was then performed by ELISA in the same manner as in (a) above, and three hybridoma clones, 4C6-1, 5D9-3, and 6B4-2, which reacted specifically only with the AT peptide were obtained. FIG. 1 shows the binding activity of clones 4C6-1, 5D9-3, and 6B4-2 to the APOA2-AT peptide and the APOA2-ATQ peptide.

[0125] Sequence analysis of the genes encoding the monoclonal antibodies revealed that the CDR sequences of 4C6-1, 5D9-3, and 6B4-2 were the amino acid sequences shown in SEQ ID NOs: 33 to 38, 39 to 44, and 45 to 50, respectively.

[0126] (Example 2) Detection of APOA2-AT protein by sandwich ELISA method Using the monoclonal antibodies 4C6-1, 5D9-3, and 6B4-2 obtained in Example 1, the anti-APOA2-AT terminal polyclonal antibody, and the anti-APOA2 non-terminal monoclonal antibody MAB1, detection of APOA2-AT protein was carried out by sandwich ELISA.

[0127] In this study, POD labeling of anti-APOA2-AT terminal antibody was performed using the PEROXIDASE LABELING KIT-SH (Dojindo Laboratories), and details were followed according to the accompanying protocol. Anti-APOA2 non-terminal antibody was prepared at 4 μg / mL in PBS solution, and then 100 μL was added to each well of an Immunoplate Maxisorp and immobilized overnight. The next day, the solution was discarded, and 400 μL of PBS-T was added for washing. 400 μL of blocking buffer solution (1% BSA, 0.05% TWEEN 20, PBS) was added and the plate was left to stand overnight at 4°C. The solution was then discarded, and the antibody-immobilized plate was prepared. Next, 100 μL of APOA2-AT protein serially diluted using diluent was added to each well and allowed to react at room temperature for 1 hour. After discarding the solution in the wells, the wells were washed with PBS-T, and 100 μL of POD-labeled anti-APOA2-AT terminal antibody diluted with diluent was added and allowed to react for 1 hour at room temperature. After washing with PBS-T, 100 μL of TMB solution was added to carry out the enzyme reaction. Finally, 100 μL of 2N sulfuric acid solution was added to stop the reaction, and the absorbance at 450 nm was measured.

[0128] Figure 2 shows the results of detecting APOA2-AT protein at various concentrations by sandwich ELISA using anti-APOA2 non-terminal monoclonal antibody MAB1 and anti-APOA2-AT terminal monoclonal antibody or anti-APOA2-AT terminal polyclonal antibody. As shown in Figure 2, it was confirmed that the absorbance increased in a concentration-dependent manner of APOA2-AT protein. This demonstrated that changes in APOA2AT protein concentration can be detected using ELISA.

[0129] In the following examples and comparative examples, the performance of the method for distinguishing between malignant pancreatic cystic tumors and benign pancreatic cystic tumors using APOA2-AT protein, APOA2-ATQ protein, and CA19-9 was verified using plasma obtained from patients with intraductal papillary mucinous neoplasm (IPMN), a type of pancreatic cystic tumor.

[0130] (Example 3) Determination of malignant pancreatic cystic tumor group using two APOA2 variants (i) Plasma samples from IPMN patients The concentrations of APOA2-AT protein and APOA2-ATQ protein were measured by ELISA in plasma samples collected from 15 benign IPMN patients and 7 malignant IPMN patients after obtaining informed consent.

[0131] (ii) Measurement of APOA2-AT Protein Amount The amount of APOA2-AT protein in plasma was measured using the above-mentioned plasma as the target by sandwich ELISA using an anti-APOA2-AT terminal polyclonal antibody and a POD-labeled version of the above-mentioned anti-APOA2 non-terminal monoclonal antibody MAB1. POD labeling of the anti-APOA2 non-terminal monoclonal antibody was performed using a PEROXIDASE LABELING KIT-SH, and details were followed in accordance with the attached protocol. The anti-APOA2-AT terminal polyclonal antibody was adjusted to 2 μg / mL in PBS solution, and then 100 μL was added to each well of an Immunoplate Maxisorp (NUNC) and immobilized overnight. The next day, the solution was discarded, and 400 μL of PBS-T (0.05% TWEEN 20, PBS) was added for washing. 400 μL of blocking buffer (1% BSA, 0.05% TWEEN 20, PBS) solution was added and allowed to stand at room temperature for 1 hour. The solution was then discarded, and an antibody-immobilized plate was prepared. Next, 100 μL of plasma diluted with diluent was added to each well and allowed to react at room temperature for 1 hour. The plasma was diluted 1:10,000. After discarding the antigen solution in the wells, the plate was washed with PBS-T, and 100 μL of the POD-labeled anti-APOA2 non-terminal polyclonal antibody diluted to 0.2 μg / mL with diluent was added and allowed to react at room temperature for 1 hour. After washing with PBS-T (Pierce (now Thermo-Fisher Scientific)), 100 μL of TMB solution was added to carry out the enzyme reaction, and 100 μL of 0.5 N sulfuric acid solution was added to stop the reaction, and the absorbance at 450 nm was measured. The protein concentration in the blood was calculated from the obtained measurement value using an antigen solution of the recombinant human-derived protein APOA2-AT protein as a standard.

[0132] (iii) Measurement of APOA2-ATQ Protein Amount The measurement of the APOA2-ATQ protein amount in plasma was performed using the same plasma as described above, in the same manner as in the measurement of the APOA2-AT protein amount, by sandwich ELISA using a POD-labeled anti-APOA2-ATQ terminal monoclonal antibody 7F2 and the anti-APOA2 non-terminal polyclonal antibody. POD labeling of the 7F2 antibody and sandwich ELISA were also performed in the same manner as described above. The protein concentration in blood was calculated from the obtained measurement values ​​using an antigen solution of the recombinant human-derived protein APOA2-ATQ protein as a standard.

[0133] (iv) Determination of malignant pancreatic cystic tumor group Figures 4 and 5 show the results of plotting the measured values ​​of APOA2-AT protein and APOA2-ATQ protein for each specimen as box plots, respectively. It was found that the mean APOA2-AT protein concentration was lower in the malignant group than in the benign group, and the mean APOA2-ATQ protein concentration was higher in the malignant group than in the benign group.

[0134] Table 1 shows two cutoff values ​​for APOA2-AT protein and APOA2-ATQ protein calculated from the plasma concentrations in each sample from seven malignant IPMN patients and the plasma concentrations in 15 benign IPMN patients or seven malignant IPMN patients. Cutoff value A of APOA2-AT protein calculated from the plasma concentrations in 15 benign IPMN patients was set at 39.7 μg / ml, obtained by subtracting 0.5×SD from the mean plasma concentration of 15 benign IPMN patient plasma samples. Cutoff value B of APOA2-AT protein calculated from the plasma concentrations in seven malignant IPMN patients was set at 57.9 μg / ml, obtained by adding 0.5×SD to the mean plasma concentration of 7 malignant IPMN patient plasma samples. The cutoff value A of APOA2-ATQ protein calculated from the plasma concentrations of 15 benign IPMN patients was set to 162.2 μg / ml, which was the mean value of benign IPMN patients plus 0.5×SD. The cutoff value B of APOA2-ATQ protein calculated from the plasma concentrations of 7 malignant IPMN patients was set to 120.7 μg / ml, which was the mean value of the plasma concentrations of 7 malignant IPMN patient plasma samples minus 0.5×SD.

[0135] When cutoff value A calculated from the plasma concentrations of 15 benign IPMN patients was used, in the judgment using APOA2-AT protein, 6 out of 7 malignant IPMN cases (malignant A, malignant C, malignant D, malignant E, malignant F, malignant G) were below the cutoff value and were correctly judged to be malignant, whereas in the judgment using APOA2-ATQ protein, 3 cases (malignant A, malignant C, malignant D) were above the cutoff value and were correctly judged to be malignant. Furthermore, when cutoff value B calculated from the plasma concentrations of seven malignant IPMN patients was used, in the judgment using APOA2-AT protein, six of the seven malignant IPMN cases (malignant A, malignant C, malignant D, malignant E, malignant F, and malignant G) were below the cutoff value and were correctly judged to be malignant, whereas in the judgment using APOA2-ATQ protein, five cases (malignant A, malignant C, malignant D, malignant E, and malignant F) were above the cutoff value and were correctly judged to be malignant.

[0136] (Comparative Example 1) Discrimination between malignant pancreatic cystic tumor group and benign pancreatic cystic tumor group using CA19-9 (i) Plasma samples from IPMN patients Plasma concentrations of CA19-9 were measured by ELISA for 15 benign IPMN patient cases and 7 malignant IPMN patient cases used in Example 3.

[0137] (ii) Determination of malignant pancreatic cystic tumors using CA19-9 Figure 3 shows the results of plotting the measured values ​​for each sample as a boxplot, revealing that the mean CA19-9 concentration was comparable between the benign and malignant groups. Table 1 shows the plasma concentrations of CA19-9 in each sample from seven malignant IPMN patients, as well as two cutoff values ​​calculated from the plasma concentrations of CA19-9 in 15 benign IPMN patients and seven malignant IPMN patients. Cutoff value A was set as the mean value of benign IPMN patients plus 0.5 × SD (25.3 U / ml), and cutoff value B was set as the mean value of malignant IPMN patients minus 0.5 × SD (11.1 U / ml).

[0138] When cutoff value A calculated from the plasma concentrations of 15 benign IPMN patients was used, only two of the seven malignant IPMN cases exceeded the cutoff value and were judged to be malignant (malignant C and malignant G).When cutoff value B calculated from the plasma concentrations of seven malignant IPMN patients was used, four of the seven malignant IPMN cases exceeded the cutoff value and were judged to be malignant (malignant B, malignant C, malignant F, malignant G).

[0139] From the results of Example 3 and Comparative Example 1, it became clear that both the APOA2-AT protein and the APOA2-ATQ protein can detect malignant pancreatic cystic tumors with higher sensitivity than CA19-9.

[0140]

[0141] (Comparative Example 2) Discrimination between malignant pancreatic cystic tumor group and benign pancreatic cystic tumor group using CA19-9 Following Comparative Example 1, the discrimination performance of CA19-9 was evaluated for 15 benign IPMN patient cases and 7 malignant IPMN patient cases. As a result of ROC analysis, the AUC value was 0.58, confirming that the discrimination performance was low.

[0142] Example 4: Discrimination between malignant pancreatic cystic tumor group and benign pancreatic cystic tumor group using APOA2 protein variants in blood Following Example 3, the discrimination performance of each of the markers APOA2-AT protein and APOA2-ATQ protein in blood between 15 benign IPMN patient cases and 7 malignant IPMN patient cases was evaluated. As a result of ROC analysis, the discrimination performance of APOA2-AT protein was AUC value = 0.762, and the discrimination performance of APOA2-ATQ protein was AUC value = 0.648, confirming that higher accuracy of discrimination is possible compared to the discrimination method using CA19-9.

[0143] From the results of the above examples and comparative examples, it was found that the method for assisting in the diagnosis of malignant pancreatic cystic tumors using the APOA2-AT protein and APOA2-ATQ protein of the present invention is useful for distinguishing between benign pancreatic cystic tumors and malignant pancreatic cystic tumors using non-invasive methods, which has been considered difficult to do until now.

[0144] The present invention provides a simple and non-invasive method for assisting in determining whether a pancreatic cystic tumor is benign or malignant, and enables the detection of malignant pancreatic cystic tumors that are candidates for further examination or treatment. The present invention is an invention that can be widely used in the medical and pharmaceutical fields, etc.

[0145] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method for assisting in the determination of malignant pancreatic cystic tumor in a subject suffering from pancreatic cystic tumor, comprising the steps of measuring in vitro the amount of APOA2-AT protein, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 30 at its carboxyl terminus, or / and APOA2-ATQ protein, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 31 at its carboxyl terminus, present in a body fluid sample obtained from the subject, and assisting in the determination of whether the subject's pancreatic cystic tumor is benign or malignant based on the amount.

2. The method of claim 1, wherein the pancreatic cystic tumor is an intraductal papillary mucinous neoplasm (IPMN) or a mucinous cystic neoplasm (MCN).

3. The method of claim 1 or 2, which aids in determining that a pancreatic cystic tumor is malignant when the amount of APOA2-AT protein present in a body fluid sample obtained from the subject is less than the amount of APOA2-AT protein present in a body fluid sample obtained from a subject with a known benign pancreatic cystic tumor.

4. The method of claim 3, which assists in determining that a pancreatic cystic tumor is malignant when the amount of APOA2-AT protein present in a body fluid sample obtained from the subject is less than a cutoff value established based on the amount of APOA2-AT protein present in a body fluid sample obtained from a subject with a known benign pancreatic cystic tumor or the amount of APOA2-AT protein present in a sample obtained from a subject with a known malignant pancreatic cystic tumor.

5. The method of any one of claims 1 to 4, which aids in determining that a pancreatic cystic tumor is malignant when the amount of APOA2-ATQ protein present in a body fluid sample obtained from the subject is greater than the amount of APOA2-ATQ protein present in a body fluid sample obtained from a subject with a known benign pancreatic cystic tumor.

6. The method of claim 5, which assists in determining that a pancreatic cystic tumor is malignant when the amount of APOA2-ATQ protein present in a body fluid sample obtained from the subject is greater than a cutoff value set based on the amount of APOA2-ATQ protein present in a body fluid sample obtained from a subject with a known benign pancreatic cystic tumor or the amount of APOA2-ATQ protein present in a sample obtained from a subject with a known malignant pancreatic cystic tumor.

7. The method of any one of claims 1 to 6, wherein the body fluid sample is blood, plasma, or serum.

8. The method according to any one of claims 1 to 7, wherein the subject's prevalence of pancreatic cystic tumor has been determined in advance by an imaging test and / or the following steps (A) to (C): (A) a first step of measuring the amount of APOA2-ATQ protein in a sample using an anti-APOA2-ATQ terminal antibody that specifically binds to the carboxyl-terminal region of the APOA2-ATQ protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and an anti-APOA2-ATQ non-terminal antibody that binds to an amino acid sequence other than the carboxyl-terminal region; (B) a second step of measuring the amount of APOA2-AT protein in a sample using an anti-APOA2-ATQ terminal antibody that specifically binds to the carboxyl-terminal region of the APOA2-AT protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and an anti-APOA2-AT non-terminal antibody that binds to an amino acid sequence other than the carboxyl-terminal region; (C) A third step for determining the presence or absence of pancreatic cystic tumor, in which the measured values ​​of APOA2-ATQ protein amount obtained in the first step and the measured values ​​of APOA2-AT protein amount obtained in the second step are input into a predetermined discriminant formula to obtain a discriminant value, which is then compared with the discriminant value of a known healthy subject.

9. A kit for assisting in the diagnosis of malignant pancreatic cystic tumor, for measuring in vitro the amount of APOA2-AT protein, a polypeptide containing the amino acid sequence shown in SEQ ID NO: 30 at its carboxyl terminus, or / and APOA2-ATQ protein, a polypeptide containing the amino acid sequence shown in SEQ ID NO: 31 at its carboxyl terminus, contained in a body fluid sample obtained from a subject.

10. The kit according to claim 9, comprising an anti-APOA2-AT end antibody or a binding fragment thereof, and / or an anti-APOA2-ATQ end antibody or a binding fragment thereof.

11. The kit according to claim 10, wherein the anti-APOA2-ATQ terminal antibody or binding fragment thereof specifically binds to the carboxyl-terminal region of the APOA2-ATQ protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and the heavy chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, or 10, 11, and 12, respectively, and the light chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, or 13, 14, and 15, respectively, and is an anti-APOA2-ATQ terminal monoclonal antibody or binding fragment thereof.

12. The kit according to claim 10 or 11, comprising an anti-APOA2-AT terminus monoclonal antibody or binding fragment thereof that specifically binds to the carboxyl-terminal region of the APOA2-AT protein consisting of the amino acid sequence set forth in SEQ ID NO:1, and wherein the heavy chain CDR1, CDR2, and CDR3 consist of the amino acid sequences set forth in SEQ ID NOs:33, 34, and 35, 39, 40, and 41, or 45, 46, and 47, respectively, and the light chain CDR1, CDR2, and CDR3 consist of the amino acid sequences set forth in SEQ ID NOs:36, 37, and 38, 42, 43, and 44, or 48, 49, and 50, respectively.

13. The kit according to any one of claims 10 to 12, further comprising an anti-APOA2 non-terminal monoclonal antibody or a binding fragment thereof that specifically binds to an amino acid sequence other than the carboxyl-terminal region of an APOA2-AT protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or an APOA2-ATQ protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and wherein the heavy chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, or 22, 23, and 24, respectively, and the light chain CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NOs: 19, 20, and 21, or 25, 26, and 27, respectively.

14. An anti-APOA2-AT terminal monoclonal antibody or binding fragment thereof which specifically binds to the carboxyl-terminal region of the APOA2-AT protein consisting of the amino acid sequence set forth in SEQ ID NO:1, wherein the CDR1, CDR2, and CDR3 of the heavy chain consist of the amino acid sequences set forth in SEQ ID NOs:33, 34, and 35, SEQ ID NOs:39, 40, and 41, or SEQ ID NOs:45, 46, and 47, respectively, and the CDR1, CDR2, and CDR3 of the light chain consist of the amino acid sequences set forth in SEQ ID NOs:36, 37, and 38, SEQ ID NOs:42, 43, and 44, or SEQ ID NOs:48, 49, and 50, respectively.